CN118510802A - CD20-PD1 binding molecules and methods of use thereof - Google Patents

Abstract

The present disclosure relates to molecules capable of binding to both CD20 and PD1, as well as pharmaceutical compositions comprising such molecules and methods of use thereof.

Description

CD20-PD1 binding molecules and methods of use thereof

1. Cross-reference to related applications

This application claims the benefit of priority to U.S. Provisional Application No. 63/278,454, filed on November 11, 2021, and U.S. Provisional Application No. 63/278,374, filed on November 11, 2021, the contents of each of which are incorporated herein by reference in their entirety.

2. Sequence Listing

This application contains a sequence listing that has been submitted electronically and is hereby incorporated by reference in its entirety. Said copy, created on November 8, 2022, is named RGN-012WO_SL.xml and is 63,928 bytes in size.

3. Background technology

Autoimmune diseases occur when an organism mounts an abnormal immune response against its own cells and tissues. For decades, efforts have been made to understand autoimmunity. During this time, it has become clear that the immune system has evolved multiple mechanisms for controlling self-reactivity. Defects in one or more of these mechanisms can lead to a breakdown in tolerance and the potential for autoimmune disease to develop.

The initial trigger for both systemic autoimmune disorders and organ-specific autoimmune disorders may involve recognition of self or foreign molecules by innate sensors. This recognition triggers an inflammatory response and the engagement of previously quiescent autoreactive T and B cells. Theofilopolous, Kono, and Baccala, 2017, Nat Immunol, 18(17):716-724. The range of autoreactivity ranges from low "physiological" levels of autoreactivity necessary for lymphocyte selection and immune system homeostasis to moderate levels of autoimmunity manifested as circulating autoantibodies and minor tissue infiltration without clinical consequences, to pathogenic autoimmunity associated with immune-mediated organ damage. Theofilopolous, Kono, and Baccala, 2017, Nat Immunol, 18(17):716-724. Autoimmune diseases are divided into organ-specific (e.g., type 1 diabetes (T1D), multiple sclerosis (MS), inflammatory bowel disease (IBD), myasthenia gravis) and systemic (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome ( syndrome) and can be mediated by autoantibodies or cytotoxic T cells, but in all cases requires helper T cells. Theofilopolous, Kono, and Baccala, 2017, Nature Immunology, 18(17): 716-724.

Most autoimmune diseases show clinical heterogeneity, polygenic properties, and multifactorial effects that usually require genetic and environmental factors. Four mechanisms contribute to the control of escaped autoreactive T cells and B cells: inhibitory molecules, anergy, ignorance, and active inhibition. Kono and Theofilopolous, Kono and Baccala, 2017, "Nature Immunology", 18 (17): 716-724. Several inhibitory molecules (e.g., CTLA-4, PD-1, LAG-3, TIM3, VISTA, TIGIT, FcγRIIb, certain Siglecs) are expressed on the surface of T cells and B cells to inhibit excessive immune responses, both normal immune responses and anti-autoimmune responses. The lack of some of these molecules leads to autoimmunity, which provides strong evidence that autoreactive lymphocytes exist in peripheral reservoirs but are usually under control. See Paterson and Sharpe, 2010, Nature Immunology, 11: 109-111; Okazaki et al., 2013, Nature Immunology, 14: 1212-1218; Pincetic et al., 2014, Nature Immunology, 15: 707-716; Macauley, Crocker and Paulson, 2914, Nature Reviews Immunology, 14: 653-666; Ceeraz et al., 2016, Arthritis Rheumatol 69(4): 814-825; and Schmitt et al., 2016, J Exp Med, 213: 1627-1644. Various immune-related adverse events often occur due to unchecked autoreactivity. Michot et al., 2016, Eur J Cancer, 54:139-148.

Existing treatments for autoimmune diseases have only achieved limited success. For example, organ-specific autoimmune diseases may usually be corrected by metabolic control. In the case of loss of function and inability to recover, mechanical substitutes or tissue transplants may be suitable. Although some of the symptoms may be alleviated using this method, there is no effective long-term curative treatment for some of the most disabling autoimmune disorders. Although many compounds including insulin, corticosteroids and modified beta interferon can improve some of the symptoms of autoimmune diseases, the compounds may have serious side effects and/or require long-term use. General immunosuppressive drug therapy, such as chronic treatment with cyclosporin A, FK506 and rapamycin (rapamycin), also cannot cure these diseases, and the use of the therapy is accompanied by many harmful side effects. The effects include increased nephrotoxicity, increased susceptibility to infectious diseases and increased incidence of neoplasia.

Therefore, novel therapeutic compositions and regimens are sought that can be used to treat autoimmune diseases.

4. Summary of the invention

The present disclosure provides novel CD20-PD1 binding molecules. The CD20-PD1 binding molecules of the present disclosure generally include or consist of CD20-PD1 monomers, which monomers contain one or more CD20 targeting moieties and/or one or more PD1 agonist moieties. The CD20-PD1 binding molecules of the present disclosure include a protein, which includes at least one CD20 targeting moiety, at least one PD1 agonist moiety, at least one dimerization moiety, and optionally one or more linker moieties, which separate one or more parts of the protein. In some embodiments where the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of PDL1, and the dimerization moiety is an Fc domain, optionally i) the light chain of the Fab is not fused to the extracellular domain of PDL1 or its PD1 binding portion; ii) the PD1 agonist moiety is not located at the N-terminus of the VH of the anti-CD20 Fab; iii) the PD1 agonist moiety is not located at the C-terminus of the Fc domain; iv) the protein is monovalent for the CD20 targeting moiety and/or the PD1 agonist moiety; iv) the protein is asymmetric; v) the protein comprises an Fc heterodimer; or any combination of two or more of the foregoing (i) to (vi).

Exemplary CD20-PD1 binding molecules are disclosed in Section 6.2 and Numbered Examples 1 to 142. Exemplary CD20 targeting moieties are disclosed in Section 6.2.1 and Numbered Examples 2 to 6. Exemplary PD1 agonist moieties are disclosed in Section 6.4 and Numbered Examples 7 to 22.

The present disclosure further provides nucleic acids encoding the CD20-PD1 binding molecules, the CD20-PD1 monomers, and the CD20 targeting moieties and PD1 agonist moieties. The nucleic acids encoding the CD20-PD1 binding molecules and CD20-PD1 monomers composed of two or more polypeptide chains can be single nucleic acids (e.g., vectors encoding all polypeptide chains) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains). The present disclosure further provides host cells and cell lines, which are engineered to express nucleic acids and CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting moieties and PD1 agonist moieties of the present disclosure. The present disclosure further provides methods for producing CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting moieties and PD1 agonist moieties of the present disclosure. Exemplary nucleic acids, host cells, cell lines, and methods of producing the CD20-PD1 binding molecules, the CD20-PD1 monomers, the CD20 targeting moieties, and the PD1 agonist moieties are described in Section 6.8 and numbered Examples 150 to 152, below.

The present disclosure further provides pharmaceutical compositions comprising a CD20-PD1 binding molecule of the present disclosure, a CD20-PD1 monomer, a CD20 targeting moiety, and a PD1 agonist moiety. Exemplary pharmaceutical compositions are described in Section 6.9 and Numbered Example 153 below.

Further provided herein are methods of using the CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting moieties, PD1 agonist moieties, and pharmaceutical compositions disclosed herein, for example, for treating autoimmune diseases, inhibiting cellular autoimmune responses, or inhibiting a subject's immune system. Exemplary methods are described in Section 6.10 below and in numbered Examples 154 to 165.

5. Description of the drawings

Figures 1A-1L are a series of sketches showing various formats of CD20-PD1 binding molecules according to certain embodiments. The heavy chain variable domain of the CD20 targeting portion is shown in a striped pattern, the light chain variable domain is shown in a dashed pattern, and the PD1 agonist portion (e.g., the extracellular domain of PDL1 or PDL2 or a PD1 binding portion thereof) is shown in a circle with a dashed line.

Figures 2A and 2B present a series of sketches representing the murine CD20-PD1 binding molecules PD1 (anti-mCD20 x mPDL1) tested (molecules A-L; Figure 2A) and controls (molecules M-S; Figure 2B).

FIG3A depicts 3-dimensional models of hPDL1 (left) and mPDL1 (right), highlighting the unpaired cysteine residue (C113) at the surface of mPDL1.

Figure 3B depicts a partial sequence alignment (78-137aa) of mPDL1 (top sequence) and hPDL1 (bottom sequence). The figure discloses SEQ ID NOs: 54-55, respectively, in order of appearance.

Figures 4A and 4B depict traces from flow binding studies and represent mPDL1 binding (upper panels) or anti-mCD20 binding (lower panels) by the indicated CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain) on Jurkat/mPD1 and MC38/mCD20 or HEK293/mCD20 cells, respectively.

Figures 5A-5B depict luciferase assay schemes. Figure 5A is a schematic depiction of a luciferase reporter assay, and Figure 5B is a sketch representation depicting the interactions between the key players depicted in Figure 5A.

Figures 6A-6E depict a test molecule (Figure 6A) and a series of traces using the test molecule (Figures 6B-6E). The traces depict mPD1 agonism measured using the bioassay depicted in Figure 5. Cells and molecules were used as indicated for each individual trace.

FIG. 7 depicts the experimental design for dose titration efficacy testing in prediabetic non-obese diabetic (NOD) mice.

Figures 8A-8I present a series of traces depicting individual animal data demonstrating spontaneous onset of diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 xmPDL1 ectodomain).

Figures 9A and 9B depict graphs demonstrating the ability of a CD20-PD1 binding molecule (anti-mCD20 x mPDL1 extracellular domain) (top: molecule L of Figure 2A; bottom: molecule G of Figure 2B) to modulate the onset of diabetes in NOD mice.

10A-10C are box plots depicting the reduction of activated autoreactive islet-specific CD8+ T cell infiltration into the pancreas of NOD mice following treatment with a CD20-PD1 binding molecule (anti-mCD20 x mPDL1 ectodomain). ^* p<0.05.

Figures 11A-11C are box plots depicting the reduction of activated autoreactive CD3+, CD4+, and CD8+ T cell infiltration into the spinal cord of EAE-MS mice following treatment with CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001.

6. Specific implementation methods

6.1. Definitions

About, approximately : Throughout the specification, the terms "about,""approximately," and the like are used in front of a number to indicate that the number is not necessarily exact (e.g., to account for fractions, changes in measurement accuracy and/or precision, timing, etc.). It should be understood that disclosure of "about X" or "approximately X" where X is a number is also disclosure of "X." Thus, for example, disclosure of embodiments where one sequence has "about X% sequence identity" to another sequence is also disclosure of embodiments where the sequence has "X% sequence identity" to another sequence.

And and or : Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logical operator, which encompasses the characteristics of selection alternatives (A or B, where selections A and B are mutually exclusive) and the characteristics of selection conjunctions (A or B, where both A and B are selected). In some places in the text, the term "and/or" is used for the same purpose, which should not be interpreted as implying that "or" is used with reference to mutually exclusive alternatives.

Antigen binding domain or ABD and antigen binding fragments : As used herein, the terms "antigen binding domain" or "ABD" and "antigen binding fragment" refer to the portion of a targeting moiety that is capable of specific, non-covalent and reversible binding to a target molecule.

Association : In the context of CD20-PD1 binding molecules or components thereof (e.g., CD20 targeting moiety; PD1 agonist moiety; dimerization moiety), the term "association" refers to a functional relationship between two or more polypeptide chains or portions of polypeptide chains. Specifically, the term "association" means that two or more polypeptides associate with each other, such as non-covalently by molecular interactions or covalently by one or more disulfide bridges or chemical cross-links, thereby producing a functional CD20-PD1 binding molecule. Examples of associations that may exist in the CD20-PD1 binding molecules of the present disclosure include, but are not limited to, associations between homodimeric or heterodimeric Fc domains in the Fc region, associations between VH and VL regions in Fab or scFv, associations between CH1 and CL in Fab, and associations between CH3 and CH3 in domain-substituted Fab.

Bivalent : The term "bivalent" as used herein with respect to a CD20-PD1 binding molecule with respect to a CD20 targeting moiety and/or a PD1 agonist moiety means that the CD20-PD1 binding molecule has two CD20 targeting moieties (e.g., two antigen binding fragments of an anti-CD20 antibody) and/or two PD1 agonist moieties (e.g., two PDL1 agonist moieties, two PDL2 agonist moieties, or a combination thereof), respectively. The CD20-PD1 binding molecule can be bivalent for one type of moiety (e.g., CD20 targeting moiety) and can be monovalent for another type of moiety (e.g., PD1 agonist moiety).

CD20-PD1 binding molecule : The term "CD20-PD1 binding molecule" refers to a molecule that includes at least one CD20 targeting moiety and at least one PD1 agonist moiety. Typically, a CD20-PD1 binding molecule is a molecule composed of one or more polypeptide chains (e.g., one, two, three or four polypeptide chains) that together include at least one CD20 targeting moiety and at least one PD1 agonist moiety.

In the context of the CD20-PD1 binding molecules of the present disclosure, the term "CD20-PD1 binding molecule" sometimes refers to the core components of the molecule, i.e., the CD20 targeting portion and the PD1 agonist portion, and sometimes also refers to the dimerization portion, such as the Fc domain and any/or associated linker portion. It should be understood that the term "CD20-PD1 binding molecule" also extends to molecules that include additional features, such as one or more stabilizing portions, one or more dimerization portions, one or more linker portions, and any combination of the foregoing, unless the context dictates otherwise.

CD20 targeting moiety : The term "CD20 targeting moiety" refers to any molecule or binding portion thereof (e.g., an immunoglobulin or antigen binding fragment thereof) that can bind to CD20. In some embodiments, the CD20 targeting moiety comprises an antigen binding fragment of an anti-CD20 antibody. The CD20 binding fragment of an anti-CD20 antibody can be in the form of a Fab, Fv, or scFv. The term "CD20 targeting moiety" comprises a molecule that can bind to any domain or region of CD20 (including a topological domain or a transmembrane domain). In some embodiments, the CD20 targeting moiety is a molecule that can bind to an extracellularly displayed CD20 region on the surface of a cell (e.g., a B cell). CD20 targeting moieties are further described in Section 6.2.

Complementarity determining region or CDR : As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR1-L1, CDR-L2, CDR-L3) in each light chain variable region. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. See, e.g., Kabat, 1991, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABM numbering scheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). Public databases can also be used to identify CDR sequences within antibodies.

Dimerization moiety : The term "dimerization moiety" refers to a polypeptide chain or amino acid sequence that is capable of promoting the association between two polypeptide chains to form a dimer. A first dimerization moiety can associate with a second dimerization moiety that is the same as the first dimerization moiety, or can associate with a second dimerization moiety that is different from the first dimerization moiety. In some embodiments, the dimerization moiety is an Fc domain, wherein the association of two Fc domains forms an Fc region. Thus, the Fc region can be a homodimer or a heterodimer.

EC50 : The term "EC50" refers to the half-maximal effective concentration of a molecule (such as a CD20-PD1 binding molecule) that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody or CD20-PD1 binding molecule at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of the CD20-PD1 binding molecule that gives half-maximal activation in an assay as described in Section 7.1.3.3.

Epitope : An epitope or antigenic determinant is a portion of an antigen (eg, CD20) that is recognized by an antibody or other antigen binding moiety as described herein. An epitope can be linear or conformational.

Fab : In the context of the CD20 targeting moiety of the present disclosure, the term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising the variable heavy (VH) domain of the antibody, which is located at the N-terminus of the first constant domain (referred to herein as C1), and the second polypeptide chain comprising the variable light (VL) domain of the antibody, which is located at the N-terminus of the second constant domain (referred to herein as C2) capable of pairing with the first constant domain. In a natural antibody, the VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and the VL is located at the N-terminus of the constant domain (CL) of the light chain. The Fab of the present disclosure can be arranged according to the natural orientation or contain domain substitutions or exchanges that promote correct VH and VL pairing. For example, the CH1 and CL domain pairs in Fab can be replaced with CH3 domain pairs to promote correctly modified Fab chain pairing in heterodimer molecules. CH1 and CL can also be reversed so that CH1 is connected to VL and CL is connected to VH, and this configuration is generally referred to as Crossmab, a type of "domain exchange".

Fc domain and Fc region : The term "Fc domain" refers to a portion of a heavy chain that pairs with the corresponding portion of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region can be identical to or different from each other. In native antibodies, the Fc domains are typically identical, but one or both Fc domains can be advantageously modified to allow heterodimerization (e.g., by knob-and-hole interactions) and/or purification (e.g., by star mutations).

Host cell or recombinant host cell : As used herein, the terms "host cell" and "recombinant host cell" refer to cells that are genetically engineered, for example, by the introduction of heterologous nucleic acids. It should be understood that such terms are intended to refer not only to specific subject cells, but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parental cells, but are still included in the scope of the term "host cell" as used herein. Host cells can carry heterologous nucleic acids transiently (e.g., on extrachromosomal heterologous expression vectors) or stably (e.g., by integrating heterologous nucleic acids into the host cell genome). For the purpose of expressing CD20-PD1 binding molecules, host cells can be cell lines of mammalian origin or mammalian-like properties, such as monkey kidney cells (COS, such as COS-1, COS-7), HEK293, baby hamster kidney (BHK, such as BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and/or engineered variants thereof. Engineered variants include, for example, modified glycan profiles and/or site-specific integration site derivatives.

Monomer and CD20-PD1 Monomer : As used herein, the terms "monomer" and "CD20-PD1 monomer" refer to a molecule comprising a first polypeptide chain that (a) comprises at least one CD20 targeting moiety and is capable of associating with a second polypeptide chain; (b) comprises at least one PD1 agonist moiety and is capable of associating with a second polypeptide chain; (c) comprises a dimerization moiety (e.g., an Fc domain) and is capable of associating with a corresponding dimerization moiety on a second polypeptide chain (e.g., another Fc domain); or (d) any combination of (a), (b), and (c) above. A monomer is capable of associating with other monomers by pairing with a dimerization moiety (e.g., an Fc domain). In some embodiments, one or more associations between monomers are stabilized by a hinge sequence or other portion of an Fc domain. Thus, a monomer of the present disclosure is capable of associating with another monomer to form a dimer. A dimer can be a homodimer (in which each constituent monomer is identical) or a heterodimer (in which case each constituent monomer is different). As used herein, reference to a "monomer" is for convenience and does not exclude the presence of one or more additional polypeptide chains, such as one or more light chains of one or more Fab domains. Thus, a "dimer" of two monomers may contain more than two polypeptide chains, such as three, four or more polypeptide chains, and reference to a monomer or dimer is not intended to imply any temporal order of association between polypeptide chains.

Monovalent : The term "monovalent" as used herein with respect to a CD20-PD1 binding molecule with respect to a CD20 targeting moiety and/or a PD1 agonist moiety means that the CD20-PD1 binding molecule has one CD20 targeting moiety (e.g., one antigen binding domain of an anti-CD20 antibody) and/or one PD1 agonist moiety (e.g., one PDL1 agonist moiety or one PDL2 agonist moiety), respectively. The CD20-PD1 binding molecule can be monovalent for one type of moiety (e.g., PD1 agonist moiety) and can be bivalent for another type of moiety (e.g., CD20 targeting moiety).

Multivalent : The term "multivalent" as used herein with respect to a CD20-PD1 binding molecule with respect to a CD20 targeting moiety and/or a PD1 agonist moiety refers to a CD20-PD1 binding molecule having two or more CD20 targeting moieties (e.g., two antigen-binding fragments of an anti-CD20 antibody) and/or two or more PD1 agonist moieties (e.g., two PDL1 agonist moieties, two PDL2 agonist moieties, or a combination thereof), respectively. A CD20-PD1 binding molecule can be multivalent with respect to one type of moiety (e.g., a CD20 targeting moiety) and can be monovalent with respect to another type of moiety (e.g., a PD1 agonist moiety).

