OA21499A

OA21499A - Immunocytokine for activating human IL10RA receptor and use thereof.

Info

Publication number: OA21499A
Application number: OA1202300217
Authority: OA
Inventors: Dmitry Valentinovich MOROZOV; Pavel Andreevich IAKOVLEV; Aleksey Vladimirovich KONONOV; luliia Viktorovna EVDOKIMOVSKAIA; lana Andreevna SMIRNOVA; Stanislav Rudolfovich EVDOKIMOV; Elena Sergeevna KOLOSOVA; Sergei Andreevich AGEEV; Vladimir Sergeevich TSYMPILOV; Valéry Vladimirovich SOLOVYEV
Original assignee: Joint Stock Company ‘‘Biocad’’
Priority date: 2020-12-10
Filing date: 2021-12-10
Publication date: 2024-07-31

Abstract

The present invention relates to the field of biotechnology and medicine, in particular to an immunocytokine for activating the human IL-10Ra receptor. The invention further relates to nucleic acids encoding said immunocytokine, expression vectors, host cells and methods for producing same, methods for producing the immunocytokine, pharmaceutical compositions comprising the above immunocytokine, pharmaceutical compositions comprising the above immunocytokine and other therapeutically active compounds, methods for treating an oncological disease and the uses of the immunocytokine or pharmaceutical compositions thereof for treating an oncological disease.

Description

IMMUNOCYTOKINE FOR ACTIVATING HUMAN IL-10RA RECEPTOR AND USE THEREOF

Field of the invention

The présent invention relates to the field of biotechnology and medicine, in particular 10 to an immunocytokine for activating the human IL-lORa receptor. The invention further relates to nucleic acids encoding said immunocytokine, expression vectors, host cells and methods for producing same, methods for producing the immunocytokine, pharmaceutical compositions comprising the above immunocytokine, pharmaceutical compositions comprising the above immunocytokine and other therapeutically active compounds, methods 15 for treating an oncological disease and the uses of the immunocytokine or pharmaceutical compositions thereof for treating an oncological disease.

Background of the invention

Interleùkin 10 (or IL-10) is a cytokine and until recently was viewed as an anti20 . inflammatory cytokine. It is a homodimer with a molecular weight of approximately 37-39 kDa. Human and interleùkin 10 has high homology with murine interleùkin 10, which is about 80%.

IL-10 is able to abrogate angiogenesis, metastasis, and hâve a direct antitumor effect (Martin Oft, IL-10: Master Switch from Tumor-Promoting Inflammation to Antitumor 25 Immunity, Cancer Immunology at the Crossroads: Experimental Immunothérapies, March 2014, Volume 2, Issue 3,10.1158/2326-6066. CIR-13-0214).

It has been shown that one of the likely antitumor effects of IL-10 is its activating effect on PD-1-expressing CD8 lymphocytes. IL-10 leads to potent antiapoptotic intracellular cascades, which fact contributes to the survival of cytotoxic lymphocytes in the tumor 30 microenvironment (Martin Oft, Immune régulation and cytotoxic T cell activation of IL-10 agonists - Preclinical and clinical expérience, Seminars in Immunology, Volume 44, August 2019, DOI: 10.1016/j.smim.2019.101325).

. IL-10 is characterized by a short plasma half-life due to its small size, which is approximately 37-39 kDa, resulting in rapid rénal clearance; in fact, its half-life in the 35 systemic compartment is 2.5 hours (Braat H. et al., Interleùkin-10-based therapy for inflammatory bowel disease, Expert Opin. Biol. Ther. 3(5), 2003, pp. 725-731). Pegylation of this cytokine has been employed by some authors to increase rétention time in the bloodstream, exposure, efficacy, and to reduce rénal absorption (Mattos A. et al., PEGylation of interleukin-10 improves the pharmacokinetic profile and enhances the antifibrotic effectivity in CClü-induced fibrogenesis in mice, J. Control Release 162, 2012, pp. 84-91 and Mumm J.B. et al., IL-10 elicits IFNy-dependent tumor immune surveillance, Cancer Cell 20(6), 2011, pp. 781-796). However, the disadvantages of pegylation may include potential ‘ risks such as immunogenicity, decreased biological activity of the pegylated molécule and heterogeneity of pegylation, which fact may lead to the production of therapeutic molécules with different properties (size, charge, etc.), and the formation of a hydrophilic coating surrounding the therapeutic molécule. Furthermore, this approach can extend the half-life of the formulation in the bloodstream to no more than 24 hours.

Another method for extending the half-life of IL-10 in plasma is to use fusion proteins 15 comprising an antibody or a fragment thereof and IL-10 for the directional transport of the cytokine to inflamed tissues and accumulation of the cytokine therein.

The prior art provides articles (Zheng, X.X. ET AL., A noncytolytic IL-10/Fc fusion . protein prevents diabètes, blocks autoimmunity, and promûtes suppressor phenomena in NOD mice. J. Immunol. 1997, 158, 4507^1513 h Zheng, X.X. ET AL., Administration of 20 noncytolytic IL-10/Fc in murine models of lipopolysaccharide-induced septic shock and . allogeneic islet transplantation. J. Immunol. 1995, 154, 5590-5600), which describe an immunocytokine based on IL-10 and an Fc fragment.

International applications WO2012045334 and WO2014023673 further provide a fusion protein comprising a heterodimeric complex based on IL-10 and an Fc fragment.

International application WO2015117930 provides a fusion protein comprising an . IgG-class antibody and a mutant IL-10 molécule, wherein the fusion protein comprises two identical heavy chain polypeptides and two identical light chain polypeptides, and wherein the mutant IL-10 molécule comprises an amino acid substitution that reduces the binding affinity of the mutant IL-10 molécule to the IL-10 receptor, as compared to a wild- type IL30 10 molécule.

The above fusion proteins (immunocytokines) based on IL-10 and Fc fragment hâve a . common disadvantage, in particular insufficient stability in the blood, since they are prone to dégradation by sérum proteases.

In connection with the above, there is a need to develop fusion proteins 35 (immunocytokines) based on IL-10 and Fc fragment, which will show an extended half-life in blood plasma as compared to IL-10 and will be stable due to résistance to sérum protease dégradation.

Brief description of the invention

As found by the authors, the immunocytokine comprising a homodimeric complex based on IL-10 and human IgGl Fc fragment provided in the présent invention has an extended plasma half-life as compared to that of IL-10 and is stable due to résistance to sérum protease dégradation thanks to the unique structure thereof.

In one aspect, the présent invention relates to an immunocytokine for activating the human IL-lORa receptor, which comprises a homodimeric complex based on IL-10 and human IgGl Fc fragment, wherein the monomer based on IL-10 and human IgGl Fc fragment comprises the amino acid sequence of SEQ ID NO:1.

In one aspect, the présent invention relates to an isolated nucleic acid that encodes the 15 above immunocytokine.

In some embodiments, the isolated nucleic acid is DNA.

In some embodiments, the isolated nucleic acid includes the nucléotide sequence with SEQIDNO:3.

In some embodiments, the isolated nucleic acid includes the nucléotide sequence with

SEQIDNO:4.

In one aspect, the présent invention relates to an expression vector comprising any of . the above nucleic acids.

In one aspect, the présent invention relates to a method for production of a host cell for production of the above immunocytokine of the invention, which comprises 25 transformation of the cell with the above vector.

In one aspect, the présent invention relates to a host cell for production of the above immunocytokine of the invention, the host cell comprises any of the above nucleic acids.

In one aspect, the présent invention relates to a method for preparing a formulation . comprising the above immunocytokine of the invention, which comprises culturing of the 30 above host cell in a culture medium under conditions sufficient to produce said . immunocytokine, if necessary, followed by isolation and purification of the resulting immunocytokine of the invention.

In one aspect, the présent invention relates to a pharmaceutical composition for activating the human IL-lORa receptor, which comprises the above immunocytokine of the 35 invention and one or more pharmaceutically acceptable excipients.

In one aspect, the présent invention relates to a pharmaceutical composition for activating the human IL-lORa receptor, which comprises the above immunocytokine of the invention and at least one other therapeutically active compound.

In some embodiments, the pharmaceutical composition is used for the treatment of an oncological disease.

In some embodiments, the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast 10 cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small . cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite . . instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell 15 lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone . B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higherrisk myelodysplastic syndrome.

. In some embodiments of the pharmaceutical composition, other therapeutically active compound is used that is an antibody, chemotherapeutic agent, or hormone therapy agent.

In some embodiments of the pharmaceutical composition, other therapeutically active compound is used that is an immune checkpoint inhibitor.

In some embodiments of the pharmaceutical composition, an immune checkpoint inhibitor is used that is selected from a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor.

In some embodiments of the pharmaceutical composition, a PD-L1 inhibitor is used 25 that is an antibody that specifically binds to PD-L1.

In some embodiments of the pharmaceutical composition, an antibody is used that specifically binds to PD-L1 and is selected from the group comprising durvalumab, avelumab, atezolizumab, manelimab.

. In some embodiments of the pharmaceutical composition, a PD-1 inhibitor is used that is an antibody that specifically binds to PD-1.

In some embodiments of the pharmaceutical composition, an antibody is used that specifically binds to PD-1 and is selected from the group comprising prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the pharmaceutical composition, a CTLA-4 inhibitor is used 35 that is an antibody that specifically binds to CTLA-4.

In some embodiments of the pharmaceutical composition, an antibody is used that specifically binds to CTLA-4 and is ipilimumab.

In one aspect, the présent invention relates to a pharmaceutical combination for activating the human IL-lORa receptor, which comprises the above immunocytokine of the . invention and at least one other therapeutically active compound.

In some embodiments, the pharmaceutical combination is used for the treatment of an oncological disease.

In some embodiments, the oncological disease is selected from the group comprising:

HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown . primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher- risk myelodysplastic syndrome.

In some embodiments of the pharmaceutical combination, other therapeutically active compound is used that is an antibody, chemotherapeutic agent, or hormone therapy agent.

In some embodiments of the pharmaceutical combination, other therapeutically active compound is used that is an immune checkpoint inhibitor.

In some embodiments of the pharmaceutical combination, an immune checkpoint inhibitor is used that is selected from a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor.

In some embodiments of the pharmaceutical combination, a PD-L1 inhibitor is used that is an antibody that specifically binds to PD-L1.

