CD20-PD1 binding molecules and methods of use thereof

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

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 278,454, filed November 11, 2021, and U.S. Provisional Application No. 63 / 278,374, filed November 11, 2021, the contents of each of which are incorporated by reference herein in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically, which is incorporated herein by reference in its entirety. The copy created on November 8, 2022 is named RGN-012WO_SL.xml and is 63,928 bytes in size. [Background technology]

[0003] 3.Background Autoimmune diseases occur when an organism has an abnormal immune response against its own cells and tissues. Efforts to understand autoimmunity have been ongoing for decades. During this time, it has become clear that the immune system has evolved multiple mechanisms to control self-reactivity. Defects in one or more of these mechanisms can lead to a breakdown in tolerance, resulting in autoimmune diseases.

[0004] The initial trigger of both systemic and organ-specific autoimmune diseases likely involves the recognition of self or foreign molecules by innate sensors. This recognition leads to an inflammatory response and engagement of previously quiescent autoreactive T and B cells. Theofilopolous, Kono, and Baccala, 2017, Nat Immunol, 18(17):716-724. Autoreactivity ranges from low, "physiologic" levels of autoreactivity essential for lymphocyte selection and immune system homeostasis, to intermediate levels of autoimmunity manifested as circulating autoantibodies and minor tissue infiltration without clinical consequences, to pathogenic autoimmunity associated with immune-mediated organ injury. Theofilopolous, Kono, and Baccala, 2017, Nat Immunol, 18(17):716-724. Autoimmune diseases are divided into organ-specific (e.g., type I diabetes (T1D), multiple sclerosis (MS), inflammatory bowel disease (IBD), myasthenia gravis) and systemic (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome) and can be mediated by autoantibodies or cytotoxic T cells, but in all cases helper T cells are required. Theofilopolous, Kono, and Baccala, 2017, Nat Immunol, 18(17):716-724.

[0005] Most autoimmune diseases show clinical heterogeneity, polygenicity, and multifactorial contributions that often require both genetic and environmental factors. Four mechanisms contribute to the control of escape of autoreactive T and B cells: inhibitory molecules, anergy, ignorance, and active suppression. Kono and Theofilopolous, Kono, and Baccala, 2017, Nat Immunol, 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 and B cells to suppress excessive immune responses, both normal and anti-self. Deficiency of some of these molecules leads to autoimmunity, providing strong evidence that autoreactive lymphocytes are present in the peripheral repertoire but are usually under control. See Paterson and Sharpe, 2010, Nat Ummunol, 11:109-111; Okazaki et al., 2013, Nat Immunol, 14:1212-1218; Pintec et al., 2014, Nat Immunol, 15:707-716; Macauley, Crocker, and Paulson, 2914, Nat Rev Immunol, 14:653-666; Ceeraz et al., 2016, Arthritis Rheumatol 69(4):814-825; and Schmitt et al., 2016, J Exp Med, 213:1627-1644. A wide range of immune-related adverse events resulting from unchecked autoreactivity occur frequently. Michot et al., 2016, Eur J Cancer, 54:139-148.

[0006] Existing therapies for autoimmune diseases have only achieved limited success. For example, it is often possible to correct organ-specific autoimmune diseases through metabolic control. When function is lost and cannot be restored, mechanical replacements or tissue grafts may be appropriate. Although some of the symptoms may be alleviated using this approach, for some of the most disabling autoimmune diseases, there is no effective long-term curative treatment. Many compounds, including insulin, corticosteroids, and modified beta interferon, can ameliorate some symptoms of autoimmune diseases, but they may have severe side effects and / or require long-term use. Common immunosuppressive drug therapies, such as chronic treatment with cyclosporine A, FK506, and rapamycin, also fail to provide treatment for these diseases, and their use is associated with many adverse side effects. Such effects include nephrotoxicity, increased predisposition to infections, and increased incidence of neoplasms.

[0007] Thus, there is a need for new therapeutic compositions and protocols that can be used to treat autoimmune diseases. Summary of the Invention

[0008] 4. Overview The present disclosure provides novel CD20-PD1 binding molecules. The CD20-PD1 binding molecules of the present disclosure typically comprise or consist of a CD20-PD1 monomer comprising one or more CD20 targeting moieties and / or one or more PD1 agonist moieties. The CD20-PD1 binding molecules of the present disclosure comprise a protein, the protein comprising at least one CD20 targeting moiety, at least one PD1 agonist moiety, at least one dimerization moiety, and optionally one or more linker moieties separating the one or more moieties in the protein. In some embodiments where the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain of PDL1, and the dimerization moiety is an Fc domain, optionally: i) the light chain of the Fab is not fused to the ectodomain of PDL1 or a PD1-binding portion thereof; ii) the PD1 agonist moiety is not at the N-terminus of the VH of the anti-CD20 Fab; iii) the PD1 agonist moiety is not at the C-terminus of the Fc domain; iv) the protein is monovalent with respect to 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)-(vi).

[0009] Exemplary CD20-PD1 binding molecules are disclosed in Section 6.2 and numbered embodiments 1 through 142. Exemplary CD20 targeting moieties are disclosed in Section 6.2.1 and numbered embodiments 2 through 6. Exemplary PD1 agonist moieties are disclosed in Section 6.4 and numbered embodiments 7 through 22.

[0010] The present disclosure further provides nucleic acids encoding CD20-PD1 binding molecules, CD20-PD1 monomers, and CD20 targeting moieties and PD1 agonist moieties. Nucleic acids encoding CD20-PD1 binding molecules and CD20-PD1 monomers that are composed of more than one polypeptide chain can be a single nucleic acid (e.g., a vector encoding all of the 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 engineered to express the 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 of producing the 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 for producing CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting moieties, and PD1 agonist moieties are described below in Section 6.8 and in numbered embodiments 150-152.

[0011] The present disclosure further provides pharmaceutical compositions comprising the CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting moieties, and PD1 agonist moieties of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.9, below, and in numbered Example 153.

[0012] Further provided herein are methods of using the CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting moieties, PD1 agonist moieties, and pharmaceutical compositions of the present disclosure, e.g., for treating an autoimmune disease, suppressing a cellular autoimmune response, or suppressing the immune system of a subject. Exemplary methods are described in Section 6.10, below, and in numbered embodiments 154-165. [Brief description of the drawings]

[0013] 5. Brief description of the drawings [Figure 1]1A-1L are a series of cartoons depicting various formats of CD20-PD1 binding molecules according to certain embodiments. The heavy chain variable domain of the CD20 targeting moiety is shown in a striped pattern, the light chain variable domain is shown in a dotted pattern, and the PD1 agonist moiety (e.g., the ectodomain of PDL1 or PDL2 or a PD1 binding portion thereof) is shown as a circle with a dashed line. [Figure 2A] A series of cartoons depicting the tested murine CD20-PD1 binding molecules PD1 (anti-mCD20 × mPDL1) (molecules A–L, Figure 2A) and controls (molecules M–S, Figure 2B) are presented. [Figure 2B] A series of cartoons depicting the tested murine CD20-PD1 binding molecules PD1 (anti-mCD20 × mPDL1) (molecules A–L, Figure 2A) and controls (molecules M–S, Figure 2B) are presented. [Figure 3A] Three-dimensional models of hPDL1 (left) and mPDL1 (right) are shown, highlighting the unpaired cysteine ​​residue (C113) on the surface of mPDL1. [Figure 3B] The partial sequence alignment (78-137 aa) of mPDL1 (upper sequence) and hPDL1 (lower sequence) is shown. The figure discloses SEQ ID NOs: 54-55, respectively, in order of appearance. [Figure 4A] Traces from flow binding studies are shown, representing 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. [Figure 4B] Traces from flow binding studies are shown, representing 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. [Figure 5A]Luciferase assay protocol: Figure 5A is a schematic description of the luciferase reporter assay, and Figure 5B is a cartoon representation showing the interactions between the key players described in Figure 5A. [Figure 5B] Luciferase assay protocol: Figure 5A is a schematic description of the luciferase reporter assay, and Figure 5B is a cartoon representation showing the interactions between the key players described in Figure 5A. [Figure 6] Shown is a test molecule (FIG. 6A) and a series of traces using it (FIGS. 6B-6E). The traces show mPD1 agonism as measured using the bioassay shown in FIG. 5. Cells and molecules used as indicated for each individual trace. [Figure 7] 1 shows the experimental design for a dose-escalation efficacy study in pre-diabetic non-obese diabetic (NOD) mice. [Figure 8A] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8B] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8C] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8D] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8E] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8F]A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8G] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8H] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 8I] A series of tracings are presented showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecules (anti-mCD20 x mPDL1 ectodomain). [Figure 9A] Graphs are shown demonstrating the ability of CD20-PD1 binding molecules (anti-mCD20×mPDL1 ectodomain) (top: molecule L in FIG. 2A, bottom: molecule G in FIG. 2B) to modulate the development of diabetes in NOD mice. [Figure 9B] Graphs are shown demonstrating the ability of CD20-PD1 binding molecules (anti-mCD20×mPDL1 ectodomain) (top: molecule L in FIG. 2A, bottom: molecule G in FIG. 2B) to modulate the development of diabetes in NOD mice. [Figure 10A] Box plots showing reduction of activated autoreactive islet-specific CD8+ T cell infiltration into NOD mouse pancreas following treatment with CD20-PD1 binding molecule (anti-mCD20×mPDL1 ectodomain). *p<0.05. [Figure 10B] Box plots showing reduction of activated autoreactive islet-specific CD8+ T cell infiltration into NOD mouse pancreas following treatment with CD20-PD1 binding molecule (anti-mCD20×mPDL1 ectodomain). *p<0.05. [Figure 10C]Box plots showing reduction of activated autoreactive islet-specific CD8+ T cell infiltration into NOD mouse pancreas following treatment with CD20-PD1 binding molecule (anti-mCD20×mPDL1 ectodomain). *p<0.05. [Figure 11A] Box plots showing 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 molecule (anti-mCD20 x mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001. [Figure 11B] Box plots showing 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 molecule (anti-mCD20 x mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001. [Figure 11C] Box plots showing 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 molecule (anti-mCD20 x mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 6. Detailed Description 6.1.Definition About, Approximately: The terms "about," "approximately," and the like are used throughout the specification preceding a numerical value to indicate that the numerical value is not necessarily precise (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, is understood to also be a disclosure of "X." Thus, for example, a disclosure of an embodiment in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of an embodiment in which the sequence has "X% sequence identity" to the other sequence.

[0015] And and Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (A or B, where the selection of A is mutually exclusive of B) and the selection of conjoint features (A or B, where both A and B are selected). In several places in the text, the term "and / or" is used with the same purpose and should not be interpreted to mean that "or" is used in reference to mutually exclusive alternatives.

[0016] Antigen-binding domain or ABD, and antigen-binding fragment: As used herein, the terms "antigen-binding domain" or "ABD" and "antigen-binding fragment" refer to a portion of a targeting moiety that can specifically, non-covalently, and reversibly bind to a target molecule.

[0017] Associated: The term "associated" in reference to a CD20-PD1 binding molecule or a component thereof (e.g., a CD20 targeting moiety, a PD1 agonist moiety, a dimerization moiety) refers to a functional relationship between two or more polypeptide chains or portions of polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with one another, e.g., non-covalently through molecular interactions or covalently through one or more disulfide or chemical bridges, to generate 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, an association between homodimeric or heterodimeric Fc domains within an Fc region, an association between a VH region and a VL region within a Fab or scFv, an association between a CH1 and a CL within a Fab, and an association between a CH3 and a CH3 within a domain-substituted Fab.

[0018] 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, the term "bivalent" 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. A CD20-PD1 binding molecule may be bivalent with respect to one type of moiety (e.g., a CD20 targeting moiety) and monovalent with respect to another type of moiety (e.g., a PD1 agonist moiety).

[0019] CD20-PD1 binding molecule: The term "CD20-PD1 binding molecule" refers to a molecule that comprises at least one CD20 targeting moiety and at least one PD1 agonist moiety. Generally, a CD20-PD1 binding molecule is a molecule that is composed of one or more polypeptide chains (e.g., one, two, three, or four polypeptide chains) that together comprise at least one CD20 targeting moiety and at least one PD1 agonist moiety.

[0020] In the context of the CD20-PD1 binding molecules of the present disclosure, the term "CD20-PD1 binding molecule" can refer to the core components of the molecule, i.e., the CD20 targeting moiety and the PD1 agonist moiety, and can also refer to a dimerization moiety, such as an Fc domain and any / or associated linker moieties. 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 moieties, one or more dimerization moieties, one or more linker moieties, and any combination of the foregoing, unless the context dictates otherwise.

[0021] 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) capable of binding 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" includes molecules capable of binding to any domain or region of CD20, including the topological domain or the transmembrane domain. In some embodiments, the CD20 targeting moiety is a molecule capable of binding to a region of CD20 that is extracellularly displayed on the surface of a cell (e.g., a B cell). CD20 targeting moieties are further described in Section 6.2.

[0022] Complementarity determining region or CDR: The term "complementarity determining region" or "CDR" as used herein refers to a sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary rules that can be used to identify the boundaries of CDRs 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 are also available to identify CDR sequences within antibodies.

[0023] Dimerization moiety: The term "dimerization moiety" refers to a polypeptide chain or an amino acid sequence that can promote association between two polypeptide chains to form a dimer. A first dimerization moiety can associate with an identical second 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, and the association of two Fc domains forms an Fc region. Thus, the Fc region can be a homodimer or a heterodimer.

[0024] 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 a CD20-PD1 binding molecule that gives half-maximal activation in the assay described in Section 7.1.3.3.

[0025] Epitope: An epitope, or antigenic determinant, is the part of an antigen (e.g., CD20) that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational.

[0026] Fab: The term "Fab" in the context of the CD20 targeting moiety of the present disclosure refers to a pair of polypeptide chains, the first polypeptide chain comprising the variable heavy chain (VH) domain at the antibody N-terminus to a first constant domain (referred to herein as C1), and the second polypeptide chain comprising the variable light chain (VL) domain at the antibody N-terminus to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, the VH is N-terminal to the first constant domain (CH1) of the heavy chain, and the VL is N-terminal to the constant domain (CL) of the light chain. The Fabs of the present disclosure can be arranged according to the native orientation or can include domain substitutions or swaps that promote correct VH and VL pairing. For example, the CH1 and CL domain pair in the Fab can be replaced with a CH3 domain pair to promote correct engineered Fab-chain pairing in a heterodimeric molecule. It is also possible to reverse CH1 and CL, such that CH1 is attached to VL and CL to VH, a configuration commonly known as a crossmab, a type of "domain swapping".

[0027] Fc domain and Fc region: The term "Fc domain" refers to the 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 may be identical to each other or different. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, e.g., via knob-in-hole interactions, and / or to allow purification, e.g., via a star mutation.

[0028] Host cell or recombinant host cell: The terms "host cell" and "recombinant host cell", as used herein, refer to cells that have been genetically engineered, for example, by the introduction of heterologous nucleic acid. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such cells. Since certain modifications may occur in successive generations, either by mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Host cells can harbor heterologous nucleic acid transiently, for example, on an extrachromosomal heterologous expression vector, or stably, for example, by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing CD20-PD1 binding molecules, the host cell may be a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., HepG2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Engineered variants include, for example, glycan profile modifications and / or site-specific integration site derivatives.

[0029] 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 (e.g., another Fc domain) on the second polypeptide chain, or (d) comprises any combination of (a), (b), and (c) above. A monomer can associate with another monomer through a pair of dimerization moieties (e.g., an Fc domain). In some embodiments, the association between one or more monomers is stabilized through a hinge sequence or other portion of the Fc domain. Thus, a monomer of the present disclosure can associate with another monomer to form a dimer. A dimer can be a homodimer, where each constituent monomer is identical, or a heterodimer, where 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, e.g., one or more light chains of one or more Fab domains. Thus, a "dimer" of two monomers may contain more than two polypeptide chains, e.g., three, four or more polypeptide chains, and reference to a monomer or dimer is not intended to imply a temporal order of association between the polypeptide chains.

[0030] Monovalent: As used herein, the term "monovalent" 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. A CD20-PD1 binding molecule may be monovalent with respect to one type of moiety (e.g., a PD1 agonist moiety) or bivalent with respect to another type of moiety (e.g., a CD20 targeting moiety).

[0031] 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, the term "multivalent" means that the CD20-PD1 binding molecule has 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 may be multivalent with respect to one type of moiety (e.g., a CD20 targeting moiety) and monovalent with respect to another type of moiety (e.g., a PD1 agonist moiety).

