[go: up one dir, main page]

WO2025242835A1 - Molecules comprising masking linkers and uses thereof for the treatment of cancer - Google Patents

Molecules comprising masking linkers and uses thereof for the treatment of cancer

Info

Publication number
WO2025242835A1
WO2025242835A1 PCT/EP2025/064209 EP2025064209W WO2025242835A1 WO 2025242835 A1 WO2025242835 A1 WO 2025242835A1 EP 2025064209 W EP2025064209 W EP 2025064209W WO 2025242835 A1 WO2025242835 A1 WO 2025242835A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cytokine
peptide linker
group
multifunctional molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/064209
Other languages
French (fr)
Inventor
Amandine GEORGES
Nicolas Poirier
Aurore MORELLO
Margaux SEITÉ
Caroline Mary
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
OSE Immunotherapeutics SA
Original Assignee
OSE Immunotherapeutics SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by OSE Immunotherapeutics SA filed Critical OSE Immunotherapeutics SA
Publication of WO2025242835A1 publication Critical patent/WO2025242835A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the invention pertains to the field of medicine, in particular immunotherapy. It relates to a molecule comprising an antigen binding domain derived from an antibody and a cytokine having a pro- inflammatory effect, the molecule comprising linker(s) suitable for masking the effect of the cytokine. It further relates to the use of the molecule for the treatment of cancer.
  • Multifunctional molecules are currently the object of developments in immunology, especially in the field of oncology.
  • Such multifunctional molecules typically comprise an antigen binding domain directed to a particular target (e.g., on immune cells or tumoral cells) and a cargo (e.g., an immunoactive molecule such as a cytokine).
  • a cargo e.g., an immunoactive molecule such as a cytokine.
  • the design of multifunctional molecules implies several key attributes such as binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, compatibility with the attachment of additional domains but also production yield and cost compatible with a clinical development.
  • bifunctional molecules based on an antibody antagonizing PD-1 and linked to various interleukins (IL2, IL15, ...), or other immunotherapeutic agents have been disclosed, in particular in WO2018/184964 or W02018071919.
  • Such molecules provide the advantages to target tumor tissues thanks to the PD1-PDL1 interaction, and in the same time allow to deliver interleukins into the tumoral site.
  • optimal targeting of intra-tumoral cells remains difficult to achieve.
  • the multifunctional molecules are directed to cells expressing the receptor rather than to the tumor. To circumvent this inconvenient, different solutions have been proposed.
  • a first strategy is based on the masking of the immunotherapeutic agents by a molecule, protein or peptide, such as antigen binding domains or receptors, that have a binding affinity for the immunotherapeutic agents.
  • this strategy comprises the inclusion of additional moieties in the multifunctional molecule and the risks of insufficient demasking once onsite. Additional moieties increase the size and weight of the multifunctional molecule, affecting its pharmacokinetic characteristics and distribution, which in turn may render the multifunctional molecule less effective than a standard medication. Circumventing this issue with a double binding antibody is complicated as it needs to search, find and test multiple potential antigen binding domain for each pair of target and immunotherapeutic agent to assess efficiency and safety.
  • Other strategies are based on cleavable linkers, i.e., linkers which mask the cargo of bifunctional molecules until its arrival at a delivery site. Once onsite, the linker is cleaved to release its cargo.
  • this strategy still comprises the risks of insufficient activation in the tumor environment and of undesired activation in non-tumor tissues. Indeed, this solution has the inconvenient to be dependent to the type and quantity of proteases present in the tumor microenvironment for its efficacy.
  • cleavable linkers have been designed to allow the delivery of the effector only into the tumor.
  • the effector part of the compound thus released may generate secondary uncontrolled effects, notably for immunotherapeutic agents like cytokines that can induce toxicity.
  • WO2022/117692 discloses pH-dependent IL-2 mutants having a reduced systemic activity at neutral pH and a full activity in the tumor environment at acidic pH.
  • the applicant has developed an innovative solution allowing to control the activity of cytokine by using masking peptide linkers that are not cleavable, and thereby provides a reliable platform usable with a large array of immunotherapeutic multifunctional molecules with minimal to no modification whatsoever.
  • linker peptides according to the invention is their ability to mask a cytokine, in particular its activity and/or binding to its cognate receptor.
  • the multifunctional molecules of the invention it is the binding of the targeting moiety (e.g., antibody) to its target that enables unmasking/demasking of the cytokine and its binding to its receptor.
  • These masking and demasking properties are intrinsic to the composition/sequence of the linkers of the invention. This is why the inventor have thought of the name "cytomask” or "cytomasking" linkers when referring to the linkers of the invention.
  • Such platform allows to include molecules having an effect too important to be safely used alone, especially because of a narrow therapeutic index or an off-target systemic toxicity.
  • wild type cytokines such as IL2 can be used in the multifunctional molecule of the invention, without further modification, even though their use is usually discouraged because of their pleiotropic side effect.
  • an antigen binding domain (ABD) which targets activated immune cells, tumor cells or infected cells.
  • a multifunctional molecule of the invention comprising an anti-PDl binding domain bearing a cytokine having a pro-inflammatory effect will only be activated in the tumoral microenvironment.
  • the lack of PD1 positive cells would trigger masking the effect of the cytokine until the recognition of a PD1 positive cell.
  • non-cleavable linkers are their increased plasma stability when compared to cleavable linkers.
  • the resistance to cleavage outside of targeted cells may actually increase the specificity of drug release.
  • non-cleavable linked ADCs outperform their cleavable counterparts (indicated in Combs et al, AAPS J. 2015 Mar;17(2):339-51). It is thus believed that the peptide linkers provided herein will increase therapeutic efficiency of multifunctional molecules.
  • Another major advantage of the molecule of the invention is its capacity to act via cis-targeting (i.e., on the same cell), and exert a synergistic effect on the targeted cells.
  • Cleavable linkers by releasing the cytokine in the media, are incapable of engaging an additional target.
  • the multifunctional molecules of the invention have particularly advantageous and surprising properties: cis-masking of the pro-inflammatory cytokine until the recognition/binding of the multifunctional molecules to the targeted cells (interaction between the antigen binding domain of the multifunctional molecule with a targeted antigen on the targeted cell); cis-demasking of said pro-inflammatory cytokine consecutively to said recognition/binding, without uncontrolled liberation and/or loss of efficiency; the consecutive cis-activation of the target cell with synergy of i) the effect of the antigen binding domain (ABD) and ii) the effect of the pro-inflammatory cytokine, for instance the synergistic activation of a T cell by both i) PD1 (via the antigen binding domain of the multifunctional molecule) and ii) a cytokine (such as IL-2, IL-12, IL-15 or IL-21).
  • a cytokine such as IL-2, IL-12, IL-15 or IL-21.
  • the multifunctional molecule of the invention Compared to some molecule of the prior art, the multifunctional molecule of the invention also features a pharmacokinetic profile similar to unmasked multifunctional molecules, hence avoiding any risk of poor distribution or toxicity.
  • the invention relates to a multifunctional molecule comprising or consisting essentially of: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, said cytokine, variant or fragment being covalently linked to the N-terminal and/or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker covalently linked to the N-terminal and/or C-terminal end of the cytokine; wherein the peptide linker is 5 to 30 amino acids in length and consists of
  • the peptide linker is a masking peptide linker, particularly that allows the masking and/or demasking of the cytokine.
  • the peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; a sequence having 5 to 30 consecutive amino acids N.
  • said peptide linker comprises, essentially consists of or consists of:
  • the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12),
  • DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
  • DDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNNNNNN (SEQ ID NO: 37),
  • the peptide linker is covalently linked to the N-terminal end of the cytokine and is connecting the antigen binding domain and the cytokine.
  • Said peptide linker is called the "masking" peptide linker.
  • the multifunctional molecule comprises two peptide linkers, a first peptide linker being as described herein (i.e., a "masking peptide linker") and a second peptide linker, wherein:
  • the second peptide linker has an amino acid sequence as defined for the first linker (i.e., a "masking peptide linker");
  • the second peptide linker is identical to the first peptide linker
  • the second peptide linker is between 10 and 30 amino acids in length and consists of less than 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
  • the first peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine
  • the second peptide linker is linked to the C -terminal end of the cytokine.
  • the second peptide linker is covalently linked to the N- terminal end of the cytokine and connects the antigen binding domain and the cytokine
  • the first peptide linker is linked to the C-terminal end of the cytokine.
  • the multifunctional molecule comprises a first peptide linker comprising or consisting of EEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5) and a second peptide linker comprising or consisting of EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), preferably wherein:
  • the N-terminal end of the first peptide linker is covalently linked to the C-terminal end of the antigen binding domain, preferably in C-terminal end of a Fc domain;
  • the C-terminal end of the first peptide linker is covalently linked to the N-terminal end of the cytokine
  • the N-terminal end of the second peptide linker is covalently linked to the C-terminal end of the cytokine.
  • the multifunctional molecule comprises a single antigen binding domain, optionally a Fc domain, and a single cytokine.
  • the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of antigen binding domain and of cytokine; and b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) either
  • first peptide linker and the single cytokine or variant or fragment thereof; said first peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine; or - the first and second peptide linkers and the single cytokine or variant or fragment thereof; wherein the first peptide linker is covalently linked to the N-terminal end of the cytokine and the second peptide linker is linked to the C-terminal end of the cytokine.
  • the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15, IL-16, IL-17A, IL17B, IL-18, IL- 21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine, preferably a cytokine selected from the group consisting of IL-2, IL-12, IL-15, I L18 and IL-21 or a variant thereof.
  • the cytokine is selected from the group consisting of IL-2, I LIO, IL-12, IL-15, IL-18 and IL-21 or a variant thereof.
  • the antigen binding domain binds to a target expressed: on immune cells surface and is preferably selected from the group consisting of PD-1, BCMA/TNFRSF17, BTLA, CD101/IGSF2, CD119, CD137/4-1BB/TNFRSF9, CD150/SLAMF1, CD153, CD226, CD25, CD254, CD26, CD27, CD275/ICOSL, CD39/ENTPD1, CD40L/CD154, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3/CD183, CXCR4, CXCR5, CXCR6, FasL/TNFSF6, GITR/TNFRSF18/CD357, GPR32, TIM3/HAVCR2, ICOS, IL18Rl/CXCRl/CD218a, ITGAE/CD103, LAG3/CD223, TRAIL
  • Fibroblast Activation Protein FAP
  • FAP Fibroblast Activation Protein
  • TNC Al Al domain of Tenascin-C
  • TC A2 A2 domain of Tenascin-C
  • EDB Extra Domain B of Fibronectin
  • CEA or CEACAM Carcinoembryonic Antigen
  • MCSP Melanoma- associated Chondroitin Sulphate Proteoglycan
  • Her2/Neu human epidermal growth factor receptor 2
  • CD22, EpCAM (CD326) CDEGFR
  • PSMA Prostate Carcinoma
  • FAP Fibroblast Activation Protein
  • the antigen binding domain binds to an immune checkpoint inhibitor selected from the group consisting of PD-1, TIGIT, TIM3, LAG3, VISTA, HVEM, BTLA and CTLA-4.
  • an immune checkpoint inhibitor selected from the group consisting of PD-1, TIGIT, TIM3, LAG3, VISTA, HVEM, BTLA and CTLA-4.
  • the antigen binding domain is a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, preferably comprising or consisting a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 74 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 75; ii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 96 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 97; and iii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 104 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 105.
  • VH heavy chain variable domain
  • VL
  • the antigen binding domain is an anti-human TIGIT antibody or antigenbinding fragment thereof, preferably comprising or consisting of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 129 and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 130.
  • the invention also relates to an isolated nucleic acid molecule, a group of isolated nucleic acid molecules or a vector encoding the multifunctional molecule as described herein.
  • the invention also relates to a host cell comprising the isolated nucleic acid molecule and/or the group of isolated nucleic acid molecules and/or the vector as described herein.
  • the invention also relates to a pharmaceutical composition comprising a multifunctional molecule as described herein. Said pharmaceutical composition may further comprise an additional therapeutic agent, preferably an anti-cancer agent or anti-infection agent.
  • the invention also relates to the multifunctional molecule or the pharmaceutical composition as described herein for use as a medicament, preferably in the treatment of a cancer or an infection. It also relates to the use of the multifunctional molecule or the pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of a cancer or an infection. It further relates to a method for treating a cancer or an infection in a subject in need thereof, comprising administering a therapeutically effective amount of the multifunctional molecule or the pharmaceutical composition as described herein to said subject. Said method may further comprise the administration of an additional therapeutic agent, preferably an anti-cancer agent or anti-infection agent.
  • an additional therapeutic agent preferably an anti-cancer agent or anti-infection agent.
  • antibody describes a type of immunoglobulin molecule and is used in its broadest sense.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2) or subclass.
  • antibody includes intact immunoglobulins and "antibody fragment” or "antigen binding fragment” (such as Fab, Fab', F(ab')2, Fv), single chain (scFv or scFab), CrossMAb, mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies.
  • antibody refers to a humanized antibody.
  • CH domains in the context of IgG are as follows: “CHI” refers to positions 118-215 according to the EU index as in Kabat. "Hinge” refers to positions 216-230 according to the EU index as in Kabat. "CH2” refers to positions 231-340 according to the EU index as in Kabat, and “CH3” refers to positions 341- 447 according to the EU index as in Kabat.
  • an “antibody heavy chain” as used herein, refers to the larger of the two types of polypeptide chains present in antibody conformations.
  • the CDRs of the antibody heavy chain are typically referred to as “HCDR1”, “HCDR2” and “HCDR3”.
  • the framework regions of the antibody heavy chain are typically referred to as “HFR1”, “HFR2”, “HFR3” and “HFR4".
  • An antibody heavy chain is typically structured as follows : HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.
  • an “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations; K and X light chains refer to the two major antibody light chain isotypes.
  • the CDRs of the antibody light chain are typically referred to as “LCDR1”, “LCDR2” and “LCDR3”.
  • the framework regions of the antibody light chain are typically referred to as “LFR1”, “LFR2”, “LFR3” and “LFR4".
  • An antibody light chain is typically structured as follows : LFR1-LCDR1-LFR2-LCDR2- LFR3-LCDR3-LFR4.
  • amino acid change or “amino acid modification” is meant herein a change in the amino acid sequence of a polypeptide.
  • amino acid modifications include substitution, insertion and/or deletion in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • amino acid insertion or “insertion” is meant the addition of an amino acid at a particular position in a parent polypeptide sequence.
  • amino acid deletion or “deletion” is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative.
  • the first amino acid in the amino acids sequence i.e., starting from the N terminus
  • a conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and/or hydrophobicity).
  • R-group residue having a side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:
  • sequence identity between two sequences is described by the parameter “sequence identity”, “sequence similarity” or “sequence homology”.
  • sequence identity is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods in the art, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle” for pairwise sequence alignment of proteins providing by EMBL-EBI and available on: www.ebi.ac.
  • Sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with the default settings.
  • disorder refers to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors.
  • these terms refer to a health disorder or disease e.g., an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to immune and/or inflammatory diseases that affect animals and/or humans, such as cancer.
  • agonist refers to a substance that activates the functionality of an activating receptor. Particularly, this term refers to an antibody that binds to a cellular activating receptor as a reference substance, and have at least partially the same effect of the biologically natural ligand (e.g., inducing the activator effect of the receptor).
  • antagonist refers to a substance that binds to a specific target, typically a receptor or a protein, and inhibits or blocks its normal biological activity.
  • this term refers to an antibody that binds to a receptor or protein as a reference substance, and have at least partially the same effect of the biologically natural ligand (e.g., inducing the inhibition of the receptor).
  • this term refers to an antibody that binds to a cellular receptor as a reference substance (e.g. biologically natural ligand of the receptor), preventing it from producing all or part of its usual biological effects (e.g., thereby inhibiting the effect of the receptor when it is bound by its biologically natural ligand).
  • trans-targeting and “trans-activity” refer to the capacity of a multifunctional molecule to bond to at least two different targets expressed on different cells and/or cell types.
  • cis-targeting and “cis-activity” refer to the capacity of a multifunctional molecule to bond to at least two different targets expressed on the same cell.
  • PK Pharmacokinetics
  • PD pharmacodynamics
  • PK refers to the body's biological response to drugs.
  • PK describes a drug's exposure by characterizing absorption, distribution, bioavailability, metabolism, and excretion as a function of time.
  • PD describes drug response in terms of biochemical or molecular interactions.
  • PK and PD Analyses are used to characterize drug exposure, predict and assess changes in dosage, estimate rate of elimination and rate of absorption, assess relative bioavailability / bioequivalence of a formulation, characterize intra- and inter-subject variability, understand concentration-effect relationships, and establish safety margins and efficacy characteristics.
  • improving PK it is meant that one of the above characteristics is improved, for example, such as an increased half-life of the molecule, in particular a longer serum half-life of the molecule when injected to a subject.
  • Pharmacokinetics and “PK” are used interchangeably and refer to the fate of compounds, substances or drugs administered to a living organism.
  • Pharmacokinetics particularly comprise the ADME or LADME scheme, which stands for Liberation (i.e., the release of a substance from a composition), Absorption (i.e., the entrance of the substance in blood circulation), Distribution (i.e., dispersion or dissemination of the substance through the body) Metabolism (i.e., transformation or degradation of the substance) and Excretion (i.e., the removal or clearance of the substance from the organism).
  • ADME Adsorption
  • Distribution i.e., dispersion or dissemination of the substance through the body
  • Metabolism i.e., transformation or degradation of the substance
  • Excretion i.e., the removal or clearance of the substance from the organism.
  • the two phases of metabolism and excretion can also be grouped together under the title elimination.
  • pharmacokinetics parameters can be monitored by the man skilled in the art, such as elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma cleared of the drug per unit time), Cmax (Maximum serum concentration), and Drug exposure (determined by Area under the curve, see Scheff et al, Pharm Res. 2011 May;28(5):1081-9) among others.
  • the term “isolated” indicates that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment.
  • the term “and/or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.
  • a or “an” can refer to one of or a plurality of the elements it modifies (e.g., "a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
  • a multifunctional molecule refers to a molecule that comprises the recited elements (e.g., antigen binding domain(s), immunoactive molecule(s) and peptide linker(s)), and optionally includes other elements that do not particular interfere with the structure or function of the molecule, such as peptide spacers.
  • elements e.g., antigen binding domain(s), immunoactive molecule(s) and peptide linker(s)
  • other elements that do not particular interfere with the structure or function of the molecule, such as peptide spacers.
  • At least one means "one or more” or “one or several”. For instance, it refers to one, two, three or more.
  • the multifunctional molecules envisioned herein comprise or consist of one or more antigen binding domain(s), one or more cytokine(s) having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, and one or more peptide linker(s).
  • the terms "multifunctional molecule” or “polyfunctional molecule” are interchangeable and refer to hybrid or conjugated drugs or as chimeric drugs from two or more drugs having different pharmacological activities.
  • the multifunctional molecule can be a bifunctional molecule, in which a first function is hold by the pro-inflammatory cytokine(s) and a second function is hold by the antigen binding domain(s) derived from an antibody.
  • the multifunctional molecule can present additional functions such as another binding moiety, another immunoactive molecule or any other active function such as a cytotoxic agent, a antiproliferative agent or an pro-inflammatory agent that have additional or different pharmacological activities.
  • the multifunctional molecule of the invention does not comprise a moiety able to bind the cytokine.
  • the multifunctional molecule does not comprise a moiety that presents a binding affinity to the cytokine, such as an antibody-antigen or receptor-ligand interaction.
  • Such moiety can be selected from the group consisting of molecules, peptides, proteins, antibodies, antigen binding domain, receptors and fragment thereof that have a binding affinity for the cytokine.
  • the multifunctional molecule of the invention does not comprise an antigen binding domain that is able to bind the cytokine comprised in the multifunctional molecule, especially such as a Fab, a scFv or a VHH.
  • the multifunctional molecule of the invention when the multifunctional molecule of the invention comprises IL-2, the multifunctional molecule does not comprise an antigen binding domain, such as an scFv that is able to bind/recognize IL-2. Indeed, in the multifunctional molecule of the invention, it is the peptide linker that is responsible for the masking/demasking of the cytokine. Typically, the multifunctional molecule does not comprise an additional masking moiety or domain (such as an antigen binding domain able to bind the cytokine). Preferably, the multifunctional molecule does not comprise a moiety that present a binding affinity equal or lower than IO -6 M, IO -7 M, 10' 8 M, 10' 9 M or IO 10 M for the cytokine.
  • the multifunctional molecule does not comprise an anti-IL2 antigen binding domain.
  • the multifunctional molecule does not comprise a receptor or a fragment thereof that is able to interact with the cytokine.
  • the multifunctional molecule does not comprise IL-2R or a fragment thereof able to bind IL-2.
  • the multifunctional molecule comprises or consists essentially of: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, said cytokine, variant or fragment being covalently linked to the N-terminal and/or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker (i.e., a "masking" peptide linker) covalently linked to the N- terminal and/or C-terminal end of the cytokine; wherein the peptide linker is 5 to 30 amino acids in length and consists of
  • the multifunctional molecule may comprise or essentially consist of: one or more antigen binding domain(s), one or more cytokine(s), variant(s) or fragment(s) thereof having a pro-inflammatory effect covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain(s), and one or more peptide linker(s) covalently linked to the N-terminal end and/or C-terminal end of the cytokine(s).
  • the peptide linkers can be identical or different but at least one peptide linker is a "masking" peptide linker as defined above with the ratio of amino acids (i.e., at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
  • amino acids i.e., at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
  • the multifunctional molecule comprises at least one cytokine or variant or fragment thereof having a pro-inflammatory effect covalently linked to at least one antigen binding domain, wherein one or two peptide linker(s) are covalently linked to the cytokine.
  • the linkers are linked to the cytokine so as the latter is flanked by a first linker at one end and by a second linker at the other end and at least one of them is a "masking" peptide linker.
  • the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain(s), one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain(s) or the Fc domain, and one to eight peptide linker(s) covalently linked to the N-terminal end and/or C-terminal end of the cytokine(s) or variant(s) or fragment(s) thereof, wherein each cytokine is linked to at least one peptide linker as defined above as a "masking" peptide linker (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S,
  • the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain(s), one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain(s) or the Fc domain, and two to eight peptide I inker(s), wherein a first linker is covalently linked to the N-terminal end of the cytokine(s), variant(s) or fragment(s) thereof and a second linker is linked to the C-terminal end of the cytokine(s), variant(s) or fragment(s) thereof (i.e., two peptide linkers per cytokine) but with at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic
  • the multifunctional molecule may comprise an IgG antibody linked to one, two, three or four cytokine(s), variant(s) or fragment(s) thereof.
  • the cytokine(s) can be the same or different.
  • the C-terminal end of the IgG antibody chain are covalently linked to the cytokine, preferably to the N-terminal end of the cytokine, preferably through a peptide linker.
  • Such multifunctional molecule particularly comprises:
  • a first peptide linker covalently linked to N-terminus of each of the cytokines and a second peptide linker linked to the C-terminus of each of the cytokines, said peptide linkers being the same or different and with at least one peptide linker being a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
  • the multifunctional molecule comprises an antigen binding domain which is an antibody, in particular an IgG antibody
  • said antibody is covalently linked to a cytokine or variant or fragment thereof at the C-terminal end of one light chain, at the C-terminal end of both light chains, at the C-terminal end of one heavy chain, at the C-terminal end of both heavy chains, at the C-terminal end of one light chain and at the C-terminal end of one heavy chain, at the C-terminal end of one light chain and at the C-terminal end of both heavy chains, at the C-terminal end of both light chains and at the C-terminal end of one heavy chain, or at the C-terminal end of both light chains and at the C- terminal end of both heavy chains of said antibody.
  • the cytokine is covalently linked to the antibody at the C-terminal end of one or both heavy chains.
  • Such antibody may comprise heterodimeric Fc domains which promote heterodimeric formation and/or facilitate purification of heterodimers over the homodimers.
  • the multifunctional molecule may comprise a diabody (comprising two chains including each a VH domain and a VL domain of an antibody) linked to one or two cytokine(s), variants or fragment(s) thereof.
  • the multifunctional molecule may comprise a diabody in which each of the two chains are linked to a cytokine or a variant or fragment thereof (i.e., the multifunctional molecule comprises two cytokines, said cytokines being the same or different).
  • the C- terminal end of the diabody's chain is covalently linked to the N-terminal end of a cytokine, preferably by a peptide spacer or a peptide linker of the invention.
  • Such multifunctional molecule may comprise:
  • peptide linkers covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end, and another peptide linker linked to the C-terminus of the cytokine, said peptide linkers being the same or different;
  • At least one peptide linker is a "masking peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
  • the multifunctional molecule may comprise a diabody in which only one of the two chains is linked to the cytokine, preferably by a peptide linker or spacer.
  • the cytokine can be linked to two diabodies, said diabodies can be the same or different and being placed either on each side of the cytokine or both at one side.
  • the multifunctional molecule comprises only one antigen binding domain, such as a Fab, a scFv or a VHH.
  • the multifunctional molecule comprises only one cytokine or variant or fragment thereof.
  • the multifunctional molecule comprises only one antigen binding domain and only one cytokine or variant or fragment thereof.
  • the multifunctional molecule may particularly comprise, essentially consist or consist of: a single antigen binding domain, optionally a Fc domain linked to the C terminal end of the antigen binding domain, a single cytokine or variant or fragment thereof covalently linked to N-terminus and/or C- terminus to the antigen binding domain or Fc domain, and i) one peptide linker covalently linked to the N-terminal end or C-terminal end of the cytokine; or ii) two peptide linkers; wherein a first linker is covalently linked to the N-terminal end of the cytokine and a second linker is linked to the C-terminal end of the cytokine, wherein at least one peptide linker is a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from
  • the multifunctional molecule is a polypeptide or protein produced as a recombinant protein. Accordingly, the multifunctional molecule is a fusion protein.
  • the multifunctional molecule from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a "masking" peptide linker as defined herein and a cytokine or a variant or fragment thereof.
  • the multifunctional molecule from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a cytokine or a variant or fragment thereof and a "masking" peptide linker as defined herein .
  • the multifunctional molecule from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a first peptide linker, a cytokine or a variant or fragment thereof and a second peptide linker, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein.
  • the multifunctional molecule from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a peptide spacer, a cytokine or a variant or fragment thereof and a "masking" peptide linker as defined herein.
  • the multifunctional molecule when the multifunctional molecule comprises a single antigen binding domain, the multifunctional molecule comprises an antigen binding domain covalently linked, optionally through a peptide spacer, by its C-terminal end to N-terminal end of a first Fc chain, a complementary second Fc chain forming with the first Fc chain a Fc domain, said first Fc chain being covalently linked by its C-terminal end to the cytokine or variant or fragment thereof and a "masking" peptide linker as defined herein being either between the first Fc chain and the cytokine or variant or fragment thereof or at the C-terminal end of the cytokine or variant or fragment thereof.
  • the complementary second Fc chain is devoid of binding domain, devoid of cytokine or devoid of a binding domain and of a cytokine.
  • the antigen binding domain is a Fab domain, a Fab', a single-chain variable fragment (scFV) or a single domain antibody (sdAb).
  • Such multifunctional molecule comprises:
  • peptide linker as defined herein covalently linked to N-terminus or C-terminus of the cytokine; or - one peptide linker covalently linked to N-terminus of the cytokine and another peptide linker linked to the C-terminus of the cytokine, said peptide linkers being the same or different and at least one of them being a "masking" peptide linker as defined herein; or
  • the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv)
  • peptide linker as defined herein and a single cytokine or variant or fragment thereof; said peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine; or
  • first and second peptide linkers being a "masking" peptide linker as defined herein, optionally both; or
  • a peptide spacer is covalently linked to the N-terminus of the cytokine and a peptide linker is linked to the C-terminus of the cytokine.
  • the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first linker is covalently linked to the C-terminus of the second Fc chain and to the N-terminus of the cytokine and a second linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein, optionally both.
  • entity refers to part or moiety of the multifunctional molecule.
  • the multifunctional molecule when the multifunctional molecule comprises a single antigen binding domain, the multifunctional molecule comprises an antigen binding domain which is a single-chain variable fragment (scFV), covalently linked, optionally through a peptide spacer, by its C-terminal end to N- terminal end of a first Fc chain, a complementary second Fc chain forming with the first Fc chain a Fc domain, said first Fc chain being covalently linked by its C-terminal end to the cytokine or variant or fragment thereof and a "masking" peptide linker as defined herein being either between the first Fc chain and the cytokine or variant or fragment thereof or at the C-terminal end of the cytokine or variant or fragment thereof.
  • the complementary second Fc chain is i) devoid of binding domain, ii) devoid of cytokine or iii) devoid of a binding domain and of a cytokine.
  • the multifunctional molecule comprises or consists of: a) an antigen binding domain which is a single-chain variable fragment (scFV), b) optionally a Fc domain; and c) two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first linker is covalently linked to the C-terminus of the single-chain variable fragment and to the N- terminus of the cytokine and a second linker is linked to the C-terminus of the cytokine, and wherein at least one of the two peptide linkers is a "masking" peptide linker as defined herein, optionally both.
  • scFV single-chain variable fragment
  • FcFV single-chain variable fragment
  • two peptide linkers and a single cytokine or variant or fragment thereof wherein a first linker is covalently linked to the C-terminus of the single-chain variable fragment and to the N- terminus of the cytokine and a second linker is linked to
  • the multifunctional molecules envisioned herein comprise or consist of one or more antigen binding domain(s), one or more cytokine(s), one or more "masking" peptide linker(s) as defined herein and one or more additional peptide linker(s).
  • the multifunctional molecule may comprise or consist of: one or more antigen binding domain(s), one or more cytokine(s) or variants or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain, optionally through a peptide spacer, one or more "masking" peptide linker(s) as defined herein covalently linked to the N-terminal end and/or C-terminal end of the cytokine, and one or more additional peptide linker(s) covalently linked to the N-terminal end and/or C- terminal end of the cytokine.
  • the multifunctional molecule comprises at least one cytokine or variant or fragment thereof covalently linked to at least one antigen binding domain, wherein one peptide linker is covalently linked to the cytokine and one additional peptide linker is covalently linked to the cytokine, wherein at least one of the peptide linkers is a "masking" peptide linker as defined herein, optionally both.
  • the linkers are linked to the cytokine so as the latter is flanked by a first linker (the peptide linker or the additional peptide linker) at one end and by a second linker (the additional peptide linker or the peptide linker, respectively) at the other end.
  • the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain, one to four cytokine(s) or variant(s) or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain or the Fc domain, and one to four peptide I inker(s), covalently linked to the N-terminal end and/or C-terminal end of the cytokine, and one to four additional peptide linker(s), covalently linked to the N-terminal end and/or C- terminal end of the cytokine, wherein a first linker (peptide linker or additional peptide linker) is covalently linked to the N-terminal end of the cytokine and a second linker (additional peptide linker or peptide linker, respectively) is linked to the C-terminal end of the cytokine,
  • Examples of the multifunctional molecules are particularly provided herein below under the section "Examples of multifunctional molecules”.
  • the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures A, B, Cl, C2, C3, C4, C5, C6, C7, C8 and of Figure 1C.
  • the multifunctional molecule envisioned herein comprises one or more peptide linker(s) and at least one "masking" peptide linker as defined herein.
  • the peptide linker is linked to N-terminus and/or C- terminus of the cytokine.
  • masking peptide linker refers to a short peptide sequence that is strategically designed and incorporated into a larger protein molecule such as an antibody or multifunctional molecule disclosed herein to hide or mask specific functional proteins or epitopes such as a cytokine.
  • Masking peptide linker generally allow avoiding immunogenicity, controlling activation and/or stabilization of the masked proteins or epitopes. In the context of the invention, the masking peptide linker allows the masking or inactivation of the cytokine until the antigen binding domain binds to its target.
  • the "masking peptide linker" is typically both able to mask the cytokine or the cytokine activity and/or is able to unmask or demask the cytokine or cytokine activity, especially when the antigen biding domain of the multifunctional molecule binds to its target.
  • the peptide linker of the invention allows to mask and/or unmask or demask the cytokine, preferably the cytokine activity, even more preferably the binding of the cytokine to its receptor.
  • covalently linked or “covalently bound”, it is meant a chemical bond that involves the sharing of electrons to form electron pairs between atoms between two moieties, e.g., a cytokine and a peptide linker.
  • covalent links are not reversible.
  • the covalent links are amide bond.
  • non-cleavable peptide linker refers to an amino acid sequence that is designed to remain intact without being cleaved or broken down under certain conditions, such as physiological conditions (e.g. pH), enzymatic or chemical cleavage.
  • Such peptide linkers typically ensure that the linked molecules (e.g., an antigen binding domain and a cytokine such as disclosed herein) remain connected throughout the intended application, whether it is in vivo or in vitro.
  • non-cleavable peptide linkers do not comprise a metalloprotease cleavage site.
  • the masking peptide linker particularly does not specifically bind to or recognize the cytokine to which it is covalently linked.
  • the number of peptide linker(s) may vary depending on the number of cytokine(s).
  • the molecule comprises one or two peptide linkers per cytokine, but at least one "masking" peptide linker as defined herein.
  • the molecule comprises two "masking" peptide linkers as defined herein per cytokine.
  • the multifunctional molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 peptide linker(s).
  • the multifunctional molecule may comprise one or two peptide linker(s) if the multifunctional molecule comprises one cytokine, and at least one "masking" peptide linker as defined herein.
  • the multifunctional molecule may comprise two to four peptide linkers if the multifunctional molecule comprises two cytokines, and at least two "masking" peptide linkers as defined herein.
  • the multifunctional molecule may comprise three to six peptide linkers if the multifunctional molecule comprises three cytokines, and at least three "masking" peptide linkers as defined herein.
  • the multifunctional molecule may comprise four to eight peptide linkers if the multifunctional molecule comprises four cytokines, and at least four "masking" peptide linkers as defined herein.
  • the multifunctional molecule comprises at least one "masking" peptide linker by cytokine, said "masking" peptide linker being 5 to 30 amino acids in length and consists of
  • the peptide linker comprises a ratio or percentage of the amino acids, it is meant that the number of occurrences of this particular amino acid or these particular amino acids to the total number of amino acids in the peptide linker.
  • the percentage of acidic or amidic amino acids is 100% (4 D + 16 E /20 aa)
  • the percentage of basic amino acids is 0% and there is no remaining amino acids.
  • a peptide linker EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), the percentage of acidic or amidic amino acids is 80% (16 E /20 aa), the percentage of basic amino acids is 0% and 100% of the remaining amino acids are A.
  • the percentage of acidic or amidic amino acids is 80% (12 E /15 aa)
  • the percentage of basic amino acids is 20% (3 K /15 aa)
  • linker peptide comprises 5, 10 or 20% basic amino acids selected from the group consisting of H, K and R, it has to comprise at least 15, 30 or 60% of acidic or amidic amino acids; respectively.
  • the peptide linker comprises at least 5, 6, 7, 8, 9 or 10 amino acids.
  • the peptide linker is from 5 to 30 amino acids in length or 10 to 25 amino acids in length, preferably from 10 to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids in length. Even more preferably, the peptide linker is from 10 to 20 amino acids in length.
  • the peptide linker is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • the peptide linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.
  • the peptide linker is 15 amino acids in length.
  • the "masking" peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50, 60, 70, 80 or 90% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; and a sequence having 5 to 30 consecutive amino acids N.
  • a peptide linker EEEEDEEEEDEEEEEEDEEEED (SEQ ID NO: 15) the percentage of polar, amidic and acidic amino acids is 100% (4 D + 16 E /20 aa) and 0% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T.
  • the percentage of polar, amidic and acidic amino acids is 80% (16 E /20 aa) and 20% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T (4 A /20 aa).
  • a sequence has at least 50, 60, 70, 80 or 90% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and 10, 20, 30, 40 or 50% of remaining amino acids are independently selected from the group consisting of G, P, A, V, S, and T.
  • the "masking" peptide linker comprises, essentially consists of or consists of:
  • the "masking" peptide linker(s) envisioned herein essentially consist(s) of or consist(s) of the same amino acid residue selected in the group consisting of Asparagine (N), Glutamate (E) and Aspartic acid (D).
  • the same amino acid is selected from the group consisting of the group consisting of N, E and D. More preferably, the same amino acid is selected from the group consisting of E, D and N. Even more preferably, the same amino acid is E.
  • the multifunctional molecule comprises at least one "masking" peptide linker having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% of the amino acid E, D and N, preferably E.
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ. ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), 1
  • EEEEDEEEED SEQ ID NO: 13
  • EEEEDEEEEDEEEED SEQ ID NO: 14
  • EEEEDEEEEDEEEEEEDEEEED SEQ ID NO: 15
  • EDDDDEDDDDEDDDD SEQ ID NO: 41
  • EEEETEEEET SEQ ID NO: 19
  • EEEETEEEETEEEET SEQ ID NO: 20
  • EEEETEEEETEEEETEEEET SEQ ID NO: 21
  • ETETETETETETETETE SEQ ID NO: 44
  • the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5) and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), optionally with 1, 2 or 3 amino acid modifications, in particular an amino acid substitution with an amino acid selected from the group consisting of D, N, Q, T, S, G, P, A, V, K, H and R, preferably D, N, Q, T, A and K, even more preferably A, T, D and N.
  • the multifunctional molecule comprises 1, 2, 3, 4, 5, 6, 7 or 8 "masking" peptide linker(s) as defined above.
  • the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41
  • the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
  • a "masking" peptide linker as defined herein is linked at the N-terminal end of the cytokine.
  • the peptide linker is covalently linked to the N-terminal end of the cytokine and is covalently linking or connecting the antigen binding domain and the cytokine.
  • the multifunctional molecule comprises only one "masking" peptide linker per cytokine.
  • the multifunctional molecule comprises two peptide linkers per cytokine, a first peptide linker being a "masking" peptide linker as described herein and a second peptide linker, wherein:
  • the second peptide linker has an amino acid sequence as defined herein for a "masking" linker
  • the second peptide linker is identical to the first peptide linker
  • the second peptide linker is between 10 and 30 amino acids in length and consists of less than 10, 20, 30, 40 or 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
  • the percentage of basic amino acids is 7% (1 H /15 aa), and the percentage of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T is 93% (14 A/15 aa).
  • the percentage of basic amino acids is 0%, and the percentage of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T is 100% (16 P + 4 S /15 aa).
  • the second or additional peptide linker consists of less than 10, 20, 30, 40 or 50% of basic amino acids and the remaining amino acids are independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
  • the second or additional peptide linker(s) is/are not cleavable or does/do not contain any protease cleavage site.
  • the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I H (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I I I
  • the peptide linkers may have the same length or they can have a length slightly different, for instance having a difference in length of at most 10 to 20%, preferably 10% to 15%, in number of amino acids.
  • the multifunctional molecule comprises two peptide linkers per cytokine and a first peptide linker being a "masking" peptide linker as defined herein is linked at the N-terminal end of the cytokine and a second peptide linker is linked at the C-terminal end of the cytokine, said second linker being as defined above.
  • the multifunctional molecule comprises two peptide linkers per cytokine and a first peptide linker is linked at the N-terminal end of the cytokine and a second peptide linker is linked at the C-terminal end of the cytokine, the first and second peptide linkers are being a "masking" peptide linker as defined herein.
  • the multifunctional molecule comprises linker(s) and a cytokine with one of the following arrangements:
  • the multifunctional molecule comprises linker(s) and a cytokine with one of the following arrangements:
  • the multifunctional molecule comprises linker(s) and a dimeric cytokine with one of the following arrangements:
  • the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, peptide linker(s) and at least one or two peptide linkers are "masking" peptide linkers selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • DDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNNNNNN (SEQ ID NO: 37),
  • the multifunctional molecule comprises at least one or two peptide linkers per cytokine selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12),
  • DDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16),
  • DDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNNNNNN (SEQ ID NO: 37),
  • the multifunctional molecule comprises one or more cytokine(s) or variants()s or fragment(s) thereof having a pro-inflammatory effect.
  • the cytokine(s) has or have a narrow therapeutic index or a systemic toxicity, especially an off-target systemic toxicity. Examples of such cytokines are provided here below.
  • the peptide linkers of the invention enable to mask and/or demask the cytokine.
  • the cytokine is not able to bind to its cognate receptor and/or does not exhibit its biological activity.
  • the cytokine is able to bind to its cognate receptor and/or is able to exhibit its biological activity.
  • the receptors and biological activity of a particular cytokine have been described in the art and are well known to the man skilled in the art.
  • the biological activity of a cytokine typically refers to the effects a cytokine exerts on target cells, tissues, or organs, especially by binding to its receptor and/or triggering intracellular signalling pathways. These activities can include regulating immune responses, inflammation, cell growth, differentiation, survival, or death.
  • cytokines for the masking technology are those that have a high systemic toxicity known such as illustrative IL2 or IL15 cytokines.
  • W02024/068705 describes the toxicity of IL2 associated to Vascular Leak Syndrom, and the insufficient results obtained even with fusion targeted molecules comprising an anti-PDl antibody and IL2 variants of the prior art and the critical need to develop new molecules with wider therapeutic index.
  • toxicity of IL-15 is illustrated for instance in Guo et al (J Immunol, 2015, 195(5):2353-64. doi:
  • the present invention is suitable to overcome this problem because the use of "masking linker” improves the therapeutic index of the cytokines.
  • the cytokine has its effect only or preferably when the ABD is bond to its target, leading to the specific activity of the cytokine.
  • the monofunctional molecule of the invention avoids interactions of the cytokine with its receptor in absence of the ABD target and reduces unintended cytokine pathway activations.
  • the cytokine has a narrow therapeutic index.
  • Therapeutic index (Tl) is the range of doses at which a medication is effective without unacceptable adverse events.
  • Narrow therapeutic index (NTI) cytokines are defined as those cytokines where small differences in dose or blood concentration may lead to dose and blood concentration dependent, serious therapeutic failures or adverse drug reactions. Serious events are those which are persistent, irreversible, slowly reversible, or life-threatening, possibly resulting in hospitalization, disability, or even death.
  • narrow therapeutic index, narrow therapeutic window, narrow therapeutic range and narrow therapeutic ratio can be used interchangeably.
  • a “narrow therapeutic index” refers to a range between a drug's therapeutic dose (e.g., producing a clinical/therapeutic effect) and its toxic dose (e.g., causing harmful side effects) that is limited (narrow).
  • a drug product such as a cytokine typically has a narrow therapeutic index when:
  • Particularly preferred cytokines beneficiating from the masking effect described herein are those having a narrow therapeutic index, in particular a therapeutic index which is the same or narrower that the therapeutic index of a cytokine selected from the group consisting of IL-2, IL-12 or IL-15.
  • the cytokine has a narrow therapeutic index as defined among the possible definition set by the FDA, in particular when there is less than a two-fold difference in median lethal dose (LD50) and median effective dose values (ED50), or there is less than a two-fold difference in the minimum toxic concentrations (MTC) and minimum effective concentrations (MEC) in the blood.
  • LD50 median lethal dose
  • ED50 median effective dose
  • MTC minimum toxic concentrations
  • MEC minimum effective concentrations
  • the cytokine having a narrow therapeutic index is a cytokine with two fold or at least two-fold difference in median lethal dose (LD50) and median effective dose (ED50) values.
  • the cytokine has a systemic toxicity, especially off-target systemic toxicity.
  • the invention also applies to such cytokines when are pro- inflammatory and exhibit systemic toxicity.
  • cytokines which are not in the scope of the present application are those for which the therapeutic index is much less critical and raises no or only low or very low problem of toxicity at usual clinical doses.
  • Cytokines are small protein messengers secreted primarily by immune cells that transmit signals between cells to regulate and orchestrate various immune and inflammatory processes within the body.
  • cytokine having a pro-inflammatory effect e.g., pro-inflammatory cytokine
  • inflammatory cytokine e.g., IL-6, ESR, WBC
  • cytokine having a pro-inflammatory effect e.g., CRP, ESR, WBC
  • CT or MRI imaging studies
  • Some specific cytokines such as interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-a), may be measured to assess inflammation in certain conditions.
  • IL-6 interleukin-6
  • TNF-a tumor necrosis factor-alpha
  • the cytokine has a stimulatory effect. More specifically, the cytokine has a stimulatory effect on cells activating the immune response. Alternatively, the cytokine can have an inhibitory effect on cells inhibiting the immune response, such as T reg.
  • the cytokine having a pro-inflammatory is an immuno-stimulating cytokine.
  • the immuno-stimulating cytokine is capable of stimulating or activating an immune cell.
  • the immune cell can be selected in the non-exhaustive list comprising B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes.
  • the immune cells are T cells, more specifically CD8+ T cells, effector T cells or exhausted T cells.
  • the immune cells are effector memory stem like T cells.
  • the immuno-stimulating cytokine can be selected from: T-cell growth factors, in particular growth factors to increase number and repertoire of naive T cells, growth factors to increase the number of dendritic cells (DCs), agonists to activate DCs and other antigen-presenting cells (APCs), adjuvants to allow and augment cancer vaccines, agonists to activate and stimulate T cells, inhibitors of T-cell checkpoint blockade, T-cell growth factors to increase the growth and survival of immune T cells, agents to inhibit, block, or neutralize cancer cell and immune cell-derived immunosuppressive cytokine.
  • the cytokine is able to activate and stimulate effector memory stem like T cell.
  • the cytokine may have a dual effect.
  • dual effect it is intended to refer to a cytokine which can have both a pro-inflammatory effect and an anti-inflammatory effect, depending on the biological context.
  • cytokines may exhibit either a pro-inflammatory effect or an anti-inflammatory effect depending on several factors.
  • IL-2 may have a pro-inflammatory effect when directed to Th through signal amplification.
  • Treg when addressed to Treg, it will activate those cells and switch them toward an anti-inflammatory pathway, thus triggering the resolution of the inflammation or the resolution of the auto-immune condition.
  • the cytokine may be mutated or altered so that the biological activity of the cytokine is altered, e.g., the biological activity is increased or decreased.
  • the cytokine is modified to decrease its biological activity, e.g., its affinity for its receptor.
  • the cytokine consists in a fragment thereof retaining the biological activity of the cytokine.
  • the cytokine or variant or fragment thereof has a size of at least 10 kDa, at least, 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa or at least 50kDa.
  • the cytokine or variant or fragment thereof has a size comprised between 10 kDa and 50 kDa, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 kDa and 20 kDa, between 20 kDa and 50 kDa, between 20 kDa and 40 kDa or between 20 kDa and 30 kDa.
  • the cytokine is from human or derived from human.
  • the cytokine is an interleukin.
  • the cytokine is a monomer.
  • the cytokine comprises a plurality of subunits.
  • the cytokine is a dimer, either a homodimer and a heterodimer, and comprises two subunits.
  • said subunits are linked together by a peptide linker or spacer as described herein.
  • the peptide linker or spacer is linked to the N-terminal end of a first subunit of the cytokine, and to the C-terminal end of a second subunit of the cytokine.
  • the peptide linker or spacer is linked to the C-terminal end of a first subunit of the cytokine, and to the N-terminal end of a second subunit of the cytokine.
  • the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15, IL-17A, IL17B, IL-18, IL-21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-6, IL-15, IL-17A, IL17B, IL-18, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of IL-2, IL-10, IL-12, IL-21, IL-22 and IL-23, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, I L18 and IL-21 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the cytokine can be selected in the list of Table D below, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • Table D List of immunostimulating or pro-inflammatory cytokines
  • the cytokine is a monomeric cytokine and is selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-15, IL-17A, IL17B, IL-18, IL-21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the cytokine is a monomeric cytokine and is selected from the group consisting of IL-2, IL-15, IL-18 and IL-21 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the cytokine is selected from the group consisting of IL-2, IL-12 and IL15 or a variant or fragment thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the immuno-stimulating cytokine is IL-2 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the immuno-stimulating cytokine is IL-12 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the immuno-stimulating cytokine is IL-15 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the immuno-stimulating cytokine is IL-18 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the immuno-stimulating cytokine is IL-21 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the cytokines with a NTI could be selected in the non-exhaustive list comprising IL-2, IL- 12, IL-15, IL-18 and IL-21.
  • the multifunctional molecule of the invention comprises a cytokines or variant or fragment thereof selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21, preferably from the group consisting of IL-2, IL-12 and IL-15.
  • the cytokine variant is a function conservative variant.
  • the cytokine variant or fragment described herein retains substantially equivalent biological property and/or function in comparison to a wild-type cytokine For instance, it retains comparable biological property/function as the full-length or wild-type protein, respectively.
  • the cytokine variant or fragment maintains biological activity of at least 10 %, 20%, 30%, 40%, 50%, 60% in comparison with the wild type cytokine.
  • the biological activity is the bind of the cytokine to its receptor and/or inhibition of said receptor.
  • the immuno-stimulating cytokine is lnterleukin-2 (IL-2), preferably a human IL-2, for example as disclosed under the UniProt accession number P60568 or a mutant or variant thereof.
  • IL-2 lnterleukin-2
  • IL-2 preferably a human IL-2, for example as disclosed under the UniProt accession number P60568 or a mutant or variant thereof.
  • the IL-2 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 50 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 50.
  • IL-2 can be mutated in various ways to reduce its toxicity and/or increase its efficacy.
  • Hu et al. (Blood 101, 4853-4861 (2003), US Pat. Publ. No. 2003/0124678) have substituted the arginine residue in position 38 of IL-2 by tryptophan to eliminate IL-2's vasopermeability activity.
  • Shanafelt et al. (Nature Biotechnol 18, 1 197-1202 (2000)) have mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells.
  • Heaton et al. (Cancer Res 53, 2597-602 (1993); US Pat. No.
  • cytokine is an IL-2 mutant for example as described in WO 2012/107417 or WO 2018/184964.
  • the IL-2 variant may comprise one or several substitutions at positions of human IL-2 (without the signal peptide; SEQ ID NO: 50) selected from the group consisting of Qll, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, SI 27 , 1129, and S130, the numbering being preferably the one of SEQ ID NO: 50.
  • the IL-2 variant may comprise the substitution relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) F42A or F42K.
  • the IL-2 variant may further comprise one or several substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) selected from the group consisting of:
  • the IL-2 variant may comprise one of the following substitutions combination relative to human IL-2 (without the signal peptide; SEQ ID NO: 50): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and V91E; R38E, F42A, and D84H; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and V91E; R38D, F42A, and D84H; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88
  • the IL-2 variant may comprise one of the following substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50), in particular at least one of the substitutions selected in the group comprising K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K; or a combination thereof, preferably the three substitutions F42A, Y45A,
  • Mutants of human IL-2 (hlL-2) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 3, 35, 38, 42, 43, 45 or 72 or combinations thereof, corresponding to residues position of human IL-2 (without the signal peptide; SEQ ID NO: 50).
  • the mutant IL-2 is a human IL-2 molecule comprising the amino acid substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) T3A, F42A, Y45A, L72G and/or C125A, preferably F42A, Y45A and L72G, more preferably T3A, F42A, Y45A, L72G and C125A, for example as disclosed in WO 2018/184964.
  • the immuno-stimulating cytokine is an IL-2 mutant having the substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) F42A, Y45A and L72G, preferably T3A, F42A, Y45A, L72G and C125A, the numbering being preferably the one of SEQ ID NO: 50.
  • the bifunctional molecule of the invention comprises an IL2 variant comprising or consisting of a sequence as set forth in SEQ ID NO:110.
  • the IL-2 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-2. Additionally or alternatively, the IL-2 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-2R, in comparison to a wild-type IL-2.
  • the immuno-stimulating cytokine is Interleukin-12 (IL-12), preferably a human IL-12, or a mutant or variant thereof.
  • IL-12 is a heterodimeric cytokine including two subunits, IL-12A and IL-12B as disclosed in Uniprot reference P29459 and P29460, respectively.
  • IL-12 may comprise IL-12A or IL-12B of SEQ ID NOs: 51 and 52 (sequences without signal peptide), respectively, or a variant thereof having a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity therewith or a sequence having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NOs: 51 and 52, respectively.
  • IL-12 may include a variant of IL-12A having one or several substitutions with respect to the wildtype IL-12A selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q, the amino acid numbering being preferably the one of SEQ ID NO: 51.
  • the variant of IL-12A has one of the following substitutions combinations: N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D, the amino acid numbering being preferably the one of SEQ ID NO: 51..
  • IL-12 may include a variant of IL-12B having one or several substitutions with respect to the wildtype IL-12B selected from the group consisting of E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D,
  • the variant of IL-12B has one of the following substitutions combinations: N103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,
  • E59K/K99E/K264E E59K/K99Y, E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E,
  • E59K/K99E/Q144K E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,
  • E59K/K99E/C252S/K264E E59K/K99E/C252S, N103D/N113D, N103D/N200D, N103D/N281D,
  • N113D/N200D N113D/N281D, N200D/N281D, N103D/N113D/N200D, N103D/N113D/N281D,
  • N103D/N200D/N281D N113D/N200D/N281D, N103Q/N113Q, N103Q/N200Q, N103Q/N281Q,
  • amino acid numbering being preferably the one of SEQ ID NO: 52.
  • IL-12 may include a variant of IL-12A and a variant of IL-12B as disclosed above.
  • the two subunits of IL-12 are covalently linked by a peptide linker or peptide spacer.
  • they are covalently linked by a "masking" peptide linker as defined herein.
  • the IL-12 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-12. Additionally or alternatively, the IL-12 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-12R, in comparison to a wild-type IL-12.
  • the immuno-stimulating cytokine is IL-15 or a variant of IL-15.
  • IL-15 is a human IL-15, for example as disclosed under the UniProt accession number P40933 or a mutant or variant thereof.
  • the IL-15 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 53 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 53.
  • the IL-15 variant may comprise one or several substitutions at positions of human IL-15 (without the signal peptide; SEQ ID NO: 53) selected from the group consisting of N1D,V3I, V3M, V3R, N4D, D8N, D8A, K11L, K11M, KIIR, D30N, D61N, E64Q, N65D, N71D, N71S, N72D, N72A, N72R, N72Y, S73I, N77A, N79D, N79E, N79S, Q108E, N112D, N112H, N112M and N112Y, preferably N4D, D61N, N65D, and Q108E, the amino acid numbering being preferably the one of SEQ ID NO: 53.
  • the IL-15 variant may comprise one of the following substitutions combination relative to human IL-15 (without the signal peptide; SEQ ID NO: 53): N4D/N65D, D30N/N65D, D30N/E64Q, D30N/E64Q/N65D, N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N1D/D61N, N1D/E64Q, N4D, D61N, N4D/E64Q, D8N/D61N, D8N/E64Q, D61N/E64Q, N1D/D30N, E64Q/Q108E, N1D/N4D/D8N, D61N/E64Q/N65Q, N1D/D61N/E64Q/Q108E, N4D/D61N, N4D/D61N/E64Q/Q108E, N4D/D61N, N4D/D61
  • the IL-15 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-15. Additionally or alternatively, the IL-15 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-15R, in comparison to a wild-type IL-15.
  • the immuno-stimulating cytokine is Interleukin-18 (IL-18), preferably a human IL-18, for example as disclosed under the UniProt accession number Q14116, or a mutant or variant thereof.
  • IL-18 Interleukin-18
  • IL-18 preferably a human IL-18, for example as disclosed under the UniProt accession number Q14116, or a mutant or variant thereof.
  • the IL-18 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 55 or has 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 55 or has 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID
  • IL-18 variants are known in the field. For instance, IL-18 variants are disclosed in W004091517, W019051015, WO22038417, WO22094473, WO22172944, WO23056193, WO23118497, WO24044780, WO24051728, the disclosure of which being incorporated herein by reference.
  • IL-18 variants may include one substitution or a set of substitutions combination selected from the group consisting of G3P, E6K, D54H, Q56D, P57R, N91V, and R104V; G3S, E6K, D54W, Q56P, P57D, N91G, and R104T; G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y; G3P, E6K, D54W, Q56G, P57V, N91V, and R104F; G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K; G3P, E6R, D54W, Q56T, N91V, and R104T; G3P, E6R, D54H, Q56T, P57A, and N91A; G3P, E6R, D54W, Q56P, P57A, N91A, and R104V; G3
  • IL-18 variant may include an N91K substitution and at least one substitution mutation selected from one of the following groups consisting of: E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C68D, C76A, M86V, N111R, N111K, S117C, C127A, and N155T; and E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N111R, N111K, S117C, C127A, and N155T, the amino acid numbering being preferably the one of SEQ. ID NO: 55.
  • IL-18 variant may include a N91K substitution and at least one substitution mutation selected from the group consisting of : E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C68D, C76A, M86V, N111R, N111K, S117C, C127A, and N155T; and E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N111R, N111K, S117C, C127A, and N155T, and any combination thereof;
  • IL-18 variants may include one substitution or a set of substitution combination selected from the group consisting of E6K, Vlll, C38A, K53A, T63A, C76A and C127A.
  • IL-18 variant includes the combination of substitution comprising or consisting of E6K, Vlll, C38A, K53A, T63A, C76A and C127A,the amino acid numbering being preferably the one of SEQ ID NO: 55.
  • the variant may have the amino acid sequence as disclosed in SEQ ID NO: 56.
  • the IL-18 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-18. Additionally or alternatively, the IL-18 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-18R, in comparison to a wild-type IL-18.
  • the immuno-stimulating cytokine is IL-21 or a variant of IL-21.
  • IL-21 is a human IL-21, for example as disclosed under the UniProt accession number Q9HBE4 or a mutant or variant thereof.
  • the IL-21 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 54 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 54.
  • L-21 variants are known in the field. For instance, IL-21 variants are disclosed in WO05035565, WO06111524, W008049920, WO08074863, W010103038, EP3885357, EP4273158, W024002170, the disclosure of which being incorporated herein by reference.
  • the IL-21 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-21. Additionally or alternatively, the IL-21 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-21R, in comparison to a wild-type IL-21.
  • the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-10 and IL-18 or a variant thereof, typically such as described here above.
  • the multifunctional molecule of the invention can comprise 1, 2, 3 or 4 cytokine molecules, that can be the same or different.
  • the multifunctional molecule comprises more than one cytokine
  • the cytokines are attached in series to one another and connected by a peptide linker or spacer according to the invention.
  • the multifunctional molecule of the invention comprises a single cytokine molecule.
  • the cytokine molecule(s) comprised in the multifunctional molecule of the invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: SO- 56, 110, 112, 118 and 128 and any variant thereof, preferably selected from the group consisting of SEQ ID NO: 50, 53, 56, 110 and 112.
  • the antigen binding domain or targeting moiety of the multifunctional molecule is an antibody or an antigen binding fragment thereof or a derivative thereof.
  • the antigen binding domain comprised in the multifunctional molecule of the invention specifically binds to a target expressed on immune cells surface, particularly targets that are only or specifically expressed on immune cells.
  • the antigen binding domain comprised in the multifunctional molecule of the invention is directed towards a target expressed on tumoral cells.
  • the antigen binding domain is not directed towards a target expressed on tumoral cells.
  • the antigen binding domain comprised in the multifunctional molecule does not bind or have affinity to the cytokine.
  • an "antigen-binding fragment” or “antigen-binding domain” of an antibody means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for a particular antigen, possibly in its native form; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody.
  • the antigen-binding fragments have a similar binding affinity as the corresponding 4- chain antibodies.
  • antigen-binding fragment that have a reduced antigen-binding affinity with respect to corresponding 4-chain antibodies are also encompassed within the invention.
  • the antigenbinding capacity can be determined by measuring the affinity between the antibody and the target fragment.
  • These antigen-binding fragments may also be designated as "functional fragments" of antibodies.
  • Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof.
  • Antigen binding domain fragment typically includes Fab domain, Fab', (Fab')2.
  • Antigen binding domain derivatives typically includes single-chain variable fragment (scFV), scFab, diabody and CrossMAb.
  • the terms “bind” or “binding” refer to antibodies including antigen binding fragments thereof and derivatives that recognize and contact another peptide, polypeptide, protein or molecule.
  • the terms “specific binding”, “specifically binds to,” “specific for,” “selectively binds” and “selective for” a particular target mean that the antigen binding domain recognizes and binds a specific target, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically (or preferentially) binds to an antigen is an antibody that binds the antigen for example with greater affinity, avidity, more readily, and/or with greater duration than it binds to other molecules.
  • the term "specific binding” means the contact between an antibody and an antigen with a binding affinity equal or lower than 10’ 7 M.
  • antibodies bind with affinities equal or lower than IO -8 M, IO -9 M or IO 10 M.
  • target of the antigen binding domain refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted by the antigen binding domain according to the invention and expressed on the external surface of immune cells.
  • expression of a target on the surface of immune cells the term “expressed” refers to a target, such as carbohydrates, lipids, peptides, polypeptides, proteins, antigens or epitopes that are present or presented at the outer surface of a cell, preferably an immune cell.
  • the antigen binding domain of the multifunctional molecule can be a Fab domain, a Fab', a F(ab')2, a single-chain variable fragment (scFV), a Fv, a diabody, a scFab, a CrossMAb, a single domain antibody (sdAb) or a VHH.
  • the antigen-binding domain is a Fab domain, a single-chain variable fragment (scFV) or a VHH.
  • the antigen-binding domain is a scFV.
  • the antigen binding domain can be an antibody such as an IgG antibody that may include modifications, in particular in the Fc domain; a Fab, a Fab', a F(ab')2, a Fv, a diabody, a single domain antibody (sdAb), a VHH, a scFab, a CrossMAb or a ScFv, optionally linked to a Fc domain.
  • an antibody such as an IgG antibody that may include modifications, in particular in the Fc domain; a Fab, a Fab', a F(ab')2, a Fv, a diabody, a single domain antibody (sdAb), a VHH, a scFab, a CrossMAb or a ScFv, optionally linked to a Fc domain.
