CD20-PD1 binding molecule and method of use

Abstract

This disclosure relates to molecules capable of binding to both CD20 and PD1, as well as nucleic acids encoding such molecules, pharmaceutical compositions comprising such molecules, and methods of using the same. In certain embodiments, a CD20-PD1 binding molecule is disclosed comprising at least one CD20 targeting moiety and at least one PD1 agonist moiety, wherein in certain embodiments, the PD1 agonist moiety is a PDL1 or PDL2 ectodomain, or its PD1 binding moiety.

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 501,252, filed on 10 May 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML sequence listing, created on April 25, 2024, is named RGN-030WO_SL and has a size of 139,033 bytes. [Background technology]

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

[0004] Both systemic and organ-specific autoimmune diseases are likely to be initially triggered by the recognition of self or foreign molecules by innate sensors. This recognition triggers an inflammatory response and involvement of previously quiescent autoreactive T and B cells (Non-Patent Literature 1). Autoreactivity ranges from low "physiological" levels of autoreactivity essential for lymphocyte selection and immune system homeostasis, to intermediate levels of autoimmunity that manifest as circulating autoantibodies and minor tissue infiltration without clinical consequences, and to pathogenic autoimmunity associated with immune-mediated organ injury (Non-Patent Literature 1). Autoimmune diseases are divided into organ-specific (e.g., type 1 diabetes (T1D), multiple sclerosis (MS), inflammatory bowel disease (IBD), myasthenia gravis) and systemic (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome) and can be mediated by autoantibodies or cytotoxic T cells, but all cases require helper T cells (Non-Patent Literature 1).

[0005] Most autoimmune diseases exhibit clinical heterogeneity, polygeneity, and multifactorial contributions often involving both genetic and environmental factors. Four mechanisms contribute to the control of autoreactive T and B cell escape: inhibitory molecules, anergy, neglect, and active suppression (Non-Patent Literature 1). Several inhibitory molecules (e.g., CTLA-4, PD-1, LAG-3, TIM3, VISTA, TIGIT, FcγRIIb, and certain Siglec) are expressed on the surface of T and B cells to suppress excessive immune responses, both normal and anti-auto. Deficiencies in some of these molecules result in autoimmunity, providing strong evidence that autoreactive lymphocytes are present in the peripheral repertoire but are normally under control. See Non-Patent Literature 2, 3, 4, 5, 6, and 7. Widespread immune-related adverse events frequently occur due to unchecked autoreactivity (Non-Patent Literature 8).

[0006] Existing treatments for autoimmune diseases have yielded limited results. For example, correcting organ-specific autoimmune diseases through metabolic regulation is often possible. When function is lost and cannot be recovered, mechanical replacements or tissue grafts may be appropriate. While this approach may alleviate some symptoms, there are no effective long-term curative treatments for some of the most debilitating autoimmune diseases. Many compounds, including insulin, corticosteroids, and modified beta-interferons, can improve some symptoms of autoimmune diseases, but they can have serious 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 a cure for these diseases, and their use is associated with many harmful side effects. These effects include nephrotoxicity, increased susceptibility to infection, and increased incidence of neoplasms.

[0007] Therefore, there is a need for novel therapeutic compositions and protocols that can be used to suppress immune responses and treat autoimmune diseases. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Theofilopolous,Kono,and Baccala,2017,Nat Immunol,18(17):716-724 [Non-Patent Document 2] Paterson and Sharpe,2010,Nat Immunol,11:109-111 [Non-Patent Document 3] Okazaki et al.,2013,Nat Immunol,14:1212-1218 [Non-Patent Document 4] Pincetic et al.,2014,Nat Immunol,15:707-716 [Non-Patent Document 5] Macauley, Crocker, and Paulson, 2914, Nat Rev Immunol, 14:653-666 [Non-Patent Document 6] Ceeraz et al.,2016,Arthritis Rheumatol 69(4):814-825 [Non-Patent Document 7] Schmitt et al.,2016,J Exp Med,213:1627-1644 [Non-Patent Document 8] Michot et al.,2016,Eur J Cancer,54:139-148 [Overview of the Initiative]

[0009] 4. Outline of the Invention This disclosure provides novel CD20-PD1 binding molecules. The CD20-PD1 binding molecules of this 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. In certain embodiments, the CD20-PD1 monomer also comprises one or more antigen-binding fragments of an agonist anti-PD1 antibody.

[0010] The CD20-PD1 binding molecules of the present disclosure are typically proteins 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 that separate one or more moieties in the protein. In some embodiments, the protein further comprises at least one antigen-binding fragment of an agonist anti-PD1 antibody. In certain embodiments, the CD20 targeting moiety is an anti-CD20 Fab or scFv, the PD1 agonist moiety is the IgV domain of PDL1 (or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions), and the dimerization moiety is an Fc domain. Thus, the CD20-PD1 binding domain is typically a dimer composed of two polypeptide chains each comprising (a) an Fc domain, (b) at least one CD20 targeting moiety, (c) at least one PD1 agonist moiety, and optionally, (d) an antigen-binding fragment of an agonist anti-PD1 antibody and / or (e) one or more linker moieties that separate one or more moieties in the polypeptide chain.