[0032] Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, where the two or more regions are linked to produce a functional polypeptide or two or more nucleic acid sequences, e.g., to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0033] PD1 agonist moiety: The term "PD1 agonist moiety" refers to any molecule or portion thereof that can bind to and stimulate PD1. In some embodiments, the PD1 agonist moiety comprises an extracellular domain of programmed death ligand 1 (PDL1) or a PD1-binding portion thereof, preferably an amino acid sequence having at least 70% sequence identity with a mammalian PDL1 (e.g., human PDL1 or mouse PDL1). In other embodiments, the PD1 agonist moiety comprises an extracellular domain of programmed death ligand 2 (PDL2) or a PD1-binding portion thereof, preferably an amino acid sequence having at least 70% sequence identity with a mammalian PDL2 (e.g., human or mouse PDL2). The extracellular domains of PDL1 and PDL2 are sometimes known as the "PDL1 ectodomain" and the "PDL2 ectodomain," respectively. The terms "PDL1 ectodomain" and "PDL2 ectodomain" are used for convenience herein to refer not only to the PDL1 and PDL2 ectodomains, but also to fragment and variant sequences that additionally have PD1 binding activity. Thus, reference herein to the terms "PDL1 ectodomain" and "PDL2 ectodomain" is intended to encompass the PD1-binding portions of the PDL1 and PDL2 ectodomains, as well as variants thereof that have PD1 binding function, e.g., amino acid sequences that have at least 70% or more sequence identity to PD1 or PD2 and retention of PDL1 binding.

[0034] The CD20-PD1 binding molecule can comprise 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 mouse PDL1 ectodomain containing a C113S substitution.

[0035] PD1 agonist moieties are 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.

[0036] Subject: The term "subject" includes human 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" and "subject" are used interchangeably herein.

[0037] Treat, Treatment, Treating: As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of a disorder described herein, the amelioration of one or more symptoms (preferably one or more discernible symptoms) of a condition or disorder described herein, or the prevention of a condition or disorder described herein, e.g., an autoimmune or inflammatory condition or disorder, resulting from the administration of a molecule or composition (e.g., one or more CD20-PD1 binding molecules of the present disclosure). In certain embodiments, the terms "treat", "treatment" and "treating" refer to the amelioration of at least one measurable physical parameter of a disorder (e.g., an autoimmune disorder), which is not necessarily discernible by the patient. In other embodiments, the terms "treat", "treatment" and "treating" refer to the inhibition of progression or development of a disorder, either physically (e.g., by stabilization of a discernible symptom), physiologically (e.g., by stabilization of a physical parameter), or both.

[0038] Universal light chain: The term "universal light chain" as used herein in connection with a targeting moiety refers to a light chain polypeptide that can pair with a heavy chain region of a targeting moiety and can also pair with other heavy chain regions. A universal light chain is also known as a "common light chain."

[0039] VH: The term "VH" refers to the variable region of an antibody immunoglobulin heavy chain, including the heavy chain of an scFv or Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an scFv or Fab.

[0040] 6.2.CD20-PD1 binding molecule 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.

[0041] The CD20-PD1 binding molecules of the present disclosure typically comprise or consist of a CD20-PD1 monomer comprising one or more CD20 targeting moieties and / or one or more PD1 agonist moieties. The CD20-PD1 binding molecules of the present disclosure comprise a protein comprising at least one CD20 targeting moiety, at least one PD1 agonist moiety, at least one dimerization moiety, and optionally one or more linker moieties separating the one or more moieties in the protein.

[0042] 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 at the N-terminus of the dimerization moiety, the CD20-PD1 monomer thus has an N-terminus to C-terminus 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 at the C-terminus of the dimerization moiety, the CD20-PD1 monomer thus has an N-terminus to C-terminus orientation of dimerization moiety-PD1 agonist moiety-CD20 targeting moiety.

[0043] In some embodiments, e.g., in embodiments where the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain 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 ectodomain of PDL1 or a PD1-binding portion thereof, ii) the PD1 agonist moiety is not at the N-terminus of the VH of the anti-CD20 Fab, iii) the PD1 agonist moiety is not at the C-terminus of the Fc domain, iv) the protein is monovalent with respect to the CD20 targeting moiety and / or the PD1 agonist moiety, v) the protein is asymmetric, vi) the protein comprises an Fc heterodimer, or any combination of two or more of the foregoing (i)-(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 ectodomain of PDL1 or a PD1-binding portion thereof). In some embodiments, the CD20-PD1 binding molecule has feature ii) (i.e., the PD1 agonist moiety is not 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 moiety is not 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 with respect to the CD20 targeting moiety and / or the PD1 agonist moiety). 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 comprises 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 foregoing features. For example, in some embodiments, the CD20-PD1 binding molecules disclosed herein have feature ii) (i.e., the PD1 agonistic portion is not at the N-terminus of the VH of the anti-CD20 Fab) and feature iii) (i.e., the PD1 agonistic portion is not at the C-terminus of the Fc domain).

[0044] Exemplary dimerization moieties are described in Section 6.5 and include an Fc domain that confers homodimerization or heterodimerization capability to the CD20-PD1 binding molecule. A CD20-PD1 binding molecule may be composed of one or more polypeptides. In some embodiments, a CD20-PD1 binding molecule is composed of multiple (e.g., two) monomers that include at least one CD20 targeting moiety and / or at least one PD1 agonist moiety, and in some embodiments also includes a dimerization moiety. In some embodiments, a CD20-PD1 binding molecule of the present disclosure is composed of two monomers, optionally associated with one or more additional polypeptide chains (e.g., a polypeptide chain that includes a light chain of an anti-CD20 Fab moiety). The monomers may be identical, thereby forming a homodimer, or different, thereby forming a heterodimer. The dimerization moieties of each monomer of a CD20-PD1 binding molecule may be configured to dimerize together. Exemplary dimerization moieties are described in Section 6.5.

[0045] The one or more CD20 targeting moieties and the one or more PD1 agonist moieties can be included in the same arm of a CD20-PD1 binding molecule (e.g., if the CD20 targeting moiety comprises an anti-CD20 Fab and the PD1 agonist moiety comprises a PDL1-based PD1 agonist moiety, then the variable heavy or variable light chain of the anti-CD20 Fab and the PDL1-based PD1 agonist moiety are on the same polypeptide chain) or can be included in different arms of a bispecific CD20-PD1 agonist (e.g., if the CD20 targeting moiety comprises an anti-CD20 Fab and the PD1 agonist moiety comprises a PDL1-based PD1 agonist moiety, then the variable heavy or variable light chain of the anti-CD20 Fab and the PDL1-based PD1 agonist moiety are on different polypeptide chains). Exemplary configurations of CD20-PD1 binding molecules of the present disclosure are disclosed, inter alia, in Figures 1A-1L and in numbered embodiments 31-106.

[0046] A CD20-PD1 binding molecule can be monovalent with respect to the CD20 targeting moiety (i.e., has a single CD20 targeting moiety) or multivalent with respect to the CD20 targeting moiety (i.e., has multiple CD20 targeting moieties). Similarly, a CD20-PD1 binding molecule can be monovalent with respect to the PD1 agonist moiety (i.e., has a single PD1 agonist moiety) or multivalent with respect to the PD1 agonist moiety (i.e., has multiple PD1 agonist moieties). In some embodiments, a CD20-PD1 binding molecule is bivalent with respect to the CD20 targeting moiety (i.e., has two CD20 targeting moieties). When a CD20-PD1 binding molecule is multivalent with respect to 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.

[0047] In some embodiments, a CD20-PD1 binding molecule can comprise one or more linker sequences connecting various components of its one or more polypeptide chains, for example, (1) a CD20 targeting moiety or portion thereof (e.g., a heavy or light chain of an anti-CD20 Fab) and a PD1 agonist moiety or 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.

[0048] Most CD20-PD1 binding molecules are multimeric due to the association of dimerization moieties (e.g., Fc domains) that are configured to associate with each other. CD20-PD1 binding molecules may comprise two, three, four, or more polypeptide chains, some of which associate via dimerization moieties and others that associate via VH-VL interactions. For convenience and illustrative purposes only, the present disclosure generally refers to a polypeptide that comprises a CD20 targeting moiety, a PD1 agonist moiety, and / or a dimerization moiety (e.g., a first Fc domain) that can associate with another polypeptide chain that comprises a CD20 targeting moiety, a PD1 agonist moiety, and / or a corresponding dimerization moiety (e.g., a second Fc domain), respectively, as a "monomer." A monomer may comprise one, two, three, or more polypeptide chains. For example, in one embodiment, a monomer may be comprised of (a) a first polypeptide chain comprising an anti-CD20 VH, a PD1 agonist moiety, and an Fc domain, and (b) a second polypeptide chain comprising a VL capable of pairing with the anti-CD20 VH. In another embodiment, a monomer may be comprised 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.

[0049] Below are some illustrative examples of monomers of the present disclosure, listed in N-terminal to C-terminal orientation: The individual elements of each monomer are described in detail herein, for example, in the subsections and numbered embodiments below.

[0050] (1) Exemplary Monomer 1: CD20 targeting moiety-optional linker-dimerization moiety (see, e.g., Figures 1A, 1E, 1F, 1G, and 1H, left monomer).

[0051] (2) Exemplary Monomer 2: PD1 agonist moiety-optional linker-dimerization moiety (see, e.g., FIG. 1A, right-hand monomer). (3) Exemplary Monomer 3: Optional Linker-Dimerization Moiety (see, e.g., Figures 1B, 1C, and 1D, left monomer).

[0052] (4) Exemplary Monomer 4: PD1 agonist moiety-optional linker-CD20 targeting moiety-optional linker-dimerization moiety (see, e.g., the right-hand monomer in FIG. 1B and FIG. 1E, both monomers in FIG. 1I).

[0053] (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, both monomers).

[0054] (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, both monomers).

[0055] (7) Exemplary Monomer 7: PD1 agonist moiety-optional linker-PD1 agonist moiety-optional linker-dimerization moiety (see, e.g., Figure 1H, right-hand monomer).

[0056] (8) Exemplary Monomer 8: CD20 targeting moiety-optional linker-dimerization moiety-optional linker-CD20 targeting moiety (see, e.g., FIG. 1K, left-hand monomer).

[0057] (9) Exemplary Monomer 9: PD1 agonist moiety-optional linker-dimerization moiety-optional linker-PD1 agonist moiety (see, e.g., Figure 1K, right-hand monomer).

[0058] In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 1 and exemplary monomer 2 (see, e.g., FIG. 1A). In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 3 and exemplary monomer 4 (see, e.g., FIG. 1B).

[0059] In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 3 and exemplary monomer 5 (see, e.g., FIG. 1C). In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 3 and exemplary monomer 6 (see, e.g., FIG. 1D).

[0060] In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 1 and exemplary monomer 4 (see, e.g., FIG. 1E). In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 1 and exemplary monomer 5 (see, e.g., FIG. 1F).

[0061] In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 1 and exemplary monomer 6 (see, e.g., FIG. 1G). In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 1 and exemplary monomer 7 (see, e.g., FIG. 1H).

[0062] In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising two monomers according to exemplary monomer 4 (see, e.g., FIG. 1I). In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising two monomers according to exemplary monomer 5 (see, e.g., FIG. 1J).

[0063] In some embodiments, the disclosure provides a CD20-PD1 binding molecule comprising exemplary monomer 8 and exemplary monomer 9 (see, e.g., FIG. 1K). In some embodiments, the disclosure provides a CD20-PD1 binding molecule that includes two monomers according to exemplary monomer 6 (see, e.g., FIG. 1L).

[0064] 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 having a heavy chain variable region and the other polypeptide chain(s) having a light chain variable region. The CD20 targeting moiety may comprise 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 comprise, for example, from N-terminus to C-terminus, the heavy chain variable domain of the CD20 targeting moiety-optional linker-PD1 agonist moiety-optional linker-dimerization moiety, and polypeptide B may comprise the light chain variable domain of the CD20 targeting moiety. When the monomer is bivalent with respect to the CD20 targeting moiety, the monomer may comprise a third polypeptide chain (polypeptide C) comprising another light chain variable domain of the CD20 targeting moiety.

[0065] Alternatively, the CD20 targeting moiety can be in the form of an 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 provided below.

[0066] 6.2.1. Biochemical Characterization of CD20-PD1 Binding Molecules In vivo, large antibody complexes may be rapidly cleared by phagocytosis, resulting in reduced antibody efficacy. Large complexes may also increase the immunogenicity of therapeutic antibodies. See, e.g., WO2020047067A1. During manufacturing, aggregation is a common problem that compromises the quality, safety, and efficacy of antibodies. The CD20-PD1 binding molecules of the present disclosure may be less prone to aggregation in vivo or ex vivo, e.g., compared to the parent antibody from which the CD20 targeting moiety was derived and / or compared to other antibody formats that include a CD20 targeting moiety and a PD1 agonist moiety. Thus, in some embodiments, the CD20-PD1 binding molecules of the present disclosure aggregate during recombinant production in mammalian cell lines 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 molecules can be determined, for example, by size-exclusion ultra-performance liquid chromatography. The majority of the CD20-PD1 binding molecules showed more than 85% monomeric species without additional size-exclusion chromatography (SEC) (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) increased to 99% after two column purifications including a SEC step (see section 7.2.2).

[0067] The CD20-PD1 binding molecules of the present disclosure also exhibit good thermal stability. High thermal stability and low aggregation tendency facilitates the manufacture and storage of the antibody and promotes a long serum half-life. Carter and Merchant, 1997, Curr Opin Biotechnol, 8(4):449-454. Thermal stability may 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 had similar thermal stability as measured by DSF, with melting temperature 1 (Tm1), which represents the initial unfolding midpoint of the protein, being approximately 60° C. (see Section 7.2.2).

[0068] 6.3.CD20 targeting moiety Incorporation of a CD20 targeting moiety in the CD20-PD1 binding molecules of the present disclosure, in some embodiments, provides for the delivery of high concentrations of the 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, as well as for the treatment of graft-versus-host disease (GVHD). In some embodiments, in addition to facilitating the local delivery of the PD1 agonist moiety, the anti-CD20 moiety provides an additional therapeutic route for such autoimmune diseases.

[0069] In certain embodiments of the present disclosure, each CD20 targeting moiety of the CD20-PD1 binding molecule comprises an antigen-binding domain of an anti-CD20 antibody. In some embodiments, the CD20-PD1 binding molecule of the present disclosure comprises a single CD20 targeting moiety (e.g., in embodiments in which the CD20-PD1 binding molecule is monovalent with respect to the CD20 targeting moiety, a CD20 targeting moiety on the first monomer or on the second monomer). In some embodiments, the CD20-PD1 binding molecule of the present disclosure comprises two CD20 targeting moieties (e.g., in embodiments in which the CD20-PD1 binding molecule is bivalent with respect to the CD20 targeting moiety, a first CD20 targeting moiety on the first monomer and a second CD20 targeting moiety on the second monomer, or both the first and second CD20 targeting moieties can be on either the first monomer or the second monomer). In such embodiments, the two CD20 targeting moieties can be the same or different. If different, the two CD20 targeting moieties may be orthogonal, may bind to distinct epitopes of CD20, and / or may be non-competitive.

[0070] In some embodiments, the CD20 targeting moiety comprises the 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, ibritumomab ituxetan, 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 six CDR sequences of the reference CD20 antibody. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the reference CD20 antibody and the light chain CDR sequences of the universal light chain. In further aspects, the CD20 targeting moiety comprises a VH that comprises the amino acid sequence of the VH of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety further comprises a VL that comprises the amino acid sequence of the VL of the reference CD20 antibody. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0071] In other embodiments, the CD20 targeting moiety comprises an antigen-binding domain that binds to the same CD20 epitope as rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, or veltuzumab and / or competes with them for binding to CD20. Assays for measuring antibody competition are known in the art. For example, a sample of CD20 can be bound to a solid support. A first antibody and a second antibody are then added. One of the two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to separate and distinct sites on CD20, the labeled antibody will bind at the same level regardless of whether the unlabeled antibody is present. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete and the amount of labeled antibody that binds to the antigen will be reduced. When unlabeled antibodies are present in excess, the labeled antibody will bind very little, if at all. In some embodiments, a competing 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%. Details of the procedures for carrying out 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 performed by using purified antibodies. A standard curve can be established by titrating one antibody against itself, i.e., by using the same antibody as both the label and the competitor. The ability of the unlabeled competing antibody to inhibit the binding of the labeled antibody to the plate is titrated. 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 using a real-time, label-free biolayer interferometry assay, for example on an Octet HTX biosensor platform (Pall ForteBio Corp.).

[0072] 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, Fv fragment as described in Section 6.3.1.1, or an scFv as described in Section 6.3.1.2.

[0073] The CD20 targeting moiety can be incorporated into a CD20-PD1 binding molecule having any of the configurations described herein. A 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 described in Section 6.2, the CD20 targeting moiety can be incorporated into any one 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 detailed in Section 6.2.

[0074] 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 immunoglobulin molecule of the IgG class, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include VH (or V H ) fragment, VL (or V L ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0075] 6.3.1.1.Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the CD20-PD1 binding molecules of the present disclosure, the Fab domain is typically recombinantly expressed as part of the CD20-PD1 binding molecule.

[0076] The Fab domain can include constant and variable region sequences from any suitable species, and thus can be murine, chimeric, human, or humanized. In some embodiments, the variable and / or constant domain sequences are derived from a known anti-CD20 antibody. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, and veltuzumab.