In some embodiments of the pharmaceutical combination, an antibody is used that specifically binds to PD-L1 and is selected from the group comprising durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the pharmaceutical combination, a PD-1 inhibitor is used that is an antibody that specifically binds to PD-1.

In some embodiments of the pharmaceutical combination, an antibody is used that specifically binds to PD-1 and is selected from the group comprising prolgolimab, , pembrolizumab, nivolumab.

In some embodiments of the pharmaceutical combination, a CTLA-4 inhibitor is used ; that is an antibody that specifically binds to CTLA-4.

I

In some embodiments of the pharmaceutical combination, an antibody is used that specifically binds to CTLA-4 and is ipilimumab.

In one aspect, the présent invention relates to a method for treatment of an oncological disease, which comprises administering to a subject in need of such treatment the above immunocytokine of the invention or any above pharmaceutical composition, in 10. . a therapeutically effective amount.

, In some embodiments of the method for treatment, the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC . (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, 15 melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, . glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, 20 follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

In one aspect, the présent invention relates to a method for activating the human IL10Ra receptor in a subject in need of such activation, which comprises administering to a subject in need of such treatment an effective amount of the above immunocytokine of the 25 invention or any above pharmaceutical composition, in a therapeutically effective amount.

In one aspect, the présent invention relates to the use of the above immunocytokine of the invention or any above pharmaceutical composition for the treatment of an oncological disease in a subject in need of such treatment.

In one aspect, the présent invention relates to the use of the above immunocytokine of 30 the invention or at least one other therapeutically active compound for the treatment in a subject in need of such treatment of an oncological disease.

In some embodiments of the use, the oncological disease is selected from the group comprising: HNSCC (head,and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary,. glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative 35 breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non- • . small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higherrisk myelodysplastic syndrome.

In some embodiments of the use, other therapeutically active compound is used that is 10 an antibody, chemotherapeutic agent, or hormone therapy agent.

In some embodiments of the use, other therapeutically active compound is used that is an immune checkpoint inhibitor.

In some embodiments of the use, an immune checkpoint inhibitor is used that is ., selected from a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor.

In some embodiments of the use, a PD-L1 inhibitor is used that is an antibody that specifically binds to PD-L1.

: In some embodiments of the use, an antibody is used that specifically binds to PD-L1 / and is selected from the group comprising durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the use, a PD-1 inhibitor is used that is an antibody that 20 specifically binds to PD-1.

In some embodiments of the use, an antibody is used that specifically binds to PD-1 , and is selected from the group comprising prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the use, a CTLA-4 inhibitor is used that is an antibody that specifically binds to CTLA-4.

In some embodiments of the use, an antibody is used that specifically binds to CTLA4 and is ipilimumab.

Brief description of drawings

Figure 1 is a map of the plasmid pIntA-IL-10Ra-exc-hum-EPEA comprising a 30 sequence ofthe extracellular portion of the human IL-10R receptor subunit alpha with an ΕΡΕΑ tag linked to C terminal.

AmpR is a gene that provides résistance to ampicillin pUC is a bacterial origin of réplication : cmv-enhancer is a cytomégalovirus enhancer hCMV promoter is a human cytomégalovirus eukaryotic promoter

Intron A is a sequence of intron A

Kozak is a Kozak consensus sequence for translation initiation mlgK Leader is a leader peptide

Sali is a Sali restriction site . IL-lORa-exc-hum is the extracellular portion of the human IL-10 receptor subunit alpha

/. EPEA-tag is an ΕΡΕΑ tag sequence for protein purification . Kpnl is a Kpnl restriction site

Stop is a stop codon . SV40 PA term is a poly(A) SV40 transcription terminator EBV ori is an eukaryotic origin of réplication

Figure 2 is a map of the plasmid pIntA-N-Fc-LALA-link-hIL-10 comprising a .·..· · sequence of the N-terminal human IgGl Fc fragment having mutations LALA, del GK and 15 cross-linkedto humanIL-10 viaalinker.

\ AmpR is a gene that provides résistance to ampicillin pUC is a bacterial origin of réplication cmv-enhancer is a cytomégalovirus enhancer hCMV promoter is a human cytomégalovirus eukaryotic promoter

Intron A is a sequence of intron A

Kozak is a Kozak consensus sequence for translation initiation . mlgK Leader is a leader peptide

Sali is a Sali restriction site . Fc-LALA-DELGK is a sequence of the human IgGl Fc fragment with a LALA mutation (L19A, L20A according to sequential numbering) and deleted GK (G231, K232 according to sequential numbering) at the C-terminal end

Linker is a linker sequence hIL-10 is a human IL-10 sequence Xbal is an Xbal restriction site

Stop is a stop codon .

SV40_PA term is a poly(A) SV40 transcription terminator EBV bri is an eukaryotic origin of réplication •Y· Figure 3 is PAAG electrophoresis of the extracellular portion of human, cynomolgus and rabbit IL-10 receptors derived and isolated from CHO cells.

1) Protein molecular weight markers

2) Human excIL-lORa.

) Cynomolgus excIL-1 ORa

4) Protein molecular weight markers

5) Rabbit excIL-lORa

Figure 4 is PAAG electrophoresis of the recombinant immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10-Fc) derived and isolated from CHO cells.

1) Protein molecular weight markers

2) IL10-Fc 4.5 pg under reducing conditions . 3) ILIO-Fc 4.5 pg under non-reducing conditions.

Figure 5 is a graph showing the stability of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10+Fc) when stored in human sérum for a long time at +37C. X-axis is the storage time, Y-axis is the relative 15 content of stable molécules in sérum (percent of the original sample).

Figure 6 is a graph showing the ability of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10+Fc) to exhibit proliférative activity as compared to that of IL-10. X-axis is the concentration of IL-10 (positive control human recombinant IL-10 from Peprotech), Y-axis is the relative fluorescence reflecting the 20 number of viable cells. The half maximal effective concentration for the control sample was 91.7 pM, that for IL-10+Fc of the invention was 53.3 pM.

Figure 8 is a graph showing the effect of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10+Fc) on the cytotoxicity of CD8+ T cells against Raji target cells as compared to that of recombinant human IL-10 from 25 Peprotech and control.

Figure 9 is a graph showing the absence of CDC activity in the test immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10+Fc) in an assay employing the Jurkat cell line. The control antibody Rituximab induces complement-mediated lysis of target cells.

Figure 10 is a graph showing the absence of antibody-dependent cellular cytotoxicity (ADCC activity),in the test immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10+Fc) in an assay employing the Jurkat target cells. Anti-PDl antibody was used as a positive control.

Figure 11 is a histogram showing the absence of autocytotoxicity in the 35 immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10+Fc) with respect to subpopulations of human peripheral blood cells at concentrations ofO.Ol pg/ml, 0.1 μg/ml, 1 pg/ml, 10 pg/ml:

Figure 11 A) CD4+ T cells,

Figure 11 B) CD8+ T cells,

Figure 11 C) T cells,

Figure 11 D) NK cells,

Figure 11 E) B cells.

Anti-CD20 antibody inducing autocytotoxicity against B cells and anti-CD47' 10 antibody inducing autocytotoxicity against NK cells were used as a positive control. ;

. Figure 12 is a histogram reflecting the ability of the immunocytokine (fusion protein), based on IL-10 and human IgGl Fc fragment of the invention (IL-10-Fc) and a combination of IL-10-Fc of the invention with anti-mPDl antibody to exert antitumor activity in Balb/c mice vaccinated with CT26 carcinoma. Anti-MPDl antibody was used as a control. '

Figure 13 is a histogram reflecting the ability of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10-Fc) and a combination of IL-10-Fc of the invention with anti-mPDl antibody to exert antitumor activity in Balb/c mice vaccinated with CT26 carcinoma. Anti-MPDl antibody was used as a control. Y-axis is the index of tumor growth (ITG).

Figure 14 is a histogram reflecting the ability of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention (IL-10-Fc) and a combination of IL-10-Fc of the invention with anti-mPDl antibody to exert antitumor activity in Balb/c mice vaccinated with CT26 carcinoma; the histogram shows the indicator of tumor growth inhibition (TGI). Anti-MPDl antibody was used as a control.

Figure 15 is a schematic représentation of the format of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention

Description of the invention . Définitions and general methods ' Unless defïned otherwise herein, ail technical and scientific terms used in connection with the présent invention will hâve the same meaning as is commonly understood by those skilled in the. art. '

Further, unless otherwise required by context, singular terms shall include plural terms, and the plural terms shall include the singular terms. Typically, the présent 35 classification and methods' of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known by those skilled and widely used in the art. Enzyme reactions and . . i.

purification methods are performed according to the manufacturées guidelines, as is common in the art, or as described herein.

Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molécule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, binding affinity refers to intrinsic 10 (characteristic, true) binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molécule X for its binding partner Y can generally be represented by the dissociation constant (Kd). The preferred Kd value is; about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM,' 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by common methods known in the art, 15 including those described in the présent description. Low-affînity antibodies generally bind an antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind an antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the présent invention. -

The term in vitro, refers to a biological entity, a biological process, or a biological reaction outside the body under artificial conditions. For example, a cell grown in vitro is to be understood as a cell grown in an environment outside the body, e.g. in a test tube, a culture vial, or a microtiter plate.

The term IC50 (inhibitory Concentration 50%), as used herein, refers to 25 concentrations of a formulation, at which a measurable activity or response, for example, growth/proliferation of cells such as tumor cells, is inhibited by 50%. IC50 value can be calculated using appropriate dose-response curves, using spécial statistical software for curve fitting.

The term ED50 (EC50) (50% effective dose/concentration) refers to concentrations 30 of a formulation producing 50% biological effect (which may include cytoxicity).

As used in the présent description and daims that follow, unless otherwise dictated by . the context, the words hâve, include, and comprise or variations thereof such as has, having, includes, including, comprises, or comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer 35 or group of integers.

Immunocytokine ।

As surprisingly found by the authors, the immunocytokine comprising a homodimeric complex based on IL-10 and human IgGl Fc fragment provided in the présent invention has an extended plasma half-life as compared to that of IL-10 and is stable due to résistance to· sérum protease dégradation thanks to the unique structure thereof.

. The format of the above immunocytokine is shown in Figure 15.

The term “immunocytokine” means a molécule comprising an antibody or fragments

·. thereof directly or indirectly linked by covalent bonds to a cytokine or dérivatives thereof.

Said antibody and said cytokine can be linked by a linker peptide.