[0011] Exemplary CD20-PD1 binding molecules are disclosed in Sections 6.2 and Numbered Embodiments 1-159 below. Exemplary CD20 targeting moieties are disclosed in Section 6.3. Exemplary PD1 agonist moieties are disclosed in Section 6.4. Exemplary antigen-binding fragments of agonist anti-PD1 antibodies are disclosed in Section 6.5.

[0012] The present disclosure further provides a CD20-PD1 binding molecule, a CD20-PD1 monomer, and a nucleic acid encoding a CD20 targeting portion and a PD1 agonist portion. Nucleic acids encoding a CD20-PD1 binding molecule and a CD20-PD1 monomer composed of two or more polypeptide chains can be a single nucleic acid (e.g., a vector encoding all polypeptide chains) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting portions, and PD1 agonist portions of the present disclosure. The present disclosure further provides methods for producing the CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting portions, and PD1 agonist portions of the present disclosure. Exemplary nucleic acids, host cells, cell lines, and methods for producing CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting portions, and PD1 agonist portions are described in Sections 6.9 and numbered embodiments 160-162 below.

[0013] The present disclosure further provides a pharmaceutical composition comprising the CD20-PD1 binding molecule, CD20-PD1 monomer, CD20 targeting portion, and PD1 agonist portion of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.10 and numbered embodiment 163 below.