Operably linked : As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are linked to produce a functional polypeptide, or two or more nucleic acid sequences, for example to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

PD1 agonist portion : The term "PD1 agonist portion" refers to any molecule or portion thereof that can bind to and agonize PD1. In some embodiments, the PD1 agonist portion includes an amino acid sequence having at least 70% sequence identity with the extracellular domain of programmed death ligand 1 (PDL1) or its PD1 binding portion, preferably mammalian PDL1 (e.g., human or murine PDL1). In other embodiments, the PD1 agonist portion includes an amino acid sequence having at least 70% sequence identity with the extracellular domain of programmed death ligand (PDL2) or its PD1 binding portion, preferably mammalian PDL2 (e.g., human or murine PDL2). The extracellular domains of PDL1 and PDL2 are sometimes referred to as "PDL1 extracellular domain" and "PDL2 extracellular domain", respectively. The terms "PDL1 extracellular domain" and "PDL2 extracellular domain" are conveniently used in this specification to refer not only to the PDL1 and PDL2 extracellular domains, but also to fragments and variant sequences having PD1 binding activity. Therefore, the terms "PDL1 extracellular domain" and "PDL2 extracellular domain" mentioned in this specification are intended to cover the PD1 binding portions of the PDL1 and PDL2 extracellular domains and variants thereof having PD1 binding function, such as amino acid sequences that have at least 70% or greater sequence identity with PD1 or PD2 and retain PDL1 binding.

The CD20-PD1 binding molecule can include a PD1 agonist portion having one or more amino acid substitutions, deletions and/or insertions compared to the corresponding wild-type sequence. For example, in some embodiments, the PD1 agonist portion is a murine PDL1 extracellular domain comprising a C113S substitution.

The PD1 agonist moiety is further described in Section 6.2.1.

Single-chain Fv or scFv : As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

Subject : The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

Treat, Treatment, Treating : As used herein, the terms "treat,""treatment," and "treating" refer to a reduction or improvement in the progression, severity, and/or duration of a condition as described herein, an improvement in one or more symptoms (preferably, one or more discernible symptoms) of a condition or disorder as described herein, or the prevention of a condition or disorder as described herein (e.g., an autoimmune or inflammatory condition or disorder caused by administration of a molecule or composition (e.g., one or more CD20-PD1 binding molecules of the present disclosure). In specific embodiments, the terms "treat,""treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a condition (e.g., an autoimmune condition) that is not necessarily discernible by the patient. In other embodiments, the terms "treat,""treatment," and "treating" refer to inhibiting the progression or onset of a condition physically, for example, by stabilizing discernible symptoms, physiologically, for example, by stabilizing physical parameters, or both.

Universal light chain : As used herein, in the context of a targeting moiety, the term "universal light chain" refers to a light chain polypeptide that is capable of pairing with the heavy chain region of the targeting moiety and is also capable of pairing with other heavy chain regions. Universal light chains are also referred to as "common light chains."

VH : The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of a scFv or Fab.

VL : The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of a scFv or Fab.

6.2.CD20-PD1 binding molecules

The present disclosure provides CD20-PD1 binding molecules comprising at least one CD20 targeting moiety and at least one PD1 agonist moiety. In some embodiments, the CD20-PD1 binding molecule further comprises a dimerization moiety.

The CD20-PD1 binding molecules of the present disclosure generally include or consist of CD20-PD1 monomers, which monomers contain one or more CD20 targeting moieties and/or one or more PD1 agonist moieties. The CD20-PD1 binding molecules of the present disclosure include a protein, which includes at least one CD20 targeting moiety, at least one PD1 agonist moiety, at least one dimerization moiety, and optionally one or more linker moieties, which separate one or more moieties in the protein.

In some embodiments, the PD1 agonist moiety is located between the CD20 targeting moiety and the dimerization moiety of the CD20-PD1 monomer. In such embodiments, when both the CD20 targeting moiety and the PD1 agonist moiety are located at the N-terminus of the dimerization moiety, the CD20-PD1 monomer thus has an N-terminal to C-terminal orientation of CD20 targeting moiety-PD1 agonist moiety-dimerization moiety. In such embodiments, when both the CD20 targeting moiety and the PD1 agonist moiety are located at the C-terminus of the dimerization moiety, the CD20-PD1 monomer thus has an N-terminal to C-terminal orientation of dimerization moiety-PD1 agonist moiety-CD20 targeting moiety.

In some embodiments (e.g., embodiments in which the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an extracellular domain of PDL1, and the dimerization moiety is an Fc domain), the CD20-PD1 binding molecule optionally has one or more of the following features: i) the light chain of the Fab is not fused to the extracellular domain of PDL1 or its PD1 binding portion; ii) the PD1 agonist moiety is not located at the N-terminus of the VH of the anti-CD20 Fab; iii) the PD1 agonist moiety is not located at the C-terminus of the Fc domain; iv) the protein is monovalent for the CD20 targeting moiety and/or the PD1 agonist moiety; v) the protein is asymmetric; vi) the protein includes an Fc heterodimer; or any combination of two or more of the foregoing (i) to (vi). In some embodiments, the CD20-PD1 binding molecule has feature i) (i.e., the light chain of the Fab is not fused to the extracellular domain of PDL1 or its PD1 binding portion). In some embodiments, the CD20-PD1 binding molecule has feature ii) (i.e., the PD1 agonist portion is not located at the N-terminus of the VH of the anti-CD20 Fab). In some embodiments, the CD20-PD1 binding molecule has feature iii) (i.e., the PD1 agonist portion is not located at the C-terminus of the Fc domain). In some embodiments, the CD20-PD1 binding molecule has feature iv) (i.e., the protein is monovalent for the CD20 targeting portion and/or the PD1 agonist portion). In some embodiments, the CD20-PD1 binding molecule has feature v) (i.e., the protein is asymmetric). In some embodiments, the CD20-PD1 binding molecule has feature vi) (i.e., the protein includes an Fc heterodimer). The CD20-PD1 binding molecules of the present disclosure may have any combination of two, three, four, five or all of the aforementioned features. For example, in some embodiments, the CD20-PD1 binding molecules disclosed herein have feature ii) (i.e., the PD1 agonist portion is not located at the N-terminus of the VH of the anti-CD20 Fab) and feature iii) (i.e., the PD1 agonist portion is not located at the C-terminus of the Fc domain).

Exemplary dimerization moieties are described in Section 6.5 and comprise an Fc domain that confers homodimerization or heterodimerization capability to the CD20-PD1 binding molecule.

The CD20-PD1 binding molecule can be composed of one or more polypeptides. In some embodiments, the CD20-PD1 binding molecule is composed of a plurality of (e.g., two) monomers, the monomers including at least one CD20 targeting moiety and/or at least one PD1 agonist moiety and in some embodiments also including a dimerization moiety. In some embodiments, the CD20-PD1 binding molecule of the present disclosure is composed of two monomers, the monomers optionally associated with one or more additional polypeptide chains (e.g., polypeptide chains including light chains of anti-CD20 Fab moieties). The monomers may be identical, thereby forming homodimers, or the monomers may be different, thereby forming heterodimers. The dimerization moiety of each monomer of the CD20-PD1 binding molecule may be configured to dimerize together. Exemplary dimerization agent moieties are described in Section 6.5.

The one or more CD20 targeting moieties and the one or more PD1 agonist moieties may be included on the same arm of the CD20-PD1 binding molecule (e.g., wherein the CD20 targeting moiety comprises an anti-CD20 Fab and the PD1 agonist moiety comprises a PDL1-based PD1 agonist moiety, the variable heavy chain or variable light chain of the anti-CD20 Fab and the PDL1-based PD1 agonist moiety are on the same polypeptide chain), or may be included on different arms of the bispecific CD20-PD1 agonist (e.g., wherein the CD20 targeting moiety comprises an anti-CD20 Fab and the PD1 agonist moiety comprises a PDL1-based PD1 agonist moiety, the variable heavy chain or variable light chain of the anti-CD20 Fab and the PDL1-based PD1 agonist moiety are on different polypeptide chains). Exemplary configurations of the CD20-PD1 binding molecules of the present disclosure are disclosed, inter alia, in Figures 1A-1L and numbered Examples 31 to 106.

The CD20-PD1 binding molecule can be monovalent for the CD20 targeting moiety (i.e., having a single CD20 targeting moiety) or can be multivalent for the CD20 targeting moiety (i.e., having multiple CD20 targeting moieties). Similarly, the CD20-PD1 binding molecule can be monovalent for the PD1 agonist moiety (i.e., having a single PD1 agonist moiety) or can be multivalent for the PD1 agonist moiety (i.e., having multiple PD1 agonist moieties). In some embodiments, the CD20-PD1 binding molecule is bivalent for the CD20 targeting moiety (i.e., having two CD20 targeting moieties). When the CD20-PD1 binding molecule is multivalent for the CD20 targeting moiety and/or the PD1 agonist moiety, the multiple CD20 targeting moieties can be the same or different from each other, and/or the multiple PD1 agonist moieties can be the same or different from each other.

In some embodiments, the CD20-PD1 binding molecule may comprise one or more linker sequences connecting various components of one or more polypeptide chains thereof, such as (1) a CD20 targeting moiety or a portion thereof (e.g., a heavy chain or light chain of an anti-CD20 Fab) and a PD1 agonist moiety or a portion thereof (e.g., PDL1 or PDL2) (when present on the same polypeptide chain), (2) a CD20 targeting moiety and a dimerization domain (e.g., an Fc domain), (3) a PD1 agonist moiety and a dimerization domain (e.g., an Fc domain), or (4) any combination of the foregoing. Exemplary linkers are described in Section 6.7.

Most CD20-PD1 binding molecules are multimeric due to the association of dimerization moieties (e.g., Fc domains) configured to associate with each other. The CD20-PD1 binding molecules may comprise two, three, four or more polypeptide chains, some of which are associated through dimerization moieties and other polypeptide chains are associated through VH-VL interactions. For convenience and descriptive purposes only, the present disclosure generally refers to a polypeptide containing a CD20 targeting moiety, a PD1 agonist moiety and/or a dimerization moiety (e.g., a first Fc domain) as a "monomer," which can associate with another polypeptide chain containing a CD20 targeting moiety, a PD1 agonist moiety and/or a corresponding dimerization moiety (e.g., a second Fc domain), respectively. A monomer may comprise one, two, three or more polypeptide chains. For example, in one embodiment, a monomer may be composed of (a) a first polypeptide chain containing an anti-CD20 VH, a PD1 agonist moiety and an Fc domain and (b) a second polypeptide chain containing a VL that can be paired with an anti-CD20 VH. In another embodiment, the monomer may be composed of (a) a first polypeptide chain comprising a first anti-CD20 VH, a second anti-CD20 VH and an Fc domain, (b) a second polypeptide chain comprising a first VL capable of pairing with the first anti-CD20 VH, and (c) a third polypeptide chain comprising a second VL capable of pairing with the second anti-CD20 VH.

The following are some illustrative examples of monomers of the present disclosure described in an N-terminal to C-terminal orientation. The individual elements of each monomer are described in detail herein (eg, in subsequent subsections and numbered examples).

(1) Exemplary Monomer 1 : CD20 targeting moiety - optional linker - dimerization moiety (see, e.g., Figures 1A, 1E, 1F, 1G and 1H, left monomer).

(2) Exemplary Monomer 2 : PD1 agonist moiety - optional linker - dimerization moiety (see, eg, FIG. 1A , right monomer).

(3) Exemplary Monomer 3 : Optional linker-dimerization moiety (see, eg, Figures 1B, 1C and 1D, left monomer).

(4) Exemplary monomer 4 : PD1 agonist moiety-optional linker-CD20 targeting moiety-optional linker-dimerization moiety (see, e.g., Figures 1B and 1E, right monomer; Figure 1I, two monomers).

(5) Exemplary monomer 5 : CD20 targeting moiety - optional linker - dimerization moiety - PD1 agonist moiety (see, e.g., Figures 1C and 1F, right monomer; Figure 1J, two monomers).

(6) Exemplary Monomer 6 : CD20 targeting moiety-optional linker-PD1 agonist moiety-optional linker-dimerization moiety (see, e.g., Figures 1D and 1G, right monomer; Figure 1L, two monomers).

(7) Exemplary monomer 7 : PD1 agonist moiety-optional linker-PD1 agonist moiety-optional linker-dimerization moiety (see, eg, FIG. 1H , right monomer).

(8) Exemplary monomer 8 : CD20 targeting moiety-optional linker-dimerization moiety-optional linker-CD20 targeting moiety (see, e.g., FIG. 1K , left monomer).

(9) Exemplary monomer 9 : PD1 agonist moiety-optional linker-dimerization moiety-optional linker-PD1 agonist moiety (see, eg, FIG. 1K , right monomer).

In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising Exemplary Monomer 1 and Exemplary Monomer 2 (see, e.g., FIG. 1A ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 3 and exemplary monomer 4 (see, e.g., FIG. 1B ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 3 and exemplary monomer 5 (see, e.g., FIG. 1C ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 3 and exemplary monomer 6 (see, e.g., FIG. 1D ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 1 and exemplary monomer 4 (see, e.g., FIG. 1E ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 1 and exemplary monomer 5 (see, e.g., FIG. 1F ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 1 and exemplary monomer 6 (see, e.g., FIG. 1G ).

In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 1 and exemplary monomer 7 (see, e.g., FIG. 1H ).

In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising two monomers according to exemplary monomer 4 (see, e.g., FIG. 1I ).

In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising two monomers according to exemplary monomer 5 (see, e.g., FIG. 1J ).

In some embodiments, the present disclosure provides CD20-PD1 binding molecules including exemplary monomer 8 and exemplary monomer 9 (see, e.g., FIG. 1K ).

In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising two monomers according to exemplary monomer 6 (see, e.g., FIG. 1L ).

In the CD20-PD1 binding molecules of the present disclosure, when the CD20 targeting moiety is an antigen binding domain ("ABD") of an antibody, each monomer may be composed of two or more polypeptide chains, one polypeptide chain carrying a heavy chain variable region, and the other polypeptide chain carrying a light chain variable region. The CD20 targeting moiety may include heavy and light chain variable domains on separate polypeptide chains. For example, a monomer may be composed of polypeptide A and polypeptide B. Polypeptide A may include, for example, from N-terminus to C-terminus: a heavy chain variable domain of a CD20 targeting moiety-an optional linker-PD1 agonist moiety-an optional linker-dimerization moiety; and polypeptide B may include a light chain variable domain of a CD20 targeting moiety. In the case where the monomer is divalent for the CD20 targeting moiety, the monomer may include a third polypeptide chain (polypeptide C) comprising another light chain variable domain of the CD20 targeting moiety.

Alternatively, the CD20 targeting moiety may be in the form of a scFv, in which the heavy and light chain variable regions of the CD20 targeting moiety are fused to each other in a single polypeptide.

Further details of the components of the CD20-PD1 binding molecules of the present disclosure are presented below.

6.2.1. Biochemical properties of CD20-PD1 binding molecules

In vivo, large complexes of antibodies can be rapidly eliminated by phagocytosis, resulting in reduced antibody efficacy. Large complexes can also increase the immunogenicity of therapeutic antibodies. See, for example, WO2020047067A1. During manufacturing, aggregation is a common problem that compromises the quality, safety, and efficacy of antibodies. Compared to the parent antibody from which the CD20 targeting portion is derived and/or compared to other antibody forms including a CD20 targeting portion and a PD1 agonist portion, the CD20-PD1 binding molecules of the present disclosure may be, for example, not prone to aggregation in vivo or ex vivo. Therefore, in some embodiments, during recombinant production in a mammalian cell line, the aggregation of the CD20-PD1 binding molecules of the present disclosure is at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, or at least 99% less than the parent antibody. As described in Section 7.1.4, the oligomerization state of the CD20-PD1 binding molecule can be determined, for example, by size exclusion ultra-high performance liquid chromatography. Without additional size exclusion chromatography (SEC), most CD20-PD1 binding molecules show greater than 85% monomeric species (see Section 7.2.2). Column purification can then be used to further purify the monomeric species. For example, the monomer percentage of 2+2m20_mPL_4 (molecule L in Figure 2A) was increased to 99% after two column purifications, including a SEC step (see Section 7.2.2).

The CD20-PD1 binding molecules disclosed herein also exhibit good thermal stability. High thermal stability and low aggregation tendency promote antibody manufacturing and storage, and promote long serum half-life. Carter and Merchant, 1997, Curr Opin Biotechnol, 8(4): 449-454. Thermal stability can be measured by methods known in the art, including differential scanning fluorimetry (DSF) (see, e.g., Section 7.1.5). All tested CD20-PD1 molecules have thermal stability similar to that measured by DSF, with a melting temperature 1 (Tm1) (which represents the first unfolding midpoint of the protein) of about 60°C (see Section 7.2.2).

6.3.CD20 Targeting Moiety

In some embodiments, incorporation of a CD20 targeting moiety into the CD20-PD1 binding molecules of the present disclosure provides for delivery of a high concentration of a local PD1 agonist moiety for the treatment of autoimmune disorders, including but not limited to type 1 diabetes, systemic lupus erythematosus, and Crohn's disease, and for the treatment of graft-versus-host disease (GVHD). In some embodiments, in addition to facilitating local delivery of a PD1 agonist moiety, an anti-CD20 moiety provides an additional therapeutic approach for such autoimmune diseases.

In certain embodiments of the present disclosure, each CD20 targeting portion of the CD20-PD1 binding molecule includes an antigen binding domain of an anti-CD20 antibody. In some embodiments, the CD20-PD1 binding molecule of the present disclosure includes a single CD20 targeting portion (e.g., a CD20 targeting portion on the first monomer or on the second monomer in an embodiment where the CD20-PD1 binding molecule is monovalent for the CD20 targeting portion). In some embodiments, the CD20-PD1 binding molecule of the present disclosure includes two CD20 targeting portions (e.g., a first CD20 targeting portion on the first monomer and a second CD20 targeting portion on the second monomer in an embodiment where the CD20-PD1 binding molecule is bivalent for the CD20 targeting portion; or both the first CD20 targeting portion and the second CD20 targeting portion can be on the first monomer or the second monomer). In such embodiments, the two CD20 targeting portions may be the same, or the two CD20 targeting portions may be different. When different, the two CD20 targeting portions may be orthogonal, bind to different epitopes of CD20, and/or be non-competitive.

In some embodiments, the CD20 targeting moiety comprises an antigen binding domain of a known anti-CD20 antibody. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomabituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, and veltuzumab (each a "reference CD20 antibody"). In further embodiments, the CD20 targeting moiety comprises a CDR having a CDR sequence of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety comprises all 6 CDR sequences of a reference CD20 antibody. In other embodiments, the targeting moiety comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of a reference CD20 antibody and a light chain CDR sequence of a universal light chain. In other aspects, the CD20 targeting moiety comprises a VH comprising an amino acid sequence of a VH of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety further comprises a VL comprising an amino acid sequence of a VL of a reference CD20 antibody. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

In other embodiments, the CD20 targeting moiety comprises an antigen binding domain that binds to the same CD20 epitope and/or competes with the following for binding to CD20: rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, utoximab, okatuzumab, TRU-015 or veltuzumab. Assays for measuring antibody competition are known in the art. For example, a sample of CD20 can be bound to a solid support. Then, a first antibody and a second antibody are added. One of the two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to separate and discrete sites on CD20, the labeled antibody will bind at the same level, regardless of whether the unlabeled antibody is present. However, if the sites of interaction are identical or overlapping, the unlabeled antibody will compete, and the amount of the labeled antibody bound to the antigen will be reduced. If the unlabeled antibody is present in excess, very little (if any) labeled antibody will bind. In some embodiments, the competitive antibody is an antibody that reduces the binding of another antibody to CD20 by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. The details of the procedures for performing such competitive assays are well known in the art and can be found, for example, in Greenfield, ed., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2014. Such assays can be quantitatively determined using purified antibodies. A standard curve can be established by titrating an antibody itself, i.e., the same antibody is used for both the marker and the competitor. The unlabeled competitive antibody is titrated to inhibit the ability of the labeled antibody to bind to the plate. The results can be plotted and the concentrations required to achieve the desired degree of binding inhibition can be compared. In some embodiments, competition for binding to a target molecule can be determined, for example, using real-time, label-free biolayer interferometry on an Octet HTX biosensor platform (Pall ForteBioCorp.).