The immunocytokine of the invention is contemplated to refer to an isolated immunocytokine.

15,· The immunocytokine of the invention refers to a fusion protein based on IL-10 and human IgGl Fc fragment.

The term isolated used to describe various immunocytokines of this description means an immunocytokine which has been identified and separated and/or regenerated from a cell or cell culture, in which the immunocytokine is expressed. Impurities (contaminant 20 components) from natural environment are materials which typically interfère with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutés. The isolated polypeptide is typically prepared by at least one purification step.

The immunocytokine of the invention is a recombinant immunocytokine.

The term recombinant immunocytokine is intended to refer to an immunocytokine that is expressed in a cell or cell line comprising nucléotide sequence(s) encoding the immunocytokine, wherein said nucléotide sequence(s) is not naturally associated with the cell.

The fragment crystallizable région (Fc région, Fc fragment) of an immunoglobulin 30 is the tail région of an immunoglobulin molécule that interacts with cell surface Fc. receptor, as well as some proteins of the complément System. This property allows antibodies to activate the immune System. In IgG, IgA and IgD isotypes, the Fc région is composed of two identical protein fragments, respectively, from the second and third constant domains of the two heavy chains; in IgM and IgE isotypes, the Fc contains three heavy chain constant 35 domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

The receptor for IL-10 is a heterotetramer complex comprising two IL-lORa (also referred to as IL-10R1) molécules encoded by the IL-lOra gene and two ILIORP (also referred to as IL-10R2) molécules encoded by the IL-1 Orb gene.

The human IL-lORa receptor (or CD210a, or IL-10R1) refers to interleukin 10 . receptor, alpha subunit. The molecular weight of IL-lORa is 90-120 kDa and serves as a . ligand-binding subunit of the receptor complex. ·

In one aspect, the présent invention relates to an immunocytokine for activating the' human IL-lORa receptor, which comprises a homodimeric complex based on IL-10 and a human IgGl Fc fragment, i.e. a dimer that includes two identical monomers based on IL-10 and a human IgGl Fc fragment, wherein the monomer comprises the amino acid sequence; EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV i \ KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG ¹

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGGGSGGGPGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKD QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENL KTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMT MKIRN (SEQ IDNO:1).

In one embodiment, the présent invention relates to an immunocytokine for activating the human IL-lORa receptor, which comprises a homodimeric complex based on IL-10 and a human IgGl Fc fragment, i.e. a dimer that includes two identical monomers based on IL-10 . and a human IgGl Fc fragment, wherein the monomer has the amino acid sequence of SEQ IDNO:1.

The immunocytokine of the invention includes the L234A and L235A (LALA) mutations in the Fc fragment of a human IgGl antibody to provide effectorless properties of the immunocytokine. These mutations are présent in the above immunocytokine, which includes the amino acid sequence with SEQ ID NO: 1.

In one embodiment; the immunocytokine of the invention comprises a leader peptide.

In one embodiment of the invention, the immunocytokine of the invention with a leader peptide comprises à homodimeric complex based on a leader peptide, IL-10 and a human IgGl Fc fragment, i.e. a dimer that includes two identical monomers based on a leader peptide, IL-10 and human IgGl Fc fragment, wherein the monomer has the amino acid sequence h

MMSFVSLLLVGILFHATQAEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS rtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvl . HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV . ¹³ . ___________i________________________________________________________________________________________

FSCSVMHEALHNHYTQKSLSLSPGGGSGGGPGSGGSPGQGTQSENSCTHFPGNLPNM LRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMP QAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEK .

. . GIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID N0:2). ;

The terms “immunocytokine of the invention”, “immunocytokine based on IL-10 and 10 Fc”, “fusion protein of the invention”, “IL-10-Fc” and “IL-10+Fc” are used interchangeably . herein and refer to the above immunocytokine of the invention. ;

Nucleic acid

In one aspect, the présent invention relates to an isolated nucleic acid that encodes the 15 above immunocytokine of the invention.

The terms nucleic acid, nucleic sequence, nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucléotide sequence, used interchangeably in the présent description, mean a précisé sequence of nucléotides, ' modified or not, determining a fragment or a région of a nucleic acid, containing unnatural 20 nucléotides or not, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs.

It should also be included here that the présent invention does not relate to nucléotide . sequences in their naturel chromosomal environment, i.e. in a naturel State. The sequences of . . the présent invention hâve been isolated and/or purified, i.e., they were sampled directly or indirectly, for example by copying, their environment having been at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by Chemical synthesis should also be mentioned here.

• An isolated nucleic acid molécule is one which is identifïed and separated from at least one nucleic acid molecule-impurity, which the former is bound to in the naturel source.

An isolated nucleic acid molécule is different from the form or set in which it is found under naturel conditions. Thus, an isolated nucleic acid molécule is different from a nucleic acid molécule that exists in cells under naturel conditions. An isolated nucleic acid molécule however includes a nucleic acid molécule located in cells in which the immunocytokine is normally expressed, for example, if the nucleic acid molécule has a chromosomal localization 35 that is different from its localization in cells under naturel conditions.

. In some embodiments, the isolated nucleic acid is DNA.

In one embodiment, the présent invention relates to a nucleic acid molécule comprising a nucléotide sequence that encodes a monomer of a fusion protein based on IL-10 and a human IgGl Fc fragment, wherein the monomer comprises the amino acid sequence of SEQIDNO:1.

In one embodiment, the présent invention relates to a nucleic acid molécule. . comprising a nucléotide sequence that encodes a monomer of a fusion protein based on IL-10 and a human IgGl Fc fragment, wherein the monomer has the amino acid sequence of SEQ 10 IDNO: 1.

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the fusion protein with the amino acid sequence of SEQ ID NO: 1. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such 15 variant DNA sequences are within the scope of the présent invention.

A reference to a nucléotide sequence encompasses the complément thereof unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

In some embodiments, the isolated nucleic acid that encodes the monomer of the fusion protein based on IL-10 and a human IgGl Fc fragment, wherein the monomer . comprises the amino acid sequence of SEQ ID NO: 1, comprises the nucléotide sequence with SEQ ID NO: 3.

In some embodiments, the isolated nucleic acid that encodes the monomer of the 25 fusion protein based on IL-10 and human IgGl Fc fragment, wherein the monomer has the amino acid sequence of SEQ ID NO: 1, has the nucléotide sequence with SEQ ID NO: 3.

In some embodiments, the isolated nucleic acid that encodes the monomer of the . fusion protein based on IL-10 and a human IgGl Fc fragment, wherein the monomer comprises the amino acid sequence of SEQ ID NO: 1, comprises the nucléotide sequence 30 with SEQ ID NO: 4. . <

In some embodiments, the isolated nucleic acid that encodes the monomer of the fusion protein based on IL-10 and human IgGl Fc fragment, wherein the monomer has the amino acid sequence of SEQ ID NO: 1, has the nucléotide sequence with SEQ ID NO: 4.

In some embodiments, the nucleic acid of the invention encodes the monomer ofthe 35 fusion protein based on a leader peptide, IL-10, and human IgGl Fc fragment.

In some embodiments, the nucleic acid that encodes the monomer of the fusion protein based on a leader peptide, IL-10, and human IgGl Fc fragment, wherein the monomer ¹⁵ has the amino acid sequence of SEQ ID NO: 2, has the nucléotide sequence with SEQ ID NO: 5.

In some embodiments, the nucleic acid that encodes the monomer of the fusion, protein based on a leader peptide, IL-10, and human IgGl Fc fragment, wherein the monomer. has the amino acid sequence of SEQ ID NO: 2, has the nucléotide sequence with SEQ ID' 10 NO: 6. i

The above nucleic acid molécules may be used to express the immunocytokine of the; invention. '

Expression vector :

In one aspect, the présent invention relates to an expression vector comprising any of . the above nucléotide sequences.

The présent invention relates to a vector suitable for the expression of any of - nucléotide sequences described herein.

The term vector as used herein means a nucleic acid molécule capable of 20 transporting another nucleic acid to which it has been linked. In some embodiments, a vector is a plasmid, i.e., a circulai^- double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, a vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, vectors are capable of autonomous réplication in a host cell into 25 which they are introduced (e.g. bacterial vectors having a bacterial origin site of réplication and episomal mammalian vectors). In further embodiments, vectors (e.g. non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host gene. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors 30 are referred to herein as recombinant expression vectors (or simply, expression vectors).

, The: présent invention relates to vectors comprising nucleic acid molécules that encode the amino acid sequence of the immunocytokine (fusion protein) of the invention, portions thereof (e.g., IL-10 sequences or sequences of the second and third constant domains ,.· · of the Fc fragment), as described herein. The invention further relates to vectors comprising 35 nucleic acid molécules encoding the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment of the invention or fragments thereof.

Expression vectors . include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. DNA molécules may be ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended fonction of regulating the transcription and translation of the DNA. An.

. expression vector and expression control sequences may be chosen to be compatible with the;

. expression host cell used. DNA molécules can be introduced into an expression vector by standard methods (e.g. ligation of complementary restriction sites on an immunocytokine gene fragment and vector, or blunt end ligation if no restriction sites are présent).

The term control sequences refers to DNA sequences necessary for the expression! of an operably linked coding sequence in a particular host organism. The control sequences; that are suitable for prokaryotes are, for example, a promoter, optionally an operator sequence: 15 and a ribosome binding site. Eukaryotic cells are known to include promoters, polyadenylation signais, and enhancers.

Nucleic acid is operably linked when it is placed into a fonctional relationship with another nucleic acid sequence. For example,'DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that 20 participâtes in the sécrétion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Typically, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader sequence, are contiguous and in reading phase.

However, enhancers do not hâve to be contiguous.

A recombinant expression vector can also encode a signal (leader) peptide that facilitâtes the sécrétion of the immunocytokine from a host cell. The immunocytokine gene may be cloned into a vector such that the signal (leader) peptide is linked in-frame to the amino terminus of the immunocytokine chain. The signal (leader) peptide can be an 30 immunoglobulin signal (leader) peptide or a heterologous signal peptide (i.e., a signal (leader) peptide from a non-immunoglobulin protein).