[0014] The present disclosure further provides methods of using the CD20-PD1 binding molecule, CD20-PD1 monomer, CD20 targeting portion, PD1 agonist portion, and pharmaceutical composition of the present disclosure, for example, for the treatment of autoimmune diseases, suppression of cellular autoimmune responses, or suppression of the immune system of a subject. Exemplary methods are described in Section 6.11 and numbered embodiments 164-186 below. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 5. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A]These are a series of sketches representing an exemplary format of a CD20-PD1 binding molecule according to a particular embodiment. The heavy chain variable domain of the CD20 targeting moiety is shown as a dotted line, the VH domain of the agonist anti-PD1 antibody is shown as a striped pattern, and the IgV domain of the PDL1 or PDL2 ectodomain is shown as a semicircle. The CD20 and PD1 targeting moieties are shown as Fab for convenience, but one or more Fabs shown in Figures 1A-1E may be replaced with any targeting moiety described herein, such as an Fv fragment or scFv. [Figure 1B] These are a series of sketches representing an exemplary format of a CD20-PD1 binding molecule according to a particular embodiment. The heavy chain variable domain of the CD20 targeting moiety is shown as a dotted line, the VH domain of the agonist anti-PD1 antibody is shown as a striped pattern, and the IgV domain of the PDL1 or PDL2 ectodomain is shown as a semicircle. The CD20 and PD1 targeting moieties are shown as Fab for convenience, but one or more Fabs shown in Figures 1A-1E may be replaced with any targeting moiety described herein, such as an Fv fragment or scFv. [Figure 1C] These are a series of sketches representing an exemplary format of a CD20-PD1 binding molecule according to a particular embodiment. The heavy chain variable domain of the CD20 targeting moiety is shown as a dotted line, the VH domain of the agonist anti-PD1 antibody is shown as a striped pattern, and the IgV domain of the PDL1 or PDL2 ectodomain is shown as a semicircle. The CD20 and PD1 targeting moieties are shown as Fab for convenience, but one or more Fabs shown in Figures 1A-1E may be replaced with any targeting moiety described herein, such as an Fv fragment or scFv. [Figure 1D]These are a series of sketches representing an exemplary format of a CD20-PD1 binding molecule according to a particular embodiment. The heavy chain variable domain of the CD20 targeting moiety is shown as a dotted line, the VH domain of the agonist anti-PD1 antibody is shown as a striped pattern, and the IgV domain of the PDL1 or PDL2 ectodomain is shown as a semicircle. The CD20 and PD1 targeting moieties are shown as Fab for convenience, but one or more Fabs shown in Figures 1A-1E may be replaced with any targeting moiety described herein, such as an Fv fragment or scFv. [Figure 1E] These are a series of sketches representing an exemplary format of a CD20-PD1 binding molecule according to a particular embodiment. The heavy chain variable domain of the CD20 targeting moiety is shown as a dotted line, the VH domain of the agonist anti-PD1 antibody is shown as a striped pattern, and the IgV domain of the PDL1 or PDL2 ectodomain is shown as a semicircle. The CD20 and PD1 targeting moieties are shown as Fab for convenience, but one or more Fabs shown in Figures 1A-1E may be replaced with any targeting moiety described herein, such as an Fv fragment or scFv. [Figure 2A] Traces from flow-binding studies are shown, representing mPDL1 binding (upper panel) or anti-mCD20 binding (lower panel) by the indicated CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain) on Jurkat / mPD1 and MC38 / mCD20 or HEK293 / mCD20 cells, respectively. [Figure 2B] Traces from flow-binding studies are shown, representing mPDL1 binding (upper panel) or anti-mCD20 binding (lower panel) by the indicated CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain) on Jurkat / mPD1 and MC38 / mCD20 or HEK293 / mCD20 cells, respectively. [Figure 3A] The luciferase assay protocol is shown. Figure 3A is a schematic representation of the luciferase reporter assay, and Figures 3B and 3C are graphic representations showing the interactions between the key players described in Figure 3A, each representing either the 2+1 or 2+2 alternative format molecules. [Figure 3B] The luciferase assay protocol is shown. Figure 3A is a schematic representation of the luciferase reporter assay, and Figures 3B and 3C are graphic representations showing the interactions between the key players described in Figure 3A, each representing either the 2+1 or 2+2 alternative format molecules. [Figure 3C] The luciferase assay protocol is shown. Figure 3A is a schematic representation of the luciferase reporter assay, and Figures 3B and 3C are graphic representations showing the interactions between the key players described in Figure 3A, each representing either the 2+1 or 2+2 alternative format molecules. [Figure 4-1] The test molecule (Figure 4A) and a series of traces using it are shown (Figures 4B-4E). The traces show mPD1 agonism measured using the bioassay shown in Figure 3. Cells and molecules used, as shown in each individual trace. [Figure 4-2] The test molecule (Figure 4A) and a series of traces using it are shown (Figures 4B-4E). The traces show mPD1 agonism measured using the bioassay shown in Figure 3. Cells and molecules used, as shown in each individual trace. [Figure 4-3] The test molecule (Figure 4A) and a series of traces using it are shown (Figures 4B-4E). The traces show mPD1 agonism measured using the bioassay shown in Figure 3. Cells and molecules used, as shown in each individual trace. [Figure 5] This document outlines the experimental design for a dose-escalation efficacy study in non-obese diabetic (NOD) mice before the onset of diabetes. [Figure 6A] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6B]We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6C] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6D] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6E] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6F] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6G] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6H] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 6I] We present a series of traces showing individual animal data demonstrating the development of spontaneous diabetes in the presence of the indicated control or the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). [Figure 7A]The graphs demonstrate the ability of the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain) (top: molecule L in Figure 2A, bottom: molecule G in Figure 2B) to regulate the development of diabetes in NOD mice. [Figure 7B] The graphs demonstrate the ability of the CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain) (top: molecule L in Figure 2A, bottom: molecule G in Figure 2B) to regulate the development of diabetes in NOD mice. [Figure 8A] This box plot shows the reduction in activated autoreactive islet-specific CD8+ T cell infiltration into NOD mouse pancreas after treatment with a CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). *p<0.05. [Figure 8B] This box plot shows the reduction in activated autoreactive islet-specific CD8+ T cell infiltration into NOD mouse pancreas after treatment with a CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). *p<0.05. [Figure 8C] This box plot shows the reduction in activated autoreactive islet-specific CD8+ T cell infiltration into NOD mouse pancreas after treatment with a CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). *p<0.05. [Figure 9A] This box plot shows the reduction in activated autoreactive CD3+, CD4+, and CD8+ T cell infiltration into the spinal cord of EAE-MS mice treated with a CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001. [Figure 9B] This box plot shows the reduction in activated autoreactive CD3+, CD4+, and CD8+ T cell infiltration into the spinal cord of EAE-MS mice treated with a CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001. [Figure 9C]This box plot shows the reduction in activated autoreactive CD3+, CD4+, and CD8+ T cell infiltration into the spinal cord of EAE-MS mice treated with a CD20-PD1 binding molecule (anti-mCD20 × mPDL1 ectodomain). *p<0.05, **p<0.01, ***p<0.001. [Figure 10] These are sketches representing exemplary constructs of CD20-PD1 binding molecules containing mouse PDL1-IgV ectodomains. Figure 10A shows the anti-mCD20×mPDL1 IgV ectodomain construct, AF-12.1, comprising anti-mCD20 Fab-10 aa linker-mPDL1 IgV ectodomain-5 aa linker-mIgG1. Figure 10B shows the anti-mCD20×mPDL1 IgV ectodomain construct, AF-12.2, comprising anti-mCD20 Fab-10 aa linker-mPDL1 IgV ectodomain-5 aa linker-hIgG1. Figure 10C shows the control mPDL1 IgV ectodomain construct, AF-17.1, comprising mPDL1 IgV ectodomain-5 aa linker-mIgG1. The heavy chain variable domain of the mouse anti-CD20 Fab domain is shown as a striped pattern, the light chain variable domain as a broken wave pattern, and the mouse PDL1 ectodomain as a semicircle with dots. [Figure 11]This is a schematic diagram representing exemplary constructs of CD20-PD1 binding molecules containing human PDL1-IgV ectodomains. Figure 11A shows the anti-hCD20×hPDL1 IgV ectodomain construct, AF-16.1, comprising anti-hCD20 Fab-10 aa linker-hPDL1 IgV ectodomain-5 aa linker-hIgG1. Figure 11B shows the anti-hCD20×hPDL1 IgV ectodomain construct, AF-16.2, comprising anti-hCD20 Fab-10 aa linker-hPDL1 IgV ectodomain-5 aa linker-hIgG1. Figure 11C shows the hPDL1 IgV ectodomain construct, AF-24.1, comprising anti-hPDL1 IgV ectodomain-5 aa linker-hIgG1. Figure 11D shows the hPDL1 IgV-anti-hPD1-anti-hCD20 construct, AF-69, comprising the components hPDL1 IgV ectodomain-18 aa linker-anti-hPD1 Fab-15 aa linker-anti-hCD20 Fab-hIgG1. Figure 11E shows the hPDL1 IgV-anti-hPD1-anti-hCD20 construct, AF-70, comprising the components anti-hPD1 Fab-10 aa linker-hPDL1 IgV ectodomain-10 aa linker-anti-hCD20 Fab-hIgG1. The heavy chain variable domain of the anti-hCD20 Fab domain is shown with a diamond pattern, the heavy chain variable domain of the anti-hPD1 Fab domain is shown with a striped pattern, and the human PDL1 ectodomain is shown with a semicircle with a blank circle pattern. [Figure 12A] Graphs showing the binding efficacy of mPDL1 IgV and mPDL1 full-length ectodomain-containing molecules are shown. Figures 12A and 12B show the binding efficacy of the evaluated constructs to mPD1-overexpressing Jurkat cells and mCD20-overexpressing MC38 cells, respectively, while Figures 12C and 12D show the binding efficacy of the same constructs to parental Jurkat cells and MC38 cells, respectively. [Figure 12B]Graphs showing the binding efficacy of mPDL1 IgV and mPDL1 full-length ectodomain-containing molecules are shown. Figures 12A and 12B show the binding efficacy of the evaluated constructs to mPD1-overexpressing Jurkat cells and mCD20-overexpressing MC38 cells, respectively, while Figures 12C and 12D show the binding efficacy of the same constructs to parental Jurkat cells and MC38 cells, respectively. [Figure 12C] Graphs showing the binding efficacy of mPDL1 IgV and mPDL1 full-length ectodomain-containing molecules are shown. Figures 12A and 12B show the binding efficacy of the evaluated constructs to mPD1-overexpressing Jurkat cells and mCD20-overexpressing MC38 cells, respectively, while Figures 12C and 12D show the binding efficacy of the same constructs to parental Jurkat cells and MC38 cells, respectively. [Figure 12D] Graphs showing the binding efficacy of mPDL1 IgV and mPDL1 full-length ectodomain-containing molecules are shown. Figures 12A and 12B show the binding efficacy of the evaluated constructs to mPD1-overexpressing Jurkat cells and mCD20-overexpressing MC38 cells, respectively, while Figures 12C and 12D show the binding efficacy of the same constructs to parental Jurkat cells and MC38 cells, respectively. [Figure 13] Figure 3 shows graphs illustrating the PD1 agonism of mPDL1 IgV and mPDL1 full-length ectodomain-containing molecules, measured using the bioassay shown. [Modes for carrying out the invention]