  • the antigen binding domain can be a Fab domain, a Fab', a (Fab')2, a single-chain variable fragment (scFV), a scFab, a diabody, a CrossMAb, or a single domain antibody (sdAb) or a VHH.
  • the antigen-binding domain preferably comprises a heavy chain variable region (VH) and a light chain variable region (VL).
  • the antigen-binding domain is a Fab, a single-chain variable fragment (scFV) or a VHH.
  • the antigen binding domain is a CrossMAb.
  • CrossMAb refers to antigen binding domains with an inversion of CL and CHI domains, in particular in one binding arm of antibodies.
  • binding domain comprises a VH domain linked to a CL domain and a VL domain linked to a CHI domain.
  • Such format reduces the byproduct formation caused by a mismatch of a light chain of a first binding domain that specifically binds to a first antigen with the wrong heavy chain of a second binding domain that specifically binds to a second antigen (when compared to approaches without such CL-CH1 domain exchanges).
  • CrossMAb are for example described in WO 2009/080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191, the disclosure of which being incorporated herein by reference.
  • the antigen binding domain is a recombinant antibody or an antigen binding fragment thereof.
  • the term "recombinant antibody” refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences.
  • Recombinant antibodies include, for example, chimeric and humanized antibodies.
  • the antigen binding domain is or derived from an humanized antibody.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., chimeric antibodies that contain minimal sequence derived from a non-human antibody).
  • a "humanized form" of an antibody e.g., a non- human antibody, also refers to an antibody that has undergone humanization.
  • a humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody.
  • humanized antibody has a T20 humanness score greater than 80%, 85% or 90%.
  • "Humanness" of an antibody can for example be measured using the T20 score analyzer to quantify the humanness of the variable region of antibodies as described in Gao S H, Huang K, Tu H, Adler A S. BMC Biotechnology. 2013: 13:55 or via a web-based tool to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http://abAnalyzer.lakepharma.com.
  • the antigen binding domain is or derived from an chimeric antibody.
  • chimeric antibody an antibody made by combining genetic material from a nonhuman source, preferably such as a mouse, with genetic material from a human being. Such antibody derives from both human and non-human antibodies linked by a chimeric region. Chimeric antibodies generally comprise constant domains from human and variable domains from another mammalian species, reducing the risk of a reaction to foreign antibodies from a non-human animal when they are used in therapeutic treatments.
  • the target is specifically expressed by immune cells in a healthy subject or in a subject suffering from a disease, in particular such as a cancer.
  • a disease in particular such as a cancer.
  • the target has a higher expression level in immune cells than in other cells or that the ratio of immune cells expressing the target by the total immune cells is higher than the ratio of other cells expressing the target by the total other cells.
  • the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100. More specifically, it can be determined for a particular type of immune cells, for instance T cells, more specifically CD8+ T cells, effector T cells or exhausted T cells, or in a particular context, for instance a subject suffering of a disease such as a cancer or an infection.
  • Immuno cells refers to cells involved in innate and adaptive immunity for example such as white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells and Natural Killer T cells (NKT) and myeloid-derived cells (granulocytes such as neutrophil, eosinophil, basophil, or other cells such as monocyte, macrophage, dendritic cells).
  • the immune cell can be selected in the non- exhaustive list comprising B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes.
  • T cell as used herein includes for example CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells and inhibitory T cells.
  • activated immune cells immune cells that are involved or have been activated during an immune response towards the presence of non-self cells such as pathogens or cancer cells, or toward self cells in the case of autoimmune diseases. Activated immune cells are particularly recruited in the localization wherein the inflammation, triggered by the presence of non-self cells, occurs. Particular markers of immune cells of activation that can be targeted by the antigen binding domain are particularly described here after.
  • T effector cell As used herein, the term “T effector cell”, “T eff” or “effector cell” describes a group of immune cells that includes several T cells types that actively respond to a stimulus, such as co-stimulation. It particularly includes T cells which function to eliminate antigen (e.g., by producing cytokines which modulate the activation of other cells or by cytotoxic activity). It notably includes CD4+, CD8+, cytotoxic T cells and helper T cells (Thl and Th2). As used herein, the term “Tumor Infiltrating lymphocytes (TILs)” refers to immune cells, especially ? cells, which are located in a tumor or in its stroma.
  • TILs Tums
  • Treg cells As used herein, the term "regulatory ? cell”, Treg cells” or “T reg” refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells.
  • exhaustted T cell refers to a population of T cell in a state of dysfunction (i.e., "exhaustion”). T cell exhaustion is characterized by progressive loss of function, changes in transcriptional profiles and sustained expression of inhibitory receptors. Exhausted T cells lose their cytokines production capacity, their high proliferative capacity and their cytotoxic potential, which eventually leads to their deletion. Exhausted T cells typically indicate higher levels of CD43, CD69 and inhibitory receptors combined with lower expression of CD62L and CD127.
  • effector memory stem like T cell refers to a subset of tumor-reactive intra-tumoral T cells bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcfl. These cells can be called Tcfl+PD-1+CD8+ T cells. These cells reside in the tumor microenvironment and are critical for immune control of cancer promoted by immunotherapy. They are critical for maintaining the T cell response during chronic viral infection and cancer, and provide the proliferative burst seen after PD-1 immunotherapy. These cells undergo a slow self-renewal and also give rise to the more terminally differentiated exhausted CD8 T cells.
  • immune response refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complements) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • the antigen binding domain specifically binds to a target expressed immune cells selected from the group consisting of B-cells, T-cells, Natural killer, dendritic cells, monocytes and innate lymphoid cells (ILCs).
  • a target expressed immune cells selected from the group consisting of B-cells, T-cells, Natural killer, dendritic cells, monocytes and innate lymphoid cells (ILCs).
  • the immune cell is a T cell.
  • T cell or "T lymphocytes” as used herein includes for example CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T regulator, T helper 17 type T cells and inhibitory T cells.
  • the immune cell is an exhausted T cell.
  • the immune cell is a TIL.
  • the immune cell is an effector memory stem like T cell.
  • the target can be a receptor expressed at the surface of the immune cells, especially T cells.
  • the receptor can be an inhibitor receptor.
  • the receptor can be an activating receptor.
  • the antigen binding domain binds to a target selected from the group consisting of PD-1, CD28, CD80, CTLA-4, LAG-3/CD223, BTLA, TIG IT, CD160, CD40L/CD154, ICOS, CD27, 0X40, 4-1BB, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30/TNFRSF8, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, CD28H, CD38, CXCR5, CD3, PDL2, CD4, CD8, IGSF2, CD119, CD150/SLAMF1, CD153, CD226, CD25, CD254, CD26,
  • a target selected from the group consisting of PD-1, CD28, CD80, CTLA-4, LAG-3/CD223, BTLA, TIG IT, CD160, CD40L/CD154, ICOS, CD27, 0X40, 4-1BB, HVEM, Tim-1, LFA-1, TIM3, CD39
  • CD275/ICOSL ENTPD1, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CXCR4, CXCR6, FasL, GPR32, CXCRl/IL18Rl/CD218a, ITGAE/CD103, TRAILR, OX40L, LY108, PDCD1, PTPN22, RGS1, L0X1, SIGLEC 6, TACI, CD163, CD206, LTBR, TNFSF14, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), FCRL4, TNFRSF18/GITR/CD357, CD78, TRAF1, TRAF2, TRAF3, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1, MED24, TNFR2, CDCR3,
  • the antigen binding domain binds to a target expressed on activated T cells surface, preferably selected from the group consisting of CD101/IGSF2, CD119, CD137/4-1BB/TNFRSF9, CD183/CXCR3, CD25, CD254, CD26, CD275/ICOSL, CD40L/CD154, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR6, FasL/TNFSF6, GITR/TNFRSF18/CD357, GPR32, ICOS, IL18Rl/CXCRl/CD218a, ITGAE/CD103, LY108 /SlamF6, OX40/TNFRSF4, RGS1, PD-1, LTBR/CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3/TNFRSF6B, RELT/TNFRSF19L, TNFRSF19/TRO
  • the antigen binding domain binds to a target selected from the group consisting of PD-1, TIM3, LAG3, CD101/IGSF2, CD40L/CD154, VISTA, HVEM/TNFRSF14/CD270, BTLA, TIG IT, CD137 and CTLA-4.
  • the antigen binding domain binds to a target expressed on Tumor Infiltrating lymphocytes (TILs) surface, preferably selected from the group consisting of CD101, CD137 (Tnfrsf9/4-lBBL), CRTAM, CST7, CTLA4, CXCR3/CD183, FAS, IL18R1/CXCR1/CD218A, LAG-3, PTPN22, RGS1, TNFSF14 and PD-1.
  • TILs Tumor Infiltrating lymphocytes
  • the antigen binding domain binds to a target expressed on activated B cells surface, preferably selected from the group consisting of BCMA/TNFRSF17, CD150/SLAMF1, CD86, OX40L, LOX1, TACI/TNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3/CD40BP, TRAP1,
  • the antigen binding domain binds to a target expressed on activated myeloid cells surface, preferably selected from the group consisting of CD163, CD206, SIGLEC 6, TRAF1, TRAF4, TRAF7, TRAP100/MED24, TNFRSF12A/FN14/TWEAKR, CD301, IL4R, and CLEC-1A.
  • the antigen binding domain binds to a target expressed on activated natural killer cells surface, preferably selected from the group consisting of CST7, CXCR4, NKG2A, TRAF3IP1, and CMKLR1.
  • the antigen binding domain binds to a target expressed on exhausted T cells or effector memory stem like T cells and the target of the antigen binding domain is a factor expressed on the surface of exhausted T cells or effector memory stem like T cells.
  • T cell exhaustion is a state of T cell progressive loss of function, proliferation capacity and cytotoxic potential, eventually leading to their deletion.
  • T cell exhaustion can be triggered by several factors such as persistent antigen exposure or inhibitory receptors including PD-1, TIM3, CTLA-4, LAG-3, BTLA, TIGIT and CD160.
  • PD-1 persistent antigen exposure or inhibitory receptors
  • TIM3, CTLA-4 LAG-3
  • BTLA BTLA
  • TIGIT TIGIT
  • CD160 BTLA- 4
  • LAG3 LAG-3
  • BTLA TIGIT
  • CD160 is selected from the group consisting of PD-1, TIM3, CTLA- 4, LAG3, BTLA, TIGIT and CD160.
  • the antigen binding domain binds to PD-1.
  • the antigen binding domain may have an agonistic or antagonist activity on the target. In a preferred aspect, the antigen binding domain has an antagonist activity on the target.
  • Numerous antibodies directed against PD-1, TIM3, CTLA-4, LAG-3, BTLA, TIG IT and CD160 have already been described in the art.
  • the terms “Programmed Death 1”, “Programmed Cell Death 1”, “PD-1”, “PDCD1”, “PD- 1 antigen”, “human PD-1”, “hPD-1” and “hPD-1” are used interchangeably and refer to the Programmed Death-1 receptor, also known as CD279, and include variants and isoforms of human PD- 1, and analogs having at least one common epitope with PD-1.
  • PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene.
  • PD-1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC).
  • PBMC Peripheral blood mononuclear cells
  • PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD-1, such as PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4.
  • the terms include any variant and, isoform of human PD-1 that are naturally expressed by PBMC, or that are expressed by cells transfected with a PD-1 gene.
  • the antigen binding domain binds to PD-1 and has an antagonistic effect.
  • the anti-PD-1 antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO- 4538), OSE-279 (see WO2020/127366), Pidilizumab (CT-011), Cemiplimab (Libtayo), Camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (Tisleizumab), PDR001 (spartalizumab), MK-3477, SCH- 900475, PF-06801591, JNJ-63723283, Genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT- 1306, AK-103 (HX-008), MEDI-06
  • BI-754091 CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see WO 2014/194302), AGEN2034 (see WO 2017/040790), MGA012 (see WO 2017/19846), or IBI308 (see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540, the disclosure thereof being incorporated herein by reference), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168, the disclosure thereof being incorporated herein by reference.
  • Antibodies directed against TIM-3 targeting TIM-3 are also known such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367.
  • the TFM-3 antibody is as disclosed in International Patent Application Publication Nos. W02013006490, W02016/161270, WO 2018/085469, or WO 2018/129553, WO 2011/155607, U.S. 8,552,156, EP 2581113 and U.S 2014/044728, the disclosure thereof being incorporated herein by reference.
  • Antibodies directed against CTLA-4 targeting CTLA-4 are also known such as Ipilimumab, Tremelimumab, MK-1308, AGEN-1884, MEDI5752 (AstraZeneca).
  • Anti-CTLA-4 antibodies are also disclosed in WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232,
  • Antibodies directed against LAG-3 targeting LAG-3 are also known such as BMS- 986016, IMP701 or MGD012.
  • Anti-LAG-3 antibodies are also disclosed in W02008132601, EP2320940, WO19152574, the disclosure thereof being incorporated herein by reference.
  • Antibodies directed against BTLA are also known in the art such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mabl9A7, or hu Mab4C7.
  • the antibody TAB004 against BTLA are currently under clinical trial in subjects with advanced malignancies.
  • Anti-BTLA antibodies are also disclosed in W008076560, WQ10106051 (e.g., BTLA8.2), WO11014438 (e.g., 4C7), W017096017 and WO17144668 (e.g., 629.3), the disclosure thereof being incorporated herein by reference.
  • the antigen binding domain comprised in the multifunctional molecule of the invention binds to TIGIT.
  • Antibodies directed against TIGIT are known in the art, such as BMS-986207 or AB154, BMS-986207
  • Anti-TIGIT antibodies are also disclosed in WO16028656, W016106302, WO16191643,
  • the anti-TIGIT binding domain comprises or consists of:
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-TIGIT binding domain is a bivalent antibody comprising or consisting of: (a) a first heavy chain, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 121, or variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion,
  • a second heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 122, or variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion,
  • two light chains comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 123, variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion.
  • the target is CD127 or IL-7R and the antigen binding domain is specific to CD127, preferably human CD127.
  • the antigen binding domain is an antagonist of CD127.
  • Antibodies directed against CD127 or IL7-R are also known in the art, such as GSK2618960, RN168, AbD11590, MAB306-100, R34.34, A019D5, eBioRDR5, 40131, 1A12, M21, 47H4, HIL-7R-M21, eBioYL8, RDR5.
  • Anti- CD127 antibodies are also disclosed in WQ14102430, WQ20077190, WQ04000238, WQ11104687, WQ16059512 and WO17062748, the disclosure thereof being incorporated herein by reference.
  • the target is CLEC-1A and the antigen binding domain is specific to CLEC-1A, preferably human CLEC-1A.
  • the antigen binding domain is an antagonist of CLEC-1A.
  • CLEC-1 A relates to a C- type lectin-like receptor- 1 A from a mammal species, preferably a human CLEC-1 A.
  • a reference sequence of the human CLEC-1 A corresponds to the sequence associated to the Accession number Q8NC01 Uniprot.
  • CLEC-1 antagonist has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CLEC-1. In particular, the CLEC-1 antagonist inhibits the interactions between the CLEC-1 and at least one of its ligands.
  • Antibodies directed against CLEC-1A are also known in the art, such as MAB1704, ABIN526589, AF1704 and ABIN526590.
  • the target is PD-1 and the antigen binding domain of the multifunctional molecule is an antibody, or an antigen binding fragment or a derivative thereof or an antibody mimic that is specific to PD-1.
  • the antigen binding domain comprised in the multifunctional molecule according to the invention is an anti-PDl antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-PDl antibody or antigen binding fragment thereof.
  • the antigen binding domain is an antagonist of PD-1.
  • the anti-PD-1 antibody is Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538) or OSE-279 (such as described in WQ2020/127366, the disclosure thereof being incorporated herein by reference).
  • the antigen binding domain targets PD-1 and is derived from the antibody disclosed in WO2020/127366, the disclosure thereof being incorporated herein by reference.
  • the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
  • the heavy chain CDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 60, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but position 3 of SEQ ID NO: 60;
  • the heavy chain CDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 61, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 13, 14 and 16 of SEQ ID NO: 61;
  • HCDR3 the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 62; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 62;
  • the light chain CDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 63, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 63;
  • LCDR2 the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 64, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof;
  • LCDR3 the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 65, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 65.
  • the anti-PD-1 antigen-binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61 and a CDR3 of SEQ ID NO: 62; and (ii) a light chain variable region (VL) comprising a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64 and a CDR3 of SEQ ID NO: 65.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PDl antibody or antigen binding fragment according to the invention comprises framework regions, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4 and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and
  • the anti-PD-1 antigen-binding domain comprises or consists essentially of:
  • VH heavy chain variable region
  • HFR1 of SEQ ID NO : 66 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • a HCDR1 of SEQ ID NO: 60 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • a HCDR2 of SEQ ID NO: 61 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • a HCDR3 of SEQ ID NO: 62 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • VL light chain variable region
  • a LFR1 of SEQ ID NO : 70 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • a LCDR1 of SEQ ID NO: 63, a LFR2 of SEQ ID NO : 71 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • a LCDR2 of SEQ ID NO: 64, a LFR3 of SEQ ID NO : 72 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof
  • a LCDR3 of SEQ ID NO: 65 and a LFR4 of SEQ ID NO : 73 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the anti-PD-1 antigen-binding domain comprises or consists essentially of:
  • VH heavy chain variable region
  • VL light chain variable region
  • the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).
  • the anti-PD-1 antigen-binding domain comprises or consists essentially of:
  • VH heavy chain variable region
  • the anti-PD-1 antigen-binding domain comprises or consists essentially of:
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antigen-binding domain comprises VH, VL, CHI and a CL domain, so that the antigen binding domain is a Fab.
  • the heavy chain constant domain comprises or consists essentially of SEQ ID NO: 76, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the anti-PD-1 antigen-binding domain comprises a heavy chain that comprises or consists of a VH of SEQ ID 74 and a CHI of SEQ ID NO:76.
  • the light chain constant domain (CL) comprises or consists essentially of SEQ ID NO: 77 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the anti-PD-1 antigen-binding domain comprises a light chain that comprises or consists of a VL of SEQ ID 75 and a CL of SEQ ID NO:77.
  • the anti-PD-1 antigen-binding domain is a Fab or a Fab', a Fab or a F(ab')2 and comprises i) a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID No: 78, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and ii) a VL domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID No: 79, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).
  • the antigen binding domain is an anti-PD-1 Fab or F(ab')2, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID Nos: 74 and 76 respectively and ii) a chain comprising or consisting of VL and CL domains, said domains having the amino acid sequence as set forth in SEQ ID Nos: 75 and 77, respectively.
  • the antigen binding domain is an anti-PD-1 Fab or F(ab')2, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 78 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 79 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).
  • the antigen binding domain comprises an anti-PD-1 CrossMAb comprising or consisting of i) a chain comprising or consisting of VH and CL domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 74 and 77, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 75 and 76, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the antigen binding domain comprises an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID NOs: 74 and 77, respectively, and ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 75 and 76, respectively.
  • the antigen binding domain is an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 80 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 81 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
  • the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
  • HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 90,
  • HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 91,
  • HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 92,
  • LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 93,
  • LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 94, and
  • LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 95,
  • the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
  • VH heavy chain variable region
  • VL light chain variable region
  • HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 98
  • HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 99
  • HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 100
  • LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 101
  • LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 102
  • LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 103
  • the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
  • VH heavy chain variable region
  • VL light chain variable region
  • the target is specifically expressed by a tumor cell.
  • the target has a higher expression level in tumor cells than in other cells, especially healthy cells, or that the ratio of cancer cells expressing the target by the total cancer cells is higher than the ratio of other cells expressing the target by the total other cells.
  • the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100.
  • the target can be a tumor antigen.
  • the antigen can be selected from the group consisting of Fibroblast Activation Protein (FAP), the Al domain of Tenascin-C (TNC Al), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).
  • FAP Fibroblast Activation Protein
  • TMCSP Melanoma-associated Chondroitin Sulfate Proteoglycan
  • a target specifically expressed by a tumor cell allows to direct the multifunctional molecule comprising the cytokine and the antigen binding domain targeting it specifically to a cancer cell, rather than the tumor microenvironment.
  • the cytokine in conjunction with the target specifically expressed by a tumor cell, the cytokine may be selected so that its adverse effect is directed toward said cancer cell rather than a healthy cell, turning said adverse effect to the advantage of the patient.
  • tumor associated antigens include: Her2/Neu (human epidermal growth factor receptor 2); CD22; EpCAM (CD326); EGFR; PSMA (Prostate Carcinoma); CD30; CD20; CD33; membrane IgE; IgE Receptor (CD23), CD80; CD86; CD2; CA125 (cancer antigen- 125); Carbonic Anhydrase IX; CD70; CD74; CD56; CD40; CD19; c-met/HGFR; DRS; PD-1; PDL1; IGF-1R; VEGF and VEGFR (Solid tumor and eye AMD), VEGF-R2; Prostate stem cell antigen (PSCA); MUC1; CanAg; Mesothelin; P-cadherin; Myostatin (GDF8); Cripto (TDGF1); ACVRL 1/ALK1 (activin a receptor type 1); MUC5AC; CEA, in particular CEACAM (carcinoembryonic antigen); CD137; CX
  • the antigen binding domain as described above may be associated with antibody constant regions, in particular from IgA, IgM, IgE, IgD or IgG such as IgGl, lgG2, lgG3, lgG4, preferably IgGl, lgG2, or lgG4.
  • the antigen binding domain comprises an IgG Fc region, preferably an IgGl, lgG2, or lgG4 Fc region.
  • the Fc domain includes all or a portion of a hinge region.
  • the hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes.
  • the hinge region is derived from a human or humanized IgGl, lgG2, lgG3 or lgG4, preferably from a human or humanized IgGl, lgG2, or lgG4.
  • the molecules envisioned herein optionally comprise a Fc domain.
  • fragment crystallizable region As used herein, the terms “fragment crystallizable region” "Fc region” or “Fc domain” are interchangeable and refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors.
  • the Fc region or domain is typically composed of two domains, optionally identical, derived from the second and third constant domains of the antibody's two heavy chains (i.e., CH2 and CH3 domains). Portion of the Fc domain refers to the CH2 or the CH3 domain.
  • the Fc region or domain may optionally comprise all or a portion of the hinge region between CHI and CH2. Accordingly, the Fc domain may comprise the hinge, the CH2 domain and the CH3 domain.
  • the Fc domain is that from IgGl, lgG2, lgG3 or lgG4, optionally with IgGl hinge-CH2-CH3 and lgG4 hinge-CH2-CH3.
  • the Fc domain of the multifunctional molecule can form together with a part of the antigen binding domain a heavy chain of an IgG immunoglobulin.
  • the antigen binding domain is a Fab
  • the multifunctional molecule may comprise one heavy chain, including the variable heavy chain (VH), CHI, hinge, CH2 and CH3 domains.
  • VH variable heavy chain
  • CHI variable heavy chain
  • CH2 and CH3 domains the multifunctional molecule may also have other structures such as scFv, or diabody
  • the Fc domain can be from a heavy chain constant domain of a human immunoglobulin heavy chain, for example, IgGl, lgG2, lgG3, lgG4, or other classes.
  • the multifunctional molecule comprises an IgGl or an lgG4 heavy chain constant domain.
  • the Fc domain comprises CH2 and CH3 domains.
  • it can include all or a portion of the hinge region, the CH2 domain and/or the CH3 domain.
  • the CH2 and/or a CH3 domains are derived from a human lgG4 or IgGl heavy chain.
  • the domain derived from lgG4 or IgGl heavy chain is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the lgG4 or IgGl heavy chain or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the Fc domain includes all or a portion of a hinge region.
  • the hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes.
  • the hinge region is derived from human IgGl, lgG2, lgG3, lgG4.
  • the hinge region derived from IgGl, lgG2, lgG3, lgG4 heavy chain is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the IgGl, lgG2, lgG3, lgG4 heavy chain or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the hinge region is derived from a human or humanized IgGl or lgG4 heavy chain.
  • the hinge region has a sequence of SEQ ID NO: 89 or is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity thereto or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the IgGl hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines permit efficient and consistent disulfide bonding formation between Fc portions. Therefore, a preferred hinge region of the present invention is derived from IgGl, more preferably from human IgGl. In some aspects, the first cysteine within the human IgGl hinge region is mutated to another amino acid, preferably serine. For instance, the sequence including CHI, CH2 and CH3 based on an IgGl is disclosed in SEQ ID NO: 83 including a N298A substitution.
  • the hinge region of lgG4 is known to form interchain disulfide bonds inefficiently.
  • a suitable hinge region for the present invention can be derived from the lgG4 hinge region, preferably containing a mutation that enhances correct formation of disulfide bonds between heavy chain-derived moieties (Angal S, et al. (1993) Mol. Immunol., 30:105-8). More preferably, the hinge region is derived from a human lgG4 heavy chain. For instance, the sequence including CHI, CH2 and CH3 based on an lgG4 is disclosed in SEQ ID NO: 82 including a S228P substitution.
  • the heavy chain constant domains comprise or consist of the sequence as set forth in SEQ ID NO: 82 or 83 or is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with SEQ ID NO: 82 or 83 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
  • the mutations are outside the CDRs, i.e., are in the framework regions.
  • the multifunctional molecule comprises a dimeric Fc domain. Accordingly, two monomers comprise each one a Fc chain, the Fc chains being able to form a dimeric Fc domain.
  • the dimeric Fc domain can be an homodimer, each Fc monomer being identical or essentially identical. Alternatively, the dimeric Fc domain can be a heterodimer, each Fc monomer being different and complementary in order to promote the formation of the heterodimeric Fc domain.
  • the Fc domain is a heterodimeric Fc domain.
  • Heterodimeric Fc domains are made by altering the amino acid sequence of each monomer.
  • the heterodimeric Fc domains rely on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and/or allow for ease of purification of heterodimers over the homodimers.
  • these mechanisms can be combined to ensure high heterodimerization.
  • amino acid variants that lead to the production of heterodimers are referred to as "heterodimerization variants”.
  • Heterodimerization variants can include steric variants (e.g., the "knobs and holes” or “skew” variants described below and the “charge pairs” variants described below) as well as “pi variants”, which allows purification of homodimers away from heterodimers.
  • WQ2014/145806 hereby incorporated by reference in its entirety, discloses useful mechanisms for heterodimerization include “knobs and holes”, “electrostatic steering” or “charge pairs”, pi variants, and general additional Fc variants. See also, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; US Patent No.
  • the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain based on the "knobs and holes” technology.
  • the first Fc chain is a "knob” or K chain, meaning that it comprises the substitution characterizing a knob chain
  • the second Fc chain is a "hole” or H chain, meaning that it comprises the substitution characterizing a hole chain.
  • the first Fc chain is a "hole” or H chain, meaning that it comprises the substitution characterizing a hole chain
  • the second Fc chain is a "knob” or K chain, meaning that it comprises the substitution characterizing a knob chain.
  • the first Fc chain is a "hole” or H chain and the second Fc chain is a "knob” or K chain.
  • a Fc domain comprising a knob into hole preferably comprises a N297A substitution.
  • the heterodimeric Fc domain may comprise one heterodimeric Fc chain which comprises the substitutions as shown in the following Table F and the other heterodimeric Fc chain comprising the substitutions as shown in the following Table F.
  • one Fc chain is a "hole” or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and one Fc chain is a "knob” or K chain and comprises the substitutions T366W/S354C.
  • the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv)
  • peptide linker and a single cytokine or variant or fragment thereof; said peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine and being a "masking" peptide linker as defined herein; or
  • first and second peptide linkers are covalently linked to the N-terminus of the cytokine and a second peptide linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a “masking” peptide linker as defined herein; or
  • first Fc chain comprises or consists of a "knob” chain
  • second Fc chain comprises or consists of a "hole” chain.
  • the Fc chain may further comprise additional substitutions.
  • amino acid modifications may be introduced into the Fc region to generate an Fc region variant.
  • the Fc region variant possesses some, but not all, effector functions.
  • Such multifunctional molecules may be useful, for example, in applications in which the half-life of the antibody in vivo is important, yet certain effector functions are unnecessary or deleterious. Numerous substitutions or substitutions or deletions with altered effector function are known in the art.
  • the constant region of the Fc domain contains a mutation that reduces affinity for an Fc receptor or reduces Fc effector function.
  • the constant region can contain a mutation that eliminates the glycosylation site within the constant region of an IgG heavy chain.
  • the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain.
  • the Fc domain is modified to increase the binding to FcRn, thereby increasing the half-life of the multifunctional molecule.
  • the Fc domain is modified to decrease the binding to FcyR, thereby reducing ADCC or CDC, or to increase the binding to FcyR, thereby increasing ADCC or CDC.
  • the junction region of a protein or polypeptide of the present invention can contain alterations that, relative to the naturally-occurring sequences of an immunoglobulin heavy chain and erythropoietin, preferably lie within about 10 amino acids of the junction point. These amino acid changes can cause an increase in hydrophobicity.
  • the constant region is derived from an IgG sequence in which the C-terminal lysine residue is replaced.
  • the C-terminal lysine of an IgG sequence is replaced with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life.
  • the constant region of the Fc domain has one of the mutations described in the Table G below, or any combination thereof.
  • Table G Suitable human engineered Fc domain of an antibody, numbering of residues in the heavy chain constant region is according to EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63,
  • the multifunctional molecule comprises a human IgGl heavy chain constant domain or an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; P329G; N297A + M252Y/S254T/T256E; K322A, K444A, K444A, K444E, K444D, K444G, K444S, K444L, K444Q, K444
  • the multifunctional molecule comprises a human IgGl heavy chain constant domain or an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; P329G; N297A + M252Y/S254T/T256E; K322A,K444A, K444E, K444D, K444G, K444S, M428L, L309D, Q311H, N43434L; T250
  • the multifunctional molecule comprising a human IgGl heavy chain constant domain or an IgGl Fc domain with the combination of substitutions L234A/L235A/P329G greatly reduces or altogether suppresses ADCC, ADCP and/or CDC caused by said multifunctional molecule, thus reducing nonspecific cytotoxicity.
  • the multifunctional molecule comprises a human lgG4 heavy chain constant domain or a human lgG4 Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A/L235A, P329G, S228P + M252Y/S254T/T256E and K444A.
  • the multifunctional molecule comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4.
  • the multifunctional molecule comprises a human lgG4 heavy chain constant domain or a human lgG4 Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A/L235A; L234A/L235A/P329G, P329G, S228P + M252Y/S254T/T256E, K444A K444E, K444D, K444G and K444S.
  • the multifunctional molecule preferably the multifunctional molecule according to the invention comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4.
  • the and "+" refer to mutations that are cumulative.
  • S228P + M252Y/S254T/T256E it is meant the following mutations: S228P, M252Y, S254T and T256E.
  • the multifunctional molecule comprising a human lgG4 heavy chain constant domain or an lgG4 Fc domain with the substitution P329G reduces ADCC and/or CDC caused by said multifunctional molecule, thus reducing nonspecific cytotoxicity.
  • All subclass of Human IgG carries a C-terminal lysine residue of the antibody heavy chain (K444) that are susceptible to be cleaved off in circulation. This cleavage in the blood may compromise or decrease the bioactivity of the multifunctional molecule by releasing the linked immune-stimulating moiety to the multifunctional molecule.
  • K444 amino acid in the IgG domain can be substituted by another amino acid to reduce proteolytic cleavage, a mutation commonly used for antibodies.
  • the multifunctional molecule comprises at least one further amino acid substitution consisting of K444A, K444E, K444D, K444G, K444L, K444Q, K444N, K444I, K444P, K444V, K444T or K444S, preferentially K444A or K444L.
  • K444 amino acid in the IgG domain can be substituted by an alanine or a lysine to reduce proteolytic cleavage, a mutation commonly used for antibodies.
  • the multifunctional molecule comprises at least one further amino acid substitution consisting of K444A or K444L.
  • the multifunctional molecule comprises an additional cysteine residue at the C-terminal domain of the Fc domain to create an additional disulfide bond and potentially restrict the flexibility of the multifunctional molecule.
  • the multifunctional molecule according to the invention comprises a heterodimer of Fc domains that comprises the "knob into holes" modifications such as described above.
  • Fc domains are IgGl or lgG4 Fc domain such as described above, even more preferably an IgGl Fc domain comprising the mutation N297A such as disclosed above.
  • the first Fc chain is a "knob” or K chain and comprises the substitutions T366W/S354C and optionally N297A and the second Fc chain is a "hole” or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and optionally N297A.
  • the first Fc chain is a "knob” or K chain and comprises the substitutions T366W/S354C and N297A
  • the second Fc chain is a "hole” or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and N297A.
  • knob Fc chain may comprise or consist in SEQ ID NO: 84 and/or the hole Fc chain may comprise or consist in SEQ ID NO: 85.
  • an object of the present invention relates to a polypeptide comprising, from the N- terminal to the C-terminal, an antigen binding domain or a part thereof, a Fc chain (knob or hole Fc chain), preferably the hole-chain of the Fc domain, and either:
  • peptide linker and a single cytokine or variant or fragment thereof; said peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine and being a "masking" peptide linker as defined herein; or
  • first linker is covalently linked to the N-terminal end of the cytokine and a second linker is linked to the C-terminal end of the cytokine, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a “masking” peptide linker as defined herein.
  • the antigen binding domain according to the invention is linked to the knob-chain and/or the hole chain of the heterodimeric Fc domain.
  • the multifunctional molecule comprises or consists of:
  • a first entity comprising or consisting of a first Fc chain which is a knob Fc chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine
  • a second entity comprising i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; iii) a "masking" peptide linker as defined herein, and iv) a cytokine.
  • the multifunctional molecule comprises or consists of:
  • a first entity comprising or consisting of a first Fc chain which is a knob Fc chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine
  • the multifunctional molecule comprises or consists of:
  • a first entity comprising or consisting of a first Fc chain which is a knob chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine
  • a second entity comprising i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; iii) a first peptide linker, iv) a cytokine and v) a second peptide linker, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein.
  • the multifunctional molecule may comprise one or more spacers, especially peptide spacers, particularly between the antigen binding domain and the Fc domain. Such a spacers may be useful to prevent steric hindrances.
  • the spacer is usually 3-44 amino acid residues in length. Preferably, the spacer has 3-30 amino acid residues. In some aspects, the spacer has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acid residues.
  • the spacer sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the spacer is preferably non-immunogenic in the subject to which the multifunctional molecule is administered.
  • One useful group of spacer sequences are derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine sequences.
  • spacer sequences are Gly/Ser linkers of different length including (Gly4Ser) 4 (SEQ ID NO: 107), (Gly4Ser) 3 (SEQ ID NO: 106), (Gly4Ser) 2 (SEQ ID NO: 108), Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2) 3 (SEQ ID NO: 109).
  • the multifunctional molecule does not comprise a glycine-serine linker, preferably such as defined above, especially that links the cytokine to the antigen binding domain, typically between the Fc domain and the cytokine.
  • the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures A, B, Cl, C2, C3, C4, C5, C6, C7, C8 and D of Figure 1C.
  • the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures Cl, C2, C3, C4, C5, C6, C7 and C8 of Figure 1C.
  • the structure and/or sequence of the multifunctional molecule is selected from the structure and/or sequence disclosed in Table 4 of the Example section, when a peptide linker of the invention is comprised.
  • the multifunctional molecule comprises one of the following arrangements, said arrangements being described from the N-terminus to the C-terminus: antigen binding domain heavy chain (VH or VH+CH1)- optionally a Fc chain - "masking" linker
  • the multifunctional molecule according to the invention comprises or consists of an antibody (monovalent or bivalent) - a first masking linker - a cytokine - a second masking linker, preferably this structure being read from N-terminal to C-terminal.
  • the C- terminal end of the Fc domain of the antibody preferably only the hole Fc chain, is covalently linked to the N-terminal end of the first peptide linker.
  • the multifunctional molecule according to the invention comprises or consists of an antibody (monovalent or bivalent), preferably an anti-PDl or anti-TIGIT antibody - a first EEEEEEEEEEEEEEEEEEE linker (SEQ ID NO: 5) - a cytokine, preferably selected from the group consisting of IL-2, IL-15, IL-10 and IL-18 - a second EEEEEEEEEEEEEEE linker (SEQ ID NO: 5), preferably this structure being read from N-terminal to C-terminal.
  • the C-terminal end of the Fc domain of the antibody preferably only the hole Fc chain, is covalently linked to N-terminal end of the first peptide linker.
  • the multifunctional molecule according to the invention comprises or consists of:
  • cytokine(s) or fragment(s) thereof covalently linked to the N- terminal and/or C-terminal end of the antibody
  • peptide linker(s) as described above covalently linked to the N- terminal and/or C-terminal end of the cytokine(s), wherein at least one peptide linker per cytokine is a "masking" peptide linker as defined herein.
  • the multifunctional molecule comprises or consists of:
  • peptide linker covalently linked to the N-terminal and/or C-terminal end of the cytokine(s) as described above, wherein at least one peptide linker is a "masking" peptide linker as defined herein and preferably both.
  • the structure of the multifunctional molecule, from N-terminal to C-terminal is selected from the group consisting of: anti-PDl antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; anti-PDl antigen binding domain, preferably antibody - first peptide linker-cytokine; anti-TIGIT antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; and anti- TIG IT antigen binding domain, preferably antibody - first peptide linker-cytokine.
  • the antibody can be bivalent or monovalent.
  • the peptide linker and/or cytokine are covalently linked to the C-terminal end of the Fc chain of the antibody, preferably the hole Fc chain.
  • the first peptide linker is E15 (EEEEEEEEEEEEEEE (SEQ ID NO: 5). More preferably, the first and the second peptide linkers are E15 ( EEEEE EEEEE EEEEE (SEQ ID NO: 5).
  • the cytokine is a pro- inflammatory cytokine, preferably IL-2, IL-12, IL-15, IL-18 or IL-21 or a variant thereof, preferably IL-2, IL-15, IL-21 or a variant thereof as described above.
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • DDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNNNNNN (SEQ ID NO: 37),
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41),
  • the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
  • the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I I H (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I (SEQ ID NO: 8), I I I I I I I I I I I I I THTTTTT (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 1), AAAAHAAAAAAAA (SEQ
  • the multifunctional molecule comprises or consists of: a Fc domain, preferably an heterodimeric Fc domain comprising a "knob" chain and a "hole” chain, a single antigen binding domain covalently linked to the N-terminus of the Fc domain, said antigen binding domain comprising or consisting of a VH and a VL domains (e.g., Fab) of an anti-PD-1 or anti-TIGIT antibody as described above, a single cytokine covalently linked to the C-terminus of the Fc domain, and one or two peptide linker(s), preferably two peptide linkers, covalently linked to N-terminus and/or C-terminus of the cytokine, as described above, wherein at least one peptide linker is a "masking" peptide linker as defined herein and preferably both.
  • a Fc domain preferably an heterodimeric Fc domain comprising a "knob" chain and a "hole” chain
  • the antigen binding domain and the cytokine are on the same Fc chain.
  • the antigen binding domain and the cytokine are on the "hole" Fc chain.
  • the molecule comprises or consists of: a) a first entity comprising a first Fc chain ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains of an anti-PD-1 or anti-TIGIT antibody as described above, ii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) either - one "masking" peptide linker as defined herein and a single cytokine; said "masking" peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine; or
  • first linker is covalently linked to the N-terminus of the cytokine and a second linker is covalently linked to the C-terminus of the cytokine, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; or
  • the cytokine is a pro-inflammatory cytokine, preferably! L-2, IL-12, IL- 15, IL-18 or IL-21 or a variant thereof, preferably IL-2, IL-15, IL-21 or a variant thereof such as described above.
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48),
  • DDDDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEEEDEEEED (SEQ ID NO: 15),
  • the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41),
  • the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
  • the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I TH (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I HT
  • the first Fc chain is a knob chain and the second Fc chain is a hole chain.
  • the molecule comprises or consists of: a) a first entity comprising a first Fc chain ; said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-PD-1 antibody as described herein, ii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferablylL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-termin
  • the multifunctional molecule comprises or consists of: a) a first chain comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH of SEQ ID NO : 74 and VL of SEQ ID NO : 75 of an anti-PD-1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two "masking" peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (S
  • the first Fc chain is a knob chain and the second Fc chain is a hole chain.
  • the multifunctional molecule comprises or consists of: a) a first chain comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH of SEQ ID NO :74 and VL of SEQ ID NO : 75 of an anti-PD-1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the first Fc chain is a knob chain and the second Fc chain is a hole chain.
  • the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH of SEQ ID NO :74 and VL of SEQ ID NO : 75 of an anti-PD-1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the first Fc chain is a knob chain and the second Fc chain is a hole chain.
  • the multifunctional molecule comprises or consists of: a) a first entity comprising or consisting of a polypeptide chain comprising or consisting of a first Fc chain, preferably of SEQ ID NO : 84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising or consisting of: a first polypeptide chain comprising or consisting of i) a VH of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO : 74 ii) a second Fc chain, preferably of SEQ ID NO : 85, complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5
  • the first Fc chain is a knob chain and the second Fc chain is a hole chain.
  • the multifunctional molecule comprises or consists of: c) a first polypeptide chain comprising or consisting of a first Fc chain, preferably of SEQ ID NO : 84, said Fc chain being devoid of an antigen binding domain and of a cytokine; d) a second polypeptide chain comprising or consisting of i) a VH of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO :74 ii) a second Fc chain, preferably of SEQ ID NO : 85, complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the first Fc chain is a knob chain and the second Fc chain is a hole chain.
  • the multifunctional molecule comprises or consists of: a) a first polypeptide chain comprising or consisting of a first Fc chain, preferably of SEQ ID NO : 84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second polypeptide chain comprising or consisting of i) a VH of an anti-PD-1 antibody linked to a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; preferably as described in SEQ ID NO: 88 and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEE
  • the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-TAA antibody as described herein, ii) a first Fc chain; said Fc chain being devoid of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-PD-1 antibody as described herein, ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described here
  • the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-TAA antibody as described herein, ii) a first Fc chain; said Fc chain being devoid of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-CD3 antibody as described herein, ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described here
  • the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-CD28 antibody as described herein, ii) a first Fc chain; and iii) an anti-TAA antibody or fragment thereof as described herein, wherein the anti-TAA antibody is fused to the C-terminus of the first Fc chain; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-PD-1 antibody as described herein, ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL
  • the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEE
  • the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEE
  • the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of SEQ ID NO: 88-5-(50, 53, 56, 110 or 112)-5 from N-terminal end to the C-terminal end, preferably of SEQ ID NO: 111 a second heavy chain comprising or consisting of SEQ ID NO: 86; and a first and second light chains comprising or consisting of SEQ ID NO: 79.
  • the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-TIGIT VH domain preferably comprising or consisting of SEQ ID NO: 129, ii) optionally a CHI domain of an antibody , iii) optionally a hinge domain, iv) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
  • the multifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 121-5-50-5, from N-terminal end to the C-terminal end; a second heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 122; and a first and second light chains comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 123.
  • nucleic acid sequences or group of nucleic acid sequences coding for the multifunctional molecule are subcloned into one or more expression vectors.
  • Such vectors are generally used to transfect mammalian cells.
  • General techniques for producing molecules comprising antibody sequences are described in Coligan et al. (eds.), Current protocols in immunology, at pp. 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are hereby incorporated by reference and in "Antibody engineering: a practical guide” from W. H. Freeman and Company (1992), in which commentary relevant to production of molecules is dispersed throughout the respective texts.
  • the multifunctional molecule is a polypeptide or protein produced as a recombinant protein.
  • such method comprises the following steps of: (1) transfecting or transforming appropriate host cells with the polynucleotide(s) encoding the recombinant multifunctional molecule of the invention or the vector containing the polynucleotide(s);
  • the invention further relates to a nucleic acid encoding a multifunctional molecule as disclosed above, a vector, preferably an expression vector, comprising the nucleic acid of the invention, a genetically engineered host cell transformed with the vector of the invention or directly with the sequence encoding the recombinant multifunctional molecule, and a method for producing the multifunctional molecule of the invention by recombinant techniques.
  • the nucleic acid, the vector and the host cells are more particularly described hereafter.
  • Nucleic acid molecules encoding the molecules or multifunctional molecules of the present invention.
  • Recombinant Expression Vectors and Host Cells comprising such
  • the invention also relates to an isolated nucleic acid molecule encoding the multifunctional molecule as defined above or to a group of isolated nucleic acid molecules encoding the multifunctional molecule as defined above.
  • Nucleic acid encoding the multifunctional molecule disclosed herein can be amplified by any techniques known in the art, such as PCR. Such nucleic acids may be readily isolated and sequenced using conventional procedures.
  • nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:
  • a first nucleic acid encoding a polypeptide comprising, from N to C-terminus i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer iii) a first Fc chain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;
  • a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; and
  • nucleic acid molecule encoding the light chain of the antibody.
  • nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:
  • a first nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody ii) optionally a peptide spacer and iii) a first Fc chain;
  • a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;
  • nucleic acid molecule encoding the light chain of the antibody.
  • nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:
  • a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;
  • nucleic acid molecule encoding the light chain of the antigen-binding domain.
  • the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule.
  • the invention relates to a vector comprising the nucleic acid molecule or the group of nucleic acid molecules as defined above.
  • a "vector” is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell.
  • the term “vector” encompasses plasmids, viruses, cosmids and artificial chromosomes.
  • engineered vectors comprise an origin of replication, a multicloning site and a selectable marker.
  • the vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the "backbone” of the vector.
  • Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag.
  • Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.
  • the nucleic acid molecule encoding the multifunctional molecule, the fusion protein can be cloned into a vector by those skilled in the art, and then transformed into host cells. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The methods known to the artisans in the art can be used to construct an expression vector containing the nucleic acid sequence of the multifunctional molecule, variant described herein and appropriate regulatory components for transcription/translation. Accordingly, the present invention also provides a recombinant vector, which comprises a nucleic acid molecule encoding the multifunctional molecule according to the present invention.
  • the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug-resistance gene for screening.
  • the expression vector may further comprise a ribosome -binding site for initiating the translation, transcription terminator and the like.
  • Suitable expression vectors typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like.
  • transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants.
  • the invention relates to a lipid-based nanoparticle comprising the multifunctional molecule of the invention or a nucleic acid sequence or vector encoding such.
  • the lipid-based nanoparticle according to the invention is particularly formulated either as a liposome or a lipid nanoparticle (LNP), especially a lipid nanoparticle comprising a mixture of lipids.
  • LNP lipid nanoparticle
  • the lipid-based nanoparticle also encompasses similar nanoparticles such as but not limited to micelles and nano-emulsions.
  • Lipid-based nanoparticles typically comprise helper lipid, sterol and/or PEG lipid components along with the mRNA of interest.
  • the elements of a LNP may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements.
  • the particular formulation of a lipid-based nanoparticle may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements.
  • the lipid-based nanoparticles is a lipid nanoparticle comprising one or more ionizable or cationic lipid(s), one or more helper lipid(s), one or more sterol(s), and/or one or more polyethylene glycol (PEG)-modified lipid(s).
  • the invention in another aspect, relates to a host cell comprising a vector or a nucleic acid molecule or group of nucleic acid molecules as defined above, for example for multifunctional molecule production purposes.
  • a host cell is intended to include any individual cell or cell culture that can be or has been recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the multifunctional molecule according to the present invention.
  • the term "host cell” is also intended to include progeny or potential progeny of a single cell.
  • Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.
  • Suitable hosts cells are especially eukaryotic hosts cells which provide suitable post-translational modifications such as glycosylation.
  • suitable eukaryotic host cell may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; insect cell such as Mythimna separate; plant cell such as tobacco, and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells and COS cells.
  • the host cell of the present invention is selected from the group consisting of CHO cell, COS cell, NSO cell, and HEK cell.
  • host cells stably or transiently express the multifunctional molecule according to the present invention.
  • Such expression methods are known by the man skilled in the art.
  • a method of production of the multifunctional molecule comprises culturing a host cell comprising a nucleic acid encoding the multifunctional molecule as provided above, under conditions suitable for its expression, and optionally recovering the multifunctional molecule from the host cell (or host cell culture medium).
  • nucleic acid encoding a multifunctional molecule e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • the molecules or multifunctional molecules are then isolated and/or purified by any methods known in the art.
  • multifunctional molecule isolation techniques may particularly include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography and ion exchange chromatography. Protein A preferably is used to isolate the molecules or multifunctional molecules of the invention.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a multifunctional molecule as described herein, the isolated nucleic acid molecule, the group of isolated nucleic acid molecules, the vector and/or the host cells as described hereabove, preferably as the active ingredient or compound.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers, excipients, salts, anti-oxidant and/or stabilizers which do not deleteriously interact with the multifunctional molecule of the invention, nucleic acid, vector and/or host cell of the invention and does not impart any undesired toxicological effects.
  • the pharmaceutical composition may further comprise an additional therapeutic agent.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active agents, such as comprising a multifunctional molecule according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism.
  • a “composition” typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • an "acceptable vehicle” or “acceptable carrier” as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • compositions of the present invention can be in a form suitable for any conventional route of administration or use.
  • the pharmaceutical composition according to the invention can be formulated for any conventional route of administration including a topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
  • the multifunctional molecule as described herein can be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005).
  • the pharmaceutical composition may be prepared by mixing a multifunctional molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, anti-oxidant, and/or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • suitable carriers, excipients, anti-oxidant, and/or stabilizers are well known in the art and have been for example described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • any of the multifunctional molecules or its encoding nucleic acids can be conjugated with a chaperon agent.
  • the chaperon agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipid. It can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polypeptide.
  • compositions according to the invention may be formulated to release the active ingredients (e.g., the multifunctional molecule of the invention) substantially immediately upon administration or at any predetermined time or time period after administration.
  • the pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Means known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
  • the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood.
  • Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer.
  • composition typically is sterile and stable under the conditions of manufacture and storage. Prevention of presence of microorganisms may be ensured both by sterilization procedures (for example by microfiltration), and/or by the inclusion of various antibacterial and antifungal agents.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.
  • multifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the present invention have numerous in vitro and in vivo utilities and applications.
  • any of multifunctional molecules, nucleic acid molecules, group of nucleic acid molecules, vectors, host cells or pharmaceutical composition provided herein may be used in therapeutic methods and/or for therapeutic purposes, in particular for the treatment of diseases or as a medicament or vaccine.
  • treatment refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and/or a prophylactic treatment of a disease.
  • a curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and/or eliminating, reducing and/or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly.
  • a prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and/or delaying the progression and/or the incidence of a disease or the risk of its occurrence.
  • such a term refers to the improvement or eradication of a disease, a disorder, an infection or symptoms associated with it. In other aspects, this term refers to minimizing the spread or the worsening of cancers.
  • Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
  • the term "treatment” refers to the application or administration of a composition including one or more active agents to a subject who has a disorder/disease.
  • the term “medicament” refers to any substance or composition with curative or preventive properties against disorders or diseases.
  • the present invention particularly relates to a multifunctional molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition comprising such for use in the treatment of a disease in a subject and/or for use as a medicament or vaccine. It also relates to the use of a multifunctional molecule as described herein; a nucleic acid or a vector encoding such, or a pharmaceutical composition comprising such for treating a disease and/or disorder in a subject.
  • a multifunctional molecule a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition comprising such as disclosed herein in the manufacture of a medicament for treating a disease and/or disorder in a subject, such as cancer or infections.
  • diseases to be treated are more particularly described hereafter.
  • a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule to the subject, or a nucleic acid or group of nucleic acid molecules or a vector encoding such.
  • the treatment method comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a multifunctional molecule, nucleic acid, vector or pharmaceutical composition as described herein.
  • a subject in need of a treatment may be a human having, at risk for, or suspected of having a disease. Such a patient can be identified by routine medical examination.
  • the invention particularly relates to a method of treatment of a cancer or an infectious disease in a subject in need thereof comprising administering to said subject an effective amount of a multifunctional molecule or pharmaceutical composition as defined above.
  • the multifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to enhance immunity, preferably in order to treat a disorder and/or disease.
  • the invention provides a method of modifying an immune response in a subject comprising administering to the subject a multifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention such that the immune response in the subject is modified.
  • the immune response is enhanced, increased, stimulated or up-regulated.
  • the multifunctional molecule or pharmaceutical composition can be used to enhance immune responses such as T cell activation in a subject in need of a treatment.
  • the multifunctional molecule or pharmaceutical composition can be used to reduce T cells exhaustion or to reactivate exhausted T cells.
  • the invention particularly provides a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutic effective amount of any of the multifunctional molecules, nucleic acid, vector or pharmaceutical composition comprising such described herein, such that an immune response in the subject is enhanced.
  • the multifunctional molecule or the pharmaceutical composition as described herein can be used to reduce T cells exhaustion or to reactivate exhausted T cells.
  • the invention provides the use of a multifunctional molecule or a pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a cancer, for instance for inhibiting growth of tumor cells in a subject.
  • the invention particularly provides a multifunctional molecule or a pharmaceutical composition as disclosed herein for use in the treatment of cancer.
  • cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
  • the invention provides a method of treating a cancer, for instance for inhibiting growth of tumor cells, in a subject, comprising administering to the subject a therapeutically effective amount of multifunctional molecule or a pharmaceutical composition according to the invention.
  • the present invention relates to the treatment of a subject using a multifunctional molecule such that growth of cancerous cells is inhibited.
  • the cancer to be treated is associated with exhausted T cells.
  • Any suitable cancer may be treated with the provided herein can be hematopoietic cancer or solid cancer.
  • Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, stomach cancer, urethral cancer environmentally induced cancers and any combinations of said cancers.
  • the invention includes refractory or recurrent malignancies.
  • the cancer to be treated or prevented is selected from the group consisting of metastatic or not metastatic, Melanoma, malignant mesothelioma, Non-Small Cell Lung Cancer, Renal Cell Carcinoma, Hodgkin's Lymphoma, Head and Neck Cancer, Urothelial Carcinoma, Colorectal Cancer, Hepatocellular Carcinoma, Small Cell Lung Cancer Metastatic Merkel Cell Carcinoma, Gastric or Gastroesophageal cancers and Cervical Cancer.
  • the cancer is a hematologic malignancy or a solid tumor.
  • Such a cancer can be selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia.
  • the cancer is a cancer induced by virus or associated with immunodeficiency.
  • a cancer can be selected from the group consisting of Kaposi sarcoma (e.g., associated with Kaposi sarcoma herpes virus); cervical, anal, penile and vulvar squamous cell cancer and oropharyngeal cancers (e.g., associated with human papilloma virus); B cell non-Hodgkin lymphomas (NHL) including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classic Hodgkin lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and/or Kaposi sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis B and/or C viruses); Merkel cell carcinoma (e.
  • Preferred cancers for treatment include cancers typically responsive to immunotherapy.
  • preferred cancers for treatment are cancers non-responsive to immunotherapy.
  • the multifunctional molecule, nucleic acid, group of nucleic acid, vector, host cells or pharmaceutical compositions of the invention can be used to treat patients that have been exposed to particular toxins or pathogens. Accordingly, an aspect of the invention provides a method of treating an infectious disease in a subject comprising administering to the subject a multifunctional molecule according to the present invention, or a pharmaceutical composition comprising such, preferably such that the subject is treated for the infectious disease.
  • the invention particularly concerns a multifunctional molecule or a pharmaceutical composition as disclosed herein for use in the treatment of an infectious disease.
  • Any suitable infection may be treated with a multifunctional molecule, nucleic acid, group of nucleic acid, vector, host cells or pharmaceutical composition as provided herein.
  • pathogenic viruses causing infections treatable by methods of the invention include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.
  • herpes virus e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus
  • adenovirus e.g., influenza virus, flaviviruses, echovirus, rhinovirus, coxsacki
  • pathogenic bacteria causing infections treatable by methods of the invention include chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria.
  • pathogenic fungi causing infections treatable by methods of the invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • pathogenic parasites causing infections treatable by methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.
  • the multifunctional molecule or pharmaceutical composition according to the invention can be combined with some other potential strategies for overcoming immune evasion mechanisms with agents in clinical development or already on the market (see table 1 from Antonia et al. Immunooncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258-6268, 2014). Such combination with the multifunctional molecule according to the invention may be useful notably for:
  • any of the multifunctional molecules or a pharmaceutical composition comprising such, as described herein and a suitable second agent, for the treatment of a disease or disorder.
  • the multifunctional molecule and the second agent can be present in a unique pharmaceutical composition as described above.
  • the terms "combination therapy” or “combined therapy”, as used herein embrace administration of these two agents (e.g., a multifunctional molecule as described herein and an additional or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route.
  • the agents can be administered by the same route or by different routes.
  • a first agent e.g., a multifunctional molecule
  • an additional therapeutic agent e.g., an anti-cancer agent, an anti-infection agent; or an immune modulator
  • an agent of the combination selected may be administered by intravenous injection while the other agents of the combination may be administered orally.
  • the multifunctional molecule or pharmaceutical composition of the invention is used or is for use in combination with an additional/second therapeutic agent, preferably an anticancer agent, an anti-infection agent or an immune modulator.
  • an additional/second therapeutic agent preferably an anticancer agent, an anti-infection agent or an immune modulator.
  • the multifunctional molecule or pharmaceutical composition of the invention is used or is for use in combination with an additional/second therapeutic treatment, preferably an anticancer treatment or an anti-infection treatment or an immune modulator.
  • the additional therapeutic agent can be selected in the non-exhaustive list comprising alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic, antiproliferative, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunotherapies, inhibitors of inhibitors of apoptosis proteins (l)-bind
  • the additional therapeutic agent can be selected in the group consisting of chemotherapy, radiotherapy, targeted therapy, antiangiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes from tumor antigens, myeloid checkpoints inhibitors, other immunotherapies, and HDAC inhibitors.
  • the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is particularly selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular of adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates.
  • the second therapeutic agent is particularly selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular of adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates.
  • suitable agents for co-use with any of the multifunctional molecule or with the pharmaceutical composition according to the invention include an antibody binding to a co-stimulatory receptor (e.g., 0X40, CD40, ICOS, CD27, HVEM or GITR), an agent that induces immunogenic cell death (e.g., a chemotherapeutic agent, a radio-therapeutic agent, an anti- angiogenic agent, or an agent for targeted therapies), an agent that inhibits a checkpoint molecule (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), a cancer vaccine, an agent that modifies an immunosuppressive enzyme (e.g., IDO1 or iNOS), an agent that targets Treg cells, an agent for adoptive cell therapy, or an agent that modulates myeloid cells.
  • a co-stimulatory receptor e.g., 0X40, CD40, ICOS, CD27, HVEM or GITR
  • the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti- HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.
  • T and B lymphocytes adaptive immune cells
  • the present invention also relates to a method for treating a disease in a subject comprising administering to said subject a therapeutically effective amount of the multifunctional molecule or the pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent.
  • the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapy agents, cytokines, cell therapy agents (such as CAR-T cells), antibiotics and probiotics.
  • Combination therapy could also rely on the combination of the administration of multifunctional molecule with surgery.
  • the present invention relates to a multifunctional molecule as disclosed herein, a nucleic acid molecule or group of nucleic acid molecules or a vector encoding such, a host cell or a pharmaceutical composition for use as a medicament or for use in the treatment of a disease or for administration in a subject in need thereof. It also relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule to a subject.