[0077] In some embodiments, the CD20 targeting moiety comprises a Fab that binds to the same CD20 epitope as the Fabs of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, or veltuzumab (each a "reference CD20 antibody") and / or competes with these Fabs for binding to CD20. In further embodiments, the CD20 targeting moiety comprises a CDR having the CDR sequences of the reference CD20 antibody. In some embodiments, the CD20 targeting moiety comprises all six CDR sequences of the reference CD20 antibody. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the reference CD20 antibody and the light chain CDR sequences of the universal light chain. In further aspects, the CD20 targeting moiety comprises a VH that comprises the amino acid sequence of the VH of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety further comprises a VL that comprises the amino acid sequence of the VL of the reference CD20 antibody. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0078] A Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0079] 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 correct association of Fab domains belonging to the same ABD and minimize aberrant pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategy shown in Table 1 below can be used:

[0080] [Table 1]

[0081] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is promoted by swapping the VL and VH domains of the Fab with one another, or swapping the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0082] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. 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 other components of the Fab.

[0083] In one embodiment, the one or more amino acid modifications are restricted to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0084] In one embodiment, the modifications introduced in the VH and CH1 domains and / or the VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of different interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of structural and chemical correspondence between the two interacting surfaces.

[0085] In one embodiment, the one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, the one or more introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0086] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and a 114A and 137K substitution in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0087] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0088] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, the 39K, 62E modifications are introduced in the VH domain, the H172A, F174G modifications are introduced in the CH1 domain, the 1R, 38D, (36F) modifications are introduced in the VL domain, and the L135Y, S176W modifications are introduced in the CL domain. In another embodiment, the 39Y modification is introduced in the VH domain and the 38R modification is introduced in the VL domain.

[0089] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0090] Fab domains can also be modified by replacing the CH1 and CL domains 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 a T cell receptor and the CL domain with the b domain of a T cell receptor, 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.

[0091] Alternatively, or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, a VL of a common light chain (also referred to as a universal light chain) can be used in each FabVL region of the CD20-PD1 binding molecules of the present disclosure. In various embodiments, using a common light chain as described herein reduces the number of incorrect species of the CD20-PD1 binding molecules compared to using the original cognate VL. In various embodiments, the VL domain of the CD20-PD1 binding molecule is identified from a monospecific antibody that comprises a common light chain. In various embodiments, the VH region of the CD20-PD1 binding molecule comprises a limited human light chain repertoire or human heavy chain variable gene segments that have been rearranged in vivo in mouse B cells previously engineered to express a single human light chain that is cognate to a human heavy chain, and in response to exposure to an antigen of interest, generates an antibody repertoire that comprises one of two possible human VLs or multiple human VHs that are cognate to one, which antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatically mutated (e.g., affinity matured) forms. See, e.g., U.S. Patent No. 10,412,940.

[0092] 6.3.1.2.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as single-chain polypeptides, and retain the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding. Examples of linkers suitable for linking the VH and VL chains of an scFv are the linkers identified in Section 6.7.

[0093] As used herein, unless otherwise specified, an scFv may have the VL variable region and the VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0094] The scFv can comprise VH and VL sequences from any suitable species, such as mouse, human, or humanized VH and VL sequences. In some embodiments, the scFv can comprise VH and VL sequences from a known anti-CD20 antibody. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, and veltuzumab.

[0095] In some embodiments, the CD20 targeting moiety comprises an scFv that binds to the same CD20 epitope as and / or competes for binding to CD20 with an scFv derived from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, or veltuzumab.

[0096] To generate nucleic acids encoding scFvs, DNA fragments encoding the VH and VL can be operably linked to another fragment encoding a linker, e.g., a fragment encoding any of the linkers described in Section 6.7 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:1)), and the VH and VL sequences can be expressed as VL and VH regions joined 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).

[0097] 6.4. PD1 Agonist Moiety In certain embodiments of the present disclosure, the PD1 agonist moiety of the CD20-PD1 binding molecule comprises 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 comprises a single PD1 agonist moiety (e.g., a PD1 agonist moiety on the first monomer or on the second monomer in embodiments where the CD20-PD1 binding molecule is monovalent with respect to the PD1 agonist moiety). In some embodiments, the CD20-PD1 binding molecule of the present disclosure comprises two PD1 agonist moieties (e.g., a first PD1 agonist moiety on the first monomer and a second PD1 agonist moiety on the second monomer, or both a first PD1 agonist moiety and a second PD1 agonist moiety on either the first monomer or the second monomer). In such embodiments, the two PD1 agonist moieties may be the same or different. If different, the two PD1 agonist moieties may interact with PD1 differently (eg, with different affinities).

[0098] The PD1 agonist moiety can be incorporated into a CD20-PD1 binding molecule having any of the configurations described herein. A 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 described in Section 6.2, the PD1 agonist moiety can be incorporated into any one of the exemplary monomers 2, 4, 5, 6, 7, and 9. Exemplary CD20-PD1 binding molecules incorporating one or more of the exemplary monomers 2, 4, 5, 6, 7, and 9 are described in detail in Section 6.2. In some embodiments, the PD1 agonist moiety is a PDL1-based agonist moiety. In other embodiments, the wet PD1 agonist is a PDL2-based agonist moiety.

[0099] 6.4.1. PDL1-Based PD1 Agonist Moieties PDL1 plays an important role in the induction and maintenance of immune tolerance to self. 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, in which at least one PD1 agonist moiety comprises an amino acid sequence that includes or is homologous to a PDL1 amino acid sequence described herein. Such PD1 agonist moieties are referred to herein as "PDL1-based PD1 agonist moieties" or similar terms.

[0100] The human PDL1 protein is synthesized as a 290 amino acid precursor polypeptide from which 18 amino acids are removed to generate mature hPDL1, with amino acids 19-238 (numbering based on the precursor protein) forming the hPDL1 extracellular domain, or ectodomain. The sequence of human PDL1 has Uniprot identifier Q9NZQ7 (uniprot.org / uniprot / Q9NZQ7). The sequence of mouse PDL1 has Uniprot identifier Q9EP73 (uniprot.org / uniprot / Q9EP73).

[0101] The precursor human PDL1 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold):

[0102] [ka]

[0103] Mouse PDL1 polypeptide is synthesized as a 290 amino acid precursor polypeptide from which 18 amino acids are removed to generate mature mPDL1. Amino acids 19-239 (numbering based on the precursor protein) form the mPDL1 extracellular domain, or ectodomain. The precursor mouse PDL1 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold):

[0104] [ka]

[0105] In some embodiments, the PD1 agonist moiety is a PDL1-based agonist moiety that comprises an amino acid sequence that comprises at least 70% sequence identity, 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, with the PD1-binding portion of a mammalian (e.g., human or mouse) PDL1, or with the entire ectodomain of a mammalian (e.g., human or mouse) PDL1. In certain aspects, the PD1-binding portion of PDL1 comprises the IgV domain of human PDL1 or mouse PDL1. In certain embodiments, the PD1-binding portion of PDL1 comprises amino acids 19-134 of human PDL1 or amino acids 19-134 of mouse PDL1.

[0106] In certain embodiments, the PDL1-based PD1 agonist moiety 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%, at least 99%) sequence identity with the ectodomain of PDL1 or a PD1-binding portion thereof, and one or more amino acid substitutions compared to wild-type PDL1. In some embodiments, the one or more amino acid substitutions increase the stability of the PDL1-based PD1 agonist moiety. For example, in some embodiments, mPDL1 comprises the amino acid substitution C113S (numbering based on the precursor protein).

[0107] In some embodiments, the PDL1-based PD1 agonist moiety is fused directly or indirectly to the CD20 targeting moiety, optionally via a linker (e.g., as described in Section 6.7). If present on the same monomer, the PDL1-based PD1 agonist moiety can be at the N-terminus or C-terminus of the CD20 targeting moiety. If the PDL1-based PD1 agonist moiety is "directly" fused to the CD20 targeting moiety, the PDL1-based PD1 agonist moiety and the CD20 targeting moiety are adjacently located on the same monomer, separated only by a linker, if present. If the PDL1-based PD1 agonist moiety is "indirectly" fused to the CD20 targeting moiety, the PDL1-based PD1 agonist moiety and the CD20 targeting moiety are separated by one or more other domains (e.g., dimerization moieties) on the same monomer, or are located on separate monomers.

[0108] 6.4.2. PDL2-Based PD1 Agonist Moieties 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 moiety comprises an amino acid sequence that includes or is homologous to a PDL2 amino acid sequence described herein. Such PD1 agonist moieties are referred to herein as "PDL2-based PD1 agonist moieties" or similar terms.

[0109] The human PDL2 protein is synthesized as a 273 amino acid precursor polypeptide from which 19 amino acids are removed to generate mature hPDL2, with amino acids 20-220 (numbering based on the precursor protein) forming the hPDL2 extracellular domain, or ectodomain. The sequence of human PDL2 has Uniprot identifier Q9BQ51 (uniprot.org / uniprot / Q9BQ51). The sequence of mouse PDL2 has Uniprot identifier Q9WUL5 (uniprot.org / uniprot / Q9WUL5).

[0110] The precursor human PDL2 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold):

[0111] [ka]

[0112] Mouse PDL2 polypeptide is synthesized as a 247 amino acid precursor polypeptide from which 19 amino acids are removed to generate mature mPDL2. Amino acids 20-221 (numbering based on the precursor protein) form the mPDL2 extracellular domain, or ectodomain. The precursor mouse PDL2 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold):

[0113] [ka]

[0114] In some embodiments, the PD1 agonist moiety is a PDL2-based agonist moiety that comprises an amino acid sequence that comprises at least 70% sequence identity, 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, with the PD1-binding portion of a mammalian (e.g., human or mouse) PDL2, or with the entire ectodomain of a mammalian (e.g., human or mouse) PDL1. In certain aspects, the PD1-binding portion of PDL2 comprises the IgV domain of human PDL2 or mouse PDL2. In certain embodiments, the PD1-binding portion of PDL2 comprises amino acids 20-121 of human PDL2 or amino acids 20-121 of mouse PDL2.

[0115] 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%, at least 99%) sequence identity to the ectodomain of PDL2 or a PD1-binding portion thereof, and one or more amino acid substitutions compared to wild-type PDL2.

[0116] In some embodiments, the PDL2-based PD1 agonist moiety is fused directly or indirectly to the CD20 targeting moiety, optionally via a linker (e.g., as described in Section 6.7). If present on the same monomer, the PDL2-based PD1 agonist moiety can be at the N-terminus or C-terminus of the CD20 targeting moiety. If the PDL2-based PD1 agonist moiety is "directly" fused to the CD20 targeting moiety, the PDL2-based PD1 agonist moiety and the CD20 targeting moiety are adjacently located on the same monomer, separated only by a linker, if present. If the PDL2-based PD1 agonist moiety is "indirectly" fused to the CD20 targeting moiety, the PDL2-based PD1 agonist moiety and the CD20 targeting moiety are separated by one or more other domains (e.g., dimerization moieties) on the same monomer, or are located on separate monomers.

[0117] 6.5. Dimerization moiety 6.5.1.Fc Domain In some embodiments, the CD20-PD1 binding molecules and CD20-PD1 monomers of the disclosure comprise one or more dimerization moieties, e.g., one or more dimerization moieties that are or include an Fc domain. In certain embodiments, the CD20-PD1 monomers of the disclosure comprise a single dimerization moiety (e.g., a single Fc domain) and / or the CD20-PD1 binding molecules of the disclosure comprise two dimerization moieties (e.g., two Fc domains that can associate to form an Fc region).

[0118] The CD20-PD1 binding molecules and CD20-PD1 monomers of the present disclosure can comprise an Fc domain, or a pair of Fc domains that associate 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.

[0119] The CD20 targeting moiety and / or the PD1 agonist moiety may be fused to the N-terminus or C-terminus of the IgGFc domain. One embodiment of the present disclosure is directed to a dimer comprising two Fc fusion polypeptides created by fusing one or more CD20 targeting moieties and / or PD1 agonist moieties to an Fc domain, e.g., by fusing both a CD20 targeting moiety and a PD1 agonist moiety to an Fc domain that can form a CD20-PD1 monomer that can homodimerize upon expression, or by fusing one or more CD20 targeting moieties and / or one or more PD1 agonist moieties to a first Fc domain and one or more CD20 targeting moieties and / or one or more PD1 agonist moieties to a second Fc domain, forming two different CD20-PD1 monomers that can heterodimerize upon expression. Dimers can be produced, for example, by inserting a gene fusion encoding the fusion protein(s) into an appropriate expression vector, expressing the gene fusion(s) in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein(s) to assemble in the same manner as antibody molecules, whereupon interchain bonds form between the Fc portions to produce dimers.

[0120] The Fc domain that can be incorporated into the CD20-PD1 monomer can be derived from any suitable class of antibody, 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.

[0121] The two Fc domains within the Fc region may be identical or different from one another. In natural antibodies, the Fc domains are typically identical, however, for purposes of producing multispecific binding molecules, such as the CD20-PD1 binding molecules of the present disclosure, the Fc domains may be advantageously different to allow for heterodimerization, as described in Section 6.5.1 below.

[0122] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG is composed of two heavy chain constant domains (CH2 and CH3), while the domain of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0123] In the CD20-PD1 binding molecules of the disclosure, the Fc region, and / or the Fc domains therein, can comprise heavy chain constant domains derived from one or more different classes of antibodies, e.g., one, two, or three different classes.

[0124] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG1. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG2. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3.

[0125] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4. In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain.

[0126] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from an IgG and the CH4 domain derived from an IgM. It will be understood that the heavy chain constant domains for use in producing the Fc region of the CD20-PD1 binding molecules of the present disclosure may include variants of the naturally occurring constant domains described above. Such variants may include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will 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.

[0127] IgM and IgA naturally occur in humans as covalent multimers of a common H2L2 antibody unit. IgM exists as a pentamer when the J chain is incorporated and as a hexamer when the J chain is lacking. IgA exists as a monomer and a dimeric form. The heavy chains of IgM and IgA have an 18 amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between the heavy chains in the polymer and is believed to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the CD20-PD1 binding molecules of the present disclosure do not contain a tail.

[0128] The Fc domain incorporated into the CD20-PD1 binding molecules of the present disclosure may contain one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, altered disulfide bond structure, or altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.5.1.1.

[0129] 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 preferential pairing of non-identical Fc domains over identical Fc domains. Heterodimerization allows for the production of CD20-PD1 binding molecules in which different polypeptide components are connected to each other by Fc regions that contain Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.5.1.2.

[0130] It will be appreciated that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or can be combined with other modifications to alter the properties of the CD20-PD1 binding molecule.

[0131] 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.

[0132] In a particular embodiment, the Fc receptor is an Fc gamma receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fc gamma receptor, more specifically, human Fc gamma RIIIa, Fc gamma RI or Fc gamma RIIa, most specifically, human Fc gamma RIIIa. In one embodiment, the effector function is one or more selected from the group of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0133] In one embodiment, the Fc domain (e.g., the Fc domain of a CD20-PD1 monomer) or Fc region (e.g., one or both 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 of E233, L234, L235, G237, N297, A330, P331, and P329 (numbering according to 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 of L234, L235, and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to 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, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG").

[0134] In some embodiments, the Fc domain or Fc region comprises amino acid substitutions at positions L234, L235, G237, A330, and P331 (numbering according to the Kabat EU index). In more specific embodiments, the amino acid substitutions are L234A, L235E, G237A, A330S, and P331S (numbering according to the Kabat EU index).

[0135] 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 particular 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 and second Fc domains 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) (Kabat EU index numbering). In another specific embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235E, G237A, A330S, and P331S (numbering according to Kabat EU index), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), the glycine residue at position 237 is substituted with an alanine residue (G237A), the alanine residue at position 330 is substituted with a serine residue (A330S), and the proline residue at position 331 is substituted with a serine residue (P331S) (numbering according to Kabat EU index).

[0136] In one embodiment, the Fc domain is an IgG1 Fc domain, e.g., a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 that includes D265A and N297A mutations (EU numbering) to reduce effector function. In other embodiments, the IgG1 Fc domain is a variant IgG1 that includes L234A, L235E, G237A, A330S, and P331S mutations (numbering according to Kabat EU index) to provide an effector-null IgG1 (IgG1EN). The amino acid substitutions L234A, L235E, and G237A reduce binding to FcγRI, FcγRIIa, and FcγRIII, while the substitutions A330S and P331S reduce C1q-mediated complement binding.

[0137] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table 2 below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below:

[0138] [Table 2-1]

[0139] [Table 2-2]

[0140] [Table 2-3]

[0141] In certain embodiments, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, and is sometimes referred to herein as IgG4s or hIgG4s.

[0142] For heterodimeric Fc regions, it is possible to incorporate combinations of the variant IgG4 Fc sequences mentioned above, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or the bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or the bolded portion thereof).

[0143] 6.5.1.2.Fc Heterodimerization Variants Certain CD20-PD1 binding molecules, unlike native immunoglobulins, involve dimerization between two Fc domains that are operably linked to non-identical N-terminal regions (e.g., one Fc domain is connected to a Fab and the other Fc domain is connected to a PD1 agonist moiety). Inefficient heterodimerization of the two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule, presenting a purification challenge. Various approaches 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 disclosure, for example, as disclosed in EP 1870459 A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493 A1; and PCT Publication No. WO 2009 / 089004 A1.