[0016] 6. Modes for Carrying Out the Invention 6.1.Definition Approximately, roughly: Terms such as "approximately" and "roughly" are used throughout the specification before numerical values ​​to indicate that the numerical values ​​are not necessarily exact (for example, to account for fractions, measurement accuracy and / or variations in accuracy, timing, etc.). It should be understood that a disclosure of "approximately X" or "roughly X," where X is a numerical value, is also a disclosure of "X." Therefore, for example, a disclosure of an embodiment in which one sequence has "approximately X% sequence identity" with another sequence is also a disclosure of an embodiment in which that sequence has "X% sequence identity" with that other sequence.

[0017] And and Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of a feature in a choice (choice A is mutually exclusive from choice B, A or B) and the selection of a combined feature (both A and B are selected, A or B). In some parts of the text, the terms "and / or" are used for the same purpose and should not be interpreted as meaning that "or" is used to refer to mutually exclusive choices.

[0018] 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 portions of a targeting moiety that can bind specifically, non-covalently, and reversibly to a target molecule.

[0019] Antigen-binding fragments of agonist anti-PD1 antibodies: The term “antigen-binding fragments of agonist anti-PD1 antibodies” refers to the binding portion of an immunoglobulin or its antigen-binding fragment that can bind to PD1 and stimulate PD1 (i.e., activate PD1 signaling when bound to PD1 expressed on the surface of immune cells). Antigen-binding fragments of agonist anti-PD1 antibodies can be in the form of Fab, Fv, or scFv. Antigen-binding fragments of agonist anti-PD1 antibodies are further described in Section 6.5.

[0020] Associated: In the context of a CD20-PD1 binding molecule or its components (e.g., CD20 targeting moiety, PD1 agonist moiety, dimerizing moiety), the term “associated” refers to a functional relationship between two or more polypeptide chains or parts of polypeptide chains. In particular, the term “associated” means that two or more polypeptides associate with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide crosslinks or chemical crosslinks, to form a functional CD20-PD1 binding molecule. Examples of associations that may exist in the CD20-PD1 binding molecules of this disclosure include, but are not limited to, associations between homodimer or heterodimer Fc domains within an Fc domain, associations between VH and VL domains within a Fab or scFv, associations between CH1 and CL within a Fab, and associations between CH3 and CH3 within a domain-substituted Fab.