  • an effective amount or a “therapeutic effective amount” as used herein refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect.
  • the “effective amount” will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • the subject to treat may be a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old or of at least 70 years old.
  • the subject can be immunosuppressed (e.g., the subject has an auto-immune disease or has a transplant) or immunocompromised (e.g., the subject has a cancer or infections such as AIDS).
  • the multifunctional molecule or the pharmaceutical composition as disclosed herein can be used to administer the multifunctional molecule or the pharmaceutical composition as disclosed herein to a subject, depending upon the type of diseases to be treated or the site of the disease e.g., administered orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the multifunctional molecule or the pharmaceutical composition as disclosed herein is administered via subcutaneous, intra-cutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-tumoral, intra-sternal, intra-thecal, intra-lesion, and intracranial injection or infusion techniques.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.
  • kits for use in enhancing immune responses and/or treating diseases or disorders e.g., cancer and/or infection
  • kit means two or more components (one of which corresponding to the multifunctional molecule, the nucleic acid molecule, the vector or the cell of the invention) packaged in a container, recipient or otherwise.
  • a kit can hence be described as a set of products and/or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit.
  • the kits of this invention are in suitable packaging.
  • nucleic acid molecule or a group of nucleic acid molecules encoding said multifunctional molecule
  • the kit may thus include, in suitable container means, the pharmaceutical composition, fusion proteins or multifunctional molecules, and/or host cells of the present invention, and/or vectors encoding the nucleic acid molecules of the present invention, and/or nucleic acid molecules or related reagents of the present invention.
  • suitable container means the pharmaceutical composition, fusion proteins or multifunctional molecules, and/or host cells of the present invention, and/or vectors encoding the nucleic acid molecules of the present invention, and/or nucleic acid molecules or related reagents of the present invention.
  • means of taking a sample from an individual and/or of assaying the sample may be provided.
  • the compositions comprised in the kit according to the invention may particularly be formulated into a syringe compatible composition.
  • the kit further includes an additional agent for treating cancer or an infectious disease
  • the additional agent may be combined with the pharmaceutical composition, fusion proteins or multifunctional molecules, and/or host cells of the present invention, and/or vectors encoding the nucleic acid molecules of the present invention, and/or nucleic acid molecules, or other components of the kit of the present invention or may be provided separately in the kit.
  • the kit described herein may include one or more additional therapeutic agents such as those described in the "Combined Therapy" described hereabove.
  • the kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual as described hereabove.
  • the instructions related to the use of the multifunctional molecule or pharmaceutical composition described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, means for reconstituting the multifunctional molecule and/or means for diluting the multifunctional molecule of the invention.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual).
  • FIGURES Figure 1 Schematic representation of an embodiment of the multifunctional molecule according to the invention (A), cis demasking and cis activation technology of the bifunctional molecule constructed with optimized linker (B) and example of bifunctional antibody format designed with the linkers (C).
  • bifunctional antibody format designed with the linkers are: A: antibody with a single antigen binding domain fused in C-terminal of the Hole Fc chain to a cytokine and a peptide linker, B: antibody with a single antigen biding domain fused in C-terminal of the Hole Fc chain to a peptide linker and a cytokine, Cl : antibody fused in C-terminal of the Hole Fc chain to a first peptide linker, a cytokine and a second peptide linker; C2 antibody fused in C-terminal of one light chain to a first peptide linker, a cytokine and a second peptide linker; C3 : Monovalent Fab and a Fc domain, wherein a first peptide linker, a cytokine and a second peptide linker is fused to the N-terminal of one chain of the Fc domain; C4 : Bivalent scFv linked to a F
  • FIG. 2 Optimized linkers allow a cytokine masking effect on PD-l-cell lines while inducing a specific cis-potentiation on PD-1+ T cell lines.
  • IL15Rbg PD-1- CD122+CD132+
  • PD-1+CD122+CD132+ black line
  • IL15 wild-type cytokine IL15 wild-type cytokine
  • X isotype-E15-IL15wt-E15
  • ⁇ anti-PDl-E15-IL15wt-E15
  • B anti-PDl-(G4S)3-IL15wt (0).
  • FIG. 3 Optimized linkers in multifunctional molecules with wild-type cytokine (IL2) allow to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines.
  • IL2Rbg PD-1- CD122+CD132+
  • IL2Rbg PD-1+CD122+CD132+
  • B PD-1+CD122+CD132+
  • T cells were activated during 4 days with agonist anti-msCD3 (145-2C11) and anti-msCD28 (37.51) then treated with mouse I L12 wild-type cytokine (• dashed line), with anti PD-l-(G4S)3-mslL12p40-(G4S)3- mslL12p35 (• plain line), anti-PDl-E15-mslL12p40-E15-mslL12p35-E15 ( ⁇ ) hPDl+ and hPDl- T cells were separately incubated with the different constructions at escalating doses for 15min at 37°C and percentage of pSTAT4 signal was quantified by flow cytometry.
  • C A ratio of EC50 pSTA4 value on PD- 1- negative T cells vs PD1 + T cells reported on Figure. A mouse IL-12 cytokine was used for the design of the construction.
  • N 2 independent experiments
  • Figure 5 The optimized linkers of anti PD-1 bifunctional molecule mask cytokine activity on naive T cells and allow cis-demasking and cis-potentiation of activated T cells expressing PD-1+ cells.
  • Naive cells PBMC were isolated from whole blood of human healthy volunteers by Ficoll, and after 2 stimulations on anti-CD3/ anti-CD28 coating, LT activated were recovered and incubated with the different constructions at escalating doses.
  • B pSTAT5 evaluation on naive versus activated T cells by flow cytometry after treatment with Anti-PDl-E15-IL15wt-E15.
  • D pSTAT5 evaluation on naive versus activated T cells by flow cytometry after treatment with Anti-PDl-E15-IL2wt-E15. Naive ( A ) versus activated T cells ( ⁇ ).
  • EC50 (nM) was calculated to evaluate difference of activation between both constructions.
  • FIG. 6 Trans-activation assay on PD-1 presentation by antigen coating or expressing cells alone or in co-culture on PD-l-cells lines and PD-1+ cells lines.
  • A pStat5 signalling measured on Jurkat cells PD-1- CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone ( ⁇ ) or pre-incubated on PD1 coating ( ⁇ ) and Jurkat cells PD-1+CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone (•)
  • B pStat5 signalling measured on Jurkat cells PD-1- CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone ( ⁇ ) or pre-incubated on U937 PD1+ cells ( ⁇ ) and Jurkat cells PD-1+CD122+CD132+ added to anti-PDl-E15- IL15wt-E15 alone (•).
  • C Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye incubated with IL15wt alone (o) or anti-PDl-E15-IL15wt-E15 (V) and Jurkat PD- 1+ IL15Rbg+ cells incubated with IL15wt alone (•) or anti-PDl-E15-IL15wt-E15 ( ⁇ ) at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • MFI Median Fluorescent Intensity
  • Figure 7 Study assay of one or two optimized linker in construct to mask cytokine activity on PD-l- cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines.
  • A Schematic representation of the different constructions used in (B) and (C) is depicted in the figure: Anti PD-1 construction is monovalent and IL15wt cytokine was fused with either a (G4S)3 linker, two optimized E15 linker or a single optimized E15 linker fused to the cytokine, either between the cytokine and the anti-PDl or to the cytokine alone.
  • Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD-1+CD122+CD132+ (IL15Rbg) (B) were incubated with anti PD-l-(G4S)3-IL15wt (square), anti-PDl- E15-IL15wt-E15 (black circle), anti-PDl-E15-IL15wt (stars) or anti-PDl-IL15wt-E15 (triangle).
  • Each PD- 1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • MFI Median Fluorescent Intensity
  • (C) pSTAT5 bioassay on Jurkat PD-1 negative CD122+CD132+ (IL15Rbg) was performed with escalating dose of monovalent anti PD-l-IL15wt molecules constructed with one (G4S)3 flexible linker ((G4S)3-IL15n), Two (G4S)3 flexible linkers ((G4S)3-IL15-(G4S)3 ( ⁇ ), two (EAAAK)3 rigid linker ((EAAAK)3) ( ⁇ ) or the optimized E15 linker(E15-IL15wt-E15 ( ⁇ ).
  • FIG. 8 Peptide linkers of different amino-acid sequences tested on PD-l-cells and PD-1+ cells lines.
  • Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD- 1+CD122+CD132+ (IL15Rbg) (B) were incubated with IL15 wild-type cytokine (grey dot), anti PD-1- (G4S)3-IL15wt (square), isotype-E15-IL15wt-E15 (white dot), anti-PDl-E15-IL15wt-E15 (grey diamond), anti-PDl-D15-IL15wt-D15 (triangle) or anti-PDl-N15-IL15wt-N15 (cross).
  • IL15 wild-type cytokine grey dot
  • anti PD-1- (G4S)3-IL15wt square
  • isotype-E15-IL15wt-E15
  • PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • C EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells for each compound.
  • Jurkat cells expressing PD1+ CD122+ or expressing PD1+ and CD122+ but blocked with lmg/mL of an anti-PDl were incubated with I L15 alone, anti-PD-1 (G4S)3-IL15, or anti-PDl X15-IL15- X15, where X is a combination of different proportion of amino acid Glu (E) and Asp (D) (A), Glu and Thr (T) (Fig 9BGIu and Ala (A) (Fig 9C)) or Glu and Lys (K) (Fig. 9D).
  • PD-1- (PD1+ cells saturated with anti-PD-1) and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • MFI Median Fluorescent Intensity
  • Figure 10 Different length of optimized linkers tested to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cell lines.
  • Jurkat cells expressing PD-1-CD122+ (A) or expressing PD-1+ CD122+cells (B) were incubated with anti PD-1-(G4S)3-IL15 (white square); anti- PD1-E6-IL15-E6 (light grey diamond), anti-PDl-E10-IL15-E10 (triangle), anti-PDl-E15-IL15-E15 (grey circle), anti-PDl-E20-IL15-E20 (grey diamond) or anti-PDl-E25-IL15-E20 (black square).
  • PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • MFI Median Fluorescent Intensity
  • Figure 11 Binding PD1 assay of the anti PD-1 bifunctional molecule with optimized linker or conventional linker. Binding ELISA assay was performed on PD1 coating with incubation at different concentration of either anti PD-1-(G4S)3-IL15 (0) or anti-PDl-E15-IL15-E15 ( ⁇ ) for 2h at 37°C. Antibodies were detected with peroxidase-labelled anti-human IgG polyclonal antibody and revealed by TMB. Optical Density (DO) was measured at 450nm by spectrophotometer (Tecan).
  • DO optical Density
  • FIG. 12 Optimized linkers are capable of masking and demasking cytokine with one or multiple valences of anti PD-1.
  • A Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye (dotted lines) and Jurkat PD-1+ IL15Rbg+ cells (solid lines) were incubated with a monovalent anti-PDl-E15-IL15-E15 (triangle), a bivalent anti-PDl-E15-IL15-E15 (diamond) or an isotype-E15-IL15-E15 (circle) at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • B Median Fluorescent Intensity (MFI) of pStat5 activation in PD-1- or PD1+ cells during co-
  • FIG. 13 Anti PD-1 E15-IL2-E15 enhances safety as evaluated in toxicity mouse model in vivo.
  • FIG. 14 Anti PD-1-IL15 constructed with optimized linker E15 enhances safety in toxicity mouse model in vivo.
  • CT26 bearing BALB/c mice were intravenously injected with 4 doses (Day 4, 8, 12 and 15 post tumor inoculation) of PBS, anti PD-1 E15 IL15 wild type E15 or anti PD-1 (G4S)3 IL15wt (5mg/kg).
  • Anti PD-1 construction targeting mouse PD-1 (clone 17D2) were used for these experiments.
  • Monovalent anti PD-1 IL15wt constructions with (G4S)3 or E15-E15 linker (A) or bivalent anti PD-1 IL15wt constructions with (G4S)3 or E15-E15 linker (B) were used for these experiments.
  • mouse weight was monitored and Median survival reported. Survival represents mouse death due to injected drug toxicity.
  • One mouse was excluded in Bivalent Anti PD-1 (G4S)3 group due to tumor necrosis high tumor volume.
  • FIG. 15 Anti PD-1-IL15 constructed with optimized linker E15 demonstrated in vivo efficacy in monotherapy.
  • BalbC mice were inoculated with CT26 tumor subcutaneously (le6 cell/mouse) then treated with bivalent anti PD-1 E15- IL15wtE15, Anti PD-1(G4S)3 IL15wt construction on Day 4, 8, 12 and 15 (5 mg/kg) or PBS.
  • Anti PD-1 construction is an antagonist targeting mouse PD-1 (clone 17D2) Tumor growth (mm3) and survival were evaluated. Tumor growth could not be measured for this group.
  • N 7 to 8 mice/group
  • FIG. 16 Anti PD-l-IL2v or Anti PD-l-IL2wt constructed with optimized linker E15 demonstrated in vivo efficacy in monotherapy.
  • N 2 independent experiments
  • PanC02 (pancreas) bearing hPDIKI mice were injected intraperitoneal with 4 doses (day 7 , 11,15 and 19 post-tumor inoculation) of PBS or anti-PD-l-E15-IL2v-E15 (0,5 or lmg/kg).
  • Anti PD1 molecule used is a bivalent anti PD-1 format with one IL-2v molecule. Survival was monitoring during these experiments.
  • Statistical analysis was performed using Graph Pad prism using Log-Rank (Mantel Cox) test ** p ⁇ 0.002
  • FIG. 17 Anti PD-1-IL2 constructed with optimized linker E15 improved the therapeutic index of toxic immunocytokine.
  • Bifunctional molecules comprising anti-PD-l/IL-2v constructed with an optimized linker have high anti-tumor efficacy without lethal toxicity.
  • hPDKl Knock in mice inoculated with MC38 cells (ectopic model) were treated on Day 10, 12 and 14 with anti PD-1/ IL-2v molecule (Anti PD-1 bivalent constructed with IgGl N297A KIH isotype).
  • n 5 to 6 mice per group after treatment with Anti PD-l/IL- 2v constructed with optimized (E15-E15) linker.
  • D Therapeutic index of bifunctional molecules comprising anti-PD-l/IL-2v constructed either with an (E15-E15) optimized linker or with a conventional linker (Gly4-Ser)3. Therapeutics indexes were calculated with Maximum tolerated Dose versus EC50 efficacy.
  • E and F Therapeutic index calculated with all dose responses efficacy versus lethal dose of the construction anti PD-1 (G4S)3 IL2v or anti PD-1 E15-IL2v-E15.
  • EC50 was calculated for Efficacy and Lethal dose to evaluate the therapeutic window (fold change versus TD50 and LD50)
  • FIG. 18 pStat5 activation in co-culture assay with PD-1- IL15Rbg+ cells lines and PD-1+ IL15Rbg+cells lines.
  • Co-culture assay with ratio 1:1 of Jurkat PD-1- CD122+CD132+ cells stained with CellTrackerGreen dye (left panel) and Jurkat PD-1+ CD122+CD132+ cells (right panel) were incubated with IL15wt alone (black circles), anti-PDl-(G4S)3-IL15wt (grey circles, conventional linker) or anti-PDl- E15-IL15wt-E15 (white circles, cytomask linker) at escalating doses for 30min at 37°C.
  • Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • FIG. 19 Optimized linker (E15-E15) masked activity of mutated 11-15 cytokine (IL15v) on primary naive PD-1 negative cells and showed higher CIS activity on PD-1+ vs PD-1- compared to (G4S)3 conventional flexible linker (A) pSTAT5 activity measurement of human PBMCs cells sing flow cytometry of the monovalent Anti PD-l/IL-15v constructed with (G4S)3 linker or E15-E15 linker (IL15v ad set forth in SEQ ID NO: 112).
  • B Co-culture assay with ratio 1:1 of U937 PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye incubated at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • EC50 (nM) was calculated for each cell line and Ratio was obtained with EC50 PD-1+/EC50 PD-1 neg for each molecule tested.
  • FIG. 20 Optimized linker E15 allowed a masking effect of IL-15/IL15ra RLI construction on PD1- cell lines while inducing a specific cis-potentiation on PD1+ cell lines to higher extend compared to (G4S)3 conventional linker (A) Construction used for the Bioassay.
  • Bivalent Anti PD-1 antibody was fused to I L-15ra sushi domain, (G4S)3 linker and IL-15 wt cytokine (as set forth in SEQ ID NO: 113) or bivalent antibody was fused to IL-15Ra sushi domain, E15, IL-15wt and E15 linker (as set forth in SED ID NO: 114)
  • B pSTAT5 activity on PD-1+ and PD-1- CD122 transduced U937 cells using coculture assay (ratio 1:1 of U937 PD-1-/PD-1+) PD-1+ CD122+ cells were stained with CellTrackerGreen dye and cocultivated with PD-1+ CD122+ cells.
  • MFI Median Fluorescent Intensity
  • Optimized linker allowed a masking effect of IL-10 on naive PD-1 low T cells while maintaining high CIS activity on stimulated PD-1+ T cells
  • A PD-1 expression on human T cells after 2 stimulations using CD3/CD28. Expression was evaluated by flow cytometry
  • B pSTAT3 activity measurement of naive human T cells using flow cytometry after stimulation with the bivalent Anti PD- l/IL-10 constructed with (G4S)3 linker or E15 linker.
  • pSTAT3 % (left graph) and MFI (right graph) was quantified after permeabilization and intranuclear staining into CD3 T cell population.
  • FIG. 22 Optimized linker (E15) induced IL18 signaling only on targeted cells.
  • NKL cell line expressing +/- PD-1 were incubated with IL-12 (lOng/mL) and escalating doses of IL18, anti PD-1-(G4S)3-IL18 or anti PD-1-E15-IL18 for 24hours at 37°C (IL18 variant being as set forth in SEQ ID NO: 56).
  • N 2 independent experiments.
  • FIG 23 Multifunctional molecule activity constructed with an Optimized linker (E15-E15), an antimouse PD-1 and IL15.
  • Mouse T cells were isolated from C57BL6 mouse spleen and were +/- stimulated overnight with anti-CD3 agonist antibody to overexpress PD-1.
  • A Mouse PD-1 expression were quantified by flow cytometry for each T cell population (activated vs non activated).
  • Mouse T cells expressing +/- PD-1- were incubated at escalating doses for 15min at 37°C with bivalent anti msPD-1- E15-hlL15-E15; (B) or bivalent anti msPD-l-E15-IL2-E15; (C) or monovalent anti msPD-l-E15-hlL15-E15; (D) and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each cell population.
  • MFI Median Fluorescent Intensity
  • Optimized linker (E15-E15) allowed a masking effect of IL15 cytokine even fused to another antibody backbone as an anti-CD3 scFv on CD3negative mouse cells while inducing a specific cis-potentiation on CD3+ cells.
  • Mouse splenocytes were isolated from C57BL6 mouse spleen and were +/- presaturated with anti-msCD3 antibody (50pg/ml) to have no access to CD3 (representing CD3neg cells).
  • Mouse T cells expressing +/- CD3+ were incubated with a high dose (300nM) of human IL-15, anti msCD3-(G4S)3-hlL15 or anti msCD3-E15-hlL15-E15 and % pSTAT5 activation was quantified by flow cytometry after intracellular/lntranuclear staining.
  • Optimized linker (E15-E15) was also functional on monocytes cell line.
  • U937 monocyte cell line expressing CD122 and +/- PD-1 were incubated at escalating doses for 15min at 37°C with anti PD- l-(G4S)3-IL2v or anti PD-l-E15-IL2v-E15 and Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry for each cell population.
  • the right graph represent MFI of pSTAT5 activation at the highest dose tested (lOnM).
  • IL2v sequence is as set forth in SEQ ID NO: 110. Ill
  • FIG. 26 Optimized linker (E15-E15) maintained its integrity and activity in vivo.
  • A hPDIKI mice were injected intravenously with 5mg/kg of Anti-PD-l*2-E15-IL2v-E15 (SEQ. ID No: 110 - IL2v). Sera were sampled at different time point and pharmacokinetics were studied and concentration dosed using 2 ELISA with a anti human Kappa antibody and revelation with an anti-hlgG or an anti-hl L2 to evaluate quantity of antibody only or the full intact molecule comprising IL-2 cytokine.
  • (B) MC38 bearing hPDIKI mice were injected intraperitoneal with 4mg/kg of Anti-PD-l*l-(G4S)3-IL15 or Anti- PD-1*1-E15-IL15-E15. Organs were sampled 24hours post injection. Antibodies were dosed in organs after digestion by anti-human kappa / anti-hlL15 sandwich ELISA.
  • Sera containing antibodies were diluted at 1/8 in medium and used to stimulated U937 monocyte cell line expressing CD122 +/- PD-1.
  • pSTAT5 signaling on U937 were studied by flow cytometry after 15min of stimulation and intracellular staining with an anti pSTAT5- A647 and revealed by flow cytometry.
  • FIG. 27 Immunogenicity of E15-E15 or D15-D15 optimized linkers are similar to conventional (G4S)3 linker.
  • A Immunogenicity score in silico prediction (IEDB tools) as measured on binding properties of the sequence IL-15 +linker on MHC class II
  • B hPDIKI mice were injected intravenously with 5mg/kg of bivalent anti-PD-l*2-(G4S)3-IL15wt , anti-PD-l*2-D15-IL15wt-D15 or anti-PD-l*2-E15- IL15wt-E15.
  • Sera were sampled before injection and Dll post injection.
  • ADA anti-drugs antibodies
  • FIG. 28 Multifunctional immunocytokine anti PD-1/IL2 constructed with the optimized linker showed better IL-2 signaling on PD1+ T cells than other prior art masking strategy anti PD-l-IL-2ra- IL2.
  • CD122 and PD-1+/-- were stimulated with anti PD-l-IL2ra-IL2wt cytokine (right graph) or the anti PD-1 E15-IL2wt-E15 construction (left graph).
  • Cis activity was calculated with fold EC50 difference between PD-1+ ( ⁇ Black) and PD-1 negative (• grey ) cells.
  • hPBMCs were isolated from buffy coat and T cells were stimulated twice with agonist anti-CD3 or anti-CD28 to express PD1 (85% of expression). Cells were incubated at escalating doses for 15min at 37°C with anti PD-1 I L-2ra- IL2wt ( ⁇ ) or Anti PD-1 -E15 IL2wt-E15 (o) and Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry.
  • MFI Median Fluorescent Intensity
  • Optimized linker (E15-E15) is functional with an anti-TIGIT antagonist antibody Jurkat expressing CD122 and +/- TIG IT were incubated at escalating doses for 15min at 37°C with anti-TIGIT- G4S3-IL2wt molecules ( ⁇ ) or anti TIGIT-E15-IL2wt-E15 molecules (•).
  • Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry for each Jurkat T cell population TIGIT+ (plain line) orTIGIT negative (Dashed line).
  • the present invention describes the design and use of a novel specific linker between the targeting moiety (e.g. anti PD-1 antibody) and the fused protein (i.e., a cytokine) ( Figure 1).
  • the specific linker allows an inactivation/masking of the fused protein on non-specific naive T cells (PD-1 negative) and induces a specific cis-demasking and cis-activation after targeting/binding the right T cells with antibody moiety (e.g. targeting PD-1 + tumor specific T cells) as schematized on Figure 1A.
  • the present invention demonstrates superior efficacy than conventional flexible linker (G4S)3 or rigid linker (EAAAK)3 used for the construction of bifunctional molecule comprising immunocytokine and does not require metalloprotease cleavage for demasking a protein nor the use of mutated cytokine to decrease affinity of the cytokine. Mutated cytokines may still be used to fine tune affinity of the cytokine, but is not mandatory.
  • the inventors designed multiple bifunctional molecules comprising the optimized masking linker with a targeting moiety (i.e., antigen binding domain, such as monovalent, bivalent, scFv, VHH) and a cytokine (Figure IB). Multiple molecules can be targeted with targeting moiety without altering efficacy of the present invention.
  • the monovalent anti PD-l-X15-protein-X15 used in the examples, is a multifunctional molecule comprising a cytokine fused to the C-terminal domain of an anti PD-1 antibody with one linker between the Fc domain of the antibody and the cytokine, and one linker after the cytokine in C- terminal.
  • the two linkers X15/X15 were optimized linkers and were able to mask the molecule on PD1 negative cells. All constructions were engineered with an IgGl N297A isotype and amino acid sequences were mutated in the Fc portion to create a knob on the CH2 and CH3 of the Heavy chains A and a hole on the CH2 and CH3 of the Heavy chains B.
  • X15 is selected from the group consisting of SEQ ID NO : 32, 37, 41,42,14,43,44,20,45,17,58,59 and 46.
  • the monovalent anti PD-l-E15-protein-E15 used in the examples, is a multifunctional molecule comprising a cytokine fused to the C-terminal domain of an anti PD-1 antibody with one linker between the Fc domain of the antibody and the cytokine, and one linker after the cytokine in C-terminal.
  • the two linkers E15/E15 (EEEEEEEEEEEEEEEEE, SEQ ID NO: 5) were optimized linkers and were able to mask the molecule on PD1 negative cells.
  • a bivalent anti-PD-1 X15-protein-X15 was also tested.
  • the isotype-E15-IL15-E15 corresponds to a bifunctional molecule that comprise an antibody that does not target/bind PD-1.
  • the VH and VL sequences of the anti-PD-1 antibody used in Figures 2 to 9 are as set forth in SEQ ID NO: 74 and 75, respectively.
  • the VH and VL sequences of the anti-PD-1 antibody used can be as set forth in SEQ ID NOs: 74 and 75, respectively.
  • SEQ ID NO: 86 for anti-PD-1 VH with a knob IgGl Fc and N297A substitution and SEQ ID NO: 88 for anti-PD- 1 VH with a hole IgGl Fc and N297A substitution can also be used.
  • the cytokine is bound to the hole chain.
  • SEQ ID NO: 88 for anti-PD-1 VH with a hole IgGl Fc and N297A substitution and SEQ ID NO: 84 for the KIH Fc knob portion are used.
  • Linkers and cytokine are fused to the C-terminal end of the anti-PD-1 VH with a hole IgGl Fc and N297A substitution.
  • Anti-PD-1-1* or Mono-PDl refers to a monovalent construct whereas Anti-PD-1-2* or Biv-PDl refers to a bivalent construct.
  • Light chain of anit-PD-1 is as set forth in SEQ ID NO: 79.
  • the anti-PD-1 antibody used in Figures 14 to 16 is the antibody 17D2 that is disclosed in US2019/0336496.
  • the VH of 17D2 with a knob IgGl Fc and N297A substitution is disclosed in SEQ ID NO: 124.
  • the VH of 17D2 with a hole IgGl Fc is disclosed in SEQ ID NO: 125.
  • the light chain of 17D2 is disclosed in SEQ ID NO: 126.
  • SEQ ID NO: 125 for anti-PD-1 VH with a hole IgGl Fc and N297A substitution and SEQ ID NO: 84 for the KIH Fc knob portion are used.
  • Linkers and cytokine are fused to the C-terminal end of the anti-PD-1 VH with a hole IgGl Fc and N297A substitution.
  • the VH and VL sequences of the anti-TIGIT antibody used can be as set forth in SEQ ID NOs: 129 and 130, respectively.
  • SEQ ID NO: 120 for anti-TIGIT VH with a knob IgGl Fc and N297A substitution and SEQ ID NO: 121 for anti- TIGIT VH with a hole IgGl F and N297A substitution can also be used.
  • the cytokine is bound to the hole chain.
  • Anti-TIGIT-2* refers to a bivalent construct.
  • Light chain of anti-TIGIT is as set forth in SEQ ID NO: 130.
  • I L15 sequence is as set forth in SEQ ID NO: 53. Sequence of I L15 variant is as set forth in SEQ ID NO: 112. IL2 sequence is as set forth in SEQ ID NO: 50. Sequence of IL2 variant is as set forth in SEQ ID NO: 110. The two subunits of I L12 are as set forth in SEQ ID NOs: 51 and 52. IL-15Ra sushi-(G4S)3-IL15 is as set forth in SEQ ID NO: 113 whereas IL-15Ra sushi-E15-IL15-E15 is as set forth in SEQ ID NO: 114. IL-10 is as set forth in SEQ ID NO: 128. 1 L18 sequence is as set forth in SEQ ID NO: 55. Sequence of I L18 variant is as set forth in SEQ ID NO: 56.
  • Example 1 Bifunctional molecule constructed with an optimized linker E15-E15 (Anti PD-l/E-15- IL15-E15, Anti PD-1/E-15-IL2-E15) allow a specific cis-demasking and cis-activation of PD-1 + expressing T cells while sparing PD-1- T cells.
  • Optimized E15 linker show superior specific activity than the conventional flexible linker (G4S)3 commonly used in the immunocytokine prior art.
  • the inventors assessed the capacity of the optimized linkers to decrease activity of the cytokine on PD- 1- cells and allows a preferential cis-demasking and cis-activation of the cytokine on PD-1+ cells without inducing cleavage and with possibility to use wild-type or mutated cytokine.
  • Jurkat cells expressing only CD122+CD132+ (IL15RPy) (IL-15 complex signalling receptor), no PD-1 expression or co-expressing CD122+CD132+ (I LISRPy) and PD-1+ were incubated with I L15 wild-type cytokine, anti-PDl-E15-IL15wt-E15 (anti-PDl fused to I L15 with the E15 optimized linker), isotype-E15- IL15wt-E15 (isotype control fused to IL15 with the E15 optimized linker) and anti-PDl-(G4S)3-IL15wt (anti-PDl fused to I L15 with the conventional linker (G4S)3).
  • I L15 wild-type cytokine anti-PDl-E15-IL15wt-E15 (anti-PDl fused to I L15 with the E15 optimized linker)
  • isotype-E15- IL15wt-E15 isotype control fused to IL15 with the E15
  • Each Jurkat PD-1- 1 L15 RPy+ cells or Jurkat PD-1+ I L15RPy+ cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C.
  • the IL-15R signalling (pSTAT5) was quantified by flow cytometry into each PD-1 + and PD-1- cell population.
  • Figure 2A shows that the optimized linkers E15-E15 fused to Fc domain of the anti-PD-1 antibody allows to mask wild-type IL15 compared to IL15wt cytokine alone, which can induce high pStat5 activation into PD-1+ and PD-1 negative cells with similar efficacy.
  • Isotype-E15-IL15-E15 or anti PD-1-E15-IL15-E15 has low to no effect on inducing pSTAT5 signalling into PD-1 negative cells while the construction induces surprisingly a strong activation of IL15R signalling pStat5 into PD-1+ cells, similar to IL15wt cytokine alone, whereas the isotype-E15-IL15-E15, without anti-PDl targeting, has similar efficacy in PD-1+ and PD-1- cells with low activation of both cells.
  • the anti PD-1 domain of the anti-PD-l-E15-IL15wt-E15 molecule allows the targeting and cis- demasking of IL-15 on PD-1+ cells, and the cis-activation of IL15 signalling only on PD-1+ cells.
  • the multifunctional molecule with the specific optimized linker E15-E15 added in N-terminal and C- terminal of I L15 and fused to an anti-PDl binding domain is able to mask the cytokine activity on PD-1 negative cells.
  • the binding of the multifunctional molecule on PD-1+ cells restore the full activity of the cytokine in cis-manner by demasking the cytokine on PD-1+ cells.
  • immunocytokines or bifunctional antibodies are generally constructed with a conventional flexible linker GGGGS (more preferably (G4S)3) (for example Shen S, Sckisel G, Sahoo A, Lalani A, Otter DD, Pearson J, DeVoss J, Cheng J, Casey SC, Case R, Yang M, Low R, Daris M, Fan B, Agrawal NJ, Ali K. Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity. Front Immunol. 2020 May 8;11:832, or Xu, Yuanming et al.
  • Example 2 The intrinsic properties of the optimized linker to mask the fused cytokine and cisdemask and cis-activate PD-1 targeted T cells are conserved across multiple cytokines.
  • the inventors assessed the capacity of the optimized linkers to decrease activity of the fused cytokine on PD-1- cells and allows a preferential cis-demasking and cis- activation of the cytokine to PD-1+ cells.
  • Multifunctional molecules comprising optimized linkers has been tested with cytokines, namely an IL15 wild type cytokine, an IL-2 wild-type cytokine and an IL-12 wild type cytokine.
  • Jurkat expressing CD122+CD132+ (IL-2 complex receptor) only (no PD- 1 expression) (A) or co-expressing CD122+CD132+ and PD-1+ (B) were incubated with IL2 wild-type cytokine alone (naked recombinant cytokine), anti PD-1-(G4S)3-IL2 (anti-PDl binding domain fused to wild type IL2 with conventional linker) or anti-PDl-E15-IL2-E15 (anti-PDl binding domain fused to IL2 with E15 optimized linkers).
  • Jurkat cell PD-1+ or PD-1- population was respectively incubated with the different constructions at escalating doses.
  • pStat5 signalling for each cell lines was quantified by flow cytometry.
  • EC50 (nM) of the pSTAT5 activation was determined for each construction and regrouped in Table 2 for each PD-1+ and PD-1- cell population.
  • FIG. 3 on PD-1- cells shows that the optimized linkers fused to cytokine (anti-PDl-E15- protein-E15) masks the cytokine activity in decreasing pStat5 activity of wild-type IL2 anti PD-1 fused bifunctional antibody in similar manner as IL-15 cytokine.
  • the optimized linker outperforms the conventional (G4S)3 linker by decreasing to higher extend cytokine activity on PD-1 negative cells, as tested with construct named anti-PDl-(G4S)3-IL2.
  • Figure 3 on PD-1+ cells shows that the E15 optimized linker (anti-PDl-E15-IL2-E15) conserved its property to demask IL-2 cytokine on PD-1+ expressing cells allowing high signalling activation (EC50 in Table 3).
  • Optimized linker intrinsic property to mask the cytokine on PD-1- cells was confirmed for wild-type IL2 in addition to IL-15, suggesting that the optimized linker disclosed herein can be fused to various cytokines for a similar efficacy.
  • Example 3 Optimized linkers in multifunctional molecules with wild-type IL12 allow to mask cytokine activity on PD-l-cells, cis-demasking and cis-potentiation of PD-1+ cells lines, as well as enhancing safety as evaluated in vivo in toxicity mouse model.
  • Anti PD-1 monovalent antibody was fused to E15-IL-12 p40 -E15-IL12 p35-E15 or to E15-IL12p40-(G4S)3-E15 as schematized on Figure 4A.
  • Naive splenocytes PD-1- IL12R+ extracted from wild-type mouse (A) or activated splenocytes extracted from human PD1-KI mouse and activated CD3/CD28 to obtain activated PD-1+ IL12R+ cells (B) were incubated with mslL12 wild-type cytokine, with anti PD-1-(G4S)3- mslL12p40-(G4S)3-mslL12p35, anti- PDl-E15-mslL12p40-(G4S)3-mslL12p35-E15 (vl) or anti-PDl-E15-IL12p40-E15-IL12p35-E15.
  • PD1- naives and PD1+ activated cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C and Median Fluorescent Intensity (MFI) of pStat4 was quantified by flow cytometry for each cell population.
  • MFI Median Fluorescent Intensity
  • Anti PD-l-Cytokine bifunctional molecule comprising the optimized linker E15/E15 induce specific cis-demasking and cis-activation of human primary activated exhausted T cells while sparing naive PD-1 negative T cells.
  • PD-1 receptor is induced at the surface of T cells.
  • Chronic stimulation of T cells by tumor antigen induces exhaustion of the cells characterized by a loss of effector functions and anti-tumor activity and increased T cell death.
  • Tumor specific exhausted T cells can be reinvigorated through activation of a costimulatory signal (e.g. cytokine) into T cells and blockage of PD-1 inhibitory receptor.
  • a costimulatory signal e.g. cytokine
  • Anti-PD-1 bifunctional molecule constructed with an immunocytokine aimed to target PD-1+ tumor specific T cells and cis-deliver the cytokine to fully reactivate exhausted T cells.
  • pSTAT5 signalling following treatment was assessed into activated /exhausted T cells (PD-1+) and compared to the activation of naive T cells (PD-1-).
  • the inventors assessed the capacity of the optimized linkers to decrease activity of the fused cytokine on naive PD-1- T cells and allows a preferential cis-demasking of the cytokine and cis-activation on activated T cells expressing PD-1.
  • the aim is to reduce toxicity of the cytokine in periphery of the tumor where naive T cells are present and increase cytokine activity to re-activate specifically PD-1+ tumor specific activated/exhausted T cell.
  • Experiments were performed with a wild-type human IL15 or IL2 cytokine.
  • naive T cells from PBMCs of peripheral whole blood of human healthy volunteers were isolated by Ficoll and incubated with the different constructions at escalating doses.
  • pStat5 signalling was evaluated by flow cytometry into CD3+ T cell population.
  • Activated/exhausted T cells were generated by stimulating naive PBMCs two times with an anti-CD3/anti-CD28 antibody to mimic chronic antigen stimulation occurring into the tumor microenvironment and T cell exhaustion. After stimulation, T cells were incubated with the different constructions added at different concentrations.
  • Anti-PDl- E15-IL15wt-E15 activity was tested on naive and activated T cells, and EC50 was compared to nontargeted wild type IL15 recombinant cytokine or to anti-PDl-(G4S)3-IL15wt constructed with the conventional linker (Figure 5B). Same experiment was performed with the anti-PDl-E15-IL2wt-E15 construction and pStat5/3 was analyzed into T cell population (Figure 5C).
  • Figure 5B shows the anti-PDl-E15-IL15wt-E15 activity on naive T cells (PD-1-) compared to activated T cells (PD-1+).
  • the optimized linker fused to IL15 wild type cytokine inactivate the cytokine pSTAT5 activity on naive T cells (EC50 > 50nM) whereas non targeted recombinant IL15wt cytokine efficiently activates naive T cells (EC50 close to 0,lnM), the anti-PDl fused to IL15wt with conventional linker has an EC50 equal to 0.3nM.
  • optimized linker is able to decrease cytokine binding more than 500-fold on naive T cells compared to wild type non targeted cytokine.
  • anti-PDl-E15-IL15wt-E15 molecule binds activated T cells on PD-1 receptor and is able to cis-demask IL15 and promote efficient IL15 signalling to similar extend than recombinant IL-15wt non targeted cytokine.
  • An EC50 close to 0.1 nM is obtained with anti-PDl-E15-IL15wt-E15, similar to recombinant IL15.
  • Binding of anti PDl-E15-IL15wt-E15 to PD1 on activated T cells can promote conformational change of the cytokine only in cis-manner. More than 500-fold increased activity in PD-1 activated T cells versus naive T cells is observed with the optimized linker, demonstrating the advantage of the construction to reduce toxicity and cytokine activity on periphery cells, compared to activated T cells localized in the tumor microenvironment. Similar data were obtained with an anti PD-1 bifunctional antibody constructed with an IL-2wt cytokine.
  • pStat3/5 cis activation was reproduced with IL2 fused to anti-PDl with the optimized linker (Figure 5C) on naive and activated T cells.
  • Very low activation of naive T cells is observed with the anti-PDl-E15-IL2wt-E15 construction, whereas on activated T cells PD-1+, cis-activation was highly efficient by binding to PD-1 and increased more than 5000-fold potency of the drug into activated /exhausted T cells (EC50 close to 0.02nM with anti-PDl-E15-IL2wt-E15).
  • the construction comprising the optimized linker induce specific activation of activated PD-1+ exhausted T cells while sparing naive peripheral PD-l- T cells by binding with PD-1.
  • the inventors also evaluated the efficacy of anti-PD-l-E15-IL15wt-E15 to reinvigorate and cis-activate exhausted T cell infiltrating the tumor.
  • Human kidney tumor explant was dissociated, and T cells were isolated +/- stimulation with the anti PD-1 E15-IL15wt-E15 or non-targeted isotype E15- IL15wt-E15 and proliferation (Ki67 expression) was analyzed after 6 days of culture.
  • Figure 5D shows that the multifunctional molecule comprising anti-PD-l-E15-IL15wt-E15 efficiently promotes proliferation of exhausted T cells infiltrating the tumor to higher extend than Isotype E15-IL15-E15, demonstrating that the optimized linker allows to cis-activate T cells upon PD-1 targeting.
  • the fused cytokine can be presented to all surrounding T cells that can be tumor specific (PD-1+) or non-tumor specific T cells, thereby increasing unwanted toxicity (see Vincent, Marie et al. "Antitumor activity of an immunocytokine composed of an anti-GD2 antibody and the IL-15 superagonist RLI.” Oncoimmunology vol. 2,11 (2013): e26441).
  • the inventors assessed the capacity of the optimized linkers to strictly induce cis-activation of PD-1 + T cells, without inducing trans-activation of non PD-1 expressing cells via presentation by another PD-1+ T cells.
  • pStat5 signalling into Jurkat PD-1- expressing IL15RPy+ (CD122+CD132+) was analyzed after anti-PDl-E15-IL15wt-E15 pre-incubation on recombinant PD1 coated plate
  • B pSTAT5 signalling was analyzed into Jurkat PD-1- expressing IL15RPy+ (CD122+CD132+) after coculture with U937 PD1+ cells ( I LISRPy negative) cells.
  • Another coculture assay was performed by mixing Jurkat T cells PD-1- CD122+CD132+ stained with cell dye (Cell Tracker Green, Thermofisher) and Jurkat PD-1+ CD122+CD132+ cells (ratio 1:1). Mixed cells were then treated with the bifunctional anti PD1-E15-IL15-E15 for 30 min to analyze pStat5 signalling of each population PD-1- and PD-1+ cells.
  • Figure 6A represents pStat5 signalling into Jurkat PDl-IL15RPy+ with anti-PDl-E15-IL15wt-E15 molecule pre-incubated or not on PD1 coating. No higher pStat5 signal into Jurkat PDl-IL15Rbg+ was observed whether the molecule binds to PD1 coated plate compared to without coating. These data demonstrate that there is no trans-activation.
  • Figure 6B shows same results were obtained on Figure 6B with anti-PDl- E15-IL15wt-E15 pre-incubated or not on the surface of U937 PD-1+ (IL15RPy-) on pStat5 signalling of Jurkat PD-1- CD122+CD132+, which was similar between the conditions with or without PD-1+ presentation by cells, whereas, a strong signal was observed on Jurkat PD-l+IL15RPy+ cells.
  • Figure 6C- D shows pStat5 signalling of each population of PD-1- or PD-1+ cells obtained during co-culture assay with anti-PDl-E15-IL15wt-E15 compared to non-targeted IL15 recombinant cytokine.
  • Anti-PD1-E15- IL15wt-E15 multifunctional molecule activates PD-1+ cells while having a very low effect PD-1- cells, even in a co-culture model.
  • pSTAT5 EC50 (nM) is equivalent on PD-1- cells whether the cells are alone or co-cultivated with PD-1+ cells, demonstrating in another assay that no trans- activation of anti-PDl-E15-IL15wt-E15 is induced. The same is true for PD-1+ cells.
  • a preferential cis-activation on PD-1+ cells, and no-transactivation due to PD1 targeting on two different cell types with the multifunctional molecule comprising a E15/E15 optimized linker is observed.
  • the potentiation of IL15 signalling (cis-demasking and cis-activation) was strictly dependent on PD-1 binding on the same cells, thus, activation of IL15 pathway was restricted to PD-1+ cells present on the same cell.
  • linkers were designed and tested by the inventors to specifically induce inactivation of cytokine to PD-1 negative cells while allowing cis-demasking and activation of the molecule on PD-1+ T cells.
  • Different linkers tested were modified on the amino acid composition (type of amino-acid in the sequence of the linker or percentage of the same amino acid in the sequence of the linker), length and/or positioning (N-terminus and/or C-terminus of the cytokine) of the optimized linker.
  • Example 6 Design of the E15 Linker 1 +/- E15 Linker 2 to allow superior efficacy to mask fused cytokine: one or two E15 optimized linker fused in N-terminal or C-terminal of the cytokine mask cytokine activity to PD-1- cells to higher extend than conventional linker while efficiently allowing activation of PD-1+ cells
  • bifunctional molecules comprising (1) optimized linker E15 between Fc domain and cytokine IL15wt, named anti-PDl-E15-IL15*; (2) a molecule with one optimized linker E15 in C-terminal of the cytokine I L15, where the IL15wt cytokine is directly fused (without linker) to the Fc domain and the E15 linker is fused after the cytokine, named anti-PDl-nolink-IL15wt-E15 and compared to the anti PD-1 fused to two linkers before and after the IL15wt cytokine, named anti-PDl-E15-IL15wt-E15, and a bifunctional molecule comprising a linker of the prior art, namely an anti-PDl-(G4S)3-
  • the inventors evaluated the advantage of the optimized linker E15 versus another linker of the prior art, (EAAAK)3 (SEQ. ID NO 57)having a rigid structure (for example such as disclosed in Chen et al., 2013 PMID: 23026637).
  • Figure 7A on PD-1- cells shows that construction with only one E15 linker in C-terminal of the cytokine or between the Fc domain and the cytokine is sufficient to decrease pStat5 activity of the cytokine IL15, while the conventional linker (G4S)3 is more potent to activate the PD-1 negative cells ( Figure 7A).
  • Either one E15 linker, regardless of its position, or two E15 linkers are effective to mask cytokine and inactivate the cytokine ( Figure 7B).
  • the inventors constructed the Anti PD-1 immunocytokine with two (G4S)3 linker (a first (G4S)3 linker in N-terminal of the cytokine and a second (G4S)3 linker in C-terminal of the cytokine, (G4S)3-IL15-(G4S)3).
  • Figure 7C shows that 2 (G4S)3 linkers are not sufficient to mask the IL-15 activity on PD-1 negative cells while the optimized linker efficiently mask IL-15 activity on PD-1 negative cells.
  • linkers with other amino acids and tested their capacity to mask and cis-demask cytokines on PD1-/+ cells.
  • Two linkers were constructed solely with Asp (D) or Asn (N), and compared to the linker E15.
  • Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD- 1+CD122+CD132+ (IL15Rbg) (B) were incubated with IgG isotype-E15-IL15wt-E15 (white dot), anti- PDl-E15-IL15wt-E15 (grey diamond), anti-PDl-D15-IL15wt-D15 (triangle) or anti-PDl-N15-IL15wt-N15 (cross).
  • linkers can be constructed from a mix of amino acids without impact on their
  • the inventors investigated the necessity to have optimized linkers constructed solely of the same amino acid residue, and whether a mix between different group of amino acid has an effect on the masking and demasking ability.
  • Jurkat cells expressing PD1+ CD122+ or expressing PD1+ CD122+ but blocked with lmg/mL of an anti- PD1 were incubated with IL15 alone, anti-PD-1 (G4S)3-IL15, or anti-PDl X15-IL15-X15, where X is a combination of different proportion of amino acid Glu (E) and Asp (D) ( Figure 9A), Glu and Thr (T) ( Figure 9B), Glu and Ala (A) ( Figure 9C) or Glu and Lys (K) ( Figure 9D).
  • PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
  • MFI Median Fluorescent Intensity
  • the optimized linkers constructed with about half or 80% of positively charged amino acid could not mask the cytokine, resulting in an unspecific activation of the IL15 receptor.
  • a linker comprising more than 60% of negatively charged amino acid retained its masking/demasking ability (e.g., E12K3-IL15 E1K3 linker as shown in Figure 9D).
  • the inventors have proven that a linker constructed with a minimum portion of either negatively charged residue or amino acid residue with a polar uncharged side chain were capable of masking and demasking their cytokine, as long as the linker does not contain more than a maximum amount of positively charged amino acid.
  • Example 9 An optimized linker of 6 to 25 amino acid length is capable of fully masking cytokine on PD-1- cells and cis-demasking on PD-1+ cells
  • linker E Different length of the optimized linker was also tested to evaluate whether a specific size of the linker E is efficient for its activity.
  • New constructions with different length of linker composed of 6, 10, 15, 20 or 25 amino-acids Glu (E) amino acid.
  • linker 1 and linker 2 having the same length (a first linker between the Fc domain and the cytokine and a second linker in C-terminal of the cytokine), except for the 25 aa long linker which was coupled with a 20 aa long linker. All constructs tested in this assay comprise a monovalent anti-PDl binding domain and a wild-type IL15 cytokine.
  • Jurkat cells expressing CD122+ and not PD-1 (A) or co expressing CD122+PD-1+ cells (B) were incubated with the different constructions.
  • pSTAT5 activation was assessed for each construction by flow cytometry on PD-1- or PD-1+ cells.
  • Figure 10 shows that the length of the linker does not impact the property of the linker to mask the cytokine on PD-1 negative cells. All linkers demonstrated superior efficacy to inactivate/mask the cytokine compared to conventional ((G4S)3 linker) which are not sufficient to decrease cytokine activity. 10. Bifunctional antibody constructed with optimized linker efficiently bind to PD-1 on activated T cells
  • the inventors have verified that optimized linkers that are able to mask cytokine activation/binding does not modify the overall structure of the antibody and has no effect on the binding capacity anti- PD1 paratope of the antibody. Indeed, it is believed that the cis-demasking properties of the multifunctional molecules of the invention rely on the binding of the anti-PDl binding domain to PD- 1+ receptor, so that the paratope must be preserved in the construction for an optimal efficacy.
  • the binding to PD-1 was assessed by ELISA assay by coating PD-1 recombinant protein, binding of the antibody was revealed by a peroxidase-labelled anti-human Fc polyclonal antibody. Signal was quantified by Optical Density (OD) measured by a spectrophotometer (TECAN system).
  • Figure 11 shows that anti-PDl-E15-IL15wt-E15 molecule efficiently binds to PD-1 receptor to a similar manner than the anti-PDl-(G4S)3-IL15wt molecule comprising the conventional linker, demonstrating that the present invention with the optimized linker does not interfere in the binding of the anti PD-1 antibody paratope, and preserve the integrity of the targeting moiety of the multifunctional molecule while only masking the activity of the fused cytokine.
  • Optimized linkers are capable of masking and demasking cytokine with one or multiple valences of anti PD-1.
  • the inventors set to assess whether the number of valence of the antigen binding domain has an influence on the masking and demasking properties of the optimized linkers.
  • Figure 12A illustrates that the pSTAT5 activation of the PD1 negative and PD1 positive cells are equivalent between the molecule constructed with a single valence of an anti-PDl binding domain (i.e., a multifunctional molecule comprising a single anti-PD-1 binding domain) and those constructed with a double valence of anti-PDl biding domains(i.e., a multifunctional molecule comprising two anti- PD-1 binding domains).
  • EC50 calculated from this experiment confirms this observation, with a very high EC50 on PD1 negative cells and low EC50 on PD1 positive cells.
  • Example 12 Anti PD-1 E15 IL2 E15 and anti PD-1 E15 IL15 E15 molecules enhanced safety as evaluated in vivo in toxicity mouse model while maintaining efficient anti-tumor efficacy in vivo.
  • Cytokine therapy development in clinic is limited by the severe toxicity induced through activation of peripheral non-specific immune cells. Although the fusion of an antibody to the cytokine allows to redirect activity of the cytokine on immune cells, off-target related toxicity is observed across multiple immunocytokines and constructions. High systemic toxicity is induced by cytokine, in particular for IL- 2, limiting dose injected into patient of conventional immunocytokine. Since optimized linker improves masking of cytokine, the inventors next assessed effect of anti PD-1/IL2 wild type constructed with optimized E15 linker versus conventional linker. Mouse weight was followed every day after one (A) or multiple injection (B) (every day) at 2mg/kg.
  • Figure 13 shows that Anti PD-1 (G4S)3 IL2 multifunctional molecule induces toxicity associated with loss of overall weight, whereas anti PD-1 E15 IL2 E15 did not demonstrated significant toxicity. This data confirms the advantage of the use of the optimize linker to reduce peripheral systemic toxicity.
  • Anti PD-1 IL-15 constructed with E15 linker (Anti PD-1 E15-IL15-E15) is not toxic even after multiple injections at high doses (4 injections, 5mg/kg). No weight loss or death was observed (Figure 14) similar to control group (PBS injection). Altogether these data confirm the advantage of the E15 linker versus conventional linker to reduce off-target toxicity in vivo by masking cytokine activity/binding on non-targeted PD-1 negative cells.
  • the inventors next evaluated in vivo anti-tumor efficacy of the Anti PD-1 E15-IL2wt-E15 or the Anti PD- 1 E15-IL15-E15 in several mouse models.
  • Colon ectopic CT26 model BalbC mice were injected subcutaneously with CT26 tumor cells then Anti PD-1 E15- IL15wt-E15 (Bivalent anti PD-1) or PBS control was intraperitoneally injected on Day 5, 8, 12 and 15 at 5mg/kg and tumor growth was monitored using caliper.
  • melanoma ectopic B16 model C57bl6 mice were injected subcutaneously with B16-OVA tumor cells then Anti PD-1 E15- IL2wt-E15 (monovalent anti PD-1) or PBS (control) was intraperitoneally injected on Day 10, 14, 18 and 23 at 2mg/kg and tumor growth was monitored using caliper.
  • Pancreatic orthotopic model PanC02 cells were directly injected into the Pancreas and mice were treated on Day 7, 11, 15 and 19 with 2mg/kg of Anti PD-1 E15 IL2wt E15 construction. Tumor growth was quantified by Bioluminescence and survival monitored. 1 7
  • monovalent Anti PD-1 1 L2wild type form constructed with optimized linker E15 demonstrated a significant and good therapeutic response in immune checkpoint refractory PanCO2 model (decrease tumor load and improved survival (Figure 16A).
  • mice treated with a bivalent anti PD-1 antibody fused to E15- IL2v-E15 at 0.5 and 1 mg/kg (3 doses) induced significant anti-tumor response in vivo in this PanCO2 model (Figure 16B).
  • Example 13 The optimized linkers allow to improve therapeutic index of toxic immunocytokine by decreasing toxicity and maintaining high in vivo efficacy
  • IL-2v a single mutated IL-2 (IL-2v) (SEQ ID NO: 110) was used as well as an anti PD-1 targeting human receptor (VH/VL of SEQ ID NO: 74 and 75, respectively), with two optimized E15 linkers (i.e., each of SEQ ID NO: 5).
  • the bifunctional molecule tested thus comprises a bivalent anti- PD-1 antibody wherein the C-terminal of only one Fc chain is covalently linked to E15 linker- IL2v - E15 linker.
  • the bifunctional molecule constructed with the optimized linkers comprises: a first heavy chain of SEQ ID NO: 111 (i.e., comprising SEQ ID NO: 88-5-110-5), a first light chain of SEQ ID NO: 79 associated with the first heavy chain. a second heavy chain of SEQ ID NO: 86, and a second light chain of SEQ ID NO: 79 associated with the second heavy chain.
  • mice expressing human PD-1 receptor were inoculated subcutaneously with 0.5e6 MC38 cells.
  • tumor volume (mm3) was measured, and mice were randomized in the different groups to ensure a similar mean tumor volume across treatment groups.
  • 12 and 14 mice were treated with Anti PD-l/IL-2v molecule constructed with optimized linker (anti-PDl- E15-IL-2v-E15).
  • the immunocytokine IL-2v is mutated to attenuate binding to CD25 receptor. Treatment was performed intraperitoneally at 30, 10, 5 or 2 mg/kg. Tumor volume was measured at multiple time point as well as clinical score was assessed to evaluate potential toxic effect of the drug (lethal dose).
  • C, D and F anti-PD-1 bifunctional molecule constructed with an optimized linker and IL2 does not promote severe toxicity (mouse death) at the high doses tested (i.e., 15, 20, 10, 5, 2, 1 and 0.5mg/kg).
  • severe toxicity mi.e., 15, 20, 10, 5, 2, 1 and 0.5mg/kg.
  • a high anti-tumor efficacy was observed at all doses with a complete tumor regression. Only at the highest dose of 30 and 50mg/kg tested that sign of severe toxicity was observed, at 30 mg/kg one mice out of 5 died due to toxicity and at the dose of 50 mg/kg, 3 out 4 mice died following the 3 injections of the anti PD-1 IL-2v constructed with optimized linker.
  • Figure 17C illustrates the survival of the mice after injection of the drugs with a significant reduction of toxicity with optimized linker demonstrating the advantage of the present invention to optimize the therapeutic index of the immunocytokines.
  • Figure 17D, 17E and 17F show the Therapeutic index of the molecules at the doses tested, demonstrating once again the superiority of a multifunctional molecule comprising an optimized linker of the invention. Different methods to evaluate the therapeutic window were used for the calculation. The first method shown Figure 17C evaluates difference between the MTD (Maximum Tolerated Dose) and EC50 of therapeutic dose efficacy (TD50).
  • MTD Maximum Tolerated Dose
  • TD50 therapeutic dose efficacy
  • Example 14 The optimized linkers mask cytokine activity on PD-1- T cells even in the presence of PD-1+ cells.
  • Jurkat cells transduced with CD122+CD132+ and PD-1 were mixed with Jurkat cells transduced with CD122+CD132+ only (PD-1- cells) stained with cell tracker dye (CellTracker Green, Thermofisher ref C7025), mixed at a ratio 1:1 and treated with different molecules (i.e., IL15 wild type (wt) alone, an anti-PDl-(G4S)3- IL15wt multifunctional molecule or an anti-PDl-E15-IL15wt-E15 multifunctional molecule) during 30 minutes at 37°C.
  • IL15 wild type (wt) alone, an anti-PDl-(G4S)3- IL15wt multifunctional molecule or an anti-PDl-E15-IL15wt-E15 multifunctional molecule i.e., IL15 wild type (wt) alone, an anti-PDl-(G4S)3- IL15wt multifunctional molecule or an anti-PDl-E15-IL15wt-
  • pSTAT5 activation was evaluated into Jurkat PD1+ CD122+CD132+ cells and Jurkat PD1- CD122+CD132+ cells stained with CellTrackerGreen. The pStat5 signalling for each cell was quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction for each PD-1+ and PD-1- cell population. Cytomask linker is E15 and conventional linker is (G4S)3.
  • Figure 18 shows that the optimized linker E15-E15 fused to the Fc domain of an anti-PD-1 antibody allowed to mask wild-type IL15 on PD-1 negative cells compared to IL15wt cytokine alone, even if the construct was bound on PD1 positive cells and trans-presented by the PD-1 positive cells.
  • the anti PD-1-E15-IL15-E15 multifunctional molecule had no effect on inducing pSTAT5 signalling into PD-1 negative cells, and on the other hand, the anti PD-1-E15-IL15-E15 multifunctional molecule surprisingly induced a strong activation of IL15R signalling into PD-1+ cells, similar to IL15wt cytokine alone.
  • Optimized linkers can be fused to different cytokines while maintaining masking and CIS-demasking properties.
  • hPBMCs were isolated from healthy donors buffy coat using Ficoll gradient. Immune cells were stimulated 15min with treatments at 37°C then fix, permeabilized and stained with anti-CD3- Pacific blue and anti-pSTAT5-AF647. Median Fluorescent Intensity (MFI) of pStat5 was determined for naive T cells ( Figure 19A).
  • PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was determined for each PD-1 and PD-1+ cell type.
  • MFI Median Fluorescent Intensity
  • hPBMCs were +/- stimulated with agonist anti-CD3 and anti-CD28 antibodies for 3 days to promote overexpression of PD-1 on T cells.
  • Activated T cells were +/- incubated with high dose of Anti-PD-1 to block the receptor. Then, cells were stimulated 15min with anti-PD-1- (G4S)3-IL10 or anti-PD-l-E15-IL10 and expression of pSTAT3 was evaluated (Figure 21).
  • NKL cell line were treated with low dose of IL-12 and escalating dose of I L18, anti- PD-l-(G4S)3-IL18v or anti-PD-l-E15-IL18v-E15 during 24hours at 37°C.
  • IFNy secretion were dosed by ELISA in supernatant using I FNg ELISA ( Figure 22).
  • Example 1 and 2 The inventors have fused different cytokines to the optimized linkers and evaluated their masking and CIS-Demasking properties. As shown in Example 1 and 2, optimized linkers enhanced masking activity of IL-15 and IL-2 (wild-type form). In the Example 13 and Figure 17, the inventors have also demonstrated the advantage of optimized linker to mask activity of mutated IL-2 cytokine (F42A, Y45A, L72G, as set forth in SEQ ID NO: 110). IL-2 mutation used in these experiments is a common mutation described to decrease binding to CD25 (IL-2Ra) to spare activation of Tregs that express high level of CD25.
  • IL-2Ra mutated IL-2 cytokine
  • anti PD-1 antibody was fused to the optimized and IL-15 cytokine mutated (N1G-D30N-E46G-V49R-E64Q, as set forth in SEQ ID NO: 112).
  • pSTAT5 signalling Median fluorescence MFI analysis was evaluated on naive primary T cells and on PD-1+ versus PD-1 negative transduced cell lines and compared to the same anti/PD-IL15v molecule constructed with the (G4S)3 flexible linker ( Figure 19A and B).
  • Optimized linker E15-E15 inhibited activity of the cytokine on naive primary T cells that do not express PD-1 or very low expression, showing high masking properties of the E15-E15 linker for mutated IL-15 to higher extent compared to Anti PD-1 (G4S)3 IL-15v.
  • Anti-PD-1 E15-IL15v-E15 has low activity on PD-1 negative CD122+ transduced Jurkat cell line while showing high CIS-activity on PD-1+ CD122+ transduced cell line.
  • the inventors have also evaluated the effect of the optimized linker on Anti PD-l/IL-15 Immunocytokine constructed with a sushi domain.
  • the human IL-15 covalently linked to the human IL- 15Ra sushi(+) domain to create a superagonist of IL-15 and to bypass the need of endogenous IL-15Ra for transpresentation of IL-15 cytokine to CD122+/CD132+ T cells.
  • This construction has been previously described as potent molecule for the cancer therapy as non-targeted format or fused to an anti PD-1 to target and cis activate PD-1+ activated Tcells.
  • E15-E15 optimized linker decreased pSTAT activity of IL-15 superagonist compared to (G4S)3 flexible linker while maintaining high activity on PD-1+ cells with similar to (G4S)3 linker as shown on Figure 20B.
  • the optimized linker showed higher CIS-activity fold change between PD-1 neg cells and PD-1+ targeted cells .
  • cytokines as IL-10 as set forth in SEQ ID NO: 128) and IL-18 (as set forth in SEQ ID NO: 56) were also fused to the anti PD-1 antibody to evaluate effect of the optimized E15 linker (One linker) ( Figure 21 and 22).
  • E15 linker masking activity on the cytokine on untargeted cells compared to conventional (G4S)3 linker and good capacity to demask cytokine activity on targeted cells as observed by pSTAT3 signaling (IL-10 stimulation) or IFNg secretion (IL-18 stimulation).
  • IL-10 is an homodimeric cytokine (2 similar subunits)
  • IL-12 is an heterodimeric cytokine (2 different subunits)
  • IL-2, IL-15 or IL-18 are monomeric cytokines.
  • E15 linker decreases activity of the cytokine on non-targeted cells while maintaining a good cytokine activity on targeted cells, demonstrating versatile property of this linker for multiple cytokines regardless of the structure and properties of the cytokine.
  • fusion of receptor domain can be inserted in the construction with E15 linker while maintain CIS- activity of the cytokine.
  • Optimized linker can be fused to different targeting antibody moiety while maintaining masking and CIS-demasking properties on non-targeted cells versus targeted cells.
  • mice T cells were isolated from C57BL6 mouse spleen using mechanical digestion and magnetic sorting. T cells were +/- stimulated overnight with anti-CD3 agonist antibody to overexpress PD-1. Non-stimulated (naive, dotted line) and stimulated T cells (solid line) were incubated at escalating dose of anti-PD-l-E15-hlL15-E15 or anti-PD-l-E15-hlL2v- E15. Median Fluorescent Intensity (MFI) of pStat5 was determined for each PD-1 and PD-1+ cells ( Figure 23).
  • MFI Median Fluorescent Intensity
  • mouse splenocytes were isolated from C57BL/6 mouse using mechanical digestion. Cells were +/- pre-saturated with ant-mouse CD3 antibody to block access to the CD3 antigen (same clone as Immunocytokine construction (clone 145.2C11). Cells were treated with a high dose (300nM) of human IL-15, anti msCD3-(G4S)3-hlL15 or anti- msCD3-E15-hlL15-E15 for 15 minutes then stained with anti-pSTAT5. Expression of pSTAT5 was evaluated on both CD3 "negative” or CD3 positive cells ( Figure 24).
  • the inventors have tested different antibody backbones to evaluate the capacity of optimized linkers to be used with different antibodies having different formats or targets. Different constructions have been designed with another anti PD-1 backbone (mouse 17D2 clone). First, another anti PD-1 backbone in bivalent format (2 Fab arm) was used and fused to E15 optimized linker and IL15wt cytokine (Anti msPD-l-E15-IL-15wt-E15). The anti PD-1 clone used targets mouse PD- 1 receptor and published to target Activated PD-1+ T cells with PD-L1 antagonist properties in vitro and in vivo (Chan et al., 2022, Nat. cancer, PMID: 35256819).
  • an anti-mouse CD3 antibody (clone 145.2C11) (Alegre et al, JI, 1997, PMID 7636216).
  • This antibody used in the construction is a single chain (scFv domain) targeting CD3+ mouse T cells, fused to lgGlN297A isotype, an E15 linker, the IL-15wt human cytokine and a E15 linker compared to a scFv CD3, fused to lgGlN297A isotype, (G4S)3, IL-15wt.
  • the inventors also tested the usefulness of the optimized linker with other checkpoint inhibitory receptor, by fusing an anti TIG IT antibody used in clinic (Tiragolumab) to the optimized linker E15 and IL-2wt cytokine.
  • Data of Figure 29 shows that a multifunctional molecule comprising E15 optimized linker mask activity of IL2 cytokine on TIGIT negative cell line, while maintaining high CIS activation of TIGIT + cells similar to the anti TIGIT-(G4S)3-IL2wt molecule.
  • pSTAT5 signaling ex-vivo was studied.
  • hPDIKI mice were treated with intravenously with lOmg/kg of Anti-PD-1*2-(G4S)3-IL15 or Anti-PD-1*2-E15-IL15-E15 and sera were sampled 24hours after.
  • Sera containing antibodies were diluted at 1/8 in medium and used to stimulate U937 monocyte cell line expressing CD122 +/- PD-1.
  • pSTAT5 signaling on U937 were studied by flow cytometry after 15min of stimulation and intracellular staining with pSTAT5-A647 ( Figure 26C). Results.
  • the intact molecules (anti PD-1 fused to IL-15) was quantified in the spleen, tumor nodule, liver using an antihuman kappa antibody coated and revelation was performed using an anti-human IL-15 (Figure 27B).
  • Identical quantity of the Anti PD-l/IL-15 molecule between the 2 constructions were obtained by ELISA confirming that E15 linker is not cleaved in vivo, thereby no 11-2 will be released into tumor microenvironment.
  • Optimized linker has no immunogenic properties as evaluated bv in silico prediction in human and in mice.
  • ADA anti-drugs antibodies
  • hPDIKI mice were treated intravenously with 5mg/kg of anti-PD-l-(G4S)3-IL15wt, anti-PD-l-D15- IL15wt-D15 or anti-PD-l-E15-IL15wt-E15.
  • Sera were sampled before and 11 days after injection.
  • ADA in sera were quantified by ELISA by coating of drugs at 5pg/ml overnight at 4°C, capture of mouse sera for 2h at 37°C and revelation with donkey ant-mouse IgG peroxidase for lh at 37°C and conventional methods for reading optical density.
  • Efficacy and CIS activity of the present invention were compared to another immunocytokine comprising a non-cleavable masking strategy.
  • the described immunocytokine is constructed with an Anti PD-1 fused to IL-2Ra receptor and IL-2wt cytokine and use a flexible linker fused between the Fc domain, the cytokine and the receptor.
  • This construction termed PDl-IL2Ra-IL2 was recently published and its activity described (Wu et al., Cell Rep Med, . 2024 Oct 15;5(10):101747, PMID: 39326410).
  • the inventors constructed the same immunocytokine IL-2/IL-2ra structure as disclosed in Wu et al., with the same anti-PD-1 backbone as the anti PD-1 E15-IL2wt-E15 molecule used herein, to have a direct comparison of the masking strategies regardless of the targeting domain affinity.
  • SEQ ID NO: 120 describes the sequence of G4S)3-IL2Ra-(G4S)5-IL2wt.
  • Figure 28A shows that the Anti PD-l-IL2wt constructed with E15 linkers showed a strong activation of PD-1+ T cells while low activation of PD-1 negative U937 cells is observed, a high CIS-activity was observed (not calculable due to high activation).
  • Figure 28B confirms in primary human T cells that Anti PD-l-IL2wt constructed with optimized linker outperformed to promote activation of IL-2 signaling into activated PD-1 + T cells, while the PDl-IL2Ra-IL2 construction showed less capacity to restimulate this PD-1+ T cells.
  • plasmid (Knob :Hole :VL) were co-transfected by polyethylenimine method. After 7 days incubation, supernatant was recovered and purified by affinity on Protein A chromatography (HiTrap, GeHealthcare) (with citric acid 0.1M pH 3 elution buffer) and full antibody was isolated by gel filtration chromatography (preparative SEC column, Hiload 16/600 Superdex 200, Cytiva). Purified antibody was dialyzed in PBS and concentrated. They were quantified by UV at 280nm and tested in several test.
  • Each PD-1- cells or PD-1+ cells were respectively incubated with the different constructions at escalating doses for 15 to 30min at 37°C, 5%CC>2 in TexMacs medium.
  • A647-labelled anti-pStat5 (clone 47/Stat5(pY694, BD Biosciences ref 612599) was used to stain pStat5 for 30min at RT.
  • the pStat5 signaling for each cell lines were quantified by flow cytometry.
  • EC50 (nM) of the pSTAT5 activation was determined for each construction for each PD-1+ and PD-1- cell populations.
  • human PDl-Fc (SinoBiological ref 10377-H02H) was coated overnight at 4°C on P96-plate before adding Jurkat PD1- 1 L15RP/+ cells and the different constructions to analyse pStat5 signaling.
  • Jurkat or U937 transduced with CD122+CD132+ and PD-1 were mixed with Jurkat or U937 transduced with CD122+CD132+ only stained with cell tracker dye (CellTracker Green, Thermofisher ref C7025) mixed at a ratio 1:1 and treated with the tested molecule during 30 minutes at 37°C.
  • cell tracker dye CellTracker Green, Thermofisher ref C7025
  • pSTAT5 activation was evaluated into Jurkat or U937 PD1+ CD122+CD132+ cells and Jurkat or U937 PD1- CD122+CD132+ cells stained with CellTrackerGreen. The pStat5 signaling for each cell were quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction for each PD-1+ and PD-1- cell population. pSTAT5 analysis in human naive and activated T cell
  • PBMCs isolated from peripheral blood of human healthy volunteers were incubated 30 minutes with the different constructions at 37°C.
  • pSTAT5 or pSTAT3 activation was evaluated into CD3+ T cell population.
  • Activated/exhausted T cells were generated by stimulating naive PBMCs two times with an anti-CD3 (clone OKT3) / anti-CD28 (clone CD28.2) coating at 3pg/ml for 2-3days at 37°C, 5%CO2 for each stimulation.
  • pSTAT5 or PSTAT3 activation was evaluated into CD3+ T cell population with staining with Pacific Blue anti-CD3 antibody (BD Biosciences, reference B220).
  • activated T cells can be pretreated with high dose of anti-PDl (50pg/ml) to block the receptor.
  • TexMacs medium Miltenyi ref 130-097-196
  • cytokine or the different constructions were incubated with cytokine or the different constructions at escalating doses for 15 to 30min at 37°C, 5%CC>2 in TexMacs medium.
  • Mouse T cells were isolated from C57BI/6 mouse spleen and were +/- stimulated overnight with anti- CD3 agonist antibody (clone 145-2C11, BD Biosciences, reference 553057) to overexpress PD-1. Cells were incubated 15 minutes with the different constructions at 37°C. For mouse T cells, pSTAT5 activation was evaluated into CD3+ T cell population, like described above. In some experiment, activated mouse T cells can be pretreated with high dose of anti-mouse CD3 antibody (50pg/ml) (clone 145-2C11, BD Biosciences, reference 553057) to block the receptor. NKL activation with I L18
  • NKL cell line was transduced to express human PD1+.
  • NKL PDl- or NKL PD1+ were cultivated during test in TexMacs medium (Miltenyi ref 130-097-196) and were treated with low dose of IL-12 (lOng/ml) (Miltenyi, reference 130-129-722) and escalating dose of I L18, anti-PD-l-(G4S)3-IL18 or anti- PD-1-E15-IL18-E15 during 24hours at 37°C.
  • IFNy secretion were dosed by ELISA in supernatant using commercial human I FNg ELISA assay (Biolegend, reference 430104).
  • mice were intravenously injected with anti PD-1 bifunctional antibodies (5mg/kg).
  • Plasma drug concentration was determined by 2 methods ELISA : the first to measure IgG using an immobilized anti-human light chain antibody (clone NaM76- 5F3, OSE Immunotherapeutics), then serum-containing antibodies were added. Detection was performed with a peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) and revealed by conventional methods.
  • mice were subcutaneously injected with MC38 tumor. Around Day 10 when the tumor was palpable, mice were treated with Anti PD-1 E15-IL-15-E15 (4mg/kg, intraperitoneal administration, one dose). After 24h, mice were sacrificed, organs were harvested and crushed with FastPrep beads Matrix D (MP Biomedical, reference 1169130-CF) in Ripa buffer and molecules were quantified in lysate by ELISA using an immobilized anti-human light chain antibody (clone NaM76-5F3, OSE Immunotherapeutics), then lysate-containing antibodies were added.
  • FastPrep beads Matrix D MP Biomedical, reference 1169130-CF
  • Detection was performed with biotinylated-labeled anti-human IL15 (Biolegend, reference 515104, clone BH1543), followed by peroxidase-labelled streptavidin (Jackson Immunoresearch, reference 016-030-084) and revealed by conventional methods. Concentrations obtained were normalized to total protein concentration measured by BCA assay (Interchim, reference UP40840A). pStat5 activation was measured in lysate as described above on U937 expressed express CD122+ PD- 1- or to express CD122+PD-1+ cells. pSTAT5 signaling on U937 were studied by flow cytometry after 15min of stimulation and intracellular staining with pSTAT5-A647 with similar methods than described above.
  • Immunogenicity For prediction of immunogenicity in silico , we use IEDB software tools (NetMHCpanll method), which analyzed the antibody sequence and the potential of peptide binding to HLA-DR (MHC classll, through more predominant alleles: DRBl*01:01; DRBl*03:01; DRBl*04:01; DRBl*07:01; DRBl*08:01; DRB1*11:O1; DRB1*13:O1; DRB1*15:O1) molecule hence predicting activation of adaptive immune response and immunogenicity of the drug.
  • IEDB analysis ranks peptide binders depending on the predicted affinity to MHCII and homology with human sequences. Mean of percentile rank of all different peptides is calculated for each allele. Values equal to 100 corresponds to absence of immunogenicity, value ⁇ 1 corresponds to a a high immunogenicity score and value between 1-30 is considered as medium immunogenicity.
  • hPDIKI mice were injected intravenously with 5mg/kg of bivalent anti-PD-l*2-(G4S)3-IL15wt , anti-PD-l*2-D15-IL15wt-D15 or anti-PD-l*2-E15-IL15wt-E15.
  • Sera were sampled before injection and Dll post injection.
  • ADA anti-drugs antibodies
  • ELISA enzyme-activated immunosorbentase-labelect-associated anti-derived anti-derived anti-derived anti-derived anti-derived anti-IL15wt-E15.
  • Sera were sampled before injection and Dll post injection.
  • ADA anti-drugs antibodies
  • ELISA in incubating on coated-plate with the drug and in detecting with peroxidase- labelled anti-mouse IgG antibody (Jackson Immunoresearch; USA; reference 715-036-151) and revealed by conventional methods.
  • PanC02 pancreas cancer cell line bearing hPDIKI mice were injected intraperitoneally with 4 doses (day 7, 11,15 and 19 post-tumor inoculation) of PBS or anti-PD-l-E15-IL2v-E15 (0,5 or lmg/kg).
  • Anti PD1 molecule used is a bivalent anti PD-1 format with one IL-2v molecule. Survival was monitoring during these experiments.
  • Statistical analysis was performed using Graph Pad prism using Log-Rank (Mantel Cox) test ** p ⁇ 0.002
  • hPDIKI mice expressing human PD-1 receptor were inoculated subcutaneously with 0.5e6 MC38 cells.
  • tumor volume (mm3) was measured, and mice were randomized in the different groups to ensure a similar mean tumor volume across treatment groups.
  • 12 and 14 mice were treated with Anti PD-l/IL-2v molecule constructed with optimized linker (anti-PDl-E15-IL-2v-E15).
  • Treatment was performed intraperitoneally at 30, 10, 5 or 2 mg/kg. Tumor volume was measured at multiple time point as well as clinical score was assessed to evaluate potential toxic effect of the drug (lethal dose).
  • E15 it is meant 15 repetition of the amino acid E (i.e., EEEEEEEEEEEEEEEEEEEEEEE as described in SEQ ID NO: 5).
  • G4S3 it is meant a (G4S)3 linker (i.e., as described in SEQ. ID NO: 106).
  • Example section and Table 4 may refer to "*1", meaning one valency of the antigen binding domain.
  • a construction or molecule refers for example to anti-PDl*l, it means a monovalent/single antigen binding domain against PD-1, typically comprising a single VH and a single VL against PD-1.
  • Monovalent molecule particularly comprises a first heavy chain devoid of antigen binding domain, a heavy chain with an antigen binding domain and a single light chain.
  • bivalent i.e., has two antigen binding domains (e.g., two Fab)
  • the Example section and Table 4 may refer to "*2" , meaning two valencies of the antigen binding domain.
  • a construction or molecule refers for example to anti-PDl*2, it means a bivalent molecule (i.e., with two antigen biding domains, such as two Fab, against PD-1).
  • Bivalent molecule particularly comprises two heavy chains and two light chains.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to a molecule comprising an antigen binding domain derived from an antibody; one or more cytokines having a pro-inflammatory effect covalently linked to the antigen binding domain; and one or more peptide linker(s) covalently linked to the cytokine, the peptide linker(s) being suitable for masking the effect of the cytokine. It also relates to the therapeutic uses of said molecule for the treatment of cancer.