Suitable CD20 targeting moiety formats are described in Section 6.3.1. The CD20 targeting moiety is preferably a CD20 binding fragment of an anti-CD20 antibody, such as a Fab as described in Section 6.3.1.1, an Fv fragment as described in Section 6.3.1.2, or a scFv.

The CD20 targeting moiety can be incorporated into a CD20-PD1 binding molecule having any of the configurations described herein. The CD20-PD1 binding molecule is typically composed of multiple polypeptide chains, such as represented by the exemplary monomers described in Section 6.2. As shown in Section 6.2, the CD20 targeting moiety can be incorporated into any of the exemplary monomers 1, 4, 5, 6, and 8. Exemplary CD20-PD1 binding molecules incorporating one or more of the exemplary monomers 1, 4, 5, 6, and 8 are described in detail in Section 6.2.

6.3.1. CD20 Targeting Moiety Format

In certain aspects, the CD20 targeting moiety can be any type of antibody or fragment thereof that retains specific binding to CD20. In some embodiments, the antigen binding moiety is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgG ₁ or IgG ₄ immunoglobulin molecule. Antibody fragments include but are not limited to VH (or _VH ) fragments, VL (or _VL ) fragments, Fab fragments, F(ab') ₂ fragments, scFv fragments, Fv fragments, minibodies, bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies.

6.3.1.1.Fab

Fab domains are traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the CD20-PD1 binding molecules of the present disclosure, the Fab domains are typically recombinantly expressed as part of the CD20-PD1 binding molecules.

The Fab domain may include constant domains and variable region sequences from any suitable species, and thus may be murine, chimeric, human or humanized. In some embodiments, the variable region sequences and/or constant domain region sequences are derived from known anti-CD20 antibodies. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, utoximab, okatuzumab, TRU-015 and veltuzumab.

In some embodiments, the CD20 targeting moiety comprises a Fab that binds to the same CD20 epitope as and/or competes with the following for binding to CD20: a Fab of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, utoximab, okatuzumab, TRU-015, or veltuzumab (each a "reference CD20 antibody"). In further embodiments, the CD20 targeting moiety comprises a CDR having a CDR sequence of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety comprises all 6 CDR sequences of a reference CD20 antibody. In other embodiments, the targeting moiety comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of a reference CD20 antibody and a light chain CDR sequence of a universal light chain. In further aspects, the CD20 targeting moiety comprises a VH comprising the amino acid sequence of a VH of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety further comprises a VL comprising the amino acid sequence of the VL of a reference CD20 antibody. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

The Fab domain generally includes a CH1 domain connected to a VH domain, which is paired with a CL domain connected to a VL domain. In a wild-type immunoglobulin, the VH domain is paired with the VL domain to form an Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding module. The disulfide bond between the two constant domains can further stabilize the Fab domain.

For the CD20-PD1 binding molecules of the present disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same ABD and minimize abnormal pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategy shown in Table 1 below can be used:

Thus, in certain embodiments, the correct association between the two polypeptides of the Fab is promoted by exchanging the VL and VH domains of the Fab with each other or exchanging the CH1 and CL domains with each other, for example, as described in WO 2009/080251.

Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain of the Fab and one or more amino acid modifications in the CL domain and/or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other rather than with components of other Fabs.

In one embodiment, the one or more amino acid modifications are limited to the conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains as shown in the Kabat residue numbering. Almagro, 2008, Frontiers In Bioscience 13: 1619-1633 provides a definition of framework residues based on the Kabat, Chothia and IMGT numbering schemes.

In one embodiment, the modifications introduced in the VH and CH1 and/or VL and CL domains are complementary to each other. The complementarity of the heavy and light chain interfaces can be achieved based on steric and hydrophobic contacts, electrostatic/charge interactions, or a combination of multiple interactions. Complementarity between protein surfaces is extensively described in the literature in terms of lock and key fits, knobs and sockets, protrusions and cavities, donors and acceptors, etc., all of which imply the nature of the structural and chemical match between the two interacting surfaces.

In one embodiment, one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO 2014/150973 and WO 2014/082179, the contents of which are hereby incorporated by reference.

In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain that introduce a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J. Immunol. 196:3199-211).

In some embodiments, the Fab domain includes 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which are used to exchange the hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J. Immunol. 196:3199-211).

In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modifications are introduced in the VH domain, and 38R modifications are introduced in the VL domain.

The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby improving the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

The Fab domain can also be modified by replacing the CH1 domain and the CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7: 364-76 describe replacing the CH1 domain with the constant domain of the T cell receptor and replacing the CL domain with the b domain of the T cell receptor, and pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

Instead of or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each Fab VL region of the CD20-PD1 binding molecule of the present disclosure. In various embodiments, the use of a common light chain as described herein reduces the number of CD20-PD1 binding molecules of inappropriate species compared to the use of the original homologous VL. In various embodiments, the VL domain of the CD20-PD1 binding molecule is identified from a monospecific antibody comprising a common light chain. In various embodiments, the VH region of the CD20-PD1 binding molecule includes a human heavy chain variable gene segment rearranged in vivo in a mouse B cell, the mouse B cell having previously been engineered to express a limited human light chain library or a single human light chain homologous to a human heavy chain, and in response to exposure to an antigen of interest, an antibody library containing multiple human VHs homologous to one or more of two possible human VLs is produced, wherein the antibody library is specific to the antigen of interest. The common light chain is a light chain derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence and comprises a somatically mutated (eg, affinity matured) version. See, eg, US Pat. No. 10,412,940.

6.3.1.2.scFv

Single-chain Fv or "scFv" antibody fragments include the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as a single-chain polypeptide, and retain the specificity of the intact antibody from which it is derived. Typically, the scFv polypeptide further includes a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of the scFv are the linkers identified in Section 6.7.

Unless otherwise indicated, as used herein, a scFv can have the VL and VH variable regions in any order, for example, relative to the N-terminal and C-terminal ends of the polypeptide, a scFv can include VL-linker-VH or can include VH-linker-VL.

The scFv may include VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences. In some embodiments, the scFv may include VH and VL sequences from known anti-CD20 antibodies. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, utoximab, okatuzumab, TRU-015 and veltuzumab.

In some embodiments, the CD20 targeting moiety comprises a scFv that binds to the same CD20 epitope as and/or competes for binding to CD20 with a scFv derived from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015, or veltuzumab.

To generate a nucleic acid encoding an scFv, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, e.g., any of the linkers described in Section 6.7 (typically a repeating sequence of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser) ₃ (SEQ ID NO:1), such that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH regions are connected by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

6.4.PD1 agonist part

In certain embodiments of the present disclosure, the PD1 agonist portion of the CD20-PD1 binding molecule includes a wild-type or variant PD1 binding domain of programmed death ligand 1 (PDL1) or programmed death ligand 2 (PDL2). In some embodiments, the CD20-PD1 binding molecule of the present disclosure includes a single PD1 agonist portion (e.g., a PD1 agonist portion on the first monomer or on the second monomer in an embodiment where the CD20-PD1 binding molecule is monovalent for the PD1 agonist portion). In some embodiments, the CD20-PD1 binding molecule of the present disclosure includes two PD1 agonist portions (e.g., a first PD1 agonist portion on the first monomer and a second PD1 agonist portion on the second monomer, or both a first PD1 agonist portion and a second PD1 agonist portion on the first monomer or the second monomer). In such embodiments, the two PD1 agonist portions may be the same, or the two PD1 agonist portions may be different. When different, the two PD1 agonist portions may interact differently with PD1 (e.g., with different affinities).

The PD1 agonist portion can be incorporated into a CD20-PD1 binding molecule having any of the configurations described herein. The CD20-PD1 binding molecule is typically composed of multiple polypeptide chains, for example, as represented by the exemplary monomers described in Section 6.2. As shown in Section 6.2, the PD1 agonist portion can be incorporated into any of the exemplary monomers 2, 4, 5, 6, 7, and 9. Exemplary CD20-PD1 binding molecules incorporating one or more exemplary monomers of exemplary monomers 2, 4, 5, 6, 7, and 9 are described in detail in Section 6.2. In some embodiments, the PD1 agonist portion is an agonist portion based on PDL1. In other embodiments, the PD1 agonist portion is an agonist portion based on PDL2.

6.4.1. PDL1-based PD1 agonist

PDL1 plays a key role in inducing and maintaining immune tolerance to oneself. As a ligand for the inhibitor receptor PD1, PDL1 regulates the activation threshold of T cells and limits T cell effector responses. The present disclosure provides CD20-PD1 binding molecules, wherein at least one PD1 agonist portion comprises an amino acid sequence comprising a PDL1 amino acid sequence as described herein or homologous thereto. Such PD1 agonist portions are referred to herein as "PD1 agonist portions based on PDL1" or similar terms.

The human PDL1 protein is synthesized as a precursor polypeptide of 290 amino acids, from which 18 amino acids are removed to produce mature hPDL1, of which amino acids 19-238 (numbered based on the precursor protein) form the hPDL1 extracellular domain or ectodomain. The sequence of human PDL1 has the Uniprot identifier Q9NZQ7 (uniprot.org/uniprot/Q9NZQ7). The sequence of murine PDL1 has the Uniprot identifier Q9EP73 (uniprot.org/uniprot/Q9EP73).

The precursor human PDL1 polypeptide has the following amino acid sequence (signal sequence = underlined ; extracellular domain = bold):

Murine PDL1 polypeptide is synthesized as a precursor polypeptide of 290 amino acids, from which 18 amino acids are removed to produce mature mPDL1. Amino acids 19-239 (numbered based on the precursor protein) form the mPDL1 extracellular domain or ectodomain. The precursor murine PDL1 polypeptide has the following amino acid sequence (signal sequence = underlined ; extracellular domain = bold):

In some embodiments, the PD1 agonist portion is an agonist portion based on PDL1, which includes an amino acid sequence having at least 70% sequence identity with the PD1 binding portion PDL1 of a mammal (e.g., human or murine) or the entire extracellular domain PDL1 of a mammal (e.g., human or murine), such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity. In certain aspects, the PD1 binding portion of PDL1 includes the IgV domain of human or mouse PDL1. In certain embodiments, the PD1 binding portion of PDL1 includes amino acids 19-134 of human PDL1 or amino acids 19-134 of murine PDL1.

In certain embodiments, the PD1 agonist portion based on PDL1 includes an amino acid sequence having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) sequence identity and one or more amino acid substitutions with the extracellular domain of PDL1 or its PD1 binding portion compared to wild-type PDL1. In some embodiments, the one or more amino acid substitutions increase the stability of the PD1 agonist portion based on PDL1. For example, in some embodiments, mPDL1 includes the amino acid substitution C113S (numbered based on the precursor protein).

In some embodiments, the PDL1-based PD1 agonist portion is optionally fused directly or indirectly to the CD20 targeting portion via a linker (e.g., as described in Section 6.7). When present on the same monomer, the PDL1-based PD1 agonist portion can be located at the N-terminus or C-terminus of the CD20 targeting portion. When the PDL1-based PD1 agonist portion is "directly" fused to the CD20 targeting portion, the PDL1-based PD1 agonist portion and the CD20 targeting portion are positioned adjacent to each other on the same monomer, separated only by a linker (if present). When the PDL1-based PD1 agonist portion is "indirectly" fused to the CD20 targeting portion, the PDL1-based PD1 agonist portion and the CD20 targeting portion are separated by one or more other domains (e.g., a dimerization portion) on the same monomer, or are located on separate monomers.

6.4.2. PDL2-based PD1 agonist

The interaction of PDL2 with PD1 inhibits T cell proliferation by blocking cell cycle progression and cytokine production. The present disclosure provides CD20-PD1 binding molecules, wherein at least one PD1 agonist portion comprises an amino acid sequence that comprises or is homologous to the PDL2 amino acid sequence described herein. Such PD1 agonist portions are referred to herein as "PD1 agonist portions based on PDL2" or similar terms.

The human PDL2 protein is synthesized as a precursor polypeptide of 273 amino acids, from which 19 amino acids are removed to produce mature hPDL2, of which amino acids 20-220 (numbered based on the precursor protein) form the hPDL2 extracellular domain or ectodomain. The sequence of human PDL2 has the Uniprot identifier Q9BQ51 (uniprot.org/uniprot/Q9BQ51). The sequence of murine PDL2 has the Uniprot identifier Q9WUL5 (uniprot.org/uniprot/Q9WUL5).

The precursor human PDL2 polypeptide has the following amino acid sequence (signal sequence = underlined ; extracellular domain = bold):

Murine PDL2 polypeptides are synthesized as a precursor polypeptide of 247 amino acids, from which 19 amino acids are removed to produce mature mPDL2. Amino acids 20-221 (numbered based on the precursor protein) form the mPDL2 extracellular domain or ectodomain. The precursor murine PDL2 polypeptide has the following amino acid sequence (signal sequence = underlined ; extracellular domain = bold):

In some embodiments, the PD1 agonist portion is an agonist portion based on PDL2, which includes an amino acid sequence having at least 70% sequence identity with the PD1 binding portion PDL2 of a mammal (e.g., human or murine) or the entire extracellular domain PDL1 of a mammal (e.g., human or murine), such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity. In certain aspects, the PD1 binding portion of PDL2 includes the IgV domain of human or mouse PDL2. In certain embodiments, the PD1 binding portion of PDL2 includes amino acids 20-121 of human PDL2 or amino acids 20-121 of murine PDL2.

In certain embodiments, the PDL2-based PD1 agonist portion comprises an amino acid sequence having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) sequence identity and one or more amino acid substitutions with the extracellular domain of PDL2 or its PD1 binding portion, compared to wild-type PDL2.

In some embodiments, the PDL2-based PD1 agonist portion is optionally fused directly or indirectly to the CD20 targeting portion via a linker (e.g., as described in Section 6.7). When present on the same monomer, the PDL2-based PD1 agonist portion can be located at the N-terminus or C-terminus of the CD20 targeting portion. When the PDL2-based PD1 agonist portion is "directly" fused to the CD20 targeting portion, the PDL2-based PD1 agonist portion and the CD20 targeting portion are adjacently positioned on the same monomer, separated only by a linker (if present). When the PDL2-based PD1 agonist portion is "indirectly" fused to the CD20 targeting portion, the PDL2-based PD1 agonist portion and the CD20 targeting portion are separated by one or more other domains (e.g., dimerization moieties) on the same monomer, or are located on separate monomers.

6.5. Dimerization

6.5.1. Fc domain

In some embodiments, the CD20-PD1 binding molecules and CD20-PD1 monomers of the present disclosure comprise one or more dimerization moieties, such as one or more dimerization moieties that are or include an Fc domain. In certain embodiments, the CD20-PD1 monomers of the present disclosure comprise a single dimerization moiety (e.g., a single Fc domain) and/or the CD20-PD1 binding molecules of the present disclosure comprise two dimerization moieties (e.g., two Fc domains that can associate to form an Fc region).

The CD20-PD1 binding molecules and CD20-PD1 monomers disclosed herein may comprise an Fc domain or a pair of Fc domains associated to form an Fc region, derived from any suitable species and operably linked to a CD20 targeting moiety and/or a PD1 agonist moiety. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the Fc domain is derived from a human IgG Fc domain.

The CD20 targeting moiety and/or the PD1 agonist moiety can be fused to the N-terminus or C-terminus of the IgG Fc domain.

One embodiment of the present disclosure is directed to a dimer comprising two Fc fusion polypeptides produced by fusing one or more CD20 targeting moieties and/or PD1 agonist moieties to an Fc domain, for example, by fusing both a CD20 targeting moiety and a PD1 agonist moiety to an Fc domain, which when expressed can form a CD20-PD1 monomer capable of homodimerization, or by fusing one or more CD20 targeting moieties and/or one or more PD1 agonist moieties to a first Fc domain and fusing one or more CD20 targeting moieties and/or one or more PD1 agonist moieties to a second Fc domain, which when expressed form two different CD20-PD1 monomers capable of heterodimerization. The dimer can be prepared, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble into a very similar antibody molecule, thereby forming an interchain bond between the Fc moieties to produce a dimer.

The Fc domain that can be incorporated into the CD20-PD1 monomer can be derived from any suitable antibody class, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc domain is derived from IgG1. In some embodiments, the Fc domain is derived from IgG4.

The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are usually identical, but for the purpose of generating multispecific binding molecules (e.g., CD20-PD1 binding molecules of the present disclosure), the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.5.1 below.

In natural antibodies, the heavy chain Fc domain of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3), and the heavy chain Fc domain of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerize to form the Fc region.

In the CD20-PD1 binding molecules of the present disclosure, the Fc region and/or the Fc domain therein may include heavy chain constant domains from one or more different classes (e.g., one, two, or three different classes) of antibodies.

In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG1.

In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG2.

In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG3.

In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG4.

In one embodiment, the Fc region includes a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

It should be understood that the heavy chain constant domain of the Fc region used to generate the CD20-PD1 binding molecules of the present disclosure may include variants of the above-mentioned naturally occurring constant domains. Such variants may include one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that is different in sequence from the wild-type constant domain. It should be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

IgM and IgA naturally exist in humans as covalent polymers of the common H2L2 antibody unit. When IgM incorporates the J chain, it appears as a pentamer, or when the J chain is lacking, it appears as a hexamer. IgA exists in monomeric and dimer forms. The heavy chains of IgM and IgA have 18 amino acids extending to the C-terminal constant domain, which is called the tailpiece. The tailpiece contains cysteine residues that form disulfide bonds between the heavy chains in the polymer and are believed to play an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the CD20-PD1 binding molecules of the present disclosure do not include a tailpiece.

The Fc domain incorporated into the CD20-PD1 binding molecules of the present disclosure may include one or more modifications that alter the functional properties of the protein (e.g., binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond structure, or altered glycosylation patterns). Exemplary Fc modifications that alter effector function are described in Section 6.5.1.1.

The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric CD20-PD1 binding molecules, for example by allowing heterodimerization, which is the pairing of non-identical Fc domains in preference to identical Fc domains. Heterodimerization allows the generation of CD20-PD1 binding molecules in which the different polypeptide components are linked to each other via Fc regions containing Fc domains of different sequences. Examples of heterodimerization strategies are exemplified in Section 6.5.1.2.

It will be appreciated that any of the above modifications may be combined in any suitable manner to achieve desired functional properties and/or combined with other modifications to alter the properties of the CD20-PD1 binding molecule.

6.5.1.1. Fc domains with altered effector functions

In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function.

In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the following groups: complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and cytokine secretion. In a specific embodiment, the effector function is ADCC.

In one embodiment, the Fc domain (e.g., the Fc domain of a CD20-PD1 monomer) or Fc region (e.g., one or two Fc domains of a CD20-PD1 binding molecule that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, G237, N297, A330, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, amino acid substitution is P329A or P329G, particularly P329G (numbered according to the Kabat EU index). In one embodiment, Fc domain or Fc region include amino acid substitution at position P329 and include other amino acid substitution at a position selected from: E233, L234, L235, N297 and P331 (numbered according to the Kabat EU index). In a more specific embodiment, other amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, Fc domain or Fc region include amino acid substitution at position P329, L234 and L235 (numbered according to the Kabat EU index). In a more specific embodiment, Fc domain includes amino acid mutation L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG").

In some embodiments, the Fc domain or Fc region comprises amino acid substitutions at positions L234, L235, G237, A330, and P331 (numbered according to the Kabat EU index). In more specific embodiments, the amino acid substitutions are L234A, L235E, G237A, A330S, and P331S (numbered according to the Kabat EU index).

Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in a specific embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first Fc domain and the second Fc domain of the Fc region, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index). In another specific embodiment, each Fc domain of the Fc region comprises amino acid substitutions L234A, L235E, G237A, A330S and P331S (numbering according to the Kabat EU index), i.e., in each of the first Fc domain and the second Fc domain of the Fc region, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), the glycine residue at position 237 is replaced by an alanine residue (G237A), the alanine residue at position 330 is replaced by a serine residue (A330S), and the proline residue at position 331 is replaced by a serine residue (P331S) (numbering according to the Kabat EU index).