[0021] Divalent: As used herein with respect to CD20-PD1 binding molecules with respect to the CD20 targeting moiety and / or PD1 agonist moiety, the CD20-PD1 binding molecule means that it 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). The CD20-PD1 binding molecule may be divalent with respect to one type of moiety (e.g., the CD20 targeting moiety) and monovalent with respect to the other type of moiety (e.g., the PD1 agonist moiety).

[0022] CD20-PD1 binding molecule: The term "CD20-PD1 binding molecule" refers to a molecule that contains at least one CD20 targeting moiety and at least one PD1 agonist moiety. Generally, a CD20-PD1 binding molecule is a molecule composed of one or more polypeptide chains (e.g., one, two, three, or four polypeptide chains) that together contain at least one CD20 targeting moiety and at least one PD1 agonist moiety.

[0023] In relation to the CD20-PD1 binding molecules of this disclosure, the term “CD20-PD1 binding molecule” may refer to the core components of the molecule, namely the CD20 targeting moiety and the PD1 agonist moiety, as well as dimerizing moieties such as the Fc domain and any / or associated linker moiety. Unless otherwise indicated in the context, the term “CD20-PD1 binding molecule” should be understood to also extend to molecules containing additional features, such as one or more stabilizing moieties, one or more dimerizing moieties, one or more linker moieties, and any combination thereof.

[0024] CD20 Targeting Molecule: The term “CD20 targeting molecule” refers to any molecule or its binding portion that can bind to CD20 (e.g., an immunoglobulin or its antigen-binding fragment). In some embodiments, the CD20 targeting molecule includes an antigen-binding fragment of an anti-CD20 antibody. The CD20-binding fragment of an anti-CD20 antibody may be in the form of a Fab, Fv, or scFv. The term “CD20 targeting molecule” includes any molecule that can bind to any domain or region of CD20, including a topological domain or a transmembrane domain. In some embodiments, the CD20 targeting molecule is a molecule that can bind to an extracellularly exposed region of CD20 on the surface of a cell (e.g., a B cell). CD20 targeting molecules are further described in Section 6.3.

[0025] Complementarity-Determining Regions or CDRs: The term “complementarity-determining region” or “CDR” as used herein refers to the sequence of amino acids within the antibody variable region that confer 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. For example, see 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.USA86:9268-9272 (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.

[0026] Dimerizing moiety: The term “dimerizing moiety” refers to a polypeptide chain or amino acid sequence that can facilitate association between two polypeptide chains to form a dimer. A first dimerizing moiety can associate with the same second dimerizing moiety, or with a second dimerizing moiety different from the first. In some embodiments, the dimerizing 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.

[0027] 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 midway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody or CD20-PD1 binding molecule at which 50% of its maximum effect is observed. In certain embodiments, the EC50 value is equal to the concentration of the CD20-PD1 binding molecule that gives the maximum half of the activation in a luciferase reporter assay.

[0028] Ectodomain: As used herein, the term “ectodomain” refers to a region of a transmembrane protein (e.g., PDL1 or PDL2) that lies outside the membrane when expressed on the surface of a cell. Ectodomains often contain a binding domain that specifically binds to a ligand or cell surface receptor. The term “ectodomain” is used herein for convenience to refer not only to the ectodomain of a transmembrane protein, but also, additionally, to fragments and variant sequences that possess target-binding activity.

[0029] Epitope: An epitope, or antigenic determinant, is a portion of an antigen (e.g., CD20) recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or three-dimensional.

[0030] Fab: In relation to the CD20-targeting portion of this disclosure, the term “Fab” refers to a pair of polypeptide chains, the first polypeptide chain comprising the variable heavy chain (VH) domain at the N-terminus of the antibody to the first constant domain (referred to herein as C1), and the second polypeptide chain comprising the variable light chain (VL) domain at the N-terminus of the antibody to the second constant domain (referred to herein as C2) that can pair with the first constant domain. In natural antibodies, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is located at the N-terminus of the constant domain (CL) of the light chain. The Fab of this disclosure may be positioned according to the natural orientation, or may include domain substitutions or swaps to facilitate correct VH and VL pairing. For example, the CH1 and CL domain pairs in the Fab may be replaced with CH3 domain pairs to facilitate correct modified Fab-chain pairing in a heterodimeric molecule. It is also possible to reverse CH1 and CL, attaching CH1 to VL and CL to VH, a configuration generally known as Crossmab, a type of "domain exchange."

[0031] Fc domains and Fc regions: The term "Fc domain" refers to a portion of a heavy chain that pairs with a corresponding portion of another heavy chain. The term "Fc region" refers to a 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 or distinct from one another. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified, for example, via knob-in-hole interactions to enable heterodimerization and / or via star mutations to enable purification.