Description

MOLECULES COMPRISING MASKING LINKERS AND USES THEREOF FOR THE TREATMENT OF CANCER
FIELD OF THE INVENTION
The invention pertains to the field of medicine, in particular immunotherapy. It relates to a molecule comprising an antigen binding domain derived from an antibody and a cytokine having a pro- inflammatory effect, the molecule comprising linker(s) suitable for masking the effect of the cytokine. It further relates to the use of the molecule for the treatment of cancer.
BACKGROUND OF THE INVENTION
Multifunctional molecules are currently the object of developments in immunology, especially in the field of oncology. Such multifunctional molecules typically comprise an antigen binding domain directed to a particular target (e.g., on immune cells or tumoral cells) and a cargo (e.g., an immunoactive molecule such as a cytokine). Even though they present many advantages, these molecules may also present inconveniences. The design of multifunctional molecules implies several key attributes such as binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, compatibility with the attachment of additional domains but also production yield and cost compatible with a clinical development.
For instance, bifunctional molecules based on an antibody antagonizing PD-1 and linked to various interleukins (IL2, IL15, ...), or other immunotherapeutic agents have been disclosed, in particular in WO2018/184964 or W02018071919. Such molecules provide the advantages to target tumor tissues thanks to the PD1-PDL1 interaction, and in the same time allow to deliver interleukins into the tumoral site. However, optimal targeting of intra-tumoral cells remains difficult to achieve. For instance, due to the high affinity of IL-2 for its receptor, the multifunctional molecules are directed to cells expressing the receptor rather than to the tumor. To circumvent this inconvenient, different solutions have been proposed.
A first strategy is based on the masking of the immunotherapeutic agents by a molecule, protein or peptide, such as antigen binding domains or receptors, that have a binding affinity for the immunotherapeutic agents. However, this strategy comprises the inclusion of additional moieties in the multifunctional molecule and the risks of insufficient demasking once onsite. Additional moieties increase the size and weight of the multifunctional molecule, affecting its pharmacokinetic characteristics and distribution, which in turn may render the multifunctional molecule less effective than a standard medication. Circumventing this issue with a double binding antibody is complicated as it needs to search, find and test multiple potential antigen binding domain for each pair of target and immunotherapeutic agent to assess efficiency and safety. Other strategies are based on cleavable linkers, i.e., linkers which mask the cargo of bifunctional molecules until its arrival at a delivery site. Once onsite, the linker is cleaved to release its cargo.
Multiple linkers with physicochemical properties were used to cleave cytokine by proteases into tumors and to release cytokines specifically into tumor environments (Hsu et al, 2021, Nat. Commun., 12, 2768; Cao et al, 2021, Nat. Commun., 12, 5866 ; Desnoyers et al, 2013, Sci. Transl. Med., 5). This strategy improves cytokine biodisponibility and prevents off-target in periphery. Protease activable masked cytokines have been reported by using a variety of masking moieties.
Nevertheless, this strategy still comprises the risks of insufficient activation in the tumor environment and of undesired activation in non-tumor tissues. Indeed, this solution has the inconvenient to be dependent to the type and quantity of proteases present in the tumor microenvironment for its efficacy.
Finally, attempts have been made in the prior art to develop compounds able to release the effector (immunotherapeutic agent) at the lower intratumor pH. In particular, cleavable linkers have been designed to allow the delivery of the effector only into the tumor. However, the effector part of the compound thus released may generate secondary uncontrolled effects, notably for immunotherapeutic agents like cytokines that can induce toxicity. As illustration of this strategy, WO2022/117692 discloses pH-dependent IL-2 mutants having a reduced systemic activity at neutral pH and a full activity in the tumor environment at acidic pH.
There remains therefore a significant need in the art for new and improved multifunctional molecule for safe immunotherapy against cancer. The present inventors have made a significant step forward with the invention disclosed herein.
SUMMARY OF THE INVENTION
The applicant has developed an innovative solution allowing to control the activity of cytokine by using masking peptide linkers that are not cleavable, and thereby provides a reliable platform usable with a large array of immunotherapeutic multifunctional molecules with minimal to no modification whatsoever.
The advantage of the linker peptides according to the invention is their ability to mask a cytokine, in particular its activity and/or binding to its cognate receptor. In the multifunctional molecules of the invention, it is the binding of the targeting moiety (e.g., antibody) to its target that enables unmasking/demasking of the cytokine and its binding to its receptor. These masking and demasking properties are intrinsic to the composition/sequence of the linkers of the invention. This is why the inventor have thought of the name "cytomask" or "cytomasking" linkers when referring to the linkers of the invention. Such platform allows to include molecules having an effect too important to be safely used alone, especially because of a narrow therapeutic index or an off-target systemic toxicity. For example, wild type cytokines such as IL2 can be used in the multifunctional molecule of the invention, without further modification, even though their use is usually discouraged because of their pleiotropic side effect.
It is especially efficient to target site of infection or tumor microenvironment, by selecting an antigen binding domain (ABD) which targets activated immune cells, tumor cells or infected cells. For example, a multifunctional molecule of the invention comprising an anti-PDl binding domain bearing a cytokine having a pro-inflammatory effect will only be activated in the tumoral microenvironment.
If, for any reason, the molecule was to exit the tumoral microenvironment to return to the blood flow or to access healthy tissue, the lack of PD1 positive cells would trigger masking the effect of the cytokine until the recognition of a PD1 positive cell.
Moreover, the greatest advantage of using non-cleavable linkers is their increased plasma stability when compared to cleavable linkers. Despite the limited "bystander" effect, the resistance to cleavage outside of targeted cells may actually increase the specificity of drug release. For example, several in vivo studies and clinical data have shown that non-cleavable linked ADCs outperform their cleavable counterparts (indicated in Combs et al, AAPS J. 2015 Mar;17(2):339-51). It is thus believed that the peptide linkers provided herein will increase therapeutic efficiency of multifunctional molecules.
This solution leads to a multifunctional molecule which gathers the advantages of both cleavable and non-cleavable linkers: great plasma stability, excellent specificity to a specific environment and independence from proteases to uncover the pro-inflammatory cytokine when the multifunctional molecule is onsite.
Another major advantage of the molecule of the invention is its capacity to act via cis-targeting (i.e., on the same cell), and exert a synergistic effect on the targeted cells. Cleavable linkers, by releasing the cytokine in the media, are incapable of engaging an additional target.
The multifunctional molecules of the invention have particularly advantageous and surprising properties: cis-masking of the pro-inflammatory cytokine until the recognition/binding of the multifunctional molecules to the targeted cells (interaction between the antigen binding domain of the multifunctional molecule with a targeted antigen on the targeted cell); cis-demasking of said pro-inflammatory cytokine consecutively to said recognition/binding, without uncontrolled liberation and/or loss of efficiency; the consecutive cis-activation of the target cell with synergy of i) the effect of the antigen binding domain (ABD) and ii) the effect of the pro-inflammatory cytokine, for instance the synergistic activation of a T cell by both i) PD1 (via the antigen binding domain of the multifunctional molecule) and ii) a cytokine (such as IL-2, IL-12, IL-15 or IL-21). The absence of trans-activity is yet another advantage of the invention, as it limits or suppresses the possibility of a binding and activation of cells out of the desired environment, thus limiting the risk of an off-target effect and possible toxicity.
Compared to some molecule of the prior art, the multifunctional molecule of the invention also features a pharmacokinetic profile similar to unmasked multifunctional molecules, hence avoiding any risk of poor distribution or toxicity.
The invention relates to a multifunctional molecule comprising or consisting essentially of: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, said cytokine, variant or fragment being covalently linked to the N-terminal and/or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker covalently linked to the N-terminal and/or C-terminal end of the cytokine; wherein the peptide linker is 5 to 30 amino acids in length and consists of
- at least 20% acidic or amidic amino acids independently selected from the group consisting of E, D and N,
- up to 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and
- at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.
Typically, the peptide linker is a masking peptide linker, particularly that allows the masking and/or demasking of the cytokine.
Optionally, the peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; a sequence having 5 to 30 consecutive amino acids N.
Optionally, said peptide linker comprises, essentially consists of or consists of:
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids selected independently from the group consisting of E, D and N; or - 3, 4, 5, &, , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being E; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being D; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being N.
Optionally, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12),
EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),
DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),
DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),
NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).
Optionally, the peptide linker is covalently linked to the N-terminal end of the cytokine and is connecting the antigen binding domain and the cytokine. Said peptide linker is called the "masking" peptide linker.
Optionally, the multifunctional molecule comprises two peptide linkers, a first peptide linker being as described herein (i.e., a "masking peptide linker") and a second peptide linker, wherein:
- the second peptide linker has an amino acid sequence as defined for the first linker (i.e., a "masking peptide linker"); or
- the second peptide linker is identical to the first peptide linker; or
- the second peptide linker is between 10 and 30 amino acids in length and consists of less than 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
Optionally, the first peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the second peptide linker is linked to the C -terminal end of the cytokine. Alternatively, the second peptide linker is covalently linked to the N- terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the first peptide linker is linked to the C-terminal end of the cytokine.
Preferably, the multifunctional molecule comprises a first peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5) and a second peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5), preferably wherein:
- the N-terminal end of the first peptide linker is covalently linked to the C-terminal end of the antigen binding domain, preferably in C-terminal end of a Fc domain;;
- the C-terminal end of the first peptide linker is covalently linked to the N-terminal end of the cytokine; and
- the N-terminal end of the second peptide linker is covalently linked to the C-terminal end of the cytokine.
Optionally, the multifunctional molecule comprises a single antigen binding domain, optionally a Fc domain, and a single cytokine.
Optionally, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of antigen binding domain and of cytokine; and b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) either
- the first peptide linker and the single cytokine or variant or fragment thereof; said first peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine; or - the first and second peptide linkers and the single cytokine or variant or fragment thereof; wherein the first peptide linker is covalently linked to the N-terminal end of the cytokine and the second peptide linker is linked to the C-terminal end of the cytokine.
Optionally, the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15, IL-16, IL-17A, IL17B, IL-18, IL- 21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine, preferably a cytokine selected from the group consisting of IL-2, IL-12, IL-15, I L18 and IL-21 or a variant thereof.
Preferably, the cytokine is selected from the group consisting of IL-2, I LIO, IL-12, IL-15, IL-18 and IL-21 or a variant thereof.
Optionally, the antigen binding domain binds to a target expressed: on immune cells surface and is preferably selected from the group consisting of PD-1, BCMA/TNFRSF17, BTLA, CD101/IGSF2, CD119, CD137/4-1BB/TNFRSF9, CD150/SLAMF1, CD153, CD226, CD25, CD254, CD26, CD27, CD275/ICOSL, CD39/ENTPD1, CD40L/CD154, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3/CD183, CXCR4, CXCR5, CXCR6, FasL/TNFSF6, GITR/TNFRSF18/CD357, GPR32, TIM3/HAVCR2, ICOS, IL18Rl/CXCRl/CD218a, ITGAE/CD103, LAG3/CD223, TRAILR, OX40L, LY108 /SlamF6, NKG2D, OX40/TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI/TNFRSF13B, TIGIT, CD163, CD206, LTBR/CD70, TNFSF14, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30/TNFRSF8, CD78, TRAF1, TRAF2, TRAF3/CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1/TNFRSF1A/CD120A, TRAP100/MED24, TNFR2/TNFRSF1811/CD120B, CDCR3/TNFRSF6B, TNFRSF12A/FN14/TWEAKR,
BAFFR/TNFRSF13C/CD268, HVEM/TNFRSF14/CD270, RELT/TNFRSF19L, TNFRSF19/TROY, TNFRSF21/DR6, TNFRSF25/DR3/TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD74, CD165, 4-1BBL/CD137L, CD127 and CD160; or
- on tumoral cells surface and is preferably selected from the group consisting of Fibroblast Activation Protein (FAP), the Al domain of Tenascin-C (TNC Al), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA or CEACAM) and the Melanoma- associated Chondroitin Sulphate Proteoglycan (MCSP), Her2/Neu (human epidermal growth factor receptor 2), CD22, EpCAM (CD326), EGFR, PSMA (Prostate Carcinoma), CD30, CD20, CD33, membrane IgE, IgE Receptor (CD23), CD80, CD86, CD2, CA125 (cancer antigen- 125), Carbonic Anhydrase IX, CD70, CD74, CD56, CD40, CD19, c-met/HGFR, DRS, PD-1, PDL1, IGF-1R, VEGF and VEGFR (Solid tumour and eye AMD), VEGF-R2, Prostate stem cell antigen (PSCA), MUC1, CanAg, Mesothelin, P-cadherin, Myostatin (GDF8), Cripto (TDGF1), ACVRL 1/ALK1 (activin a receptor type 1), MUC5AC, CD137, CXCR4, Neuropilin 1, Glypicans, HER3, platelet derived growth factor receptor alpha (PDGFRa), EphA2 (Ephrin type- A receptor 2), nucleolin, CD38, CD138, a4-integrin, C5 complement, C3 complement, MASP-2, C5aR, CR1, C3b, CA19-9, calretinin, epithelial membrane protein (EMA), epithelial tumour antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, melanoma antigen recognized by T lymphocytes (MART-1), myo-DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumour M2-PK), an abnormal Ras protein, an abnormal p53 protein, diganglioside GD2, interleukin 13 receptor a 2 (IL13Ra2), CD133, natural-killer group 2, member D (NKG2D), chondroitin sulphate proteoglycan 4 (CSPG4), CS-I, LI cell adhesion molecule (L1CAM), BCMA (-cell maturation antigen), alpha-fetoprotein (AFP), and CFH.
Preferably, the antigen binding domain binds to an immune checkpoint inhibitor selected from the group consisting of PD-1, TIGIT, TIM3, LAG3, VISTA, HVEM, BTLA and CTLA-4.
In a very particular aspect, the antigen binding domain is a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, preferably comprising or consisting a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 74 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 75; ii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 96 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 97; and iii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 104 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 105.
In a very particular aspect, the antigen binding domain is an anti-human TIGIT antibody or antigenbinding fragment thereof, preferably comprising or consisting of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 129 and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 130.
The invention also relates to an isolated nucleic acid molecule, a group of isolated nucleic acid molecules or a vector encoding the multifunctional molecule as described herein. The invention also relates to a host cell comprising the isolated nucleic acid molecule and/or the group of isolated nucleic acid molecules and/or the vector as described herein. The invention also relates to a pharmaceutical composition comprising a multifunctional molecule as described herein. Said pharmaceutical composition may further comprise an additional therapeutic agent, preferably an anti-cancer agent or anti-infection agent.
The invention also relates to the multifunctional molecule or the pharmaceutical composition as described herein for use as a medicament, preferably in the treatment of a cancer or an infection. It also relates to the use of the multifunctional molecule or the pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of a cancer or an infection. It further relates to a method for treating a cancer or an infection in a subject in need thereof, comprising administering a therapeutically effective amount of the multifunctional molecule or the pharmaceutical composition as described herein to said subject. Said method may further comprise the administration of an additional therapeutic agent, preferably an anti-cancer agent or anti-infection agent.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.
As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2) or subclass. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Unless specifically noted otherwise, the term "antibody" includes intact immunoglobulins and "antibody fragment" or "antigen binding fragment" (such as Fab, Fab', F(ab')2, Fv), single chain (scFv or scFab), CrossMAb, mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to a humanized antibody.
In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CHI" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341- 447 according to the EU index as in Kabat.
An "antibody heavy chain" as used herein, refers to the larger of the two types of polypeptide chains present in antibody conformations. The CDRs of the antibody heavy chain are typically referred to as "HCDR1", "HCDR2" and "HCDR3". The framework regions of the antibody heavy chain are typically referred to as "HFR1", "HFR2", "HFR3" and "HFR4". An antibody heavy chain is typically structured as follows : HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.
An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations; K and X light chains refer to the two major antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as "LCDR1", "LCDR2" and "LCDR3". The framework regions of the antibody light chain are typically referred to as "LFR1", "LFR2", "LFR3" and "LFR4". An antibody light chain is typically structured as follows : LFR1-LCDR1-LFR2-LCDR2- LFR3-LCDR3-LFR4.
By "amino acid change" or "amino acid modification" is meant herein a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitution, insertion and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and/or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acids sequence, generally specified with the one letter codes for the amino acids. The first amino acid in the amino acids sequence (i.e., starting from the N terminus) should be considered as having position 1.
A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and/or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:
Table A - Amino Acid Residue
Table B - Alternative Conservative Amino Acid Residue Substitution Groups
Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues As used herein, the "sequence identity" between two sequences is described by the parameter "sequence identity", "sequence similarity" or "sequence homology". For purposes of the present invention, the "percentage identity" between two sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods in the art, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle" for pairwise sequence alignment of proteins providing by EMBL-EBI and available on: www.ebi.ac. uk/Tools/services/web/toolform.ebi?tool=emboss_needle&context=protein, for example using default settings: (I) Matrix : BLOSUM62, (ii) Gap open : 10, (iii) gap extend : 0.5, (iv) output format : pair, (v) end gap penalty : false, (vi) end gap open : 10, (vii) end gap extend : 0.5.
Alternatively, Sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with the default settings.
The terms "derive from" and "derived from" as used herein refers to a compound having a structure derived from the structure of a parent compound or protein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar properties, activities and utilities as the claimed compounds.
As used herein, the terms "disorder" or "disease" refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease e.g., an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to immune and/or inflammatory diseases that affect animals and/or humans, such as cancer.
The term "agonist" as used herein, refers to a substance that activates the functionality of an activating receptor. Particularly, this term refers to an antibody that binds to a cellular activating receptor as a reference substance, and have at least partially the same effect of the biologically natural ligand (e.g., inducing the activator effect of the receptor).
The term "antagonist" as used herein, refers to a substance that binds to a specific target, typically a receptor or a protein, and inhibits or blocks its normal biological activity. Particularly, this term refers to an antibody that binds to a receptor or protein as a reference substance, and have at least partially the same effect of the biologically natural ligand (e.g., inducing the inhibition of the receptor). Preferably, this term refers to an antibody that binds to a cellular receptor as a reference substance (e.g. biologically natural ligand of the receptor), preventing it from producing all or part of its usual biological effects (e.g., thereby inhibiting the effect of the receptor when it is bound by its biologically natural ligand).
As used herein, "trans-targeting" and "trans-activity" refer to the capacity of a multifunctional molecule to bond to at least two different targets expressed on different cells and/or cell types.
As used herein, "cis-targeting" and "cis-activity" refer to the capacity of a multifunctional molecule to bond to at least two different targets expressed on the same cell.
"Pharmacokinetics" (PK) refers to the movement of drugs through the body, whereas pharmacodynamics (PD) refers to the body's biological response to drugs. PK describes a drug's exposure by characterizing absorption, distribution, bioavailability, metabolism, and excretion as a function of time. PD describes drug response in terms of biochemical or molecular interactions. PK and PD Analyses are used to characterize drug exposure, predict and assess changes in dosage, estimate rate of elimination and rate of absorption, assess relative bioavailability / bioequivalence of a formulation, characterize intra- and inter-subject variability, understand concentration-effect relationships, and establish safety margins and efficacy characteristics. By "improving PK" it is meant that one of the above characteristics is improved, for example, such as an increased half-life of the molecule, in particular a longer serum half-life of the molecule when injected to a subject.
As used herein, the terms "pharmacokinetics" and "PK" are used interchangeably and refer to the fate of compounds, substances or drugs administered to a living organism. Pharmacokinetics particularly comprise the ADME or LADME scheme, which stands for Liberation (i.e., the release of a substance from a composition), Absorption (i.e., the entrance of the substance in blood circulation), Distribution (i.e., dispersion or dissemination of the substance through the body) Metabolism (i.e., transformation or degradation of the substance) and Excretion (i.e., the removal or clearance of the substance from the organism). The two phases of metabolism and excretion can also be grouped together under the title elimination. Different pharmacokinetics parameters can be monitored by the man skilled in the art, such as elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma cleared of the drug per unit time), Cmax (Maximum serum concentration), and Drug exposure (determined by Area under the curve, see Scheff et al, Pharm Res. 2011 May;28(5):1081-9) among others.
As used herein, the term "isolated" indicates that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. The term "and/or" as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.
The term "a" or "an" can refer to one of or a plurality of the elements it modifies (e.g., "a reagent" can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
The term "about" as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to +/- 10% (e.g., +/- 0.5%, +/-1 %, +/-1 .5%, +/- 2%, +/- 2.5%, +/- 3%, +/- 3.5%, +/- 4%, +/- 4.5%, +/- 5%, +/- 5.5%, +/- 6%, +/- 6.5%, +/- 7%, +/- 7.5%, +/- 8%, +/- 8.5%, +/- 9%, +/-9.5%). The use of the term "about" at the beginning of a string of values modifies each of the values (i.e., "about 1, 2 and 3" refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). As used herein, the term "consist essentially of" refers to those elements required for a given embodiment. This term indicates the inclusion of any recited characteristics and permits the optional presence of elements that do not materially affect nor change the characteristics or functions of said embodiment. Preferably, in the context of a multifunctional molecule, it refers to a molecule that comprises the recited elements (e.g., antigen binding domain(s), immunoactive molecule(s) and peptide linker(s)), and optionally includes other elements that do not particular interfere with the structure or function of the molecule, such as peptide spacers.
The term "at least one" means "one or more" or "one or several". For instance, it refers to one, two, three or more.
Multifunctional molecule structure
The multifunctional molecules envisioned herein comprise or consist of one or more antigen binding domain(s), one or more cytokine(s) having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, and one or more peptide linker(s).
As used herein, the terms "multifunctional molecule" or "polyfunctional molecule" are interchangeable and refer to hybrid or conjugated drugs or as chimeric drugs from two or more drugs having different pharmacological activities. In particular, the multifunctional molecule can be a bifunctional molecule, in which a first function is hold by the pro-inflammatory cytokine(s) and a second function is hold by the antigen binding domain(s) derived from an antibody. However, optionally, the multifunctional molecule can present additional functions such as another binding moiety, another immunoactive molecule or any other active function such as a cytotoxic agent, a antiproliferative agent or an pro-inflammatory agent that have additional or different pharmacological activities.
The multifunctional molecule of the invention does not comprise a moiety able to bind the cytokine. Preferably, the multifunctional molecule does not comprise a moiety that presents a binding affinity to the cytokine, such as an antibody-antigen or receptor-ligand interaction. Such moiety can be selected from the group consisting of molecules, peptides, proteins, antibodies, antigen binding domain, receptors and fragment thereof that have a binding affinity for the cytokine. Typically, the multifunctional molecule of the invention does not comprise an antigen binding domain that is able to bind the cytokine comprised in the multifunctional molecule, especially such as a Fab, a scFv or a VHH. For example, when the multifunctional molecule of the invention comprises IL-2, the multifunctional molecule does not comprise an antigen binding domain, such as an scFv that is able to bind/recognize IL-2. Indeed, in the multifunctional molecule of the invention, it is the peptide linker that is responsible for the masking/demasking of the cytokine. Typically, the multifunctional molecule does not comprise an additional masking moiety or domain (such as an antigen binding domain able to bind the cytokine). Preferably, the multifunctional molecule does not comprise a moiety that present a binding affinity equal or lower than IO-6 M, IO-7 M, 10'8 M, 10'9 M or IO 10 M for the cytokine. For example, when the cytokine is IL-2, the multifunctional molecule does not comprise an anti-IL2 antigen binding domain. Alternatively, or additionally, the multifunctional molecule does not comprise a receptor or a fragment thereof that is able to interact with the cytokine. For example, when the cytokine is IL-2, the multifunctional molecule does not comprise IL-2R or a fragment thereof able to bind IL-2.
The multifunctional molecule comprises or consists essentially of: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, said cytokine, variant or fragment being covalently linked to the N-terminal and/or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker (i.e., a "masking" peptide linker) covalently linked to the N- terminal and/or C-terminal end of the cytokine; wherein the peptide linker is 5 to 30 amino acids in length and consists of
- at least 20% acidic or amidic amino acids independently selected from the group consisting of E, D and N,
- up to 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and - at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.
In some aspects, the multifunctional molecule may comprise or essentially consist of: one or more antigen binding domain(s), one or more cytokine(s), variant(s) or fragment(s) thereof having a pro-inflammatory effect covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain(s), and one or more peptide linker(s) covalently linked to the N-terminal end and/or C-terminal end of the cytokine(s). As further details herein, the peptide linkers can be identical or different but at least one peptide linker is a "masking" peptide linker as defined above with the ratio of amino acids (i.e., at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
More particularly, the multifunctional molecule comprises at least one cytokine or variant or fragment thereof having a pro-inflammatory effect covalently linked to at least one antigen binding domain, wherein one or two peptide linker(s) are covalently linked to the cytokine. When two peptide linkers are present, the linkers are linked to the cytokine so as the latter is flanked by a first linker at one end and by a second linker at the other end and at least one of them is a "masking" peptide linker.
The multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain(s), one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain(s) or the Fc domain, and one to eight peptide linker(s) covalently linked to the N-terminal end and/or C-terminal end of the cytokine(s) or variant(s) or fragment(s) thereof, wherein each cytokine is linked to at least one peptide linker as defined above as a "masking" peptide linker (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
Preferably, the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain(s), one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain(s) or the Fc domain, and two to eight peptide I inker(s), wherein a first linker is covalently linked to the N-terminal end of the cytokine(s), variant(s) or fragment(s) thereof and a second linker is linked to the C-terminal end of the cytokine(s), variant(s) or fragment(s) thereof (i.e., two peptide linkers per cytokine) but with at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
In particular, the multifunctional molecule may comprise an IgG antibody linked to one, two, three or four cytokine(s), variant(s) or fragment(s) thereof. In a multifunctional molecule, the cytokine(s) can be the same or different. Preferably, the C-terminal end of the IgG antibody chain are covalently linked to the cytokine, preferably to the N-terminal end of the cytokine, preferably through a peptide linker. Such multifunctional molecule particularly comprises:
- one "masking" peptide linker as defined herein covalently linked to N-terminus or C-terminus of each of the cytokines; or
- a first peptide linker covalently linked to N-terminus of each of the cytokines and a second peptide linker linked to the C-terminus of each of the cytokines, said peptide linkers being the same or different and with at least one peptide linker being a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
In a particular aspect, when the multifunctional molecule comprises an antigen binding domain which is an antibody, in particular an IgG antibody, said antibody is covalently linked to a cytokine or variant or fragment thereof at the C-terminal end of one light chain, at the C-terminal end of both light chains, at the C-terminal end of one heavy chain, at the C-terminal end of both heavy chains, at the C-terminal end of one light chain and at the C-terminal end of one heavy chain, at the C-terminal end of one light chain and at the C-terminal end of both heavy chains, at the C-terminal end of both light chains and at the C-terminal end of one heavy chain, or at the C-terminal end of both light chains and at the C- terminal end of both heavy chains of said antibody.
Preferably, the cytokine is covalently linked to the antibody at the C-terminal end of one or both heavy chains. Such antibody may comprise heterodimeric Fc domains which promote heterodimeric formation and/or facilitate purification of heterodimers over the homodimers.
Alternatively, the multifunctional molecule may comprise a diabody (comprising two chains including each a VH domain and a VL domain of an antibody) linked to one or two cytokine(s), variants or fragment(s) thereof. For instance, the multifunctional molecule may comprise a diabody in which each of the two chains are linked to a cytokine or a variant or fragment thereof (i.e., the multifunctional molecule comprises two cytokines, said cytokines being the same or different). Preferably, the C- terminal end of the diabody's chain is covalently linked to the N-terminal end of a cytokine, preferably by a peptide spacer or a peptide linker of the invention. Such multifunctional molecule may comprise:
- one peptide linker covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end; or
- one peptide linker covalently linked to the C-terminus of the cytokine; or
- one peptide linker covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end, and another peptide linker linked to the C-terminus of the cytokine, said peptide linkers being the same or different; or
- one peptide spacer covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end, and one peptide linker linked to the C-terminus of the cytokine; wherein at least one peptide linker is a "masking peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
Alternatively, the multifunctional molecule may comprise a diabody in which only one of the two chains is linked to the cytokine, preferably by a peptide linker or spacer. In another alternative, the cytokine can be linked to two diabodies, said diabodies can be the same or different and being placed either on each side of the cytokine or both at one side.
In another aspect, the multifunctional molecule comprises only one antigen binding domain, such as a Fab, a scFv or a VHH. In a particular aspect, the multifunctional molecule comprises only one cytokine or variant or fragment thereof. Optionally, the multifunctional molecule comprises only one antigen binding domain and only one cytokine or variant or fragment thereof.
The multifunctional molecule may particularly comprise, essentially consist or consist of: a single antigen binding domain, optionally a Fc domain linked to the C terminal end of the antigen binding domain, a single cytokine or variant or fragment thereof covalently linked to N-terminus and/or C- terminus to the antigen binding domain or Fc domain, and i) one peptide linker covalently linked to the N-terminal end or C-terminal end of the cytokine; or ii) two peptide linkers; wherein a first linker is covalently linked to the N-terminal end of the cytokine and a second linker is linked to the C-terminal end of the cytokine, wherein at least one peptide linker is a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).
In a particular aspect, the multifunctional molecule is a polypeptide or protein produced as a recombinant protein. Accordingly, the multifunctional molecule is a fusion protein.
In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a "masking" peptide linker as defined herein and a cytokine or a variant or fragment thereof.
In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a cytokine or a variant or fragment thereof and a "masking" peptide linker as defined herein .
In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a first peptide linker, a cytokine or a variant or fragment thereof and a second peptide linker, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein.
In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a peptide spacer, a cytokine or a variant or fragment thereof and a "masking" peptide linker as defined herein.
In a particular aspect, when the multifunctional molecule comprises a single antigen binding domain, the multifunctional molecule comprises an antigen binding domain covalently linked, optionally through a peptide spacer, by its C-terminal end to N-terminal end of a first Fc chain, a complementary second Fc chain forming with the first Fc chain a Fc domain, said first Fc chain being covalently linked by its C-terminal end to the cytokine or variant or fragment thereof and a "masking" peptide linker as defined herein being either between the first Fc chain and the cytokine or variant or fragment thereof or at the C-terminal end of the cytokine or variant or fragment thereof. Optionally, the complementary second Fc chain is devoid of binding domain, devoid of cytokine or devoid of a binding domain and of a cytokine. Preferably, the antigen binding domain is a Fab domain, a Fab', a single-chain variable fragment (scFV) or a single domain antibody (sdAb).
Such multifunctional molecule comprises:
- one "masking" peptide linker as defined herein covalently linked to N-terminus or C-terminus of the cytokine; or - one peptide linker covalently linked to N-terminus of the cytokine and another peptide linker linked to the C-terminus of the cytokine, said peptide linkers being the same or different and at least one of them being a "masking" peptide linker as defined herein; or
- one peptide spacer covalently linked to N-terminus of the cytokine and a "masking" peptide linker as defined herein linked to the C-terminus of the cytokine.
Preferably, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv)
- one "masking" peptide linker as defined herein and a single cytokine or variant or fragment thereof; said peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine; or
- two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein, optionally both; or
- one "masking" peptide linker as defined herein, one peptide spacer and a single cytokine or variant or fragment thereof; wherein a peptide spacer is covalently linked to the N-terminus of the cytokine and a peptide linker is linked to the C-terminus of the cytokine.
Preferably, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first linker is covalently linked to the C-terminus of the second Fc chain and to the N-terminus of the cytokine and a second linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein, optionally both.
The term "entity" envisioned herein refers to part or moiety of the multifunctional molecule.
In a particular aspect, when the multifunctional molecule comprises a single antigen binding domain, the multifunctional molecule comprises an antigen binding domain which is a single-chain variable fragment (scFV), covalently linked, optionally through a peptide spacer, by its C-terminal end to N- terminal end of a first Fc chain, a complementary second Fc chain forming with the first Fc chain a Fc domain, said first Fc chain being covalently linked by its C-terminal end to the cytokine or variant or fragment thereof and a "masking" peptide linker as defined herein being either between the first Fc chain and the cytokine or variant or fragment thereof or at the C-terminal end of the cytokine or variant or fragment thereof. Optionally, the complementary second Fc chain is i) devoid of binding domain, ii) devoid of cytokine or iii) devoid of a binding domain and of a cytokine.
Preferably, the multifunctional molecule comprises or consists of: a) an antigen binding domain which is a single-chain variable fragment (scFV), b) optionally a Fc domain; and c) two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first linker is covalently linked to the C-terminus of the single-chain variable fragment and to the N- terminus of the cytokine and a second linker is linked to the C-terminus of the cytokine, and wherein at least one of the two peptide linkers is a "masking" peptide linker as defined herein, optionally both.
In a specific aspect of the invention, the multifunctional molecules envisioned herein comprise or consist of one or more antigen binding domain(s), one or more cytokine(s), one or more "masking" peptide linker(s) as defined herein and one or more additional peptide linker(s).
In some aspects, the multifunctional molecule may comprise or consist of: one or more antigen binding domain(s), one or more cytokine(s) or variants or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain, optionally through a peptide spacer, one or more "masking" peptide linker(s) as defined herein covalently linked to the N-terminal end and/or C-terminal end of the cytokine, and one or more additional peptide linker(s) covalently linked to the N-terminal end and/or C- terminal end of the cytokine.
Particularly, the multifunctional molecule comprises at least one cytokine or variant or fragment thereof covalently linked to at least one antigen binding domain, wherein one peptide linker is covalently linked to the cytokine and one additional peptide linker is covalently linked to the cytokine, wherein at least one of the peptide linkers is a "masking" peptide linker as defined herein, optionally both. When two peptide linkers are present, the linkers are linked to the cytokine so as the latter is flanked by a first linker (the peptide linker or the additional peptide linker) at one end and by a second linker (the additional peptide linker or the peptide linker, respectively) at the other end.
Preferably, the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain, one to four cytokine(s) or variant(s) or fragment(s) thereof covalently linked to the N-terminal end and/or C-terminal end of the antigen binding domain or the Fc domain, and one to four peptide I inker(s), covalently linked to the N-terminal end and/or C-terminal end of the cytokine, and one to four additional peptide linker(s), covalently linked to the N-terminal end and/or C- terminal end of the cytokine, wherein a first linker (peptide linker or additional peptide linker) is covalently linked to the N-terminal end of the cytokine and a second linker (additional peptide linker or peptide linker, respectively) is linked to the C-terminal end of the cytokine (i.e., one peptide linker and one additional peptide linker per cytokine) and wherein at least one peptide linker is a "masking" peptide linker as defined herein.
Examples of the multifunctional molecules are particularly provided herein below under the section "Examples of multifunctional molecules".
Examples of each element (i.e., antigen binding domain, Fc domain, cytokine and peptide linker, especially "masking" peptide linker as defined herein) comprised in the multifunctional molecules according to the invention are described herein below in their respective sections and apply to any of the multifunctional molecules disclosed herein. Some general illustrative and non-limiting structures are disclosed in Figure 1C.
In some aspects, the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures A, B, Cl, C2, C3, C4, C5, C6, C7, C8 and of Figure 1C.
Peptide Linkers
The multifunctional molecule envisioned herein comprises one or more peptide linker(s) and at least one "masking" peptide linker as defined herein. The peptide linker is linked to N-terminus and/or C- terminus of the cytokine.
By "masking peptide linker" it is meant refers to a short peptide sequence that is strategically designed and incorporated into a larger protein molecule such as an antibody or multifunctional molecule disclosed herein to hide or mask specific functional proteins or epitopes such as a cytokine. Masking peptide linker generally allow avoiding immunogenicity, controlling activation and/or stabilization of the masked proteins or epitopes. In the context of the invention, the masking peptide linker allows the masking or inactivation of the cytokine until the antigen binding domain binds to its target.
In a multifunctional molecule of the invention, the "masking peptide linker" is typically both able to mask the cytokine or the cytokine activity and/or is able to unmask or demask the cytokine or cytokine activity, especially when the antigen biding domain of the multifunctional molecule binds to its target. Typically, the peptide linker of the invention allows to mask and/or unmask or demask the cytokine, preferably the cytokine activity, even more preferably the binding of the cytokine to its receptor.
By "covalently linked" or "covalently bound", it is meant a chemical bond that involves the sharing of electrons to form electron pairs between atoms between two moieties, e.g., a cytokine and a peptide linker. In the context of the invention, covalent links are not reversible. Preferably, the covalent links are amide bond.
The "masking" peptide linker is not cleavable or does not contain any protease cleavage site. As used herein, the term "non-cleavable peptide linker" or "uncleavable linker" refers to an amino acid sequence that is designed to remain intact without being cleaved or broken down under certain conditions, such as physiological conditions (e.g. pH), enzymatic or chemical cleavage. Such peptide linkers typically ensure that the linked molecules (e.g., an antigen binding domain and a cytokine such as disclosed herein) remain connected throughout the intended application, whether it is in vivo or in vitro. Preferably, non-cleavable peptide linkers do not comprise a metalloprotease cleavage site.
The masking peptide linker particularly does not specifically bind to or recognize the cytokine to which it is covalently linked.
In the multifunctional molecule of the invention, the number of peptide linker(s) may vary depending on the number of cytokine(s). The molecule comprises one or two peptide linkers per cytokine, but at least one "masking" peptide linker as defined herein. Optionally, the molecule comprises two "masking" peptide linkers as defined herein per cytokine.
Preferably, the multifunctional molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 peptide linker(s). In particular, the multifunctional molecule may comprise one or two peptide linker(s) if the multifunctional molecule comprises one cytokine, and at least one "masking" peptide linker as defined herein.
The multifunctional molecule may comprise two to four peptide linkers if the multifunctional molecule comprises two cytokines, and at least two "masking" peptide linkers as defined herein.
The multifunctional molecule may comprise three to six peptide linkers if the multifunctional molecule comprises three cytokines, and at least three "masking" peptide linkers as defined herein.
The multifunctional molecule may comprise four to eight peptide linkers if the multifunctional molecule comprises four cytokines, and at least four "masking" peptide linkers as defined herein.
The multifunctional molecule comprises at least one "masking" peptide linker by cytokine, said "masking" peptide linker being 5 to 30 amino acids in length and consists of
- at least 20, 30, 40, 50, 60, 70, 80 or 90% acidic or amidic amino acids independently selected from the group consisting of E, D and N, - up to 5, 10 or 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and
- at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.
When the peptide linker comprises a ratio or percentage of the amino acids, it is meant that the number of occurrences of this particular amino acid or these particular amino acids to the total number of amino acids in the peptide linker. For instance, in a peptide linker EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), the percentage of acidic or amidic amino acids is 100% (4 D + 16 E /20 aa), the percentage of basic amino acids is 0% and there is no remaining amino acids. In an additional example, in a peptide linker EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), the percentage of acidic or amidic amino acids is 80% (16 E /20 aa), the percentage of basic amino acids is 0% and 100% of the remaining amino acids are A. In a last example, in a peptide linker EEEEKEEEEKEEEEK (SEQ ID NO: 46), the percentage of acidic or amidic amino acids is 80% (12 E /15 aa), the percentage of basic amino acids is 20% (3 K /15 aa), and there is no remaining amino acids.
More particularly, when the linker peptide comprises 5, 10 or 20% basic amino acids selected from the group consisting of H, K and R, it has to comprise at least 15, 30 or 60% of acidic or amidic amino acids; respectively.
The peptide linker comprises at least 5, 6, 7, 8, 9 or 10 amino acids. Preferably, the peptide linker is from 5 to 30 amino acids in length or 10 to 25 amino acids in length, preferably from 10 to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids in length. Even more preferably, the peptide linker is from 10 to 20 amino acids in length.
Optionally, the peptide linker is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In a particular aspect, the peptide linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In a very particular aspect, the peptide linker is 15 amino acids in length.
In a preferred aspect, the "masking" peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50, 60, 70, 80 or 90% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; and a sequence having 5 to 30 consecutive amino acids N.
For instance, in a peptide linker EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), the percentage of polar, amidic and acidic amino acids is 100% (4 D + 16 E /20 aa) and 0% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T. In an additional example, in a peptide linker EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), the percentage of polar, amidic and acidic amino acids is 80% (16 E /20 aa) and 20% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T (4 A /20 aa). In a last example, in a peptide linker EEEETEEEETEEEETEEEET (SEQ ID NO: 21), the percentage of polar, amidic and acidic amino acids is 100% (16 E + 4 T /20 aa) and 20% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T (4 T /20 aa). In a particular aspect, a sequence has at least 50, 60, 70, 80 or 90% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and 10, 20, 30, 40 or 50% of remaining amino acids are independently selected from the group consisting of G, P, A, V, S, and T.
In a particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of:
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids selected independently from the group consisting of E, D and N; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being E; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being D; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being N.
The "masking" peptide linker(s) envisioned herein essentially consist(s) of or consist(s) of the same amino acid residue selected in the group consisting of Asparagine (N), Glutamate (E) and Aspartic acid (D).
Preferably, the same amino acid is selected from the group consisting of the group consisting of N, E and D. More preferably, the same amino acid is selected from the group consisting of E, D and N. Even more preferably, the same amino acid is E.
In the "masking" peptide linker, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% or 100% of the amino acids of the linker can be the same amino acid. In a particular aspect, the multifunctional molecule comprises at least one "masking" peptide linker having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% of the amino acid E, D and N, preferably E.
In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ. ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46).
In a preferred aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).
In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), 1
EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).
In a even more particular aspect, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
In a particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5) and EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), optionally with 1, 2 or 3 amino acid modifications, in particular an amino acid substitution with an amino acid selected from the group consisting of D, N, Q, T, S, G, P, A, V, K, H and R, preferably D, N, Q, T, A and K, even more preferably A, T, D and N.
Even more preferably, the multifunctional molecule comprises 1, 2, 3, 4, 5, 6, 7 or 8 "masking" peptide linker(s) as defined above.
In some aspects, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46).
In a preferred aspect, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).
In a very particular aspect, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).
In an even more particular aspect, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
In a preferred aspect, a "masking" peptide linker as defined herein is linked at the N-terminal end of the cytokine. Preferably, the peptide linker is covalently linked to the N-terminal end of the cytokine and is covalently linking or connecting the antigen binding domain and the cytokine. Optionally, the multifunctional molecule comprises only one "masking" peptide linker per cytokine.
Optionally, the multifunctional molecule comprises two peptide linkers per cytokine, a first peptide linker being a "masking" peptide linker as described herein and a second peptide linker, wherein:
- the second peptide linker has an amino acid sequence as defined herein for a "masking" linker; or
- the second peptide linker is identical to the first peptide linker; or
- the second peptide linker is between 10 and 30 amino acids in length and consists of less than 10, 20, 30, 40 or 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
For instance, in a peptide linker AAAAHAAAAAAAAAA (SEQ ID NO: 3), the percentage of basic amino acids is 7% (1 H /15 aa), and the percentage of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T is 93% (14 A/15 aa). In an additional example, in a peptide linker PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), the percentage of basic amino acids is 0%, and the percentage of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T is 100% (16 P + 4 S /15 aa).
In a particular aspect, the second or additional peptide linker consists of less than 10, 20, 30, 40 or 50% of basic amino acids and the remaining amino acids are independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
Preferably, the second or additional peptide linker(s) is/are not cleavable or does/do not contain any protease cleavage site.
In some aspect, the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I I I I H (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I I TH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).
When the multifunctional molecule comprises more than one peptide linker, the peptide linkers may have the same length or they can have a length slightly different, for instance having a difference in length of at most 10 to 20%, preferably 10% to 15%, in number of amino acids.
In a preferred aspect, the multifunctional molecule comprises two peptide linkers per cytokine and a first peptide linker being a "masking" peptide linker as defined herein is linked at the N-terminal end of the cytokine and a second peptide linker is linked at the C-terminal end of the cytokine, said second linker being as defined above. In a more preferred, the multifunctional molecule comprises two peptide linkers per cytokine and a first peptide linker is linked at the N-terminal end of the cytokine and a second peptide linker is linked at the C-terminal end of the cytokine, the first and second peptide linkers are being a "masking" peptide linker as defined herein.
Optionally, the multifunctional molecule comprises linker(s) and a cytokine with one of the following arrangements:
"masking" linker - cytokine - "masking" linker,
"masking" linker - cytokine, cytokine - "masking" linker, masking" linker - cytokine - linker, and linker - cytokine - "masking" linker. Preferably, the multifunctional molecule comprises linker(s) and a cytokine with one of the following arrangements:
"masking" linker - cytokine - "masking" linker,
"masking" linker - cytokine, and masking" linker - cytokine - linker.
When the cytokine is a dimer, either a homodimer or a heterodimer cytokine, the two monomers of the cytokine are covalently linked by a peptide linker or spacer, optionally a "masking" peptide linker as defined herein. Accordingly, the multifunctional molecule comprises linker(s) and a dimeric cytokine with one of the following arrangements:
"masking" linker - 1st monomer of the cytokine - peptide linker / spacer - 2nd monomer of the cytokine - "masking" linker,
"masking" linker - 1st monomer of the cytokine - peptide linker / spacer - 2nd monomer of the cytokine - linker,
"masking" linker - 1st monomer of the cytokine - peptide linker / spacer - 2nd monomer of the cytokine,
"masking" linker - 1st monomer of the cytokine - "masking" linker - 2nd monomer of the cytokine - "masking" linker,
"masking" linker - 1st monomer of the cytokine - "masking" linker - 2nd monomer of the cytokine - linker, and
"masking" linker - 1st monomer of the cytokine - "masking" linker - 2nd monomer of the cytokine.
In some aspects, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, peptide linker(s) and at least one or two peptide linkers are "masking" peptide linkers selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),
DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),
NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43), and more preferably EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44), and optionally an additional linker selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), TT I I I I I I I I I I TTH (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), TT I I I I I I I I I I I I I I I I TT (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I I TH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).
In a preferred aspect, the multifunctional molecule comprises at least one or two peptide linkers per cytokine selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12),
EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),
DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),
DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),
NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43), and more preferably EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).
In the present invention, the multifunctional molecule comprises one or more cytokine(s) or variants()s or fragment(s) thereof having a pro-inflammatory effect. In addition, the cytokine(s) has or have a narrow therapeutic index or a systemic toxicity, especially an off-target systemic toxicity. Examples of such cytokines are provided here below.
As described above, in the multifunctional molecule described herein, the peptide linkers of the invention enable to mask and/or demask the cytokine. Preferably, when the cytokine is masked, the cytokine is not able to bind to its cognate receptor and/or does not exhibit its biological activity. Preferably, when the cytokine is unmasked, the cytokine is able to bind to its cognate receptor and/or is able to exhibit its biological activity. The receptors and biological activity of a particular cytokine have been described in the art and are well known to the man skilled in the art. The biological activity of a cytokine typically refers to the effects a cytokine exerts on target cells, tissues, or organs, especially by binding to its receptor and/or triggering intracellular signalling pathways. These activities can include regulating immune responses, inflammation, cell growth, differentiation, survival, or death.
Particularly preferred cytokines for the masking technology are those that have a high systemic toxicity known such as illustrative IL2 or IL15 cytokines. As an illustration, W02024/068705 describes the toxicity of IL2 associated to Vascular Leak Syndrom, and the insufficient results obtained even with fusion targeted molecules comprising an anti-PDl antibody and IL2 variants of the prior art and the critical need to develop new molecules with wider therapeutic index. Similarly, toxicity of IL-15 is illustrated for instance in Guo et al (J Immunol, 2015, 195(5):2353-64. doi:
10.4049/jimmunol.1500300). Similarly, WO2024102693 notes that high dose treatment with IL-18 could results in systemic toxicities.
The present invention is suitable to overcome this problem because the use of "masking linker" improves the therapeutic index of the cytokines. Indeed, the cytokine has its effect only or preferably when the ABD is bond to its target, leading to the specific activity of the cytokine. Thereby and non- exhaustively, the monofunctional molecule of the invention avoids interactions of the cytokine with its receptor in absence of the ABD target and reduces unintended cytokine pathway activations.
Accordingly, in a particular aspect, the cytokine has a narrow therapeutic index. Therapeutic index (Tl) is the range of doses at which a medication is effective without unacceptable adverse events. Narrow therapeutic index (NTI) cytokines are defined as those cytokines where small differences in dose or blood concentration may lead to dose and blood concentration dependent, serious therapeutic failures or adverse drug reactions. Serious events are those which are persistent, irreversible, slowly reversible, or life-threatening, possibly resulting in hospitalization, disability, or even death. Several terms such as narrow therapeutic index, narrow therapeutic window, narrow therapeutic range and narrow therapeutic ratio can be used interchangeably. Typically, a "narrow therapeutic index" refers to a range between a drug's therapeutic dose (e.g., producing a clinical/therapeutic effect) and its toxic dose (e.g., causing harmful side effects) that is limited (narrow).
A drug product such as a cytokine typically has a narrow therapeutic index when:
(a) there is less than a 5-fold, 4-fold, 3-fold or 2-fold difference in median lethal dose (LD50) and median effective dose values (ED50) and/or
(b) there is less than a 5-fold, 4-fold, 3-fold or 2-fold difference in the minimum toxic concentrations (MTC) and minimum effective concentrations (MEC) in the blood and/or
(c) safe and effective use of the drug requires careful titration and patient monitoring. Preferably, there is (a) less than a 2-fold difference in median lethal dose (LD50) and median effective dose values (ED50) and/or (b) less than a 2-fold difference in the minimum toxic concentrations (MTC) and minimum effective concentrations (MEC) in the blood.
Particularly preferred cytokines beneficiating from the masking effect described herein are those having a narrow therapeutic index, in particular a therapeutic index which is the same or narrower that the therapeutic index of a cytokine selected from the group consisting of IL-2, IL-12 or IL-15.
In a very specific and preferred aspect, the cytokine has a narrow therapeutic index as defined among the possible definition set by the FDA, in particular when there is less than a two-fold difference in median lethal dose (LD50) and median effective dose values (ED50), or there is less than a two-fold difference in the minimum toxic concentrations (MTC) and minimum effective concentrations (MEC) in the blood. In addition, and safe and effective use of the cytokine requires careful titration and patient monitoring. Preferably, the cytokine having a narrow therapeutic index is a cytokine with two fold or at least two-fold difference in median lethal dose (LD50) and median effective dose (ED50) values.
In an alternative or addition aspect, the cytokine has a systemic toxicity, especially off-target systemic toxicity.
In view of the growing knowledge of cytokines, with newly discovered cytokines which are not or not yet well described in the prior art, the invention also applies to such cytokines when are pro- inflammatory and exhibit systemic toxicity.
On the opposite, other cytokines which are not in the scope of the present application are those for which the therapeutic index is much less critical and raises no or only low or very low problem of toxicity at usual clinical doses.
Cytokines are small protein messengers secreted primarily by immune cells that transmit signals between cells to regulate and orchestrate various immune and inflammatory processes within the body. As used herein, the terms "cytokine having a pro-inflammatory effect", "pro-inflammatory cytokine" or "inflammatory cytokine" are used interchangeably and refer to a cytokine that is secreted by immune cells to initiate, promote or amplify inflammation. Detecting inflammation can be assessed by a variety of techniques known to the man skilled in the art, such as clinical assessment, biopsy, laboratory tests (e.g., CRP, ESR, WBC), imaging studies (CT or MRI). Some specific cytokines, such as interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-a), may be measured to assess inflammation in certain conditions.
In a specific aspect, the cytokine has a stimulatory effect. More specifically, the cytokine has a stimulatory effect on cells activating the immune response. Alternatively, the cytokine can have an inhibitory effect on cells inhibiting the immune response, such as T reg. In a particular aspect, the cytokine having a pro-inflammatory is an immuno-stimulating cytokine. The immuno-stimulating cytokine is capable of stimulating or activating an immune cell. The immune cell can be selected in the non-exhaustive list comprising B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes. In a preferred aspect, the immune cells are T cells, more specifically CD8+ T cells, effector T cells or exhausted T cells. In a particular preferred aspect, the immune cells are effector memory stem like T cells.
For instance, the immuno-stimulating cytokine can be selected from: T-cell growth factors, in particular growth factors to increase number and repertoire of naive T cells, growth factors to increase the number of dendritic cells (DCs), agonists to activate DCs and other antigen-presenting cells (APCs), adjuvants to allow and augment cancer vaccines, agonists to activate and stimulate T cells, inhibitors of T-cell checkpoint blockade, T-cell growth factors to increase the growth and survival of immune T cells, agents to inhibit, block, or neutralize cancer cell and immune cell-derived immunosuppressive cytokine. In a particular aspect, the cytokine is able to activate and stimulate effector memory stem like T cell.
Optionally, the cytokine may have a dual effect. By dual effect, it is intended to refer to a cytokine which can have both a pro-inflammatory effect and an anti-inflammatory effect, depending on the biological context.
Indeed, some cytokines may exhibit either a pro-inflammatory effect or an anti-inflammatory effect depending on several factors. For example, IL-2 may have a pro-inflammatory effect when directed to Th through signal amplification. On the other hand, when addressed to Treg, it will activate those cells and switch them toward an anti-inflammatory pathway, thus triggering the resolution of the inflammation or the resolution of the auto-immune condition. Other examples exist: IL-10, which may activate B cells and T DC8+ cells which exert a pro-inflammatory effect, or activate myeloid cells and triggering an anti-inflammatory response. It is well known for the person skilled in the art, based on the literature, which combination of cytokine and antigen binding domain may be used to trigger one or the other pathway.
Optionally, the cytokine may be mutated or altered so that the biological activity of the cytokine is altered, e.g., the biological activity is increased or decreased. In a particular aspect, the cytokine is modified to decrease its biological activity, e.g., its affinity for its receptor.
Optionally, the cytokine consists in a fragment thereof retaining the biological activity of the cytokine. In one aspect, the cytokine or variant or fragment thereof has a size of at least 10 kDa, at least, 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa or at least 50kDa. Preferably, the cytokine or variant or fragment thereof has a size comprised between 10 kDa and 50 kDa, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 kDa and 20 kDa, between 20 kDa and 50 kDa, between 20 kDa and 40 kDa or between 20 kDa and 30 kDa. Preferably, the cytokine is from human or derived from human.
In some aspects, the cytokine is an interleukin.
In one aspect, the cytokine is a monomer. In an alternative aspect, the cytokine comprises a plurality of subunits. Preferably, the cytokine is a dimer, either a homodimer and a heterodimer, and comprises two subunits. Preferably, if the cytokine has a plurality of subunits, said subunits are linked together by a peptide linker or spacer as described herein. In a preferred aspect, the peptide linker or spacer is linked to the N-terminal end of a first subunit of the cytokine, and to the C-terminal end of a second subunit of the cytokine. In an alternative aspect, the peptide linker or spacer is linked to the C-terminal end of a first subunit of the cytokine, and to the N-terminal end of a second subunit of the cytokine.
In a particular aspect, the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15, IL-17A, IL17B, IL-18, IL-21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
Optionally, the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-6, IL-15, IL-17A, IL17B, IL-18, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
Optionally, the pro-inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of IL-2, IL-10, IL-12, IL-21, IL-22 and IL-23, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In a very particular aspect, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, I L18 and IL-21 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In particular, the cytokine can be selected in the list of Table D below, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
Table D: List of immunostimulating or pro-inflammatory cytokines
Optionally, the cytokine is a monomeric cytokine and is selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-15, IL-17A, IL17B, IL-18, IL-21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof. In a particular aspect, the cytokine is a monomeric cytokine and is selected from the group consisting of IL-2, IL-15, IL-18 and IL-21 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
Optionally, the cytokine is selected from the group consisting of IL-2, IL-12 and IL15 or a variant or fragment thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In a very specific aspect, the immuno-stimulating cytokine is IL-2 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In another very specific aspect, the immuno-stimulating cytokine is IL-12 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In another very specific aspect, the immuno-stimulating cytokine is IL-15 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In another very specific aspect, the immuno-stimulating cytokine is IL-18 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In another very specific aspect, the immuno-stimulating cytokine is IL-21 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
In particular, the cytokines with a NTI could be selected in the non-exhaustive list comprising IL-2, IL- 12, IL-15, IL-18 and IL-21. In some aspects, the multifunctional molecule of the invention comprises a cytokines or variant or fragment thereof selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21, preferably from the group consisting of IL-2, IL-12 and IL-15. Preferably, the cytokine variant is a function conservative variant. Preferably, the cytokine variant or fragment described herein retains substantially equivalent biological property and/or function in comparison to a wild-type cytokine For instance, it retains comparable biological property/function as the full-length or wild-type protein, respectively. Preferably, the cytokine variant or fragment maintains biological activity of at least 10 %, 20%, 30%, 40%, 50%, 60% in comparison with the wild type cytokine. Preferably, the biological activity is the bind of the cytokine to its receptor and/or inhibition of said receptor.