The recombinant vector expression of the invention can carry regulatory sequences that control the expression of immunocytokine genes in a host cell. It will be understood by those skilled in the art that the design of an expression vector, 35 including the sélection of regulatory sequences, may dépend on such factors as the choice of a host cell to be transformed, the level of expression of a desired protein, and so forth. Preferred control sequences for an expression host cell in mammals include viral éléments that ensure high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomégalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g. the major laté promoter adenovirus (AdMLP)), polyomavirus and strongmammalian promoters such as native immunoglobulin promoter or actin promoter. I . Methods for expressing polypeptides in bacterial cells or fungal cells, e.g. yeast cells, are also well known in the art. ,

In addition to the immunocytokine genes and regulatory sequences, the recombinant . expression vectors of the invention may carry additional sequences, such as sequences that regulate réplication of a vector in host cells (e.g. origins of réplication) and selectable marker genes. The selectable marker gene facilitâtes the sélection of host cells into which a vector 15 has been introduced.

The term expression control sequence as used in the présent description refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA 20 processing signais such as splicing and polyadenylation signais; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein sécrétion. The nature of such control sequences differs depending upon the . host organism; in prokaryotes, such control sequences generally include the promoter of 25 ribosome binding site, and transcription termination sequences; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences. The term control sequences includes at least ail components, the presence of which is essential for expression and processing, and can also include additional components, the presence of . which is advantageous,' for example, leader sequences and fusion partner sequences.

_: . !’·'

Host cells and method for production thereof

In one aspect, the présent invention relates to a method for production of a host cell for production of the immunocytokine of the invention, which comprises transformation of the cell with the above vector.

In one aspect, the présent invention relates to a host cell for production of the immunocytokine of the invention, the host cell comprises any of the above nucleic acids.

The term recombinant host cell (or simply host cell) as used herein refers to a cell into which a recombinant expression vector has been introduced. The présent invention relates to host cells, which may include, for example, a vector according to the invention described above. The présent invention further relates to host cells that include any of the nucleic acids encoding the immunocytokine of the présent invention. It should be understood that recombinant host cell and host cell refer not only to a particular subject cell but to the^!progeny of such a cell as well. Since modifications may occur in succeeding générations due 10 . to either mutation or environmental influences, such progeny may not, in fact, be identical to a parental cell; however, such cells are still included within the scope of the terni host cell as used herein. ;

Nucleic acid molécules encoding immunocytokines of the invention and vectors: comprising these nucleic acid molécules can be used for transfection of a suitable mammaliah or cell thereof, plant or cell thereof, bacterial or yeast host cell. Transformation can be carried out by any known technique of introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextranD-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate précipitation, polybreneQmediated transfection, protoplast fusion, encapsulation of the; polynucleotide(s) in liposomes, and direct microinjection of DNA into nuclei. In addition, nucleic acid molécules may be introduced into mammalian ·, cells by viral vectors.

Mammalian cell lines used as hosts for transformation are well known in the art and include a plurality of immortalized cell lines available. These include, e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell dînes are selected by determining which cell lines hâve high expression levels and provide for necessary characteristics of the protein produced. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When the recombinant expression vectors encoding the immunocytokine of the invention are introduced into mammalian host cells, the immunocytokine is produced by culturing the host cells for a period of time sufficient to express the immunocytokine of the invention in the host cells, or, more preferably, secrete the immunocytokine into the culture medium in which the host cells are cultured. The immunocytokine of the invention can be isolated from culture medium using standard. protein purification techniques. Plant host cells include, e.g. Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and ; ^; . I. . ' ' , ¹⁹ ¹ _______éÙ

Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Furthermore, level of production of the immunocytokine of the invention from a producing cell line can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression System (the GS System) is a common approach for 10 enhancing expression under certain conditions.

It is likely that the immunocytokine of the invention in different cell lines or transgenic animais will hâve different glycosylation patterns from each other. However, the . immunocytokine of the invention encoded by nucleic acid molécules described herein, or . comprising amino acid sequences provided herein are part of the présent invention, regardless 15, of the glycosylation of the binding molécules, and, in general, regardless of the presence or absence of post-translational modifications.

The above host cell does not refer to a host cell produced using human embryos.

The above host cell does not refer to a host cell produced by modifying the genetic 20 integrity of human germline cells.

In one aspect, the présent invention relates to a method for preparing a product comprising the immunocytokine of the invention, which comprises culturing of the above host cell in a culture medium under conditions sufficient to produce said immunocytokine, if necessary, followed by isolation and purification of the resulting immunocytokine.

The présent invention relates to methods for producing the immunocytokine of the invention. One embodiment of the invention relates to a method for producing the Y immunocytokine as defined herein, comprising preparing a recombinant host cell capable of expressing the immunocytokine of the invention, culturing said host cell under conditions suitable for expression/production of the immunocytokine of the invention, and isolating the resulting immunocytokine. An immunocytokine produced by such expression in such recombinant host cells is' referred to herein as a recombinant immunocytokine. The invention also relates to the· progeny of cells from such host cells and the immunocytokine produced analogously. i

Pharmaceutical composition and pharmaceutical combination

In one aspect, the présent invention relates to a pharmaceutical composition for activating the human IL-lORa receptor, which comprises the immunocytokine of the invention and one or nlore pharmaceutically acceptable excipients.

. ²⁰_ ί, ।

Pharmaceutical composition refers to a composition comprising an immunocytokine of the invention and at least one of components selected from the group comprising pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, distributing and sensing agents, delivery agents, such as preservatives, stabilizers, filler, disintegrators, moisteners, emulsifiers, suspending agents, thickeners, 10 sweeteners, flavouring agents, aromatizing agents, antibacterial agents, fungicides, lubricants, . and prolonged delivery controllers, the choice and suitable proportions of which dépend on the type and way of administration and dosage. Examples of suitable suspending agents are . ethoxylated isostearyl alcohol, polyoxyethene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacant and their mixtures as 15 well. Protection against action of microorganisms can be provided by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanole, sorbic acid, and similar compounds. The composition may also contain isotonie agents, such as, for example, sugars, polyols, sodium chloride, and the like. Prolonged action of the composition may be achieved by agents slowing down absorption«of active ingrédient, for example, aluminum 20 monostearate and gélatine; Examples of suitable carriers, solvents, diluents and delivery agents include water, éthanol; polyalcohols and their mixtures, naturel oils (such as olive oil) and organic esters (such as ethyl oleate) for injections. Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, and the like. Examples of disintegrators and distributors are starch, alginic acid and its salts, silicates and the like.

Examples of suitable lubricants are magnésium stéarate, sodium lauryl sulfate, talc and polyethylene glycol of high. molecular weight. The pharmaceutical composition for pérorai, sublingual, transdermal, intraocular, intramuscular, intravenous, subeutaneous, local or rectal administration of active ingrédient, alone or in combination with another active compound may be administered to human and animais in a standard administration form, in a mixture 30 with traditional pharmaceutical carriers. Suitable standard administration forms include pérorai forms such as tablets, gelatin capsules, pills, powders, granules, chewing-gums and pérorai solutions or suspensions; sublingual and transbuccal administration forms; aérosols; implants; local, transdermal, subeutaneous, intramuscular, intravenous, intranasal or intraocular forms and rectal administration forms.

The term excipient is used herein to describe any ingrédient other than the immunocytokine of the invention. These are substances of inorganic or organic nature which are used in the pharmaceutical manufacturing in order to give drug products the necessary physicochemical properties..

The terni pharmaceutically acceptable refers to one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably a human.

. The term buffer, buffer composition, buffering agent refers to a solution, which is capable of resisting changes in pH by the action of its acid-base conjugate components, and which allows the immunocytokine product to resist changes in pH. Generally, the 10 pharmaceutical composition preferably has a pH in the range from 4.0 to 8.0. Examples of buffers used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, . etc. buffer solutions.

> : The tenus tonie agent, osmolyte or osmotic agent, as used herein, refer to an . excipient that can adjust the osmotic pressure of a liquid formulation of the immunocytokine 15 of the invention. Isotonie drug is a drug that has an osmotic pressure équivalent to that of human blood. Isotonie drugs typically hâve an osmotic pressure from about 250 to 350 mOsm/kg. Isotonie agents used include, but are not limited to, polyols, saccharides and sucrose, amino acids, métal salts, for example, sodium chloride, etc.

Stabilizer refers to an excipient or a mixture of two or more excipients that provide 20 the physical and/or Chemical stability of the active agent. Stabilizers include amino acids, for example, but are mot limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, for example, but are not limited to, polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), polyethylene-polypropylene glycol and copolymers . thereof (trade names: Poloxamer, Pluronic, sodium dodecyl sulfate (SDS); antioxidants, for 25 example, but are not limited to, méthionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfurons acid salts, etc.; chelating agents, for example, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, etc. '

In one aspect, the . présent invention relates to a pharmaceutical composition for 30 activating the human IL-lORa receptor, which comprises the immunocytokine of the invention and at least one other therapeutically active compound.

In some embodiments of the pharmaceutical composition, other therapeutically active compound is used that is an antibody, chemotherapeutic agent, or hormone therapy agent.

In some embodiments of the pharmaceutical composition, other therapeutically active 35 compound is used that is an immune checkpoint inhibitor.

In some embodiments of the pharmaceutical composition, a PD-L1 inhibitor is used that is an antibody that specifically binds to PD-L1.

In some embodiments of the pharmaceutical composition, an antibody is used that specifically binds to PD-L1 and is selected from the group comprising durvalumab, . avelumab, atezolizumab, manelimab.

In some embodiments of the pharmaceutical composition, a PD-1 inhibitor is used that is an antibody that specifically binds to PD-1.

In some embodiments of the pharmaceutical composition, an antibody is used that specifically binds to PD-1 and is selected from the group comprising prolgolimab, . pembrolizumab, nivolumab.

In some embodiments of the pharmaceutical composition, a CTLA-4 inhibitor is used that is an antibody that specifically binds to CTLA-4.

In one aspect, the présent invention relates to a pharmaceutical combination for 20 activating the human IL-lORa receptor, which comprises the above immunocytokine of the invention and at least one other therapeutically active compound.

In some embodiments of the pharmaceutical combination, other therapeutically active 25 compound is used that is an immune checkpoint inhibitor.

In some embodiments of the pharmaceutical combination, an antibody is used that specifically binds to .PD-1 and is selected from the group comprising prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the pharmaceutical combination, a CTLA-4 inhibitor is used that is an antibody that specifically binds to CTLA-4.

In some embodiments, the pharmaceutical composition or pharmaceutical 10 combination is used for the treatment of an oncological disease.

“Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to 15 “treatment” include references to curative, palliative and prophylactic treatment.

The terms oncological disease, cancer and cancerous refer to a physiological condition or describe a physiological condition in mammals that is typically characterized by unregulated growth/proliferation of cells, Examples of oncological diseases include, but are : not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular 20 examples of such cancerous diseases include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer including stomach cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder . cancer, breast cancer, colon cancer, colorectal cancer, endométrial or uterine carcinoma, 25 salivary gland carcinoma, kidney cancer (rénal cell carcinoma), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, and varions head and neck cancers.

. In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any âge.

The immunocytokine of the invention within the pharmaceutical composition or combination is présent in a therapeutically effective amount.

“Therapeutically ' effective amount” refers to that amount of the therapeutic agent being administered during treatment which will relieve to some extent one or more of the symptoms of the disease being treated.

HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer,, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small ' : I ' ' ’ I cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone

B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higherrisk myelodysplastic syndrome.

Pharmaceutical compositions or pharmaceutical combinations of the présent invention and methods of préparation thereof will be undoubtedly apparent to those skilled in the art. The pharmaceutical compositions or pharmaceutical combinations should preferably be 15 manufactured in compliance with the GMP (Good Manufacturing Practice) requirements. The composition or combination may comprise a buffer composition, tonicity agents, stabilizers and solubilizers. Prolonged action of the composition or combination may be achieved by agents slowing down absorption of active pharmaceutical ingrédient, for example, aluminum monostearate and gélatine. Examples of suitable carriers, solvents, 20 diluents and delivery agents include water, éthanol, polyalcohols and their mixtures, oils, and organic esters for injections.

Any method for administering an immunocytokine accepted in the art may be suitably employed for the immunocytokine of the invention.

The pharmaceutical composition or pharmaceutical combination is stable if the 25 active agent retains physical stability and/or Chemical stability and/or biological activity thereof during the: specified shelf life at storage température, for example, of 2-8 °C. Preferably, the active agent retains both physical and Chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or naturel aging conditions. '

A pharmaceutical composition or pharmaceutical combination of the invention can be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready formulation. The term single unit dose as used herein refers to discrète quantity of a pharmaceutical composition containing a predetermined quantity of an active ingrédient. The quantity of the active ingrédient typically equals the 35 dose of the active ingrédient to be administered in a subject, or a convenient portion of such dose, for example, half or a third of such dose.

Pharmaceutical compositions or pharmaceutical combinations according to the présent invention are typically suitable for parentéral administration as stérile formulations intended for administration in a human body through the breach in skin or mucosal barriers, bypassing the gastrointestinal tract by virtue of injection, infusion and implantation. In . . particular, parentéral administration includes, inter alia, subcutaneous, intraperitoneal, . intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial, transdermal injection or infusion, and kidney dialytic infusion 10 techniques. Intra-tumor delivery, for example, intra-tumor injection, can also be employed.

Régional perfusion is also contemplated.

Preferred embodiments include intravenous and subcutaneous routes. Any method for / administering peptides or proteins accepted in the art may be suitably employed for the immunocytokine of the invention.

Injectable formulations may be prepared, packaged, or sold, without limitation, in unit dosage form, such as in ampoules, vials, in plastic containers, pre-filled syringes, autoinjection devices. Formulations for parentéral administration include, inter alia, . suspensions, solutions, émulsions in oily or aqueous bases, pastes, and the like.

In another embodiment, the invention provides a médicinal formulation for parentéral 20 . administration, wherein the pharmaceutical composition or pharmaceutical combination is provided in dry (i.e. powder or granular) form for reconstitution with a suitable base (e.g. stérile pyrogen-free water) prior to administration. Such médicinal formulation may be prepared by, for example, lyophilization, i.e. a process, which is known in the art as freeze drying, and which involves freezing a product followed by removal of solvent from frozen 25 material. f . An immunocytokine of the invention can also be administered intranasally or by inhalation, either alone, as a mixture with a suitable pharmaceutically acceptable excipient from an inhaler, such as a pressurized aérosol container, pump, spray, atomiser, or nebuliser, wherein a suitable propellant is used or not used, or as nasal drops, or spray.

Dosage forms for parentéral administration may be formulated to be immédiate or modified release. Modified release médicinal formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

Method for treatment/Use for treatment

In one aspect, the présent invention relates to a method for treatment of an . oncological disease, which comprises administering to a subject in need of such treatment the immunocytokine of the invention or any above pharmaceutical composition of the invention, . in a therapeutically effective amount.

i

In one aspect, the présent invention relates to the use of the immunocytokine of the invention or any above pharmaceutical composition of the invention for the treatment in a subject in need of such treatment of an oncological disease.

In some embodiments of the method for treatment, the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical 10 cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, 15 hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

In the case of a tumor (for example, cancer), the therapeutically effective amount of 20 an immunocytokine of the invention may reduce the number of cancer cells; reduce the initial tumor size;: inhibit (i.e. slow to some extent and preferably stop) cancer cell infiltration into . . surrounding organs; inhibit (i.e. slow to some extent and preferably stop) tumor metastasis; . inhibit to some extent tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disease. The immunocytokine of the invention may to some 25 extent prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

For cancer therapy, in¹ vivo effîcacy can, for example, be measured by assessing survival, . time to tumor progression (progression-free survival), tumor response rate to treatment (TRR), duration of response and/or quality of life.

. The immunocytokine of the invention can be administered without further therapeutic treatment, i.e. as an independent therapy. Furthermore, treatment by the immunocytokine of the invention may comprise at least one additional therapeutic treatment (combination therapy). In some embodiments, the immunocytokine of the invention may be administered jointly or formulated with another medication/preparation for the treatment of cancer.

As used herein, the terms co-administration, co-administered and in combination 35 with, referring to the immunocytokine of the invention and one or more other therapeutic agents, are contemplated to mean, refer to or include the following:

1) simultaneous administration of such combination of an immunocytokine of the invention and a therapeutic agent to a patient «in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially . the same time to said patient,

2) simultaneous administration of such combination of an immunocytokine of the invention and a therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient,

3) sequential administration of such combination of an immunocytokine of the . invention and a therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and

4) sequential administration of such combination of an immunocytokine of the invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled 20 manner, whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said patient, where each part may be administered by either the same or a different route, i _;

In one aspect, the . présent invention relates to the use of the immunocytokine of the invention or at least one other therapeutically active compound for the treatment in a subject 25 in need of such treatment of an oncological disease.

The immunocytokine of the présent invention can be combined with a therapeutic agent selected from the group comprising: a cytotoxic agent, a chemotherapeutic agent, a hormone therapy agent, or another therapeutic antibody.

The term cytotoxic agent as used herein refers to a substance that inhibits or 30 prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molécule toxins or enzymatically active, toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants. thereof. ¹

A chemotherapeutic agent is a Chemical compound useful in the treatment of a malignant neoplasm. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan;. aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylmelamine; acetogenins (e.g. bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol MARINOL®); , beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), 10 acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (e.g. cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as 15 chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, . mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, . chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, e.g. calicheamicin gamma II and calicheamicin oméga II (see, e.g. Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; esperamicin; as well as neocarzinostatin chromophore and related chromoprotein : enediyne i antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including · ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin, doxorubicin· HCl liposome injection (DOXOL®), liposomal · doxorubicin TLC . D-99 (MYOCET®), peglylated liposomal doxorubicin . (CAELYX®), and. deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, . mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, 30 . peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); folie acid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, 35 thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as aminoglutéthimide', mitotane, trilostane; folie acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;

bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane;

rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albuminengineered nanoparticle formulation of paclitaxel (ABRAXANE), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vinca alkaloids, which prevent tubulin polymerization from forming microtubules, including Vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate;

daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); biphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), étidronate (DIDROCAL®), NE- 58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAJX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, e.g. those that inhibit expression of genes in signaling pathways implicated in abeirant cell prolifération, such as for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and

VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 . inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen, sodium (GENASENSE®); pixantrone; EGFR inhibitors (see définition below);

tyrosine kinase inhibitors (see définition below); and pharmaceutically acceptable salts, acids or dérivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovovin.

Also included in this définition are hormone therapy agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxifene, toremifene 10 (FARESTON®), idoxifene, droloxifene, raloxifene (EVTSTA®), trioxifene, keoxifene, and sélective estrogen receptor modulators (SERMs), such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including stéroïdal aromatase inhibitors, 15 such as formestane and exerhestane (AROMASIN®), and nonsteroidal aromatase inhibitors, such as anastrazole (AREVIIDEX®), letrozole (FEMARA®) and aminoglutéthimide, and other aromatase inhibitors including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazole; lutenizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including 20 progestines, such as megestrol acetate and medroxyprogesterone acetate, estrogens, such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, fiilly transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor downregulators (ERDs); anti-androgens, such as . flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or dérivatives of any of the above;

as well as combinations of two or more of the above.

The, other therapeutic agent that can be used in combination with the immunocytokine : of the présent invention may be a therapeutic antibody selected from the group comprising antibodies to PD-1 (e.g. prolgolimab, pembrolizumab or nivolumab), antibodies to PD-L1, antibodies to CTLA4, antibodies to 4-1BB, antibodies to 0X40, antibodies to GITR, 30 antibodies to CD20 (e.g. rituximab), antibodies to HER2 (e.g. trastuzumab or pertuzumab), antibodies to VEGF (e.g. bevacizumab), or combinations thereof.

The term “immune, checkpoint inhibitor” (or checkpoint inhibitor) refers to 35 compounds inhibiting the activity of immune checkpoints. Inhibition includes réduction of function and full blockade. Examples of inhibitory checkpoint molécules include B7-H3, B7H4, BTLA, CTLA-4, KIR, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, TIGIT, and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically

1,1’

X.

. . .

recognizes an immune checkpoint protein. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative < immune checkpoint inhibitors may be developed in the near future. The immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molécules, and low molecular weight compounds.

In some embodiments of the use, an immune checkpoint inhibitor is used that is selected from a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor.

In some embodiments of the use, a PD-L1 inhibitor is used that is an antibody that ’ ; specifïcally binds to PD-L1.

In some embodiments of the use, an antibody is used that specifïcally binds to PD-L1 and is selected from the group comprising durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the use, a PD-1 inhibitor is used that is an antibody that specifïcally binds to PD-1.