[0144] The present disclosure provides CD20-PD1 binding molecules that comprise Fc heterodimers, i.e., Fc regions that comprise heterologous, non-identical Fc domains. Typically, each Fc domain in an Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the preceding section.

[0145] Heterodimerization of two different heavy chains at the CH3 domain will result in the desired CD20-PD1 binding molecule, whereas homodimerization of the same heavy chain will reduce the yield of the desired CD20-PD1 binding molecule. Thus, in a preferred embodiment, the polypeptides that assemble to form the CD20-PD1 binding molecules of the present disclosure will contain a CH3 domain with a modification that favors heterodimeric association compared to an unmodified Fc domain.

[0146] 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, which includes a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, allowing the protrusion to be positioned within the cavity, to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusion are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0147] Thus, in some embodiments, amino acid residues in the CH3 domain of a first subunit of an Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protuberance in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of the second subunit, and amino acid residues in the CH3 domain of a second subunit of an Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit into which the protuberance in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0148] In a specific such embodiment, in the first Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), 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) (particularly, the serine residue at position 354 is replaced by a cysteine ​​residue). The second Fc domain additionally has the tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

[0149] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote association of the first and second Fc domains of the Fc region.

[0150] As an alternative or in addition to the use of modified Fc domains to promote heterodimerization, the Fc domain may be modified to allow for purification strategies that allow for the selection of Fc heterodimers. In one such embodiment, one polypeptide comprises a modified Fc domain that abrogates its binding to Protein A, thus allowing for a purification method that results in a heterodimeric protein. See, e.g., U.S. Pat. No. 8,586,713. Such CD20-PD1 binding molecules comprise a first CH3 domain and a second IgCH3 domain, the first and second IgCH3 domains differing from each other by at least one amino acid, the at least one amino acid difference reducing binding of the CD20-PD1 binding molecule to Protein A compared to a corresponding CD20-PD1 binding molecule lacking the amino acid difference. In one embodiment, the first CH3 domain binds Protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates Protein A binding, e.g., a H95R modification (according to IMGT exon numbering, H435R in EU numbering). The second CH3 may further comprise a Y96F modification (Y436F according to IMGT, according to EU). This class of modifications is referred to herein as "star" mutations.

[0151] In some embodiments, the Fc may contain one or more mutations to promote heterodimerization (e.g., knob and hole mutations) and a star mutation to facilitate purification. 6.6. Stabilization part The CD20-PD1 binding molecules of the present disclosure can include a stabilizing moiety that can increase the serum half-life of the molecule in vivo. Serum half-life is often divided into an alpha phase and a beta phase. Either or both phases can be significantly improved by the addition of an appropriate stabilizing moiety. For example, a stabilizing moiety can increase the serum half-life of a CD20-PD1 binding molecule by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800, 1000% or more compared to a corresponding CD20-PD1 binding molecule that does not contain a stabilizing moiety. For purposes of the present disclosure, serum half-life can refer to half-life in humans or other mammals (e.g., mice or non-human primates).

[0152] Stabilizing moieties include polyoxyalkylene moieties (e.g., polyethylene glycol), sugars (e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc and fragments and variants thereof, transferrin, or serum albumin).

[0153] Other stabilizing moieties that may be used in the CD20-PD1 binding molecules of the present disclosure include those 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., Adnectins PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimics 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.

[0154] Thus, in some embodiments, the disclosure provides CD20-PD1 binding molecules that include a stabilizing moiety that is a polymeric sugar. Serum albumin can also participate in half-life extension through modules that have the ability to non-covalently interact with albumin. Thus, the CD20-PD1 binding molecules of the present disclosure can include an albumin binding protein as a stabilizing moiety. The albumin binding protein may 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 from certain bacteria. For example, streptococcal protein G contains several small albumin binding domains composed of about 50 amino acid residues (6 kDa). Additional examples of serum albumin binding proteins, such as those described in US Publication Nos. 2007 / 0178082 and 2007 / 0269422. Fusion of albumin binding domains to proteins results in significantly extended half-lives (see Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76).

[0155] In other embodiments, the stabilizing moiety is human serum albumin, hi other embodiments, the stabilizing moiety is transferrin. In some embodiments, the stabilizing moiety is an Fc domain, such as any of the Fc domains described in Section 6.5.1 and its subsections, which are incorporated herein by reference. The Fc domains described in Section 6.5.1 are generally capable of dimerizing. However, for stabilization purposes, the Fc domain may be a soluble monomeric Fc domain with reduced ability to self-associate. See, for example, Helm et al., 1996, J. Biol. Chem. 271:7494-7500 and Ying et al., 2012, J Biol Chem. 287(23):19399-19408. An example of a soluble monomeric Fc domain includes amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3, as described in US Patent Publication No. 2019 / 0367611. The monomeric Fc domain may be any Ig subtype and may include additional substitutions that reduce effector function, as described in Section 6.5.1 and its subsections.

[0156] In yet other embodiments, the stabilizing moiety is a polyethylene glycol moiety or another polymer, as described below in Section 6.6.1. The stabilizing moieties can be connected 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.

[0157] Polyethylene glycol In some embodiments, the CD20-PD1 binding molecule comprises polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing moiety, such as ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and copolymers thereof. The polymer may be of any molecular weight and may be branched or unbranched.

[0158] 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, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or modification at the termini of the PEG, and can be represented by the following formula: X-O(CH2CH2O). n -1CH2CH2OH, where n is 20 to 2300, and X is H or a terminal modification, such as C 1-4 (The PEG is an alkyl group.) PEG can contain additional chemical groups necessary for the conjugation reaction resulting from the chemical synthesis of the molecule or that serve as spacers for optimal distance of the parts of the molecule. In addition, such PEG can consist of one or more PEG side chains linked together. PEGs with multiple PEG chains are called multi-armed or branched PEGs. Branched PEGs are described, for example, in European Application No. 473084A and U.S. Pat. No. 5,932,462.

[0159] One or more PEG molecules may be attached at different positions on the CD20-PD1 binding molecule, and such attachment may be accomplished by reaction with an amine, thiol, or other suitable reactive group. The amine moiety may 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.

[0160] PEGylation can be achieved by site-specific PEGylation, where a suitable reactive group is introduced into the protein to create a site where PEGylation will preferentially occur. In some embodiments, the CD20-PD1 binding molecule is modified to introduce a cysteine ​​residue at a desired position, allowing site-specific PEGylation on the cysteine. A mutation can be introduced into the coding sequence of the CD20-PD1 binding molecule of the present disclosure to generate a cysteine ​​residue. This can be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutating to cysteine ​​residues include serine, threonine, alanine, and other hydrophilic residues. Preferably, the residue mutated to cysteine ​​is a surface-exposed residue. Algorithms for predicting surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. PEGylation of cysteine ​​residues can be performed, for example, using PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, or PEG-orthopyridyldisulfide.

[0161] PEG is typically activated with a suitable activating group suitable for coupling to a desired site on a 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, JM Harris, Plenus Press, New York (1992), and Zalipsky, 1995, Advanced Drug Reviews 16:157-182.

[0162] The PEG moiety can vary widely in molecular weight and can be branched or linear. Typically, the weight average molecular weight of PEG is about 100 Daltons to about 150,000 Daltons. Exemplary weight average molecular weights of PEG include 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. Branched PEGs having any of the total molecular weights listed above can also be used. In some embodiments, the PEG has two branches. In other embodiments, the PEG has four branches. In another embodiment, the PEG is a bis-PEG (NOF Corporation, DE-200MA).

[0163] Conventional separation and purification techniques known in the art can be used to purify PEGylated CD20-PD1 binding molecules, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. Products can also be separated using SDS-PAGE. Products that can be separated include mono-, di-, tri-, poly-, and non-PEGylated CD20-PD1 binding molecules, as well as free PEG. The percentage of mono-PEG conjugates can be controlled by pooling broader fractions around the elution peak to increase the percentage of mono-PEG in the composition. Approximately 90% mono-PEG conjugates represents a good balance of yield and activity.

[0164] In some embodiments, PEGylated CD20-PD1 binding molecules will preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the biological activity associated with an unmodified CD20-PD1 binding molecule. D , k on , or k off "CD20" refers to its ability to bind to CD20, PD1, or both CD20 and PD1, as assessed by:

[0165] Linker In certain aspects, the present disclosure provides CD20-PD1 binding molecules in which two or more components of the CD20-PD1 binding molecule are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect (a) a CD20 targeting moiety and a dimerization moiety, (b) a CD20 targeting moiety and a PD1 agonist moiety, (c) a PD1 agonist moiety and a dimerization moiety, or (d) different domains within a CD20 targeting moiety (e.g., the VH and VL domains within an scFv).

[0166] The peptide linker can be in the range of 2 to 60 or more amino acids, and in certain embodiments, the peptide linker can be in the range of 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0167] In certain embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0168] In some of the aforementioned embodiments, the linker is in the range of 5 amino acids to 50 amino acids in length, e.g., 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, or 5-20 amino acids in length. In other of the aforementioned embodiments, the linker is in the range of 6 amino acids to 50 amino acids in length, e.g., 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, or 6-20 amino acids in length. In yet other of the aforementioned embodiments, the linker is in the range of 7 amino acids to 50 amino acids in length, e.g., 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, or 7-20 amino acids in length.

[0169] Charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that may be used in the CD20-PD1 binding molecules of the present disclosure include those disclosed in 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 include repeats of glycine and serine, e.g., G n S (SEQ ID NO: 12) or SG n(SEQ ID NO: 13), where 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, for example, (GGGGS) n (SEQ ID NO: 14), or which comprises a monomer or multimer of G4S repeats.

[0170] Polyglycine linkers may be suitable for use in the CD20-PD1 binding molecules of the present disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 15), five consecutive glycines (5Gly) (SEQ ID NO: 16), six consecutive glycines (6Gly) (SEQ ID NO: 17), seven consecutive glycines (7Gly) (SEQ ID NO: 18), eight consecutive glycines (8Gly) (SEQ ID NO: 19), or nine consecutive glycines (9Gly) (SEQ ID NO: 20).

[0171] 6.7.1. Hinge arrangement In some embodiments, the CD20-PD1 binding molecules of the present disclosure include a linker that is a hinge region. In particular, a hinge can be used to connect a CD20 targeting moiety, e.g., a Fab domain, to a dimerization domain, e.g., an Fc domain. The hinge region can be a natural hinge region or a modified hinge region. Hinge regions are typically found at the N-terminus of an Fc region. The term "hinge region", unless otherwise indicated by context, refers to a monomeric hinge domain in the context of a single or monomeric polypeptide chain, and to a naturally or non-naturally occurring hinge sequence that can include two associated hinge sequences on separate polypeptide chains in the context of a dimeric polypeptide (e.g., a homodimeric or heterodimeric CD20-PD1 binding molecule formed by the association of two Fc domains).

[0172] A native hinge region is the hinge region that is usually found between the Fab and Fc domains in naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can 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 can include a complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a portion or repeat unit of a native hinge, with each unit of the repeat being derived from a native hinge region. In yet another method, the native hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0173] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0174] In one embodiment, a CD20-PD1 binding molecule of the disclosure comprises an Fc region, wherein one or both Fc domains have an intact hinge region at their N-terminus. In various embodiments, positions 233-236 in the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, and the positions are numbered according to EU numbering.

[0175] In some embodiments, a CD20-PD1 binding molecule of the disclosure comprises a modified hinge region that reduces binding affinity to an Fcγ receptor compared to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0176] In one embodiment, the CD20-PD1 binding molecule of the present disclosure comprises an Fc region, where each Fc domain has an intact hinge region at its N-terminus, and each Fc domain and hinge region is derived from IgG4, and each hinge region comprises the modified sequence CPPC (SEQ ID NO:21). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO:22) compared to IgG1, which contains the sequence CPPC (SEQ ID NO:29). The serine residues present in the IgG4 sequence provide increased flexibility in this region, such that a proportion of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than crosslinking to other heavy chains within the IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to prolines to obtain the same core sequence as IgG1 allows complete formation of the interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0177] 6.7.1.1. Chimeric hinge sequences The hinge region may be a chimeric hinge region. For example, a chimeric hinge may comprise 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.

[0178] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO:23) (previously disclosed as SEQ ID NO:8 in WO2014 / 121087, incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO:24) (previously disclosed as SEQ ID NO:9 in WO2014 / 121087). Such chimeric hinge sequences may be suitably linked to an IgG4 CH2 region (which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., by incorporation into an IgG4 Fc domain, e.g., a human Fc domain or a mouse Fc domain, e.g., as described in Section 6.5.1.1).

[0179] 6.7.1.2. Hinge sequences with reduced effector function In further embodiments, the hinge region can be modified to reduce effector function, for example, as described in WO2016161010A2, which is incorporated by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering (as shown in FIG. 1 of WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, with "-" representing an empty position.

[0180] Position 236 is vacant in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0181] Hinge modifications in positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous to stabilize IgG4 antibodies and reduce heavy-light chain pair exchange 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).

[0182] An exemplary hinge region has 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 comprises CPPCPAPGGG-GPSVF (SEQ ID NO:25) (previously disclosed as SEQ ID NO:1 in WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO:26) (previously disclosed as SEQ ID NO:2 in WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO:27) (previously disclosed as SEQ ID NO:3 in WO2016161010A2), or CPPCPAP----GPSVF (SEQ ID NO:28) (previously disclosed as SEQ ID NO:4 in WO2016161010A2).

[0183] The modified hinge regions described above can be incorporated into a heavy chain constant region, which typically includes CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinges) flanking the designated regions. Such additional constant region segments present are typically of the same isotype, preferably human isotype, but may also be hybrids 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 hybrids thereof, where the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016161010A2.

[0184] In certain embodiments, a modified hinge sequence may be linked to an IgG4 CH2 region (which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., by incorporation into an IgG4 Fc domain, e.g., a human Fc domain or a murine Fc domain, e.g., as described in Section 6.5.1.1).

[0185] 6.8. Nucleic Acids and Host Cells In another aspect, the disclosure provides nucleic acids encoding the CD20-PD1 binding molecules of the 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 that include a CD20 targeting moiety composed of more than one polypeptide chain, the CD20-PD1 binding molecules can be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0186] A single nucleic acid can encode a CD20-PD1 binding molecule that comprises a single polypeptide chain, a CD20-PD1 binding molecule that comprises two or more polypeptide chains, or a CD20-PD1 binding molecule that comprises a portion of a CD20-PD1 binding molecule that comprises three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a CD20-PD1 binding molecule that comprises three, four or more polypeptide chains, or three polypeptide chains of a CD20-PD1 binding molecule that comprises four or more polypeptide chains). To separately control expression, open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory elements and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0187] In some embodiments, a CD20-PD1 binding molecule that comprises two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the CD20-PD1 binding molecule can be equal to or less than the number of polypeptide chains in the CD20-PD1 binding molecule (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0188] The nucleic acids of the 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 may be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in more detail herein below.

[0189] Vectors The present disclosure provides vectors comprising a nucleotide sequence encoding one or two of the polypeptide chains of a CD20-PD1 binding molecule or CD20-PD1 binding molecule components described herein, e.g., a CD20-PD1 monomer. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0190] A number of vector systems can be used. For example, one class of vectors 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 class of vectors utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern equine encephalitis virus, and flaviviruses.

[0191] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0192] Once the DNA sequence containing expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, 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 based on the present specification depending on the specific expression vector and mammalian host cell used.

[0193] 6.8.2.Cells The disclosure also provides a host cell comprising a nucleic acid of the disclosure. In one embodiment, the host cell is genetically engineered to contain one or more of the nucleic acids described herein.

[0194] In one embodiment, the host cell is genetically engineered by using an expression cassette. The term "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without an intron, and a termination signal. Additional factors necessary or helpful in effecting expression, such as an inducible promoter, can also be used.

[0195] The present disclosure also provides a host cell comprising the vector described herein. The cell may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. 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.

[0196] 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 the CD20-PD1 binding molecule and one or more carriers, excipients and / or diluents. The composition may be formulated for a particular use, such as veterinary use or pharmaceutical use in 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, in the case of therapeutic use, the mode of administration.

[0197] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition that includes a pharma- ceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0198] The pharmaceutical composition can be conveniently presented in a unit dosage form containing a predetermined amount of the CD20-PD1 binding molecule of the present disclosure per dose. The amount of CD20-PD1 binding molecule contained in a unit dose will depend on the disease being treated as well as other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing an amount of CD20-PD1 binding molecule suitable for single administration, or in the form of a liquid. The dry powder unit dosage form can be packaged in a kit with a syringe, an appropriate amount of diluent, and / or other components useful for administration. The unit dosage in liquid form can be conveniently supplied in the form of a syringe pre-filled with an amount of CD20-PD1 binding molecule suitable for single administration.