IL-2 and IL-2 variants
In a very specific aspect, the immuno-stimulating cytokine is lnterleukin-2 (IL-2), preferably a human IL-2, for example as disclosed under the UniProt accession number P60568 or a mutant or variant thereof.
The IL-2 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 50 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 50.
IL-2 can be mutated in various ways to reduce its toxicity and/or increase its efficacy. Hu et al. (Blood 101, 4853-4861 (2003), US Pat. Publ. No. 2003/0124678) have substituted the arginine residue in position 38 of IL-2 by tryptophan to eliminate IL-2's vasopermeability activity. Shanafelt et al. (Nature Biotechnol 18, 1 197-1202 (2000)) have mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); US Pat. No. 5,229, 109) have introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of proinflammatory cytokines from NK cells. Gillies et al. (US Pat. Publ. No. 2007/0036752) have substituted three residues of IL-2 (Asp20Thr, Asn88Arg, and Glnl26Asp) that contribute to affinity for the intermediate-affinity IL- 2 receptor to reduce VLS. Gillies et al. (WO 2008/0034473) have also mutated the interface of IL-2 with CD25 by amino acid substitution Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells for enhancing efficacy. In one aspect, the cytokine is an IL-2 mutant for example as described in WO 2012/107417 or WO 2018/184964.
Optionally, the IL-2 variant may comprise one or several substitutions at positions of human IL-2 (without the signal peptide; SEQ ID NO: 50) selected from the group consisting of Qll, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, SI 27 , 1129, and S130, the numbering being preferably the one of SEQ ID NO: 50. Optionally, the IL-2 variant may comprise the substitution relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) F42A or F42K. Optionally, the IL-2 variant may further comprise one or several substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) selected from the group consisting of:
- R38A, R38D, R38E, E62Q, E68A, E68Q, E68K and E68R, and/or - H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, SI 27 A, S127E, S127K, and S127Q, and/or
- C125A the numbering being preferably the one of SEQ ID NO: 50.
Optionally, the IL-2 variant may comprise one of the following substitutions combination relative to human IL-2 (without the signal peptide; SEQ ID NO: 50): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and V91E; R38E, F42A, and D84H; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and V91E; R38D, F42A, and D84H; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and V91E; R38A, F42K, and D84H; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and V91E; F42A, E62Q, and D84H; H16D, F42A, and E62Q; H16E, F42A, and E62Q; F42A, E62Q, and Q126S; R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; F42A, C125A and Q126S; F42A, Y45A and L72G; and T3A, F42A, Y45A, L72G and C125A.
Optionally, the IL-2 variant may comprise one of the following substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50), in particular at least one of the substitutions selected in the group comprising K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K; or a combination thereof, preferably the three substitutions F42A, Y45A and L72G, the numbering being preferably the one of SEQ ID NO: 50.
Mutants of human IL-2 (hlL-2) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 3, 35, 38, 42, 43, 45 or 72 or combinations thereof, corresponding to residues position of human IL-2 (without the signal peptide; SEQ ID NO: 50). Preferably, the mutant IL-2 is a human IL-2 molecule comprising the amino acid substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) T3A, F42A, Y45A, L72G and/or C125A, preferably F42A, Y45A and L72G, more preferably T3A, F42A, Y45A, L72G and C125A, for example as disclosed in WO 2018/184964. Even more preferably, the immuno-stimulating cytokine is an IL-2 mutant having the substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) F42A, Y45A and L72G, preferably T3A, F42A, Y45A, L72G and C125A, the numbering being preferably the one of SEQ ID NO: 50.
In some embodiments, the bifunctional molecule of the invention comprises an IL2 variant comprising or consisting of a sequence as set forth in SEQ ID NO:110.
Preferably, the IL-2 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-2. Additionally or alternatively, the IL-2 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-2R, in comparison to a wild-type IL-2.
IL-12 and IL-12 variants
In a very particular aspect, the immuno-stimulating cytokine is Interleukin-12 (IL-12), preferably a human IL-12, or a mutant or variant thereof. IL-12 is a heterodimeric cytokine including two subunits, IL-12A and IL-12B as disclosed in Uniprot reference P29459 and P29460, respectively. IL-12 may comprise IL-12A or IL-12B of SEQ ID NOs: 51 and 52 (sequences without signal peptide), respectively, or a variant thereof having a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity therewith or a sequence having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NOs: 51 and 52, respectively.
For instance, IL-12 may include a variant of IL-12A having one or several substitutions with respect to the wildtype IL-12A selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q, the amino acid numbering being preferably the one of SEQ ID NO: 51. Optionally, the variant of IL-12A has one of the following substitutions combinations: N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D, the amino acid numbering being preferably the one of SEQ ID NO: 51..
For instance, IL-12 may include a variant of IL-12B having one or several substitutions with respect to the wildtype IL-12B selected from the group consisting of E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D,
N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,
K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q, the amino acid numbering being preferably the one of SEQ ID NO: 52.. Optionally, the variant of IL-12B has one of the following substitutions combinations: N103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,
Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,
E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q, S43E/E59Q, S43K/E49Q,
E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E, E59K/K99E, D18K/E59K/K99E,
E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E,
E59K/K99E/Q144K, E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,
DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,
E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D, N103D/N281D,
N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D, N103D/N113D/N281D,
N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q, N103Q/N200Q, N103Q/N281Q,
N113Q/N200Q, N113Q/N281Q, N200Q/N281Q, N103Q/N113Q/N200Q, N103Q/N113Q/N281Q,
N103Q/N200Q/N281Q, N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q,
E59K/K99E/N103Q/C252S/K264E, E59K/K99E/N113Q/C252S/K264E,
E59K/K99E/N200Q/C252S/K264E, E59K/K99E/N281Q/C252S/K264E,
E59K/K99E/N103Q/N113Q/C252S/K264E, E59K/K99E/N103Q/N200Q/C252S/K264E,
E59K/K99E/N103Q/N281Q/C252S/K264E, E59K/K99E/N113Q/N200Q/C252S/K264E,
E59K/K99E/N113Q/N281Q/C252S/K264E, E59K/K99E/N200Q/N281Q/C252S/K264E,
E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,
E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, and
E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E, the amino acid numbering being preferably the one of SEQ ID NO: 52.
IL-12 may include a variant of IL-12A and a variant of IL-12B as disclosed above.
Preferably, the two subunits of IL-12 are covalently linked by a peptide linker or peptide spacer. Preferably, they are covalently linked by a "masking" peptide linker as defined herein.
Preferably, the IL-12 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-12. Additionally or alternatively, the IL-12 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-12R, in comparison to a wild-type IL-12.
IL-15 and IL-15 variants In a particular aspect, the immuno-stimulating cytokine is IL-15 or a variant of IL-15. Preferably, IL-15 is a human IL-15, for example as disclosed under the UniProt accession number P40933 or a mutant or variant thereof.
The IL-15 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 53 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 53.
Optionally, the IL-15 variant may comprise one or several substitutions at positions of human IL-15 (without the signal peptide; SEQ ID NO: 53) selected from the group consisting of N1D,V3I, V3M, V3R, N4D, D8N, D8A, K11L, K11M, KIIR, D30N, D61N, E64Q, N65D, N71D, N71S, N72D, N72A, N72R, N72Y, S73I, N77A, N79D, N79E, N79S, Q108E, N112D, N112H, N112M and N112Y, preferably N4D, D61N, N65D, and Q108E, the amino acid numbering being preferably the one of SEQ ID NO: 53.
Optionally, the IL-15 variant may comprise one of the following substitutions combination relative to human IL-15 (without the signal peptide; SEQ ID NO: 53): N4D/N65D, D30N/N65D, D30N/E64Q, D30N/E64Q/N65D, N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N1D/D61N, N1D/E64Q, N4D, D61N, N4D/E64Q, D8N/D61N, D8N/E64Q, D61N/E64Q, N1D/D30N, E64Q/Q108E, N1D/N4D/D8N, D61N/E64Q/N65Q, N1D/D61N/E64Q/Q108E, N4D/D61N, N4D/D61N/E64Q/Q108E, N1D/N65D, N1D/Q108E, N4D/D30N, D30N/Q108E, N65D/Q108E, D30N/Q180E, E64Q/N65D, D61N/E64Q/N65D, N1D/N4D/N65D, N71S/N72A/N77A, and N4D/D61N/N65D, preferably D30N/E64Q/N65D, the amino acid numbering being preferably the one of SEQ ID NO: 53.
Preferably, the IL-15 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-15. Additionally or alternatively, the IL-15 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-15R, in comparison to a wild-type IL-15.
IL-18 or IL-18 variants
In a very particular aspect, the immuno-stimulating cytokine is Interleukin-18 (IL-18), preferably a human IL-18, for example as disclosed under the UniProt accession number Q14116, or a mutant or variant thereof.
The IL-18 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 55 or has 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID
NO: 55. IL-18 variants are known in the field. For instance, IL-18 variants are disclosed in W004091517, W019051015, WO22038417, WO22094473, WO22172944, WO23056193, WO23118497, WO24044780, WO24051728, the disclosure of which being incorporated herein by reference.
Non-exhaustively, IL-18 variants may include one substitution or a set of substitutions combination selected from the group consisting of G3P, E6K, D54H, Q56D, P57R, N91V, and R104V; G3S, E6K, D54W, Q56P, P57D, N91G, and R104T; G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y; G3P, E6K, D54W, Q56G, P57V, N91V, and R104F; G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K; G3P, E6R, D54W, Q56T, N91V, and R104T; G3P, E6R, D54H, Q56T, P57A, and N91A; G3P, E6R, D54W, Q56P, P57A, N91A, and R104L; G3P, E6R, D54W, Q56R, P57A, N91S, and R104S; G3P, E6R, D54Q, Q56L, P57W, and N91S; G3P, E6R, D54S, Q56R, P57N, N91G, and R104T; G3P, E6K, D54G, Q56G, P57A, and N91T; G3P, E6K, D54Q, Q56I, and P57W; G3N, E6K, D54P, Q56S, P57S, N91R, and R104A; G3P, E6R, D54S, Q56Y, P57T, and N91G; G3P, E6R, D54S, Q56R, P57N, N91G, and R104S; G3P, E6R, D54L, Q56T, P57A, and N91G; G3P, E6R, D54S, Q56R, P57R, N91G, and R104S; G3P, E6K, D54H, Q56E, P57Q, and N91A; G3P, E6R, D54S, Q56R, P57S, N91G, and R104S; G3P, E6R, D54S, Q56S, P57T, N91G, and R104S; G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S; G3P, E6R, D54Y, Q56T, and P57R; G3P, E6R, D54Y, Q56T, and P57S; G3P, E6R, D54L, Q56T, P57T, and N91R; G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y; G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y; G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y; G3P, E6M, D54F, Q56D, P57R, and N91P; G3P, E6L, D54H, Q56T, P57V, and N91S; G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y; G3P, E6G, D54S, Q56S, and P57R; G3E, E6H, D54R, Q56T, and P57H; G3P, E6R, D54H, Q56R, P57N, N91V, and R104E; G3P, E6R, D54G, Q56G, P57A, and N91G; G3P, E6S, D54A, Q56D, P57Q, and N91G; G3P, E6G, D54Q, Q56V, and P57W; G3P, E6S, D54W, Q56G, P57A, N91V, and R104I; G3P, E6R, D54W, Q56P, P57G, N91V, and R104L; G3D, E6K, D54P, Q56S, P57W, and N91W; G3P; G3P, E6K, D54G, Q56G, and P57A; G3P, E6R, D54L, Q56G, P57S, and N91V; G3P, E6R, Vlll, D54G, Q56G, P57A, and N91G; G3P, E6K, D54H, Q56Y, and P57S; E6R, D54W, Q56S, and P57Q; G3P, E6K, D54L, Q56T, P57Q, and N91V; G3A, E6Y, D54R, Q56S, P57L, and N91G; G3P, E6R, D54L, Q56T, P57I, and N91G; E6G, D54L, Q56T, P57E, N91G, and R104S; G3A, E6Y, D54R, Q56S, P57L, and N91A; M60K, and K96D; G3P, E6R, and K96E; M60K; G3P, and E6R; G3P, and E6K; G3D, E6K, and N91S; G3S, and I149M; G3P, E6R, and N91S; E6R;) E6R, and N91S; Vlll; and G3S, and K140R. IL-18 variant may include an N91K substitution and at least one substitution mutation selected from one of the following groups consisting of: E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C68D, C76A, M86V, N111R, N111K, S117C, C127A, and N155T; and E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N111R, N111K, S117C, C127A, and N155T, the amino acid numbering being preferably the one of SEQ. ID NO: 55.
IL-18 variant may include a N91K substitution and at least one substitution mutation selected from the group consisting of : E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C68D, C76A, M86V, N111R, N111K, S117C, C127A, and N155T; and E6K, N14C, C38S, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N111R, N111K, S117C, C127A, and N155T, and any combination thereof;
- a set of substitutions E6K, M51V, K53G, P57S, M60Y, M86V, N91K, N111R, and N155T; an K93N substitution and at least one substitution mutation selected from of the group consisting of : 1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C68D, C76A, M86V, N91R, D98E, DI ION, N111H, SI 17C, and C127A; and Y1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C76A, M86V, N91R, D98E, DI ION, N111H, S 117C, and C127A, and any combination thereof; an N91R substitution and at least one substitution mutation selected from the group consisting of Y1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C76A, M86V, K93N, D98E, D110N, N111H, S117C, and C127A;
- a set of substitutions E6D, M51R, S55P, Q56N, M60I, M86V, N91R, K93N, D98E, D 110N, and N111H; a set of substitutions K53A, P57T, and M60A; and a set of substitutions G3Y, S10K, M51Q, and K53A.
In a particular aspect, IL-18 variants may include one substitution or a set of substitution combination selected from the group consisting of E6K, Vlll, C38A, K53A, T63A, C76A and C127A. In a very particular aspect, IL-18 variant includes the combination of substitution comprising or consisting of E6K, Vlll, C38A, K53A, T63A, C76A and C127A,the amino acid numbering being preferably the one of SEQ ID NO: 55. The variant may have the amino acid sequence as disclosed in SEQ ID NO: 56.
Preferably, the IL-18 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-18. Additionally or alternatively, the IL-18 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-18R, in comparison to a wild-type IL-18.
IL-21 or IL-21variants
In a particular aspect, the immuno-stimulating cytokine is IL-21 or a variant of IL-21. Preferably, IL-21 is a human IL-21, for example as disclosed under the UniProt accession number Q9HBE4 or a mutant or variant thereof.
The IL-21 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 54 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 54. L-21 variants are known in the field. For instance, IL-21 variants are disclosed in WO05035565, WO06111524, W008049920, WO08074863, W010103038, EP3885357, EP4273158, W024002170, the disclosure of which being incorporated herein by reference.
Preferably, the IL-21 variant or fragment retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the pro-inflammatory capacity of a wild-type IL-21. Additionally or alternatively, the IL-21 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-21R, in comparison to a wild-type IL-21.
Preferably, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-10 and IL-18 or a variant thereof, typically such as described here above.
In some aspects, the multifunctional molecule of the invention can comprise 1, 2, 3 or 4 cytokine molecules, that can be the same or different. When the multifunctional molecule comprises more than one cytokine, the cytokines are attached in series to one another and connected by a peptide linker or spacer according to the invention.
Preferably, the multifunctional molecule of the invention comprises a single cytokine molecule.
In some aspects, the cytokine molecule(s) comprised in the multifunctional molecule of the invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: SO- 56, 110, 112, 118 and 128 and any variant thereof, preferably selected from the group consisting of SEQ ID NO: 50, 53, 56, 110 and 112.
Antigen binding domain
The antigen binding domain or targeting moiety of the multifunctional molecule is an antibody or an antigen binding fragment thereof or a derivative thereof.
In a first aspect, the antigen binding domain comprised in the multifunctional molecule of the invention specifically binds to a target expressed on immune cells surface, particularly targets that are only or specifically expressed on immune cells.
In another particular aspect, the antigen binding domain comprised in the multifunctional molecule of the invention is directed towards a target expressed on tumoral cells. Alternatively, the antigen binding domain is not directed towards a target expressed on tumoral cells.
In particular, the antigen binding domain comprised in the multifunctional molecule does not bind or have affinity to the cytokine.
As used herein, an "antigen-binding fragment" or "antigen-binding domain" of an antibody means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for a particular antigen, possibly in its native form; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragments have a similar binding affinity as the corresponding 4- chain antibodies. However, antigen-binding fragment that have a reduced antigen-binding affinity with respect to corresponding 4-chain antibodies are also encompassed within the invention. The antigenbinding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be designated as "functional fragments" of antibodies. Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof. Antigen binding domain fragment typically includes Fab domain, Fab', (Fab')2. Antigen binding domain derivatives typically includes single-chain variable fragment (scFV), scFab, diabody and CrossMAb.
With regard to the "binding" capacity of the antigen binding domain, the terms "bind" or "binding" refer to antibodies including antigen binding fragments thereof and derivatives that recognize and contact another peptide, polypeptide, protein or molecule. The terms "specific binding", "specifically binds to," "specific for," "selectively binds" and "selective for" a particular target mean that the antigen binding domain recognizes and binds a specific target, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically (or preferentially) binds to an antigen is an antibody that binds the antigen for example with greater affinity, avidity, more readily, and/or with greater duration than it binds to other molecules. Preferably, the term "specific binding" means the contact between an antibody and an antigen with a binding affinity equal or lower than 10’ 7 M. In certain aspects, antibodies bind with affinities equal or lower than IO-8 M, IO-9 M or IO 10 M.
As used herein, the term "target" of the antigen binding domain refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted by the antigen binding domain according to the invention and expressed on the external surface of immune cells. With regards to the expression of a target on the surface of immune cells, the term "expressed" refers to a target, such as carbohydrates, lipids, peptides, polypeptides, proteins, antigens or epitopes that are present or presented at the outer surface of a cell, preferably an immune cell.
The antigen binding domain of the multifunctional molecule can be a Fab domain, a Fab', a F(ab')2, a single-chain variable fragment (scFV), a Fv, a diabody, a scFab, a CrossMAb, a single domain antibody (sdAb) or a VHH. Preferably, the antigen-binding domain is a Fab domain, a single-chain variable fragment (scFV) or a VHH. In some aspects, the antigen-binding domain is a scFV.
Optionally, the antigen binding domain can be an antibody such as an IgG antibody that may include modifications, in particular in the Fc domain; a Fab, a Fab', a F(ab')2, a Fv, a diabody, a single domain antibody (sdAb), a VHH, a scFab, a CrossMAb or a ScFv, optionally linked to a Fc domain.
Optionally, the antigen binding domain can be a Fab domain, a Fab', a (Fab')2, a single-chain variable fragment (scFV), a scFab, a diabody, a CrossMAb, or a single domain antibody (sdAb) or a VHH. The antigen-binding domain preferably comprises a heavy chain variable region (VH) and a light chain variable region (VL). Preferably, the antigen-binding domain is a Fab, a single-chain variable fragment (scFV) or a VHH.
In some aspects, the antigen binding domain is a CrossMAb. As used herein, the term "CrossMAb" refers to antigen binding domains with an inversion of CL and CHI domains, in particular in one binding arm of antibodies. Thus, such binding domain comprises a VH domain linked to a CL domain and a VL domain linked to a CHI domain. Such format reduces the byproduct formation caused by a mismatch of a light chain of a first binding domain that specifically binds to a first antigen with the wrong heavy chain of a second binding domain that specifically binds to a second antigen (when compared to approaches without such CL-CH1 domain exchanges). CrossMAb are for example described in WO 2009/080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191, the disclosure of which being incorporated herein by reference.
In some aspects, the antigen binding domain is a recombinant antibody or an antigen binding fragment thereof. As used herein, the term "recombinant antibody" refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.
In some aspects, the antigen binding domain is or derived from an humanized antibody.
As used herein, the term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., chimeric antibodies that contain minimal sequence derived from a non-human antibody). A "humanized form" of an antibody, e.g., a non- human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. Additional framework region modifications may be made within the human framework sequences. Preferably humanized antibody has a T20 humanness score greater than 80%, 85% or 90%. "Humanness" of an antibody can for example be measured using the T20 score analyzer to quantify the humanness of the variable region of antibodies as described in Gao S H, Huang K, Tu H, Adler A S. BMC Biotechnology. 2013: 13:55 or via a web-based tool to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http://abAnalyzer.lakepharma.com. In some aspects, the antigen binding domain is or derived from an chimeric antibody.
By "chimeric antibody" is meant an antibody made by combining genetic material from a nonhuman source, preferably such as a mouse, with genetic material from a human being. Such antibody derives from both human and non-human antibodies linked by a chimeric region. Chimeric antibodies generally comprise constant domains from human and variable domains from another mammalian species, reducing the risk of a reaction to foreign antibodies from a non-human animal when they are used in therapeutic treatments.
Antigen binding domain targeting immune cells
In one aspect, the target is specifically expressed by immune cells in a healthy subject or in a subject suffering from a disease, in particular such as a cancer. This means that the target has a higher expression level in immune cells than in other cells or that the ratio of immune cells expressing the target by the total immune cells is higher than the ratio of other cells expressing the target by the total other cells. Preferably the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100. More specifically, it can be determined for a particular type of immune cells, for instance T cells, more specifically CD8+ T cells, effector T cells or exhausted T cells, or in a particular context, for instance a subject suffering of a disease such as a cancer or an infection.
"Immune cells" as used herein refers to cells involved in innate and adaptive immunity for example such as white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells and Natural Killer T cells (NKT) and myeloid-derived cells (granulocytes such as neutrophil, eosinophil, basophil, or other cells such as monocyte, macrophage, dendritic cells). In particular, the immune cell can be selected in the non- exhaustive list comprising B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes. "T cell" as used herein includes for example CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells and inhibitory T cells.
By "activated immune cells" it is meant immune cells that are involved or have been activated during an immune response towards the presence of non-self cells such as pathogens or cancer cells, or toward self cells in the case of autoimmune diseases. Activated immune cells are particularly recruited in the localization wherein the inflammation, triggered by the presence of non-self cells, occurs. Particular markers of immune cells of activation that can be targeted by the antigen binding domain are particularly described here after.
As used herein, the term "T effector cell", "T eff" or "effector cell" describes a group of immune cells that includes several T cells types that actively respond to a stimulus, such as co-stimulation. It particularly includes T cells which function to eliminate antigen (e.g., by producing cytokines which modulate the activation of other cells or by cytotoxic activity). It notably includes CD4+, CD8+, cytotoxic T cells and helper T cells (Thl and Th2). As used herein, the term "Tumor Infiltrating lymphocytes (TILs)" refers to immune cells, especially ? cells, which are located in a tumor or in its stroma.
As used herein, the term "regulatory ? cell", Treg cells" or "T reg" refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells.
The term "exhausted T cell" refers to a population of T cell in a state of dysfunction (i.e., "exhaustion"). T cell exhaustion is characterized by progressive loss of function, changes in transcriptional profiles and sustained expression of inhibitory receptors. Exhausted T cells lose their cytokines production capacity, their high proliferative capacity and their cytotoxic potential, which eventually leads to their deletion. Exhausted T cells typically indicate higher levels of CD43, CD69 and inhibitory receptors combined with lower expression of CD62L and CD127.
The term "effector memory stem like T cell" refers to a subset of tumor-reactive intra-tumoral T cells bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcfl. These cells can be called Tcfl+PD-1+CD8+ T cells. These cells reside in the tumor microenvironment and are critical for immune control of cancer promoted by immunotherapy. They are critical for maintaining the T cell response during chronic viral infection and cancer, and provide the proliferative burst seen after PD-1 immunotherapy. These cells undergo a slow self-renewal and also give rise to the more terminally differentiated exhausted CD8 T cells. These cells and their characteristics are further defined in the following articles, the disclosure thereof being incorporated herein by reference: Siddiqui et al, 2019, Immunity, 50, 195-211; and Jadhav et al, 2019, PNAS, 116, 14113-14118).
The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complements) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
Preferably, the antigen binding domain specifically binds to a target expressed immune cells selected from the group consisting of B-cells, T-cells, Natural killer, dendritic cells, monocytes and innate lymphoid cells (ILCs).
Even more preferably, the immune cell is a T cell. "T cell" or "T lymphocytes" as used herein includes for example CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T regulator, T helper 17 type T cells and inhibitory T cells. In a very particular aspect, the immune cell is an exhausted T cell.
In a particular aspect, the immune cell is a TIL.
In a particular aspect, the immune cell is an effector memory stem like T cell. The target can be a receptor expressed at the surface of the immune cells, especially T cells. The receptor can be an inhibitor receptor. Alternatively, the receptor can be an activating receptor.
In some aspects, the antigen binding domain binds to a target selected from the group consisting of PD-1, CD28, CD80, CTLA-4, LAG-3/CD223, BTLA, TIG IT, CD160, CD40L/CD154, ICOS, CD27, 0X40, 4-1BB, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30/TNFRSF8, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, CD28H, CD38, CXCR5, CD3, PDL2, CD4, CD8, IGSF2, CD119, CD150/SLAMF1, CD153, CD226, CD25, CD254, CD26,
CD275/ICOSL, ENTPD1, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CXCR4, CXCR6, FasL, GPR32, CXCRl/IL18Rl/CD218a, ITGAE/CD103, TRAILR, OX40L, LY108, PDCD1, PTPN22, RGS1, L0X1, SIGLEC 6, TACI, CD163, CD206, LTBR, TNFSF14, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), FCRL4, TNFRSF18/GITR/CD357, CD78, TRAF1, TRAF2, TRAF3, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1, MED24, TNFR2, CDCR3, TWEAKR, BAFFR, RELT, TNFRSF19, DR6, TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC- 9A, CD180, CD59, CD54, CD71, CD35, CD74, CD165, 4-1BBL, CD183/CXCR3 and CD127.
Such targets are more particularly described in the Table E below.
Table E: Examples of target expressed on immune cells. For instance, the antigen binding domain binds to a target expressed on activated T cells surface, preferably selected from the group consisting of CD101/IGSF2, CD119, CD137/4-1BB/TNFRSF9, CD183/CXCR3, CD25, CD254, CD26, CD275/ICOSL, CD40L/CD154, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR6, FasL/TNFSF6, GITR/TNFRSF18/CD357, GPR32, ICOS, IL18Rl/CXCRl/CD218a, ITGAE/CD103, LY108 /SlamF6, OX40/TNFRSF4, RGS1, PD-1, LTBR/CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3/TNFRSF6B, RELT/TNFRSF19L, TNFRSF19/TROY, TNFRSF21/DR6, TNFRSF25/DR3/TNFRSF12 and CD160.
In a particular aspect, the antigen binding domain binds to a target selected from the group consisting of PD-1, TIM3, LAG3, CD101/IGSF2, CD40L/CD154, VISTA, HVEM/TNFRSF14/CD270, BTLA, TIG IT, CD137 and CTLA-4.
For instance, the antigen binding domain binds to a target expressed on Tumor Infiltrating lymphocytes (TILs) surface, preferably selected from the group consisting of CD101, CD137 (Tnfrsf9/4-lBBL), CRTAM, CST7, CTLA4, CXCR3/CD183, FAS, IL18R1/CXCR1/CD218A, LAG-3, PTPN22, RGS1, TNFSF14 and PD-1.
For instance, the antigen binding domain binds to a target expressed on activated B cells surface, preferably selected from the group consisting of BCMA/TNFRSF17, CD150/SLAMF1, CD86, OX40L, LOX1, TACI/TNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3/CD40BP, TRAP1,
BAFFR/TNFRSF13C/CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CXCRl/IL18Rl/CD218a, CD74, and CD165.
For instance, the antigen binding domain binds to a target expressed on activated myeloid cells surface, preferably selected from the group consisting of CD163, CD206, SIGLEC 6, TRAF1, TRAF4, TRAF7, TRAP100/MED24, TNFRSF12A/FN14/TWEAKR, CD301, IL4R, and CLEC-1A.
For instance, the antigen binding domain binds to a target expressed on activated natural killer cells surface, preferably selected from the group consisting of CST7, CXCR4, NKG2A, TRAF3IP1, and CMKLR1. In a particular aspect, the antigen binding domain binds to a target expressed on exhausted T cells or effector memory stem like T cells and the target of the antigen binding domain is a factor expressed on the surface of exhausted T cells or effector memory stem like T cells. T cell exhaustion is a state of T cell progressive loss of function, proliferation capacity and cytotoxic potential, eventually leading to their deletion. T cell exhaustion can be triggered by several factors such as persistent antigen exposure or inhibitory receptors including PD-1, TIM3, CTLA-4, LAG-3, BTLA, TIGIT and CD160. Preferably, such factor, in particular such exhaustion factor, is selected from the group consisting of PD-1, TIM3, CTLA- 4, LAG3, BTLA, TIGIT and CD160.
In a very specific aspect, the antigen binding domain binds to PD-1.
In an aspect, the antigen binding domain may have an agonistic or antagonist activity on the target. In a preferred aspect, the antigen binding domain has an antagonist activity on the target. Numerous antibodies directed against PD-1, TIM3, CTLA-4, LAG-3, BTLA, TIG IT and CD160 have already been described in the art.
As used herein, the terms "Programmed Death 1", "Programmed Cell Death 1", "PD-1", "PDCD1", "PD- 1 antigen", "human PD-1", "hPD-1" and "hPD-1" are used interchangeably and refer to the Programmed Death-1 receptor, also known as CD279, and include variants and isoforms of human PD- 1, and analogs having at least one common epitope with PD-1. PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. As an example, the amino acid sequence of a human PD-1 is disclosed under GenBank accession number NP_005009. PD-1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC). Accordingly, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD-1, such as PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4. Unless specified otherwise, the terms include any variant and, isoform of human PD-1 that are naturally expressed by PBMC, or that are expressed by cells transfected with a PD-1 gene.
In a very specific aspect, the antigen binding domain binds to PD-1 and has an antagonistic effect.
Several anti-PD-1 are already clinically approved, and others are still in clinical developments. For instance, the anti-PD-1 antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO- 4538), OSE-279 (see WO2020/127366), Pidilizumab (CT-011), Cemiplimab (Libtayo), Camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (Tisleizumab), PDR001 (spartalizumab), MK-3477, SCH- 900475, PF-06801591, JNJ-63723283, Genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT- 1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), JS001 (see Si-Yang Liu et al., J. Hematol. Oncol.10:136 (2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see WO 2014/194302), AGEN2034 (see WO 2017/040790), MGA012 (see WO 2017/19846), or IBI308 (see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540, the disclosure thereof being incorporated herein by reference), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168, the disclosure thereof being incorporated herein by reference.
Antibodies directed against TIM-3 targeting TIM-3 are also known such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367. In some aspects, the TFM-3 antibody is as disclosed in International Patent Application Publication Nos. W02013006490, W02016/161270, WO 2018/085469, or WO 2018/129553, WO 2011/155607, U.S. 8,552,156, EP 2581113 and U.S 2014/044728, the disclosure thereof being incorporated herein by reference.
Antibodies directed against CTLA-4 targeting CTLA-4 are also known such as Ipilimumab, Tremelimumab, MK-1308, AGEN-1884, MEDI5752 (AstraZeneca). Anti-CTLA-4 antibodies are also disclosed in WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232,
WO19056281, WO19023482, WQ18209701, WO18165895, WO18160536, WO18156250,
WO18106862, WO18106864, WO18068182, WQ18035710, WO18025178, WO17194265,
WO17106372, WQ17084078, WO17087588, WO16196237, WO16130898, WO16015675,
WO12120125, WQ09100140 and WQ07008463, the disclosure thereof being incorporated herein by reference.
Antibodies directed against LAG-3 targeting LAG-3 are also known such as BMS- 986016, IMP701 or MGD012. Anti-LAG-3 antibodies are also disclosed in W02008132601, EP2320940, WO19152574, the disclosure thereof being incorporated herein by reference.
Antibodies directed against BTLA are also known in the art such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mabl9A7, or hu Mab4C7. The antibody TAB004 against BTLA are currently under clinical trial in subjects with advanced malignancies. Anti-BTLA antibodies are also disclosed in W008076560, WQ10106051 (e.g., BTLA8.2), WO11014438 (e.g., 4C7), W017096017 and WO17144668 (e.g., 629.3), the disclosure thereof being incorporated herein by reference.
In some aspects, the antigen binding domain comprised in the multifunctional molecule of the invention binds to TIGIT.
Antibodies directed against TIGIT are known in the art, such as BMS-986207 or AB154, BMS-986207
CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083,
CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5,
CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,
CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3,
CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1,
CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8 as disclosed in
WO19232484. Anti-TIGIT antibodies are also disclosed in WO16028656, W016106302, WO16191643,
W017030823, W017037707, WO17053748, WO17152088, WO18033798, WO18102536,
WO18102746, WQ18160704, WQ18200430, WO18204363, WQ19023504, WO19062832,
WO19129221, WO19129261, WO19137548, WO19152574, WO19154415, WO19168382 and
WO19215728, the disclosure thereof being incorporated herein by reference.
Preferably, the anti-TIGIT binding domain comprises or consists of:
(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID
NO: 129 and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 130.
Preferably, the anti-TIGIT binding domain is a bivalent antibody comprising or consisting of: (a) a first heavy chain, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 121, or variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion,
(b) a second heavy chain, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 122, or variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion,
(c) two light chains, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 123, variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion.
In some aspects, the target is CD127 or IL-7R and the antigen binding domain is specific to CD127, preferably human CD127. Preferably, the antigen binding domain is an antagonist of CD127. Antibodies directed against CD127 or IL7-R are also known in the art, such as GSK2618960, RN168, AbD11590, MAB306-100, R34.34, A019D5, eBioRDR5, 40131, 1A12, M21, 47H4, HIL-7R-M21, eBioYL8, RDR5. Anti- CD127 antibodies are also disclosed in WQ14102430, WQ20077190, WQ04000238, WQ11104687, WQ16059512 and WO17062748, the disclosure thereof being incorporated herein by reference.
In some aspects, the target is CLEC-1A and the antigen binding domain is specific to CLEC-1A, preferably human CLEC-1A. Preferably, the antigen binding domain is an antagonist of CLEC-1A.
As used herein, the term "CLEC-1 A" relates to a C- type lectin-like receptor- 1 A from a mammal species, preferably a human CLEC-1 A. A reference sequence of the human CLEC-1 A corresponds to the sequence associated to the Accession number Q8NC01 Uniprot. As used herein, the term "CLEC-1 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CLEC-1. In particular, the CLEC-1 antagonist inhibits the interactions between the CLEC-1 and at least one of its ligands.
Antibodies directed against CLEC-1A are also known in the art, such as MAB1704, ABIN526589, AF1704 and ABIN526590.
In another particular aspect, the target is PD-1 and the antigen binding domain of the multifunctional molecule is an antibody, or an antigen binding fragment or a derivative thereof or an antibody mimic that is specific to PD-1. Then, in a particular aspect, the antigen binding domain comprised in the multifunctional molecule according to the invention is an anti-PDl antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-PDl antibody or antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of PD-1. In some aspects, the anti-PD-1 antibody is Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538) or OSE-279 (such as described in WQ2020/127366, the disclosure thereof being incorporated herein by reference). In a very specific aspect of the present disclosure, the antigen binding domain targets PD-1 and is derived from the antibody disclosed in WO2020/127366, the disclosure thereof being incorporated herein by reference.
Then, in an aspect, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
(i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and
(ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:
- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 60, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but position 3 of SEQ ID NO: 60;
- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 61, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 13, 14 and 16 of SEQ ID NO: 61;
- the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 62; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 62;
- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 63, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 63;
- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 64, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and
- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 65, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 65.
In another aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61 and a CDR3 of SEQ ID NO: 62; and (ii) a light chain variable region (VL) comprising a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64 and a CDR3 of SEQ ID NO: 65.
In one embodiment, the anti-PDl antibody or antigen binding fragment according to the invention comprises framework regions, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4 and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and
LFR4. Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:
(i) a heavy chain variable region (VH) comprising a HFR1 of SEQ ID NO : 66, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR1 of SEQ ID NO: 60, a HFR2 of SEQ ID NO : 67, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR2 of SEQ ID NO: 61, a HFR3 of SEQ ID NO : 68, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR3 of SEQ ID NO: 62; and a HFR4 of SEQ ID NO : 69, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, and
(ii) a light chain variable region (VL) comprising a LFR1 of SEQ ID NO : 70, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR1 of SEQ ID NO: 63, a LFR2 of SEQ ID NO : 71, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR2 of SEQ ID NO: 64, a LFR3 of SEQ ID NO : 72, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR3 of SEQ ID NO: 65 and a LFR4 of SEQ ID NO : 73, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
In an aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of:
(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence SEQ ID NO:
74, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO: 74;
(b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO:
75, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO: 74.
Preferably, the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).
In another aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of:
(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 74; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof outside of the CDRs (i.e., in the framework region only);
(b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 75 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof outside of the CDRs (i.e., in the framework region only). Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:
(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 74 and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 75.
In an aspect, the anti-PD-1 antigen-binding domain comprises VH, VL, CHI and a CL domain, so that the antigen binding domain is a Fab.
In such aspect, the heavy chain constant domain (CHI) comprises or consists essentially of SEQ ID NO: 76, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Particularly, the anti-PD-1 antigen-binding domain comprises a heavy chain that comprises or consists of a VH of SEQ ID 74 and a CHI of SEQ ID NO:76.
Preferably, the light chain constant domain (CL) comprises or consists essentially of SEQ ID NO: 77 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Particularly, the anti-PD-1 antigen-binding domain comprises a light chain that comprises or consists of a VL of SEQ ID 75 and a CL of SEQ ID NO:77.
In an embodiment, the anti-PD-1 antigen-binding domain is a Fab or a Fab', a Fab or a F(ab')2 and comprises i) a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID No: 78, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and ii) a VL domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID No: 79, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Preferably, the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).
Preferably, the antigen binding domain is an anti-PD-1 Fab or F(ab')2, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID Nos: 74 and 76 respectively and ii) a chain comprising or consisting of VL and CL domains, said domains having the amino acid sequence as set forth in SEQ ID Nos: 75 and 77, respectively.
In an aspect, the antigen binding domain is an anti-PD-1 Fab or F(ab')2, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 78 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 79 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Preferably, the amino acids are outside of the CDRs, i.e., are in the framework regions (FR). In an aspect, the antigen binding domain comprises an anti-PD-1 CrossMAb comprising or consisting of i) a chain comprising or consisting of VH and CL domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 74 and 77, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 75 and 76, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
Preferably, the antigen binding domain comprises an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID NOs: 74 and 77, respectively, and ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 75 and 76, respectively.
In an aspect, the antigen binding domain is an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 80 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 81 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.
Alternatively, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
(i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and
(ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein
HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 90,
HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 91,
HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 92,
LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 93,
LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 94, and
LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 95,
Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 96;
(b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 97.
(i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and
(ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein
HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 98, HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 99, HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 100, LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 101, LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 102, and LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 103, Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:
(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 104;
(b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 105.
Antigen binding domain targeting tumoral cells
In another aspect, the target is specifically expressed by a tumor cell. This means that the target has a higher expression level in tumor cells than in other cells, especially healthy cells, or that the ratio of cancer cells expressing the target by the total cancer cells is higher than the ratio of other cells expressing the target by the total other cells. Preferably the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100. For instance, the target can be a tumor antigen. For instance, the antigen can be selected from the group consisting of Fibroblast Activation Protein (FAP), the Al domain of Tenascin-C (TNC Al), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).
Selecting a target specifically expressed by a tumor cell allows to direct the multifunctional molecule comprising the cytokine and the antigen binding domain targeting it specifically to a cancer cell, rather than the tumor microenvironment. Conveniently, in conjunction with the target specifically expressed by a tumor cell, the cytokine may be selected so that its adverse effect is directed toward said cancer cell rather than a healthy cell, turning said adverse effect to the advantage of the patient.
Additional examples of tumor associated antigens include: Her2/Neu (human epidermal growth factor receptor 2); CD22; EpCAM (CD326); EGFR; PSMA (Prostate Carcinoma); CD30; CD20; CD33; membrane IgE; IgE Receptor (CD23), CD80; CD86; CD2; CA125 (cancer antigen- 125); Carbonic Anhydrase IX; CD70; CD74; CD56; CD40; CD19; c-met/HGFR; DRS; PD-1; PDL1; IGF-1R; VEGF and VEGFR (Solid tumor and eye AMD), VEGF-R2; Prostate stem cell antigen (PSCA); MUC1; CanAg; Mesothelin; P-cadherin; Myostatin (GDF8); Cripto (TDGF1); ACVRL 1/ALK1 (activin a receptor type 1); MUC5AC; CEA, in particular CEACAM (carcinoembryonic antigen); CD137; CXCR4; Neuropilin 1; Glypicans; HER3; PDGFRa (platelet derived growth factor receptor alpha); EphA2 (Ephrin type- A receptor 2); nucleolin; CD38; CD138; a4-integrin, C5 complement, C3 complement, MASP-2, C5aR, CR1, C3b, CA19-9, calretinin, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, an abnormal p53 protein, diganglioside GD2, interleukin 13 receptor a 2 (IL13Ra2), fibroblast activation protein (FAP), CD133, natural-killer group 2, member D (NKG2D), chondroitin sulfate proteoglycan 4 (CSPG4), CS-I, LI cell adhesion molecule (L1CAM), BCMA (-cell maturation antigen), alpha-fetoprotein (AFP), and CFH. Some other tumor associated antigen targets have been reviewed (Gerber, et al, mAbs 2009 1 :247-253; Novellino et al, Cancer Immunol Immunother. 2005 54: 187-207, Franke, et al, Cancer Biother Radiopharm. 2000, 15:459-76, Guo, et al., Adv Cancer Res. 2013; 119: 421-475, Parmiani et al. J Immunol. 2007 178: 1975-9).
Fc domain
In a particular aspect of the disclosure, the antigen binding domain as described above may be associated with antibody constant regions, in particular from IgA, IgM, IgE, IgD or IgG such as IgGl, lgG2, lgG3, lgG4, preferably IgGl, lgG2, or lgG4. Preferably, the antigen binding domain comprises an IgG Fc region, preferably an IgGl, lgG2, or lgG4 Fc region. Preferably, the Fc domain includes all or a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from a human or humanized IgGl, lgG2, lgG3 or lgG4, preferably from a human or humanized IgGl, lgG2, or lgG4.
The molecules envisioned herein optionally comprise a Fc domain.
As used herein, the terms "fragment crystallizable region" "Fc region" or "Fc domain" are interchangeable and refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors. The Fc region or domain is typically composed of two domains, optionally identical, derived from the second and third constant domains of the antibody's two heavy chains (i.e., CH2 and CH3 domains). Portion of the Fc domain refers to the CH2 or the CH3 domain. Optionally, the Fc region or domain may optionally comprise all or a portion of the hinge region between CHI and CH2. Accordingly, the Fc domain may comprise the hinge, the CH2 domain and the CH3 domain. Optionally, the Fc domain is that from IgGl, lgG2, lgG3 or lgG4, optionally with IgGl hinge-CH2-CH3 and lgG4 hinge-CH2-CH3.
The Fc domain of the multifunctional molecule can form together with a part of the antigen binding domain a heavy chain of an IgG immunoglobulin. Indeed, when the antigen binding domain is a Fab, the multifunctional molecule may comprise one heavy chain, including the variable heavy chain (VH), CHI, hinge, CH2 and CH3 domains. However, the multifunctional molecule may also have other structures such as scFv, or diabody
The Fc domain can be from a heavy chain constant domain of a human immunoglobulin heavy chain, for example, IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the multifunctional molecule comprises an IgGl or an lgG4 heavy chain constant domain.
Preferably, the Fc domain comprises CH2 and CH3 domains. Optionally, it can include all or a portion of the hinge region, the CH2 domain and/or the CH3 domain. In some aspects, the CH2 and/or a CH3 domains are derived from a human lgG4 or IgGl heavy chain.
Specifically, the domain derived from lgG4 or IgGl heavy chain is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the lgG4 or IgGl heavy chain or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
Preferably, the Fc domain includes all or a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from human IgGl, lgG2, lgG3, lgG4. The hinge region derived from IgGl, lgG2, lgG3, lgG4 heavy chain is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the IgGl, lgG2, lgG3, lgG4 heavy chain or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
More preferably, the hinge region is derived from a human or humanized IgGl or lgG4 heavy chain. Particularly, the hinge region has a sequence of SEQ ID NO: 89 or is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity thereto or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.
The IgGl hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines permit efficient and consistent disulfide bonding formation between Fc portions. Therefore, a preferred hinge region of the present invention is derived from IgGl, more preferably from human IgGl. In some aspects, the first cysteine within the human IgGl hinge region is mutated to another amino acid, preferably serine. For instance, the sequence including CHI, CH2 and CH3 based on an IgGl is disclosed in SEQ ID NO: 83 including a N298A substitution.
The hinge region of lgG4 is known to form interchain disulfide bonds inefficiently. However, a suitable hinge region for the present invention can be derived from the lgG4 hinge region, preferably containing a mutation that enhances correct formation of disulfide bonds between heavy chain-derived moieties (Angal S, et al. (1993) Mol. Immunol., 30:105-8). More preferably, the hinge region is derived from a human lgG4 heavy chain. For instance, the sequence including CHI, CH2 and CH3 based on an lgG4 is disclosed in SEQ ID NO: 82 including a S228P substitution. Specifically, the heavy chain constant domains (CH1+CH2+CH3) comprise or consist of the sequence as set forth in SEQ ID NO: 82 or 83 or is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with SEQ ID NO: 82 or 83 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof. Preferably, the mutations are outside the CDRs, i.e., are in the framework regions.
The multifunctional molecule comprises a dimeric Fc domain. Accordingly, two monomers comprise each one a Fc chain, the Fc chains being able to form a dimeric Fc domain. The dimeric Fc domain can be an homodimer, each Fc monomer being identical or essentially identical. Alternatively, the dimeric Fc domain can be a heterodimer, each Fc monomer being different and complementary in order to promote the formation of the heterodimeric Fc domain.
More specifically, the Fc domain is a heterodimeric Fc domain. Heterodimeric Fc domains are made by altering the amino acid sequence of each monomer. The heterodimeric Fc domains rely on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and/or allow for ease of purification of heterodimers over the homodimers. There are a number of mechanisms that can be used to generate the heterodimers of the present invention. In addition, as will be appreciated by those in the art, these mechanisms can be combined to ensure high heterodimerization. Thus, amino acid variants that lead to the production of heterodimers are referred to as "heterodimerization variants". Heterodimerization variants can include steric variants (e.g., the "knobs and holes" or "skew" variants described below and the "charge pairs" variants described below) as well as "pi variants", which allows purification of homodimers away from heterodimers. WQ2014/145806, hereby incorporated by reference in its entirety, discloses useful mechanisms for heterodimerization include "knobs and holes", "electrostatic steering" or "charge pairs", pi variants, and general additional Fc variants. See also, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, Merchant et al., Nature Biotech. 16:677 (1998), all of which are hereby incorporated by reference in their entirety. For "electrostatic steering" see Gunasekaran et al., J. Biol. Chem. 285(25): 19637 (2010), hereby incorporated by reference in its entirety. For pi variants, see US 2012/0149876 hereby incorporated by reference in its entirety.
Then, in a preferred aspect, the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain based on the "knobs and holes" technology. For instance, the first Fc chain is a "knob" or K chain, meaning that it comprises the substitution characterizing a knob chain, and the second Fc chain is a "hole" or H chain, meaning that it comprises the substitution characterizing a hole chain. And vice versa, the first Fc chain is a "hole" or H chain, meaning that it comprises the substitution characterizing a hole chain, and the second Fc chain is a "knob" or K chain, meaning that it comprises the substitution characterizing a knob chain. In a preferred aspect, the first Fc chain is a "hole" or H chain and the second Fc chain is a "knob" or K chain. A Fc domain comprising a knob into hole preferably comprises a N297A substitution.
Optionally, the heterodimeric Fc domain may comprise one heterodimeric Fc chain which comprises the substitutions as shown in the following Table F and the other heterodimeric Fc chain comprising the substitutions as shown in the following Table F.
Table F (the numbering being according to EU index)
In a preferred aspect, one Fc chain is a "hole" or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and one Fc chain is a "knob" or K chain and comprises the substitutions T366W/S354C. In a yet preferred embodiment, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv)
- a peptide linker and a single cytokine or variant or fragment thereof; said peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine and being a "masking" peptide linker as defined herein; or
- two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein; or
- one peptide linker and a single cytokine or variant or fragment thereof; said peptide linker being linked to the C-terminus of the cytokine and being a "masking" peptide linker as defined herein wherein the first Fc chain comprises or consists of a "knob" chain and the second Fc chain comprises or consists of a "hole" chain.
Optionally, the Fc chain may further comprise additional substitutions.
In particular, for multifunctional molecules that target cell-surface molecules, especially those on immune cells, abrogating effector functions may be required. Engineering Fc regions may also be desired to either reduce or increase the effector function of the multifunctional molecules.
In certain aspects, amino acid modifications may be introduced into the Fc region to generate an Fc region variant. In certain aspects, the Fc region variant possesses some, but not all, effector functions. Such multifunctional molecules may be useful, for example, in applications in which the half-life of the antibody in vivo is important, yet certain effector functions are unnecessary or deleterious. Numerous substitutions or substitutions or deletions with altered effector function are known in the art.
In one aspect, the constant region of the Fc domain contains a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region can contain a mutation that eliminates the glycosylation site within the constant region of an IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain.
In a particular aspect, the Fc domain is modified to increase the binding to FcRn, thereby increasing the half-life of the multifunctional molecule. In another aspect or additional aspect, the Fc domain is modified to decrease the binding to FcyR, thereby reducing ADCC or CDC, or to increase the binding to FcyR, thereby increasing ADCC or CDC.
The alteration of amino acids near the junction of the Fc portion and the non-Fc portion can dramatically increase the serum half-life of the Fc fusion protein as shown in WO 01/58957. Accordingly, the junction region of a protein or polypeptide of the present invention can contain alterations that, relative to the naturally-occurring sequences of an immunoglobulin heavy chain and erythropoietin, preferably lie within about 10 amino acids of the junction point. These amino acid changes can cause an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is replaced. Preferably, the C-terminal lysine of an IgG sequence is replaced with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life.
In one embodiment, the constant region of the Fc domain has one of the mutations described in the Table G below, or any combination thereof. Table G: Suitable human engineered Fc domain of an antibody, numbering of residues in the heavy chain constant region is according to EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63,
78-85 (1969); www. imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber. html#refs)
In a particular aspect, the multifunctional molecule comprises a human IgGl heavy chain constant domain or an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; P329G; N297A + M252Y/S254T/T256E; K322A, K444A, K444A, K444E, K444D, K444G, K444S, K444L, K444Q, K444N, K444I, K444P, K444V, K444T, L234A/L235A/P329G, M428L, L309D, Q311H, N434S, M428L + N434S (LS) and L309D + Q311H + N434S (DHS), preferably selected from the group consisting of N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A or L234A/L235A/P329G. Preferably, the substitution or combination of substitutions selected from the group consisting of T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the group consisting of N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A.
In a particular aspect, the multifunctional molecule comprises a human IgGl heavy chain constant domain or an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; P329G; N297A + M252Y/S254T/T256E; K322A,K444A, K444E, K444D, K444G, K444S, M428L, L309D, Q311H, N434S, M428L + N434S and L309D + Q311H + N434S, preferably selected from the group consisting of N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A optionally with P329G. The multifunctional molecule comprising a human IgGl heavy chain constant domain or an IgGl Fc domain with the combination of substitutions L234A/L235A/P329G greatly reduces or altogether suppresses ADCC, ADCP and/or CDC caused by said multifunctional molecule, thus reducing nonspecific cytotoxicity.
In another aspect, the multifunctional molecule comprises a human lgG4 heavy chain constant domain or a human lgG4 Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A/L235A, P329G, S228P + M252Y/S254T/T256E and K444A. Even more preferably, the multifunctional molecule, comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4.
In another aspect, the multifunctional molecule comprises a human lgG4 heavy chain constant domain or a human lgG4 Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A/L235A; L234A/L235A/P329G, P329G, S228P + M252Y/S254T/T256E, K444A K444E, K444D, K444G and K444S. Even more preferably, the multifunctional molecule, preferably the multifunctional molecule according to the invention comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4.
As mentioned herein the and "+" refer to mutations that are cumulative. Thus, by the mutation S228P + M252Y/S254T/T256E, it is meant the following mutations: S228P, M252Y, S254T and T256E. The multifunctional molecule comprising a human lgG4 heavy chain constant domain or an lgG4 Fc domain with the substitution P329G reduces ADCC and/or CDC caused by said multifunctional molecule, thus reducing nonspecific cytotoxicity.
All subclass of Human IgG carries a C-terminal lysine residue of the antibody heavy chain (K444) that are susceptible to be cleaved off in circulation. This cleavage in the blood may compromise or decrease the bioactivity of the multifunctional molecule by releasing the linked immune-stimulating moiety to the multifunctional molecule. To circumvent this issue, K444 amino acid in the IgG domain can be substituted by another amino acid to reduce proteolytic cleavage, a mutation commonly used for antibodies. Then, in one aspect, the multifunctional molecule comprises at least one further amino acid substitution consisting of K444A, K444E, K444D, K444G, K444L, K444Q, K444N, K444I, K444P, K444V, K444T or K444S, preferentially K444A or K444L. Particularly, K444 amino acid in the IgG domain can be substituted by an alanine or a lysine to reduce proteolytic cleavage, a mutation commonly used for antibodies. Then, in one aspect, the multifunctional molecule comprises at least one further amino acid substitution consisting of K444A or K444L.
Optionally, the multifunctional molecule comprises an additional cysteine residue at the C-terminal domain of the Fc domain to create an additional disulfide bond and potentially restrict the flexibility of the multifunctional molecule. In one particular aspect, the multifunctional molecule according to the invention comprises a heterodimer of Fc domains that comprises the "knob into holes" modifications such as described above. Preferably, such Fc domains are IgGl or lgG4 Fc domain such as described above, even more preferably an IgGl Fc domain comprising the mutation N297A such as disclosed above.
For instance, the first Fc chain is a "knob" or K chain and comprises the substitutions T366W/S354C and optionally N297A and the second Fc chain is a "hole" or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and optionally N297A. Preferably, the first Fc chain is a "knob" or K chain and comprises the substitutions T366W/S354C and N297A and the second Fc chain is a "hole" or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and N297A.
More particularly, the knob Fc chain may comprise or consist in SEQ ID NO: 84 and/or the hole Fc chain may comprise or consist in SEQ ID NO: 85.
Accordingly, an object of the present invention relates to a polypeptide comprising, from the N- terminal to the C-terminal, an antigen binding domain or a part thereof, a Fc chain (knob or hole Fc chain), preferably the hole-chain of the Fc domain, and either:
- a peptide linker and a single cytokine or variant or fragment thereof; said peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine and being a "masking" peptide linker as defined herein; or
- two peptide linkers and a single cytokine or variant or fragment thereof; wherein a first linker is covalently linked to the N-terminal end of the cytokine and a second linker is linked to the C-terminal end of the cytokine, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein.
More specifically, the antigen binding domain according to the invention is linked to the knob-chain and/or the hole chain of the heterodimeric Fc domain.
In a first aspect, the multifunctional molecule comprises or consists of:
- a first entity comprising or consisting of a first Fc chain which is a knob Fc chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine
- a second entity comprising i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; iii) a "masking" peptide linker as defined herein, and iv) a cytokine.
In a second aspect, the multifunctional molecule comprises or consists of:
- a first entity comprising or consisting of a first Fc chain which is a knob Fc chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine
- a second entity comprising or consisting of i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; optionally, a peptide spacer, iii)) a cytokine, and iv) a "masking" peptide linker as defined herein.
In a third aspect, the multifunctional molecule comprises or consists of:
- a first entity comprising or consisting of a first Fc chain which is a knob chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine
- a second entity comprising i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; iii) a first peptide linker, iv) a cytokine and v) a second peptide linker, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein.
Preferably, in the multifunctional molecule of the invention comprising a Fc domain, particularly a heterodimeric Fc domain, the peptide linker(s) and the cytokine are linked to the hole Fc chain, preferably to the C-terminal end of the hole Fc chain.
The multifunctional molecule may comprise one or more spacers, especially peptide spacers, particularly between the antigen binding domain and the Fc domain. Such a spacers may be useful to prevent steric hindrances. The spacer is usually 3-44 amino acid residues in length. Preferably, the spacer has 3-30 amino acid residues. In some aspects, the spacer has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acid residues.
The spacer sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the spacer is preferably non-immunogenic in the subject to which the multifunctional molecule is administered. One useful group of spacer sequences are derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine sequences. Further preferred examples of spacer sequences are Gly/Ser linkers of different length including (Gly4Ser)4(SEQ ID NO: 107), (Gly4Ser)3 (SEQ ID NO: 106), (Gly4Ser)2 (SEQ ID NO: 108), Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2)3 (SEQ ID NO: 109).
In some embodiments, when the multifunctional molecule comprises only one peptide linker at the C- terminus of the cytokine, the multifunctional molecule may further comprise a peptide spacer between the antigen binding domain or the Fc domain and the cytokine. In addition, if the cytokine is a dimer, a peptide spacer can be used to covalently linked the two subunits of the cytokine.
In some embodiments, the multifunctional molecule does not comprise a glycine-serine linker, preferably such as defined above, especially that links the cytokine to the antigen binding domain, typically between the Fc domain and the cytokine.
In some embodiments, the multifunctional molecule of the invention does not comprise a cleavable linker, in particular comprising a protease cleavage site, typically a metalloprotease cleavage site. Examples of multifunctional molecules
Examples of multifunctional molecules of the invention are provided below.
In some aspects, the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures A, B, Cl, C2, C3, C4, C5, C6, C7, C8 and D of Figure 1C.
In some aspects, the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures Cl, C2, C3, C4, C5, C6, C7 and C8 of Figure 1C.
In some aspect, the structure and/or sequence of the multifunctional molecule is selected from the structure and/or sequence disclosed in Table 4 of the Example section, when a peptide linker of the invention is comprised.
In some aspects, the multifunctional molecule comprises one of the following arrangements, said arrangements being described from the N-terminus to the C-terminus: antigen binding domain heavy chain (VH or VH+CH1)- optionally a Fc chain - "masking" linker
- cytokine - "masking" linker, antigen binding domain light chain (VL or VL+CL) -"masking" linker - cytokine - "masking" linker, antigen binding domain heavy chain (VH or VH+CH1)- optionally a Fc chain - "masking" linker
- cytokine, antigen binding domain light chain- "masking" linker - cytokine, antigen binding domain heavy chain(VH or VH+CH1) — optionally a Fc chain - cytokine - "masking" linker, antigen binding domain light chain (VL or VL+CL)- - cytokine - "masking" linker, antigen binding domain heavy chain(VH or VH+CH1) — optionally a Fc chain - "masking" linker
- cytokine - linker, antigen binding domain light chain (VL or VL+CL)- -"masking" linker - cytokine - linker, antigen binding domain heavy chain (VH or VH+CH1)- -- optionally a Fc chain - linker - cytokine
- "masking" linker, and an antigen binding domain light chain (VL or VL+CL)- - linker - cytokine - "masking" linker.
These arrangements may be combined i) with each other as long as the antibody comprises a VH and a VL, ii) with a Fc domain devoid of a cytokine; iii) ii) with a Fc domain devoid of a cytokine and of antigen binding domain or iv) with an antigen binding domain light chain or heavy chain that is not linked to a cytokine.
In some preferred aspects, the multifunctional molecule according to the invention comprises or consists of an antibody (monovalent or bivalent) - a first masking linker - a cytokine - a second masking linker, preferably this structure being read from N-terminal to C-terminal. Especially, the C- terminal end of the Fc domain of the antibody, preferably only the hole Fc chain, is covalently linked to the N-terminal end of the first peptide linker.
In some preferred aspects, the multifunctional molecule according to the invention comprises or consists of an antibody (monovalent or bivalent), preferably an anti-PDl or anti-TIGIT antibody - a first EEEEEEEEEEEEEEE linker (SEQ ID NO: 5) - a cytokine, preferably selected from the group consisting of IL-2, IL-15, IL-10 and IL-18 - a second EEEEEEEEEEEEEEE linker (SEQ ID NO: 5), preferably this structure being read from N-terminal to C-terminal. Especially, the C-terminal end of the Fc domain of the antibody, preferably only the hole Fc chain, is covalently linked to N-terminal end of the first peptide linker.
In some aspects, the multifunctional molecule according to the invention comprises or consists of:
- an anti-PD-1 or anti-TIGIT antibody or a fragment thereof; in particular as described above;
- one, two, three or four, preferably two, cytokine(s) or fragment(s) thereof covalently linked to the N- terminal and/or C-terminal end of the antibody;
- one to eight, preferably one to four, peptide linker(s) as described above covalently linked to the N- terminal and/or C-terminal end of the cytokine(s), wherein at least one peptide linker per cytokine is a "masking" peptide linker as defined herein.
In some aspects, the multifunctional molecule comprises or consists of:
- an anti-PD-1 or anti-TIGIT antibody or a fragment thereof; in particular as described above;
- a single cytokine or a variant or a fragment thereof covalently linked to the C-terminal end of the antibody;
- one or two peptide linker(s) covalently linked to the N-terminal and/or C-terminal end of the cytokine(s) as described above, wherein at least one peptide linker is a "masking" peptide linker as defined herein and preferably both.
Preferably, the structure of the multifunctional molecule, from N-terminal to C-terminal is selected from the group consisting of: anti-PDl antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; anti-PDl antigen binding domain, preferably antibody - first peptide linker-cytokine; anti-TIGIT antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; and anti- TIG IT antigen binding domain, preferably antibody - first peptide linker-cytokine.
In the above structures, the antibody can be bivalent or monovalent. The peptide linker and/or cytokine are covalently linked to the C-terminal end of the Fc chain of the antibody, preferably the hole Fc chain. Preferably, in said multifunctional molecules, the first peptide linker is E15 (EEEEEEEEEEEEEEE (SEQ ID NO: 5). More preferably, the first and the second peptide linkers are E15 ( EEEEE EEEEE EEEEE (SEQ ID NO: 5). Preferably, in said multifunctional molecules, the cytokine is a pro- inflammatory cytokine, preferably IL-2, IL-12, IL-15, IL-18 or IL-21 or a variant thereof, preferably IL-2, IL-15, IL-21 or a variant thereof as described above.
Preferably, in the multifunctional molecule, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46). More specifically, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),
DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),
NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43). In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).
In an even more particular aspect, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
In some aspect, in the multifunctional molecule, the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I I I I H (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), I I I I I I I I I I I I I THTTTTT (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I I TH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).
In some aspects, the multifunctional molecule comprises or consists of: a Fc domain, preferably an heterodimeric Fc domain comprising a "knob" chain and a "hole" chain, a single antigen binding domain covalently linked to the N-terminus of the Fc domain, said antigen binding domain comprising or consisting of a VH and a VL domains (e.g., Fab) of an anti-PD-1 or anti-TIGIT antibody as described above, a single cytokine covalently linked to the C-terminus of the Fc domain, and one or two peptide linker(s), preferably two peptide linkers, covalently linked to N-terminus and/or C-terminus of the cytokine, as described above, wherein at least one peptide linker is a "masking" peptide linker as defined herein and preferably both.
Preferably, the antigen binding domain and the cytokine are on the same Fc chain. Especially, the antigen binding domain and the cytokine are on the "hole" Fc chain.
In some aspects, the molecule comprises or consists of: a) a first entity comprising a first Fc chain ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains of an anti-PD-1 or anti-TIGIT antibody as described above, ii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) either - one "masking" peptide linker as defined herein and a single cytokine; said "masking" peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine; or
- two peptide linkers and a single cytokine; wherein a first linker is covalently linked to the N-terminus of the cytokine and a second linker is covalently linked to the C-terminus of the cytokine, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; or
- one "masking" peptide linker, one peptide spacer and a single cytokine; wherein a peptide spacer is covalently linked to the N-terminus of the cytokine and a "masking" peptide linker is covalently linked to the C-terminus of the cytokine; or one "masking" peptide linker, one peptide spacer and a single cytokine; wherein a peptide spacer is covalently linked to the C-terminus of the cytokine and a "masking" peptide linker is covalently linked to the N-terminus of the cytokine;.
In the multifunctional molecule, the cytokine is a pro-inflammatory cytokine, preferably! L-2, IL-12, IL- 15, IL-18 or IL-21 or a variant thereof, preferably IL-2, IL-15, IL-21 or a variant thereof such as described above.
Preferably, in the multifunctional molecule, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46). More specifically, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48),
DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43). In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).
In a even more particular aspect, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).
In some aspect, in the multifunctional molecule, the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I I I TH (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I I TH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).
Preferably, in the multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the molecule comprises or consists of: a) a first entity comprising a first Fc chain ; said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-PD-1 antibody as described herein, ii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferablylL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine; the first and/or second peptide linker(s) preferably consist of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49). Preferably, in the multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the multifunctional molecule comprises or consists of: a) a first chain comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH of SEQ ID NO : 74 and VL of SEQ ID NO : 75 of an anti-PD-1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two "masking" peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-15 and consists of SEQ ID NOs : 53; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.
Preferably, in such multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the multifunctional molecule comprises or consists of: a) a first chain comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH of SEQ ID NO :74 and VL of SEQ ID NO : 75 of an anti-PD-1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-21 and consists of SEQ ID NOs : 54; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.
Preferably, in such multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH of SEQ ID NO :74 and VL of SEQ ID NO : 75 of an anti-PD-1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-2 and consists of SEQ ID NO : 50; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.
Preferably, in such multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising or consisting of a polypeptide chain comprising or consisting of a first Fc chain, preferably of SEQ ID NO : 84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising or consisting of: a first polypeptide chain comprising or consisting of i) a VH of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO : 74 ii) a second Fc chain, preferably of SEQ ID NO : 85, complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and iii) a cytokine, preferably selected from the group consisting of IL-2, I L12, 1 L15, IL18 or IL21 or a variant thereof such as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine. a second polypeptide chain comprising or consisting of and VL of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO : 75.
Preferably, in such multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the multifunctional molecule comprises or consists of: c) a first polypeptide chain comprising or consisting of a first Fc chain, preferably of SEQ ID NO : 84, said Fc chain being devoid of an antigen binding domain and of a cytokine; d) a second polypeptide chain comprising or consisting of i) a VH of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO :74 ii) a second Fc chain, preferably of SEQ ID NO : 85, complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and iii) a cytokine, preferably selected from the group consisting of IL-2, I L12, 1 L15, 1 L18 or IL21 or a variant thereof such as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine. e) a third polypeptide chain comprising or consisting of and VL of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO : 75.
Preferably, in such multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.
In some aspects, the multifunctional molecule comprises or consists of: a) a first polypeptide chain comprising or consisting of a first Fc chain, preferably of SEQ ID NO : 84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second polypeptide chain comprising or consisting of i) a VH of an anti-PD-1 antibody linked to a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; preferably as described in SEQ ID NO: 88 and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and iii) a cytokine, preferably selected from the group consisting of IL-2, 1 L12, 1 L15, 1 L18 or IL21 or a variant thereof such as described herein; wherein a first peptide linker is covalently linked to the N- terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine. c) a third polypeptide chain comprising or consisting of and VL of an anti-PD-1 antibody such as described herein, said VH comprising or consisting preferably of SEQ ID NO : 85.
In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-TAA antibody as described herein, ii) a first Fc chain; said Fc chain being devoid of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-PD-1 antibody as described herein, ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine; the first and/or second peptide linker(s) preferably consist of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49).
In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-TAA antibody as described herein, ii) a first Fc chain; said Fc chain being devoid of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-CD3 antibody as described herein, ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine; the first and/or second peptide linker(s) preferably consist of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49).
In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-CD28 antibody as described herein, ii) a first Fc chain; and iii) an anti-TAA antibody or fragment thereof as described herein, wherein the anti-TAA antibody is fused to the C-terminus of the first Fc chain; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an anti-PD-1 antibody as described herein, ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-12, IL- 15, IL-18, IL-21 or a variant thereof, in particular as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine; the first and/or second peptide linker(s) preferably consist of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49). In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), (v) an IL2 variant, preferably comprising or consisting of SEQ ID NO: 110, and vi) a second peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), a second heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a second Fc chain complementary to the first Fc chain, the second Fc chain being preferably a knob chain such as comprising or consisting of SEQ ID NO: 84; said second heavy chain being preferably devoid of an IL-2 molecule or cytokine, and a first and second light chains of an anti-PD-1 antibody preferably comprising or consisting of SEQ ID NO: 79. In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), (v) preferably selected from the group consisting of IL-2, I L12, IL15, I L18 or IL21 or a variant thereof, typically such as described in SEQ ID NO: 50, 53, 56, 110 and 112, particularly an IL2 variant, preferably comprising or consisting of SEQ ID NO: 110, and vi) a second peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49); a second heavy chain comprising or consisting of a second Fc chain complementary to the first Fc chain, the second Fc chain being preferably a knob chain such as comprising or consisting of SEQ ID NO: 84; said second heavy chain being preferably devoid of an IL-2 molecule or cytokine, and of an anti-PD-1 antigen binding domain; and a first and second light chains of an anti-PD-1 antibody preferably comprising or consisting of SEQ ID NO: 79.
In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of SEQ ID NO: 88-5-(50, 53, 56, 110 or 112)-5 from N-terminal end to the C-terminal end, preferably of SEQ ID NO: 111 a second heavy chain comprising or consisting of SEQ ID NO: 86; and a first and second light chains comprising or consisting of SEQ ID NO: 79.
In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-TIGIT VH domain preferably comprising or consisting of SEQ ID NO: 129, ii) optionally a CHI domain of an antibody , iii) optionally a hinge domain, iv) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), (v) a cytokine, preferably selected from the group consisting of IL-2, IL12, IL15, IL18 or IL21 or a variant thereof, typically such as described in SEQ ID NO: 50, 53, 56, 110 and 112, more preferably IL2, even more preferably an IL2 comprising or consisting of SEQ ID NO: 110, and vi) a second peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49); a second heavy chain comprising or consisting of a second Fc chain complementary to the first Fc chain, the second Fc chain being preferably a knob chain such as comprising or consisting of SEQ ID NO: 84; said second heavy chain being preferably devoid of an IL-2 molecule or cytokine, and of an anti-PD-1 antigen binding domain; and a first and second light chains of an anti-TIGIT antibody preferably comprising or consisting of SEQ ID NO: 79.
In some aspects, the multifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 121-5-50-5, from N-terminal end to the C-terminal end; a second heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 122; and a first and second light chains comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 123.
Preparation of multifunctional molecule
To produce a multifunctional molecule according to the invention, in particular by mammalian cells, nucleic acid sequences or group of nucleic acid sequences coding for the multifunctional molecule are subcloned into one or more expression vectors. Such vectors are generally used to transfect mammalian cells. General techniques for producing molecules comprising antibody sequences are described in Coligan et al. (eds.), Current protocols in immunology, at pp. 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are hereby incorporated by reference and in "Antibody engineering: a practical guide" from W. H. Freeman and Company (1992), in which commentary relevant to production of molecules is dispersed throughout the respective texts.
Preferably, the multifunctional molecule is a polypeptide or protein produced as a recombinant protein.
Generally, such method comprises the following steps of: (1) transfecting or transforming appropriate host cells with the polynucleotide(s) encoding the recombinant multifunctional molecule of the invention or the vector containing the polynucleotide(s);
(2) culturing the host cells in an appropriate medium; and
(3) optionally isolating or purifying the multifunctional molecule from the medium or host cells.
The invention further relates to a nucleic acid encoding a multifunctional molecule as disclosed above, a vector, preferably an expression vector, comprising the nucleic acid of the invention, a genetically engineered host cell transformed with the vector of the invention or directly with the sequence encoding the recombinant multifunctional molecule, and a method for producing the multifunctional molecule of the invention by recombinant techniques.
The nucleic acid, the vector and the host cells are more particularly described hereafter.
Nucleic acid molecules encoding the molecules or multifunctional molecules of the present invention. Recombinant Expression Vectors and Host Cells comprising such
Nucleic acid sequence
The invention also relates to an isolated nucleic acid molecule encoding the multifunctional molecule as defined above or to a group of isolated nucleic acid molecules encoding the multifunctional molecule as defined above. Nucleic acid encoding the multifunctional molecule disclosed herein can be amplified by any techniques known in the art, such as PCR. Such nucleic acids may be readily isolated and sequenced using conventional procedures.
In a particular aspect, the nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:
- a first nucleic acid encoding a polypeptide comprising, from N to C-terminus i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer iii) a first Fc chain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;
- a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; and
- a third nucleic acid molecule encoding the light chain of the antibody.
In a particular aspect, the nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:
- a first nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody ii) optionally a peptide spacer and iii) a first Fc chain; - a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;
- a third nucleic acid molecule encoding the light chain of the antibody.
In a particular aspect, the nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:
- a first nucleic acid molecule encoding a first Fc chain; and
- a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;
- a third nucleic acid molecule encoding the light chain of the antigen-binding domain.
In one embodiment, the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule.
Vectors
In another aspect, the invention relates to a vector comprising the nucleic acid molecule or the group of nucleic acid molecules as defined above.
As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids and artificial chromosomes. In general, engineered vectors comprise an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.
The nucleic acid molecule encoding the multifunctional molecule, the fusion protein, can be cloned into a vector by those skilled in the art, and then transformed into host cells. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The methods known to the artisans in the art can be used to construct an expression vector containing the nucleic acid sequence of the multifunctional molecule, variant described herein and appropriate regulatory components for transcription/translation. Accordingly, the present invention also provides a recombinant vector, which comprises a nucleic acid molecule encoding the multifunctional molecule according to the present invention. In one preferred aspect, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug-resistance gene for screening. The expression vector may further comprise a ribosome -binding site for initiating the translation, transcription terminator and the like.
Suitable expression vectors typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants.
Lipid based nanoparticles
In another aspect, the invention relates to a lipid-based nanoparticle comprising the multifunctional molecule of the invention or a nucleic acid sequence or vector encoding such.
The lipid-based nanoparticle according to the invention is particularly formulated either as a liposome or a lipid nanoparticle (LNP), especially a lipid nanoparticle comprising a mixture of lipids.
The lipid-based nanoparticle also encompasses similar nanoparticles such as but not limited to micelles and nano-emulsions.
Lipid-based nanoparticles typically comprise helper lipid, sterol and/or PEG lipid components along with the mRNA of interest. The elements of a LNP may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a lipid-based nanoparticle may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements.
In a preferred embodiment, the lipid-based nanoparticles is a lipid nanoparticle comprising one or more ionizable or cationic lipid(s), one or more helper lipid(s), one or more sterol(s), and/or one or more polyethylene glycol (PEG)-modified lipid(s).
Host cells
In another aspect, the invention relates to a host cell comprising a vector or a nucleic acid molecule or group of nucleic acid molecules as defined above, for example for multifunctional molecule production purposes. As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the multifunctional molecule according to the present invention. The term "host cell" is also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.
Suitable hosts cells are especially eukaryotic hosts cells which provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cell may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; insect cell such as Mythimna separate; plant cell such as tobacco, and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells and COS cells.
Preferably, the host cell of the present invention is selected from the group consisting of CHO cell, COS cell, NSO cell, and HEK cell.
Then host cells stably or transiently express the multifunctional molecule according to the present invention. Such expression methods are known by the man skilled in the art.
A method of production of the multifunctional molecule is also provided herein. The method comprises culturing a host cell comprising a nucleic acid encoding the multifunctional molecule as provided above, under conditions suitable for its expression, and optionally recovering the multifunctional molecule from the host cell (or host cell culture medium). Particularly, for recombinant production of a multifunctional molecule, nucleic acid encoding a multifunctional molecule, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The molecules or multifunctional molecules are then isolated and/or purified by any methods known in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, supercentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and the combination thereof. As described, for example, by Coligan, multifunctional molecule isolation techniques may particularly include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography and ion exchange chromatography. Protein A preferably is used to isolate the molecules or multifunctional molecules of the invention.
Pharmaceutical Compositions and uses thereof
The present invention also relates to a pharmaceutical composition comprising a multifunctional molecule as described herein, the isolated nucleic acid molecule, the group of isolated nucleic acid molecules, the vector and/or the host cells as described hereabove, preferably as the active ingredient or compound. The formulations can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers, excipients, salts, anti-oxidant and/or stabilizers which do not deleteriously interact with the multifunctional molecule of the invention, nucleic acid, vector and/or host cell of the invention and does not impart any undesired toxicological effects. Optionally, the pharmaceutical composition may further comprise an additional therapeutic agent.
As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active agents, such as comprising a multifunctional molecule according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism.
In one aspect, a "composition" typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. An "acceptable vehicle" or "acceptable carrier" as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
Compositions of the present invention can be in a form suitable for any conventional route of administration or use. Particularly, the pharmaceutical composition according to the invention can be formulated for any conventional route of administration including a topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. To facilitate administration, the multifunctional molecule as described herein can be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005).
The pharmaceutical composition may be prepared by mixing a multifunctional molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, anti-oxidant, and/or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, anti-oxidant, and/or stabilizers are well known in the art and have been for example described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
To facilitate delivery, any of the multifunctional molecules or its encoding nucleic acids can be conjugated with a chaperon agent. The chaperon agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipid. It can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polypeptide.
Pharmaceutical compositions according to the invention may be formulated to release the active ingredients (e.g., the multifunctional molecule of the invention) substantially immediately upon administration or at any predetermined time or time period after administration. The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Means known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer.
Pharmaceutical composition typically is sterile and stable under the conditions of manufacture and storage. Prevention of presence of microorganisms may be ensured both by sterilization procedures (for example by microfiltration), and/or by the inclusion of various antibacterial and antifungal agents. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.
Use in the treatment of a disease
The multifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the present invention have numerous in vitro and in vivo utilities and applications. Particularly, any of multifunctional molecules, nucleic acid molecules, group of nucleic acid molecules, vectors, host cells or pharmaceutical composition provided herein may be used in therapeutic methods and/or for therapeutic purposes, in particular for the treatment of diseases or as a medicament or vaccine.
The term "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and/or a prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and/or eliminating, reducing and/or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly. A prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and/or delaying the progression and/or the incidence of a disease or the risk of its occurrence. In certain aspects, such a term refers to the improvement or eradication of a disease, a disorder, an infection or symptoms associated with it. In other aspects, this term refers to minimizing the spread or the worsening of cancers. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a composition including one or more active agents to a subject who has a disorder/disease.
As used herein, the term "medicament" refers to any substance or composition with curative or preventive properties against disorders or diseases.
The present invention particularly relates to a multifunctional molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition comprising such for use in the treatment of a disease in a subject and/or for use as a medicament or vaccine. It also relates to the use of a multifunctional molecule as described herein; a nucleic acid or a vector encoding such, or a pharmaceutical composition comprising such for treating a disease and/or disorder in a subject. It also concerns the use of a multifunctional molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition comprising such as disclosed herein in the manufacture of a medicament for treating a disease and/or disorder in a subject, such as cancer or infections. Examples of diseases to be treated are more particularly described hereafter. Finally, it relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule to the subject, or a nucleic acid or group of nucleic acid molecules or a vector encoding such.
In one aspect, the treatment method comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a multifunctional molecule, nucleic acid, vector or pharmaceutical composition as described herein.
A subject in need of a treatment may be a human having, at risk for, or suspected of having a disease. Such a patient can be identified by routine medical examination.
The invention particularly relates to a method of treatment of a cancer or an infectious disease in a subject in need thereof comprising administering to said subject an effective amount of a multifunctional molecule or pharmaceutical composition as defined above.
In another aspect, the multifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to enhance immunity, preferably in order to treat a disorder and/or disease. Accordingly, in one aspect, the invention provides a method of modifying an immune response in a subject comprising administering to the subject a multifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention such that the immune response in the subject is modified. Preferably, the immune response is enhanced, increased, stimulated or up-regulated. The multifunctional molecule or pharmaceutical composition can be used to enhance immune responses such as T cell activation in a subject in need of a treatment. In a particular embodiment, the multifunctional molecule or pharmaceutical composition can be used to reduce T cells exhaustion or to reactivate exhausted T cells.
The invention particularly provides a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutic effective amount of any of the multifunctional molecules, nucleic acid, vector or pharmaceutical composition comprising such described herein, such that an immune response in the subject is enhanced. In a particular embodiment, the multifunctional molecule or the pharmaceutical composition as described herein can be used to reduce T cells exhaustion or to reactivate exhausted T cells.
Cancer
In another aspect, the invention provides the use of a multifunctional molecule or a pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a cancer, for instance for inhibiting growth of tumor cells in a subject. The invention particularly provides a multifunctional molecule or a pharmaceutical composition as disclosed herein for use in the treatment of cancer.
The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
Accordingly, in one aspect, the invention provides a method of treating a cancer, for instance for inhibiting growth of tumor cells, in a subject, comprising administering to the subject a therapeutically effective amount of multifunctional molecule or a pharmaceutical composition according to the invention. Particularly, the present invention relates to the treatment of a subject using a multifunctional molecule such that growth of cancerous cells is inhibited.
In an aspect of the disclosure, the cancer to be treated is associated with exhausted T cells.
Any suitable cancer may be treated with the provided herein can be hematopoietic cancer or solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, stomach cancer, urethral cancer environmentally induced cancers and any combinations of said cancers. Additionally, the invention includes refractory or recurrent malignancies. Preferably, the cancer to be treated or prevented is selected from the group consisting of metastatic or not metastatic, Melanoma, malignant mesothelioma, Non-Small Cell Lung Cancer, Renal Cell Carcinoma, Hodgkin's Lymphoma, Head and Neck Cancer, Urothelial Carcinoma, Colorectal Cancer, Hepatocellular Carcinoma, Small Cell Lung Cancer Metastatic Merkel Cell Carcinoma, Gastric or Gastroesophageal cancers and Cervical Cancer. In a particular aspect, the cancer is a hematologic malignancy or a solid tumor. Such a cancer can be selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia.
In a particular aspect, the cancer is a cancer induced by virus or associated with immunodeficiency. Such a cancer can be selected from the group consisting of Kaposi sarcoma (e.g., associated with Kaposi sarcoma herpes virus); cervical, anal, penile and vulvar squamous cell cancer and oropharyngeal cancers (e.g., associated with human papilloma virus); B cell non-Hodgkin lymphomas (NHL) including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classic Hodgkin lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and/or Kaposi sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis B and/or C viruses); Merkel cell carcinoma (e.g., associated with Merkel cell polyoma virus (MPV)); and cancer associated with human immunodeficiency virus infection (HIV) infection.
Preferred cancers for treatment include cancers typically responsive to immunotherapy. Alternatively, preferred cancers for treatment are cancers non-responsive to immunotherapy.
Infectious disease
The multifunctional molecule, nucleic acid, group of nucleic acid, vector, host cells or pharmaceutical compositions of the invention can be used to treat patients that have been exposed to particular toxins or pathogens. Accordingly, an aspect of the invention provides a method of treating an infectious disease in a subject comprising administering to the subject a multifunctional molecule according to the present invention, or a pharmaceutical composition comprising such, preferably such that the subject is treated for the infectious disease. The invention particularly concerns a multifunctional molecule or a pharmaceutical composition as disclosed herein for use in the treatment of an infectious disease.
Any suitable infection may be treated with a multifunctional molecule, nucleic acid, group of nucleic acid, vector, host cells or pharmaceutical composition as provided herein.
Some examples of pathogenic viruses causing infections treatable by methods of the invention include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.
Some examples of pathogenic bacteria causing infections treatable by methods of the invention include chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria.
Some examples of pathogenic fungi causing infections treatable by methods of the invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
Some examples of pathogenic parasites causing infections treatable by methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.
Combined therapy
The multifunctional molecule or pharmaceutical composition according to the invention can be combined with some other potential strategies for overcoming immune evasion mechanisms with agents in clinical development or already on the market (see table 1 from Antonia et al. Immunooncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258-6268, 2014). Such combination with the multifunctional molecule according to the invention may be useful notably for:
1- Reversing the inhibition of adaptive immunity (blocking T-cell checkpoint pathways);
2- Switching on adaptive immunity (promoting T-cell costimulatory receptor signalling using agonist molecules, in particular antibodies),
3- Improving the function of innate immune cells;
4- Activating the immune system (potentiating immune-cell effector function), for example through vaccine-based strategies.
Accordingly, also provided herein are combined therapies with any of the multifunctional molecules or a pharmaceutical composition comprising such, as described herein and a suitable second agent, for the treatment of a disease or disorder. In an aspect, the multifunctional molecule and the second agent can be present in a unique pharmaceutical composition as described above. Alternatively, the terms "combination therapy" or "combined therapy", as used herein, embrace administration of these two agents (e.g., a multifunctional molecule as described herein and an additional or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., a multifunctional molecule) can be administered orally, and an additional therapeutic agent (e.g., an anti-cancer agent, an anti-infection agent; or an immune modulator) can be administered intravenously. Alternatively, an agent of the combination selected may be administered by intravenous injection while the other agents of the combination may be administered orally.
In some instances, the multifunctional molecule or pharmaceutical composition of the invention is used or is for use in combination with an additional/second therapeutic agent, preferably an anticancer agent, an anti-infection agent or an immune modulator.
In some instances, the multifunctional molecule or pharmaceutical composition of the invention is used or is for use in combination with an additional/second therapeutic treatment, preferably an anticancer treatment or an anti-infection treatment or an immune modulator.
In an aspect, the additional therapeutic agent can be selected in the non-exhaustive list comprising alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic, antiproliferative, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunotherapies, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.
For instance, the additional therapeutic agent can be selected in the group consisting of chemotherapy, radiotherapy, targeted therapy, antiangiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes from tumor antigens, myeloid checkpoints inhibitors, other immunotherapies, and HDAC inhibitors.
In one aspect, the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is particularly selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular of adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates. Preferably, suitable agents for co-use with any of the multifunctional molecule or with the pharmaceutical composition according to the invention include an antibody binding to a co-stimulatory receptor (e.g., 0X40, CD40, ICOS, CD27, HVEM or GITR), an agent that induces immunogenic cell death (e.g., a chemotherapeutic agent, a radio-therapeutic agent, an anti- angiogenic agent, or an agent for targeted therapies), an agent that inhibits a checkpoint molecule (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), a cancer vaccine, an agent that modifies an immunosuppressive enzyme (e.g., IDO1 or iNOS), an agent that targets Treg cells, an agent for adoptive cell therapy, or an agent that modulates myeloid cells.
In an aspect, the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti- HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.
The present invention also relates to a method for treating a disease in a subject comprising administering to said subject a therapeutically effective amount of the multifunctional molecule or the pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent.
Specific examples of additional or second therapeutic agents are provided in WO 2018/053106, pages 36-43.
In a preferred aspect, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapy agents, cytokines, cell therapy agents (such as CAR-T cells), antibiotics and probiotics.
Combination therapy could also rely on the combination of the administration of multifunctional molecule with surgery.
Subject, regimen and administration
The present invention relates to a multifunctional molecule as disclosed herein, a nucleic acid molecule or group of nucleic acid molecules or a vector encoding such, a host cell or a pharmaceutical composition for use as a medicament or for use in the treatment of a disease or for administration in a subject in need thereof. It also relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule to a subject.
"An effective amount" or a "therapeutic effective amount" as used herein refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The "effective amount" will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
The subject to treat may be a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old or of at least 70 years old.
In a particular aspect, the subject can be immunosuppressed (e.g., the subject has an auto-immune disease or has a transplant) or immunocompromised (e.g., the subject has a cancer or infections such as AIDS).
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the multifunctional molecule or the pharmaceutical composition as disclosed herein to a subject, depending upon the type of diseases to be treated or the site of the disease e.g., administered orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Preferably, the multifunctional molecule or the pharmaceutical composition as disclosed herein is administered via subcutaneous, intra-cutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-tumoral, intra-sternal, intra-thecal, intra-lesion, and intracranial injection or infusion techniques.
The form of the pharmaceutical compositions, the route of administration and the dose of administration of the pharmaceutical composition or the multifunctional molecule according to the invention can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, size, sex, and/or general physical condition. The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.
Kits
Any of the multifunctional molecules or compositions described herein may be included in a kit provided by the present invention. The present disclosure particularly provides kits for use in enhancing immune responses and/or treating diseases or disorders (e.g., cancer and/or infection)
In the context of the present invention, the term "kit" means two or more components (one of which corresponding to the multifunctional molecule, the nucleic acid molecule, the vector or the cell of the invention) packaged in a container, recipient or otherwise. A kit can hence be described as a set of products and/or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit. The kits of this invention are in suitable packaging.
Particularly, a kit according to the invention may comprise:
- a multifunctional molecule as defined above,
- a nucleic acid molecule or a group of nucleic acid molecules encoding said multifunctional molecule,
- a vector comprising said nucleic acid molecule or group of nucleic acid molecules, and/or
- a cell comprising said vector or nucleic acid molecule or group of nucleic acid molecules.
The kit may thus include, in suitable container means, the pharmaceutical composition, fusion proteins or multifunctional molecules, and/or host cells of the present invention, and/or vectors encoding the nucleic acid molecules of the present invention, and/or nucleic acid molecules or related reagents of the present invention. In some embodiments, means of taking a sample from an individual and/or of assaying the sample may be provided. The compositions comprised in the kit according to the invention may particularly be formulated into a syringe compatible composition.
In some embodiments, the kit further includes an additional agent for treating cancer or an infectious disease, and the additional agent may be combined with the pharmaceutical composition, fusion proteins or multifunctional molecules, and/or host cells of the present invention, and/or vectors encoding the nucleic acid molecules of the present invention, and/or nucleic acid molecules, or other components of the kit of the present invention or may be provided separately in the kit. Particularly, the kit described herein may include one or more additional therapeutic agents such as those described in the "Combined Therapy" described hereabove. The kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual as described hereabove.
The instructions related to the use of the multifunctional molecule or pharmaceutical composition described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, means for reconstituting the multifunctional molecule and/or means for diluting the multifunctional molecule of the invention. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual).
All the references cited in this description are incorporated by reference in the present application. Others features and advantages of the invention will become clearer in the following figures and examples which are given for purposes of illustration and not by way of limitation.
BRIEF DESCRIPTION OF THE FIGURES Figure 1: Schematic representation of an embodiment of the multifunctional molecule according to the invention (A), cis demasking and cis activation technology of the bifunctional molecule constructed with optimized linker (B) and example of bifunctional antibody format designed with the linkers (C). Examples of bifunctional antibody format designed with the linkers are: A: antibody with a single antigen binding domain fused in C-terminal of the Hole Fc chain to a cytokine and a peptide linker, B: antibody with a single antigen biding domain fused in C-terminal of the Hole Fc chain to a peptide linker and a cytokine, Cl : antibody fused in C-terminal of the Hole Fc chain to a first peptide linker, a cytokine and a second peptide linker; C2 antibody fused in C-terminal of one light chain to a first peptide linker, a cytokine and a second peptide linker; C3 : Monovalent Fab and a Fc domain, wherein a first peptide linker, a cytokine and a second peptide linker is fused to the N-terminal of one chain of the Fc domain; C4 : Bivalent scFv linked to a Fc domain, a first peptide linker, a cytokine and a second peptide linker is fused to the C-terminal of one chain of the Fc domain; C5 : antibody fused in C-terminal of each Fc chain to a first peptide linker, a cytokine and a second peptide linker; C6: antibody fused in N-terminal of each light chain to a first peptide linker, a cytokine and a second peptide linker; C7: antibody fused in C-terminal of the Holde Fc chain to a first peptide linker, a first cytokine subunit, a second peptide linker, a second cytokine subunit and a third linker; C8 : antibody fused in C-terminal of each heavy and light chain to a first peptide linker, a cytokine and a second peptide linker, D: antibody fused in C-terminal of the Hole Fc chain to a peptide linker and a cytokine. These structures are given for purposes of illustration and not by way of limitation.
Figure 2: Optimized linkers allow a cytokine masking effect on PD-l-cell lines while inducing a specific cis-potentiation on PD-1+ T cell lines. (A) Jurkat T cells expressing PD-1- CD122+CD132+ (IL15Rbg) (grey line) or PD-1+CD122+CD132+ (black line) were incubated with IL15 wild-type cytokine (•), isotype-E15-IL15wt-E15 (X), anti-PDl-E15-IL15wt-E15 ( ▼ ) and (B) anti-PDl-(G4S)3-IL15wt (0). Each Jurkat PD-1- IL15Rbg+ cells or Jurkat PD-1+ IL15Rbg+ cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
Figure 3: Optimized linkers in multifunctional molecules with wild-type cytokine (IL2) allow to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines. Jurkat cells expressing PD-1- CD122+CD132+ (IL2Rbg) (A) or PD-1+CD122+CD132+ (IL2Rbg) (B) were incubated with IL2 wild-type cytokine (•), with anti PD-l-(G4S)3-IL2wt (0) and anti-PDl-E15-IL2wt-E15 ( ▼ ). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. Figure 4: Optimized linkers in multifunctional molecules with wild-type IL12 allow to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines. (A) Constructs used in this figure (B) Splenocytes T cells from wild-type C57bl6 mice (human PD-1 negative, hPD-1-) or human PD-1 Knock in C57bl6 mice (human PD-1+, hPD-l+) were isolated and sorted prior pSTAT4 bioassay to evaluate IL-12 signalling following treatment with Anti PD-1-IL12 treatments. T cells were activated during 4 days with agonist anti-msCD3 (145-2C11) and anti-msCD28 (37.51) then treated with mouse I L12 wild-type cytokine (• dashed line), with anti PD-l-(G4S)3-mslL12p40-(G4S)3- mslL12p35 (• plain line), anti-PDl-E15-mslL12p40-E15-mslL12p35-E15 (■) hPDl+ and hPDl- T cells were separately incubated with the different constructions at escalating doses for 15min at 37°C and percentage of pSTAT4 signal was quantified by flow cytometry. (C) A ratio of EC50 pSTA4 value on PD- 1- negative T cells vs PD1 + T cells reported on Figure. A mouse IL-12 cytokine was used for the design of the construction. N= 2 independent experiments
Figure 5: The optimized linkers of anti PD-1 bifunctional molecule mask cytokine activity on naive T cells and allow cis-demasking and cis-potentiation of activated T cells expressing PD-1+ cells. Naive cells PBMC were isolated from whole blood of human healthy volunteers by Ficoll, and after 2 stimulations on anti-CD3/ anti-CD28 coating, LT activated were recovered and incubated with the different constructions at escalating doses. (A) PD-1 expression on naive T cells versus activated T cells as evaluated by flow cytometry (n=3 donors) (B) pSTAT5 evaluation on naive versus activated T cells by flow cytometry after treatment with Anti-PDl-E15-IL15wt-E15. Naive ( ▲ ) versus activated T cells (■). (C) EC50 (nM) was calculated and reported on Histogram (n=3 donors) (D) pSTAT5 evaluation on naive versus activated T cells by flow cytometry after treatment with Anti-PDl-E15-IL2wt-E15. Naive ( A ) versus activated T cells (■). (E) EC50 (nM) was calculated and reported on Histogram (n=2 donors) (F) Proliferation analysis (Ki67 staining) on isolated human T cells infiltrating the tumor (n=l kidney tumor). After 5 days of incubation with a dose response of Isotype E15 IL15wt E15 or anti PD-1 E15 IL15wtE15 (monovalent anti PD-1), T cells were stained with Ki67 and % of CD3+ Ki67+ was analyzed. EC50 (nM) was calculated to evaluate difference of activation between both constructions.
Figure 6: Trans-activation assay on PD-1 presentation by antigen coating or expressing cells alone or in co-culture on PD-l-cells lines and PD-1+ cells lines. (A) pStat5 signalling measured on Jurkat cells PD-1- CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone ( ▼ ) or pre-incubated on PD1 coating (□) and Jurkat cells PD-1+CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone (•) (B) pStat5 signalling measured on Jurkat cells PD-1- CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone ( ▼ ) or pre-incubated on U937 PD1+ cells (□) and Jurkat cells PD-1+CD122+CD132+ added to anti-PDl-E15- IL15wt-E15 alone (•). (C) Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye incubated with IL15wt alone (o) or anti-PDl-E15-IL15wt-E15 (V) and Jurkat PD- 1+ IL15Rbg+ cells incubated with IL15wt alone (•) or anti-PDl-E15-IL15wt-E15 ( ▼ ) at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (D) EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells during co-culture assay and independently cells assays with IL15 alone or with anti-PDl-E15- IL15wt-E15.
Figure 7: Study assay of one or two optimized linker in construct to mask cytokine activity on PD-l- cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines. (A) Schematic representation of the different constructions used in (B) and (C) is depicted in the figure: Anti PD-1 construction is monovalent and IL15wt cytokine was fused with either a (G4S)3 linker, two optimized E15 linker or a single optimized E15 linker fused to the cytokine, either between the cytokine and the anti-PDl or to the cytokine alone. Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD-1+CD122+CD132+ (IL15Rbg) (B) were incubated with anti PD-l-(G4S)3-IL15wt (square), anti-PDl- E15-IL15wt-E15 (black circle), anti-PDl-E15-IL15wt (stars) or anti-PDl-IL15wt-E15 (triangle). Each PD- 1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (C) pSTAT5 bioassay on Jurkat PD-1 negative CD122+CD132+ (IL15Rbg) was performed with escalating dose of monovalent anti PD-l-IL15wt molecules constructed with one (G4S)3 flexible linker ((G4S)3-IL15n), Two (G4S)3 flexible linkers ((G4S)3-IL15-(G4S)3 (♦), two (EAAAK)3 rigid linker ((EAAAK)3) ( ▼ ) or the optimized E15 linker(E15-IL15wt-E15 (♦).
Figure 8: Peptide linkers of different amino-acid sequences tested on PD-l-cells and PD-1+ cells lines. Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD- 1+CD122+CD132+ (IL15Rbg) (B) were incubated with IL15 wild-type cytokine (grey dot), anti PD-1- (G4S)3-IL15wt (square), isotype-E15-IL15wt-E15 (white dot), anti-PDl-E15-IL15wt-E15 (grey diamond), anti-PDl-D15-IL15wt-D15 (triangle) or anti-PDl-N15-IL15wt-N15 (cross). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (C) EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells for each compound. (D) Experiment was repeated on primary T cells sorted from human PBMC (n=3) donors, either on naive cells (37% PD1+ cells) cells stimulated with PHA ) (75% PD1+ cells) or cells stimulated twice with CD3/CD28 (100% PD1+ cells) . Constructions tested were designed with monovalent anti PD-1 antibody (Anti PD-1*1) and a wild type IL-15 fused optimized linkers (E15-15, D15-D15, N15-N15) Figure 9: Peptide linkers comprising different amino-acid sequences tested on PD-l-cells and PD-1+ cells lines. Jurkat cells expressing PD1+ CD122+ or expressing PD1+ and CD122+ but blocked with lmg/mL of an anti-PDl were incubated with I L15 alone, anti-PD-1 (G4S)3-IL15, or anti-PDl X15-IL15- X15, where X is a combination of different proportion of amino acid Glu (E) and Asp (D) (A), Glu and Thr (T) (Fig 9BGIu and Ala (A) (Fig 9C)) or Glu and Lys (K) (Fig. 9D). Each PD-1- (PD1+ cells saturated with anti-PD-1) and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
Figure 10: Different length of optimized linkers tested to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cell lines. Jurkat cells expressing PD-1-CD122+ (A) or expressing PD-1+ CD122+cells (B) were incubated with anti PD-1-(G4S)3-IL15 (white square); anti- PD1-E6-IL15-E6 (light grey diamond), anti-PDl-E10-IL15-E10 (triangle), anti-PDl-E15-IL15-E15 (grey circle), anti-PDl-E20-IL15-E20 (grey diamond) or anti-PDl-E25-IL15-E20 (black square). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
Figure 11: Binding PD1 assay of the anti PD-1 bifunctional molecule with optimized linker or conventional linker. Binding ELISA assay was performed on PD1 coating with incubation at different concentration of either anti PD-1-(G4S)3-IL15 (0) or anti-PDl-E15-IL15-E15 ( ▲ ) for 2h at 37°C. Antibodies were detected with peroxidase-labelled anti-human IgG polyclonal antibody and revealed by TMB. Optical Density (DO) was measured at 450nm by spectrophotometer (Tecan).
Figure 12: Optimized linkers are capable of masking and demasking cytokine with one or multiple valences of anti PD-1. (A) Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye (dotted lines) and Jurkat PD-1+ IL15Rbg+ cells (solid lines) were incubated with a monovalent anti-PDl-E15-IL15-E15 (triangle), a bivalent anti-PDl-E15-IL15-E15 (diamond) or an isotype-E15-IL15-E15 (circle) at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (B) EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells during co-culture assay and independently cells assays.
Figure 13: Anti PD-1 E15-IL2-E15 enhances safety as evaluated in toxicity mouse model in vivo. C57bl6 mice were intravenously injected with one or multiple doses of anti PD-1 E15 IL2 wild type E15 (black) or anti PD1 (G4S)3 IL2 wild type (white) (2mg/kg) n=3 to 4 per group. Weight was evaluated every day until day 3 and data normalized at the day 0 prior injection of the drug and statistical analysis performed* p<0.05 (A) One dose injected vs (B) multiple injections (every day).
Figure 14: Anti PD-1-IL15 constructed with optimized linker E15 enhances safety in toxicity mouse model in vivo. CT26 bearing BALB/c mice were intravenously injected with 4 doses (Day 4, 8, 12 and 15 post tumor inoculation) of PBS, anti PD-1 E15 IL15 wild type E15 or anti PD-1 (G4S)3 IL15wt (5mg/kg). Anti PD-1 construction targeting mouse PD-1 (clone 17D2) were used for these experiments. Monovalent anti PD-1 IL15wt constructions with (G4S)3 or E15-E15 linker (A) or bivalent anti PD-1 IL15wt constructions with (G4S)3 or E15-E15 linker (B) were used for these experiments. For each experiments mouse weight was monitored and Median survival reported. Survival represents mouse death due to injected drug toxicity. One mouse was excluded in Bivalent Anti PD-1 (G4S)3 group due to tumor necrosis high tumor volume. N=7 to 8 mice/group in (B) and N=5 in (A).
Figure 15: Anti PD-1-IL15 constructed with optimized linker E15 demonstrated in vivo efficacy in monotherapy. BalbC mice were inoculated with CT26 tumor subcutaneously (le6 cell/mouse) then treated with bivalent anti PD-1 E15- IL15wtE15, Anti PD-1(G4S)3 IL15wt construction on Day 4, 8, 12 and 15 (5 mg/kg) or PBS. Anti PD-1 construction is an antagonist targeting mouse PD-1 (clone 17D2) Tumor growth (mm3) and survival were evaluated. Tumor growth could not be measured for this group. N=7 to 8 mice/group
Figure 16: Anti PD-l-IL2v or Anti PD-l-IL2wt constructed with optimized linker E15 demonstrated in vivo efficacy in monotherapy. (A) C57bl6jRj mice were inoculated with PanC02 luc+ cells (0.5e6 cell/mouse) then treated with monovalent anti PD-1 E15- IL2wtE15, on Day 7 , 11, 15 and Day 19 (2 mg/kg) or PBS. Tumor growth was quantified by Bioluminescence and survival was evaluated. N=5 per group on experiment. IL-2 cytokine was used for the design of the construction. N= 2 independent experiments (B) PanC02 (pancreas) bearing hPDIKI mice were injected intraperitoneal with 4 doses (day 7 , 11,15 and 19 post-tumor inoculation) of PBS or anti-PD-l-E15-IL2v-E15 (0,5 or lmg/kg). Anti PD1 molecule used is a bivalent anti PD-1 format with one IL-2v molecule. Survival was monitoring during these experiments. Statistical analysis was performed using Graph Pad prism using Log-Rank (Mantel Cox) test ** p<0.002
Figure 17: Anti PD-1-IL2 constructed with optimized linker E15 improved the therapeutic index of toxic immunocytokine. Bifunctional molecules comprising anti-PD-l/IL-2v constructed with an optimized linker have high anti-tumor efficacy without lethal toxicity. hPDKl Knock in mice inoculated with MC38 cells (ectopic model) were treated on Day 10, 12 and 14 with anti PD-1/ IL-2v molecule (Anti PD-1 bivalent constructed with IgGl N297A KIH isotype). (A) Tumor volume after treatment with PBS (no treatment) or the Anti PD-l/IL-2v constructed with optimized (E15-E15) linker after treatment at 0.5, 2 mg/kg, 5 mg/kg, lOmg/kg or 30 mg/kg (B) Survival of the mice. n=5 to 6 mice per group. PBS tumor volume is illustrated in mean +/- SEM. CR= Complete response. (B) Tumor volume after treatment with PBS (no treatment) or the Anti PD-l/IL-2v constructed with conventional flexible (G4S)3 linker after treatment at 0,5 m/kg PBS tumor volume is illustrated in mean +/- SEM. CR= Complete response (C) Survival of the mice. n=5 to 6 mice per group after treatment with Anti PD-l/IL- 2v constructed with optimized (E15-E15) linker. (D) Therapeutic index of bifunctional molecules comprising anti-PD-l/IL-2v constructed either with an (E15-E15) optimized linker or with a conventional linker (Gly4-Ser)3. Therapeutics indexes were calculated with Maximum tolerated Dose versus EC50 efficacy. (E and F) Therapeutic index calculated with all dose responses efficacy versus lethal dose of the construction anti PD-1 (G4S)3 IL2v or anti PD-1 E15-IL2v-E15. EC50 was calculated for Efficacy and Lethal dose to evaluate the therapeutic window (fold change versus TD50 and LD50) Dose response curve of the anti PD-1 (G4S)3 IL2v (E) and anti PD-1 E15 IL2v E15 (F).
Figure 18: pStat5 activation in co-culture assay with PD-1- IL15Rbg+ cells lines and PD-1+ IL15Rbg+cells lines. Co-culture assay with ratio 1:1 of Jurkat PD-1- CD122+CD132+ cells stained with CellTrackerGreen dye (left panel) and Jurkat PD-1+ CD122+CD132+ cells (right panel) were incubated with IL15wt alone (black circles), anti-PDl-(G4S)3-IL15wt (grey circles, conventional linker) or anti-PDl- E15-IL15wt-E15 (white circles, cytomask linker) at escalating doses for 30min at 37°C. Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
Figure 19: Optimized linker (E15-E15) masked activity of mutated 11-15 cytokine (IL15v) on primary naive PD-1 negative cells and showed higher CIS activity on PD-1+ vs PD-1- compared to (G4S)3 conventional flexible linker (A) pSTAT5 activity measurement of human PBMCs cells sing flow cytometry of the monovalent Anti PD-l/IL-15v constructed with (G4S)3 linker or E15-E15 linker (IL15v ad set forth in SEQ ID NO: 112). pSTAT5 MFI was quantify after permeabilization and intranuclear staining into T cell population was evaluated after gating CD3 + population using CD3-Pacific blue staining (n= 2 donors tested). (B) Co-culture assay with ratio 1:1 of U937 PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye incubated at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. EC50 (nM) was calculated for each cell line and Ratio was obtained with EC50 PD-1+/EC50 PD-1 neg for each molecule tested.
Figure 20: Optimized linker E15 allowed a masking effect of IL-15/IL15ra RLI construction on PD1- cell lines while inducing a specific cis-potentiation on PD1+ cell lines to higher extend compared to (G4S)3 conventional linker (A) Construction used for the Bioassay. Bivalent Anti PD-1 antibody was fused to I L-15ra sushi domain, (G4S)3 linker and IL-15 wt cytokine (as set forth in SEQ ID NO: 113) or bivalent antibody was fused to IL-15Ra sushi domain, E15, IL-15wt and E15 linker (as set forth in SED ID NO: 114) (B) pSTAT5 activity on PD-1+ and PD-1- CD122 transduced U937 cells using coculture assay (ratio 1:1 of U937 PD-1-/PD-1+) PD-1+ CD122+ cells were stained with CellTrackerGreen dye and cocultivated with PD-1+ CD122+ cells. (• Grey) anti-PDl-sushi-(G4S)3-IL15wt (RLI) (• Black) anti-PDl- sushi-E15-IL15wt-E15 at escalating doses for 30min at 37°C. Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry after intracellular staining (anti pSTAT5 AF647) in each PD-1 + and PD-1- cell population (confirmed on n=2). CIS activity on PD-lneg cells vs PD-1+ cells was evaluated using EC50 ratio in each cell type and reported on Histogram for each construction. Figure 21. Optimized linker allowed a masking effect of IL-10 on naive PD-1 low T cells while maintaining high CIS activity on stimulated PD-1+ T cells (A) PD-1 expression on human T cells after 2 stimulations using CD3/CD28. Expression was evaluated by flow cytometry (B) pSTAT3 activity measurement of naive human T cells using flow cytometry after stimulation with the bivalent Anti PD- l/IL-10 constructed with (G4S)3 linker or E15 linker. pSTAT3 % (left graph) and MFI (right graph) was quantified after permeabilization and intranuclear staining into CD3 T cell population. (C) Human PBMCs were stimulated in vitro using CD3/CD28 coating antibody to induce expression of PD-1 at the surface of T cells. Then pSTAT3 was quantified in this population after treatment with the 2 constructions anti PD-l/IL-10. Data are reported in % pSTAT3 + (Left graph) or MFI (right graph)
Figure 22. Optimized linker (E15) induced IL18 signaling only on targeted cells. NKL cell line expressing +/- PD-1 were incubated with IL-12 (lOng/mL) and escalating doses of IL18, anti PD-1-(G4S)3-IL18 or anti PD-1-E15-IL18 for 24hours at 37°C (IL18 variant being as set forth in SEQ ID NO: 56). Cells supernatant were harvested and I FNy was quantified by ELISA. I FNy secretion were normalized to I L18 (higher dose =100% of activation). N=2 independent experiments.
Figure 23. Multifunctional molecule activity constructed with an Optimized linker (E15-E15), an antimouse PD-1 and IL15. Mouse T cells were isolated from C57BL6 mouse spleen and were +/- stimulated overnight with anti-CD3 agonist antibody to overexpress PD-1. (A) Mouse PD-1 expression were quantified by flow cytometry for each T cell population (activated vs non activated). Mouse T cells expressing +/- PD-1- were incubated at escalating doses for 15min at 37°C with bivalent anti msPD-1- E15-hlL15-E15; (B) or bivalent anti msPD-l-E15-IL2-E15; (C) or monovalent anti msPD-l-E15-hlL15-E15; (D) and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each cell population.
Figure 24. Optimized linker (E15-E15) allowed a masking effect of IL15 cytokine even fused to another antibody backbone as an anti-CD3 scFv on CD3negative mouse cells while inducing a specific cis-potentiation on CD3+ cells. Mouse splenocytes were isolated from C57BL6 mouse spleen and were +/- presaturated with anti-msCD3 antibody (50pg/ml) to have no access to CD3 (representing CD3neg cells). Mouse T cells expressing +/- CD3+ were incubated with a high dose (300nM) of human IL-15, anti msCD3-(G4S)3-hlL15 or anti msCD3-E15-hlL15-E15 and % pSTAT5 activation was quantified by flow cytometry after intracellular/lntranuclear staining.
Figure 25. Optimized linker (E15-E15) was also functional on monocytes cell line. U937 monocyte cell line expressing CD122 and +/- PD-1 were incubated at escalating doses for 15min at 37°C with anti PD- l-(G4S)3-IL2v or anti PD-l-E15-IL2v-E15 and Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry for each cell population. The right graph represent MFI of pSTAT5 activation at the highest dose tested (lOnM). IL2v sequence is as set forth in SEQ ID NO: 110. Ill
Figure 26. Optimized linker (E15-E15) maintained its integrity and activity in vivo. (A) hPDIKI mice were injected intravenously with 5mg/kg of Anti-PD-l*2-E15-IL2v-E15 (SEQ. ID No: 110 - IL2v). Sera were sampled at different time point and pharmacokinetics were studied and concentration dosed using 2 ELISA with a anti human Kappa antibody and revelation with an anti-hlgG or an anti-hl L2 to evaluate quantity of antibody only or the full intact molecule comprising IL-2 cytokine. (B) MC38 bearing hPDIKI mice were injected intraperitoneal with 4mg/kg of Anti-PD-l*l-(G4S)3-IL15 or Anti- PD-1*1-E15-IL15-E15. Organs were sampled 24hours post injection. Antibodies were dosed in organs after digestion by anti-human kappa / anti-hlL15 sandwich ELISA. (C) hPDIKI mice were injected intravenously with lOmg/kg of Anti-PD-1*2-(G4S)3-IL15 or Anti-PD-1*2-E15-IL15-E15 and sera were sampled 24hours post-injection. Sera containing antibodies were diluted at 1/8 in medium and used to stimulated U937 monocyte cell line expressing CD122 +/- PD-1. pSTAT5 signaling on U937 were studied by flow cytometry after 15min of stimulation and intracellular staining with an anti pSTAT5- A647 and revealed by flow cytometry.
Figure 27: Immunogenicity of E15-E15 or D15-D15 optimized linkers are similar to conventional (G4S)3 linker. (A) Immunogenicity score in silico prediction (IEDB tools) as measured on binding properties of the sequence IL-15 +linker on MHC class II (B) hPDIKI mice were injected intravenously with 5mg/kg of bivalent anti-PD-l*2-(G4S)3-IL15wt , anti-PD-l*2-D15-IL15wt-D15 or anti-PD-l*2-E15- IL15wt-E15. Sera were sampled before injection and Dll post injection. ADA (anti-drugs antibodies) in sera were quantified by ELISA with drugs coating / anti-mouse IgG revelation (n=2 mice)
Figure 28. Multifunctional immunocytokine anti PD-1/IL2 constructed with the optimized linker showed better IL-2 signaling on PD1+ T cells than other prior art masking strategy anti PD-l-IL-2ra- IL2. CD122 and PD-1+/-- were stimulated with anti PD-l-IL2ra-IL2wt cytokine (right graph) or the anti PD-1 E15-IL2wt-E15 construction (left graph). Cis activity was calculated with fold EC50 difference between PD-1+ (■ Black) and PD-1 negative (• grey ) cells. (B) To activate T cells, hPBMCs were isolated from buffy coat and T cells were stimulated twice with agonist anti-CD3 or anti-CD28 to express PD1 (85% of expression). Cells were incubated at escalating doses for 15min at 37°C with anti PD-1 I L-2ra- IL2wt (■) or Anti PD-1 -E15 IL2wt-E15 (o) and Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry.
Figure 29: Optimized linker (E15-E15) is functional with an anti-TIGIT antagonist antibody Jurkat expressing CD122 and +/- TIG IT were incubated at escalating doses for 15min at 37°C with anti-TIGIT- G4S3-IL2wt molecules (■) or anti TIGIT-E15-IL2wt-E15 molecules (•). Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry for each Jurkat T cell population TIGIT+ (plain line) orTIGIT negative (Dashed line). Upper graph: dose response curve; Lower graph: CIS activity evaluation on TIG IT + versus TIG IT negative cells was calculated with Ratio EC50 (nM) activation of TIGIT negative cells/ TIGIT+ cells using GraphPad Prism. EXAMPLES
The present invention describes the design and use of a novel specific linker between the targeting moiety (e.g. anti PD-1 antibody) and the fused protein (i.e., a cytokine) (Figure 1). The specific linker allows an inactivation/masking of the fused protein on non-specific naive T cells (PD-1 negative) and induces a specific cis-demasking and cis-activation after targeting/binding the right T cells with antibody moiety (e.g. targeting PD-1 + tumor specific T cells) as schematized on Figure 1A. Compared to the prior art, the present invention demonstrates superior efficacy than conventional flexible linker (G4S)3 or rigid linker (EAAAK)3 used for the construction of bifunctional molecule comprising immunocytokine and does not require metalloprotease cleavage for demasking a protein nor the use of mutated cytokine to decrease affinity of the cytokine. Mutated cytokines may still be used to fine tune affinity of the cytokine, but is not mandatory. The inventors designed multiple bifunctional molecules comprising the optimized masking linker with a targeting moiety (i.e., antigen binding domain, such as monovalent, bivalent, scFv, VHH) and a cytokine (Figure IB). Multiple molecules can be targeted with targeting moiety without altering efficacy of the present invention.
In particular, the monovalent anti PD-l-X15-protein-X15, used in the examples, is a multifunctional molecule comprising a cytokine fused to the C-terminal domain of an anti PD-1 antibody with one linker between the Fc domain of the antibody and the cytokine, and one linker after the cytokine in C- terminal. The two linkers X15/X15 were optimized linkers and were able to mask the molecule on PD1 negative cells. All constructions were engineered with an IgGl N297A isotype and amino acid sequences were mutated in the Fc portion to create a knob on the CH2 and CH3 of the Heavy chains A and a hole on the CH2 and CH3 of the Heavy chains B. Conventional linker was also tested in a construction with monovalent anti PD-l-(G4S)3-protein where the cytokine was fused to the C terminal domain of an anti PD-1 antibody with a GGGGSGGGGSGGGGS (SEQ ID NO: 106) linker between the Fc domain and the cytokine. In particular, X15 is selected from the group consisting of SEQ ID NO : 32, 37, 41,42,14,43,44,20,45,17,58,59 and 46.
More particularly, the monovalent anti PD-l-E15-protein-E15, used in the examples, is a multifunctional molecule comprising a cytokine fused to the C-terminal domain of an anti PD-1 antibody with one linker between the Fc domain of the antibody and the cytokine, and one linker after the cytokine in C-terminal. The two linkers E15/E15 (EEEEEEEEEEEEEEE, SEQ ID NO: 5) were optimized linkers and were able to mask the molecule on PD1 negative cells.
In some examples, a bivalent anti-PD-1 X15-protein-X15 was also tested. The isotype-E15-IL15-E15 corresponds to a bifunctional molecule that comprise an antibody that does not target/bind PD-1. Particularly, the VH and VL sequences of the anti-PD-1 antibody used in Figures 2 to 9 are as set forth in SEQ ID NO: 74 and 75, respectively.
Particularly, in Figures 2 to 13, the VH and VL sequences of the anti-PD-1 antibody used can be as set forth in SEQ ID NOs: 74 and 75, respectively. When only one heavy chain is linked to a cytokine, SEQ ID NO: 86 for anti-PD-1 VH with a knob IgGl Fc and N297A substitution and SEQ ID NO: 88 for anti-PD- 1 VH with a hole IgGl Fc and N297A substitution can also be used. Preferably, the cytokine is bound to the hole chain. When a monovalent anti-hPD-1 is used, SEQ ID NO: 88 for anti-PD-1 VH with a hole IgGl Fc and N297A substitution and SEQ ID NO: 84 for the KIH Fc knob portion are used. Linkers and cytokine are fused to the C-terminal end of the anti-PD-1 VH with a hole IgGl Fc and N297A substitution. Anti-PD-1-1* or Mono-PDl refers to a monovalent construct whereas Anti-PD-1-2* or Biv-PDl refers to a bivalent construct. Light chain of anit-PD-1 is as set forth in SEQ ID NO: 79.
The anti-PD-1 antibody used in Figures 14 to 16 is the antibody 17D2 that is disclosed in US2019/0336496. The VH of 17D2 with a knob IgGl Fc and N297A substitution is disclosed in SEQ ID NO: 124. The VH of 17D2 with a hole IgGl Fc is disclosed in SEQ ID NO: 125.The light chain of 17D2 is disclosed in SEQ ID NO: 126. When a monovalent anti-msPD-1 is used, SEQ ID NO: 125 for anti-PD-1 VH with a hole IgGl Fc and N297A substitution and SEQ ID NO: 84 for the KIH Fc knob portion are used. Linkers and cytokine are fused to the C-terminal end of the anti-PD-1 VH with a hole IgGl Fc and N297A substitution.
Particularly, in Figure 29, the VH and VL sequences of the anti-TIGIT antibody used can be as set forth in SEQ ID NOs: 129 and 130, respectively. When only one heavy chain is linked to a cytokine, SEQ ID NO: 120 for anti-TIGIT VH with a knob IgGl Fc and N297A substitution and SEQ ID NO: 121 for anti- TIGIT VH with a hole IgGl F and N297A substitution can also be used. Preferably, the cytokine is bound to the hole chain. Anti-TIGIT-2* refers to a bivalent construct. Light chain of anti-TIGIT is as set forth in SEQ ID NO: 130.
I L15 sequence is as set forth in SEQ ID NO: 53. Sequence of I L15 variant is as set forth in SEQ ID NO: 112. IL2 sequence is as set forth in SEQ ID NO: 50. Sequence of IL2 variant is as set forth in SEQ ID NO: 110. The two subunits of I L12 are as set forth in SEQ ID NOs: 51 and 52. IL-15Ra sushi-(G4S)3-IL15 is as set forth in SEQ ID NO: 113 whereas IL-15Ra sushi-E15-IL15-E15 is as set forth in SEQ ID NO: 114. IL-10 is as set forth in SEQ ID NO: 128. 1 L18 sequence is as set forth in SEQ ID NO: 55. Sequence of I L18 variant is as set forth in SEQ ID NO: 56.
The term « optimized linker » and « masking linker » are used interchangeably in the Example section. The sequences of the masking linkers are particularly provided in Table 1, below.
Table 1. Peptide linkers