In some embodiments of the use, an antibody is used that specifïcally binds to PD-1 and is selected from the group comprising prolgolimab, pembrolizumab, nivolumab.

.·. In some embodiments of the use, a CTLA-4 inhibitor is used that is an antibody that specifïcally binds to CTLA-4.. ,

In some embodiments of the use, an antibody is used that specifïcally binds to CTLA. 4 and is ipilimumab. । । .

In one aspect, the présent invention relates to a method for activating the human IL- lORa receptor in a subject in need of such activation, which comprises administering to a subject in need of such treatment an effective amount of the immunocytokine of the invention or any above pharmaceutical composition of the invention, in a therapeutically effective amount. ।

It is understood that an immunocytokine of the invention may be used in methods for treating, as described above, in the use for treatment, as described above, and/or in the manufacture of a médicament for the therapeutic applications described above. :

i · . T ^{: !}

Doses and: routes of administration

The immunocytokine of the présent invention will be administered in an amount that is effective in treatment of the condition in question, i.e. in doses and during the periods of time required to achieve the desired resuit. A therapeutically effective amount may vary according to factors such as the spécifie condition to be treated, âge, sex, and weight of a . ... , · ⁸ , i ³² ________________________________________________________________________________________________________________________________________________________________________________________________________________________________:______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ patient, and whether the immunocytokine is administered alone or in combination with one or more additional drugs or treatment techniques.

Dosage regimens may be adjusted to. provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the 10 exigencies of the therapeutic situation. It is especially advantageous to formulate parentéral compositions in a unit dosage form for ease of administration and uniformity of dosage. A unit dosage form as used herein is intended to refer to physically discrète units suited as . unitary dosages for patients/subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in 15 association with the desired pharmaceutical carrier. Spécification for the unit dosage forms of the invention is typically dictated by and directly dépendent on (a) the unique characteristics of a chemotherapeutic agent and particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inhérent in the art of compounding such an active compound for the treatment of sensitivity in the subjects.

Thus, a skilled artisan would appreciate,· based upon the disclosure provided herein, that the doses and dosage regimen are adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a détectable therapeutic effect to a patient may also be determined, as can the temporal requirements for administering each agent to provide a 25 détectable therapeutic effect to a patient. Thus, while certain dose and administration regimens are exemplifîed herein, these examples in no way limit the doses and administration regimen that may be provided to a patient in practicing the embodiments of the invention.

It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. Furthermore, it is to be 30. understood .that for_;any particular subject, spécifie dosage regimens should be adjusted over time according to the individual need and the judgment of a medical professional administering or supervising the administration of the compositions, and that dosage ranges set forth in the présent description are exemplary only and are not intended to limit the scope . or practice of the claimed compositions. Further, the dosage regimen with the compositions 35 of the présent invention may be based on a variety of factors, including the type of disease, the âge, weight, sex, medical condition of the patient, the severity of the condition, the route of administration,' and the particular immunocytokine of the invention. Thus, the dosage regimen can vary widelÿ, but can be determined routinely using standard methods. For , 21499 example, doses may be adjusted based on pharmacokinetic and pharmacodynamie parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the présent invention encompasses intra-patient dose-escalation as determined by the . person skilled in the art. Methods for determining appropriate dosages and regimens are wellknown in the art and would be understood by a skilled artisan once provided the ideas 10 disclosed herein.

Examples of suitable administration methods are provided above.

. It is contemplated that a suitable dose of an immunocytokine of the invention will be in the range of 0.007-200 mg/kg, preferably 0.007-100 mg/kg, including about 0.5-50 mg/kg, 15 for example about 1-20 mg/kg. The immunocytokine of the présent invention may be administered, e.g. in a dose of at least 0.25 mg/kg, such as at least 0.5 mg/kg, including at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2 mg/kg, e.g. at least 3 mg/kg, including at least 4 mg/kg, e.g. at least 5 mg/kg; and for example up to a maximum of 50 mg/kg, including up to a maximum of 30 mg/kg, e.g. up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. The administration will typically be repeated in appropriate time intervals, such as once a week, once every two weeks, once every three weeks or once every four weeks, and for as long as deemed appropriate by a responsible physician, who may, in some cases, increase or reduce the dose if necessary.

Implémentation of the invention . The following examples are provided for better understanding of the invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

Ail publications, patents, and patent applications cited in this spécification are 30 incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or . scope of the appended embodiments.

;

Materials and general methods

General information regarding the nucléotide sequences of human immunoglobulin light and heavy chains fis given in: Kabat, E.A., et al., Sequences of Proteins of

Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered according to EU numbering (Edehnan, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al,

Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the 15 manufacturer protocols.

Gene synthesis

Desired gene segments were prepared from oligonucleotides made by Chemical synthesis. The gene segments of 300-4000 kb long, which were flanked by singular 20 restriction sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirined by DNA sequencing.

DNA sequence détermination

DNA sequences were determined by Sanger sequencing.

. DNA and protein sequence analysis and sequence data management

The Infomax's Vector NT1 Advance suite version 8.0 was used for sequence création, mapping, analysis, annotation and illustration.

Expression vectors

For the expression of the described antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic cells (E.coli), transient expression in eukaryotic cells (e.g. in CHO cells) were applied. Beside the antibody expression cassette the vectors comprised: an origin of réplication which allows réplication of said plasmid in E. coli, 35 genes which confer résistance in E. coli to various antibiotics (e.g. to ampicillin and . kanamycin).

The fusion genes comprising the described antibody chains as described below were generated by PCR and/or gene synthesis and assembled with known recombinant methods and techniques by connection of the according nucleic acid segments, e.g. using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections, larger quantifies of the plasmids were prepared by plasmid préparation from transformed E. coli cultures.

Example 1

Production of sequences of human IL-lORa genes, and sequences of orthologous ILlORa genes of Macaca cynomolgus (fascicularis) and IL-lORa genes of Oryctolagus . cuniculus (Rabbit).

To clone sequences of extracellular portions of the human IL-lORa receptors 15 (https://www.uniprot.org/uniprot/Q13651) h IL-lORa Macaca cynomolgus (https://www.uniprot.org/uniprot/A0A2K5WJ66), we synthesized oligonucleotides of 60 nucléotides each, forming a completely overlapping gene sequence. Each gene was assembled using two-round PCR, which resulted in the production of a fragment of 642 bp. Next, the extracellular portion of the receptor was cloned into a vector for transient 20 expression of EPEA-tagged plntA for affinity purification of the protein.

To clone the sequence of the extracellular portion of the Oryctolagus cuniculus ILlORa receptor, total RNA was isolated from rabbit peripheral blood cells and cDNA was generated. Reverse transcription products were used as a template in the polymerase chain reaction to produce a receptor gene flanked by restriction sites. Next, the receptor gene was cloned_: into a vector for transient expression of EPEA-tagged plntA (Fig. 1) for affinity purification of the protein. The cloned nucleic acid sequences were verified by DNA sequencing. :

Example 2

Production of sequence of human IL- 10/Fc fragment fusion protein, as well as rabbit

IL-10/Fc fragment fusion protein. :

To clone a, gene comprising a human/rabbit IL-10 sequence and a human IgGl Fc fragment,' genes were; fused by PCR and/or gene synthesis and assembly using known recombination methods and protocols by way of connecting the appropriate nucleic acid

35. segments, for example, using SOE-PCR (Splicing by overlap extension). Next, the fusion protein gene was cloned into a vector for transient expression of plntA (Fig. 2). The cloned nucleic acid sequences were verified by DNA sequencing.

... ' . 1 i ¹ I

Example 3

Isolation and purification of human, monkey and rabbit IL-10R receptors from mammalian cell suspension culture.

Recombinant proteins (rabbit, human and monkey IL-lORa receptors, rabbit IL-10 receptor) were generated in the CHO-C cell culture medium. Following transfection of cells 10 with expression vectors, orbital feed-batch cultivation was performed in serum-free medium for 5-10 days. After culturing, cell culture was centrifuged under 2000 g for 20 min and filtered through 0.22 pm filter. Target proteins were isolated from the culture liquid using affînity chromatography on the Akta Pure 25 chromatographie System using an XK 16 column (GE Healthcare) with 5 ml CaptureSelect C-tag Affînity Matrix (Thermo Scientific) and Superdex 200 Increase 10/300 GL (GE Healthcare). The culture liquid was applied to the column with CaptureSelect C-tag Affînity Matrix, following which the column was washed with 20mM TrisHCl pH7 supplemented with 150mM NaCl and the protein was eluted with 2M MgC12 solution in 20mM TrisHCl pH7 supplemented with 150mM NaCl. Next, the. protein was dialyzed into 20mM TrisHCl pH7 with 150mM NaCl, following which the product.was concentrated to 1 ml and applied to a GE Superdex 200 Increase 10/300 GL . column equilibrated with a 20mM TrisHCl pH7 solution supplemented with 150mM NaCl.

The target peak.was collected and the resulting solution was filtered (0.22 pm). The products were stored at -70 °C. The purity of the resulting protein solution was evaluated by SDS gel electrophoresis (Fig. 3). . .

. .

Example 4

Isolation and purification of immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment (IL-10-Fc) from mammalian cell suspension culture.

The recombinant IL-10-Fc protein of the invention was generated in the CHO-C cell 30 culture medium. Following transfection of cells with expression vectors, orbital feed-batch cultivation was· performed in serum-free medium for 5-10 days. The sécrétion of the immunocytokine in question¹ was monitored using the Pall ForteBio's Octet RED96 System for molecular interactions' analysis on protein A biosensors.

After. culturing, cell culture was centrifuged under 2000 g for 20 min and filtered 35 through 0.22 pm filter. Target proteins were isolated from the culture liquid by affînity chromatography on the Akta. Pure 25 chromatography System using HiTrap rProtein A FF, 5ml and HiPrep 16/60 Sephacryl S-300 HR columns (GE Healthcare). The culture liquid was applied to the HiTrap rProtein A FF column, following which the column was washed with . ³⁷ , ^; i

PBS and the protein was eluted with a solution of 100 mM citrate buffer pH 3, following which the protein solution was neutralized by adding 2M acetate buffer pH6 at a ratio of 1/10 v/V. Next, the protein was dialyzed into 20 mM acetate buffer pH6, following which the product was concentrated and applied to a HiPrep 16/60 Sephacryl S-300 HR column equilibrated with a solution of 200 mM acetate buffer pH6. The target peak was collected and 10 the resulting solution was filtered (0.22 pm). The product was stored at -70 °C. The purity of the resulting protein solution was evaluated by SDS gel electrophoresis (Fig. 4).