[0199] Pharmaceutical compositions may also be supplied in bulk, since they contain an amount of the CD20-PD1 binding molecule suitable for multiple administrations. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing CD20-PD1 binding molecules having the desired purity with any pharma- ceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, isotonicity agents, non-ionic surfactants, antioxidants, and various other additives. See Remington, The Science and Practice of Pharmacy, 23rd edition (Adejare, ed. 2020). Such additives should be non-toxic to recipients at the dosages and concentrations employed.

[0200] Buffering agents serve to maintain the pH in a range close to physiological conditions. They may be present in a wide variety of concentrations, but will typically be present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, 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, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconic acid-sodium hydroxide mixtures, potassium gluconate glyconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffer (e.g., lactate-sodium lactate mixture, lactate-sodium hydroxide mixture, lactate-potassium lactate mixture, etc.), and acetate buffer (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffer, histidine buffer, and trimethylamine salt (e.g., Tris) may be used.

[0201] Preservatives may be added to retard microbial growth and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity agents, sometimes known as "stabilizers," may be added to ensure isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives and serve to solubilize the therapeutic agent or prevent it from denaturing or adhering to the container walls.Exemplary stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and the like), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and the like, including cyclitols such as inositol), polyethylene glycols, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, , thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate), low molecular weight polypeptides (e.g., peptides of 10 residues or less), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone, etc.), monosaccharides (e.g., xylose, mannose, fructose, glucose, etc.), disaccharides (e.g., lactose, maltose, sucrose, and trehalose, etc.), trisaccharides (e.g., raffinose, etc.), and polysaccharides (e.g., dextran, etc.). The stabilizer may be present in an amount ranging from 0.5 to 10% by weight per weight of the CD20-PD1 binding molecule.

[0202] Non-ionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the glycoprotein and to protect the glycoprotein from agitation-induced aggregation, which also allows the formulation to be exposed to stressful shear surfaces without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and pluronic polyols. Non-ionic surfactants can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0203] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0204] 6.9.2. PHARMACEUTICAL COMPOSITIONS FOR DELIVERY OF NUCLEIC ACIDS ENCODING CD20-PD1 BINDING MOLECULES The CD20-PD1 binding molecules of the disclosure can be delivered by any method useful for gene therapy, for example, as mRNA or via a viral vector encoding the CD20-PD1 binding molecule under the control of a suitable promoter.

[0205] 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, for example, rAd-p53, a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53 (Gendicine®, also known as Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297), for use in the treatment of cancer; Ad5_d11520 (also called H101 or ONYX-015, see, e.g., Russell et al., 2012, Nature Biotechnology 30:658-670), an adenovirus lacking the E1B gene to inactivate host p53; AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF (Cerullo et al., 2010, Cancer Res. 70:4297; rAd-HSVtk, a replication-deficient adenovirus carrying the HSV thymidine kinase gene, for use, e.g., in the treatment of cancer (Cerepro®, developed by Ark Therapeutics, see, e.g., U.S. Pat. No. 6,579,855; developed by Advantagene as ProstAtak™; International PCT Application No. WO 2005 / 049094); rAd-TNFα, a replication-deficient adenovirus vector expressing human tumor necrosis factor alpha (TNFα) under the control of the chemoradiation-inducible EGR-1 promoter, for use, e.g., in the treatment of cancer (TNFerade™, GenVec; Rasmussen et al., 2002, Cancer Gene Ther. 9:951-7; for example, but not limited to, Ad-IFNβ (BG00001 and H5.110CMVhIFN-β, Biogen), an adenovirus serotype 5 vector with deleted E1 and E3 genes that expresses the human interferon beta gene under the direction of the cytomegalovirus (CMV) immediate early promoter to treat cancer. Sterman et al., 2010, Mol. Ther. 18:852-860.

[0206] The nucleic acid molecule (e.g., mRNA) or virus may be formulated as the only pharmacoactive ingredient in the pharmaceutical composition, or may be combined with other active agents for the specific disease to be treated. Optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents may be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and perfuming agents, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Exemplary other agents and excipients that may be included in the compositions include, for example, water soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid, and the like.

[0207] When used as an adjunct therapy for adoptive cell transfer therapy, e.g., CAR-expressing cell therapy as described in Section 6.11.1, the cell therapy, e.g., CAR-expressing cells, can be engineered to express the CD20-PD1 binding molecule of the present disclosure. The CD20-PD1 binding molecule can be targeted to a specific genomic locus, e.g., a locus that is active in activated or dysfunctional lymphocytes, e.g., the PD-1 locus, or can be inserted into a non-specific genomic locus. Targeting a specific genomic locus can be achieved by gene editing, e.g., using zinc finger proteins, CRISPR / Cas9 systems, etc.

[0208] 6.10. Treatment indications and treatment methods The CD20-PD1 binding molecules of the present disclosure are useful for treating disease states in which modulation of the host's immune system is beneficial, particularly conditions in which suppression of cellular immune responses is desirable. Thus, the CD20-PD1 binding molecules of the present disclosure can be used to suppress immune responses in a variety of applications.

[0209] Conditions in which suppression of cellular immune responses is desirable may include disease conditions resulting from autoimmune responses. Disease conditions in which the CD20-PD1 binding molecules of the present disclosure may be administered include, for example, autoimmune diseases in which suppression of cellular autoimmune responses is an important mechanism. Particular disease conditions in which the CD20-PD1 binding molecules of the present disclosure may be used 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.

[0210] In one aspect, a CD20-PD1 binding molecule of the present disclosure is provided for use as a medicament. In a further aspect, a CD20-PD1 binding molecule of the present disclosure is provided for use in the treatment of a disease. In certain embodiments, a CD20-PD1 binding molecule of the present disclosure is provided for use in a method of treatment. In one embodiment, the present disclosure provides a CD20-PD1 binding molecule as described herein for use in the treatment of a disease in a subject in need of such treatment. In certain embodiments, the present disclosure provides a CD20-PD1 binding molecule for use in a method of treating a subject having an autoimmune disease, comprising administering to the individual a therapeutically effective amount of the CD20-PD1 binding molecule. In certain embodiments, the disease being treated is an autoimmune disease. In certain 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 further embodiments, the present disclosure provides CD20-PD1 binding molecule agonists for use in suppressing the immune system. In certain embodiments, the present disclosure provides CD20-PD1 binding molecules for use in a method of suppressing the immune system in a subject, comprising administering to the individual an effective amount of the CD20-PD1 binding molecule to suppress the immune system. The "individual" according to any of the above embodiments is a mammal, e.g., a human. "Suppression of the immune system" according to any of the above embodiments may include any one or more of a general reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell responsiveness, and the like. "Suppression of cellular autoimmune responses" according to any of the above embodiments may include, for example, a reduction in immune signals (e.g., secretion of immune activating cytokines), a reduction in the function of immune cells targeting autoantigens, and the like.

[0211] The disclosure further provides methods of local PD1 agonism comprising administering to a subject a CD20-PD1 binding molecule or pharmaceutical composition described herein. As used herein, the term "locally delivered" does not require local administration, but rather indicates that the CD20-PD1 binding molecule is selectively or preferentially localized at an intended site of immune modulation, e.g., a site of autoimmune activity, and / or at an intended cell type, e.g., B cells.

[0212] The disclosure further provides methods of administering PD1 agonist therapy to a subject with reduced systemic exposure and / or reduced systemic toxicity, e.g., where CD20 is expressed by a tissue for which PD1 agonist therapy is desired and / or intended, comprising administering PD1 agonist therapy to the subject in the form of a CD20-PD1 binding molecule or pharmaceutical composition described herein.

[0213] Thus, the above-described methods allow for PD1 agonist therapy with reduced off-target side effects by preferentially delivering the CD20-PD1 binding molecule to the intended site of PD1 agonist therapy.

[0214] The disclosure further provides a method of locally modulating (e.g., inhibiting) an immune response in a target tissue that expresses CD20, comprising administering to a subject a CD20-PD1 binding molecule or a pharmaceutical composition described herein.

[0215] In some embodiments, administration is not localized to a tissue. In a further aspect, the disclosure provides for the use of a CD20-PD1 binding molecule of the disclosure in the manufacture or preparation of a medicament for the treatment of a disease in a subject in need thereof. In one embodiment, the medicament is for use in a method of treating a disease comprising administering a therapeutically effective amount of the medicament to a subject having the disease. In certain embodiments, the disease being treated is an autoimmune disease. In certain 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 a further embodiment, the medicament is for suppressing the immune system. In a further embodiment, the medicament is for use in a method of suppressing the immune system in a subject comprising administering to the individual an amount of an agent effective to suppress the immune system. An "individual" according to any of the above embodiments may be a mammal, such as a human. "Suppression of the immune system" according to any of the above embodiments may include any one or more of a general reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell responsiveness, and the like.

[0216] In a further aspect, the present disclosure provides a method of clustering PD1 and / or enhancing PD1 activity in a subject, comprising administering to said subject an effective amount of a CD20-PD1 binding molecule of the present disclosure. The CD20-PD1 binding molecule of the present disclosure can induce PD1 clustering at the interface of CD20-presenting cells and T cells. This results in targeted immunosuppression, protecting the CD20-presenting cells and surrounding cells and tissues from T cell killing. High levels of CD20 are found on B cells that are abundant in draining lymph nodes and autoimmune tissues (e.g., pancreas in type 1 diabetes (T1D)). The CD20-PD1 binding molecule of the present disclosure can stimulate PD1 in a cell- and / or tissue-specific manner and inhibit autoreactive T cell activation. In T1D, the abundance of CD20+ B cells results in clustering of PD1 on autoreactive T cells, inhibiting autoreactive cytotoxic T cells from killing pancreatic islet cells. In one embodiment, a composition comprising a CD20-PD1 binding molecule of the disclosure in a pharma- ceutically acceptable form is administered to the subject.

[0217] In a further aspect, the disclosure provides a method for treating an autoimmune disease in a subject, comprising administering to the individual a therapeutically effective amount of a CD20-PD1 binding molecule of the disclosure. In one embodiment, a composition comprising a CD20-PD1 binding molecule of the disclosure in a pharma- ceutically acceptable form is administered to the individual. In certain embodiments, the disease being treated is an autoimmune disease. Autoimmune diseases treatable by the CD20-PD1 binding molecules of the present disclosure 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-Barré syndrome, IgA nephropathy ... These include G4-related sclerosing diseases, 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, Sjogren's syndrome, sperm or testicular autoimmunity, stiff-body syndrome (SPS), Takayasu's arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), ulcerative colitis, and vasculitis.

[0218] In certain 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 a further aspect, the disclosure provides a method for suppressing the immune system in a subject, 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 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.

[0219] In certain embodiments, the disease to be treated is an autoimmune disease. The CD20-PD1 binding molecules may be used to remove cells involved in immune cell-mediated disorders, autoimmunity, transplant rejection, and graft-versus-host disease. Those skilled in the art will readily recognize that in many cases, the CD20-PD1 binding molecules may not provide a cure, but may only provide a partial benefit. In some embodiments, a physiological change that has some benefit is also considered to be therapeutically beneficial. Thus, in some embodiments, the amount of the CD20-PD1 binding molecule that produces a physiological change is considered 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.

[0220] 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) for the prevention or treatment of a disease will depend on the type of disease being treated, the route of administration, the patient's weight, the particular CD20-PD1 binding molecule, the severity and course of the disease, whether the antibody is administered for prophylactic 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 event, the medical practitioner responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0221] The CD20-PD1 binding molecule is suitably administered to the patient at one time or over a series of treatments. For example, depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the CD20-PD1 binding molecule may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion. A typical daily dosage will range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or more, depending on the disease state, treatment will generally be continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the CD20-PD1 binding molecule would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, dosages can also include from 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 per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numerical values ​​recited herein, ranges such as 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., based on the numerical values ​​above can be administered. Thus, 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) can be administered to the patient. Such doses can be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about two to about twenty, or, for example, about six doses of the CD20-PD1 binding molecule). An initial high loading dose can be administered, followed by one or more lower doses. However, other dosage regimens can also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0222] The CD20-PD1 binding molecules of the disclosure will generally be used in an amount effective to achieve its intended purpose. For use in treating or preventing a disease state, the CD20-PD1 binding molecules of the disclosure or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. 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.

[0223] For systemic administration, the therapeutically effective amount can be estimated initially from in vitro assays, such as cell culture assays. The EC 50 A dose can be formulated in animal models to achieve a circulating concentration range including, but not limited to, 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0224] Initial doses can also be estimated from in vivo data, such as, for example, animal models, using techniques known in the art. Those of skill in the art would be readily able to optimize administration to humans based on the animal data.

[0225] Dosage and intervals can be adjusted individually to provide plasma levels of CD20-PD1 binding molecules sufficient to maintain therapeutic efficacy. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by multiple daily administrations. Levels in plasma can be measured, for example, by ELISA HPLC.

[0226] 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 of skill in the art will be able to optimize the therapeutically effective local dosage without undue experimentation.

[0227] A therapeutically effective dose of the CD20-PD1 binding molecules described herein will generally provide a therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the CD20-PD1 binding molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal studies, LD 50 (the dose at which 50% of the population is lethal) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of ED 0 to ED 2 . CD20-PD1 binding molecules that exhibit large therapeutic indices 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 in formulating a range of dosages suitable for use in humans. Dosages are preferably within the range of ED 0 to ED 2 with little or no toxicity. 50 The circulating concentration range includes: 100-200 mg / kg / day, 100-250 mg / kg / day, 100-300 mg / kg / day, 100-400 mg / kg / day, 100-500 mg / kg / day, 100-600 mg / kg / day, 100-700 mg / kg / day, 100-800 mg / kg / day, 100-900 mg / kg / day, 100-1500 mg / kg / day, 100-1500 mg / kg / day, 100-1500 mg / kg / day, 100-1500 mg / kg / day, 100-1500 mg / kg / day, 100-2500 mg / kg / day, 100-3000 mg / kg / day, 100-4000 mg / kg / day, 100-5000 mg / kg / day, 100-6000 mg / kg / day, 100-7000 mg / kg / day, 100-8000 mg / kg / day, 100-15 ...

[0228] The attending physician of a patient treated with a CD20-PD1 binding molecule of the present disclosure will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, if the clinical response is not adequate (excluding toxicity), the attending physician will also know how to adjust treatment to higher levels. The magnitude of an administered dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, and the like. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose and perhaps frequency of administration will also vary according to the age, weight, and response of the individual patient.

[0229] 6.10.1.1 diabetes mellitus In some embodiments, the CD20-PD1 binding molecules of the present disclosure can prevent or delay the onset or progression of type 1 diabetes. Thus, in some embodiments, the CD20-PD1 binding molecules, nucleic acids, and / or pharmaceutical compositions of the present disclosure can be administered to subjects with 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., rubella, coxsackievirus, and mumps), race / ethnicity (e.g., in the United States, whites are more susceptible to type 1 diabetes), family history, early diet, and the presence of other autoimmune conditions (e.g., Graves' 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 skill of the art.

[0230] In some embodiments, patients at risk of developing T1D are treated with the CD20-PD1 binding molecules, nucleic acids, and / or pharmaceutical compositions of the disclosure according to the methods of the disclosure. 6.11. Combination Therapy The CD20-PD1 binding molecules according to the present disclosure may be administered in combination with one or more other agents in a treatment. For example, the CD20-PD1 binding molecules of the present disclosure may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in a subject in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of a cell to an apoptosis inducer.

[0231] Such active ingredients are suitably present in the combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of CD20-PD1 binding molecule used, the type of disorder or treatment, and other factors discussed above. The CD20-PD1 binding molecule will generally be used in the same dosages and by any route of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0232] Such combination therapy as described above encompasses concomitant administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, where administration of a CD20-PD1 binding molecule of the present disclosure may occur prior to, concurrently with, and / or after administration of an additional therapeutic agent and / or adjuvant.

[0233] 6.11.1. Combination Therapy Using CD20-PD1 Binding Molecular Therapy and Immunotherapy The CD20-PD1 binding molecules of the present disclosure can be advantageously used in combination with chimeric antigen receptor ("CAR") expressing cells, e.g., CAR-expressing Treg ("CAR-Treg") cells, e.g., CAR-Treg, in the treatment of autoimmune diseases. In some embodiments, the CAR-Treg cells are recognized by the CD20 targeting moiety in the CD20-PD1 binding molecule. The CD20 targeting moiety can recognize the Treg cell receptor or another cell surface molecule on the CAR-Treg cells. In some embodiments, the CD20 targeting moiety in the CD20-PD1 binding molecule can bind to the extracellular domain, e.g., the antigen binding domain, of the 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 by reference in its entirety.

[0234] 6.12. Evaluation of CD20-PD1 binding molecules Aspects of the present disclosure relate to a luciferase-based reporter bioassay to assess the ability of CD20-PD1 binding molecules disclosed herein to stimulate PD1 on Jurkat cells in the presence of CD20 displayed on HEK293 cells. In some embodiments, HEK293 cells are transduced with CD22 and CD20.