[0032] Host cells or recombinant host cells: The terms “host cells” and “recombinant host cells,” as used herein, refer to cells that have been genetically modified, for example, by the introduction of heterologous nucleic acids. It should be understood that such terms are intended to refer not only to specific target cells but also to the offspring of such cells. Such offspring may not be identical to the parent cells in practice, as certain modifications can occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of the term “host cells” as used herein. Host cells can harbor heterologous nucleic acids, for example, transiently on extrachromosomal heterologous expression vectors, or stably, for example, by incorporating heterologous nucleic acids into the host cell genome. For the purpose of expressing CD20-PD1 binding molecules, host cells can be mammalian-derived or mammalian-like cell lines, e.g., 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.

[0033] Monomers and CD20-PD1 Monomers: 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 can associate with a second polypeptide chain, (b) comprises at least one PD1 agonist moiety and can associate with a second polypeptide chain, (c) comprises a dimerizing moiety (e.g., an Fc domain) and can associate with a corresponding dimerizing moiety (e.g., another Fc domain) on a second polypeptide chain, or (d) comprises any combination of (a), (b), and (c) above. A monomer can associate with other monomers via pairs of dimerizing moieties (e.g., Fc domains). In some embodiments, associations between one or more monomers are stabilized through a hinge sequence or other parts of the Fc domain. Thus, monomers of the present disclosure can associate with other monomers to form dimers. A dimer can be a homodimer, where each constituent monomer is identical, or a heterodimer, where each constituent monomer is different. Where used herein, references to “monomers” are for convenience only and do not preclude the presence of one or more additional polypeptide chains, e.g., one or more light chains of one or more Fab domains. Therefore, a “dimer” of two monomers may contain more than two polypeptide chains, e.g., three, four or more polypeptide chains, and references to monomers or dimers are not intended to imply a chronological order of association between polypeptide chains.

[0034] Monovalent: As used herein with respect to CD20-PD1 binding molecules with respect to the CD20 targeting moiety and / or PD1 agonist moiety, the term "monovalent" means that the CD20-PD1 binding molecule has, respectively, 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). The CD20-PD1 binding molecule may be monovalent with respect to one type of moiety (e.g., a PD1 agonist moiety) and divalent with respect to another type of moiety (e.g., a CD20 targeting moiety).

[0035] Polyvalent: As used herein with respect to CD20-PD1 binding molecules with respect to the CD20 targeting moiety and / or PD1 agonist moiety, the term “polyvalent” 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). The CD20-PD1 binding molecule may be polyvalent with respect to one type of moiety (e.g., the CD20 targeting moiety) and monovalent with respect to another type of moiety (e.g., the PD1 agonist moiety).

[0036] Operablely 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, for example, to produce an intraframe fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0037] PD1 agonist moiety: The term “PD1 agonist moiety” refers to a molecule or portion that can bind to PD1 and stimulate PD1, wherein PD1 (1) comprises an amino acid sequence having at least 70% sequence identity to the IgV domain of human or mouse programmed death ligand 1 (PDL1) or human or mouse programmed death ligand 2 (PDL2), and (2) lacks an amino acid sequence having at least 70% sequence identity to human or mouse PDL1 or the membrane-proximal portion of human mouse PDL1. As described herein, the “membrane-proximal portion” of PDL1 or PDL2 refers to the most C-terminal region of the extracellular domain of PDL1 or PDL2 following the IgV domain. In some embodiments, the membrane-proximal portion of PDL1 or PDL2 is the most C-terminal 20, 40, 60, or 80 amino acids of the extracellular domain of PDL1 or PDL2. Therefore, in certain embodiments, the PD1 agonist moiety is a molecule that (1) contains an amino acid sequence having at least 70% sequence identity to the IgV domain of human or mouse PDL1 or human or mouse PDL2, and (2) lacks an amino acid sequence having at least 70% sequence identity to the 20, 40, 60, or 80 amino acids at the very C-terminus of the extracellular domain of human or mouse PDL1 or human or mouse PDL2. In some embodiments, the PD1 agonist moiety contains an amino acid sequence having at least 70% sequence identity to the IgV domain of mammalian PDL1 (e.g., human PDL1 or mouse PDL1). In other embodiments, the PD1 agonist moiety contains an amino acid sequence having at least 70% sequence identity to the IgV domain of 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 herein for convenience to refer not only to the PDL1 and PDL2 ectodomains but also to fragments and variant sequences that additionally possess PD1-binding activity.Therefore, references to the terms "PDL1 ectodomain" and "PDL2 ectodomain" in this specification are intended to encompass the PD1-binding portions of the PDL1 and PDL2 ectodomains, as well as their variants that possess PD1-binding function.

[0038] The ectodomains of PDL1 and PDL2 consist of IgC and IgV domains. Without being constrained by theory, it is understood that PDL1 and PDL2 interact with PD1 via their IgV domains. In some embodiments, the PD1 agonist moiety of this disclosure is a PDL1 ectodomain comprising or consisting of the PDL1 IgV domain or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions. In other embodiments, the PD1 agonist moiety of this disclosure is a PDL2 ectodomain comprising or consisting of the PDL2 IgV domain or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions.