Example 5

Kinetic studies of affinity and specificity of the interaction between immunocytokine 15 (fusion protein) based on IL-10 and human IgGl Fc fragment (IL-10-Fc) and human, cynomolgus, murine and rabbit IL-lORa receptors using Forte Bio OctetRed96.

Binding of human IL-10-Fc of the invention to the human IL-10 receptor, as well as to the cynomolgus, murine and rabbit IL-lORa receptors, was assessed by bio-layer interferometry using the OctetRed96 device (Pall). IL-10-Fc was bound to AR2G sensors 20 according to the manufacturées instructions for the préparation and immobilization of AR2G sensors. The sensors with immobilized IL-10 were then immersed into wells containing the human, macaque, murine or rabbit IL-1 ORa receptor. Following the association of interleukin and the receptor, the sensors were immersed in the working solution for the subséquent dissociation stage. The resulting sensogramS, after subtracting a reference signal, were 25 analyzed using Octet Data Analysis software (Version 8.0) in accordance with the standard procedure and using 1:1 interaction model. According to the data obtained (Table 1), IL-10Fc of the invention binds to the human, cynomolgus, murine IL-10 receptors.

Table 1. . %

Affinity constants for interaction of IL-10-Fc of the invention with human, cynomolgus, murine and rabbit IL-10 receptors.

I	Affinity constant (KD), M ^!
Htiman IL-10 receptor, M	Cynomolgu s IL-10 receptor, M	Murine IL-10 receptor, M	Rabbit IL- 10 receptor, M
IL- 10-Fc	5.1±0.3 E-09	3.7±0.4 E- 09	2.1±0.2 E-09	2.03E-08

Example 6

Analysis of thermostability of immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment (IL-10-Fc).

The recombinant ILIO-Fc protein of the invention in 20mM acetate buffer, pH6, was heated using an amplifier in a plastic test tube at 50C for 48 hours, followed by a shift to +4C. After the end of the program, the samples were analyzed before and after heating using analytical gel chromatography on a TSK Gel G3000 SWxl column. The areas of the target peaks of the samples before and after heating were compared. A 7% change in the area of the dimer peak after heating for 48 hours indicates the stability of the product and the possibility of long-term storage (Table 2).

Table 2. Comparison of the ratio of the area of peaks in the chromatograms of the IL10-Fc product of the invention before and after heating

Sample	% ratio of peaks
. ; Laggregates, %	dimer, %	Lfragments, %
Before ' heating	, 1.80 .	. 97.25	0.96
Following heating	7.99	, 89.97	2.05

Éxample 7

Analysis of stability of the immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment (IL-10-Fc) during long-term storage in human sérum at 37C.

IL-10 has a relatively short half-life in the organism. For example, the half-life in mice as measured by in vitro bioassay or by efficacy in a septic shock simulation System [see Smith et al., Cellular hnmunology 173: 207-214 (1996)], is about 2 to 6 hours. In the présent study, stability was understood as a decrease in the concentration of full-length IL-10 . i ; | I candidate molécules following in vitro storage in human sérum at +37°C. To détermine the stability, the IL-10-Fc immunocytokine was diluted in human sérum to a concentration of 5 pg/ml and stored at +37 °C in a temperature-regulated chamber for 24 h to 14 days. Samples of unsupplemented human sérum used for subséquent détermination of the background concentration values of the samples in question were stored under similar conditions. After storage, the concentration of the samples was determined by enzyme immunoassay. Data calculation and plotting were performed using the MS Excel software package (Fig. 5).

Thus, IL-10+Fc of the invention has high stability in sérum due to résistance to sérum protease dégradation. The résistance is provided by the structural feature of the given fusion protein, the structure of which being shown in SEQ ID NO: 1.

Example 8

Détermination of proliférative activity of immunocytokine (fusion protein) based on IL-10 and human IgGl Fc fragment (IL-10-Fc) in a cellular assay on MC/9 mouse mast cells.

A critical step for the cytokine function is the interaction of the cytokine with its receptor. Activation of the IL-lORa receptor by the test candidates was analyzed on the MC/9 cell line in a standard prolifération assay in the presence of the costimulatory cytokine IL4. It has been shown in the literature that IL-10 stimulâtes the prolifération of MC/9 mouse mast cells in the presence of IL4 (Thompson-Snipes L et al J Exp Med. 1991 doi: 10.1084/jem.l73.2.507 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2118779/). The assay is used as a standard; assay for assessing the biological activity of commercially available formulations of recombinant human IL-10. The activity of the control product (recombinant human IL-10) is < 2.0 ng/ml in a prolifération assay on the MC/9 line, as stated by the manufacturer (Pepfotech). ¹

The assay was carried out in a 96-well culture plate for suspension cell cultures. The suspension contained per well 10,000 MC/9 cells, and the test candidates (recombinant human IL-10, Peprotech catalog number: 200-10, and IL-10+Fc of the invention) at a concentration as indicated in.the graph. The final suspension volume was 150 μΐ per well. Ail suspension components were prepared in RPMI-1640 medium supplemented with 2 mM glutamine, 10 pg/ml gentamicin, 10% inactivated fêtai bovine sérum (FBS), and 10 pg/ml IL4. After adding ail components, the plate was incubated for 72 h at 37 °C in a humid atmosphère in the presence of 5% CO2. At the end of the incubation period, 17 pL of Alamar Blue dye was added to the wells and the plate was incubated for 3-6 h at 37 °C in a humid atmosphère in the. presence of 5% CO2 until .pink staining was seen. Fluorescence was measured using a plate reader at a wavelength of 495/590 nm.

Thus, IL-10-Fc of the invention activâtes the IL-lORa receptor, as confirmed in a prolifération cellular assay bn MC/9 cells (Fig. 6). The half maximal effective concentration (EC50) of the test IL-10+Fc immunocytokine of the invention was 53.3 pM in the cellular assay, whereas that of the control recombinant human IL-10 from Peprotech was 91.7 pM.

The data obtained indicate that the activity of IL-10+Fc of the invention is higher as compared to that of the commercially available IL-10.

Example 9

Détermination of functional activity of immunocytokine (fusion protein) based on IL10 10 and human IgGl Fc fragment (IL-10-Fc) by sécrétion of IFN-γ by human CD8+ T cells.

Antigen-independent activation of TCR and CD28 on CD8+ T cells in vitro (via antiCD3 and anti-CD28 antibodies) causes PDI expression on the cell surface, which leads to suppression of the activity of cytotoxic lymphocytes followed by déplétion thereof. Concurrently, the IL-lORa receptor is expressed on the cell surface, the activation of which, 15 when interacting with IL-10, has an antiapoptotic effect, which contributes to the survival of cytotoxic T cells producing various cytokines, including IFN-γ, in the tumor microenvironment.

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from a healthy donor by Ficoll density gradient centrifugation. CD8+ T cells were then 20 isolated from the suspension of PBMCs on a magnetic column using a commercial kit.

The test was performed in a 96-well plate with anti-CD3 antibodies (1 pg/ml) preimmobilized in the wells. The suspension contained per well 30,000 CD8+ T cells, as well as an anti-CD28 antibody at a concentration of 1 pg/ml. The final volume of the cell suspension and antibodies in a well was 200 μΐ, ail components of the suspension were prepared in 25 RPMI-1640 medium comprising 10% FBS. The plate was incubated for 3 days at 37 °C with . 5% CO2. On day 4 of incubation, the cells were collected and washed from the anti-CD28 antibody by centrifugation. The resulting cell precipitate was suspended in RPMI 1640 medium supplemented with 10% FBS at a concentration of 0.3*10⁶ cells/ml. 100 μΐ of washed cells and 100 μΐ ofi candidate antibodies were introduced at a concentration as 30 specified in the graph to the wells of a new pre-prepared ELISA 96-well plate with anti-CD3 antibody immobilized onto the plastic (1 pg/ml). The plate was incubated for 3 days at 37 °C with 5% CO2. On day 4 of incubation, aliquots of culture liquid were collected from the wells and the concentration ofIFN-γ was measured.

Thus, IL-10-Fc of the invention shows the ability to induce the production of the cytokine IFN-γ by human activated cytotoxic CD8+ T cells..

Example 10 l . I · , . .. · ' .

, ⁴¹ .

I

Détermination of the effect of immunocytokine (fiised protein) based on IL-10 and human IgGl Fc fragment (IL-10-Fc) on the cytotoxicity of human CD8+ T cells against Raji target cells.

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from a healthy donor by Ficoll density gradient centrifugation. CD8+ T cells were then 10 isolated from the suspension of PBMCs on a magnetic column.

200 μΐ of a suspension containing 30,000 CD8+ T cells and 1 pg/ml of anti-CD28 . antibody was introduced to a 96-well plate with anti-CD3 antibodies (1 pg/ml) preimmobilized in the wells, and incubated for 3 days at 37°C, 5% CO2. The cells were then collected, washed by centrifugation, suspended in RPMI-1640 medium supplemented with 15 10% FBS, and injected at 30,000 cells/well into the wells of a new pre-prepared ELISA 96well plate with anti-CD3 antibody immobilized onto the plastic (1 pg/ml) and candidate antibody solutions at a concentration as specified in the graph. The plate was incubated for 3 days at 37 °C with 5% CO2. The cells were then. collected from the plate wells, washed by centrifugation, and the précipitâtes were suspended in RPMI-1640 10% medium at a 20 concentration of 0.3 * 10⁶ cells/ml.

6.5 ml of Calcein AM solution was added to 2* 10 ⁶ Raji cells. Cells with calcein were incubated for 30 minutes at 37 °C in a CO 2 incubator. At the end of incubation, the cells were washed twice from calcein by centrifugation. A suspension with a concentration of 0.3*106' cells/ml was prepared.

The assay was conducted in a 96-well culture plate. The suspension contained per well 30,000 CD8T T cells, 30,000 Raji cells labeled with calcein AM, and an anti-CD3/CD20 antibody at a final concentration of 50 ng/ml. The plate was incubated for 2.5 hours in an incubator at 37°C, 5% CO2.

minutes prior to the end of incubation, a lysis buffer solution was added to the .30 wells under control of maximum lysis. Fluorescence was measured on a plate reader at 485/538 nm.