[0235] In some embodiments, the bioassay disclosed herein involves the use of a bispecific antibody (such as CD3×CD22) in the presence of HEK293 cells to elicit an immune response from a Jurkat cell line transduced with an AP1 (activator-protein 1)-luciferase reporter, CD3 and PD1 using lentivirus. CD20-PD1 binding molecules are added to wells in the presence of Jurkat and HEK293 cells and the anti-CD3×CD22 bispecific antibody. Molecules that optimally stimulate PD1 have the ability to reduce the amount of immune response stimulated by the anti-CD3×CD22 bispecific antibody as measured by AP1-driven luciferase activity. 7. Working Example 7.1. Materials and Methods 7.1.1. Design and Production of CD20-PD1 Binding Molecules Constructs were generated that encode the bispecific CD20-PD1 agonists and controls shown in Tables 1 and 2 below. The bispecific CD20-PD1 agonists contained different configurations of mouse anti-CD20 and modified mouse PDL1 ectodomain, IgG1 effector null (EN) (L234A, L235E, G237A, A330S and P331S, EU numbering) domains, and different lengths of linkers from different repeats of G4S (SEQ ID NO: 14). A 29-amino acid signal sequence from mouse inactive tyrosine-protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the constructs. All bispecific CD20-PD1 agonists were expressed as preproteins containing the signal sequence. The signal sequence was cleaved by intracellular processing to generate the mature protein.

[0236] Knob-forming mutation: T366W (EU numbering). Hole-forming mutations: T366S, L368A, and Y407V (EU numbering). Star mutations: H435R and Y436F (EU numbering).

[0237] [Table 3]

[0238] Constructs were expressed in Expi293F™ cells by transient transfection (Thermo Fisher Scientific). Proteins in Expi293F supernatants were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with either HiTrap™ Protein G HP or MabSelect SuRe pcc columns (Cytiva). After a single-step elution, antibodies were neutralized and dialyzed into a 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 a HiPrep26 / 60 Sephacryl S-200 column was used.

[0239] The alignment and selected mutation positions in mPDL1 are shown in Figure 3B. The alignment between mPDL1 and hPDL1 was generated using MacVector. Figure 3A shows the three-dimensional structures of mPDL1 and hPDL1, including the residues that were altered in mPDL1 to improve yield and stability.

[0240] [Table 4-1]

[0241] [Table 4-2]

[0242] [Table 4-3]

[0243] [Table 4-4]

[0244] [Table 4-5]

[0245] [Table 4-6]

[0246] [Table 4-7]

[0247] Flow Cytometry Cells (HEK293, MC38 overexpressing mCD20, or Jurkat overexpressing mPD1) were cultured at 1 × 10 6 Cells were resuspended in FACS wash solution (PBS containing 1% FBS) at 1 × 10 cells / mL. Staining was performed at 1 × 10 cells / well. 5 The antibody was added at 1.3 × 10 -07 The antibodies were diluted in a 1:5 ratio from the starting concentration of 1000 mM. The diluted antibodies were then added to the wells containing the cells. The cells were stained for 30 min at 2-8 °C and washed twice with FACS wash buffer. APC-conjugated goat anti-human IgG (Jackson Immuno Research, 109-607-003, 1:400) was added and the cells were stained for 30 min at 2-8 °C. After washing, the cells were fixed in 2% paraformaldehyde for 30 min at 2-8 °C. After two washes, the stained cells were analyzed using a BD LSRFortessa™ FACS instrument. The results were analyzed by FlowJo. Mononuclear cells were selected using an FSC / SSC gate.

[0248] For spinal cord T cell infiltration flow cytometry analysis, a single cell suspension of spinal cord was first prepared by collagenase D (Roche, 11088882001) digestion and Percoll (GE Healthcare, 17-0891-02) gradient separation. Cells were resuspended in FACS wash solution and stained with LIVE / DEAD™ Fixable Blue Dead Cell Stain Kit (Thermofisher 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 protocol described above. Cells were analyzed by BD FACSymphony Cell Analyzer. Results were analyzed by OMIQ cytometry software.

[0249] 7.1.3. Luciferase Reporter Assay: Anti-mCD20 x mPDL1 Ectodomain Molecules A luciferase-based reporter assay was used to evaluate the ability of anti-mCD20×mPDL1 ectodomain molecules to stimulate mouse PD1 (mPD1) on Jurkat cells in the presence of mouse CD20 (mCD20) displayed on HEK293 cells. The overall design of the reporter assay is shown in Figure 5A and B. AP1 is a transcription factor involved in the control of gene expression during T cell activation (Samelson 2002, PMID: 11861607). A bispecific antibody (bsAb) that binds human CD3 and CD22, CD3 bsAb (REGN10551), is used to stimulate T cell activation through binding of antigens on target cells to receptors on T cells, similar to the previously described CD3×CD20 bsAb (Smith et al. 2015, PMID: 26659273). Engagement of mPD1 on Jurkat cells via the mCD20-anchored mCD20×mPDL1 leads to inhibition of PD1 agonism-driven luciferase signaling.

[0250] 7.1.3.1. Manipulation of Jurkat / AP1-luc / mPD1 cells Jurkat / AP1-luc / mPD1 cells were generated by sequential transduction of AP1 (activating protein 1)-luciferase reporter lentivirus (QIAGEN CLS-011L) followed by mPD1 ORF-containing lentivirus (mPD1NM_008798) into Jurkat E6-1 cells (ATCC no. TIB-152).

[0251] 7.1.3.2. Manipulation of HEK293 / hCD22 / mCD20 cells HEK293 / hCD22 / mCD20 cells were generated by sequential lentiviral transduction using human CD22 ORF-encoding lentivirus (NP_001762.2) followed by mCD20 ORF-encoding lentivirus (NP_031667.1).

[0252] 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 in 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% CO2. The next day, Jurkat / AP1-luc / mPD1 reporter cells were added at 30,000 or 50,000 cells / well to wells containing cultured target cells. Molecules of the present disclosure or control antibodies were then serially diluted 1:3 in assay medium to final concentrations ranging from 100 nM to 1 pM (with an additional condition lacking the test molecule) and added to the cells together with 1 nM or 2.5 nM of CD3bsAb. To obtain a range of activation, CD3bsAb was serially diluted 1:3 to final concentrations ranging from 100 nM to 1.69 pM (with an additional condition that did not contain the bispecific mAb) and added to the cells. Incubated at 37 °C / 5% CO 2After 5 h of incubation at 4°C, luciferase activity was detected in an Envision multilabel plate reader (PerkinElmer) after addition of ONE-Glo™ (Promega) reagent. All conditions were tested in duplicate.

[0253] Non-linear regression (four-parameter logistic) was used to determine EC50 / IC50 values ​​with GraphPad Prism™ software. The percentage of inhibition was calculated based on the relative luminescence unit (RLU) values ​​using the following formula:

[0254]

number

[0255] In this formula, "RLU baseline" is the luminescence value from cells treated with a fixed amount of CD3bsAb without the test molecule, "RLU inhibition" is the luminescence value at the highest concentration of test molecule with a fixed amount of CD3bsAb, and "RLU background" is the luminescence value from cells without CD3bsAb or test molecule.

[0256] 7.1.4. Determination of the Oligomerization State of Anti-mCD20 x mPDL1 Ectodomain Molecules by Size Exclusion Chromatography Size-exclusion ultra-performance liquid chromatography (SE-UPLC) was used to assess the size heterogeneity of anti-mCD20×mPDL1 ectodomain 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 (200 Å, 1.7 μm, 4.6×300 mm) and the flow rate was set at 0.3 mL / min. The mobile phase buffer contained 10 mM sodium phosphate, 500 mM NaCl, pH 7.0. Eluted samples were detected by UV absorbance at 280 nm using a photodiode array module.

[0257] 7.1.5.Thermal stability Differential scanning fluorimetry (DSF) was used to assess the thermal stability of anti-mCD20×mPDL1 ectodomain molecules. DSF analysis was performed on a ThermoFisher QuantStudio5 system. Stock solutions of each sample were diluted to 0.2 mg / mL in 1× PBS-glycerol, pH 7.4, and transferred to a 96-well plate. Excess (8×) Sypro Orange™ fluorescent dye, which preferentially binds to buried hydrophobic residues as the protein unfolds, was added to each well, followed by a linear temperature ramp from 25° C. to 95° C. over 20 min to determine the thermal stability profile.

[0258] 7.1.6. Assembly percentage Assembly of bifunctional fusion molecules was assayed by high-throughput analysis on a Cliper LabChip GX (Perkin Elmer, Waltham, MA) following the manufacturer's protocol. Briefly, sample buffer was prepared by mixing 7 ml of HT protein expression sample buffer with either 240 μl of BME (reduced) or 25 mM iodoacetamide (IAM, for non-reduced assays). Samples were normalized to 0.5 mg / ml with sample buffer and then heated at 70° C. for 10 min. 70 μl of water was added to each sample before loading into the instrument. Chips were prepared according to the manufacturer's instructions. Electropherograms of samples were analyzed using LabChip GX software. Peaks from non-reduced electropherograms indicate % of intact antibody.

[0259] 7.1.7. Activity of anti-mCD20×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, 0.1, or 0.01 mg / kg of the selected anti-mCD20×mPDL1 ectodomain molecule for the duration of the experiment (e.g., until mice were 28 weeks old). Blood glucose was monitored biweekly and body weight was monitored weekly. The overall experimental design is shown in FIG. 7.

[0260] 7.1.8. Activity of anti-mCD20×mPDL1 ectodomain molecules in an experimental autoimmune encephalomyelitis / multiple sclerosis mouse model Myelin oligodendrocyte glycoprotein (MOG) 35-55 Administration of the immunodominant 35-55 epitope of MOG produces anti-MOG antibodies that cause demyelination and chronic experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis (MS).

[0261] EAE was induced in wild-type C57BL / 6 mice (10–12 weeks, male, Jackson Laboratory) by injecting 200 mg of MOG in CFA on day 1. 35-55 EAE was induced by subcutaneous injection delivery of pertussis toxin. Considering that administration of pertussis toxin promotes T cell migration into the central nervous system by weakening the blood-brain barrier, mice were also injected intraperitoneally with 200 ng of pertussis toxin on days 1 and 2. Body weight and the occurrence of EAE were monitored on days 1, 2, 7, 10, 14, 18, and 20. EAE monitoring scores were recorded on a scale of 0 to 5 as follows: 0: healthy, 1: tail lameness, 2: abnormal gait and / or righting reflex deficit, 3: partial hind limb paralysis, 4: complete hind limb paralysis, 5: complete hind limb and partial forelimb paralysis or moribund.

[0262] Mice received intraperitoneal injections of the selected anti-mCD20×mPDL1 ectodomain molecule or appropriate control molecule twice weekly starting on day 2. Endpoint tissue harvesting 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.

[0263] 7.2. Example 1: Production and Stability of Bispecific Anti-mCD20-mPDL1 Ectodomain Agonists Overview Mammalian expression vectors for individual heavy and light chains were generated by DNA synthesis and cloning in ready-to-use constructs in the pcDNA3.4Topo Expression System from Life Technologies (Carlsbad, CA). To express the molecules, DNA of the heavy and universal light chains was co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was harvested and processed for purification via HiTrap Protein A FF or Mab Select SuRe columns (GE Healthcare). For functional validation, selected MBMs were scaled up to 2 L and subjected to a series of purification procedures, including size exclusion chromatography as the final step.

[0264] 7.2.2.Results Various anti-mCD20 x mPDL1 ectodomain molecules (Figure 2A) were expressed and purified from Expi293 Freestyle cells (Table 4) via a one-step Mab-Select SuRe column with total yields ranging from 2.7 to 7.7 mg. In general, molecules with a valency ratio of 1:1 (anti-mCD20:mPDL1 ectodomain) showed higher yields (4.1 to 7.7 mg) than those with a valency ratio of 2:1 or 2:2 (Table 4).

[0265] [Table 5]

[0266] After one-step affinity purification, the high molecular weight (HMW)% and monomer% were examined by SE-UPLC, and the thermal stability was monitored by differential scanning fluorimetry (DSF) (Table 5). The majority of anti-mCD20×mPDL1 ectodomain fusion molecules showed more than 85% monomer species without additional size-exclusion chromatography (SEC) (Table 3, molecule AL in Figure 1). For 2+2 m20_mPL_4(L), the percentage of monomer increased to 99% after two column purifications including an SEC step (Table 5). All anti-mCD20×mPDL1 ectodomain fusions had similar thermal stability measured by DSF with Tm1 at about 60°C (Table 3). Furthermore, all bifunctional fusions had excellent assembly between the heavy chains as determined by capillary electrophoresis SDS (CE-SDS) (Table 5).

[0267] [Table 6]

[0268] 7.3. Example 2: Binding Characterization of Anti-mCD20 x mPDL1 Ectodomain Molecules The ability of the anti-mCD20×mPDL1 ectodomain molecules to bind to two targets on the cell surface was assessed in a flow binding assay.

[0269] 7.3.1.Results Binding curves are shown in Figures 4A and 4B. For both mPD1 and mCD20 binding, higher potency and maximum MFI in binding was observed with bivalent molecules compared to monovalent molecules of similar format. Notably, 2+2 m20_mPL_4(L) shared similar binding to HEK293 / mCD20 cells with 2+1 m20_mPL_3(G) (Figures 4A and 4B, Table 6), but 2+2 m20_mPL_4(L) showed stronger binding to Jurkat / mPD1 cells than 2+1 m20_mPL_3(G) as a result of increased valency for mPD1 binding. Across both bivalent and monovalent molecules, binding signals appeared to be orientation dependent, with orientation of N-terminal anti-mCD20 and hPDL1 ectodomain relative to the Fc domain generally having higher potency and maximum MFI. Anti-mCD20 or mPDL1 ectodomain showed reduced binding when placed between the N-terminal portion and the hinge region in front of the Fc (FIGS. 4A and 4B, Table 6).

[0270] 7.4. Example 3: mPDL1 agonism by anti-mCD20 x mPDL1 ectodomain molecules The bioassay shown in Figure 5 and described in Section 7.1.3 was used to study mPD1 agonism by anti-mCD20 x mPDL1 ectodomain molecules.

[0271] 7.4.1.Results The results of the luciferase assay are shown in Figure 6 and Table 6. Nineteen molecules of the present disclosure were tested for PD1 agonism and its modulation 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 ​​ranging from 65 to 770 pM, with maximum inhibition ranging from 27 to 84%. Molecules 2+2 m20_MPL_4 and 2+1 m20_MPL_3 (G and L, respectively, Table 6) showed the strongest PD1 agonism with maximum inhibition of 74% to 84% (Figures 6C and 6E). Fifteen of the nineteen molecules showed weak or no inhibition, with maximum inhibition ranging from -10 to 40%. The isoform control antibody showed no inhibition of signaling. The CD3 bsAb demonstrated activation of T cell signaling with EC50 values ​​of 627 pM and 1.15 nM.

[0272] 7.5. Example 4: Summary of Data from In Vitro Assays Using Anti-mCD20 x mPDL1 Ectodomain Molecules Table 6 provides a summary of the in vitro data collected with the various anti-mCD20 x mPDL1 ectodomain molecules, including cell-based flow binding and in vitro bioassay results. Results from the luciferase assay are shown in Figures 6A-6E. Molecules 2+2 m20_mPL_4 and 2+1 m20_mPL_3 (G and L, respectively, in Table 6) showed the strongest PD1 agonism. By cell-based flow analysis, 2+2 m20_mPL_4 revealed the strongest binding to mPD1 and mCD20, whereas 2+1 m20_mPL_3 revealed only moderate binding to mPD1-expressing cells, suggesting that clustering of mPD1 via bivalent binding of mCD20 in the presence of both APCs and effector cells is required. Overall, similar cell-binding affinities (mPD1 or mCD20) did not translate to similar PD1 agonism, e.g., F vs. G and K vs. L (Figure 6A and Table 6), indicating that both the valency and structural arrangement of the CD20 and mPDL1 ectodomain arms are important for conferring activity.

[0273] [Table 7-1]

[0274] [Table 7-2]

[0275] 7.6. Example 5: In vivo efficacy of anti-mCD20 x mPDL1 ectodomain molecules The ability of selected 2+2 and 2+1 anti-mCD20×mPDL1 ectodomain molecules to prevent the onset of type 1 diabetes (T1D) was evaluated in prediabetic NOD mice. The experimental design is shown in FIG. 7 and described in section 7.1.7.

[0276] 7.6.1.Results Data for individual animals are shown in Figures 8A-8I. Figures 9A and 9B show the percentage of diabetes-free mice at each indicated time point. Typically, 80-90% of NOD mice develop diabetes around 25 weeks of age. However, in this experiment, only about 30% of mice developed diabetes by 27 weeks. Although there was a lower incidence of diabetes in control NOD mice, there was a clear trend towards protection with the higher dose of (2+2) anti-mCD20 x mPDL1 ectodomain molecule, 2+2m20_mPL_4 (molecule L in Figure 2A), but not with (2+1 anti-mCD20 x mPDL1 ectodomain molecule, 2+1 m20_mPL_3 (molecule G in Figure 2A) (Figures 9A and 9B).