In one embodiment, the Fc domain is an IgG1 Fc domain, such as a human IgG1 Fc domain. In certain embodiments, the IgG1 Fc domain is a variant IgG1, which includes D265A and N297A mutations (EU numbering) to reduce effector function. In other embodiments, the IgG1 Fc domain is a variant IgG1 including L234A, L235E, G237A, A330S and P331S mutations (numbered according to the Kabat EU index), thereby providing an effector invalid IgG1 (IgG1EN). Amino acid substitutions L234A, L235E and G237A reduce the combination with FcγRI, FcγRIIa and FcγRIII, while substitutions A330S and P331S reduce C1q-mediated complement fixation.

In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to an Fc receptor. An exemplary IgG4 Fc domain with reduced binding to an Fc receptor may include an amino acid sequence selected from Table 2 below: In some embodiments, the Fc domain comprises only the bold portion of the sequence shown below:

In specific embodiments, the IgG4 with reduced effector function includes the bold portion of the amino acid sequence of SEQ ID NO: 31 of WO2014/121087, sometimes referred to herein as IgG4 or hIgG4.

For the heterodimeric Fc region, a combination of the variant IgG4 Fc sequences described above can be incorporated, for example, an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 (or its bold portion) of WO2014/121087 and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 (or its bold portion) of WO2014/121087, or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 (or its bold portion) of WO2014/121087 and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 (or its bold portion) of WO2014/121087.

6.5.1.2. Fc Heterodimerization Variants

Certain CD20-PD1 binding molecules require dimerization between two Fc domains that, unlike native immunoglobulins, are operably linked to non-identical N-terminal regions, e.g., one Fc domain is linked to a Fab and the other Fc domain is linked to a PD1 agonist moiety. Insufficient heterodimerization of the two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and represents a purification challenge. A variety of methods available in the art can be used to enhance dimerization of Fc domains that may be present in the CD20-PD1 binding molecules of the present disclosure, for example, as disclosed in EP1870459A1; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441; U.S. Pat. No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009/089004A1.

The present disclosure provides CD20-PD1 binding molecules including Fc heterodimers, i.e., Fc regions including heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer includes a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class or subclass, and preferably the constant region of an antibody of the IgG (IgG1, IgG2, IgG3 and IgG4) class, as described in the preceding sections.

Heterodimerization of two different heavy chains at the CH3 domain produces the desired CD20-PD1 binding molecules, while homodimerization of the same heavy chain will reduce the yield of the desired CD20-PD1 binding molecules. Therefore, in a preferred embodiment, the polypeptides that associate to form the CD20-PD1 binding molecules of the present disclosure will contain a CH3 domain with a modification that favors heterodimer association relative to an unmodified Fc domain.

In a specific embodiment, the modification that promotes the formation of Fc heterodimers is a so-called "knob-into-hole" or "knob-in-hole" modification, including a "knob" modification in one Fc domain and a "hole" modification in the other Fc domain. Knob-hole technology is described in the following literature: for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, "Prot Eng" 9:617-621, and Carter, 2001, "Immunol Meth" 248:7-15. Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, so that the protrusion can be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusions are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

Therefore, in some embodiments, the amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue with a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in the cavity in the CH3 domain of the second subunit, and the amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue with a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity. Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of: alanine (A), serine (S), threonine (T) and valine (V). Protrusions and cavities can be formed by changing the nucleic acid encoding the polypeptide (e.g., by site-specific mutagenesis or by peptide synthesis). Exemplary substitutions are Y470T.

In a specific such embodiment, in the first Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first Fc domain, additionally, the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine residue (E356C) (specifically, the serine residue at position 354 is replaced by a cysteine residue), and in the second Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering according to the Kabat EU index). In specific embodiments, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the KabatEU index).

In some embodiments, electrostatic steering (eg, as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote the association of the first and second Fc domains of the Fc region.

As an alternative, or in addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to allow a purification strategy that can select Fc heterodimers. In one such embodiment, a polypeptide includes a modified Fc domain that eliminates its binding to protein A, thereby achieving a purification method for producing a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Therefore, the CD20-PD1 binding molecule includes a first CH3 domain and a second IgCH3 domain, wherein the first Ig CH3 domain and the second Ig CH3 domain differ from each other by at least one amino acid, and wherein at least one amino acid difference reduces the binding of the CD20-PD1 binding molecule to protein A compared to the corresponding CD20-PD1 binding molecule lacking amino acid differences. In one embodiment, the first CH3 domain binds to protein A, and the second CH3 domain contains a mutation/modification that reduces or eliminates protein A binding, such as H95R modification (numbered by IMGT exon; H435R by EU numbering). The second CH3 may further include a Y96F modification (by IMGT; Y436F by EU). This modification class is referred to herein as a "star" mutation.

In some embodiments, the Fc may contain one or more mutations (eg, knob and hole mutations) to promote heterodimerization, and a star mutation to promote purification.

6.6. Stable part

The CD20-PD1 binding molecules of the present disclosure may include a stabilizing portion that can extend the serum half-life of the molecule in vivo. The serum half-life is generally divided into an α phase and a β phase. Either or both phases can be significantly improved by adding an appropriate stabilizing portion. For example, relative to a corresponding CD20-PD1 binding molecule without a stabilizing portion, a stabilizing portion can increase the serum half-life of the CD20-PD1 binding molecule by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 400%, 600%, 800%, 1000% or more. For the purposes of the present disclosure, serum half-life may refer to the half-life in humans or other mammals (e.g., mice or non-human primates).

Stabilizing moieties include polyoxyalkylene moieties (eg, polyethylene glycol), sugars (eg, sialic acid), and well-tolerated protein moieties (eg, Fc and fragments and variants thereof, transferrin, or serum albumin).

Other stabilizing moieties that can be used in the CD20-PD1 binding molecules of the present disclosure include stabilizing moieties described in Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76. Such stabilizing moieties include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based on the three amino acids proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

Thus, in some embodiments, the present disclosure provides CD20-PD1 binding molecules comprising a stabilizing moiety as a polymeric sugar.

Serum albumin can also participate in half-life extension through a module having the ability to interact non-covalently with albumin. Therefore, the CD20-PD1 binding molecules of the present disclosure can include albumin binding proteins as a stabilizing portion. The albumin binding protein can be conjugated or genetically fused to one or more other components of the CD20-PD1 binding molecules of the present disclosure. Proteins with albumin binding activity are known to come from certain bacteria. For example, Streptococcus protein G contains several small albumin binding domains consisting of approximately 50 amino acid residues (6kDa). Other examples of serum albumin binding proteins, such as those described in U.S. Publication Nos. 2007/0178082 and 2007/0269422. The fusion of the albumin binding domain with the protein produces a strongly extended half-life (see Kontermann et al., 2011, Current Reviews in Biotechnology 22: 868-76).

In other embodiments, the stabilizing moiety is human serum albumin. In other embodiments, the stabilizing moiety is transferrin.

In some embodiments, the stabilizing portion is an Fc domain, such as any Fc domain in the Fc domain described in Section 6.5.1 and its subsections, which are incorporated herein by reference. The Fc domain described in Section 6.5.1 is generally capable of dimerization. However, for the purpose of stability, the Fc domain can be a soluble monomer Fc domain with reduced self-association ability. See, for example, Helm et al., 1996, Journal of Biological Chemistry (J.Biol.Chem.) 271: 7494-7500 and Ying et al., 2012, Journal of Biological Chemistry 287 (23): 19399-19408. Examples of soluble monomer Fc domains include amino acid substitutions at positions corresponding to T366 and/or Y407 in CH3, as described in U.S. Patent Publication No. 2019/0367611. The monomeric Fc domain may be of any Ig subtype, and may contain additional substitutions that reduce effector function, as described in Section 6.5.1 and its subsections.

In yet other embodiments, the stabilizing moiety is a polyethylene glycol moiety or another polymer, as described in Section 6.6.1, below.

The stabilizing moiety may be attached to one or more other components of the CD20-PD1 binding molecules of the disclosure via a linker, e.g., as described in Section 6.7, below.

6.6.1. Polyethylene glycol

In some embodiments, the CD20-PD1 binding molecule includes polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing portion, such as copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may have any molecular weight and may be branched or unbranched.

PEG is a well-known water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pp. 138-161). The term "PEG" is broadly used to encompass any polyethylene glycol molecule, regardless of size or modification at the PEG terminus, and can be represented by the formula: X--O( _CH2CH2O ) _{n - 1CH2CH2OH} , where n is 20 to ₂₃₀₀ and X is H or a terminal modification, e.g., _C1-4 alkyl. PEG may contain additional chemical groups required for the binding reaction; the chemical groups result from the chemical synthesis of the molecule; or the chemical groups act as spacers for optimal distances of parts of the molecule. Additionally, such PEGs may consist of one or more PEG side chains linked together. PEGs with more than one PEG chain are referred to as multi-arm or branched PEGs. Branched PEGs are described, for example, in European Application No. 473084A and US Pat. No. 5,932,462.

One or more PEG molecules can be attached at various positions on the CD20-PD1 binding molecule, and such attachment can be achieved by reaction with amines, thiols or other suitable reactive groups. The amine moiety can be, for example, a primary amine found at the N-terminus of the CD20-PD1 binding molecule (or a component thereof) or an amine group present in an amino acid such as lysine or arginine.

PEGylation can be achieved by site-directed PEGylation, in which a suitable reactive group is introduced into the protein to create a site where PEGylation preferentially occurs. In some embodiments, the CD20-PD1 binding molecule is modified to introduce a cysteine residue at a desired position, thereby allowing site-directed PEGylation on cysteine. Mutations can be introduced into the coding sequence of the CD20-PD1 binding molecule of the present disclosure to produce cysteine residues. This can be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutation to cysteine residues include serine, threonine, alanine and other hydrophilic residues. Preferably, the residue to be mutated to cysteine is a surface-exposed residue. Algorithms for predicting the surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. PEGylation of cysteine residues can be carried out using, for example, PEG-maleimide, PEG-vinyl sulfone, PEG-iodoacetamide or PEG-n-pyridyl disulfide.

PEG is usually activated with a suitable activating group suitable for coupling to the desired site on the polypeptide. PEGylation methods are well known in the art and are further described in Zalipsky et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J.M.Harris, Plenus Press, New York (1992); and Zalipsky, 1995, Advanced Drug Reviews 16: 157-182.

The molecular weight of the PEG part can vary widely, and can be branched or linear. Typically, the weight average molecular weight of PEG is about 100 daltons to about 150,000 daltons. The exemplary weight average molecular weight of PEG includes about 20,000 daltons, about 40,000 daltons, about 60,000 daltons and about 80,000 daltons. In certain embodiments, the molecular weight of PEG is 40,000 daltons. It is also possible to use a PEG with a total molecular weight of any of the branched forms of the foregoing. In certain embodiments, PEG has two branches. In other embodiments, PEG has four branches. In another embodiment, PEG is a double PEG (NOF Corporation, DE-200MA).

Conventional separation and purification techniques known in the art can be used to purify the PEGylated CD20-PD1 binding molecules, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. The products can also be separated using SDS-PAGE. The products that can be separated contain single, di, tri, multi and non-PEGylated CD20-PD1 binding molecules, as well as free PEG. The percentage of mono-PEG conjugates can be controlled by merging wider fractions around the elution peak to increase the percentage of mono-PEG in the composition. About 90% of mono-PEG conjugates represent a good balance of yield and activity.

In some embodiments, the PEGylated CD20-PD1 binding molecule will preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of the biological activity associated with the unmodified CD20-PD1 binding molecule. In some embodiments, biological activity refers to its ability to bind to CD20, PD1, or both CD20 and PD1, as assessed by _KD , _kon or _koff .

6.7. Connectors

In certain aspects, the present disclosure provides CD20-PD1 binding molecules, wherein two or more components of the CD20-PD1 binding molecules are connected to each other by peptide linkers. By way of example and not limitation, the linker can be used to connect (a) a CD20 targeting portion and a dimerization portion; (b) a CD20 targeting portion and a PD1 agonist portion; (c) a PD1 agonist portion and a dimerization portion; or (d) different domains within a CD20 targeting portion (e.g., VH and VL domains in a scFv).

The peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects, the peptide linker is from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, from 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

In particular aspects, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids in length.

In some of the aforementioned embodiments, the length of the linker is in the range of 5 amino acids to 50 amino acids, such as 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25 or 5 to 20 amino acids in length. In other embodiments of the aforementioned, the length of the linker is in the range of 6 amino acids to 50 amino acids, such as 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25 or 6 to 20 amino acids in length. In other embodiments of the aforementioned, the length of the linker is in the range of 7 amino acids to 50 amino acids, such as 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25 or 7 to 20 amino acids in length.

Charged (eg, charged hydrophilic linkers) and/or flexible linkers are particularly preferred.

Examples of flexible linkers that can be used in the CD20-PD1 binding molecules of the present disclosure include linkers disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers are or include a repeating sequence of glycine and serine, for example, a monomer or polymer of G _n S (SEQ ID NO: 12) or SG _n (SEQ ID NO: 13), wherein n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). In one embodiment, the linker is or includes a monomer or polymer of a repeating sequence of G ₄ S, for example (GGGGS) _n (SEQ ID NO: 14).

Suitably, polyglycine linkers can be used in the CD20-PD1 binding molecules of the present disclosure. In some embodiments, the peptide linker includes two consecutive glycine (2Gly), three consecutive glycine (3Gly), four consecutive glycine (4Gly) (SEQ ID NO: 15), five consecutive glycine (5Gly) (SEQ ID NO: 16), six consecutive glycine (6Gly) (SEQ ID NO: 17), seven consecutive glycine (7Gly) (SEQ ID NO: 18), eight consecutive glycine (8Gly) (SEQ ID NO: 19) or nine consecutive glycine (9Gly) (SEQ ID NO: 20).

6.7.1. Hinge sequence

In some embodiments, the CD20-PD1 binding molecules of the present disclosure include a linker as a hinge region. Specifically, a hinge can be used to connect a CD20 targeting portion (e.g., a Fab domain) to a dimerization domain (e.g., an Fc domain). The hinge region can be a natural or modified hinge region. The hinge region is generally found at the N-terminus of the Fc region. Unless otherwise specified in the context, the term "hinge region" refers to a hinge sequence that occurs naturally or non-naturally, which is a monomer hinge domain in the context of a single or monomeric polypeptide chain and can include two associated hinge sequences on a separate polypeptide chain in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer CD20-PD1 binding molecule formed by the association of two Fc domains).

The native hinge region is the hinge region usually found between the Fab and Fc domains of naturally occurring antibodies. The modified hinge region is any hinge whose length and/or composition are different from the native hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may include the complete hinge region of antibodies derived from categories or subclasses different from the categories or subclasses of heavy chain Fc domains or Fc regions. Alternatively, the modified hinge region may include a part of a native hinge or a repeating unit, wherein each unit in the repeating unit is derived from a native hinge region. In other alternatives, one or more cysteine or other residues may be converted into neutral residues, such as serine or alanine, or the native hinge region may be changed by converting a properly placed residue into a cysteine residue. By such means, the number of cysteine residues in the hinge region may increase or decrease. Other modified hinge regions can be fully synthetic and can be designed to have desired properties, such as length, cysteine composition, and flexibility.

Many modified hinge regions have been described in, for example, U.S. Patent Nos. 5,677,425, WO 99/15549, WO 2005/003170, WO 2005/003169, WO 2005/003170, WO 98/25971, and WO 2005/003171, and these U.S. Patents are incorporated herein by reference.

In one embodiment, the CD20-PD1 binding molecules of the present disclosure include an Fc region, wherein one or both Fc domains have an intact hinge region at their N-termini.

In various embodiments, positions 233-236 within the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, where the positions are numbered by EU numbering.

In some embodiments, the CD20-PD1 binding molecules of the present disclosure include a modified hinge region that has reduced binding affinity to an Fcγ receptor relative to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

In one embodiment, the CD20-PD1 binding molecule of the present disclosure includes an Fc region, wherein each Fc domain has a complete hinge region at its N-terminus, wherein each Fc domain and hinge region are derived from IgG4, and each hinge region includes a modified sequence CPPC (SEQ ID NO: 21). Compared with IgG1 containing the sequence CPPC (SEQ ID NO: 29), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 22). The serine residues present in the IgG4 sequence lead to increased flexibility in this region, and thus a portion of the molecules form disulfide bonds (intrachain disulfide bonds) within the same protein chain, rather than bridging with other heavy chains in the IgG molecule to form interchain disulfide bonds. (Angel et al., 1993, Mol Immunol 30 (1): 105-108). The serine residues are changed to proline to obtain the same core sequence as IgG1, allowing the interchain disulfide bonds to be fully formed in the IgG4 hinge region, thereby reducing the heterogeneity of the purified product. This altered isotype is called IgG4P.

6.7.1.1. Chimeric Hinge Sequences

The hinge region may be a chimeric hinge region.

For example, a chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 23) (previously disclosed as SEQ ID NO: 8 of WO2014/121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 24) (previously disclosed as SEQ ID NO: 9 of WO2014/121087). Suitably, such chimeric hinge sequences can be linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or murine Fc domain, which can be further modified in the CH2 and/or CH3 domains to reduce effector function, e.g., as described in Section 6.5.1.1).

6.7.1.2. Hinge sequences with reduced effector function

In other embodiments, the hinge region can be modified to reduce effector function, for example, as described in WO2016161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region are G, G, G and vacant; G, G, vacant and vacant; G, vacant, vacant and vacant; or all vacant, wherein the position is numbered by EU numbering (as shown in Figure 1 of WO2016161010A2). These sections can be expressed as GGG-, GG--, G--- or ----, where "-" represents a vacant position.

Position 236 is vacant in typical human IgG2, but is occupied in other typical human IgG isotypes. In all four human isotypes, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

Hinge modifications within positions 233-236 can be combined with position 228 occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is beneficial for stabilizing IgG4 antibodies and reducing the exchange of heavy chain light chain pairs between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P and C, respectively ("CPPC" is disclosed as SEQ ID NO: 21).

Exemplary hinge regions have residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and no G(233-236). Optionally, the hinge domain amino acid sequence includes CPPCPAP GG-GPSVF (SEQ ID NO: 25) (previously disclosed as SEQ ID NO: 1 of WO2016161010A2), CPPCPAP G--GPSVF (SEQ ID NO: 26) (previously disclosed as SEQ ID NO: 2 of WO2016161010A2), CPPCPAP---GPSVF (SEQ ID NO: 27) (previously disclosed as SEQ ID NO: 3 of WO2016161010A2) or CPPCPAP----GPSVF (SEQ ID NO: 28) (previously disclosed as SEQ ID NO: 4 of WO2016161010A2).

The above-mentioned modified hinge region can be incorporated into a heavy chain constant region, which generally includes CH2 and CH3 domains, and the heavy chain constant region may have an additional hinge segment (e.g., upper hinge) flanking a specified region. Such additional constant region segments present generally belong to the same isotype, preferably a human isotype, but may also be a hybrid of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2 or IgG3 or a hybrid thereof, wherein the domains belong to different isotypes. Exemplary sequences of human IgG1, IgG2 and IgG4 are shown in Figures 2-4 of WO2016161010A2.

In specific embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, such as a human or murine Fc domain, which can be further modified in the CH2 and/or CH3 domains to reduce effector function, e.g., as described in Section 6.5.1.1).

6.8. Nucleic Acids and Host Cells

In another aspect, the present disclosure provides nucleic acids encoding the CD20-PD1 binding molecules of the present disclosure. In some embodiments, the CD20-PD1 binding molecules are encoded by a single nucleic acid. In other embodiments, for example, in the case of heterodimeric molecules or molecules comprising a CD20 targeting moiety consisting of more than one polypeptide chain, the CD20-PD1 binding molecules may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

A single nucleic acid can encode a CD20-PD1 binding molecule comprising a single polypeptide chain, a CD20-PD1 binding molecule comprising two or more polypeptide chains, or a portion of a CD20-PD1 binding molecule comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a CD20-PD1 binding molecule comprising three, four or more polypeptide chains, or three polypeptide chains of a CD20-PD1 binding molecule comprising four or more polypeptide chains). For separate control of expression, the open reading frames encoding the two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and/or enhancers). The open reading frames encoding the two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, thereby allowing translation into separate polypeptides.

In some embodiments, a CD20-PD1 binding molecule comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding a CD20-PD1 binding molecule may be equal to or less than the number of polypeptide chains in the CD20-PD1 binding molecule (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA).