It was shown that culturing of CD8+ T cells in the presence of IL-10+Fc of the invention increases their cytotoxic activity against Raji target cells by 7 times as compared to that of the control.(Fig. 8). ,.

35i

Example 11 . 1

Measurement of CDC activity.

The CDC assay used the Jurkat cell line. The assay was conducted in a 96-well culture plate. The suspension contained per well 50,000 Jurkat cells, as well as IL-10+Fc of the invention at the specified concentration and a human sérum complément diluted to 1:4. The final volume of the cell suspension in a well was 150 μΐ, ail components of the suspension were prepared in RPMI-1640 medium comprising 0.1% BSA. The plate was then incubated for 4 h at 37 °C with 5% CO2. 15 μΐ of alamar blue reagent was added to each well and incubated at 37°C, 5% CO2 for 16 hours. An antibody with CDC activity Rituximab (CO83031118R) produced by JSC Biocad and Raji target cells, at the same concentration as Jurkat cells, were used as a positive control. Fluorescence was measured at an excitation wavelength of 544 nm and émission wavelength of 590 nm using a plate reader.

It has been shown that IL-10+Fc of the invention does not induce complementmediated lysis (CDC) of the Jurkat cell line (Fig. 9).

Example 12

Measurement of ADCC activity.

The assay used a reporter cell line Jurkat-NFAT-Luc-CD16 created on the basis of the

Jurkat cell line, stably expressing CD 16 on the surface and containing a gene encoding fîrefly luciferase, under the control of the NFAT promoter; Jurkat-PDl clone 43 was used as target cells. The assay was performed'to confirm the absence of effector properties in the test immunocytokine IL-10+Fc of the invention.

The assay was performed in a white 96-well culture plate designed for luminescence assays. The suspension contained per well 25,000 Jurkat-NFAT-Luc-CD16 effector cells, 25,000 Jurkat-PDl target cells, and IL-10-Fc of the invention at a concentration as specified in the graph. An effector anti-PDl antibody produced by JSC Biocad was used as a positive control. The final volume of the cell suspension and immunocytokine (IL-10+Fc of the invention) in a well was 75 μΐ, ail components of the suspension were prepared in RPMI1640 medium comprising 10% FBS. After adding ail the components, the plates were incubated for 5 hours at 37°C, 5%CC>2, and then, using a One-Glo luciferase assay kit (Promega), we measured j the luminescence intensity in the wells. Luminescence was measured using a plate reader.

It has been shown that IL-10+Fc of the invention does not exhibit antibody-dependent cellular cytotoxicity in the; assay using the Jurkat-NFAT-Luc-CD16-V176 reporter cell line (Fig. 10). = ’

Example 13

Measurement of autocytotoxicity.

The assay allows in vitro assessment of antibody-induced déplétion of main subpopulations of white blood cells in the blood, which in tum makes it possible to assess the safety of the therapeutic molécules being developed even before in vivo studies.

The assay was performed in a 96-well culture plate for suspension cultures. The suspension contained per well 300,000 freshly isolated PBMCs from healthy donors and IL. 10+Fc of the invention at the specified concentration, the final volume of the cell suspension in a well was 150 μΐ. Obinütuzumab (aCD20) and anti-CD47 antibody were used as a positive control. Ail suspension components were prepared in RPMI-1640 medium 15 supplemented with 10% fêtai bovine sérum. Afiter mixing PBMCs and antibodies, the plate was incubated for 16 h at 37°C, 5% CO2. The proportion of CD45+, CD56+, CD19+, CD3+, CD4+ and CD8+ subpopulations in PBMCs in suspensions was then measured by directly staining the suspensions with fluorescent-labeled antibodies against the corresponding CDs followed by analyzing the cells using a flow cytofluorometer. For the CD56+, CD19+, CD3+ cells, the graphs; show the proportion thereof relative to CD45+ cells of test suspension, whereas for theiCD4+, CD8+ cells, the graphs show the proportion thereof relative to CD45+CD3+cells. .

The in vitro autocytotoxicity assay did not show that IL-10+Fc of the invention induces significant déplétion of the NK, B and T cell populations in human PBMCs (Fig. 11 25 A, 11 B, 11 C, ll D,.ll E). Further, the control antibody Obinutuzumab (aCD20) showed almost complété déplétion of the CD20+ B cell population, and the anti-CD47 antibody showed almost 50% déplétion of NK cells.

Example 14

Study of antitumor activity in vivo.

The study was performed on Balb/c mice (males aged 4-6 weeks, with a body weight of 18-24 g). 0.1 ml CT26 tumor cell suspension was injected subcutaneously into the right latéral side of mice in an amount of 2*10⁵ cells. When the tumors reached an approximate volume (V=LW2/2) of 70 mm3, the animais were divided into groups such that the average 35 tumor volume in the groups did not differ by more than 10%. Ail of the drugs were administered by intraperitoneal (i.p.) administration twice a week for 3 weeks. In ail cases, IL-10-Fc of the invention was administered at a dose of 0.1 mg/kg, amPD-1 was administered at a dose of 10 mg/kg. In groups with the combination of IL-10-Fc of the invention, amPD-1 was administered on the day after the administration of IL-10-Fc of the invention. Animais from the tumor growth control group were administered with a buffer. The linear size of the tumor node was assessed twice a week. ITG (index of tumor growth) was calculated as the ratio of the tumor volume on the day of measurement (1-19) to the tumor volume on the day of treatment initiation.

It has been shown that IL-10+Fc of the invention has antitumor activity in monotherapy, and, further, the combination of IL-10+Fc of the invention with anti-PDl antibody has a pronounced synergy in antitumor activity as compared to monotherapy with either the immunocytokine IL-10+Fc of the invention or anti-PD-1 antibody (Fig. 12,13,14).

Claims

1. An isolated immunocytokine for activating the human IL-lORa receptor, comprising a homodimeric complex based on IL-10 and human IgGl Fc fragment, wherein the monomer based on IL-10 and human IgGl Fc fragment comprises the amino acid sequence of SEQ ID NO:1.

2. An isolated nucleic acid, which encodes the immunocytokine as claimed in claim 1.

3. The isolated nucleic acid according to claim 2, wherein the nucleic acid is DNA.

4. The isolated nucleic acid according to claim 2, wherein the nucleic acid comprises a nucléotide sequence with SEQ ID NO:3.

5. The isolated nucleic acid according to claim 2, wherein the nucleic acid comprises a nucléotide sequence with SEQ ID NO:4.

6. An expression vector comprising the nucleic acid according to any of claims 2-5.

7. A method for producing a host cell for producing the immunocytokine according to claim 1, comprising transformation of the cell with the vector according to claim 6.

8. A host cell for producing the immunocytokine according to claim 1, comprising the . nucleic acid according to any of claims 2-5.

9. A method for producing the immunocytokine according to claim 1, comprising culturing of the host cell according to claim 8 in a culture medium under conditions sufficient to produce said immunocytokine, if necessary, followed by isolation and purification of the resulting immunocytokine.

10. A pharmaceutical composition comprising the immunocytokine according to claim 1 and one or more pharmaceutically acceptable excipients.

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11. A pharmaceutical composition according to claim 1, wherein said pharmaceutical composition is for activating the human IL-lORa receptor.

12. The pharmaceutical composition according to claim 10 for treating an oncological disease.

13. The pharmaceutical composition according to claim al2, wherein the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

14. A pharmaceutical composition for activating the human IL-lORa receptor, comprising the immunocytokine according to claim 1 and at least one other therapeutically active compound.

15. The pharmaceutical composition according to claim 14 for treating an oncological disease.

16. The pharmaceutical composition according to claim 15, wherein the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

17. The pharmaceutical composition according to any of daims 14-15, wherein the other therapeutically active compound is an antibody, chemotherapeutic agent or hormone therapy agent.

18. The pharmaceutical composition according to any of daims 14-15, wherein the other therapeutically active compound is an immune checkpoint inhibitor.

19. The pharmaceutical composition according to daim 18, wherein the immune checkpoint inhibitor is selected from an antibody. that specifically binds to PD-1, an antibody that specifically binds to PD-Llor an antibody that specifically binds to CTLA-4.

20. The pharmaceutical composition according to daim 19, wherein the antibody that specifically binds to PD-1 is selected from the group comprising: prolgolimab, pembrolizumab, nivolumab.

21. The pharmaceutical composition according to daim 19, wherein the antibody that specifically binds to CTLA-4 is ipilimumab.

22. The use according to daim 19, wherein the antibody that specifically binds to PD-L1 and is selected from the group comprising: durvalumab, avelumab, atezolizumab, manelimab.

23. A method of treating an oncological disease, comprising administering to a subject the immunocytokine according to daim 1 or the pharmaceutical composition according to any of daims 10, 14, in need of such treatment, in a therapeutically effective amount.

24. The method for treating according to daim 23, wherein the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

25. A method for activating the human IL-lORa receptor in a subject in need of such activation, comprising administering to a subject an effective amount of the immunocytokine according to claim 1 or the pharmaceutical composition as according to any of daims 10, 14.

26. The use of the immunocytokine according to claim 1 or the pharmaceutical composition according to any of Claims 10, 14 for treating, in a subject in need of such treatment, an oncological disease.

27. The use according to claim 26, wherein the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triplenegative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

28. The use of the immunocytokine according to claim 1 or at least one other therapeutically active compound for the treatment, in a subject in need of such treatment, of an oncological disease.

29. The use according to claim 28, wherein the oncological disease is selected from the group comprising: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triplenegative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma,

NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, colorectal cancer with microsatellite instability, leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer, higher-risk myelodysplastic syndrome.

30. The use according to any of daims 28-29, wherein the other therapeutically active compound is an antibody, chemotherapeutic agent or hormone therapy agent.

31. The use according to any of daims 28-29, wherein the other therapeutically active compound is an immune checkpoint inhibitor.

32. The use according to daim 31, wherein the immune checkpoint inhibitor is selected from an antibody that specifically binds to PD-1, an antibody that specifically binds to PD-L1 or an antibody that specifically binds to CTLA-4.

33. The use according to daim 32, wherein the antibody that specifically binds to PD-1 is selected from the group comprising: prolgolimab, pembrolizumab, nivolumab.

34. The use according to daim 32, wherein the antibody that specifically binds to CTLA-4 is ipilimumab.

35. The use according to daim 32, wherein the antibody that specifically binds to PD-L1 and is selected from the group comprising: durvalumab, avelumab, atezolizumab, manelimab.