[0277] 7.7. Example 6: Reduction of autoimmune T cell infiltration by induction of anti-mCD20×mPDL1 ectodomain molecules T cell infiltration has been associated with 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 role of the (2+2) anti-mCD20×mPDL1 ectodomain molecule, 2+2 m20_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].

[0278] 7.7.1.Results In one evaluation, populations of proliferating (activated) and non-activated islet-specific CD8+ T cells were analyzed in NOD mice administered 0.1 or 1 mg / kg 2+2 m20_mPL_4 or control molecules as described in section 7.1.7. Treatment with 1 mg / kg 2+2 m20_mPL_4 was associated with a significant increase in the percentage of low-activated CD8+ T cell clusters (Figure 10A). While the percentage of clusters of proliferating cells did not differ across conditions (Figure 10B), the proportion of clusters of low-activated and less proliferating cells was higher in pancreatic tissue isolated from NOD mice treated with 1 mg / kg 2+2 m20_mPL_4 (Figure 10C), indicating that this treatment was able to reduce pancreatic infiltration of activated autoimmune T cells.

[0279] In another evaluation, spinal cord infiltration of T cells was evaluated in a mouse model of multiple sclerosis described in section 7.1.8. The spinal cords of mice treated with 1 mg / kg 2+2 m20_mPL_4 contained significantly fewer CD3+ (Figure 11A), CD4+ (Figure 11B), and CD8+ (Figure 11C) T cells compared to the spinal cords of mice that received control treatment at the same dosage. Thus, treatment with 2+2 m20_mPL_4 was able to reduce spinal cord infiltration of T cells in a multiple sclerosis model.

[0280] 8. Specific embodiments, references While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is exemplified by the numbered embodiments described below.

[0281] 1. A protein comprising: (a) at least one CD20 targeting moiety; (b) at least one PD1 agonist moiety; and (c) at least one dimerization moiety; and (d) optionally, one or more linker moieties separating one or more moieties in the protein; Optionally, where: (i) the portions of the protein are arranged from N-terminus to C-terminus in the following order: CD20 targeting portion-PD1 agonist portion-dimerization portion; or (ii) the portions of the protein are arranged from N-terminus to C-terminus in the following order: dimerization moiety-PD1 agonist moiety-CD20 targeting moiety; or (iii) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain 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 ectodomain of PDL1 or a PD1-binding portion thereof; or (iv) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the ectodomain of PDL1 or a PD1-binding portion thereof, the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not 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 ectodomain of PDL1 or a PD1-binding portion thereof, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not C-terminal to the Fc domain; (vi) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the ectodomain of PDL1 or a PD1-binding portion thereof, and the dimerization moiety is an Fc domain, and the protein is monovalent with respect to the CD20 targeting moiety and / or the PD1 agonist moiety; or (vii) the protein is asymmetric, (viii) the protein comprises an Fc heterodimer, or (ix) A protein that is any combination of two or more of the above (i) to (viii).

[0282] 2. The protein of embodiment 1, wherein 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 a Fab, Fv, or scFv.

[0283] 4. Anti-CD20 antibody, (a) a complementarity determining region ("CDR") having the CDR sequences of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, or veltuzumab; (b) all six CDR sequences of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, or veltuzumab; (c) at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, or veltuzumab, and the light chain CDR sequences of a universal light chain; (d) a VH comprising the amino acid sequence of the VH of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, or veltuzumab, and a VL comprising the amino acid sequence of the same antibody; or (e) The protein of embodiment 2 or embodiment 3, comprising a VH comprising the amino acid sequence of the VH of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, or veltuzumab, and a VL comprising a universal light chain VL sequence.

[0284] 5.Anti-CD20 antibody, (a) Topological domain of CD20. (b) the transmembrane domain of CD20, or (c) a region of CD20 that is extracellularly displayed on the surface of a cell (e.g., a B cell), the protein of embodiment 2 or embodiment 3 that binds to the extracellularly displayed region of CD20.

[0285] 6.Anti-CD20 antibody, (a) selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, and veltuzumab; (b) The protein of embodiment 2 or embodiment 3, which competes for binding to CD20 with an anti-CD20 antibody selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, okaratuzumab, TRU-015, and veltuzumab, and / or binds to the same epitope as an anti-CD20 antibody.

[0286] 7. A protein according to any one of embodiments 1 to 6, wherein at least one PD1 agonist portion comprises an amino acid sequence having at least 90% sequence identity to a PD1-binding portion of the extracellular domain of PDL1, and optionally, PDL1 is human PDL1 or mouse PDL1.

[0287] 8. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 95% sequence identity to a PD1-binding portion of the extracellular domain of PDL1, and optionally, PDL1 is human PDL1 or mouse PDL1.

[0288] 9. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 97% sequence identity to a PD1-binding portion of the extracellular domain of PDL1, and optionally, PDL1 is human PDL1 or mouse PDL1.

[0289] 10. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 98% sequence identity with a PD1-binding portion of the extracellular domain of PDL1, and optionally, PDL1 is human PDL1 or mouse PDL1.

[0290] 11. The protein of embodiment 7, wherein the PD1 agonist portion comprises an amino acid sequence having at least 99% sequence identity to a PD1-binding portion of the extracellular domain of PDL1, and optionally, PDL1 is human PDL1 or mouse PDL1.

[0291] 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, and optionally, PDL1 is human PDL1 or mouse PDL1.

[0292] 13. The protein according to any one of embodiments 7 to 12, wherein the PD1-binding portion of the extracellular domain of PDL1 comprises amino acids 19 to 134 of human PDL1 or amino acids 19 to 134 of mouse PDL1.

[0293] 14. A protein according to any one of embodiments 7 to 12, wherein the PD1-binding portion of the extracellular domain of PDL1 comprises or consists of the PDL1 ectodomain, and optionally, the PDL1 is human PDL1 or mouse PDL1.

[0294] 15. The protein of any one of embodiments 1 to 6, wherein at least one PD1 agonist portion comprises an amino acid sequence having at least 90% sequence identity to a PD1-binding portion of the extracellular domain of PDL2, and optionally, PDL2 is human PDL2 or mouse PDL2.

[0295] 16. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 95% sequence identity with a PD1-binding portion of the extracellular domain of PDL2, and optionally, PDL2 is human PDL2 or mouse PDL2.

[0296] 17. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 97% sequence identity to a PD1-binding portion of the extracellular domain of PDL2, and optionally, PDL2 is human PDL2 or mouse PDL2.

[0297] 18. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 98% sequence identity with a PD1-binding portion of the extracellular domain of PDL2, and optionally, PDL1 is human PDL2 or mouse PDL2.

[0298] 19. The protein of embodiment 15, wherein the PD1 agonist portion comprises an amino acid sequence having at least 99% sequence identity to a PD1-binding portion of the extracellular domain of PDL2, and optionally, PDL2 is human PDL2 or mouse PDL2.

[0299] 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, and optionally, PDL2 is human PDL2 or mouse PDL2.

[0300] 21. The protein according to any one of embodiments 15 to 20, wherein the PD1-binding portion of the extracellular domain of PDL2 comprises amino acids 20 to 121 of human PDL2 or amino acids 20 to 121 of mouse PDL2.

[0301] 22. The protein of any one of embodiments 15-20, wherein the PD1-binding portion of the extracellular domain of PDL2 comprises or consists of the PDL2 ectodomain, and optionally, PDL2 is human PDL2 or mouse PDL2.

[0302] 23. A protein according to any one of embodiments 1 to 22, comprising one or more linker moieties. 24. The protein of embodiment 23, wherein the at least one CD20 targeting moiety and the at least one PD1 agonist moiety are separated by a linker moiety.

[0303] 25. The protein according to embodiment 23 or embodiment 24, wherein at least one CD20 targeting moiety and at least one dimerization moiety are separated by a linker moiety.

[0304] 26. A protein according to 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.

[0305] 27. The protein of any one of embodiments 21 to 26, wherein each linker moiety is (a) at least 5 or at least 10 amino acids in length, (b) at most 20, at most 25 or at most 30 amino acids in length, and / or (c) between 5 and 15 amino acids or between 5 and 20 amino acids in length.

[0306] 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 G4S (sequence number 14) or a multimer thereof.

[0307] 30. The protein of embodiment 29, wherein the multimer comprises 2, 3, 4, 5 or more repeats of the amino acid sequence G4S (SEQ ID NO:56). 31. The protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 2.

[0308] 32. The protein of embodiment 31, wherein exemplary monomer 1 comprises or consists of, in N-terminal to C-terminal orientation, a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety, and a dimerization moiety.

[0309] 33. The protein of embodiment 30, wherein exemplary monomer 1 is composed of a single polypeptide chain. 34. The protein of embodiment 30, wherein exemplary monomer 1 is composed of two polypeptide chains.

[0310] 35. The protein of any one of embodiments 29 to 34, wherein exemplary monomer 2 comprises or consists of, in N-terminal to C-terminal orientation, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), an optional linker portion, and a dimerization portion.

[0311] 36. A protein according to any one of embodiments 31 to 35, having the structure shown in FIG. 1A. 37. The protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 3 and a monomer according to exemplary monomer 4.

[0312] 38. The protein of embodiment 37, wherein exemplary monomer 3 comprises or consists of, in N-terminal to C-terminal orientation, an optional linker moiety and a dimerization moiety. 39. The protein of embodiment 37 or embodiment 38, wherein exemplary monomer 4 comprises or consists of, in N-terminal to C-terminal orientation, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), 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.

[0313] 40. The protein of embodiment 39, wherein exemplary monomer 4 is composed of a single polypeptide chain. 41. The protein of embodiment 39, wherein exemplary monomer 4 is composed of two polypeptide chains.

[0314] 42. A protein according to any one of embodiments 37 to 39, having the structure shown in FIG. 1B. 43. The protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 3 and a monomer according to exemplary monomer 5.

[0315] 44. The protein of embodiment 43, wherein exemplary monomer 3 comprises or consists of, in N-terminal to C-terminal orientation, an optional linker portion and a dimerization portion. 45. The protein of embodiment 43 or embodiment 44, wherein exemplary monomer 5 comprises or consists of, in N-terminal to C-terminal orientation, 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 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 to (i) or (ii)).

[0316] 46. ​​The protein of embodiment 45, wherein exemplary monomer 5 is composed of a single polypeptide chain. 47. The protein of embodiment 45, wherein exemplary monomer 5 is composed of two polypeptide chains.

[0317] 48. A protein according to any one of embodiments 43 to 47, having the structure shown in FIG. 1C. 49. The protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 3 and a monomer according to exemplary monomer 6.

[0318] 50. The protein of embodiment 49, wherein exemplary monomer 3 comprises or consists of, in N-terminal to C-terminal orientation, an optional linker portion and a dimerization portion. 51. The protein of embodiment 49 or embodiment 50, wherein exemplary monomer 6 comprises or consists of, in 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 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 to (i) or (ii)), an optional linker portion, and a dimerization portion.

[0319] 52. A protein according to any one of embodiments 49 to 51, wherein exemplary monomer 6 is composed of a single polypeptide chain. 53. A protein according to any one of embodiments 49 to 51, wherein exemplary monomer 6 is composed of two polypeptide chains.

[0320] 54. A protein according to any one of embodiments 49 to 53, having the structure shown in FIG. 1D. 55. A protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 4.

[0321] 56. The protein of embodiment 55, wherein exemplary monomer 1 comprises or consists of, in N-terminal to C-terminal orientation, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, and a dimerization portion.

[0322] 57. The protein of embodiment 56, wherein exemplary monomer 1 is composed of a single polypeptide chain. 58. The protein of embodiment 56, wherein exemplary monomer 1 is composed of two polypeptide chains.

[0323] 59. The protein of any one of embodiments 55-58, wherein exemplary monomer 4 comprises or consists of, in N-terminal to C-terminal orientation, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), 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.

[0324] 60. The protein of embodiment 59, wherein exemplary monomer 4 is composed of a single polypeptide chain. 61. The protein of embodiment 59, wherein exemplary monomer 4 is composed of two polypeptide chains.

[0325] 62. A protein according to any one of embodiments 55 to 61, having the structure shown in Figure 1E. 63. A protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 5.

[0326] 64. The protein of embodiment 63, wherein exemplary monomer 1 comprises or consists of, in N-terminal to C-terminal orientation, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, and a dimerization portion.

[0327] 65. The protein of embodiment 64, wherein exemplary monomer 1 is composed of a single polypeptide chain. 66. The protein of embodiment 64, wherein exemplary monomer 1 is composed of two polypeptide chains.

[0328] 67. The protein of any one of embodiments 63 to 66, wherein exemplary monomer 5 comprises or consists of, in N-terminal to C-terminal orientation, 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 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 to (i) or (ii)).

[0329] 68. The protein of embodiment 67, wherein exemplary monomer 5 is composed of a single polypeptide chain. 69. The protein of embodiment 67, wherein exemplary monomer 5 is composed of two polypeptide chains.

[0330] 70. A protein according to any one of embodiments 63 to 69, having the structure shown in Figure 1F. 71. A protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 6.

[0331] 72. The protein of embodiment 71, wherein exemplary monomer 1 comprises or consists of, in N-terminal to C-terminal orientation, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, and a dimerization portion.

[0332] 73. The protein of embodiment 72, wherein exemplary monomer 1 is composed of a single polypeptide chain. 74. The protein of embodiment 72, wherein exemplary monomer 1 is composed of two polypeptide chains.

[0333] 75. The protein of any one of embodiments 71 to 74, wherein exemplary monomer 6 comprises or consists of, in 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 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 to (i) or (ii)), an optional linker portion, and a dimerization portion.

[0334] 76. The protein of embodiment 75, wherein exemplary monomer 6 is composed of a single polypeptide chain. 77. The protein of embodiment 75, wherein exemplary monomer 6 is composed of two polypeptide chains.

[0335] 78. A protein according to any one of embodiments 71 to 77, having the structure shown in FIG. 1G. 79. A protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 1 and a monomer according to exemplary monomer 7.

[0336] 80. The protein of embodiment 79, wherein exemplary monomer 1 comprises or consists of, in N-terminal to C-terminal orientation, a CD20 targeting portion (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker portion, and a dimerization portion.

[0337] 81. The protein of embodiment 80, wherein exemplary monomer 1 is composed of a single polypeptide chain. 82. The protein of embodiment 80, wherein exemplary monomer 1 is composed of two polypeptide chains.

[0338] 83. The protein of any one of embodiments 79 to 82, wherein exemplary monomer 7 comprises or consists of, in N-terminal to C-terminal orientation, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), an optional linker portion, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), an optional linker portion, and a dimerization portion.

[0339] 84. A protein according to any one of embodiments 79 to 83, having the structure shown in Figure 1H. 85. A protein according to any one of embodiments 1 to 30, comprising two monomers according to exemplary monomer 4.

[0340] 86. The protein of embodiment 85, wherein each exemplary monomer 4 comprises or consists of, in N-terminal to C-terminal orientation, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), 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.

[0341] 87. The protein of embodiment 86, wherein each exemplary monomer 4 is composed of a single polypeptide chain. 88. The protein of embodiment 86, wherein each exemplary monomer 4 is composed of two polypeptide chains.

[0342] 89. A protein according to any one of embodiments 85 to 88, having the structure shown in Figure 1I. 90. A protein according to any one of embodiments 1 to 30, comprising two monomers according to exemplary monomer 5.

[0343] 91. The protein of embodiment 90, wherein each exemplary monomer 5 comprises or consists of, in N-terminal to C-terminal orientation, 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 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 to (i) or (ii)).

[0344] 92. The protein of embodiment 91, wherein each exemplary monomer 5 is composed of a single polypeptide chain. 93. The protein of embodiment 91, wherein each exemplary monomer 5 is composed of two polypeptide chains.

[0345] 94. A protein according to any one of embodiments 90 to 93, having the structure shown in Figure 1J. 95. A protein according to any one of embodiments 1 to 30, comprising a monomer according to exemplary monomer 8 and a monomer according to exemplary monomer 9.

[0346] 96. The protein of embodiment 95, wherein exemplary monomer 8 comprises or consists of, in N-terminal to C-terminal orientation, a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV), an optional linker moiety, a dimerization moiety, an optional linker moiety, and a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv or scFV).

[0347] 97. The protein of embodiment 96, wherein each exemplary monomer 8 is composed of a single polypeptide chain. 98. The protein of embodiment 96, wherein each exemplary monomer 8 is composed of two polypeptide chains.

[0348] 99. The protein of embodiment 96, wherein each exemplary monomer 8 is composed of three polypeptide chains. 100. The protein of any one of embodiments 95 to 99, wherein exemplary monomer 9 comprises or consists of, in N-terminal to C-terminal orientation, a PD1 agonist portion (e.g., a portion comprising (i) an amino acid sequence of the extracellular domain of 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 to (i) or (ii)), 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 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 to (i) or (ii)).

[0349] 101. A protein according to any one of embodiments 95 to 100, having the structure shown in Figure 1K. 102. A protein according to any one of embodiments 1 to 30, comprising two monomers according to exemplary monomer 6.