In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure. The nucleic acids can be present in a single vector or separate vectors present in the same host cell or separate host cells, as described in more detail below.

6.8.1. Carrier

The present disclosure provides vectors comprising nucleotide sequences encoding CD20-PD1 binding molecules or CD20-PD1 binding molecule components described herein (e.g., one or two polypeptide chains of the polypeptide chains of CD20-PD1 monomers). The vectors include, but are not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs).

A variety of vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.

In addition, the cell that DNA is stably integrated into its chromosome can be selected by introducing one or more markers that allow selection of transfected host cells. The marker can, for example, provide originality, biocide resistance (for example, antibiotic) or resistance to heavy metals such as copper to auxotrophic hosts. Selectable marker gene can be directly connected to DNA sequence to be expressed, or can be introduced into the same cell by co-transformation. The optimal synthesis of mRNA may also require other elements. These elements can include splicing signals, as well as transcription promoters, enhancers and termination signals.

Once the expression vector or DNA sequence containing the construct has been prepared for expression, the expression vector can be transfected or introduced into an appropriate host cell. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. The methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can be varied or optimized according to the present specification depending on the specific expression vector and mammalian host cell used.

6.8.2. Cells

The present disclosure also provides host cells comprising the nucleic acids of the present disclosure.

In one embodiment, the host cell is genetically engineered to include one or more nucleic acids described herein.

In one embodiment, the host cell is genetically engineered using an expression cassette. The phrase "expression cassette" refers to such a sequence that can affect the expression of a gene in a host compatible with the nucleotide sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors that are necessary or helpful in affecting expression, such as inducible promoters, may also be used.

The present disclosure also provides host cells comprising the vectors described herein.

Cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

6.9. Pharmaceutical Compositions

6.9.1. Pharmaceutical compositions comprising CD20-PD1 binding molecules

The CD20-PD1 binding molecules of the present disclosure may be in the form of a composition comprising a CD20-PD1 binding molecule and one or more carriers, excipients and/or diluents. The composition may be formulated for a specific use, such as veterinary use or pharmaceutical use for humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and/or carriers used will depend on the intended use of the CD20-PD1 binding molecule, and for therapeutic use, will depend on the mode of administration.

For therapeutic use, compositions can be supplied as a part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of applying it to the patient). The pharmaceutical composition can be applied to the patient by a variety of approaches, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topically or regionally. In any given case, the most suitable route of administration will depend on the nature and severity of the specific antibody, experimenter, disease and the physical condition of the experimenter. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

The pharmaceutical composition can be conveniently present in a unit dosage form containing a predetermined amount of the CD20-PD1 binding molecule of the present disclosure per dose. The amount of the CD20-PD1 binding molecule contained in the unit dose will depend on the disease to be treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized dry powder containing a certain amount of CD20-PD1 binding molecules suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit together with a syringe, an appropriate amount of diluent and/or other components that can be used for administration. The unit dose in liquid form can be conveniently supplied in the form of a syringe, which is pre-filled with a certain amount of CD20-PD1 binding molecules suitable for a single administration.

Pharmaceutical compositions may also be supplied in bulk containing an amount of the CD20-PD1 binding molecule suitable for multiple administrations.

Pharmaceutical compositions can be prepared by mixing the CD20-PD1 binding molecules having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (all of which are referred to herein as "carriers") commonly used in the art, i.e., buffers, stabilizers, preservatives, isotonic agents, non-ionic detergents, antioxidants and other miscellaneous additives for storage as lyophilized preparations or aqueous solutions. See Remington, The Science and Practice of Pharmacy, 23rd edition (Adejare ed. 2020). Such additives should be non-toxic to the recipient at the doses and concentrations employed.

Buffers help maintain pH within a range close to physiological conditions. The buffers can be present in a variety of concentrations, but are typically present in a concentration in the range of about 2 mM to about 50 mM. Buffers suitable for use in the present disclosure include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-sodium fumarate mixtures, etc.), In some embodiments, the present invention provides the present invention relates to a buffered saline buffer (e.g., a sodium acetate-sodium acetate mixture, a sodium acetate-sodium hydroxide mixture, a potassium acetate-gluconate mixture, etc.), a gluconate buffer (e.g., a sodium acetate-sodium acetate mixture, a sodium acetate-sodium hydroxide mixture, a potassium acetate-gluconate mixture, etc.), an oxalate buffer (e.g., an oxalic acid-sodium oxalate mixture, an oxalic acid-sodium hydroxide mixture, an oxalic acid-potassium oxalate mixture, etc.), a lactate buffer (e.g., a lactic acid-sodium lactate mixture, a lactic acid-sodium hydroxide mixture, a lactic acid-potassium lactate mixture, etc.), and an acetate buffer (e.g., an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, etc.). Alternatively, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

Preservatives can be added to slow down microbial growth, and the preservatives can be added in an amount within the range of about 0.2%-1% (w/v). Preservatives suitable for the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halide (benzalconium halide) (e.g., chloride, bromide and iodide), hexamethylammonium chloride and alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic agents sometimes referred to as "stabilizers" can be added to ensure the isotonicity of the liquid composition of the present disclosure, and the isotonic agents include polyols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a large class of excipients that can range in function from fillers to additives that dissolve therapeutic agents or help prevent denaturation or adhesion to the walls of containers. Typical stabilizers can be polyhydroxy sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; amino acid polyols; Compounds; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount of 0.5 wt % to 10 wt % per weight of the CD20-PD1 binding molecule.

Can add nonionic surfactant or detergent (also referred to as " wetting agent ") to help dissolve glycoprotein and protect glycoprotein from the aggregation caused by agitation, this also allows preparation to be exposed to shear surface stress and does not cause protein denaturation.Suitable nonionic surfactant comprises polysorbate (20,80 etc.), poloxamer (184,188 etc.) and pluronic polyol.Nonionic surfactant can exist with the scope of about 0.05mg/mL to about 1.0mg/mL, for example, about 0.07mg/mL to about 0.2mg/mL.

Additional miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and solubilizing agents.

6.9.2. Pharmaceutical compositions for delivering nucleic acids encoding CD20-PD1 binding molecules

The CD20-PD1 binding molecules of the present disclosure can be delivered by any method useful for gene therapy, such as as mRNA or by a viral vector encoding the CD20-PD1 binding molecule under the control of a suitable promoter.

Exemplary gene therapy vectors include adenovirus- or AAV-based therapeutics. Non-limiting examples of adenovirus- or AAV-based therapeutics for use in the methods, uses, or compositions herein include, but are not limited to: rAd-p53, which is a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53, for example, for the treatment of cancer (also known as Qi et al., 2006, Modern Oncology, 14:1295-1297); Ad5-d11520, an adenovirus lacking the E1B gene for inactivating host p53 (also known as H101 or ONYX-015; see, e.g., Russell et al., 2012, Nature Biotechnology, 30:658-670); AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF, e.g., for the treatment of cancer (Cerullo et al., 2010, Cancer Res. 70:4297); rAd-HSVtk, a replication-deficient adenovirus with the HSV thymidine kinase gene, e.g., for the treatment of cancer (developed for Ark therapeutics, see, e.g., U.S. Pat. No. 6,579,855; ProstAtak ^™ developed by Advantagene; International PCT Application No. WO2005/049094); rAd-TNFα, a replication-defective adenoviral vector expressing human tumor necrosis factor α (TNFα) under the control of the chemoradiation-inducible EGR-1 promoter, e.g., for the treatment of cancer (TNFerade ^™ , GenVec; Rasmussen et al., 2002, Cancer Gene Therapy. Ad-IFNβ, an adenovirus serotype 5 vector expressing the human interferon-β gene under the direction of the cytomegalovirus (CMV) immediate early promoter (in which the E1 and E3 genes are deleted), is used, for example, to treat cancer (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther. 18:852-860).

Nucleic acid molecules (e.g., mRNA) or viruses can be formulated into the sole active pharmaceutical ingredient in a pharmaceutical composition, or can be combined with other active agents for a specific disease to be treated. Optionally, other pharmaceutical agents, medicaments, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate) and colorants, release agents, coating agents, sweeteners, flavoring agents and aromatics, preservatives, antioxidants, chelating agents and inert gases can also be present in the composition. Exemplary other agents and excipients that may be included in the composition include, for example: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

When used as an adjuvant therapy for adoptive cell transfer therapy (e.g., a cell therapy expressing CAR as described in Section 6.11.1), cell therapy, such as cells expressing CAR, can be engineered to express the CD20-PD1 binding molecules disclosed herein. The CD20-PD1 binding molecules can target specific genomic loci, such as loci active in activated or dysfunctional lymphocytes, such as PD-1 loci, or be inserted into non-specific genomic loci. Targeting specific genomic loci can be achieved by gene editing, such as using zinc finger proteins, CRISPR/Cas9 systems, etc.

6.10. Therapeutic indications and treatment methods

The CD20-PD1 binding molecules of the present disclosure can be used to treat disease states where modulation of the host's immune system is beneficial, particularly where inhibition of cellular immune responses is desirable. Thus, the CD20-PD1 binding molecules of the present disclosure can be used to inhibit immune responses in a variety of applications.

Situations in which inhibition of cellular immune responses is desirable may include disease states caused by autoimmune responses. Disease states in which the CD20-PD1 binding molecules of the present disclosure may be administered include, for example, autoimmune diseases, in which inhibition of cellular autoimmune responses will be an important mechanism. Specific disease states in which the CD20-PD1 binding molecules of the present disclosure may be employed include type 1 diabetes (T1D), systemic lupus erythematosus, Crohn's disease, and graft-versus-host disease (GVHD). The CD20-PD1 binding molecules of the present disclosure may be administered by themselves or in any suitable pharmaceutical composition.

On the one hand, a CD20-PD1 binding molecule of the present disclosure for use as a medicament is provided. In another aspect, a CD20-PD1 binding molecule of the present disclosure for treating a disease is provided. In certain embodiments, a CD20-PD1 binding molecule of the present disclosure for a method of treatment is provided. In one embodiment, the present disclosure provides a CD20-PD1 binding molecule as described herein, which is used to treat a disease of a subject in need thereof. In certain embodiments, the present disclosure provides a CD20-PD1 binding molecule for a method of treating a subject with an autoimmune disease, the method comprising administering a therapeutically effective amount of a CD20-PD1 binding molecule to an individual. In certain embodiments, the disease to be treated is an autoimmune disease. In specific embodiments, the disease is T1D. In other embodiments, the disease is systemic lupus erythematosus. In other embodiments, the disease is Crohn's disease. In yet other embodiments, the disease is GVHD. In certain embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to an individual. In other embodiments, the present disclosure provides a CD20-PD1 binding molecule agonist for suppressing the immune system. In certain embodiments, the present disclosure provides a CD20-PD1 binding molecule for a method of suppressing the immune system of a subject, the method comprising administering an effective amount of a CD20-PD1 binding molecule to an individual to suppress the immune system. According to any of the above embodiments, the "individual" is a mammal, such as a human. According to any of the above embodiments, the "suppression of the immune system" may include any one or more of the following: a general decrease in immune function, a decrease in T cell function, a decrease in B cell function, a decrease in T cell responsiveness, etc. According to any of the above embodiments, the "suppression of cellular autoimmune responses" may include, for example, a decrease in immune signals (e.g., secretion of immune-activating cytokines), a decrease in the function of immune cells targeting self-antigens, etc.

The present disclosure further provides a method of local PD1 agonism, the method comprising administering to a subject a CD20-PD1 binding molecule or pharmaceutical composition as described herein. As used herein, the term "local delivery" does not require local administration, but rather indicates that the CD20-PD1 binding molecule is selectively or preferentially localized at the intended site of immunomodulation, such as an autoimmune active site and/or an intended cell type, such as a B cell.

The present disclosure further provides a method of administering a PD1 agonist therapy to a subject with reduced systemic exposure and/or reduced systemic toxicity, the method comprising administering to the subject a PD1 agonist therapy in the form of a CD20-PD1 binding molecule or pharmaceutical composition as described herein, e.g., wherein CD20 is expressed by a tissue for which the PD1 agonist therapy is desirable and/or expected.

Thus, the aforementioned approach allows for PD1 agonist therapy with reduced off-target side effects by preferentially delivering the CD20-PD1 binding molecule at the site intended for PD1 agonist treatment.

The present disclosure further provides a method of locally modulating (eg, inhibiting) an immune response in a target tissue expressing CD20, the method comprising administering to a subject a CD20-PD1 binding molecule or pharmaceutical composition as described herein.

In some embodiments, the administering is not tissue local.

In a further aspect, the present disclosure provides the use of the CD20-PD1 binding molecules of the present disclosure in the manufacture or preparation of a medicament for treating a disease in a subject in need thereof. In one embodiment, the medicament is used in a method for treating a disease, the method comprising administering a therapeutically effective amount of the medicament to a subject suffering from the disease. In certain embodiments, the disease to be treated is an autoimmune disease. In a specific embodiment, the disease is T1D. In other embodiments, the disease is systemic lupus erythematosus. In other embodiments, the disease is Crohn's disease. In yet other embodiments, the disease is GVHD. In certain embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual. In a further embodiment, the medicament is used to suppress the immune system. In a further embodiment, the medicament is used in a method for suppressing the immune system of a subject, the method comprising administering an effective amount of the medicament to the individual to suppress the immune system. The "individual" according to any of the above embodiments may be a mammal, such as a human. The "suppression of the immune system" according to any of the above embodiments may include any one or more of the following: a general decrease in immune function, a decrease in T cell function, a decrease in B cell function, a decrease in T cell responsiveness, and the like.

In another aspect, the present disclosure provides a method for aggregating PD1 and/or enhancing PD1 activity in a subject, the method comprising administering an effective amount of a CD20-PD1 binding molecule of the present disclosure to the subject. The CD20-PD1 binding molecule of the present disclosure can induce PD1 aggregation at the interface of CD20 presenting cells and T cells. This provides targeted immunosuppression, in which CD20 presenting cells and surrounding cells and tissues are protected from T cell killing. High levels of CD20 can be found on B cells, which are rich in draining lymph nodes and autoimmune tissues (e.g., pancreas of type 1 diabetes (T1D)). The CD20-PD1 binding molecules of the present disclosure can excite PD1 in a cell and/or tissue-specific manner, thereby inhibiting autoreactive T cell activation. In T1D, the abundance of CD20+B cells provides PD1 aggregation on autoreactive T cells, thereby inhibiting autoreactive cytotoxic T cells from killing pancreatic islet cells. In one embodiment, a composition is administered to the subject, the composition comprising a CD20-PD1 binding molecule of the present disclosure in a pharmaceutically acceptable form.

In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering to the individual a therapeutically effective amount of a CD20-PD1 binding molecule of the present disclosure. In one embodiment, a composition is administered to the individual, the composition comprising a CD20-PD1 binding molecule of the present disclosure in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is an autoimmune disease. The autoimmune diseases that can be treated by the CD20-PD1 binding molecules of the present disclosure can include type 1 diabetes, primary biliary cholangitis (PBC), Goodpasture's syndrome, amyloidosis, ankylosing spondylitis, anti-glomerular basement membrane nephritis, anti-tubular basement membrane nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune oophoritis, graft-versus-host disease (GVHD), autoimmune pancreatitis, autoimmune retinopathy, Behcet's disease, Crohn's disease, Devic's disease, systemic lupus erythematosus (SLE), Dressler's syndrome, fibrosing alveolitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, and leukemia. syndrome), IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), multiple sclerosis, polyneuropathy, organomegaly, endocrinopathy, monoclonal syndrome (POEMS), polyarteritis nodosa, rheumatoid arthritis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome ( syndrome, sperm or testicular autoimmunity, stiff-man syndrome (SPS), Takayasu's arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), ulcerative colitis, and vasculitis.

In specific embodiments, the disease is T1D. In other embodiments, the disease is systemic lupus erythematosus. In other embodiments, the disease is Crohn's disease. In yet other embodiments, the disease is GVHD. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent. In another aspect, the present disclosure provides a method for suppressing the immune system of a subject, the method comprising administering to the individual an effective amount of a CD20-PD1 binding molecule to suppress the immune system. The "individual" according to any of the above embodiments may be a mammal, such as a human. The "suppression of the immune system" according to any of the above embodiments may include any one or more of the following: a general decrease in immune function, a decrease in T cell function, a decrease in B cell function, a decrease in T cell responsiveness, etc.

In certain embodiments, the disease to be treated is an autoimmune disease. The CD20-PD1 binding molecules can be used to eliminate cells involved in immune cell-mediated disorders, autoimmunity, transplant rejection, and graft-versus-host disease. It is readily recognized by those skilled in the art that in many cases, the CD20-PD1 binding molecules may not provide a cure, but may only provide partial benefits. In some embodiments, physiological changes with some benefits are also considered to be therapeutically beneficial. Therefore, in some embodiments, the amount of the CD20-PD1 binding molecule that provides a physiological change is considered to be an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

For the prevention or treatment of disease, the appropriate dosage of the CD20-PD1 binding molecules of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the specific CD20-PD1 binding molecule, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the CD20-PD1 binding molecule, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulse infusions.

Suitably, the CD20-PD1 binding molecule is administered to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of the CD20-PD1 binding molecule may be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may be in the range of about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired disease symptom suppression occurs. An exemplary dosage of the CD20-PD1 binding molecule will be in the range of about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, dosages may also include administration of about 1 μg/kg/body weight, about 5 μg/kg/body weight, about 10 μg/kg/body weight, about 50 μg/kg/body weight, about 100 μg/kg/body weight, about 200 μg/kg/body weight, about 350 μg/kg/body weight, about 500 μg/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, to about 1000 mg/kg/body weight or more, and any range derivable therein. In non-limiting examples of derivable ranges of the numbers listed herein, based on the above numbers, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 mg/kg/body weight, etc. may be administered. Therefore, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses or, for example, about six doses of CD20-PD1 binding molecules). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy may be easily monitored by conventional techniques and assays.

The CD20-PD1 binding molecules of the present disclosure will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease condition, the CD20-PD1 binding molecules of the present disclosure or their pharmaceutical compositions are administered or applied in a therapeutically effective amount. The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays (such as cell culture assays). The dose can then be formulated in animal models to achieve a circulating concentration range that includes the EC ₅₀ as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, such as animal models, using techniques well known in the art. One of ordinary skill in the art can easily optimize administration to humans based on animal data.

Dosage and interval can be adjusted individually to provide plasma levels of CD20-PD1 binding molecules sufficient to maintain therapeutic effect. Common patient doses administered by injection are in the range of about 0.1 mg/kg/day to 50 mg/kg/day, usually about 0.5 mg/kg/day to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by ELISA HPLC.

In cases of local administration or selective uptake, the effective local concentration of the CD20-PD1 binding molecule may not be related to plasma concentration. One skilled in the art will be able to optimize the local dosage of a therapeutically effective amount without undue experimentation.

Therapeutically effective doses of the CD20-PD1 binding molecules described herein will generally provide therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic efficacy of the CD20-PD1 binding molecules can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine LD ₅₀ (the dose that is lethal to 50% of the population) and ED ₅₀ (the dose that is therapeutically effective for 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ /ED _50. CD20-PD1 binding molecules that exhibit a large therapeutic index are preferred. In one embodiment, the CD20-PD1 binding molecules according to the present disclosure exhibit a high therapeutic index. The data obtained from cell culture assays and animal studies can be used to formulate a dosage range suitable for humans. The dose is preferably within a circulating concentration range with little or no toxicity that includes ED _50. The dose can vary within this range, depending on various factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dose can be selected by an individual physician based on the condition of the patient. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, p. 1, which is incorporated herein by reference in its entirety).

The attending physician of a patient treated with the CD20-PD1 binding molecules of the present disclosure will know how and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The size of the administered dose in the management of the condition of interest will vary with the severity of the condition to be treated and the route of administration, etc. For example, the severity of the condition can be assessed in part by standard prognostic assessment methods. In addition, the dose and possible dose frequency will also vary according to the age, weight and response of the individual patient.