Example 1. Bifunctional molecule constructed with an optimized linker E15-E15 (Anti PD-l/E-15- IL15-E15, Anti PD-1/E-15-IL2-E15) allow a specific cis-demasking and cis-activation of PD-1 + expressing T cells while sparing PD-1- T cells. Optimized E15 linker show superior specific activity than the conventional flexible linker (G4S)3 commonly used in the immunocytokine prior art.
The inventors assessed the capacity of the optimized linkers to decrease activity of the cytokine on PD- 1- cells and allows a preferential cis-demasking and cis-activation of the cytokine on PD-1+ cells without inducing cleavage and with possibility to use wild-type or mutated cytokine.
Jurkat cells expressing only CD122+CD132+ (IL15RPy) (IL-15 complex signalling receptor), no PD-1 expression or co-expressing CD122+CD132+ (I LISRPy) and PD-1+ were incubated with I L15 wild-type cytokine, anti-PDl-E15-IL15wt-E15 (anti-PDl fused to I L15 with the E15 optimized linker), isotype-E15- IL15wt-E15 (isotype control fused to IL15 with the E15 optimized linker) and anti-PDl-(G4S)3-IL15wt (anti-PDl fused to I L15 with the conventional linker (G4S)3). Each Jurkat PD-1- 1 L15 RPy+ cells or Jurkat PD-1+ I L15RPy+ cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C. The IL-15R signalling (pSTAT5) was quantified by flow cytometry into each PD-1 + and PD-1- cell population.
Results : Figure 2A shows that the optimized linkers E15-E15 fused to Fc domain of the anti-PD-1 antibody allows to mask wild-type IL15 compared to IL15wt cytokine alone, which can induce high pStat5 activation into PD-1+ and PD-1 negative cells with similar efficacy. Isotype-E15-IL15-E15 or anti PD-1-E15-IL15-E15 has low to no effect on inducing pSTAT5 signalling into PD-1 negative cells while the construction induces surprisingly a strong activation of IL15R signalling pStat5 into PD-1+ cells, similar to IL15wt cytokine alone, whereas the isotype-E15-IL15-E15, without anti-PDl targeting, has similar efficacy in PD-1+ and PD-1- cells with low activation of both cells. This data demonstrates that the anti PD-1 domain of the anti-PD-l-E15-IL15wt-E15 molecule allows the targeting and cis- demasking of IL-15 on PD-1+ cells, and the cis-activation of IL15 signalling only on PD-1+ cells. Thus, the multifunctional molecule with the specific optimized linker E15-E15 added in N-terminal and C- terminal of I L15 and fused to an anti-PDl binding domain is able to mask the cytokine activity on PD-1 negative cells. The binding of the multifunctional molecule on PD-1+ cells restore the full activity of the cytokine in cis-manner by demasking the cytokine on PD-1+ cells.
In the prior art, immunocytokines or bifunctional antibodies are generally constructed with a conventional flexible linker GGGGS (more preferably (G4S)3) (for example Shen S, Sckisel G, Sahoo A, Lalani A, Otter DD, Pearson J, DeVoss J, Cheng J, Casey SC, Case R, Yang M, Low R, Daris M, Fan B, Agrawal NJ, Ali K. Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity. Front Immunol. 2020 May 8;11:832, or Xu, Yuanming et al. "An Engineered IL15 Cytokine Mutein Fused to an Anti-PDl Improves Intratumoral T-cell Function and Antitumor Immunity." Cancer immunology research vol. 9,10 (2021): 1141-1157) linked between the Fc domain and the fused protein. The inventors thus constructed with the same format the anti PD- l/IL-15 bifunctional molecule with a conventional linker (G4S)3 as control. Figure 2B shows that anti PD-1-(G4S)3-IL15 demonstrates some cis-activity with activation of PD-1+, however, an activation of PD-1 negative cells is also observed with this construction (EC50 near 1.8nM, see Table 2. here below) while the anti PD-1-E15-IL15-15 constructed with the optimal linker does not active PD-1- cells (EC50 >100nM).
Table 2. EC50 calculation of PSTAT5 activation into Jurkat IL15R+ cells and Jurkat IL15R+ PD-1+ cells
These data clearly demonstrate that the effect observed is due to the masking linker allowing to inactivate the cytokine activity on non-specific T cells. The effect of this masking linker is particularly surprising and will allow to develop a targeted immunocytokine without non-specific T cells activation and peripheral unwanted toxicity.
Example 2. The intrinsic properties of the optimized linker to mask the fused cytokine and cisdemask and cis-activate PD-1 targeted T cells are conserved across multiple cytokines.
As shown in the previous example, the inventors assessed the capacity of the optimized linkers to decrease activity of the fused cytokine on PD-1- cells and allows a preferential cis-demasking and cis- activation of the cytokine to PD-1+ cells. Multifunctional molecules comprising optimized linkers has been tested with cytokines, namely an IL15 wild type cytokine, an IL-2 wild-type cytokine and an IL-12 wild type cytokine. On Figure 3, Jurkat expressing CD122+CD132+ (IL-2 complex receptor) only (no PD- 1 expression) (A) or co-expressing CD122+CD132+ and PD-1+ (B) were incubated with IL2 wild-type cytokine alone (naked recombinant cytokine), anti PD-1-(G4S)3-IL2 (anti-PDl binding domain fused to wild type IL2 with conventional linker) or anti-PDl-E15-IL2-E15 (anti-PDl binding domain fused to IL2 with E15 optimized linkers). Jurkat cell PD-1+ or PD-1- population was respectively incubated with the different constructions at escalating doses. The pStat5 signalling for each cell lines was quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction and regrouped in Table 2 for each PD-1+ and PD-1- cell population.
Results : Figure 3 on PD-1- cells (A) shows that the optimized linkers fused to cytokine (anti-PDl-E15- protein-E15) masks the cytokine activity in decreasing pStat5 activity of wild-type IL2 anti PD-1 fused bifunctional antibody in similar manner as IL-15 cytokine. With all cytokines tested, the optimized linker outperforms the conventional (G4S)3 linker by decreasing to higher extend cytokine activity on PD-1 negative cells, as tested with construct named anti-PDl-(G4S)3-IL2. Figure 3 on PD-1+ cells (B) shows that the E15 optimized linker (anti-PDl-E15-IL2-E15) conserved its property to demask IL-2 cytokine on PD-1+ expressing cells allowing high signalling activation (EC50 in Table 3).
Table 3. EC50 (nM) of the pSTAT5 activation
Surprisingly, signalling induced with anti PD-1-E15-IL2-E15 is highly superior to a wild-type non targeted cytokine (recombinant IL-2) on PD-1+ cells suggesting that the linker improved activity properties on PD-1 + T cells. This data shows that on PD-1+ cells, anti-PDl-E15-(cytokine)-E15 multifunctional molecule was able to demask the cytokine and induce a cis-activation of wild-type IL15 and wild-type IL2 respectively, after binding on PD-1+ cells, and better than the non-targeted cytokine. Optimized linker intrinsic property to mask the cytokine on PD-1- cells (decrease activity), and to cisdemask cytokine and restore cis-activation on PD-1+ cells was confirmed for wild-type IL2 in addition to IL-15, suggesting that the optimized linker disclosed herein can be fused to various cytokines for a similar efficacy. Example 3. Optimized linkers in multifunctional molecules with wild-type IL12 allow to mask cytokine activity on PD-l-cells, cis-demasking and cis-potentiation of PD-1+ cells lines, as well as enhancing safety as evaluated in vivo in toxicity mouse model.
In addition to their experiment on IL2 and IL15, the inventors tested their optimized linker on heterodimeric cytokine. Anti PD-1 monovalent antibody was fused to E15-IL-12 p40 -E15-IL12 p35-E15 or to E15-IL12p40-(G4S)3-E15 as schematized on Figure 4A.
Naive splenocytes PD-1- IL12R+ extracted from wild-type mouse (A) or activated splenocytes extracted from human PD1-KI mouse and activated CD3/CD28 to obtain activated PD-1+ IL12R+ cells (B) were incubated with mslL12 wild-type cytokine, with anti PD-1-(G4S)3- mslL12p40-(G4S)3-mslL12p35, anti- PDl-E15-mslL12p40-(G4S)3-mslL12p35-E15 (vl) or anti-PDl-E15-IL12p40-E15-IL12p35-E15. Each PD1- naives and PD1+ activated cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C and Median Fluorescent Intensity (MFI) of pStat4 was quantified by flow cytometry for each cell population.
Results: As shown in Figure 4B&C, a molecule comprising the pair of optimized linkers flanking the cytokine as well as an optimized linker between the subunits of the cytokine was able to mask and demask the cytokine, as evidenced by the shift of pSTAT4 activation between the PD1- and PD1+ cells. Said shift was confirmed by calculating EC50 (Figure 4C), which highlighted a 100-fold change higher cis-activity between the molecule comprising three optimized linkers and the naked cytokine, while the conventional linker was able to only induce a 20-fold change difference demonstrating less selectivity.
These data further demonstrate that intrinsic properties of the optimized linker are applicable for various cytokines including dimeric cytokines.
Example 4. Anti PD-l-Cytokine bifunctional molecule comprising the optimized linker E15/E15 induce specific cis-demasking and cis-activation of human primary activated exhausted T cells while sparing naive PD-1 negative T cells.
Following tumor antigen activation, PD-1 receptor is induced at the surface of T cells. Chronic stimulation of T cells by tumor antigen induces exhaustion of the cells characterized by a loss of effector functions and anti-tumor activity and increased T cell death. Tumor specific exhausted T cells can be reinvigorated through activation of a costimulatory signal (e.g. cytokine) into T cells and blockage of PD-1 inhibitory receptor. Anti-PD-1 bifunctional molecule constructed with an immunocytokine aimed to target PD-1+ tumor specific T cells and cis-deliver the cytokine to fully reactivate exhausted T cells. To evaluate the efficacy of the multifunctional molecule of the invention to reactivate exhausted human primary T cells, pSTAT5 signalling following treatment was assessed into activated /exhausted T cells (PD-1+) and compared to the activation of naive T cells (PD-1-).
The inventors assessed the capacity of the optimized linkers to decrease activity of the fused cytokine on naive PD-1- T cells and allows a preferential cis-demasking of the cytokine and cis-activation on activated T cells expressing PD-1. The aim is to reduce toxicity of the cytokine in periphery of the tumor where naive T cells are present and increase cytokine activity to re-activate specifically PD-1+ tumor specific activated/exhausted T cell. Experiments were performed with a wild-type human IL15 or IL2 cytokine.
For this experiment, naive T cells from PBMCs of peripheral whole blood of human healthy volunteers were isolated by Ficoll and incubated with the different constructions at escalating doses. pStat5 signalling was evaluated by flow cytometry into CD3+ T cell population. Activated/exhausted T cells were generated by stimulating naive PBMCs two times with an anti-CD3/anti-CD28 antibody to mimic chronic antigen stimulation occurring into the tumor microenvironment and T cell exhaustion. After stimulation, T cells were incubated with the different constructions added at different concentrations. Naive T cells express low level of PD-1 (10-20%), whereas activated T cells (after 2 stimulations) expressed high expression of PD-1 (~80%) n =3 donors used for the experiment (Figure 5A). Anti-PDl- E15-IL15wt-E15 activity was tested on naive and activated T cells, and EC50 was compared to nontargeted wild type IL15 recombinant cytokine or to anti-PDl-(G4S)3-IL15wt constructed with the conventional linker (Figure 5B). Same experiment was performed with the anti-PDl-E15-IL2wt-E15 construction and pStat5/3 was analyzed into T cell population (Figure 5C).
Results: Figure 5B shows the anti-PDl-E15-IL15wt-E15 activity on naive T cells (PD-1-) compared to activated T cells (PD-1+). In a multifunctional molecule, the optimized linker fused to IL15 wild type cytokine inactivate the cytokine pSTAT5 activity on naive T cells (EC50 > 50nM) whereas non targeted recombinant IL15wt cytokine efficiently activates naive T cells (EC50 close to 0,lnM), the anti-PDl fused to IL15wt with conventional linker has an EC50 equal to 0.3nM. Thus, optimized linker is able to decrease cytokine binding more than 500-fold on naive T cells compared to wild type non targeted cytokine. On activated T cells expressing PD-1+, anti-PDl-E15-IL15wt-E15 molecule binds activated T cells on PD-1 receptor and is able to cis-demask IL15 and promote efficient IL15 signalling to similar extend than recombinant IL-15wt non targeted cytokine. An EC50 close to 0.1 nM is obtained with anti-PDl-E15-IL15wt-E15, similar to recombinant IL15. Binding of anti PDl-E15-IL15wt-E15 to PD1 on activated T cells can promote conformational change of the cytokine only in cis-manner. More than 500-fold increased activity in PD-1 activated T cells versus naive T cells is observed with the optimized linker, demonstrating the advantage of the construction to reduce toxicity and cytokine activity on periphery cells, compared to activated T cells localized in the tumor microenvironment. Similar data were obtained with an anti PD-1 bifunctional antibody constructed with an IL-2wt cytokine. pStat3/5 cis activation was reproduced with IL2 fused to anti-PDl with the optimized linker (Figure 5C) on naive and activated T cells. Very low activation of naive T cells is observed with the anti-PDl-E15-IL2wt-E15 construction, whereas on activated T cells PD-1+, cis-activation was highly efficient by binding to PD-1 and increased more than 5000-fold potency of the drug into activated /exhausted T cells (EC50 close to 0.02nM with anti-PDl-E15-IL2wt-E15). Thus, the cis demasking and cis activation properties are confirmed in human primary T cells. The construction comprising the optimized linker induce specific activation of activated PD-1+ exhausted T cells while sparing naive peripheral PD-l- T cells by binding with PD-1.
Additionally, the inventors also evaluated the efficacy of anti-PD-l-E15-IL15wt-E15 to reinvigorate and cis-activate exhausted T cell infiltrating the tumor. Human kidney tumor explant was dissociated, and T cells were isolated +/- stimulation with the anti PD-1 E15-IL15wt-E15 or non-targeted isotype E15- IL15wt-E15 and proliferation (Ki67 expression) was analyzed after 6 days of culture. Figure 5D shows that the multifunctional molecule comprising anti-PD-l-E15-IL15wt-E15 efficiently promotes proliferation of exhausted T cells infiltrating the tumor to higher extend than Isotype E15-IL15-E15, demonstrating that the optimized linker allows to cis-activate T cells upon PD-1 targeting.
Example 5. Optimized linkers induce a strict cis-activity of bifunctional anti PD-1 antibody on PD-1+ T cells, no trans-activation signalling was induced on PD-1 negative cells.
Following the binding of the anti-PD-1 moiety of the bifunctional molecule, the fused cytokine can be presented to all surrounding T cells that can be tumor specific (PD-1+) or non-tumor specific T cells, thereby increasing unwanted toxicity (see Vincent, Marie et al. "Antitumor activity of an immunocytokine composed of an anti-GD2 antibody and the IL-15 superagonist RLI." Oncoimmunology vol. 2,11 (2013): e26441). In the design of the optimized linker, the inventors assessed the capacity of the optimized linkers to strictly induce cis-activation of PD-1 + T cells, without inducing trans-activation of non PD-1 expressing cells via presentation by another PD-1+ T cells. To evaluate this effect, two different methods were used to trans-present IL-15 cytokine to PD-1 negative either by (A) coating PD-1 recombinant protein to mimic cell surface presentation or by (B) coculturing PD-1+ cells and PD-1 negative cells. (A) pStat5 signalling into Jurkat PD-1- expressing IL15RPy+ (CD122+CD132+) was analyzed after anti-PDl-E15-IL15wt-E15 pre-incubation on recombinant PD1 coated plate (B) pSTAT5 signalling was analyzed into Jurkat PD-1- expressing IL15RPy+ (CD122+CD132+) after coculture with U937 PD1+ cells ( I LISRPy negative) cells. In addition, another coculture assay was performed by mixing Jurkat T cells PD-1- CD122+CD132+ stained with cell dye (Cell Tracker Green, Thermofisher) and Jurkat PD-1+ CD122+CD132+ cells (ratio 1:1). Mixed cells were then treated with the bifunctional anti PD1-E15-IL15-E15 for 30 min to analyze pStat5 signalling of each population PD-1- and PD-1+ cells.
Results: Figure 6A represents pStat5 signalling into Jurkat PDl-IL15RPy+ with anti-PDl-E15-IL15wt-E15 molecule pre-incubated or not on PD1 coating. No higher pStat5 signal into Jurkat PDl-IL15Rbg+ was observed whether the molecule binds to PD1 coated plate compared to without coating. These data demonstrate that there is no trans-activation. Same results were obtained on Figure 6B with anti-PDl- E15-IL15wt-E15 pre-incubated or not on the surface of U937 PD-1+ (IL15RPy-) on pStat5 signalling of Jurkat PD-1- CD122+CD132+, which was similar between the conditions with or without PD-1+ presentation by cells, whereas, a strong signal was observed on Jurkat PD-l+IL15RPy+ cells. Figure 6C- D shows pStat5 signalling of each population of PD-1- or PD-1+ cells obtained during co-culture assay with anti-PDl-E15-IL15wt-E15 compared to non-targeted IL15 recombinant cytokine. Anti-PD1-E15- IL15wt-E15 multifunctional molecule activates PD-1+ cells while having a very low effect PD-1- cells, even in a co-culture model. In addition, pSTAT5 EC50 (nM) is equivalent on PD-1- cells whether the cells are alone or co-cultivated with PD-1+ cells, demonstrating in another assay that no trans- activation of anti-PDl-E15-IL15wt-E15 is induced. The same is true for PD-1+ cells. Thus, a preferential cis-activation on PD-1+ cells, and no-transactivation due to PD1 targeting on two different cell types with the multifunctional molecule comprising a E15/E15 optimized linker is observed. The potentiation of IL15 signalling (cis-demasking and cis-activation) was strictly dependent on PD-1 binding on the same cells, thus, activation of IL15 pathway was restricted to PD-1+ cells present on the same cell.
To optimize linker properties, numerous linkers were designed and tested by the inventors to specifically induce inactivation of cytokine to PD-1 negative cells while allowing cis-demasking and activation of the molecule on PD-1+ T cells. Different linkers tested were modified on the amino acid composition (type of amino-acid in the sequence of the linker or percentage of the same amino acid in the sequence of the linker), length and/or positioning (N-terminus and/or C-terminus of the cytokine) of the optimized linker.
Example 6. Design of the E15 Linker 1 +/- E15 Linker 2 to allow superior efficacy to mask fused cytokine: one or two E15 optimized linker fused in N-terminal or C-terminal of the cytokine mask cytokine activity to PD-1- cells to higher extend than conventional linker while efficiently allowing activation of PD-1+ cells
First, the inventors have tested whether one or 2 linkers fused in N-terminus or C-terminus of the IL15wt cytokine is sufficient to decrease and mask cytokine activity. Additional bifunctional molecules have been generated comprising (1) optimized linker E15 between Fc domain and cytokine IL15wt, named anti-PDl-E15-IL15*; (2) a molecule with one optimized linker E15 in C-terminal of the cytokine I L15, where the IL15wt cytokine is directly fused (without linker) to the Fc domain and the E15 linker is fused after the cytokine, named anti-PDl-nolink-IL15wt-E15 and compared to the anti PD-1 fused to two linkers before and after the IL15wt cytokine, named anti-PDl-E15-IL15wt-E15, and a bifunctional molecule comprising a linker of the prior art, namely an anti-PDl-(G4S)3-IL15wt, in which (G4S)3 is the most common linker used for immunocytokine construction due to its flexible properties (on Figure 7 A-B). Additionally, the inventors evaluated the advantage of the optimized linker E15 versus another linker of the prior art, (EAAAK)3 (SEQ. ID NO 57)having a rigid structure (for example such as disclosed in Chen et al., 2013 PMID: 23026637).
For this assay, Jurkat expressing CD122+CD132+ (IL15R receptor) only no PD-1 expression (A) or coexpressing CD122+CD132+ and PD-1+ were incubated with the different constructs schematized on the top of the figure. pSTAT5 activation was determined by cytometry into PD-1- versus PD-1+ cells.
Results: Figure 7A on PD-1- cells shows that construction with only one E15 linker in C-terminal of the cytokine or between the Fc domain and the cytokine is sufficient to decrease pStat5 activity of the cytokine IL15, while the conventional linker (G4S)3 is more potent to activate the PD-1 negative cells (Figure 7A). Either one E15 linker, regardless of its position, or two E15 linkers (a first E15 linker in N- terminal of the cytokine and a second linker in C-terminal of the cytokine) are effective to mask cytokine and inactivate the cytokine (Figure 7B). To verify that the properties of the E15 linker is specific and not related to the fusion of two linkers on each extremity of the cytokine, the inventors constructed the Anti PD-1 immunocytokine with two (G4S)3 linker (a first (G4S)3 linker in N-terminal of the cytokine and a second (G4S)3 linker in C-terminal of the cytokine, (G4S)3-IL15-(G4S)3). Figure 7C shows that 2 (G4S)3 linkers are not sufficient to mask the IL-15 activity on PD-1 negative cells while the optimized linker efficiently mask IL-15 activity on PD-1 negative cells. In addition, the same construction was designed with another (EAAAK)3 linkers fused at the N and C-terminal domain of the protein. (EAAAK)3 is a well-described linker with rigid properties. Figure 7C shows that two rigid linkers are neither able to mask IL-15 cytokine activity on PD-1 negative cells. Although the flexible (G4S)3 and rigid (EAAAK)3 linkers have the same length in amino acid than E15 optimized linker, those linkers are not able to attenuate activity of the cytokine on PD-1 negative cells. Altogether, these data demonstrate that the optimized linker has new and surprising properties compared to conventional prior art linkers used for bifunctional molecules.
Example 7. Other amino acids can be used to construct optimized linkers
To assess whether the optimized construction is limited to a single amino acid, the inventors constructed linkers with other amino acids and tested their capacity to mask and cis-demask cytokines on PD1-/+ cells. Two linkers were constructed solely with Asp (D) or Asn (N), and compared to the linker E15.
Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD- 1+CD122+CD132+ (IL15Rbg) (B) were incubated with IgG isotype-E15-IL15wt-E15 (white dot), anti- PDl-E15-IL15wt-E15 (grey diamond), anti-PDl-D15-IL15wt-D15 (triangle) or anti-PDl-N15-IL15wt-N15 (cross).
Results: As shown on Figure 8A, bifunctional molecules constructed with two linkers, either E15, D15 or N15, could mask the cytokine on PD1 negative cells, as evidenced by the lack of pSTAT5 signalling, while demasking on PD1 positive cells to a similar extend as E15/E15 linker (Figure 8B). The EC50 calculated for each cell line and for each optimized linkers shows a tenfold increase between PD1+ and PD1- cells for each type of linker, confirming that the cytokine is masked on PD1- cells and demasked on PD1+ cells. As positive control of activation, recombinant IL-15wt was used while isotype E15-IL15- E15 was used as negative control (i.e., without PD-1 targeting). These data demonstrate the cis- targeting/demasking activity of the 3 different optimized linker comprising (E), (D), or (N) amino acids. Linkers constructed with Glu (E) and Asp (D), both negatively charged amino acids, are capable of masking and cis-demasking on PD1-/+ cells (respectively), as well as Asn (N) which is an amino acid residue with a polar uncharged side chain.
To confirm these results, the inventors repeated their experiments on cells sorted from human PBMC, either on naive cells (35% PD1+ cells), cells stimulated with PHA (75% PD1+ cells) or cells stimulated twice with CD3/CD28 (2 ug/mL each) (100% PD1+ cells) (Figure 8D). As evidenced by said Figures, linkers constructed with Glu (E), Asp (D) or Asn (N) were capable of masking the cytokine from PD1- cells while cis-demasking on PD1+ cells with similar effect. It is particularly interesting to mention that the greater the number of cells were expressing PD1, the greater the pSTAT5 was, suggesting the evidenced masking/demasking effect was correlated to PD1 expression on targeted cells. linkers can be constructed from a mix of amino acids without impact on their
Next, the inventors investigated the necessity to have optimized linkers constructed solely of the same amino acid residue, and whether a mix between different group of amino acid has an effect on the masking and demasking ability.
Jurkat cells expressing PD1+ CD122+ or expressing PD1+ CD122+ but blocked with lmg/mL of an anti- PD1 were incubated with IL15 alone, anti-PD-1 (G4S)3-IL15, or anti-PDl X15-IL15-X15, where X is a combination of different proportion of amino acid Glu (E) and Asp (D) (Figure 9A), Glu and Thr (T) (Figure 9B), Glu and Ala (A) (Figure 9C) or Glu and Lys (K) (Figure 9D). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.
Results: On Figure 9A, linkers constructed with only E residue, or with a mix of negatively charged amino acid (E and D residue) were efficient in masking the cytokine on PD1- cell lines and demasking the cytokine on PDl+ cell lines. The same conclusion is reached in Figure 9B with a mix of the negatively charged amino acid E and an uncharged amino acid, here T, as well as with hydrophobic amino acid (A), as evidenced by Figure 9C. A linker constructed with a combination of negatively charged amino acid (E) and positively charged amino acid (K) was however not as capable of masking as the other optimized linkers if the number of positively charged amino acid is predominant, as shown in Figure 9D. In Figure 9D, the optimized linkers constructed with about half or 80% of positively charged amino acid could not mask the cytokine, resulting in an unspecific activation of the IL15 receptor. When comprising positively charged amino acid, only a linker comprising more than 60% of negatively charged amino acid retained its masking/demasking ability (e.g., E12K3-IL15 E1K3 linker as shown in Figure 9D).
In conclusion, the inventors have proven that a linker constructed with a minimum portion of either negatively charged residue or amino acid residue with a polar uncharged side chain were capable of masking and demasking their cytokine, as long as the linker does not contain more than a maximum amount of positively charged amino acid.
Example 9. An optimized linker of 6 to 25 amino acid length is capable of fully masking cytokine on PD-1- cells and cis-demasking on PD-1+ cells
Different length of the optimized linker was also tested to evaluate whether a specific size of the linker E is efficient for its activity. New constructions with different length of linker, composed of 6, 10, 15, 20 or 25 amino-acids Glu (E) amino acid. With both linker 1 and linker 2 having the same length (a first linker between the Fc domain and the cytokine and a second linker in C-terminal of the cytokine), except for the 25 aa long linker which was coupled with a 20 aa long linker. All constructs tested in this assay comprise a monovalent anti-PDl binding domain and a wild-type IL15 cytokine. In Figure 10, Jurkat cells expressing CD122+ and not PD-1 (A) or co expressing CD122+PD-1+ cells (B) were incubated with the different constructions. pSTAT5 activation was assessed for each construction by flow cytometry on PD-1- or PD-1+ cells.
Results: Figure 10 shows that the length of the linker does not impact the property of the linker to mask the cytokine on PD-1 negative cells. All linkers demonstrated superior efficacy to inactivate/mask the cytokine compared to conventional ((G4S)3 linker) which are not sufficient to decrease cytokine activity. 10. Bifunctional antibody constructed with optimized linker efficiently bind to PD-1 on activated T cells
The inventors have verified that optimized linkers that are able to mask cytokine activation/binding does not modify the overall structure of the antibody and has no effect on the binding capacity anti- PD1 paratope of the antibody. Indeed, it is believed that the cis-demasking properties of the multifunctional molecules of the invention rely on the binding of the anti-PDl binding domain to PD- 1+ receptor, so that the paratope must be preserved in the construction for an optimal efficacy. The binding to PD-1 was assessed by ELISA assay by coating PD-1 recombinant protein, binding of the antibody was revealed by a peroxidase-labelled anti-human Fc polyclonal antibody. Signal was quantified by Optical Density (OD) measured by a spectrophotometer (TECAN system).
Results: Figure 11 shows that anti-PDl-E15-IL15wt-E15 molecule efficiently binds to PD-1 receptor to a similar manner than the anti-PDl-(G4S)3-IL15wt molecule comprising the conventional linker, demonstrating that the present invention with the optimized linker does not interfere in the binding of the anti PD-1 antibody paratope, and preserve the integrity of the targeting moiety of the multifunctional molecule while only masking the activity of the fused cytokine.
Example 11. Optimized linkers are capable of masking and demasking cytokine with one or multiple valences of anti PD-1.
To further investigate the limits of the optimized linkers, the inventors set to assess whether the number of valence of the antigen binding domain has an influence on the masking and demasking properties of the optimized linkers.
Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye (dotted lines) and Jurkat PD-1+ IL15Rbg+ cells (solid lines) were incubated with a monovalent anti-PDl-E15- IL15-E15 (triangle), a bivalent anti-PDl-E15-IL15-E15 (diamond) or an isotype-E15-IL15-E15 (circle). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was determined for each PD-1 and PD-1+ cells.
Results: Figure 12A illustrates that the pSTAT5 activation of the PD1 negative and PD1 positive cells are equivalent between the molecule constructed with a single valence of an anti-PDl binding domain (i.e., a multifunctional molecule comprising a single anti-PD-1 binding domain) and those constructed with a double valence of anti-PDl biding domains(i.e., a multifunctional molecule comprising two anti- PD-1 binding domains). EC50 calculated from this experiment confirms this observation, with a very high EC50 on PD1 negative cells and low EC50 on PD1 positive cells. Example 12. Anti PD-1 E15 IL2 E15 and anti PD-1 E15 IL15 E15 molecules enhanced safety as evaluated in vivo in toxicity mouse model while maintaining efficient anti-tumor efficacy in vivo.
Cytokine therapy development in clinic is limited by the severe toxicity induced through activation of peripheral non-specific immune cells. Although the fusion of an antibody to the cytokine allows to redirect activity of the cytokine on immune cells, off-target related toxicity is observed across multiple immunocytokines and constructions. High systemic toxicity is induced by cytokine, in particular for IL- 2, limiting dose injected into patient of conventional immunocytokine. Since optimized linker improves masking of cytokine, the inventors next assessed effect of anti PD-1/IL2 wild type constructed with optimized E15 linker versus conventional linker. Mouse weight was followed every day after one (A) or multiple injection (B) (every day) at 2mg/kg. Figure 13 (A and B) shows that Anti PD-1 (G4S)3 IL2 multifunctional molecule induces toxicity associated with loss of overall weight, whereas anti PD-1 E15 IL2 E15 did not demonstrated significant toxicity. This data confirms the advantage of the use of the optimize linker to reduce peripheral systemic toxicity.
The experiment was repeated with another wild type cytokine, IL15. Both monovalent and bivalent anti-PD-l/IL15 wild type multifunctional molecule constructed with optimized E15 linker were tested and compared to a bivalent anti-PDl-l/IL15 wild type multifunctional molecule constructed with a conventional linker. Figure 14A shows that a monovalent anti PD-1 E15-IL15wt-E15 did not demonstrated significant toxicity. In Figure 14B, 5 mice out of 7 died due to the toxicity of the bivalent Anti PD-1-(G4S)3-IL-15 injection. Surprisingly, Anti PD-1 IL-15 constructed with E15 linker (Anti PD-1 E15-IL15-E15) is not toxic even after multiple injections at high doses (4 injections, 5mg/kg). No weight loss or death was observed (Figure 14) similar to control group (PBS injection). Altogether these data confirm the advantage of the E15 linker versus conventional linker to reduce off-target toxicity in vivo by masking cytokine activity/binding on non-targeted PD-1 negative cells.
The inventors next evaluated in vivo anti-tumor efficacy of the Anti PD-1 E15-IL2wt-E15 or the Anti PD- 1 E15-IL15-E15 in several mouse models. For Colon ectopic CT26 model, BalbC mice were injected subcutaneously with CT26 tumor cells then Anti PD-1 E15- IL15wt-E15 (Bivalent anti PD-1) or PBS control was intraperitoneally injected on Day 5, 8, 12 and 15 at 5mg/kg and tumor growth was monitored using caliper. For melanoma ectopic B16 model, C57bl6 mice were injected subcutaneously with B16-OVA tumor cells then Anti PD-1 E15- IL2wt-E15 (monovalent anti PD-1) or PBS (control) was intraperitoneally injected on Day 10, 14, 18 and 23 at 2mg/kg and tumor growth was monitored using caliper. For Pancreatic orthotopic model, PanC02 cells were directly injected into the Pancreas and mice were treated on Day 7, 11, 15 and 19 with 2mg/kg of Anti PD-1 E15 IL2wt E15 construction. Tumor growth was quantified by Bioluminescence and survival monitored. 1 7
Figure 15 shows that anti PD1 E15-IL15wt-E15 molecule can eradicate CT26 ectopic tumor growth (50 % of complete response n=4/8 and 1 partial response observed) leading to significant increased survival compared to PBS treatment (Vehicule). Additionally, monovalent Anti PD-1 1 L2wild type form constructed with optimized linker E15 demonstrated a significant and good therapeutic response in immune checkpoint refractory PanCO2 model (decrease tumor load and improved survival (Figure 16A). In addition, in another experiment, mice treated with a bivalent anti PD-1 antibody fused to E15- IL2v-E15 at 0.5 and 1 mg/kg (3 doses) induced significant anti-tumor response in vivo in this PanCO2 model (Figure 16B). Altogether, these data demonstrate the advantage of the optimized linker to improve therapeutic index of immunocytokine by reducing the off-target toxicity while maintaining anti-tumor efficacy by demasking the cytokine on targeted PD-1+ T cells.
Example 13. The optimized linkers allow to improve therapeutic index of toxic immunocytokine by decreasing toxicity and maintaining high in vivo efficacy
In this experiment, a single mutated IL-2 (IL-2v) (SEQ ID NO: 110) was used as well as an anti PD-1 targeting human receptor (VH/VL of SEQ ID NO: 74 and 75, respectively), with two optimized E15 linkers (i.e., each of SEQ ID NO: 5). The bifunctional molecule tested thus comprises a bivalent anti- PD-1 antibody wherein the C-terminal of only one Fc chain is covalently linked to E15 linker- IL2v - E15 linker.
The bifunctional molecule constructed with the optimized linkers comprises: a first heavy chain of SEQ ID NO: 111 (i.e., comprising SEQ ID NO: 88-5-110-5), a first light chain of SEQ ID NO: 79 associated with the first heavy chain. a second heavy chain of SEQ ID NO: 86, and a second light chain of SEQ ID NO: 79 associated with the second heavy chain.
For this experiment, hPDIKI mice expressing human PD-1 receptor were inoculated subcutaneously with 0.5e6 MC38 cells. On day 10, tumor volume (mm3) was measured, and mice were randomized in the different groups to ensure a similar mean tumor volume across treatment groups. On Day 10, 12 and 14 mice were treated with Anti PD-l/IL-2v molecule constructed with optimized linker (anti-PDl- E15-IL-2v-E15). The immunocytokine IL-2v is mutated to attenuate binding to CD25 receptor. Treatment was performed intraperitoneally at 30, 10, 5 or 2 mg/kg. Tumor volume was measured at multiple time point as well as clinical score was assessed to evaluate potential toxic effect of the drug (lethal dose). As shown on Figure 17A, C, D and F anti-PD-1 bifunctional molecule constructed with an optimized linker and IL2 (anti-PDl-E15-IL2v-E15) does not promote severe toxicity (mouse death) at the high doses tested (i.e., 15, 20, 10, 5, 2, 1 and 0.5mg/kg). In addition, a high anti-tumor efficacy was observed at all doses with a complete tumor regression. Only at the highest dose of 30 and 50mg/kg tested that sign of severe toxicity was observed, at 30 mg/kg one mice out of 5 died due to toxicity and at the dose of 50 mg/kg, 3 out 4 mice died following the 3 injections of the anti PD-1 IL-2v constructed with optimized linker. In parallel, efficacy and toxicity of the molecule comprising an anti-PD-1 antibody linked to IL2v with a conventional linker (i.e., (G4S)3) was tested at the same doses in the same experiment for direct comparison (excepted for the doses 15, 20 and 30mg/kg that was not tested). Data with this molecule anti PD-l-(G4S)3-IL2v resulted in anti tumor efficacy response at 0.5mg/kg (4 complete response 1 partial response out of 10 animals treated; pool of 2 independent experiments); for the other higher doses at 2mg/kg and above high doses (5 mg/Kg and 10) mice died after treatment (excepted 2 mice out of 6 at the dose of 2mg/kg) (data not shown). This demonstrates the advantage of the optimized linker in masking activity of the cytokine on peripheral T cells and unwanted toxicity. Figure 17C, illustrates the survival of the mice after injection of the drugs with a significant reduction of toxicity with optimized linker demonstrating the advantage of the present invention to optimize the therapeutic index of the immunocytokines. Figure 17D, 17E and 17F show the Therapeutic index of the molecules at the doses tested, demonstrating once again the superiority of a multifunctional molecule comprising an optimized linker of the invention. Different methods to evaluate the therapeutic window were used for the calculation. The first method shown Figure 17C evaluates difference between the MTD (Maximum Tolerated Dose) and EC50 of therapeutic dose efficacy (TD50). This calculation were performed with dose (0.5, 2, 5, 10, 15 and 30mg/kg). In the second method therapeutic windows was calculated with EC50 Lethal dose and EC50 of therapeutic Dose by drawing a dose response curve of efficacy and toxicity (mouse death) with all experiments performed and all doses tested (0.5, 1, 2, 5, 10, 25, 20, 30, 50mg/kg). This second analysis is a better calculation with accurate quantification and frequently used for calculating the therapeutic window of a drug. For anti PD-l-(G4S)3-IL2v molecule, a narrow therapeutic index equal to 2 fold was calculated with Therapeutic dose TD50 equal to lmg/kg and Lethal Dose LD50 at 2mg/kg. For Anti PD-l-E15-IL2v- E15 molecules, a high therapeutic index ~ 52 fold was calculated with Therapeutic dose TD50 ~0.6mg/kg and lethal dose LD 50 ~31mg/kg. Thus, these data confirm the advantage of the optimized linker for multifunctional toxic cytokines with > 25 fold higher therapeutic index, which is associated with greater margin of safety for the drug and therapeutic clinical advantage for the patients suffering from cancer.
Example 14. The optimized linkers mask cytokine activity on PD-1- T cells even in the presence of PD-1+ cells.
To determine cis-activity of molecules in a co-culture assay, Jurkat cells transduced with CD122+CD132+ and PD-1 (PD-1+ cells) were mixed with Jurkat cells transduced with CD122+CD132+ only (PD-1- cells) stained with cell tracker dye (CellTracker Green, Thermofisher ref C7025), mixed at a ratio 1:1 and treated with different molecules (i.e., IL15 wild type (wt) alone, an anti-PDl-(G4S)3- IL15wt multifunctional molecule or an anti-PDl-E15-IL15wt-E15 multifunctional molecule) during 30 minutes at 37°C. Cells were then fixed, permeabilized and stained with an AF647 labelled anti-pSTAT5 (clone 47/Stat5(pY694), BD Bioscience ref 612599). pSTAT5 activation was evaluated into Jurkat PD1+ CD122+CD132+ cells and Jurkat PD1- CD122+CD132+ cells stained with CellTrackerGreen. The pStat5 signalling for each cell was quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction for each PD-1+ and PD-1- cell population. Cytomask linker is E15 and conventional linker is (G4S)3.
Figure 18 shows that the optimized linker E15-E15 fused to the Fc domain of an anti-PD-1 antibody allowed to mask wild-type IL15 on PD-1 negative cells compared to IL15wt cytokine alone, even if the construct was bound on PD1 positive cells and trans-presented by the PD-1 positive cells. On one hand, the anti PD-1-E15-IL15-E15 multifunctional molecule had no effect on inducing pSTAT5 signalling into PD-1 negative cells, and on the other hand, the anti PD-1-E15-IL15-E15 multifunctional molecule surprisingly induced a strong activation of IL15R signalling into PD-1+ cells, similar to IL15wt cytokine alone. These data demonstrated that the anti PD-1 domain of the anti-PD-l-E15-IL15wt-E15 molecule allowed the targeting and cis-demasking of IL-15 on PD-1+ cells, and the cis-activation of I L15 signalling only on PD-1+ cells. Thus, in a multifunctional molecule comprising an anti-PDl antibody, the presence of the optimized linked E15 in both N-terminal and C-terminal of I L15 was able to mask the cytokine activity on PD-1 negative cells, even if the product was trans-presented by PD-1 positive cells in coculture assay. This demonstrates the specificity of the multifunctional molecule of the invention towards PD-1+ cells.
Example 15. Optimized linkers can be fused to different cytokines while maintaining masking and CIS-demasking properties.
Methods. To evaluate cytokine masking and signalling, phosphorylation of STAT5 (pY694) or STAT3 (pY705) were evaluated by flow cytometry.
To evaluate I L15 signalling, hPBMCs were isolated from healthy donors buffy coat using Ficoll gradient. Immune cells were stimulated 15min with treatments at 37°C then fix, permeabilized and stained with anti-CD3-Pacific blue and anti-pSTAT5-AF647. Median Fluorescent Intensity (MFI) of pStat5 was determined for naive T cells (Figure 19A).
Co-culture assay with ratio 1:1 of U937 PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye (dotted lines) and U937 PD-1+ IL15Rbg+ cells (solid lines) were incubated with a monovalent anti-PDl-E15- IL15v-E15 (Figure 19B) or anti-PDl-IL15Ra sushi-(G4S)3-IL15wt/anti-PD-l-IL15Ra sushi-E15-IL15wt-E15 (Figure 20B). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was determined for each PD-1 and PD-1+ cell type. For IL-10 signalling evaluation, hPBMCs were +/- stimulated with agonist anti-CD3 and anti-CD28 antibodies for 3 days to promote overexpression of PD-1 on T cells. Activated T cells were +/- incubated with high dose of Anti-PD-1 to block the receptor. Then, cells were stimulated 15min with anti-PD-1- (G4S)3-IL10 or anti-PD-l-E15-IL10 and expression of pSTAT3 was evaluated (Figure 21).
For IL-18 evaluation, NKL cell line were treated with low dose of IL-12 and escalating dose of I L18, anti- PD-l-(G4S)3-IL18v or anti-PD-l-E15-IL18v-E15 during 24hours at 37°C. IFNy secretion were dosed by ELISA in supernatant using I FNg ELISA (Figure 22).
Results The inventors have fused different cytokines to the optimized linkers and evaluated their masking and CIS-Demasking properties. As shown in Example 1 and 2, optimized linkers enhanced masking activity of IL-15 and IL-2 (wild-type form). In the Example 13 and Figure 17, the inventors have also demonstrated the advantage of optimized linker to mask activity of mutated IL-2 cytokine (F42A, Y45A, L72G, as set forth in SEQ ID NO: 110). IL-2 mutation used in these experiments is a common mutation described to decrease binding to CD25 (IL-2Ra) to spare activation of Tregs that express high level of CD25. In addition, to evaluate effect of the optimized linker on other mutated cytokine, anti PD-1 antibody was fused to the optimized and IL-15 cytokine mutated (N1G-D30N-E46G-V49R-E64Q, as set forth in SEQ ID NO: 112). pSTAT5 signalling (Median fluorescence MFI analysis) was evaluated on naive primary T cells and on PD-1+ versus PD-1 negative transduced cell lines and compared to the same anti/PD-IL15v molecule constructed with the (G4S)3 flexible linker (Figure 19A and B). Optimized linker E15-E15 inhibited activity of the cytokine on naive primary T cells that do not express PD-1 or very low expression, showing high masking properties of the E15-E15 linker for mutated IL-15 to higher extent compared to Anti PD-1 (G4S)3 IL-15v. On transduced cells expressing PD-1 target or not expressing PD-1, Anti-PD-1 E15-IL15v-E15 has low activity on PD-1 negative CD122+ transduced Jurkat cell line while showing high CIS-activity on PD-1+ CD122+ transduced cell line. These data show that optimized linkers conserve their properties on mutated cytokines in addition to wild-type cytokines.
Next, the inventors have also evaluated the effect of the optimized linker on Anti PD-l/IL-15 Immunocytokine constructed with a sushi domain. The human IL-15 covalently linked to the human IL- 15Ra sushi(+) domain to create a superagonist of IL-15 and to bypass the need of endogenous IL-15Ra for transpresentation of IL-15 cytokine to CD122+/CD132+ T cells. This construction has been previously described as potent molecule for the cancer therapy as non-targeted format or fused to an anti PD-1 to target and cis activate PD-1+ activated Tcells. (Desbois et al., J Immunol, 2026, PMID: 2721758) and (Matuskova et al., 2025 , PMID 40250867). To evaluate the effect of the optimized linker in this construction, a novel construction was generated as illustrated on Figure 20A by fusing the bivalent anti PD-1, Sushi IL15Ra domain (RLI), E15 linker, IL15wt and E15 linker (See SEQ ID NO: 113 and SEQ ID NO: 114, respectively). E15-E15 optimized linker decreased pSTAT activity of IL-15 superagonist compared to (G4S)3 flexible linker while maintaining high activity on PD-1+ cells with similar to (G4S)3 linker as shown on Figure 20B. Thus, the optimized linker showed higher CIS-activity fold change between PD-1 neg cells and PD-1+ targeted cells .
Other cytokines as IL-10 (as set forth in SEQ ID NO: 128) and IL-18 (as set forth in SEQ ID NO: 56) were also fused to the anti PD-1 antibody to evaluate effect of the optimized E15 linker (One linker) (Figure 21 and 22). These data show the E15 linker masking activity on the cytokine on untargeted cells compared to conventional (G4S)3 linker and good capacity to demask cytokine activity on targeted cells as observed by pSTAT3 signaling (IL-10 stimulation) or IFNg secretion (IL-18 stimulation).
Altogether these data confirmed that activity of one or two E15 linker conserves making and demasking properties on multiple cytokines and the present invention can be used for different cytokines. IL-10 is an homodimeric cytokine (2 similar subunits), IL-12 is an heterodimeric cytokine (2 different subunits) while IL-2, IL-15 or IL-18 are monomeric cytokines. For all of these cytokines, the inventors have demonstrated that E15 linker decreases activity of the cytokine on non-targeted cells while maintaining a good cytokine activity on targeted cells, demonstrating versatile property of this linker for multiple cytokines regardless of the structure and properties of the cytokine. In addition fusion of receptor domain can be inserted in the construction with E15 linker while maintain CIS- activity of the cytokine.
Example 16. Optimized linker can be fused to different targeting antibody moiety while maintaining masking and CIS-demasking properties on non-targeted cells versus targeted cells.
Methods. To evaluate anti-mouse PD1 backbone, mouse T cells were isolated from C57BL6 mouse spleen using mechanical digestion and magnetic sorting. T cells were +/- stimulated overnight with anti-CD3 agonist antibody to overexpress PD-1. Non-stimulated (naive, dotted line) and stimulated T cells (solid line) were incubated at escalating dose of anti-PD-l-E15-hlL15-E15 or anti-PD-l-E15-hlL2v- E15. Median Fluorescent Intensity (MFI) of pStat5 was determined for each PD-1 and PD-1+ cells (Figure 23).
To evaluate anti-mouse CD3 backbone, mouse splenocytes were isolated from C57BL/6 mouse using mechanical digestion. Cells were +/- pre-saturated with ant-mouse CD3 antibody to block access to the CD3 antigen (same clone as Immunocytokine construction (clone 145.2C11). Cells were treated with a high dose (300nM) of human IL-15, anti msCD3-(G4S)3-hlL15 or anti- msCD3-E15-hlL15-E15 for 15 minutes then stained with anti-pSTAT5. Expression of pSTAT5 was evaluated on both CD3 "negative" or CD3 positive cells (Figure 24).
Results. The inventors have tested different antibody backbones to evaluate the capacity of optimized linkers to be used with different antibodies having different formats or targets. Different constructions have been designed with another anti PD-1 backbone (mouse 17D2 clone). First, another anti PD-1 backbone in bivalent format (2 Fab arm) was used and fused to E15 optimized linker and IL15wt cytokine (Anti msPD-l-E15-IL-15wt-E15). The anti PD-1 clone used targets mouse PD- 1 receptor and published to target Activated PD-1+ T cells with PD-L1 antagonist properties in vitro and in vivo (Chan et al., 2022, Nat. cancer, PMID: 35256819). Although this clone has similar properties as human anti PD-1 used in clinic, the 17D2 clone targets another epitope on mouse PD-1, as 17D2 does not interact with human PD-1 receptor (data not shown). Cis activity properties of the different constructions were evaluated on primary naive mouse T cell splenocytes (T cells with low PD-1 expression 15%) and on pre-activated mouse primary T cell splenocytes (51% PD-1 expression) (Figure 23A) . Data shown in Figure 23B demonstrate that the Anti PD-l-E15-IL15wt-E15 molecule did not activate naive T cells having low PD-1 expression while high activation of preactivated T cell expressing high level of PD-1 is observed. These data show that even with another anti-PD-1 backbone, the E15 linker conserves its masking and demasking properties and support the uses of any other anti-PD-1 backbone targeting different epitopes. Similar data were obtained with Anti PD-1 Bivalent (clone 17D2) fused to hlL-2 cytokine Figure 23C and Anti PD-1 monovalent (clone 17D2) fused to hlL-15 cytokine Figure 23D.
In addition, the inventors have also tested fusion with an anti-mouse CD3 antibody (clone 145.2C11) (Alegre et al, JI, 1997, PMID 7636216). This antibody used in the construction is a single chain (scFv domain) targeting CD3+ mouse T cells, fused to lgGlN297A isotype, an E15 linker, the IL-15wt human cytokine and a E15 linker compared to a scFv CD3, fused to lgGlN297A isotype, (G4S)3, IL-15wt. To evaluate cis activity on CD3+ T cells vs CD3 negative T cells, mouse splenocytes were pretreated with an excess of anti-mouse CD3 antibody (same clone) to mask epitope and the binding of the anti CD3/IL- 15 multifunctional molecule. Data are shown in Figure 24. E15 optimized linker was able to mask activity of the IL-15 cytokine on CD3 non targeted T cells and efficiently CIS-activate CD3+ targeted T cells as measured by activation of pSTAT5 signaling. These data demonstrate the versatility of the optimized linker to be used with different antibody moiety targeting different type of receptors on the cells. In addition, these data also demonstrate that all formats of antibody can be used (Fab monovalent, Fab Bivalent, scFv...) with the optimized linker to enhance CIS targeting properties by masking cytokine activity on non-targeted cells.
Evaluation of cis-activity was also tested on monocytic cell line to demonstrate that masking capacity of the optimized linker is not related to one specific cell type. Data shown in previous figures used mouse and human T cell (primary and cell lines) before and after activation or NK cell line (NKL), demonstrating that the optimized linker conserves its properties on different cells . A shown in Figure 25, the data confirm the masking effect on another cell line (U937 human monocytic cell). Anti PD-1 E15 IL2v E15 vs Anti PD-1 (G4S)3 IL2v constructions were tested on PD-1+ CD122+ transduced cell line vs PD-1 negative CD122 transduced cells. Low/ no activation was observed with construction comprising the optimized E15 linker while nonspecific activation was observed with (G4S)3 linker confirming masking properties of the E15-E15 linker on different cell types.
Additionally, the inventors also tested the usefulness of the optimized linker with other checkpoint inhibitory receptor, by fusing an anti TIG IT antibody used in clinic (Tiragolumab) to the optimized linker E15 and IL-2wt cytokine. Data of Figure 29 shows that a multifunctional molecule comprising E15 optimized linker mask activity of IL2 cytokine on TIGIT negative cell line, while maintaining high CIS activation of TIGIT + cells similar to the anti TIGIT-(G4S)3-IL2wt molecule. A higher ratio between activation of TIGIT negative cells versus TIGIT positive cells was obtained after treatment with the molecule anti-TIGIT-E15-IL2wt-E15 in comparison to the molecule constructed with a conventional (G4S)3 linker (Right graph).
Altogether, data shown inof Examples 15 and 16 clearly demonstrate that optimized linkers of the invention (in particular E15 linker) conserve masking and CIS-demasking properties regardless of the targeting domain, antibody structure, fused cytokine, or targeted cell type.
Example 17. Optimized linker is not cleaved in vivo
Methods. Stability of optimized linker was evaluated in vivo on humanized PD1 mice (hPDIKI). Mice were treated intravenously with anti-PD-l-E15-hlL2v-E15 (5mg/kg) and sera were sampled at different time points. Using 2 Elisas with an anti-human kappa capture antibody and an anti-hlgG or an anti-l L2 antibodies for revelation, treatments were dosed in sera to check the conformity (Figure 26A).
Biodistribution of bispecific antibodies were studied in hPDIKI bearing MC38 cancer cell subcutaneously. Mice were treated at day 10 with Anti-PD-l*l-(G4S)3-IL15 or Anti-PD-l*l-E15-IL15- E15 (4mg/kg). 24 hours post injection, organs were harvested, and antibodies were dosed in organs digest using anti-human kappa / anti-h I L15 sandwich ELISA. Concentrations obtained were normalized to total protein concentration as measured by BCA assay (Figure 26B).
To evaluate cytokine integrity in vivo, pSTAT5 signaling ex-vivo was studied. hPDIKI mice were treated with intravenously with lOmg/kg of Anti-PD-1*2-(G4S)3-IL15 or Anti-PD-1*2-E15-IL15-E15 and sera were sampled 24hours after. Sera containing antibodies were diluted at 1/8 in medium and used to stimulate U937 monocyte cell line expressing CD122 +/- PD-1. pSTAT5 signaling on U937 were studied by flow cytometry after 15min of stimulation and intracellular staining with pSTAT5-A647 (Figure 26C). Results. To demonstrate that the integrity of the multifunctional molecule is conserved with E15-E15 optimized linker even after injection in vivo, biodistribution and pharmacokinetic experiments were performed in a in vivo mouse models (Figure 26A, 26B and 26C). The immunocytokine in the sera or tissue was quantified ex vivo using two ELISA: (1) Quantification of the antibody moiety (Anti PD-1 domain) : an anti-human Kappa was coated and revelation was performed using an anti-human IgG.
Quantification of the full intact immu i PD-1 linked to IL-2) : an anti-human kappa was coated and revelation was performed with an anti-human IL-2. Figure26A shows that identical concentration were measured by the 2 ELISA methods suggesting that IL-2 is not release in vivo and that E15-E15 linker does not promote cleavage in vivo. In addition, in vivo biodistribution experiments were performed for anti PD-1 E15-IL15wt-E15 and compared to Anti PD-1 (G4S)3 IL-15wt. The intact molecules (anti PD-1 fused to IL-15) was quantified in the spleen, tumor nodule, liver using an antihuman kappa antibody coated and revelation was performed using an anti-human IL-15 (Figure 27B). Identical quantity of the Anti PD-l/IL-15 molecule between the 2 constructions were obtained by ELISA confirming that E15 linker is not cleaved in vivo, thereby no 11-2 will be released into tumor microenvironment.
The functional properties of the immunocytokine anti PD-1 E15-IL-15-E15 (e.g., masking and demasking) was conserved even after in vivo injection since Anti PD-1 E15 IL-15 -E15 molecule demonstrates low activation of PD-1 negative cells (IL-2 pSTAT5 signalling evaluation) compared to Anti PD-1-(G4S)3-IL-15 molecule, while maintaining high IL-2 pSTAT5 activity on PD-1+ cells (Figure 26C). These data confirm the in vivo stability of the linker and its capacity to conserve masking and demasking properties in vivo, i.e., without the requirement of cleavage of the linker for releasing the cytokine activity.
Example 18. Optimized linker has no immunogenic properties as evaluated bv in silico prediction in human and in mice.
Methods To evaluate immunogenicity of antibodies in vivo, anti-drugs antibodies (ADA) were studied. hPDIKI mice were treated intravenously with 5mg/kg of anti-PD-l-(G4S)3-IL15wt, anti-PD-l-D15- IL15wt-D15 or anti-PD-l-E15-IL15wt-E15. Sera were sampled before and 11 days after injection. ADA in sera were quantified by ELISA by coating of drugs at 5pg/ml overnight at 4°C, capture of mouse sera for 2h at 37°C and revelation with donkey ant-mouse IgG peroxidase for lh at 37°C and conventional methods for reading optical density.
Results. In silico evaluation of immunogenicity using IEDB software demonstrates that the immunocytokine comprising E15-E15 linker has no predictive immunogenicity in human similarly to (G4S)3 linker already used and being tested in patients. This score (percentile rank) is obtained through IEDB software that quantify of peptide sequences on most common MHC class II haplotype. (Figure 27A).
To evaluate in vivo immunogenicity of E15 optimized linker and D15 optimized linker, a pharmacokinetic experiment was performed in mice and the Anti-Drug Antibody (ADA) in the sera of the mice (Day 11) was measured by ELISA. Figure 27B shows that the immunocytokine constructed with E15 and D15 linker had the same ADA quantity as conventional (G4)3 linker demonstrating that construction PD-1/IL15 with E15 or D15 linkers were not more immunogenic than (G4S)3 in mice, thus being predicted as not immunogenic in human. Example 19. Optimized linkers demonstrate higher CIS activity compared to other prior art non cleavable masking strategies with high activation of PD-1+ T cells (primary activated T cells and PDA transduced cell lines)
Methods. To evaluate activity on cell lines PD-1+ and PD-1- CD122+ cells, U937 cells transduced with PD-1+ and CD122+ were treated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was determined for each PD-1 and PD-1+ cell type pSTAT5 was also evaluated PD-1+ activated T cells. To generate activate T cells, PBMCs were isolated from buffy coat and T cells were stimulated twice with agonist anti-CD3 or anti-CD28 to express PD1 (85% of expression). Cells were incubated at escalating doses for 15min at 37°C. Median Fluorescent Intensity (MFI) of pSTAT5 was quantified by flow cytometry for each cell population.
Results. Efficacy and CIS activity of the present invention were compared to another immunocytokine comprising a non-cleavable masking strategy. The described immunocytokine is constructed with an Anti PD-1 fused to IL-2Ra receptor and IL-2wt cytokine and use a flexible linker fused between the Fc domain, the cytokine and the receptor. This construction termed PDl-IL2Ra-IL2 (REGN10597) was recently published and its activity described (Wu et al., Cell Rep Med, . 2024 Oct 15;5(10):101747, PMID: 39326410). The inventors constructed the same immunocytokine IL-2/IL-2ra structure as disclosed in Wu et al., with the same anti-PD-1 backbone as the anti PD-1 E15-IL2wt-E15 molecule used herein, to have a direct comparison of the masking strategies regardless of the targeting domain affinity. SEQ ID NO: 120 describes the sequence of G4S)3-IL2Ra-(G4S)5-IL2wt. Figure 28A shows that the Anti PD-l-IL2wt constructed with E15 linkers showed a strong activation of PD-1+ T cells while low activation of PD-1 negative U937 cells is observed, a high CIS-activity was observed (not calculable due to high activation). In contrast, while the IL-2ra masking strategy had low activity on PD-lnegative U937 cells, this construction had also low activity on PD-1+ CD122+ cells. This construction seems to have low properties of demasking on PD-1+ cells even after targeting. Figure 28B confirms in primary human T cells that Anti PD-l-IL2wt constructed with optimized linker outperformed to promote activation of IL-2 signaling into activated PD-1 + T cells, while the PDl-IL2Ra-IL2 construction showed less capacity to restimulate this PD-1+ T cells. Thus, these data demonstrate that the optimized linkers according to the present invention allow better properties to stimulate and CIS activate PD-1+ activated exhausted T cells compared to the prior art strategy using IL-2Ra as masking strategy. This higher activation of PD-1+ T cells obtained in vitro indicates that a better T cell anti-tumor response in vivo will be induced (effector capacity, proliferation, cytokine secretion..) with the cytokine constructed with optimized linkers of the invention versus the cytokine constructed with the I L-2ra masking strategy.
MATERIAL AND METHODS
Preparation and characterisation of constructions Sequence of linkers and cytokine were synthetised and cloned by DNA assembly (NEBuilder Hifi DNA assembly kit, NEBiolabs) in pcDNA3.4 expression plasmid containing heavy variable domain of antibody (anti-PDl, anti-CD3,...) and mutated Fc domains (Hole of KIH hlgGl-N297A) digested in BsiWI (OSE immunotherapeutics plasmid).
In CHO mammalian cells, plasmid (Knob :Hole :VL) were co-transfected by polyethylenimine method. After 7 days incubation, supernatant was recovered and purified by affinity on Protein A chromatography (HiTrap, GeHealthcare) (with citric acid 0.1M pH 3 elution buffer) and full antibody was isolated by gel filtration chromatography (preparative SEC column, Hiload 16/600 Superdex 200, Cytiva). Purified antibody was dialyzed in PBS and concentrated. They were quantified by UV at 280nm and tested in several test.
ELISA binding PD1
For activity ELISA assay, recombinant hPDl (Sino Biologicals, Beijing, China; reference 10377-H08H) was immobilized on plastic at 0.5pg/ml in carbonate buffer (pH9.2) and purified antibody were added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) was added and revealed by conventional methods. pSTAT5 analysis in cell lines for IL2 or IL15 cytokine
U937 cells transduced to express CD122+ PD-1- or to express CD122+PD-1+ cells, starved in TexMacs medium (Miltenyi ref 130-097-196) overnight, were incubated with anti PD-l-(G4S)3-cytokine (construct anti-PDl fused to cytokine with conventional linker) or anti-PDl-E15-cytokine-E15 (construct anti-PDl fused to cytokine with optimized linker). Jurkat transduced to express CD122+ CD132+ (IL15/IL2RPy) PD-1- or to co-express CD122+CD132+ (IL15/IL2Rbg) and PD-1+, starved in TexMacs medium (Miltenyi ref 130-097-196) overnight, were incubated with IL15 (Miltenyi ref 130- 095-760) or IL2 wild-type cytokine alone (Miltenyi ref 130-097-743) (nu cytokine), anti PD-1-(G4S)3- IL15/IL2 (construct anti-PDl fused to I L15 or IL2 with conventional linker) or anti-PDl-E15-IL15/IL2-E15 (construct anti-PDl fused to IL15 or IL2 with optimized linker). Each PD-1- cells or PD-1+ cells were respectively incubated with the different constructions at escalating doses for 15 to 30min at 37°C, 5%CC>2 in TexMacs medium. After fixation and permeabilization with CytoFix/Cytoperm kit (BD Biosciences ref 554714) and PermBufferlll (BD Biosciences ref 558050) for 30min at 4°C, A647-labelled anti-pStat5 (clone 47/Stat5(pY694, BD Biosciences ref 612599) was used to stain pStat5 for 30min at RT. The pStat5 signaling for each cell lines were quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction for each PD-1+ and PD-1- cell populations.
For trans-presentation study, human PDl-Fc (SinoBiological ref 10377-H02H) was coated overnight at 4°C on P96-plate before adding Jurkat PD1- 1 L15RP/+ cells and the different constructions to analyse pStat5 signaling. To determine cis activity, Jurkat or U937 transduced with CD122+CD132+ and PD-1 were mixed with Jurkat or U937 transduced with CD122+CD132+ only stained with cell tracker dye (CellTracker Green, Thermofisher ref C7025) mixed at a ratio 1:1 and treated with the tested molecule during 30 minutes at 37°C. Cells were then fixed, permeabilized and stained with an AF647 labeled anti-pSTAT5 (clone 47/Stat5(pY694), BD Bioscience ref 612599). For the co-culture assay, pSTAT5 activation was evaluated into Jurkat or U937 PD1+ CD122+CD132+ cells and Jurkat or U937 PD1- CD122+CD132+ cells stained with CellTrackerGreen. The pStat5 signaling for each cell were quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction for each PD-1+ and PD-1- cell population. pSTAT5 analysis in human naive and activated T cell
PBMCs isolated from peripheral blood of human healthy volunteers were incubated 30 minutes with the different constructions at 37°C. For human PBMCs, pSTAT5 or pSTAT3 activation was evaluated into CD3+ T cell population.
Activated/exhausted T cells were generated by stimulating naive PBMCs two times with an anti-CD3 (clone OKT3) / anti-CD28 (clone CD28.2) coating at 3pg/ml for 2-3days at 37°C, 5%CO2 for each stimulation. For human activated T cells, pSTAT5 or PSTAT3 activation was evaluated into CD3+ T cell population with staining with Pacific Blue anti-CD3 antibody (BD Biosciences, reference B220). In some experiment, activated T cells can be pretreated with high dose of anti-PDl (50pg/ml) to block the receptor.
Naive or T cells activated, starved in TexMacs medium (Miltenyi ref 130-097-196) overnight, were incubated with cytokine or the different constructions at escalating doses for 15 to 30min at 37°C, 5%CC>2 in TexMacs medium. After fixation and permeabilization with CytoFix/Cytoperm kit (BD Biosciences ref 554714) and PermBufferlll (BD Biosciences ref 558050) for 30min at 4°C, stained with Pacific Blue anti-CD3 antibody (BD Biosciences, reference B220) for 30min at 4°C, then A647-labelled anti-pStat5 (clone 47/Stat5(pY694, BD Biosciences ref 612599) or A647-labelled anti-pStat3 (Stat3/pY705, BD Biosciences ref 562071) was used to stain pStat5 or pStat3 for 30min at RT. The pStat3/5 signaling for each cell were quantified by flow cytometry. EC50 (nM) of the pSTAT5 activation was determined for each construction for naive or activated cell population. pSTAT5 analysis in mouse naive and activated T cell
Mouse T cells were isolated from C57BI/6 mouse spleen and were +/- stimulated overnight with anti- CD3 agonist antibody (clone 145-2C11, BD Biosciences, reference 553057) to overexpress PD-1. Cells were incubated 15 minutes with the different constructions at 37°C. For mouse T cells, pSTAT5 activation was evaluated into CD3+ T cell population, like described above. In some experiment, activated mouse T cells can be pretreated with high dose of anti-mouse CD3 antibody (50pg/ml) (clone 145-2C11, BD Biosciences, reference 553057) to block the receptor. NKL activation with I L18
NKL cell line was transduced to express human PD1+. For assays, NKL PDl- or NKL PD1+ were cultivated during test in TexMacs medium (Miltenyi ref 130-097-196) and were treated with low dose of IL-12 (lOng/ml) (Miltenyi, reference 130-129-722) and escalating dose of I L18, anti-PD-l-(G4S)3-IL18 or anti- PD-1-E15-IL18-E15 during 24hours at 37°C. IFNy secretion were dosed by ELISA in supernatant using commercial human I FNg ELISA assay (Biolegend, reference 430104).
Pharmacokinetics and Pharmacodynamics in vivo
Pharmacokinetics and Pharmacodynamics of the product were assessed in mice following a single injection. For pharmacokinetic study humanized PD-1 Knock in mice were intravenously injected with anti PD-1 bifunctional antibodies (5mg/kg). Plasma drug concentration was determined by 2 methods ELISA : the first to measure IgG using an immobilized anti-human light chain antibody (clone NaM76- 5F3, OSE Immunotherapeutics), then serum-containing antibodies were added. Detection was performed with a peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) and revealed by conventional methods. The second to measure full antibody with anti-IL2 using an immobilized recombinant hPDl (Sino Biologicals, Beijing, China; reference 10377-H08H), then serum-containing antibodies were added. Detection was performed with a mouse anti-human IL2 (Biotechne, reference MAB2021) followed by revelation with peroxidase-labelled antimouse IgG (Jackson Immunoresearch; USA; reference 715-036-151) and revealed by conventional methods.
For biodistribution study, humanized PD-1 Knock in mice were subcutaneously injected with MC38 tumor. Around Day 10 when the tumor was palpable, mice were treated with Anti PD-1 E15-IL-15-E15 (4mg/kg, intraperitoneal administration, one dose). After 24h, mice were sacrificed, organs were harvested and crushed with FastPrep beads Matrix D (MP Biomedical, reference 1169130-CF) in Ripa buffer and molecules were quantified in lysate by ELISA using an immobilized anti-human light chain antibody (clone NaM76-5F3, OSE Immunotherapeutics), then lysate-containing antibodies were added. Detection was performed with biotinylated-labeled anti-human IL15 (Biolegend, reference 515104, clone BH1543), followed by peroxidase-labelled streptavidin (Jackson Immunoresearch, reference 016-030-084) and revealed by conventional methods. Concentrations obtained were normalized to total protein concentration measured by BCA assay (Interchim, reference UP40840A). pStat5 activation was measured in lysate as described above on U937 expressed express CD122+ PD- 1- or to express CD122+PD-1+ cells. pSTAT5 signaling on U937 were studied by flow cytometry after 15min of stimulation and intracellular staining with pSTAT5-A647 with similar methods than described above.
Immunogenicity For prediction of immunogenicity in silico , we use IEDB software tools (NetMHCpanll method), which analyzed the antibody sequence and the potential of peptide binding to HLA-DR (MHC classll, through more predominant alleles: DRBl*01:01; DRBl*03:01; DRBl*04:01; DRBl*07:01; DRBl*08:01; DRB1*11:O1; DRB1*13:O1; DRB1*15:O1) molecule hence predicting activation of adaptive immune response and immunogenicity of the drug. IEDB analysis ranks peptide binders depending on the predicted affinity to MHCII and homology with human sequences. Mean of percentile rank of all different peptides is calculated for each allele. Values equal to 100 corresponds to absence of immunogenicity, value <1 corresponds to a a high immunogenicity score and value between 1-30 is considered as medium immunogenicity.
For Anti-drug Antibody (ADA) analysis, hPDIKI mice were injected intravenously with 5mg/kg of bivalent anti-PD-l*2-(G4S)3-IL15wt , anti-PD-l*2-D15-IL15wt-D15 or anti-PD-l*2-E15-IL15wt-E15. Sera were sampled before injection and Dll post injection. ADA (anti-drugs antibodies) in sera were quantified by ELISA in incubating on coated-plate with the drug and in detecting with peroxidase- labelled anti-mouse IgG antibody (Jackson Immunoresearch; USA; reference 715-036-151) and revealed by conventional methods.
In vivo anti-tumor model
PanC02 (pancreas cancer cell line) bearing hPDIKI mice were injected intraperitoneally with 4 doses (day 7, 11,15 and 19 post-tumor inoculation) of PBS or anti-PD-l-E15-IL2v-E15 (0,5 or lmg/kg). Anti PD1 molecule used is a bivalent anti PD-1 format with one IL-2v molecule. Survival was monitoring during these experiments. Statistical analysis was performed using Graph Pad prism using Log-Rank (Mantel Cox) test ** p<0.002
For therapeutic index evaluation, hPDIKI mice expressing human PD-1 receptor were inoculated subcutaneously with 0.5e6 MC38 cells. On day 10, tumor volume (mm3) was measured, and mice were randomized in the different groups to ensure a similar mean tumor volume across treatment groups. On Day 10, 12 and 14 mice were treated with Anti PD-l/IL-2v molecule constructed with optimized linker (anti-PDl-E15-IL-2v-E15). Treatment was performed intraperitoneally at 30, 10, 5 or 2 mg/kg. Tumor volume was measured at multiple time point as well as clinical score was assessed to evaluate potential toxic effect of the drug (lethal dose).
MOLECULES DESCRIPTION
Throughout the "Examples" section, the terms "optimized linker", "masking linker", "cytomask linker" or "cytomasking linker" are used interchangeably.
When a number follows an amino acid, it means a repetition of said amino acid. For example, when referring to "E15" it is meant 15 repetition of the amino acid E (i.e., EEEEEEEEEEEEEEE as described in SEQ ID NO: 5). In the Figures, when reference is made to G4S3, it is meant a (G4S)3 linker (i.e., as described in SEQ. ID NO: 106).
When an antibody is monovalent, i.e., has a single antigen binding domain (e.g., one Fab), the Example section and Table 4 may refer to "*1", meaning one valency of the antigen binding domain. When a construction or molecule refers for example to anti-PDl*l, it means a monovalent/single antigen binding domain against PD-1, typically comprising a single VH and a single VL against PD-1. Monovalent molecule particularly comprises a first heavy chain devoid of antigen binding domain, a heavy chain with an antigen binding domain and a single light chain.
When an antibody is bivalent, i.e., has two antigen binding domains (e.g., two Fab), the Example section and Table 4 may refer to "*2" , meaning two valencies of the antigen binding domain. When a construction or molecule refers for example to anti-PDl*2, it means a bivalent molecule (i.e., with two antigen biding domains, such as two Fab, against PD-1). Bivalent molecule particularly comprises two heavy chains and two light chains.
Table 4. Summary of the constructions used in the "Examples" section and Figures, with their respective sequences.