[0350] 103. The protein of embodiment 102, wherein exemplary monomer 6 comprises or consists of, in 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 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 to (i) or (ii)), an optional linker portion, and a dimerization portion.

[0351] 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.

[0352] 106. A protein according to any one of embodiments 102 to 104, having the structure shown in Figure 1L. 107. The protein according to any one of embodiments 1 to 54 and 79 to 84, comprising one CD20 targeting moiety.

[0353] 108. The protein of embodiment 107, comprising one PD1 agonist moiety. 109. The protein of embodiment 108, having the configuration shown in Figure 1A. 110. The protein of embodiment 108, having the configuration shown in Figure 1B.

[0354] 111. The protein of embodiment 108, having the configuration shown in Figure 1C. 112. The protein of embodiment 108, having the configuration shown in Figure 1D. 113. The protein of embodiment 107, comprising two PD1 agonist moieties.

[0355] 114. The protein of embodiment 113, wherein the two PD1 agonist moieties are identical. 115. The protein according to embodiment 113 or embodiment 114, having the structure shown in Figure 1H.

[0356] 116. The protein according to 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 identical.

[0357] 118. The protein according to embodiment 116 or embodiment 117, comprising one PD1 agonist moiety. 119. The protein of embodiment 118, having the configuration shown in Figure 1E.

[0358] 120. The protein of embodiment 118, having the configuration shown in Figure 1F. 121. The protein of embodiment 118, having the configuration shown in Figure 1G. 122. The protein according to embodiment 116 or embodiment 117, comprising two PD1 agonist moieties.

[0359] 123. The protein of embodiment 122, wherein the two PD1 agonist moieties are identical. 124. The protein according to embodiment 122 or embodiment 123, having the structure shown in Figure 1I.

[0360] 125. The protein of embodiment 122 or embodiment 123, having the configuration shown in Figure 1J. 126. The protein of embodiment 122 or embodiment 123, having the structure shown in Figure 1K.

[0361] 127. The protein of embodiment 122 or embodiment 123, having the structure shown in Figure 1L. 128. The molecule according to any one of embodiments 1 to 127, wherein the CD20 targeting moiety binds to the extracellular domain of human CD20.

[0362] 129. The molecule according to any one of embodiments 1 to 128, wherein the PD1 agonist portion stimulates human PD1. 130. The molecule of any one of embodiments 1-127, wherein the CD20 targeting moiety binds to the extracellular domain of mouse CD20.

[0363] 131. The molecule according to any one of embodiments 1 to 127 and 130, wherein the PD1 agonist moiety stimulates mouse PD1. 132. The protein according to any one of the preceding embodiments, wherein at least one dimerization moiety is or comprises an Fc domain.

[0364] 133. The protein according to embodiment 132, wherein the Fc domain is a human Fc domain. 134. The protein according to embodiment 132 or embodiment 133, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0365] 135. A protein according to any one of embodiments 132 to 134, wherein the Fc domain has a reduced effector function. 136. The protein according to any one of embodiments 132 to 135, wherein the Fc domain is an IgG4 Fc domain.

[0366] 137. The protein according to any one of embodiments 132 to 136, wherein the Fc domain comprises the amino acid sequence ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 32) or a portion thereof.

[0367] 138. A protein according to any one of embodiments 132 to 137, comprising an Fc dimer. 139. The protein according to embodiment 138, wherein the Fc dimer is an Fc homodimer.

[0368] 140. The protein according to embodiment 138, wherein the Fc dimer is an Fc heterodimer. 141. The protein of embodiment 140, wherein the Fc heterodimer comprises a knob-in-hole mutation.

[0369] 142. The protein according to embodiment 140 or embodiment 141, wherein the Fc heterodimer comprises a star mutation. 143. The protein according to embodiment 1, wherein the portions of the protein are arranged from N-terminus to C-terminus in the following order: CD20 targeting portion-PD1 agonist portion-dimerization portion.

[0370] 144. The protein according to embodiment 1, wherein the portions of the protein are arranged from N-terminus to C-terminus in the following order: dimerization portion-PD1 agonist portion-CD20 targeting portion. 145. The protein of embodiment 1, wherein the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain of PDL1, the dimerization moiety is an Fc domain, and the light chain of the Fab is not fused to the ectodomain of PDL1 or to a PD1 binding portion thereof.

[0371] 146. The protein of embodiment 1, wherein the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain of PDL1, and the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not at the N-terminus of the VH of the anti-CD20 Fab.

[0372] 147. The protein of embodiment 1, wherein the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain of PDL1, the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not C-terminal to the Fc domain.

[0373] 148. The protein of embodiment 1, wherein the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is an ectodomain of PDL1, the dimerization moiety is an Fc domain, and the protein is monovalent with respect to the CD20 targeting moiety and / or the PD1 agonist moiety.

[0374] 149. The protein of embodiment 1, wherein the protein is asymmetric. 150. A nucleic acid or a plurality of nucleic acids encoding a protein according to any one of embodiments 1 to 149.

[0375] 151. A host cell engineered to express a protein according to any one of embodiments 1 to 149 or a nucleic acid(s) according to embodiment 150. 152. A method for producing a protein according to any one of embodiments 1 to 149, comprising culturing the host cell of embodiment 151 and recovering the protein expressed thereby.

[0376] 153. A pharmaceutical composition comprising a protein according to any one of embodiments 1 to 149 and an excipient. 154. A method for treating a subject suffering from an immune disorder or condition associated with T cell dysregulation, comprising administering to the subject an effective amount of a protein described in any one of embodiments 1 to 149 or a pharmaceutical composition described in embodiment 153.

[0377] 155. Immune disorders or conditions include type 1 diabetes mellitus, 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, Behçet's disease, Crohn's disease, Devic's disease, systemic lupus erythematosus (SLE), Dressler's syndrome, fibrosing alveolitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, IgA nephropathy, IgG4-related sclerosing disease, immunosuppressive agents ... 155. The method of embodiment 154, wherein the disease is infectious 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, Sjogren's syndrome, sperm or testicular autoimmunity, stiff-person syndrome (SPS), Takayasu's arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), or vasculitis.

[0378] 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.

[0379] 159. The method of embodiment 156, wherein the subject is a pediatric patient. 160. The method of any one of embodiments 156-158, wherein the subject is an adult patient.

[0380] 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.

[0381] 163. The method of embodiment 154, wherein the immune disorder or condition is graft-versus-host disease (GVHD). 164. The method according to any one of embodiments 154 to 163, wherein the protein according to any one of embodiments 1 to 149 or the pharmaceutical composition according to embodiment 153 is administered as a single dose.

[0382] 165. The method according to any one of embodiments 154 to 164, in which the administration of the protein according to any one of embodiments 1 to 149 or the pharmaceutical composition according to embodiment 153 is not repeated.

[0383] 166. The method of any one of embodiments 154 to 165, wherein the method suppresses a cellular immune response in the subject. 167. The method of embodiment 166, wherein the method suppresses the subject's immune system.

[0384] 168. The method of embodiment 167, wherein the method reduces T cell function in the subject. 169. The method of embodiment 167, wherein the method reduces B cell function in the subject.

[0385] 170. The method of embodiment 167, wherein the method reduces T cell responsiveness in the subject. 171. A method for suppressing a cellular autoimmune response in a subject, comprising administering to the subject an effective amount of a protein described in any one of embodiments 1 to 149 or a pharmaceutical composition of embodiment 153.

[0386] 172. The method of embodiment 171, wherein the method reduces T cell function in the subject. 173. The method of embodiment 171, wherein the method reduces B cell function in the subject.

[0387] 174. The method of embodiment 171, wherein the method reduces T cell responsiveness in the subject. 175. The method of any one of embodiments 154-174, further comprising administering an additional therapeutic agent to the subject.

[0388] 176. The method of embodiment 175, wherein the additional therapeutic agent is or comprises an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of the cell to an apoptosis-inducing agent.

[0389] 177. The method of embodiment 175, wherein the additional therapeutic agent is or comprises a CAR-expressing cell. 178. The method of embodiment 177, wherein the CAR-expressing cell is a CAR-expressing Treg cell.

[0390] 179. A method for local PD1 agonism, 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. 180. The method of embodiment 179, wherein administering the protein or pharmaceutical composition localizes PD1 agonism to the subject's B cells.

[0391] 181. A method for locally modulating an immune response in a target tissue or cell expressing CD20, 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.

[0392] 182. The method of embodiment 181, wherein administering the protein or pharmaceutical composition modulates an immune response in the subject's B cells. 183. A method for characterizing the ability of a molecule type I to stimulate PD1, comprising: (a) culturing type I cells stably expressing CD3, stably expressing an AP1-luciferase reporter gene, and stably expressing PD1 with type II cells stably expressing CD22 and stably expressing CD20; (b) incubating the cultured cells of step a) in the presence or absence of molecule type I and molecule type II; (c) after step b), measuring luciferase activity in the cultured cells, wherein type I of molecule is a multispecific antigen-binding molecule comprising i) a first binding specificity that binds to the ectodomain of CD20, and ii) a second binding specificity that binds to PD1, and type II of molecule is a multispecific antigen-binding molecule comprising i) a first binding specificity that binds to the ectodomain of CD3, and ii) a second binding specificity that binds to the ectodomain of CD22, wherein the presence of type II of molecule causes an increase in luciferase activity, and the presence of type I of molecule causes a reduction in luciferase activity caused by type II of molecule, and the amount of reduction in luciferase activity is indicative of the ability of type I of molecule to stimulate PD1.

[0393] 184. The method of embodiment 183, wherein PD1 is mPD1. 185. The method of embodiment 183, wherein the cell type I is Jurkat E6-1 cell.

[0394] 186. The method of embodiment 183, wherein type I cells are transduced to express the AP1-luciferase reporter gene. 187. The method of embodiment 183, wherein type I cells are transduced to express the PD1 gene.

[0395] 188. The method according to embodiment 186 or 187, wherein type I cells are transduced with a lentivirus. 189. The method of embodiment 183, wherein CD20 is mCD20.

[0396] 190. The method of embodiment 183, wherein the type II cells are HEK293 cells. 191. The method of embodiment 183, wherein type II cells are transduced to express CD22.

[0397] 192. The method of embodiment 183, wherein type II cells are transduced to express the CD20 gene. 193. The method according to embodiment 191 or 192, wherein type II cells are transduced with a lentivirus.

[0398] 194. The method according to embodiment 183, wherein cells of type II cells are seeded before cells of type I cells. 195. The method of embodiment 183, wherein the cultured cells are incubated in the presence of type II molecule, in the presence of a control antibody molecule, and in the absence of type I molecule.

[0399] 196. The method of embodiment 183, wherein the cultured cells are incubated in the presence of type I molecule and type II molecule. 197. The method of embodiment 183, wherein the type I molecule comprises the ectodomain of PDL1.

[0400] All publications, patents, patent applications, and other documents cited in this application are incorporated herein 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 a conflict between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. A protein, (a) at least one CD20 targeting moiety; (b) at least one PD1 agonist moiety; and (c) at least one dimerization moiety; and (d) optionally, one or more linker moieties separating one or more moieties in the protein; where: (i) the portions of the protein are arranged from N-terminus to C-terminus in the following order: CD20 targeting moiety-PD1 agonist moiety-dimerization moiety; or (ii) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the ectodomain 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 ectodomain of PDL1 or a PD1-binding portion thereof; or (iii) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the ectodomain of the PDL1 or a PD1-binding portion thereof, the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not at the N-terminus of the VH of the anti-CD20 Fab; (iv) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the ectodomain of PDL1 or a PD1-binding portion thereof, the dimerization moiety is an Fc domain, and the PD1 agonist moiety is not C-terminal to the Fc domain; (v) the CD20 targeting moiety is an anti-CD20 Fab, the PD1 agonist moiety is the ectodomain of PDL1 or a PD1-binding portion thereof, and the dimerization moiety is an Fc domain, and the protein is monovalent with respect to the CD20 targeting moiety and / or the PD1 agonist moiety; or (vi) or any combination of two or more of the foregoing (i) to (v).

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

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

4. the anti-CD20 antibody (a) selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, and veltuzumab; or (b) The protein of claim 3, which competes for binding to CD20 with an anti-CD20 antibody selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ublituximab, ocaratuzumab, TRU-015, and veltuzumab, and / or binds to the same epitope as the anti-CD20 antibody.

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

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

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

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

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

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

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

12. 10. The protein of claim 9, wherein the PD1-binding portion of the extracellular domain of PDL2 comprises or consists of the PDL2 ectodomain, and optionally PDL2 is human PDL2 or mouse PDL2.

13. The protein of any one of claims 1 to 4, comprising one or more linker moieties.

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

16. The protein of claim 13 , wherein at least one linker moiety comprises a glycine-serine linker.

17. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a PD1 agonist moiety, an optional linker moiety, and a dimerization moiety.

18. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, an optional linker moiety and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a PD1 agonist moiety, an optional linker moiety, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety.

19. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, an optional linker moiety and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, a dimerization moiety, and a PD1 agonist moiety.

20. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, an optional linker moiety and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, and a PD1 agonist moiety.

21. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a PD1 agonist moiety, an optional linker moiety, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety.

22. 22. The protein of claim 21, wherein the second monomer is composed of a single polypeptide chain or two polypeptide chains.

23. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, a dimerization moiety, and a PD1 agonist moiety.

24. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv, or scFv), an optional linker moiety, and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, a PD1 agonist moiety, an optional linker moiety, and a dimerization moiety.

25. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv, or scFv), an optional linker moiety, and a dimerization moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a PD1 agonist moiety, an optional linker moiety, a PD1 agonist moiety, an optional linker moiety, and a dimerization moiety.

26. 5. The protein of any one of claims 1 to 4, comprising two monomers each comprising or consisting of, in N-terminal to C-terminal orientation, a PD1 agonist portion, an optional linker portion, a CD20 targeting portion, an optional linker portion, and a dimerization portion.

27. 5. The protein of any one of claims 1 to 4, comprising two monomers each comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, a dimerization moiety, and a PD1 agonist moiety.

28. 5. The protein of any one of claims 1 to 4, comprising: (1) a first monomer comprising or consisting of, in N-terminal to C-terminal orientation, a CD20 targeting moiety, an optional linker moiety, a dimerization moiety, an optional linker moiety, and a CD20 targeting moiety; and (2) a second monomer comprising or consisting of, in N-terminal to C-terminal orientation, a PD1 agonist moiety, an optional linker moiety, a dimerization moiety, an optional linker moiety, and a PD1 agonist moiety.

29. 5. The protein of any one of claims 1 to 4, comprising two monomers each comprising or consisting of, in N-terminal to C-terminal orientation: a CD20 targeting moiety (e.g., an anti-CD20 Fab, Fv, or scFv), an optional linker moiety, and a PD1 agonist moiety.

30. The protein of any one of claims 1 to 4, wherein the CD20 targeting moiety binds to the extracellular domain of human CD20.

31. The protein of any one of claims 1 to 4, wherein the PD1 agonist portion stimulates human PD1.

32. The protein of any one of claims 1 to 4, wherein the CD20 targeting moiety binds to the extracellular domain of mouse CD20.

33. The protein of any one of claims 1 to 4, wherein the PD1 agonist portion stimulates mouse PD1.

34. The protein of any one of claims 1 to 4, wherein the at least one dimerization moiety is or comprises an Fc domain.

35. A nucleic acid or a plurality of nucleic acids encoding a protein according to any one of claims 1 to 4.

36. A host cell engineered to express a protein according to any one of claims 1 to 4 or a nucleic acid(s) encoding a protein according to any one of claims 1 to 4.

37. A method for producing a protein according to any one of claims 1 to 4, comprising culturing a host cell engineered to express the protein according to any one of claims 1 to 4, and recovering the protein expressed thereby.

38. A pharmaceutical composition comprising the protein of any one of claims 1 to 4 and an excipient.

39. 39. The pharmaceutical composition of claim 38 for treating a subject suffering from an immune disorder or condition associated with T cell dysregulation.

40. 40. The pharmaceutical composition of claim 39, wherein the immune disorder or condition is type 1 diabetes.

41. 40. The pharmaceutical composition of claim 39 for suppressing a cellular autoimmune response in a subject.

42. 42. The pharmaceutical composition of claim 41, which reduces T cell function, B cell function, or T cell responsiveness in the subject.

43. 1. A method for characterizing the ability of a molecule type I to stimulate PD1, comprising: (a) culturing type I cells stably expressing CD3, stably expressing an AP1-luciferase reporter gene, and stably expressing PD1 with type II cells stably expressing CD22 and stably expressing CD20; (b) incubating the cultured cells of step a) in the presence or absence of said type I molecule and type II molecule; (c) after step b), measuring luciferase activity in the cultured cells; the type I molecule is a multispecific antigen-binding molecule comprising i) a first binding specificity that binds to the ectodomain of CD20, and ii) a second binding specificity that binds to PD1; the type II molecule is a multispecific antigen-binding molecule comprising i) a first binding specificity that binds to the ectodomain of CD3, and ii) a second binding specificity that binds to the ectodomain of CD22; 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 the amount of decrease in luciferase activity indicates the ability of the type I molecule to stimulate PD1.

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