6.10.1.1 Diabetes mellitus type

In some embodiments, the CD20-PD1 binding molecules according to the present disclosure can prevent or slow the development or progression of type 1 diabetes. Therefore, in some embodiments, the CD20-PD1 binding molecules, nucleic acids and/or pharmaceutical compositions of the present disclosure can be administered to subjects suffering from T1D or at risk of developing T1D. Risk factors for developing T1D include, but are not limited to, genetic markers (e.g., human leukocyte antigen (HLA) complex; see Flemming and Pociot, 2016, Lancet, 387(10035):2331-2339), viral infections (e.g., German measles, coxsackievirus and mumps), race/ethnicity (e.g., in the United States, Caucasians are more susceptible to type 1 diabetes), family history, early diet and the presence of other autoimmune conditions (e.g., Grave's disease, multiple sclerosis, pernicious anemia). Cancer patients receiving immune checkpoint inhibitor therapy are also at risk of developing T1D. See de Filette et al., 2019, Eur J Endocrinol, 181(3):363-374. Identifying and selecting those individuals at risk for developing T1D is within the scope of those skilled in the art.

In some embodiments, according to the methods of the present disclosure, a patient at risk for developing T1D is treated with the CD20-PD1 binding molecules, nucleic acids and/or pharmaceutical compositions of the present disclosure.

6.11. Combination therapy

The CD20-PD1 binding molecules according to the present disclosure can be administered in combination with one or more other agents in therapy. For example, the CD20-PD1 binding molecules of the present disclosure can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent that is administered to treat the symptoms or diseases of a subject in need of such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis inducing agents.

Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of CD20-PD1 binding molecule used, the type of disorder or treatment, and other factors discussed above. The CD20-PD1 binding molecules are generally used in the same dosages and administration routes as described herein, or about 1% to 99% of the dosages described herein, or in any dosage and any route determined empirically/clinically to be appropriate.

Such combination therapies described above encompass both combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case administration of the CD20-PD1 binding molecules of the present disclosure may be performed prior to, simultaneously with, and/or after administration of the additional therapeutic agents and/or adjuvants.

6.11.1. Combination therapy using CD20-PD1 binding molecule therapy and immunotherapy

The CD20-PD1 binding molecules of the present disclosure can be advantageously used in combination with cells expressing a chimeric antigen receptor ("CAR") (e.g., Treg ("CAR-Treg") cells expressing CAR, such as CAR-Treg) for the treatment of autoimmune diseases. In some embodiments, CAR-Treg cells are recognized by the CD20 targeting portion in the CD20-PD1 binding molecule. The CD20 targeting portion can recognize a Treg cell receptor or another cell surface molecule on a CAR-Treg cell. In some embodiments, the CD20 targeting portion in the CD20-PD1 binding molecule can bind to the extracellular domain (e.g., antigen binding domain) of CAR. CAR-Treg cells are described in Fritsche et al., 2020, Trends Biotechnol, 38(10): 1099-1112; Zhang et al., 2018, Front Immunol, 9: 2359; and Mohseni et al., Front Immunol, 11: 1608, each of which is incorporated herein by reference in its entirety.

6.12. Evaluation of CD20-PD1 Binding Molecules

Aspects of the present disclosure relate to a luciferase-based reporter gene bioassay to assess the ability of the CD20-PD1 binding molecules disclosed herein to agonize PD1 on Jurkat cells in the presence of CD20 presented on HEK293 cells. In some embodiments, HEK293 cells are transduced with CD22 as well as CD20.

In some embodiments, the bioassay disclosed herein comprises using a bispecific antibody (such as CD3xCD22) to elicit an immune response from a Jurkat cell line in the presence of HEK293 cells, which has been transduced with an AP1 (activator-protein 1)-luciferase reporter gene, CD3 and PD1 using a lentivirus. In the presence of Jurkat and HEK293 cells and anti-CD3xCD22 bispecific antibodies, CD20-PD1 binding molecules are added to the wells. As measured by AP1-driven luciferase activity, the molecules that most stimulate PD1 have the ability to reduce the amount of immune response stimulated by the anti-CD3xCD22 bispecific antibodies.

7. Examples

Materials and methods

7.1.1. Design and production of CD20-PD1 binding molecules

Constructs encoding bispecific CD20-PD1 agonists and controls as listed in Tables 1 and 2 below were generated. The bispecific CD20-PD1 agonists contained different configurations of murine anti-CD20 and modified murine PDL1 extracellular domains, IgG1 effector null (EN) (L234A, L235E, G237A, A330S and P331S, EU numbering) domains, and different lengths of linkers from different repeats of G ₄ S (SEQ ID NO: 14). A 29 amino acid signal sequence from the murine inactive tyrosine-protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the construct. All bispecific CD20-PD1 agonists were expressed as pre-proteins containing signal sequences. The signal sequence was cleaved by intracellular processing to produce mature proteins.

Knob-forming mutation: T366W (EU numbering).

Hole-forming mutations: T366S, L368A and Y407V (EU numbering).

Star mutations: H435R and Y436F (EU numbering).

The construct was expressed in Expi293F ^TM cells by transient transfection (Thermo Fisher Scientific). The protein in the Expi293F supernatant was purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) with HiTrap ^TM protein G HP or MabSelect SuRe pcc columns (Cytiva). After the single-step elution, the antibody was neutralized, dialyzed into the final buffer of phosphate buffered saline (PBS) containing 5% glycerol, aliquoted and stored at -80 ° C. For some constructs, an additional step of size exclusion chromatography using HiPrep 26/60 Sephacryl S-200 columns was used.

The alignment and selected mutation positions in mPDL1 are depicted in Figure 3B. The alignment between mPDL1 and hPDL1 was generated using MacVector. Figure 3A depicts the 3-dimensional structures of mPDL1 and hPDL1, including residues that were altered in mPDL1 to improve yield and stability.

7.1.2. Flow cytometry

Cells (HEK 293, MC38 overexpressing mCD20 or Jurkat overexpressing mPD1) were resuspended in FACS wash solution (PBS with 1% FBS) at 1×10 ⁶ cells/mL. Staining was performed with 1×10 ⁵ cells per well. Antibodies were diluted from a starting concentration of 1.3x 10 ^-07 M at a ratio of 1:5. Then, the diluted antibodies were added to the wells containing cells. The cells were stained at 2-8°C for 30 minutes and washed twice with FACS wash buffer. APC-conjugated goat anti-human IgG (Jackson Immuno Research, 109-607-003, 1:400) was added to stain the cells at 2-8°C for 30 minutes. After washing, the cells were fixed in 2% paraformaldehyde at 2-8°C for 30 minutes. After two washes, the stained cells were analyzed using a BD LSRFortessa ^TM FACS instrument. The results were analyzed by FlowJo. Use FSC/SSC gate to select monocytes.

For spinal cord T cell infiltration flow cytometric analysis, single cell suspensions of spinal cord were first prepared by collagenase D (Roche, 11088882001) digestion and Percoll (GE Healthcare, 17-0891-02) gradient separation. Cells were resuspended in FACS wash buffer and stained with LIVE/DEAD ^™ fixable blue dead cell staining kit (Thermo Fisher Scientific, L34962), anti-mouse CD45-BV750 (BioLegend, 103157, 1:200 dilution), anti-mouse CD4-BUV563 (BD Bioscience, 612923, 1:200 dilution) and anti-mouse CD8a-BUV805 (BD Bioscience, 564920, 1:100 dilution) according to the above protocol. Cells were analyzed by BD FACSymphony cell analyzer. Results were analyzed by OMIQ cytometry software.

7.1.3. Luciferase reporter gene assay: anti-mCD20 x mPDL1 extracellular domain molecules

Luciferase-based reporter gene assays were used to assess the ability of anti-mCD20 x mPDL1 extracellular domain molecules to excite mouse PD1 (mPD1) on Jurkat cells in the presence of mouse CD20 (mCD20) presented on HEK293 cells. The overall design of the reporter gene assay is depicted in Figures 5A and 5B. AP1 is a transcription factor involved in the regulation of gene expression during T cell activation (Samelson 2002, PMID: 11861607). Bispecific antibodies (bsAbs) bound to human CD3 and CD22, CD3 bsAb (REGN10551) were used to stimulate T cell activation by the engagement of antigens on target cells and receptors on T cells, similar to the previously described CD3 x CD20 bsAb (Smith et al., 2015, PMID: 26659273). The engagement of mCD20xmPDL1 anchored by mCD20 with Jurkat cells resulted in the inhibition of luciferase signals driven by PD1 excitation.

7.1.3.1. Engineering of Jurkat/AP1-luc/mPD1 cells

Jurkat/AP1-luc/mPD1 cells were generated by sequentially transducing JurkatE6-1 cells (ATCC #TIB-152) with AP1 (activator-protein 1)-luciferase reporter lentivirus (QIAGEN CLS-011L) followed by lentivirus containing the mPD1 ORF (mPD1 NM_008798).

7.1.3.2. Engineering of HEK293/hCD22/mCD20 cells

HEK293/hCD22/mCD20 cells were generated by sequential lentiviral transduction using a lentivirus encoding the human CD22 ORF (NP_001762.2) followed by a lentivirus encoding the mCD20 ORF (NP_031667.1).

7.1.3.3. Luciferase assay setup

For the bioassay, HEK293/CD22/mCD20 target cells were seeded at 10,000 or 20,000 cells/well into 96-well plates in assay medium (RPMI1640 supplemented with 10% fetal bovine serum and L-glutamine-penicillin-streptomycin) and incubated overnight at 37°C in 5% CO _2. The next day, Jurkat/AP1-luc/mPD1 reporter cells were added to the wells containing the cultured target cells at 30,000 or 50,000 cells/well. Then, the molecules of the present disclosure or control antibodies were serially diluted 1:3 in assay medium to a final concentration in the range of 100 nM to 1 pM (in the absence of additional conditions for the test molecule) and added to the cells together with 1 nM or 2.5 nM CD3 bsAb. To obtain a range of activation, CD3 bsAb was serially diluted 1:3 to a final concentration in the range of 100 nM to 1.69 pM (in the absence of additional conditions of bispecific mAb) and added to cells. After incubation for 5 hours at 37°C/5% ^CO2 , luciferase activity was detected on an Envision multi-label plate reader (PerkinElmer) after addition of ONE-Glo ^™ (Promega) reagent. All conditions were tested in duplicate.

EC50/IC50 values were determined using GraphPad Prism ^™ software using nonlinear regression (4-parameter logistic). Percent inhibition was calculated based on relative luminescence unit (RLU) values using the following equation:

In this equation, "RLU Baseline" is the luminescence value from cells treated with a constant amount of CD3 bsAb without a test molecule, "RLU Inhibition" is the luminescence value at the highest concentration of the test molecule treated with a constant amount of CD3 bsAb, and "RLU Background" is the luminescence value from cells without any CD3 bsAb or test molecule.

7.1.4. Determination of the oligomerization state of anti-mCD20 x mPDL1 extracellular domain molecules by size exclusion chromatography

Size exclusion ultra-performance liquid chromatography (SE-UPLC) was used to assess the size heterogeneity of anti-mCD20 x mPDL1 extracellular domain molecules. SE-UPLC analysis was performed on a Waters Acquity UPLC H-Class system, where 10 μg of each protein sample was injected onto an Acquity BEH SEC column ( 1.7 μm, 4.6 x 300 mm) and the flow rate was set to 0.3 mL/min. The mobile phase buffer contained 10 mM sodium phosphate, 500 mM NaCl, pH 7.0. The eluted samples were detected by UV absorbance at 280 nm using a photodiode array module.

Thermal stability

Differential scanning fluorescence (DSF) was used to evaluate the thermal stability of anti-mCD20 x mPDL1 extracellular domain molecules. DSF analysis was performed on a ThermoFisher QuantStudio5 system. The stock solution of each sample was diluted to 0.2 mg/mL in 1X PBS glycerol pH 7.4 and transferred to a 96-well plate. An excess (8X) of Sypro Orange ^TM fluorescent dye was added to each well, which preferentially binds to buried hydrophobic residues when the protein is unfolded, and then the thermal stability curve was determined on a linear thermal ramp from 25°C to 95°C for 20 minutes.

7.1.6. Assembly percentage

According to the manufacturer's protocol (Perkin Elmer, Waltham, MA), the assembly of bifunctional fusion molecules was determined by high-throughput analysis on Cliper LabChip GX. In brief, sample buffer was prepared by mixing 7ml HT protein expression sample buffer with 240 μl BME (reduction) or 25mM iodoacetamide (IAM, for non-reduction determination). Samples were normalized to 0.5mg/ml with sample buffer and then heated at 70°C for 10 minutes. Before loading on the instrument, 70 μl of water was added to each sample. Chips were prepared according to the manufacturer's instructions. The electrophoretogram of the sample was analyzed using LabChip GX software. The peak from the non-reduced electrophoretogram indicated the complete antibody %.

7.1.7. Activity of anti-mCD20 x mPDL1 ectodomain molecules in prediabetic NOD mice

Ten-week-old prediabetic non-obese diabetic (NOD) mice (The Jackson Laboratory) were treated intraperitoneally twice weekly with 1 mg/kg, 0.1 mg/kg, or 0.01 mg/kg of selected anti-mCD20 x mPDL1 extracellular domain molecules for the duration of the experiment (e.g., until mice were 28 weeks old). Blood glucose levels were monitored every two weeks, while body weight was monitored weekly. The overall experimental design is depicted in FIG7 .

7.1.8. Activity of anti-mCD20 x mPDL1 extracellular domain molecules in the experimental autoimmune encephalomyelitis/multiple sclerosis mouse model

Administration of the immunodominant 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG _35-55 ) in mice produces anti-MOG antibodies that cause demyelination and chronic experimental autoimmune encephalomyelitis (EAE), a common animal model for multiple sclerosis (MS).

EAE was induced in wild-type C57BL/6 mice (10-12 weeks, male, Jackson Laboratory) by subcutaneous delivery of 200 mg CFA containing MOG _35-55 on day 1. Given that the administration of pertussis toxin promotes the migration of T cells to the central nervous system by weakening the blood-brain barrier, mice were also injected intraperitoneally with 200 ng pertussis toxin on days 1 and 2. Body weight and EAE development were monitored on days 1, 2, 7, 10, 14, 18, and 20. EAE monitoring scores were recorded on a scale of 0-5 as follows: 0: healthy; 1: tail weakness; 2: gait abnormality and/or righting reflex defect; 3: partial paralysis of the hind limbs; 4: complete paralysis of the hind limbs; and 5: complete paralysis of the hind limbs and partial paralysis of the forelimbs or dying.

Mice were injected intraperitoneally twice weekly with selected anti-mCD20 x mPDL1 ectodomain molecules or appropriate control molecules starting on day 2. Endpoint tissue collection was performed at the peak of disease on day 20. Spinal cord infiltrates were used for flow cytometry, and splenic MOG-specific T cell responses were assessed by ELISPOT.

7.2. Example 1: Generation and stability of bispecific anti-mCD20-mPDL1 extracellular domain agonists

7.2.1. Overview

Mammalian expression vectors for individual heavy and light chains were generated by DNA synthesis and cloning in ready-to-use constructs in the pcDNA3.4 Topo expression system from Life Technologies (Carlsbad, CA). To express the molecules, DNA of the heavy chain and universal light chain were cotransfected into Expi293 cells (Thermo Fisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was collected and processed for purification by HiTrap protein A FF or Mab Select SuRe columns (GE Healthcare). For functional confirmation, the selected MBM was amplified to 2 L and subjected to a series of purification procedures, including size exclusion chromatography as the final step.

Results

Various anti-mCD20 x mPDL1 extracellular domain molecules (Figure 2A) were expressed by Expi293 Freestyle cells and purified by a one-step Mab-Select SuRe column (Table 4), with total yields ranging from 2.7-7.7 mg. In general, molecules with a 1:1 valency ratio (anti-mCD20: mPDL1 extracellular domain) showed higher yields (4.1-7.7 mg) than molecules with 2:1 or 2:2 (Table 4).

After one-step affinity purification, high molecular weight (HMW)% and monomer% were checked by SE-UPLC, and thermal stability was monitored by differential scanning fluorimetry (DSF) (Table 5). Most anti-mCD20 x mPDL1 extracellular domain fusion molecules showed greater than 85% monomer species (Table 3, molecules A-L in Figure 1) without additional size exclusion chromatography (SEC). For 2+2m20_mPL_4 (L), after two column purifications including SEC steps, the monomer percentage increased to 99% (Table 5). All anti-mCD20 x mPDL1 extracellular domain fusions had similar thermal stability measured by DSF and Tm1 at about 60 ° C (Table 3). In addition, all bifunctional fusions had excellent assembly between heavy chains as determined by capillary electrophoresis SDS (CE-SDS) (Table 5).

7.3. Example 2: Binding characterization of anti-mCD20 x mPDL1 extracellular domain molecules

The ability of anti-mCD20 x mPDL1 ectodomain molecules to bind to their two targets on the cell surface was assessed in a flow binding assay.

Results

Binding curves are shown in Figures 4A and 4B. Relative to monovalent molecules of similar formats for mPD1 binding and mCD20 binding, higher efficacy and maximum MFI of bivalent molecules in binding were observed. Specifically, although 2+2m20_mPL_4 (L) and 2+1m20_mPL_3 (G) (Figures 4A and 4B; Table 6) share similar binding to HEK293/mCD20 cells, due to the increase in the valence of mPD1 binding, 2+2m20_mPL_4 (L) is more strongly bound to Jurkat/mPD1 cells than 2+1m20_mPL_3 (G). In both bivalent and monovalent molecules, the binding signal appears to be orientation-dependent, wherein relative to the Fc domain, the N-terminal anti-mCD20 and hPDL1 extracellular domain orientations generally have higher efficacy and maximum MFI. Anti-mCD20 or mPDL1 ectodomain showed reduced binding when positioned between the N-terminal portion preceding the Fc and the hinge region (Figures 4A and 4B; Table 6).

7.4. Example 3: mPDL1 agonism by anti-mCD20 x mPDL1 extracellular domain molecules

The mPD1 agonistic effects of anti-mCD20 x mPDL1 ectodomain molecules were investigated using the bioassay depicted in Figure 5 and described in Section 7.1.3.

Results

Results from luciferase assays are depicted in FIG. 6 and Table 6. Nineteen molecules of the present disclosure were tested for PD1 agonism and regulation of T cell signaling using HEK293/CD22/mCD20 and Jurkat/AP1-luc/mPD1 reporter cells with CD3 bsAb. As shown in Table 6, four of the nineteen molecules of the present disclosure showed inhibition of T cell signaling, with IC50 values in the range of 65-770 pM and maximum inhibition in the range of 27-84%. Molecules 2+2m20_MPL_4 and 2+1m20_MPL_3 (G and L, respectively; Table 6) showed the strongest PD1 agonism, with maximum inhibition of 74% to 84% (FIGS. 6C and 6E). Fifteen of the 19 molecules showed weak or no inhibition, with maximum inhibition in the range of -10% to 40%. Isotype control antibodies did not show inhibition of signaling. CD3 bsAb showed activation of T cell signaling with EC50 values of 627 pM and 1.15 nM.

7.5. Example 4: Summary of data from in vitro assays using anti-mCD20 x mPDL1 extracellular domain molecules

Table 6 provides a summary of in vitro data collected with various anti-mCD20 x mPDL1 extracellular domain molecules, including cell-based flow binding and in vitro bioassay results. Results from luciferase assays are depicted in Figures 6A-6E. Molecules 2+2m20_mPL_4 and 2+1m20_mPL_3 (G and L, respectively, in Table 6) exhibited the strongest PD1 agonism. By cell-based flow analysis, 2+2m20_mPL_4 revealed the strongest binding to mPD1 and mCD20, whereas 2+1m20_mPL_3 revealed only moderate binding to cells expressing mPD1, suggesting that in the presence of both APCs and effector cells, mPD1 needs to be aggregated by bivalent binding of mCD20. Overall, similar cell binding affinities (mPD1 or mCD20) did not translate into similar PD1 agonism, such as F vs. G and K vs. L (Figure 6A and Table 6), suggesting that both valency and structural arrangement of the CD20 and mPDL1 ectodomain arms are important for conferring activity.