[0039] CD20-PD1 binding molecules may include a PD1 agonist moiety 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 moiety is a mouse PDL1 IgV domain containing a C113S substitution.

[0040] Single-stranded Fv or scFv: As used herein, the terms "single-stranded Fv" or "scFv" refer to a polypeptide chain containing the VH and VL domains of an antibody, where these domains are present in a single-strand polypeptide.

[0041] Subject: The term “subject” includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals (non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles). Unless otherwise stated, the terms “patient” and “subject” are used interchangeably herein.

[0042] To treat, to cure, to treat: As used herein, the terms “to treat,” “to cure,” and “to treat” mean, resulting from the administration of a molecule or composition (e.g., one or more CD20-PD1 binding molecules of this disclosure), a reduction or improvement in the progression, severity, and / or duration of a disorder described herein, improvement of one or more symptoms (preferably one or more identifiable symptoms) of a condition or disorder described herein, or prevention of a condition or disorder described herein, such as an autoimmune or inflammatory condition or disorder. In certain embodiments, the terms “to treat,” “to cure,” and “to treat” mean improvement of at least one measurable physical parameter of a disorder (e.g., an autoimmune disorder) that is not necessarily recognizable by the patient. In other embodiments, the terms “to treat,” “to cure,” and “to treat” mean inhibiting the progression or onset of a disorder by physical means (e.g., by stabilization of recognizable symptoms), physiological means (e.g., by stabilization of physical parameters), or both.

[0043] Universal Light Chain: As used herein in relation to the targeted moiety, the term “universal light chain” refers to a light chain polypeptide that can pair with the heavy chain region of the targeted moiety and can also pair with other heavy chain regions. Universal light chains are also known as “common light chains.”

[0044] VH: The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, which includes the scFv or Fab heavy chain. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the scFv or Fab light chain.

[0045] 6.2.CD20-PD1 binding molecule This disclosure provides a CD20-PD1 binding molecule 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 dimerized moiety. In further embodiments, the CD20-PD1 binding molecule further comprises an antigen-binding fragment of an agonist anti-PD1 antibody. The CD20-PD1 binding molecules of this disclosure typically comprise or consist of a CD20-PD1 binding molecule monomer comprising one or more CD20 targeting moieties and / or one or more PD1 agonist moieties.

[0046] In some embodiments, the PD1 agonist moiety is located between the CD20 targeting moiety and the dimerization moiety of the CD20-PD1 binding molecule monomer. In such embodiments, the CD20-PD1 binding molecule monomer thus has an N-terminus-C-terminus orientation of CD20 targeting moiety-PD1 agonist moiety-dimerization moiety. The CD20-PD1 binding molecule may further include an antigen-binding fragment of an agonist anti-PD1 antibody located at the N-terminus relative to the PD1 agonist moiety.

[0047] In other embodiments, the CD20-targeting moiety is located between the PD1 agonist moiety and the dimerized moiety of the CD20-PD1 binding molecule monomer. In such embodiments, the CD20-PD1 binding molecule monomer thus has an N-terminus to C-terminus orientation of PD1 agonist moiety-CD20-targeting moiety-dimerized moiety. The CD20-PD1 binding molecule may further include an antigen-binding fragment of an agonist anti-PD1 antibody located between the PD1 agonist moiety and the CD20-targeting moiety.

[0048] An exemplary dimerizing moiety is described in Section 6.6 and includes an Fc domain that confers homodimerizing or heterodimerizing ability to the CD20-PD1 binding molecule. A CD20-PD1 binding molecule can consist of one or more polypeptides. In some embodiments, a CD20-PD1 binding molecule consists of multiple (e.g., two) monomers, each containing at least one CD20 targeting moiety and / or at least one PD1 agonist moiety, and in some embodiments, also includes a dimerizing moiety. In some embodiments, the CD20-PD1 binding molecule of the present disclosure consists of two monomers associated with one or more additional polypeptide chains (e.g., polypeptide chains containing the light chain of an anti-CD20Fab moiety). The monomers may be identical, thereby forming a homodimer, or they may be different, thereby forming a heterodimer. The dimerizing moieties of each monomer in the CD20-PD1 binding molecule can be configured to dimerize together. Exemplary dimerizing moieties are described in Section 6.6.

[0049] One or more CD20-targeting moieties and one or more PD1 agonist moieties can be included in the same arm of a CD20-PD1 binding molecule (for example, if the CD20-targeting moiety includes anti-CD20Fab and the PD1 agonist moiety includes a PDL1-based PD1 agonist moiety, then the variable heavy or light chain of the anti-CD20Fab and the PDL1-based PD1 agonist moiety are on the same polypeptide chain), or they can be included in different arms of a bispecific CD20-PD1 agonist (for example, if the CD20-targeting moiety includes anti-CD20Fab and the PD1 agonist moiety includes a PDL1-based PD1 agonist moiety, then the variable heavy or light chain of the anti-CD20Fab and the PDL1-based PD1 agonist moiety are on different polypeptide chains).