Claims

1. A multifunctional molecule comprising: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having a pro-inflammatory effect and a narrow therapeutic index or a systemic toxicity, said cytokine, variant or fragment being covalently linked to the N-terminal and/or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker covalently linked to the N-terminal and/or C-terminal end of the cytokine; wherein the peptide linker is of 5 to 30 amino acids in length and consists of
- at least 20% acidic or amidic amino acids independently selected from the group consisting of E, D and N,
- up to 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and
- at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.
2. The multifunctional molecule according to claim 1, wherein the peptide linker is a masking peptide linker.
3. The multifunctional molecule according to claim 1 or 2, wherein the peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; and a sequence having 5 to 30 consecutive amino acids N.
4. The multifunctional molecule according to claim 1 or 2, wherein said peptide linker comprises:
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids selected independently from the group consisting of E, D and N; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being E; or - 3, 4, 5, &, , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being D; or
- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being N.
5. The multifunctional molecule according to claim 1 or 2, wherein the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34),
DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).
6. The multifunctional molecule according to any one of the preceding claims, wherein the peptide linker is covalently linked to the N-terminal end of the cytokine and is covalently linking the antigen binding domain and the cytokine.
7. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises two peptide linkers, a first peptide linker being as described in any one of claims 1-6 and a second peptide linker, wherein:
- the second peptide linker has an amino acid sequence as defined in any one of claims 1-7 for the first linker; or
- the second peptide linker is identical to the first peptide linker; or
- the second peptide linker is between 10 and 30 amino acids in length and consists of less than 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.
8. The multifunctional molecule according to claim 7, wherein:
- the first peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the second peptide linker is linked to the C- terminal end of the cytokine; or
- the second peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the first peptide linker is linked to the C-terminal end of the cytokine.
9. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises a first peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5) and a second peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5), preferably wherein:
- the N-terminal end of the first peptide linker is covalently linked to the C-terminal end of the antigen binding domain, preferably in C-terminal end of a Fc domain;
- the C-terminal end of the first peptide linker is covalently linked to the N-terminal end of the cytokine; and
- the N-terminal end of the second peptide linker is covalently linked to the C-terminal end of the cytokine.
10. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises a single antigen binding domain, optionally a Fc domain, and a single cytokine.
11. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of antigen binding domain and of cytokine; and b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) either
- the first peptide linker and the single cytokine or variant or fragment thereof; said first peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine; or
- the first and second peptide linkers and the single cytokine or variant or fragment thereof; wherein the first peptide linker is covalently linked to the N-terminal end of the cytokine and the second peptide linker is linked to the C-terminal end of the cytokine.
12. The multifunctional molecule according to any one of the preceding claims, wherein the pro- inflammatory cytokine or variant or fragment thereof is a cytokine selected from the group consisting of TNF alpha, IFNy, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15, IL-17A, IL17B, IL-18, IL-21, IL-22, IL-23, IL-36A, IL-36B, IL-36G, LTa and LTP, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine, preferably a cytokine selected from the group consisting of IL-2, 1 LIO, IL-12, IL-15, IL-18 and IL-21 or a variant thereof, more preferably a cytokine selected from the group consisting of IL-2, IL-12, IL-15, I L18 and IL-21 or a variant thereof.
13. The multifunctional molecule according to any one of the preceding claims, wherein the antigen binding domain binds to a target expressed: on immune cells surface and being selected from the group consisting of PD-1, BCMA/TNFRSF17, BTLA, CD101/IGSF2, CD119, CD137/4-1BB/TNFRSF9, CD150/SLAMF1, CD153, CD226, CD25, CD254, CD26, CD27, CD275/ICOSL, CD39/ENTPD1, CD40L/CD154, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3/CD183, CXCR4, CXCR5, CXCR6, FasL/TNFSF6, GITR/TNFRSF18/CD357, GPR32, TIM3/HAVCR2, ICOS, IL18Rl/CXCRl/CD218a, ITGAE/CD103, LAG3/CD223, TRAILR, OX40L, LY108 /SlamF6, NKG2D, OX40/TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI/TNFRSF13B, TIGIT, CD163, CD206, LTBR/CD70, TNFSF14, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30/TNFRSF8, CD78, TRAF1, TRAF2, TRAF3/CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1/TNFRSF1A/CD120A, TRAP100/MED24, TNFR2/TNFRSF1811/CD120B, CDCR3/TNFRSF6B, TNFRSF12A/FN14/TWEAKR, BAFFR/TNFRSF13C/CD268,
HVEM/TNFRSF14/CD270, RELT/TNFRSF19L, TNFRSF19/TROY, TNFRSF21/DR6, TNFRSF25/DR3/TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD74, CD165, 4-1BBL/CD137L, CD127 and CD160; or
- on tumoral cells surface and being selected from the group consisting of Fibroblast Activation Protein (FAP), the Al domain of Tenascin-C (TNC Al), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA or CEACAM) and the Melanoma-associated Chondroitin Sulphate Proteoglycan (MCSP), Her2/Neu (human epidermal growth factor receptor 2), CD22, EpCAM (CD326), EGFR, PSMA (Prostate Carcinoma), CD30, CD20, CD33, membrane IgE, IgE Receptor (CD23), CD80, CD86, CD2, CA125 (cancer antigen- 125), Carbonic Anhydrase IX, CD70, CD74, CD56, CD40, CD19, c-met/HGFR, DRS, PD-1, PDL1, IGF- 1R, VEGF and VEGFR (Solid tumour and eye AMD), VEGF-R2, Prostate stem cell antigen (PSCA), MUC1, CanAg, Mesothelin, P-cadherin, Myostatin (GDF8), Cripto (TDGF1), ACVRL 1/ALK1 (activin a receptor type 1), MUC5AC, CD137, CXCR4, Neuropilin 1, Glypicans, HER3, platelet derived growth factor receptor alpha (PDGFRa), EphA2 (Ephrin type- A receptor 2), nucleolin, CD38, CD138, a4-integrin, C5 complement, C3 complement, MASP-2, C5aR, CR1, C3b, CA19-9, calretinin, epithelial membrane protein (EMA), epithelial tumour antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB- 45 antigen, melanoma antigen recognized by T lymphocytes (MART-1), myo-DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumour M2-PK), an abnormal Ras protein, an abnormal p53 protein, diganglioside GD2, interleukin 13 receptor a 2 (IL13Ra2), CD133, natural-killer group 2, member D (NKG2D), chondroitin sulphate proteoglycan 4 (CSPG4), CS-I, LI cell adhesion molecule (L1CAM), BCMA (-cell maturation antigen), alpha-fetoprotein (AFP), and CFH.
14. The multifunctional molecule according to any one of claims I to 11, wherein the antigen binding domain binds to an immune checkpoint inhibitor selected from the group consisting of PD-1, TIGIT, TIM3, LAG3, VISTA, HVEM, BTLA and CTLA-4.
15. The multifunctional molecule according to any one of claims I to 11, wherein the antigen binding domain is a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, preferably comprising or consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 74 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 75; ii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 96 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 97; and iii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 104 and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 105.
16. The multifunctional molecule according to any one of claims 1 to 11, wherein the antigen binding domain is an anti-human TIGIT antibody or antigen-binding fragment thereof, preferably comprising or consisting of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 129 and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 130.
17. An isolated nucleic acid molecule, a group of isolated nucleic acid molecules or a vector encoding the multifunctional molecule according to any one of claims 1 to 16.
18. A host cell comprising the isolated nucleic acid molecule and/or the group of isolated nucleic acid molecules and/or the vector according to claim 17.
19. A pharmaceutical composition comprising the multifunctional molecule according to any one of claims 1 to 16.
20. The pharmaceutical composition of claim 19, further comprising an additional therapeutic agent, preferably an anti-cancer agent or anti-infection agent.
21. The multifunctional molecule according to any one of claims 1-16 or the pharmaceutical composition according to claim 19 or 20, for use as a medicament.
22. The multifunctional molecule according to any one of claims 1-16 or the pharmaceutical composition according to claim 19 or 20, for use in the treatment of a cancer or an infection.
23. A method for treating a cancer or an infection in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the multifunctional molecule according to any one of claims 1-16 or of the pharmaceutical composition of claim 19 or 20.
24. The method of claim 23, said method further comprising the administration of an additional therapeutic agent, preferably an anti-cancer agent or anti-infection agent.
25. Use of a multifunctional molecule according to any one of claims 1-16, in the manufacture of a medicament for treating a cancer or an infection in a subject in need thereof.
PCT/EP2025/064209 2024-05-22 2025-05-22 Molecules comprising masking linkers and uses thereof for the treatment of cancer Pending WO2025242835A1 (en)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
EP24305796 2024-05-22
EP24305796.5 2024-05-22
EP24206752 2024-10-15
EP24206752.8 2024-10-15
EP25305589.1 2025-04-24
EP25305589 2025-04-24
EP25176519 2025-05-14
EP25176519.4 2025-05-14

Publications (1)

Publication Number Publication Date
WO2025242835A1 true WO2025242835A1 (en) 2025-11-27

Family

ID=95825397

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/064209 Pending WO2025242835A1 (en) 2024-05-22 2025-05-22 Molecules comprising masking linkers and uses thereof for the treatment of cancer

Country Status (1)

Country Link
WO (1) WO2025242835A1 (en)

Citations (117)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US109A (en) 1836-12-15 Erawijtg
US5229A (en) 1847-08-07 anthony
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO2001058957A2 (en) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US20030124678A1 (en) 2001-08-13 2003-07-03 University Of Southern California Interleukin-2 mutants with reduced toxicity
WO2004000238A2 (en) 2002-03-22 2003-12-31 Tittle Thomas V Utilization of mhc class ii binding motifs in immunization to produce immune serum, monoclonal antibodies and vaccines
WO2004091517A2 (en) 2003-04-15 2004-10-28 Smithkline Beecham Corporation Conjugates comprising human il-18 and substitution mutants thereof
WO2005035565A1 (en) 2003-10-10 2005-04-21 Novo Nordisk A/S Il-21 derivatives
WO2006111524A2 (en) 2005-04-18 2006-10-26 Novo Nordisk A/S Il-21 variants
WO2006121168A1 (en) 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2007008463A2 (en) 2005-07-07 2007-01-18 Coley Pharmaceutical Group, Inc. Anti-ctla-4 antibody and cpg-motif-containing synthetic oligodeoxynucleotide combination therapy for cancer treatment
US20070036752A1 (en) 2001-12-04 2007-02-15 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
WO2008034473A1 (en) 2006-09-20 2008-03-27 Dge Dr.-Ing. Günther Engineering Gmbh Method and device for separating methane and carbon dioxide from biogas
WO2008049920A2 (en) 2006-10-26 2008-05-02 Novo Nordisk A/S Il-21 variants
WO2008074863A1 (en) 2006-12-21 2008-06-26 Novo Nordisk A/S Interleukin-21 variants with altered binding to the il-21 receptor
WO2008076560A2 (en) 2006-11-15 2008-06-26 Medarex, Inc. Human monoclonal antibodies to btla and methods of use
WO2008132601A1 (en) 2007-04-30 2008-11-06 Immutep Cytotoxic anti-lag-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease
WO2009080253A1 (en) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
WO2009100140A1 (en) 2008-02-04 2009-08-13 Medarex, Inc. Anti-clta-4 antibodies with reduced blocking of binding of ctla-4 to b7 and uses thereof
WO2010103038A1 (en) 2009-03-11 2010-09-16 Novo Nordisk A/S Interleukin-21 variants having antagonistic binding to the il-21 receptor
WO2010106051A1 (en) 2009-03-17 2010-09-23 Universite De La Mediterranee Btla antibodies and uses thereof
WO2011014438A1 (en) 2009-07-31 2011-02-03 N.V. Organon Fully human antibodies to btla
EP2320940A2 (en) 2008-08-11 2011-05-18 Medarex, Inc. Human antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2011104687A1 (en) 2010-02-24 2011-09-01 Rinat Neuroscience Corporation Antagonist anti-il-7 receptor antibodies and methods
WO2011155607A1 (en) 2010-06-11 2011-12-15 協和発酵キリン株式会社 Anti-tim-3 antibody
US20120149876A1 (en) 2010-11-05 2012-06-14 Zymeworks Inc. Stable Heterodimeric Antibody Design with Mutations in the Fc Domain
US8216805B2 (en) 1995-03-01 2012-07-10 Genentech, Inc. Knobs and holes heteromeric polypeptides
WO2012107417A1 (en) 2011-02-10 2012-08-16 Roche Glycart Ag Mutant interleukin-2 polypeptides
WO2012120125A1 (en) 2011-03-09 2012-09-13 Antitope Ltd Humanised anti ctla-4 antibodies
WO2013006490A2 (en) 2011-07-01 2013-01-10 Cellerant Therapeutics, Inc. Antibodies that specifically bind to tim3
WO2014102430A1 (en) 2012-12-27 2014-07-03 Consejo Superior De Investigaciones Científicas (Csic) Therapeutic treatment of t- and b-acute lymphoblastic leukemia and human lymphomas by inhibition of the interleukin-7 receptor (il-7r)
WO2014145806A2 (en) 2013-03-15 2014-09-18 Xencor, Inc. Heterodimeric proteins
WO2014194302A2 (en) 2013-05-31 2014-12-04 Sorrento Therapeutics, Inc. Antigen binding proteins that bind pd-1
WO2016015675A1 (en) 2014-08-01 2016-02-04 中山康方生物医药有限公司 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
WO2016028656A1 (en) 2014-08-19 2016-02-25 Merck Sharp & Dohme Corp. Anti-tigit antibodies
WO2016059512A1 (en) 2014-10-18 2016-04-21 Pfizer Inc. Anti-il-7r antibody compositions
WO2016106302A1 (en) 2014-12-23 2016-06-30 Bristol-Myers Squibb Company Antibodies to tigit
WO2016130898A2 (en) 2015-02-13 2016-08-18 Sorrento Therapeutics, Inc. Antibody therapeutics that bind ctla4
WO2016161270A1 (en) 2015-04-01 2016-10-06 Anaptysbio, Inc. Antibodies directed against t cell immunoglobulin and mucin protein 3 (tim-3)
WO2016191643A2 (en) 2015-05-28 2016-12-01 Oncomed Pharmaceuticals, Inc. Tigit-binding agents and uses thereof
WO2016196237A1 (en) 2015-05-29 2016-12-08 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
WO2017019846A1 (en) 2015-07-30 2017-02-02 Macrogenics, Inc. Pd-1-binding molecules and methods use thereof
WO2017025016A1 (en) 2015-08-10 2017-02-16 Innovent Biologics (Suzhou) Co., Ltd. Pd-1 antibodies
WO2017030823A2 (en) 2015-08-14 2017-02-23 Merck Sharp & Dohme Corp. Anti-tigit antibodies
WO2017040790A1 (en) 2015-09-01 2017-03-09 Agenus Inc. Anti-pd-1 antibodies and methods of use thereof
WO2017037707A1 (en) 2015-09-02 2017-03-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human t-cell immunoglobulin and itim domain (tigit)
WO2017053748A2 (en) 2015-09-25 2017-03-30 Genentech, Inc. Anti-tigit antibodies and methods of use
WO2017062748A1 (en) 2015-10-07 2017-04-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Il-7r-alpha specific antibodies for treating acute lymphoblastic leukemia
WO2017084078A1 (en) 2015-11-19 2017-05-26 Zeling Cai Ctla-4 antibodies and uses thereof
WO2017087588A1 (en) 2015-11-18 2017-05-26 Merck Sharp & Dohme Corp. Ctla4 binders
WO2017096017A1 (en) 2015-12-02 2017-06-08 Stsciences, Inc. Antibodies specific to glycosylated btla (b- and t- lymphocyte attenuator)
WO2017106372A1 (en) 2015-12-15 2017-06-22 Oncoimmune, Inc. Chimeric and humanized anti-human ctla4 monoclonal antibodies and uses thereof
WO2017132825A1 (en) 2016-02-02 2017-08-10 华为技术有限公司 Emission power verification method, user equipment, and base station
WO2017133540A1 (en) 2016-02-02 2017-08-10 Innovent Biologics (Suzhou) Co., Ltd. Pd-1 antibodies
WO2017144668A1 (en) 2016-02-26 2017-08-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibodies having specificity for btla and uses thereof
WO2017152088A1 (en) 2016-03-04 2017-09-08 JN Biosciences, LLC Antibodies to tigit
WO2017165464A1 (en) * 2016-03-21 2017-09-28 Elstar Therapeutics, Inc. Multispecific and multifunctional molecules and uses thereof
WO2017194265A1 (en) 2016-05-10 2017-11-16 Agency For Science, Technology And Research Anti-CTLA-4 Antibodies
WO2018025178A1 (en) 2016-08-02 2018-02-08 Aduro Biotech Holdings, Europe B.V. Antibodies against human ctla-4
WO2018033798A1 (en) 2016-08-17 2018-02-22 Compugen Ltd. Anti-tigit antibodies, anti-pvrig antibodies and combinations thereof
WO2018035710A1 (en) 2016-08-23 2018-03-01 Akeso Biopharma, Inc. Anti-ctla4 antibodies
WO2018053106A1 (en) 2016-09-14 2018-03-22 Abbvie Biotherapeutics Inc. Anti-pd-1(cd279) antibodies
WO2018068182A1 (en) 2016-10-10 2018-04-19 Crown Bioscience (Taicang) Inc. Novel anti-ctla4 antibodies
WO2018071919A1 (en) 2016-10-14 2018-04-19 Xencor, Inc. IL15/IL15Rα HETERODIMERIC FC-FUSION PROTEINS
WO2018085469A2 (en) 2016-11-01 2018-05-11 Anaptysbio, Inc. Antibodies directed against t cell immunoglobulin and mucin protein 3 (tim-3)
WO2018102746A1 (en) 2016-12-02 2018-06-07 Rigel Pharmaceuticals, Inc. Antigen binding molecules to tigit
WO2018102536A1 (en) 2016-11-30 2018-06-07 Oncomed Pharmaceuticals, Inc. Methods for treatment of cancer comprising tigit-binding agents
WO2018106862A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
WO2018106864A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Antibodies and methods of use thereof
WO2018129553A1 (en) 2017-01-09 2018-07-12 Tesaro, Inc. Methods of treating cancer with anti-tim-3 antibodies
WO2018156250A1 (en) 2017-02-21 2018-08-30 Remd Biotherapeutics, Inc. Cancer treatment using antibodies that bind cytotoxic t-lymphocyte antigen-4 (ctla-4)
WO2018160704A1 (en) 2017-02-28 2018-09-07 Adimab Llc Anti-tigit antibodies
WO2018160536A1 (en) 2017-02-28 2018-09-07 Bristol-Myers Squibb Company Use of anti-ctla-4 antibodies with enhanced adcc to enhance immune response to a vaccine
WO2018165895A1 (en) 2017-03-15 2018-09-20 苏州银河生物医药有限公司 Ctla4 antibody, pharmaceutical composition and use thereof
WO2018184964A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of an anti-pd-1 antibody with a mutant il-2 or with il-15
WO2018200430A1 (en) 2017-04-26 2018-11-01 Bristol-Myers Squibb Company Methods of antibody production that minimize disulfide bond reduction
WO2018204363A1 (en) 2017-05-01 2018-11-08 Agenus Inc. Anti-tigit antibodies and methods of use thereof
WO2018209701A1 (en) 2017-05-19 2018-11-22 Wuxi Biologics (Shanghai) Co., Ltd. Novel monoclonal antibodies to cytotoxic t-lymphocyte-associated protein 4 (ctla-4)
WO2019023504A1 (en) 2017-07-27 2019-01-31 Iteos Therapeutics Sa Anti-tigit antibodies
WO2019023482A1 (en) 2017-07-27 2019-01-31 Regeneron Pharmaceuticals, Inc. Anti-ctla-4 antibodies and uses thereof
WO2019051015A1 (en) 2017-09-06 2019-03-14 Yale University Interleukin-18 variants and methods of use
WO2019056281A1 (en) 2017-09-21 2019-03-28 Eucure (Beijing) Biopharma Co., Ltd Anti-ctla4 antibodies and uses thereof
WO2019062832A1 (en) 2017-09-29 2019-04-04 江苏恒瑞医药股份有限公司 Tigit antibody, antigen-binding fragment thereof, and medical use thereof
WO2019120232A1 (en) 2017-12-20 2019-06-27 Harbour Biomed (Shanghai) Co., Ltd Antibodies binding ctla-4 and uses thereof
WO2019129221A1 (en) 2017-12-28 2019-07-04 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against tigit
WO2019129261A1 (en) 2017-12-30 2019-07-04 Beigene, Ltd. Anti-tigit antibodies and their use as therapeutics and diagnostics
WO2019137548A1 (en) 2018-01-15 2019-07-18 Nanjing Legend Biotech Co., Ltd. Antibodies and variants thereof against tigit
WO2019148444A1 (en) 2018-02-02 2019-08-08 Adagene Inc. Anti-ctla4 antibodies and methods of making and using the same
WO2019152574A1 (en) 2018-02-01 2019-08-08 Merck Sharp & Dohme Corp. Methods for treating cancer or infection using a combination of an anti-pd-1 antibody, an anti-lag3 antibody, and an anti-tigit antibody
WO2019154415A1 (en) 2018-02-06 2019-08-15 I-Mab Antibodies to t cell immunoreceptor with ig and itim domains (tigit) and uses thereof
WO2019168382A1 (en) 2018-02-28 2019-09-06 Yuhan Corporation Anti-tigit antibodies and uses thereof
WO2019174603A1 (en) 2018-03-14 2019-09-19 上海开拓者生物医药有限公司 Antibody targeting ctla-4 , preparation method therefor and use thereof
WO2019179388A1 (en) 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel anti-ctla-4 antibody polypeptide
WO2019179391A1 (en) 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel bispecific pd-1/ctla-4 antibody molecules
US20190336496A1 (en) 2018-02-16 2019-11-07 Abbvie Inc. Selective bcl-2 inhibitors in combination with an anti-pd-1 or an anti-pd-l1 antibody for the treatment of cancers
WO2019215728A1 (en) 2018-05-09 2019-11-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human nectin4
WO2019232484A1 (en) 2018-06-01 2019-12-05 Compugen Ltd Anti-pvrig/anti-tigit bispecific antibodies and methods of use
WO2020077190A1 (en) 2018-10-12 2020-04-16 Jumaa Weinacht Hassan Monoclonal antibody for treating acute lymphoblastic leukemia
WO2020127366A1 (en) 2018-12-21 2020-06-25 Ose Immunotherapeutics Humanized anti-human-pd-1 antibody
EP3885357A1 (en) 2018-11-22 2021-09-29 Hyquo Molecule (Beijing) Technology Co., Ltd Interleukin 21 protein (il21) mutant and use thereof
WO2021189139A1 (en) * 2020-03-23 2021-09-30 Blackler Ryan Masked il12 fusion proteins and methods of use thereof
WO2022038417A2 (en) 2020-08-19 2022-02-24 Bright Peak Therapeutics Ag Modified il-18 polypeptides and uses thereof
WO2022094473A1 (en) 2020-11-02 2022-05-05 Simcha IL-18, Inc. Interleukin-18 variants and methods of use
WO2022117692A2 (en) 2020-12-04 2022-06-09 F. Hoffmann-La Roche Ag Ph-dependent mutant interleukin-2 polypeptides
WO2022172944A1 (en) 2021-02-10 2022-08-18 国立大学法人 長崎大学 Novel human interleukin-18 variant and use therefor
WO2022214653A1 (en) * 2021-04-09 2022-10-13 Ose Immunotherapeutics New scaffold for bifunctional molecules with improved properties
WO2022236292A2 (en) * 2021-05-07 2022-11-10 The University Of Chicago Compositions and methods comprising protease-activated therapeutic agents
WO2023056193A2 (en) 2021-09-29 2023-04-06 Chimera Bioengineering, Inc. Il-18 variants and uses thereof
WO2023118497A1 (en) 2021-12-22 2023-06-29 Pieris Pharmaceuticals Gmbh Novel il-18 variants
WO2023164288A2 (en) * 2022-02-28 2023-08-31 Xilio Development, Inc. Targeted cytokines and methods of use thereof
EP4273158A1 (en) 2020-12-23 2023-11-08 Innovent Biologics (Suzhou) Co., Ltd. Interleukin-21 mutant and use thereof
WO2024002170A1 (en) 2022-06-29 2024-01-04 Beijing Neox Biotech Limited Il-21 polypeptides and methods of use
WO2024044780A1 (en) 2022-08-26 2024-02-29 Sutro Biopharma, Inc. Interleukin-18 variants and uses thereof
WO2024051728A1 (en) 2022-09-06 2024-03-14 I-Mab Biopharma Co., Ltd. Il-18 variant polypeptides
WO2024068705A1 (en) 2022-09-29 2024-04-04 F. Hoffmann-La Roche Ag Protease-activated polypeptides
WO2024102693A2 (en) 2022-11-07 2024-05-16 Xencor, Inc. Il-18-fc fusion proteins

Patent Citations (121)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5229A (en) 1847-08-07 anthony
US109A (en) 1836-12-15 Erawijtg
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
US8216805B2 (en) 1995-03-01 2012-07-10 Genentech, Inc. Knobs and holes heteromeric polypeptides
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO2001058957A2 (en) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US20030124678A1 (en) 2001-08-13 2003-07-03 University Of Southern California Interleukin-2 mutants with reduced toxicity
US20070036752A1 (en) 2001-12-04 2007-02-15 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
WO2004000238A2 (en) 2002-03-22 2003-12-31 Tittle Thomas V Utilization of mhc class ii binding motifs in immunization to produce immune serum, monoclonal antibodies and vaccines
WO2004091517A2 (en) 2003-04-15 2004-10-28 Smithkline Beecham Corporation Conjugates comprising human il-18 and substitution mutants thereof
WO2005035565A1 (en) 2003-10-10 2005-04-21 Novo Nordisk A/S Il-21 derivatives
WO2006111524A2 (en) 2005-04-18 2006-10-26 Novo Nordisk A/S Il-21 variants
WO2006121168A1 (en) 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2007008463A2 (en) 2005-07-07 2007-01-18 Coley Pharmaceutical Group, Inc. Anti-ctla-4 antibody and cpg-motif-containing synthetic oligodeoxynucleotide combination therapy for cancer treatment
WO2008034473A1 (en) 2006-09-20 2008-03-27 Dge Dr.-Ing. Günther Engineering Gmbh Method and device for separating methane and carbon dioxide from biogas
WO2008049920A2 (en) 2006-10-26 2008-05-02 Novo Nordisk A/S Il-21 variants
WO2008076560A2 (en) 2006-11-15 2008-06-26 Medarex, Inc. Human monoclonal antibodies to btla and methods of use
WO2008074863A1 (en) 2006-12-21 2008-06-26 Novo Nordisk A/S Interleukin-21 variants with altered binding to the il-21 receptor
WO2008132601A1 (en) 2007-04-30 2008-11-06 Immutep Cytotoxic anti-lag-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease
WO2009080253A1 (en) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
WO2009100140A1 (en) 2008-02-04 2009-08-13 Medarex, Inc. Anti-clta-4 antibodies with reduced blocking of binding of ctla-4 to b7 and uses thereof
EP2320940A2 (en) 2008-08-11 2011-05-18 Medarex, Inc. Human antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2010103038A1 (en) 2009-03-11 2010-09-16 Novo Nordisk A/S Interleukin-21 variants having antagonistic binding to the il-21 receptor
WO2010106051A1 (en) 2009-03-17 2010-09-23 Universite De La Mediterranee Btla antibodies and uses thereof
WO2011014438A1 (en) 2009-07-31 2011-02-03 N.V. Organon Fully human antibodies to btla
WO2011104687A1 (en) 2010-02-24 2011-09-01 Rinat Neuroscience Corporation Antagonist anti-il-7 receptor antibodies and methods
WO2011155607A1 (en) 2010-06-11 2011-12-15 協和発酵キリン株式会社 Anti-tim-3 antibody
US8552156B2 (en) 2010-06-11 2013-10-08 Kyowa Hakko Kirin Co., Ltd Anti-TIM-3 antibody
US20140044728A1 (en) 2010-06-11 2014-02-13 Kyushu University, National University Corporation Anti-tim-3 antibody
EP2581113A1 (en) 2010-06-11 2013-04-17 Kyowa Hakko Kirin Co., Ltd. Anti-tim-3 antibody
US20120149876A1 (en) 2010-11-05 2012-06-14 Zymeworks Inc. Stable Heterodimeric Antibody Design with Mutations in the Fc Domain
WO2012107417A1 (en) 2011-02-10 2012-08-16 Roche Glycart Ag Mutant interleukin-2 polypeptides
WO2012120125A1 (en) 2011-03-09 2012-09-13 Antitope Ltd Humanised anti ctla-4 antibodies
WO2013006490A2 (en) 2011-07-01 2013-01-10 Cellerant Therapeutics, Inc. Antibodies that specifically bind to tim3
WO2014102430A1 (en) 2012-12-27 2014-07-03 Consejo Superior De Investigaciones Científicas (Csic) Therapeutic treatment of t- and b-acute lymphoblastic leukemia and human lymphomas by inhibition of the interleukin-7 receptor (il-7r)
WO2014145806A2 (en) 2013-03-15 2014-09-18 Xencor, Inc. Heterodimeric proteins
WO2014194302A2 (en) 2013-05-31 2014-12-04 Sorrento Therapeutics, Inc. Antigen binding proteins that bind pd-1
WO2016015675A1 (en) 2014-08-01 2016-02-04 中山康方生物医药有限公司 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
WO2016028656A1 (en) 2014-08-19 2016-02-25 Merck Sharp & Dohme Corp. Anti-tigit antibodies
WO2016059512A1 (en) 2014-10-18 2016-04-21 Pfizer Inc. Anti-il-7r antibody compositions
WO2016106302A1 (en) 2014-12-23 2016-06-30 Bristol-Myers Squibb Company Antibodies to tigit
WO2016130898A2 (en) 2015-02-13 2016-08-18 Sorrento Therapeutics, Inc. Antibody therapeutics that bind ctla4
WO2016161270A1 (en) 2015-04-01 2016-10-06 Anaptysbio, Inc. Antibodies directed against t cell immunoglobulin and mucin protein 3 (tim-3)
WO2016191643A2 (en) 2015-05-28 2016-12-01 Oncomed Pharmaceuticals, Inc. Tigit-binding agents and uses thereof
WO2016196237A1 (en) 2015-05-29 2016-12-08 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
WO2017019846A1 (en) 2015-07-30 2017-02-02 Macrogenics, Inc. Pd-1-binding molecules and methods use thereof
WO2017025016A1 (en) 2015-08-10 2017-02-16 Innovent Biologics (Suzhou) Co., Ltd. Pd-1 antibodies
WO2017024465A1 (en) 2015-08-10 2017-02-16 Innovent Biologics (Suzhou) Co., Ltd. Pd-1 antibodies
WO2017030823A2 (en) 2015-08-14 2017-02-23 Merck Sharp & Dohme Corp. Anti-tigit antibodies
WO2017040790A1 (en) 2015-09-01 2017-03-09 Agenus Inc. Anti-pd-1 antibodies and methods of use thereof
WO2017037707A1 (en) 2015-09-02 2017-03-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human t-cell immunoglobulin and itim domain (tigit)
WO2017053748A2 (en) 2015-09-25 2017-03-30 Genentech, Inc. Anti-tigit antibodies and methods of use
WO2017062748A1 (en) 2015-10-07 2017-04-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Il-7r-alpha specific antibodies for treating acute lymphoblastic leukemia
WO2017087588A1 (en) 2015-11-18 2017-05-26 Merck Sharp & Dohme Corp. Ctla4 binders
WO2017084078A1 (en) 2015-11-19 2017-05-26 Zeling Cai Ctla-4 antibodies and uses thereof
WO2017096017A1 (en) 2015-12-02 2017-06-08 Stsciences, Inc. Antibodies specific to glycosylated btla (b- and t- lymphocyte attenuator)
WO2017106372A1 (en) 2015-12-15 2017-06-22 Oncoimmune, Inc. Chimeric and humanized anti-human ctla4 monoclonal antibodies and uses thereof
WO2017132825A1 (en) 2016-02-02 2017-08-10 华为技术有限公司 Emission power verification method, user equipment, and base station
WO2017133540A1 (en) 2016-02-02 2017-08-10 Innovent Biologics (Suzhou) Co., Ltd. Pd-1 antibodies
WO2017144668A1 (en) 2016-02-26 2017-08-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibodies having specificity for btla and uses thereof
WO2017152088A1 (en) 2016-03-04 2017-09-08 JN Biosciences, LLC Antibodies to tigit
WO2017165464A1 (en) * 2016-03-21 2017-09-28 Elstar Therapeutics, Inc. Multispecific and multifunctional molecules and uses thereof
WO2017194265A1 (en) 2016-05-10 2017-11-16 Agency For Science, Technology And Research Anti-CTLA-4 Antibodies
WO2018025178A1 (en) 2016-08-02 2018-02-08 Aduro Biotech Holdings, Europe B.V. Antibodies against human ctla-4
WO2018033798A1 (en) 2016-08-17 2018-02-22 Compugen Ltd. Anti-tigit antibodies, anti-pvrig antibodies and combinations thereof
WO2018035710A1 (en) 2016-08-23 2018-03-01 Akeso Biopharma, Inc. Anti-ctla4 antibodies
WO2018053106A1 (en) 2016-09-14 2018-03-22 Abbvie Biotherapeutics Inc. Anti-pd-1(cd279) antibodies
WO2018068182A1 (en) 2016-10-10 2018-04-19 Crown Bioscience (Taicang) Inc. Novel anti-ctla4 antibodies
WO2018071919A1 (en) 2016-10-14 2018-04-19 Xencor, Inc. IL15/IL15Rα HETERODIMERIC FC-FUSION PROTEINS
WO2018085469A2 (en) 2016-11-01 2018-05-11 Anaptysbio, Inc. Antibodies directed against t cell immunoglobulin and mucin protein 3 (tim-3)
WO2018102536A1 (en) 2016-11-30 2018-06-07 Oncomed Pharmaceuticals, Inc. Methods for treatment of cancer comprising tigit-binding agents
WO2018102746A1 (en) 2016-12-02 2018-06-07 Rigel Pharmaceuticals, Inc. Antigen binding molecules to tigit
WO2018106862A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
WO2018106864A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Antibodies and methods of use thereof
WO2018129553A1 (en) 2017-01-09 2018-07-12 Tesaro, Inc. Methods of treating cancer with anti-tim-3 antibodies
WO2018156250A1 (en) 2017-02-21 2018-08-30 Remd Biotherapeutics, Inc. Cancer treatment using antibodies that bind cytotoxic t-lymphocyte antigen-4 (ctla-4)
WO2018160704A1 (en) 2017-02-28 2018-09-07 Adimab Llc Anti-tigit antibodies
WO2018160536A1 (en) 2017-02-28 2018-09-07 Bristol-Myers Squibb Company Use of anti-ctla-4 antibodies with enhanced adcc to enhance immune response to a vaccine
WO2018165895A1 (en) 2017-03-15 2018-09-20 苏州银河生物医药有限公司 Ctla4 antibody, pharmaceutical composition and use thereof
WO2018184964A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of an anti-pd-1 antibody with a mutant il-2 or with il-15
WO2018200430A1 (en) 2017-04-26 2018-11-01 Bristol-Myers Squibb Company Methods of antibody production that minimize disulfide bond reduction
WO2018204363A1 (en) 2017-05-01 2018-11-08 Agenus Inc. Anti-tigit antibodies and methods of use thereof
WO2018209701A1 (en) 2017-05-19 2018-11-22 Wuxi Biologics (Shanghai) Co., Ltd. Novel monoclonal antibodies to cytotoxic t-lymphocyte-associated protein 4 (ctla-4)
WO2019023504A1 (en) 2017-07-27 2019-01-31 Iteos Therapeutics Sa Anti-tigit antibodies
WO2019023482A1 (en) 2017-07-27 2019-01-31 Regeneron Pharmaceuticals, Inc. Anti-ctla-4 antibodies and uses thereof
WO2019051015A1 (en) 2017-09-06 2019-03-14 Yale University Interleukin-18 variants and methods of use
WO2019056281A1 (en) 2017-09-21 2019-03-28 Eucure (Beijing) Biopharma Co., Ltd Anti-ctla4 antibodies and uses thereof
WO2019062832A1 (en) 2017-09-29 2019-04-04 江苏恒瑞医药股份有限公司 Tigit antibody, antigen-binding fragment thereof, and medical use thereof
WO2019120232A1 (en) 2017-12-20 2019-06-27 Harbour Biomed (Shanghai) Co., Ltd Antibodies binding ctla-4 and uses thereof
WO2019129221A1 (en) 2017-12-28 2019-07-04 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against tigit
WO2019129261A1 (en) 2017-12-30 2019-07-04 Beigene, Ltd. Anti-tigit antibodies and their use as therapeutics and diagnostics
WO2019137548A1 (en) 2018-01-15 2019-07-18 Nanjing Legend Biotech Co., Ltd. Antibodies and variants thereof against tigit
WO2019152574A1 (en) 2018-02-01 2019-08-08 Merck Sharp & Dohme Corp. Methods for treating cancer or infection using a combination of an anti-pd-1 antibody, an anti-lag3 antibody, and an anti-tigit antibody
WO2019148444A1 (en) 2018-02-02 2019-08-08 Adagene Inc. Anti-ctla4 antibodies and methods of making and using the same
WO2019154415A1 (en) 2018-02-06 2019-08-15 I-Mab Antibodies to t cell immunoreceptor with ig and itim domains (tigit) and uses thereof
US20190336496A1 (en) 2018-02-16 2019-11-07 Abbvie Inc. Selective bcl-2 inhibitors in combination with an anti-pd-1 or an anti-pd-l1 antibody for the treatment of cancers
WO2019168382A1 (en) 2018-02-28 2019-09-06 Yuhan Corporation Anti-tigit antibodies and uses thereof
WO2019174603A1 (en) 2018-03-14 2019-09-19 上海开拓者生物医药有限公司 Antibody targeting ctla-4 , preparation method therefor and use thereof
WO2019179388A1 (en) 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel anti-ctla-4 antibody polypeptide
WO2019179391A1 (en) 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel bispecific pd-1/ctla-4 antibody molecules
WO2019215728A1 (en) 2018-05-09 2019-11-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human nectin4
WO2019232484A1 (en) 2018-06-01 2019-12-05 Compugen Ltd Anti-pvrig/anti-tigit bispecific antibodies and methods of use
WO2020077190A1 (en) 2018-10-12 2020-04-16 Jumaa Weinacht Hassan Monoclonal antibody for treating acute lymphoblastic leukemia
EP3885357A1 (en) 2018-11-22 2021-09-29 Hyquo Molecule (Beijing) Technology Co., Ltd Interleukin 21 protein (il21) mutant and use thereof
WO2020127366A1 (en) 2018-12-21 2020-06-25 Ose Immunotherapeutics Humanized anti-human-pd-1 antibody
WO2021189139A1 (en) * 2020-03-23 2021-09-30 Blackler Ryan Masked il12 fusion proteins and methods of use thereof
WO2022038417A2 (en) 2020-08-19 2022-02-24 Bright Peak Therapeutics Ag Modified il-18 polypeptides and uses thereof
WO2022094473A1 (en) 2020-11-02 2022-05-05 Simcha IL-18, Inc. Interleukin-18 variants and methods of use
WO2022117692A2 (en) 2020-12-04 2022-06-09 F. Hoffmann-La Roche Ag Ph-dependent mutant interleukin-2 polypeptides
EP4273158A1 (en) 2020-12-23 2023-11-08 Innovent Biologics (Suzhou) Co., Ltd. Interleukin-21 mutant and use thereof
WO2022172944A1 (en) 2021-02-10 2022-08-18 国立大学法人 長崎大学 Novel human interleukin-18 variant and use therefor
WO2022214653A1 (en) * 2021-04-09 2022-10-13 Ose Immunotherapeutics New scaffold for bifunctional molecules with improved properties
WO2022236292A2 (en) * 2021-05-07 2022-11-10 The University Of Chicago Compositions and methods comprising protease-activated therapeutic agents
WO2023056193A2 (en) 2021-09-29 2023-04-06 Chimera Bioengineering, Inc. Il-18 variants and uses thereof
WO2023118497A1 (en) 2021-12-22 2023-06-29 Pieris Pharmaceuticals Gmbh Novel il-18 variants
WO2023164288A2 (en) * 2022-02-28 2023-08-31 Xilio Development, Inc. Targeted cytokines and methods of use thereof
WO2024002170A1 (en) 2022-06-29 2024-01-04 Beijing Neox Biotech Limited Il-21 polypeptides and methods of use
WO2024044780A1 (en) 2022-08-26 2024-02-29 Sutro Biopharma, Inc. Interleukin-18 variants and uses thereof
WO2024051728A1 (en) 2022-09-06 2024-03-14 I-Mab Biopharma Co., Ltd. Il-18 variant polypeptides
WO2024068705A1 (en) 2022-09-29 2024-04-04 F. Hoffmann-La Roche Ag Protease-activated polypeptides
WO2024102693A2 (en) 2022-11-07 2024-05-16 Xencor, Inc. Il-18-fc fusion proteins

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS
"Remington's Pharmaceutical Sciences", 1980
ANGAL S ET AL., MOL. IMMUNOL., vol. 30, 1993, pages 105 - 8
ANTONIA ET AL.: "Immuno-oncology combinations: a review of clinical experience and future prospects.", CLIN. CANCER RES. OFF. J. AM. ASSOC. CANCER RES., vol. 20, 2014, pages 6258 - 6268, XP055320194, DOI: 10.1158/1078-0432.CCR-14-1457
ATWELL ET AL., J. MOL. BIOL., vol. 270, 1997, pages 26
CAO ET AL., NAT. COMMUN, vol. 12, 2021, pages 5866
CHAN ET AL., NAT. CANCER, 2022
COMBS ET AL., AAPS J., vol. 17, no. 2, March 2015 (2015-03-01), pages 339 - 51
DESBOIS ET AL., J IMMUNOL, 2026
DESNOYERS ET AL., SCI. TRANSL. MED., 2013, pages 5
FRANKE ET AL., CANCER BIOTHER RADIOPHARM., vol. 15, 2000, pages 459 - 76
GAO S HHUANG KTU HADLER A S., BMC BIOTECHNOLOGY., vol. 13, 2013, pages 55
GERBER ET AL., MABS, vol. 1, 2009, pages 247 - 253
GUNASEKARAN ET AL., J. BIOL. CHEM., vol. 285, no. 25, 2010, pages 19637
GUO ET AL., ADV CANCER RES., vol. 119, 2013, pages 421 - 475
GUO ET AL., J IMMUNOL, vol. 195, no. 5, 2015, pages 2353 - 64
HEATON ET AL., CANCER RES, vol. 53, 1993, pages 2597 - 602
HSU ET AL., NAT. COMMUN., vol. 12, 2021, pages 2768
HU ET AL., BLOOD, vol. 101, 2003, pages 4853 - 4861
JADHAV ET AL., PNAS, vol. 116, 2019, pages 14113 - 14118
MERCHANT ET AL., NATURE BIOTECH., vol. 16, 1998, pages 677
NOVELLINO ET AL., CANCER IMMUNOL IMMUNOTHER., vol. 54, 2005, pages 187 - 207
PARMIANI ET AL., J IMMUNOL., vol. 178, 2007, pages 1975 - 9
RIDGWAY ET AL., PROTEIN ENGINEERING, vol. 9, no. 7, 1996, pages 617
S6DING, J.: "Protein homology detection by HMM-HMM comparison'.", BIOINFORMATICS, vol. 21, 2005, pages 951 - 960
SCHAEFER, W. ET AL., PNAS, vol. 108, 2011, pages 11187 - 1191
SCHEFF ET AL., PHARM RES., vol. 28, no. 5, May 2011 (2011-05-01), pages 1081 - 9
SHANAFELT ET AL., NATURE BIOTECHNOL, vol. 18, 2000, pages 1197 - 1202
SHEN SSCKISEL GSAHOO ALALANI AOTTER DDPEARSON JDEVOSS JCHENG JCASEY SCCASE R: "Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity.", FRONT IMMUNOL., vol. 11, 8 May 2020 (2020-05-08), pages 832, XP055854614, DOI: 10.3389/fimmu.2020.00832
SIDDIQUI ET AL., IMMUNITY, vol. 50, 2019, pages 195 - 211
SI-YANG LIU ET AL., J. HEMATOL. ONCOL., vol. 10, 2017, pages 136
VINCENT, MARIE ET AL.: "Antitumor activity of an immunocytokine composed of an anti-GD2 antibody and the IL-15 superagonist RLI", ONCOIMMUNOLOGY, vol. 2, no. 11, 2013, pages e26441, XP055863986, DOI: 10.4161/onci.26441
WALLES M. ET AL: "ADME and Safety Aspects of Non-cleavable Linkers in Drug Discovery and Development", CURRENT TOPICS IN MEDICINAL CHEMISTRY, vol. 17, no. 32, 23 February 2018 (2018-02-23), NL, pages 3463 - 3475, XP093218248, ISSN: 1568-0266, DOI: 10.2174/1568026618666180118153502 *
WU ET AL., CELL REP MED, ., vol. 5, no. 10, 15 October 2024 (2024-10-15), pages 101747
XU YUANMING ET AL: "An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity", CANCER IMMUNOLOGY RESEARCH, vol. 9, no. 10, 1 October 2021 (2021-10-01), US, pages 1141 - 1157, XP093054460, ISSN: 2326-6066, DOI: 10.1158/2326-6066.CIR-21-0058 *
XU, YUANMING ET AL.: "An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity", CANCER IMMUNOLOGY RESEARCH, vol. 9, no. 10, 2021, pages 1141 - 1157, XP093054460, DOI: 10.1158/2326-6066.CIR-21-0058

Similar Documents

Publication Publication Date Title
EP3897845B1 (en) Bifunctional molecule directed against human pd-1
JP7675012B2 (en) Bifunctional anti-PD-1/SIRPA molecule
CA3197935A1 (en) Interleukin-2-fc fusion proteins and methods of use
WO2021122866A1 (en) Bifunctional molecules comprising an il-7 variant
IL323895A (en) Bifunctional anti-PD-1/IL-7 molecule
US20240182571A1 (en) New scaffold for bifunctional molecules with improved properties
US20240067727A1 (en) Bifunctional anti-pd1/il-7 molecules
WO2020165374A1 (en) Bifunctional molecule comprising il-15ra
US20230303648A1 (en) Bifunctional molecules comprising an il-7 variant
JP2025525886A (en) Multifunctional molecules against CD28
CN117412989A (en) New scaffolds for bifunctional molecules with improved properties
US20240182572A1 (en) Scaffold for bifunctional molecules comprising pd-1 or cd28 and sirp binding domains
WO2025242835A1 (en) Molecules comprising masking linkers and uses thereof for the treatment of cancer
WO2025242836A1 (en) Molecules comprising masking linkers and uses thereof for the treatment of auto-immune or inflammatory diseases and disorders

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25727407

Country of ref document: EP

Kind code of ref document: A1