7.6. Example 5: In vivo efficacy of anti-mCD20 x mPDL1 extracellular domain molecules

The ability of selected 2+2 and 2+1 anti-mCD20 x mPDL1 ectodomain molecules to prevent the onset of type 1 diabetes (T1D) was evaluated in prediabetic NOD mice. The experimental design is depicted in Figure 7 and described in Section 7.1.7.

Results

Individual animal data are shown in Figures 8A-8I. Figures 9A and 9B depict the percentage of mice without diabetes at each indicated time point. Typically, 80-90% of NOD mice develop diabetes at about 25 weeks of age. However, in this experiment, only about 30% of mice developed diabetes at 27 weeks. Although there was a lower incidence of diabetes in the control NOD mice, there was a clear protective trend with a higher dose of (2+2) anti-mCD20 x mPDL1 extracellular domain molecule 2+2m20_mPL_4 (molecule L in Figure 2A) rather than with (2+1 anti-mCD20 x mPDL1 extracellular domain molecule 2+1m20_mPL_3 (molecule G in Figure 2A) (Figures 9A and 9B).

7.7. Example 6: Reduction of autoimmune T cell infiltration induced by anti-mCD20 x mPDL1 extracellular domain molecules

Infiltration of T cells has been implicated in the development of autoimmune diseases such as multiple sclerosis (Kaskow and Baecher-Allan, 2018. Cold Spring Harb Perspect Med. 8(4):a029025) and autoimmune models of diabetes (Bettini and Vignali, 2011. Curr Opinion in Immunology, 23(6):754-760). The protective effect of the (2+2) anti-mCD20 x mPDL1 extracellular domain molecule 2+2m20_mPL_4 on T cell infiltration was assessed by flow cytometry as described in Section 7.1.2 [Please supplement Section 7.1.2 with flow cytometry information relevant to experiments using cells from mouse models].

7.7.1. Results

In one evaluation, proliferative (activated) and less activated islet-specific CD8+T cell populations were analyzed in NOD mice described in Section 7.1.7, which were given 0.1 or 1 mg/kg 2+2m20_mPL_4 or a control molecule. Treatment with 1 mg/kg 2+2m20_mPL_4 was associated with a significant increase in the percentage of less activated CD8+T cell clusters (Figure 10A). Although the percentage of proliferative cell clusters did not differ under different conditions (Figure 10B), in pancreatic tissue isolated from NOD mice treated with 1 mg/kg 2+2m20_mPL_4, the ratio of less activated cell clusters to proliferative cell clusters was higher (Figure 10C), indicating that the treatment was able to reduce pancreatic infiltration of activated autoimmune T cells.

In another evaluation, spinal cord infiltration of T cells was evaluated in the multiple sclerosis mouse model described in Section 7.1.8. In the spinal cords of mice treated with 1 mg/kg 2+2m20_mPL_4, there were significantly fewer CD3+ (Figure 11A), CD4+ (Figure 11B), and CD8+ (Figure 11C) T cells relative to the spinal cords of mice treated with the same dose of control. Therefore, in the multiple sclerosis model, treatment with 2+2m20_mPL_4 can reduce spinal cord infiltration of T cells.

8. Specific Examples, References

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the present disclosure.The present disclosure is illustrated by the numbered embodiments set forth below.

1. A protein comprising

(a) at least one CD20 targeting moiety;

(b) at least one PD1 agonist moiety;

(c) at least one dimerization moiety; and

(d) optionally, one or more linker moieties that separate one or more portions of the protein,

Optionally among them:

(i) the protein portions are arranged in the order of CD20 targeting portion-PD1 agonist portion-dimerization portion from N-terminus to C-terminus;

(ii) the portions of the protein are arranged in the order of dimerization portion-PD1 agonist portion-CD20 targeting portion from N-terminus to C-terminus;

(iii) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of PDL1 or a PD1 binding portion thereof, and the dimerization moiety is an Fc domain, and the light chain of the Fab is not fused to the extracellular domain of PDL1 or a PD1 binding portion thereof;

(iv) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1 or a PD1 binding portion thereof, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not located at the N-terminus of the VH of the anti-CD20 Fab;

(v) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1 or a PD1 binding portion thereof, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not located at the C-terminus of the Fc domain;

(vi) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1 or a PD1 binding portion thereof, and the dimerization moiety is an Fc domain, and the protein is monovalent for the CD20 targeting moiety and/or the PD1 agonist moiety;

(vii) the protein is asymmetric;

(viii) the protein comprises an Fc heterodimer; or

(ix) Any combination of two or more of the above (i) to (viii).

2. The protein of embodiment 1, wherein the at least one CD20 targeting moiety is an antigen binding fragment of an anti-CD20 antibody.

3. The protein of embodiment 2, wherein the antigen-binding fragment of the anti-CD20 antibody is in the form of Fab, Fv or scFv.

4. The protein of embodiment 2 or embodiment 3, wherein the anti-CD20 antibody comprises:

(a) a complementarity determining region ("CDR") having the CDR sequence of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015, or veltuzumab;

(b) all six CDR sequences of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015, or veltuzumab;

(c) at least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, utuuximab, okatuzumab, TRU-015, or veltuzumab, and the light chain CDR sequence of a common light chain;

(d) a VH comprising the amino acid sequence of the VH of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015, or veltuzumab, and a VL comprising the amino acid sequence of the same antibody; or

(e) a VH comprising the amino acid sequence of the VH of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015, or veltuzumab, and a VL comprising a common light chain VL sequence.

5. The protein of embodiment 2 or embodiment 3, wherein the anti-CD20 antibody binds to:

(a) Topological domains of CD20;

(b) the transmembrane domain of CD20; or

(c) A CD20 region displayed extracellularly on the surface of a cell (eg, a B cell).

6. The protein according to embodiment 2 or embodiment 3, wherein:

(a) the anti-CD20 antibody is selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015 and veltuzumab,

(b) the anti-CD20 antibody competes for binding to CD20 with an anti-CD20 antibody selected from the group consisting of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ututumomab, okatuzumab, TRU-015, and veltuzumab.

7. The protein of any one of embodiments 1 to 6, wherein the at least one PD1 agonist portion comprises an amino acid sequence having at least 90% sequence identity to the PD1 binding portion of the extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

8. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 95% sequence identity to the PD1 binding portion of the extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

9. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 97% sequence identity to the PD1 binding portion of the extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

10. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 98% sequence identity to the PD1 binding portion of the extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

11. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 99% sequence identity to the PD1 binding portion of the extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

12. The protein of embodiment 7, wherein the PD1 agonist portion comprises the amino acid sequence of the PD1 binding portion of the extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

13. The protein of any one of embodiments 7 to 12, wherein the PD1 binding portion of the extracellular domain of PDL1 comprises amino acids 19-134 of human PDL1 or amino acids 19-134 of murine PDL1.

14. The protein of any one of embodiments 7 to 12, wherein the PD1 binding portion of the extracellular domain of PDL1 comprises or consists of the PDL1 extracellular domain, optionally wherein PDL1 is human or murine PDL1.

15. The protein of any one of embodiments 1 to 6, wherein the at least one PD1 agonist portion comprises an amino acid sequence having at least 90% sequence identity to the PD1 binding portion of the extracellular domain of PDL2, optionally wherein PDL2 is human or murine PDL2.

16. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 95% sequence identity to the PD1 binding portion of the extracellular domain of PDL2, optionally wherein PDL2 is human or murine PDL2.

17. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 97% sequence identity to the PD1 binding portion of the extracellular domain of PDL2, optionally wherein PDL2 is human or murine PDL2.

18. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 98% sequence identity to the PD1 binding portion of the extracellular domain of PDL2, optionally wherein PDL1 is human or murine PDL2.

19. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 99% sequence identity to the PD1 binding portion of the extracellular domain of PDL2, optionally wherein PDL2 is human or murine PDL2.

20. The protein of embodiment 15, wherein the PD1 agonist portion comprises the amino acid sequence of the PD1 binding portion of the extracellular domain of PDL2, optionally wherein PDL2 is human or murine PDL2.

21. The protein of any one of embodiments 15 to 20, wherein the PD1 binding portion of the extracellular domain of PDL2 comprises amino acids 20-121 of human PDL2 or amino acids 20-121 of murine PDL2.

22. The protein of any one of embodiments 15 to 20, wherein the PD1 binding portion of the extracellular domain of PDL2 comprises or consists of the PDL2 extracellular domain, optionally wherein PDL2 is human or murine PDL2.

23. The protein of any one of embodiments 1 to 22, comprising one or more linker moieties.

24. The protein of embodiment 23, wherein at least one CD20 targeting moiety and at least one PD1 agonist moiety are separated by a linker moiety.

25. The protein of embodiment 23 or embodiment 24, wherein at least one CD20 targeting moiety and at least one dimerization moiety are separated by a linker moiety.

26. The protein of any one of embodiments 23 to 25, wherein at least one PD1 agonist moiety and at least one dimerization moiety are separated by a linker moiety.

27. The protein of any one of embodiments 21 to 26, wherein (a) each linker portion is at least 5 or at least 10 amino acids in length, (b) each linker portion is at most 20, at most 25, or at most 30 amino acids in length, and/or (c) each linker portion is 5-15 amino acids or 5-20 amino acids in length.

28. The protein of any one of embodiments 21 to 27, wherein at least one linker moiety comprises a glycine-serine linker.

29. The protein of embodiment 28, wherein the glycine-serine linker comprises the sequence G ₄ S (SEQ ID NO: 14) or a multimer thereof.

30. The protein of embodiment 29, wherein said multimer comprises 2, 3, 4, 5 or more repeats of the amino acid sequence G ₄ S (SEQ ID NO: 56).

31. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 2.

32. The protein of embodiment 31, wherein exemplary monomer 1 comprises or consists of a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

33. The protein of embodiment 30, wherein exemplary monomer 1 consists of a single polypeptide chain.

34. The protein of embodiment 30, wherein exemplary monomer 1 is composed of two polypeptide chains.

35. A protein according to any one of embodiments 29 to 34, wherein exemplary monomer 2 comprises or consists of a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation, an optional linker portion, and a dimerization portion.

36. The protein of any one of embodiments 31 to 35, having the configuration depicted in Figure 1A.

37. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 3 and a monomer according to exemplary monomer 4.

38. The protein of embodiment 37, wherein exemplary monomer 3 comprises or consists of an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation.

39. A protein according to embodiment 37 or embodiment 38, wherein exemplary monomer 4 comprises or consists of a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation, an optional linker portion, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion and a dimerization portion.

40. The protein of embodiment 39, wherein exemplary monomer 4 consists of a single polypeptide chain.

41. The protein of embodiment 39, wherein exemplary monomer 4 is composed of two polypeptide chains.

42. The protein of any one of embodiments 37 to 39, having the configuration depicted in Figure 1B.

43. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 3 and a monomer according to exemplary monomer 5.

44. The protein of embodiment 43, wherein exemplary monomer 3 comprises or consists of an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation.

45. A protein according to embodiment 43 or embodiment 44, wherein exemplary monomer 5 comprises or consists of a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a dimerization portion and a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation.

46. The protein of embodiment 45, wherein exemplary monomer 5 consists of a single polypeptide chain.

47. The protein of embodiment 45, wherein exemplary monomer 5 is composed of two polypeptide chains.

48. The protein of any one of embodiments 43 to 47, having the configuration depicted in Figure 1C.

49. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 3 and a monomer according to exemplary monomer 6.

50. The protein of embodiment 49, wherein exemplary monomer 3 comprises or consists of an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation.

51. A protein according to embodiment 49 or embodiment 50, wherein exemplary monomer 6 comprises, in an N-terminal to C-terminal orientation, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)), an optional linker portion, and a dimerization portion or consists thereof.

52. The protein of any one of embodiments 49 to 51, wherein exemplary monomer 6 consists of a single polypeptide chain.

53. The protein of any one of embodiments 49 to 51, wherein exemplary monomer 6 is composed of two polypeptide chains.

54. The protein of any one of embodiments 49 to 53, having the configuration depicted in Figure ID.

55. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 4.

56. The protein of embodiment 55, wherein exemplary monomer 1 comprises or consists of a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

57. The protein of embodiment 56, wherein exemplary monomer 1 consists of a single polypeptide chain.

58. The protein of embodiment 56, wherein exemplary monomer 1 is composed of two polypeptide chains.

59. A protein according to any one of embodiments 55 to 58, wherein exemplary monomer 4 comprises or consists of a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation, an optional linker portion, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, and a dimerization portion.

60. The protein of embodiment 59, wherein exemplary monomer 4 consists of a single polypeptide chain.

61. The protein of embodiment 59, wherein exemplary monomer 4 is composed of two polypeptide chains.

62. The protein of any one of embodiments 55 to 61, having the configuration depicted in Figure IE.

63. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 5.

64. The protein of embodiment 63, wherein exemplary monomer 1 comprises or consists of a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

65. The protein of embodiment 64, wherein exemplary monomer 1 consists of a single polypeptide chain.

66. The protein of embodiment 64, wherein exemplary monomer 1 is composed of two polypeptide chains.

67. A protein according to any one of embodiments 63 to 66, wherein exemplary monomer 5 comprises or consists of a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a dimerization portion, and a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation.

68. The protein of embodiment 67, wherein exemplary monomer 5 consists of a single polypeptide chain.

69. The protein of embodiment 67, wherein exemplary monomer 5 is composed of two polypeptide chains.

70. The protein of any one of embodiments 63 to 69, having the configuration depicted in Figure 1F.

71. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 6.

72. A protein according to embodiment 71, wherein exemplary monomer 1 comprises or consists of a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation.

73. The protein of embodiment 72, wherein exemplary monomer 1 consists of a single polypeptide chain.

74. The protein of embodiment 72, wherein exemplary monomer 1 is composed of two polypeptide chains.

75. A protein according to any one of embodiments 71 to 74, wherein exemplary monomer 6 comprises or consists of a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)), an optional linker portion, and a dimerization portion in an N-terminal to C-terminal orientation.

76. The protein of embodiment 75, wherein exemplary monomer 6 consists of a single polypeptide chain.

77. The protein of embodiment 75, wherein exemplary monomer 6 is composed of two polypeptide chains.

78. The protein of any one of embodiments 71 to 77, having the configuration depicted in Figure 1G.

79. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 7.

80. The protein of embodiment 79, wherein exemplary monomer 1 comprises or consists of a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

81. The protein of embodiment 80, wherein exemplary monomer 1 consists of a single polypeptide chain.

82. The protein of embodiment 80, wherein exemplary monomer 1 is composed of two polypeptide chains.

83. A protein according to any one of embodiments 79 to 82, wherein exemplary monomer 7 comprises or consists of a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation, an optional linker portion, a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)), an optional linker portion, and a dimerization portion.

84. The protein of any one of embodiments 79 to 83, having the configuration depicted in Figure 1H.

85. The protein of any one of embodiments 1 to 30, comprising two monomers according to exemplary monomer 4.

86. A protein according to embodiment 85, wherein each exemplary monomer 4 comprises or consists of a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation, an optional linker portion, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion and a dimerization portion.

87. A protein according to embodiment 86, wherein each exemplary monomer 4 is composed of a single polypeptide chain.

88. A protein according to embodiment 86, wherein each exemplary monomer 4 is composed of two polypeptide chains.

89. The protein of any one of embodiments 85 to 88, having the configuration depicted in Figure II.

90. The protein of any one of embodiments 1 to 30, comprising two monomers according to exemplary monomer 5.

91. A protein according to embodiment 90, wherein each exemplary monomer 5 comprises or consists of a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a dimerization portion and a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation.

92. A protein according to embodiment 91, wherein each exemplary monomer 5 is composed of a single polypeptide chain.

93. A protein according to embodiment 91, wherein each exemplary monomer 5 is composed of two polypeptide chains.

94. The protein of any one of embodiments 90 to 93, having the configuration depicted in Figure 1J.

95. The protein of any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 8 and a monomer according to exemplary monomer 9.

96. A protein according to embodiment 95, wherein exemplary monomer 8 comprises or consists of a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a dimerization portion, an optional linker portion and a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV) in an N-terminal to C-terminal orientation.

97. A protein according to embodiment 96, wherein each exemplary monomer 8 is composed of a single polypeptide chain.

98. A protein according to embodiment 96, wherein each exemplary monomer 8 is composed of two polypeptide chains.

99. A protein according to embodiment 96, wherein each exemplary monomer 8 is composed of three polypeptide chains.

100. A protein according to any one of embodiments 95 to 99, wherein exemplary monomer 9 comprises a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) in an N-terminal to C-terminal orientation, an optional linker portion, a dimerization portion, an optional linker portion, a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)) or consists of the same.

101. The protein of any one of embodiments 95 to 100, having the configuration depicted in Figure 1K.

102. The protein of any one of embodiments 1 to 30, comprising two monomers according to exemplary monomer 6.

103. A protein according to embodiment 102, wherein each exemplary monomer 6 comprises, in an N-terminal to C-terminal orientation, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, a PD1 agonist portion (e.g., a portion comprising: (i) an amino acid sequence of the extracellular domain of said PDL1 or PDL2; (ii) a fragment of (i) capable of binding to PD1; or (iii) an amino acid sequence having at least 90% sequence identity with (i) or (ii)), an optional linker portion, and a dimerization portion or consists thereof.

104. The protein of embodiment 103, wherein each exemplary monomer 6 is composed of a single polypeptide chain.

105. The protein of embodiment 103, wherein each exemplary monomer 6 is composed of two polypeptide chains.

106. The protein of any one of embodiments 102 to 104, having the configuration depicted in Figure 1L.

107. The protein of any one of embodiments 1 to 54 and 79 to 84, comprising a CD20 targeting moiety.

108. The protein of embodiment 107, comprising a PD1 agonist portion.

109. The protein of embodiment 108, having the configuration depicted in Figure 1A.

110. The protein of embodiment 108 having the configuration depicted in Figure 1B.

111. The protein of embodiment 108 having the configuration depicted in Figure 1C.

112. The protein of embodiment 108 having the configuration depicted in Figure 1D.

113. The protein of embodiment 107, comprising two PD1 agonist portions.

114. The protein of embodiment 113, wherein the two PD1 agonist portions are identical.

115. The protein of Embodiment 113 or Embodiment 114, having the configuration depicted in Figure 1H.

116. The protein of any one of embodiments 1 to 106, comprising two CD20 targeting moieties.

117. The protein of embodiment 116, wherein the two CD20 targeting moieties are the same.

118. The protein of Embodiment 116 or Embodiment 117, comprising a PD1 agonist portion.

119. The protein of embodiment 118 having the configuration depicted in Figure IE.

120. The protein of Embodiment 118 having the configuration depicted in Figure 1F.

121. The protein of Embodiment 118, having the configuration depicted in Figure 1G.

122. The protein of Embodiment 116 or Embodiment 117, comprising two PD1 agonist portions.

123. The protein of embodiment 122, wherein the two PD1 agonist portions are identical.

124. The protein of embodiment 122 or embodiment 123, having the configuration depicted in Figure II.

125. The protein of embodiment 122 or embodiment 123, having the configuration depicted in Figure 1J.

126. The protein of Embodiment 122 or Embodiment 123, having the configuration depicted in Figure 1K.

127. The protein of Embodiment 122 or Embodiment 123, having the configuration depicted in Figure 1L.

128. A molecule according to any one of embodiments 1 to 127, wherein the CD20 targeting moiety binds to the extracellular domain of human CD20.

129. The molecule of any one of embodiments 1 to 128, wherein the PD1 agonist partially agonizes human PD1.

130. A molecule according to any one of embodiments 1 to 127, wherein the CD20 targeting moiety binds to the extracellular domain of murine CD20.

131. The molecule of any one of embodiments 1 to 127 and 130, wherein the PD1 agonist partially agonizes murine PD1.

132. The protein of any one of embodiments 1 to 131, wherein the at least one dimerization moiety is or comprises an Fc domain.

133. The protein of embodiment 132, wherein the Fc domain is a human Fc domain.

134. The protein of embodiment 132 or embodiment 133, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

135. The protein of any one of embodiments 132 to 134, wherein the Fc domain has reduced effector function.

136. The protein of any one of embodiments 132 to 135, wherein the Fc domain is an IgG4 Fc domain.

137. The protein of any one of embodiments 132 to 136, wherein the Fc domain comprises the amino acid sequence

or part thereof.

138. The protein of any one of embodiments 132 to 137, comprising an Fc dimer.

139. The protein of embodiment 138, wherein the Fc dimer is an Fc homodimer.

140. The protein of embodiment 138, wherein the Fc dimer is an Fc heterodimer.

141. The protein of embodiment 140, wherein the Fc heterodimer comprises a knob-to-hole mutation.

142. The protein of Embodiment 140 or Embodiment 141, wherein the Fc heterodimer comprises a star mutation.

143. The protein according to embodiment 1, wherein the parts of the protein are arranged in the order of CD20 targeting part-PD1 agonist part-dimerization part from N-terminus to C-terminus.

144. The protein according to embodiment 1, wherein the parts of the protein are arranged in the order of dimerization part-PD1 agonist part-CD20 targeting part from N-terminus to C-terminus.

145. A protein according to embodiment 1, wherein the CD20 targeting portion is an anti-CD20 Fab, the PD1 agonist portion is the extracellular domain of PDL1, and the dimerization portion is an Fc domain, and the light chain of the Fab is not fused to the extracellular domain of PDL1 or its PD1 binding portion.

146. A protein according to embodiment 1, wherein the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not located at the N-terminus of the VH of the anti-CD20 Fab.

147. A protein according to embodiment 1, wherein the CD20 targeting moiety is anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1, and the dimerization moiety is the Fc domain, and the PD1 agonist moiety is not located at the C-terminus of the Fc domain.

148. A protein according to embodiment 1, wherein the CD20 targeting moiety is anti-CD20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1, and the dimerization moiety is the Fc domain, and the protein is monovalent for the CD20 targeting moiety and/or the PD1 agonist moiety.

149. The protein of embodiment 1, wherein the protein is asymmetric.

150. A nucleic acid or multiple nucleic acids encoding the protein of any one of embodiments 1 to 149.

151. A host cell engineered to express the protein of any one of embodiments 1 to 149 or the nucleic acid of embodiment 150.

152. A method of producing the protein of any one of embodiments 1 to 149, the method comprising culturing the host cell of embodiment 151 and recovering the protein expressed thereby.

153. A pharmaceutical composition comprising the protein of any one of embodiments 1 to 149 and an excipient.

154. A method of treating a subject having an immune disorder or condition associated with T cell dysregulation, the method comprising administering to the subject an effective amount of a protein according to any one of embodiments 1 to 149 or a pharmaceutical composition according to embodiment 153.

155. The method of embodiment 154, wherein the immune disorder or condition is type 1 diabetes, primary biliary cholangitis (PBC), Goodpasture's syndrome, amyloidosis, ankylosing spondylitis, anti-glomerular basement membrane nephritis, anti-tubular basement membrane nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune oophoritis, graft-versus-host disease (GVHD), autoimmune pancreatitis, autoimmune retinopathy, Behcet's disease, Crohn's disease, Devic's disease, systemic lupus erythematosus (SLE), Döller's syndrome, fibrosing alveolitis, glomerulonephritis, Grave's disease, Guillain-Barre syndrome syndrome, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), multiple sclerosis, polyneuropathy, organomegaly, endocrinopathy, monoclonal syndrome (POEMS), polyarteritis nodosa, rheumatoid arthritis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm or testicular autoimmunity, stiff-man syndrome (SPS), Takayasu's arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunter syndrome (THS), or vasculitis.

156. The method of embodiment 154, wherein the immune disorder or condition is type 1 diabetes.

157. The method of embodiment 156, wherein the type 1 diabetes is childhood-onset type 1 diabetes.

158. The method of embodiment 156, wherein the type 1 diabetes is adult-onset type 1 diabetes.

159. The method of embodiment 156, wherein the subject is a pediatric patient.

160. A method according to any one of embodiments 156 to 158, wherein the subject is an adult patient.

161. The method of embodiment 154, wherein the immune disorder or condition is systemic lupus erythematosus.

162. The method of embodiment 154, wherein the immune disorder or condition is Crohn's disease.

163. The method of embodiment 154, wherein the immune disorder or condition is graft-versus-host disease (GVHD).

164. The method of any one of embodiments 154 to 163, wherein the protein of any one of embodiments 1 to 149 or the pharmaceutical composition of embodiment 153 is administered in a single dose.

165. The method of any one of embodiments 154 to 164, wherein said administration of the protein of any one of embodiments 1 to 149 or the pharmaceutical composition of embodiment 153 is not repeated.

166. The method of any one of embodiments 154 to 165, wherein said method inhibits a cellular immune response in said subject.

167. The method of embodiment 166, wherein said method suppresses the subject's immune system.

168. A method according to embodiment 167, wherein the method reduces T cell function of the subject.

169. A method according to embodiment 167, wherein the method reduces B cell function of the subject.

170. A method according to embodiment 167, wherein the method reduces T cell responsiveness of the subject.

171. A method of inhibiting a cellular autoimmune response in a subject, the method comprising administering to the subject an effective amount of a protein according to any one of embodiments 1 to 149 or a pharmaceutical composition according to embodiment 153.

172. A method according to embodiment 171, wherein the method reduces T cell function of the subject.

173. A method according to embodiment 171, wherein the method reduces B cell function of the subject.

174. A method according to embodiment 171, wherein the method reduces T cell responsiveness of the subject.

175. The method of any one of embodiments 154 to 174, further comprising administering an additional therapeutic agent to the subject.

176. A method according to embodiment 175, wherein the additional therapeutic agent is or includes an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to an apoptosis-inducing agent.

177. A method according to Example 175, wherein the additional therapeutic agent is or includes cells expressing CAR.

178. A method according to Example 177, wherein the CAR-expressing cells are CAR-expressing Treg cells.

179. A method of local PD1 agonism, the method comprising administering to a subject an effective amount of the protein of any one of embodiments 1 to 149 or the pharmaceutical composition of embodiment 153.

180. The method of embodiment 179, wherein administration of the protein or the pharmaceutical composition localizes PD1 agonism to B cells of the subject.

181. A method for locally modulating an immune response in a target tissue or a cell expressing CD20, the method comprising administering to a subject an effective amount of a protein according to any one of embodiments 1 to 149 or a pharmaceutical composition according to embodiment 153.

182. The method of embodiment 181, wherein administration of the protein or the pharmaceutical composition modulates an immune response in the subject's B cells.

183. A method for characterizing the ability of a type I molecule to agonize PD1, the method comprising:

(a) Type I cells stably expressing CD3, AP1-luciferase reporter gene, and PD1 were cultured together with type II cells stably expressing CD22 and CD20;

(b) incubating the cultured cells of step a) in the presence or absence of said type I molecule and type II molecule; and

(c) after step b), measuring luciferase activity in the cultured cells, wherein the type I molecule is a multispecific antigen-binding molecule comprising: i) a first binding specificity that binds to the extracellular domain of CD20; and ii) a second binding specificity that binds to PD1, wherein the type II molecule is a multispecific antigen-binding molecule comprising: i) a first binding specificity that binds to the extracellular domain of CD3; and ii) a second binding specificity that binds to the extracellular domain of CD22, and wherein the presence of the type II molecule causes an increase in luciferase activity, and the presence of the type I molecule causes a decrease in the luciferase activity caused by the type II molecule, and wherein the amount of decrease in luciferase activity indicates the ability of the type I molecule to agonize PD1.

184. The method of embodiment 183, wherein PD1 is mPD1.

185. A method according to embodiment 183, wherein the type I cells are Jurkat E6-1 cells.

186. A method according to embodiment 183, wherein the type I cells are transduced to express an AP1-luciferase reporter gene.

187. A method according to embodiment 183, wherein the type I cells are transduced to express the PD1 gene.

188. A method according to embodiment 186 or 187, wherein the type I cells are transduced with a lentivirus.

189. The method of embodiment 183, wherein the CD20 is mCD20.

190. A method according to embodiment 183, wherein the type II cells are HEK293 cells.

191. A method according to embodiment 183, wherein the type II cells are transduced to express CD22.

192. A method according to embodiment 183, wherein the type II cells are transduced to express the CD20 gene.

193. A method according to embodiment 191 or 192, wherein the type II cells are transduced with a lentivirus.

194. A method according to embodiment 183, wherein the cells of type II cells are inoculated before the cells of type I cells.

195. A method according to embodiment 183, wherein the cultured cells are incubated in the presence of the type II molecule, in the presence of a control antibody molecule, and in the absence of the type I molecule.

196. A method according to embodiment 183, wherein the cultured cells are incubated in the presence of the type I molecule and the type II molecule.

197. A method according to embodiment 183, wherein the type I molecule includes the extracellular domain of PDL1.

All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended to prevail.

Claims (55)

1. A protein, which comprises

(A) At least one CD20 targeting moiety;

(b) At least one PD1 agonist moiety;

(c) At least one dimerization moiety; and

(D) Optionally, one or more linker moieties, the one or more linker moieties separate one or more portions of the protein,

Wherein:

(i) The portions of the protein are arranged in the order CD20 targeting moiety-PD 1 agonist moiety-dimerization moiety from N-terminus to C-terminus;

(ii) The CD20 targeting moiety is an anti-CD 20 Fab, the PD1 agonist moiety is an extracellular domain of PDL1 or a PD1 binding moiety thereof, and the dimerization moiety is an Fc domain, and the light chain of the Fab is not fused to the extracellular domain of PDL1 or a PD1 binding moiety thereof;

(iii) The CD20 targeting moiety is an anti-CD 20 Fab, the PD1 agonist moiety is the extracellular domain of the PDL1 or a PD1 binding moiety thereof, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not located at the N-terminus of the VH of the anti-CD 20 Fab;

(iv) The CD20 targeting moiety is an anti-CD 20 Fab, the PD1 agonist moiety is the extracellular domain of PDL1 or a PD1 binding moiety thereof, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not located at the C-terminus of the Fc domain;

(v) The CD20 targeting moiety is an anti-CD 20 Fab, the PD1 agonist moiety is an extracellular domain of the PDL1 or a PD1 binding moiety thereof, and the dimerization moiety is an Fc domain, and the protein is monovalent for the CD20 targeting moiety and/or the PD1 agonist moiety;

(vi) Or any combination of two or more of the foregoing (i) through (v).

2. The protein of claim 1, wherein the at least one CD20 targeting moiety is an antigen binding fragment of an anti-CD 20 antibody.

3. The protein of claim 2, wherein the antigen binding fragment of the anti-CD 20 antibody is in the form of a Fab, fv, or scFv.

4. A protein according to claim 2 or claim 3, wherein:

(a) The anti-CD 20 antibody is selected from rituximab (rituximab), orelbizumab (ocrelizumab), obbinitron You Tuozhu mab (obinutuzumab), ofatumumab, tiumumab (ibritumomabituxetan), tositumomab (tositumomab), ulituximab (ublituximab), oxcarbatuzumab (ocaratuzumab), TRU-015, and veltuzumab (veltuzumab); or (b)

(B) The anti-CD 20 antibody competes with an anti-CD 20 antibody selected from the group consisting of binding to CD20 and/or binds to the same epitope as an anti-CD 20 antibody selected from the group consisting of: rituximab, oreb, and the obbine You Tuozhu monoclonal antibody beach You Tuozhu monoclonal antibody tositumomab rituximab (Ubbelo) Wutuxib monoclonal antibody.

5. The protein of any one of claims 1 to 4, wherein the at least one PD1 agonist moiety comprises an amino acid sequence that has at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to a PD1 binding portion of an extracellular domain of PDL1, optionally wherein PDL1 is human or murine PDL1.

6. The protein of claim 5, wherein the PD1 agonist moiety comprises an amino acid sequence of a PD1 binding portion of an extracellular domain of the PDL1, optionally wherein PDL1 is human or murine PDL1.

7. The protein of claim 5 or 6, wherein the PD1 binding portion of the extracellular domain of PDL1 comprises amino acids 19-134 of human PDL1 or amino acids 19-134 of murine PDL 1.

8. The protein of claim 5 or 6, wherein the PD1 binding portion of the extracellular domain of PDL1 comprises or consists of a PDL1 extracellular domain, optionally wherein PDL1 is human or murine PDL1.

9. The protein of any one of claims 1 to 4, wherein the at least one PD1 agonist moiety comprises an amino acid sequence that has at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to a PD1 binding portion of an extracellular domain of PDL2, optionally wherein PDL2 is human or murine PDL2.

10. The protein of claim 9, wherein the PD1 agonist moiety comprises an amino acid sequence of a PD1 binding portion of an extracellular domain of the PDL2, optionally wherein PDL2 is human or murine PDL2.

11. The protein of claim 9 or 10, wherein the PD1 binding portion of the extracellular domain of PDL2 comprises amino acids 20-121 of human PDL2 or amino acids 20-121 of murine PDL 2.

12. The protein of claim 9 or 10, wherein the PD1 binding portion of the extracellular domain of PDL2 comprises or consists of a PDL2 extracellular domain, optionally wherein PDL2 is human or murine PDL2.

13. The protein of any one of claims 1 to 12, comprising one or more linker moieties.

14. The protein of claim 13, wherein 1) at least one CD20 targeting moiety and at least one PD1 agonist moiety are separated by a linker moiety; and 2) at least one CD20 targeting moiety and at least one dimerization moiety are separated by a linker moiety.

15. The protein of claim 13 or 14, wherein (a) each linker moiety is at least 5 or at least 10 amino acids in length, (b) each linker moiety is at most 20, at most 25 or at most 30 amino acids in length, and/or (c) each linker moiety is 5-15 amino acids or 5-20 amino acids in length.

16. The protein of any one of claims 13 to 15, wherein at least one linker moiety comprises a glycine-serine linker.

17. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of a CD20 targeting moiety, optionally a linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation, and (2) a second monomer comprising or consisting of a PD1 agonist moiety, optionally a linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

18. The protein of claim 17, having the configuration depicted in fig. 1A.

19. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation and (2) a second monomer comprising or consisting of a PD1 agonist moiety, an optional linker moiety, a CD20 targeting moiety, an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation.

20. The protein of claim 19, having the configuration depicted in fig. 1B.

21. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of an optional linker moiety and a dimerization moiety in an N-terminal to C-terminal orientation and (2) a second monomer comprising or consisting of a CD20 targeting moiety, an optional linker moiety, a dimerization moiety and a PD1 agonist moiety in an N-terminal to C-terminal orientation.

22. The protein of claim 21, having the configuration depicted in fig. 1C.

23. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of an optional linker moiety and a dimerisation moiety in an N-to C-terminal orientation and (2) a second monomer comprising or consisting of a CD20 targeting moiety, an optional linker moiety, a PD1 agonist moiety in an N-to C-terminal orientation.

24. The protein of claim 23, having the configuration depicted in fig. 1D.

25. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation, and (2) a second monomer comprising or consisting of a PD1 agonist moiety, an optional linker moiety, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

26. The protein of claim 25, wherein the second monomer consists of a single polypeptide chain or two polypeptide chains.

27. The protein of claim 25 or 26, having the configuration depicted in fig. 1E.

28. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of a CD20 targeting moiety, optionally a linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation, and (2) a second monomer comprising or consisting of a CD20 targeting moiety, optionally a linker moiety, a dimerization moiety, and a PD1 agonist moiety in an N-terminal to C-terminal orientation.

29. The protein of claim 28, having the configuration depicted in fig. 1F.

30. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of a CD20 targeting moiety (e.g., anti-CD 20Fab, fv, or scFV) in an N-terminal to C-terminal orientation, an optional linker moiety, and a dimerization moiety, and (2) a second monomer comprising or consisting of a CD20 targeting moiety, an optional linker moiety, a PD1 agonist moiety, an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

31. The protein of any claim 30, having the configuration depicted in fig. 1G.

32. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of a CD20 targeting moiety (e.g., anti-CD 20Fab, fv, or scFV) in an N-terminal to C-terminal orientation, an optional linker moiety, and a dimerization moiety, and (2) a second monomer comprising or consisting of a PD1 agonist moiety, an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

33. The protein of claim 32, having the configuration depicted in fig. 1H.

34. The protein of any one of claims 1 to 16, comprising or consisting of two monomers each comprising or consisting of a PD1 agonist moiety, an optional linker moiety, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety in an N-terminal to C-terminal orientation.

35. The protein of claim 34, having the configuration depicted in fig. 1I.

36. The protein of any one of claims 1 to 16, comprising or consisting of two monomers each comprising or consisting of a CD20 targeting moiety, an optional linker moiety, a dimerization moiety and a PD1 agonist moiety in an N-terminal to C-terminal orientation.

37. The protein of claim 36, having the configuration depicted in fig. 1J.

38. The protein of any one of claims 1 to 16, comprising or consisting of (1) a first monomer comprising or consisting of a CD20 targeting moiety, an optional linker moiety, a dimerization moiety, an optional linker moiety, and a CD20 targeting moiety in an N-terminal to C-terminal orientation, and (2) a second monomer comprising or consisting of a PD1 agonist moiety, an optional linker moiety, a dimerization moiety, an optional linker moiety, a PD1 agonist moiety in an N-terminal to C-terminal orientation.

39. The protein of claim 38, having the configuration depicted in fig. 1K.

40. The protein of any one of claims 1 to 16, comprising or consisting of two monomers each comprising a CD20 targeting moiety (e.g., anti-CD 20 Fab, fv, or scFV), an optional linker moiety, a PD1 agonist moiety, in an N-terminal to C-terminal orientation.

41. The protein of claim 40, having the configuration depicted in FIG. 1L.

42. The protein of any one of claims 1 to 41, wherein the CD20 targeting moiety binds to an extracellular domain of human CD 20.

43. The protein of any one of claims 1 to 42, wherein the PD1 agonist partially agonizes human PD1.

44. The protein of any one of claims 1 to 41, wherein the CD20 targeting moiety binds to an extracellular domain of murine CD 20.

45. The protein of any one of claims 1 to 41 and 44, wherein the PD1 agonist partially agonizes murine PD1.

46. The protein of any one of claims 1 to 45, wherein the at least one dimerization moiety is or comprises an Fc domain.

47. A nucleic acid or nucleic acids encoding a protein according to any one of claims 1 to 46.

48. A host cell engineered to express a protein according to any one of claims 1 to 46 or a nucleic acid according to claim 47.

49. A method of producing a protein according to any one of claims 1 to 46, the method comprising culturing the host cell of claim 48 and recovering the protein expressed thereby.

50. A pharmaceutical composition comprising a protein according to any one of claims 1 to 46 and an excipient.

51. A method of treating a subject having an immune disorder or condition associated with a T cell disorder, the method comprising administering to the subject an effective amount of a protein according to any one of claims 1 to 46 or a pharmaceutical composition according to claim 50.

52. The method of claim 51, wherein the immune disorder or condition is type 1 diabetes.

53. A method of inhibiting a cellular autoimmune response in a subject, the method comprising administering to the subject an effective amount of a protein according to any one of claims 1 to 46 or a pharmaceutical composition according to claim 50.

54. The method of claim 53, wherein the method reduces T cell function, B cell function, or T cell responsiveness in the subject.

55. A method of characterizing the ability of a type I molecule to agonize PD1, the method comprising:

(a) Culturing a type I cell stably expressing CD3, stably expressing an AP 1-luciferase reporter gene, and stably expressing PD1 with a type II cell stably expressing CD22 and stably expressing CD 20;

(b) Incubating the cultured cells of step a) in the presence or absence of said type I and type II molecules; and

(C) Measuring luciferase activity in the cultured cells after step b),

Wherein the type I molecule is a multispecific antigen-binding molecule comprising: i) A first binding specificity for binding to the extracellular domain of CD 20; and ii) a second binding specificity for binding to PD1,

Wherein the type II molecule is a multispecific antigen-binding molecule comprising: i) A first binding specificity for binding to the extracellular domain of CD 3; and ii) a second binding specificity for binding to the extracellular domain of CD22, and

Wherein the presence of the type II molecule causes an increase in luciferase activity and the presence of the type I molecule causes a decrease in the luciferase activity caused by the type II molecule, and wherein a decreased amount of luciferase activity is indicative of the ability of the type I molecule to agonize PD 1.