[0050] A CD20-PD1 binding molecule can be monovalent with respect to the CD20 targeting moiety (i.e., having a single CD20 targeting moiety) or polyvalent with respect to the CD20 targeting moiety (i.e., having multiple CD20 targeting moieties). Similarly, a CD20-PD1 binding molecule can be monovalent with respect to the PD1 agonist moiety (i.e., having a single PD1 agonist moiety) or polyvalent with respect to the PD1 agonist moiety (i.e., having multiple PD1 agonist moieties). In some embodiments, a CD20-PD1 binding molecule is divalent with respect to the CD20 targeting moiety (i.e., having two CD20 targeting moieties). In some embodiments, a CD20-PD1 binding molecule is divalent with respect to the PD1 agonist moiety (i.e., having two PD1 agonist moieties). If the CD20-PD1 binding molecule is polyvalent with respect to the CD20 targeting moiety and / or the PD1 agonist moiety, then the multiple CD20 targeting moieties may be the same or different from one another, and / or the multiple PD1 agonist moieties may be the same or different from one another.

[0051] In some embodiments, the CD20-PD1 binding molecule may include various components of one or more polypeptide chains, for example: (1) a CD20 targeting moiety or a portion thereof (e.g., the heavy or light chain of anti-CD20 Fab) and a PD1 agonist moiety or a portion thereof (e.g., PDL1 or PDL2), if they are located on the same polypeptide chain; (2) a CD20 targeting moiety and a dimerization domain (e.g., the Fc domain); (3) a PD1 agonist moiety and a dimerization domain (e.g., the Fc domain); (4) an antigen-binding fragment of an agonist anti-PD1 antibody and a PD1 agonist moiety; (5) an antigen-binding fragment of an agonist anti-PD1 antibody and an antigen-binding fragment of a CD20 agonist moiety; or (6) one or more linker sequences connecting any of the aforementioned combinations. Exemplary linkers are described in Section 6.8.

[0052] Most CD20-PD1 binding molecules are polymers formed by the association of dimerizing moieties (e.g., Fc domains) configured to associate with each other. CD20-PD1 binding molecules may comprise two, three, four, or more polypeptide chains, some of which associate via dimerizing moieties and others via VH-VL interactions. For convenience and descriptive purposes only, this disclosure generally refers to polypeptides comprising a CD20 targeting moiety, a PD1 agonist moiety, and / or a dimerizing moiety (e.g., a first Fc domain) that can associate with another polypeptide chain comprising a CD20 targeting moiety, a PD1 agonist moiety, and / or a corresponding dimerizing moiety (e.g., a second Fc domain). A monomer may comprise one, two, three, or more polypeptide chains. For example, in one embodiment, the monomer may consist of (a) a first polypeptide chain containing an anti-CD20VH, a PD1 agonist moiety, and an Fc domain, and (b) a second polypeptide chain containing a VL that can pair with the anti-CD20VH. In another embodiment, the monomer may consist of (a) a first polypeptide chain containing a first anti-CD20VH, a second anti-CD20VH, and an Fc domain, (b) a second polypeptide chain containing a first VL that can pair with the first anti-CD20VH, and (c) a third polypeptide chain containing a second VL that can pair with the second anti-CD20VH.

[0053] The following are some exemplary examples of the monomers of this disclosure, described in N-terminal to C-terminal orientation. The CD20-PD1 bound molecules of this disclosure may comprise any two of the exemplary monomers referenced below, whether two of the same monomer or two different monomers. The individual elements of each monomer are described in detail herein, for example, in the following subsections and numbered embodiments.

[0054] (1) Exemplary monomer 1: CD20 targeting moiety - optional linker - PD1 agonist moiety - optional linker - dimerizing moiety (see, for example, Figure 1A, both monomers).

[0055] (2) Exemplary monomer 2: PD1 agonist moiety - optional linker - antigen-binding fragment of agonist anti-PD1 antibody - optional linker - CD20 targeting moiety - optional linker - dimerization moiety (see, for example, Figure 1B, both monomers).

[0056] (3) Exemplary monomer 3: PD1 agonist moiety - optional linker - CD20 targeting moiety - optional linker - antigen-binding fragment of agonist anti-PD1 antibody - optional linker - dimerization moiety (see, for example, Figure 1C, both monomers).

[0057] (4) Exemplary monomer 4: Antigen-binding fragment of agonist anti-PD1 antibody - optional linker - PD1 agonist moiety - optional linker - CD20 targeting moiety - optional linker - dimerization moiety (see, for example, Figure 1D, both monomers).

[0058] (5) Exemplary monomer 5: CD20 targeting moiety - optional linker - PD1 agonist moiety - optional linker - antigen-binding fragment of agonist anti-PD1 antibody - optional linker - dimerization moiety (see, for example, Figure 1E, both monomers).

[0059] In some embodiments, the disclosure provides a CD20-PD1-binding molecule comprising two monomers, represented by exemplary monomer 1 (see, for example, Figure 1A). In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising two monomers, represented by exemplary monomer 2 (see, for example, Figure 1B).

[0060] In some embodiments, the present disclosure provides a CD20-PD1 conjugated molecule comprising two monomers, represented by exemplary monomer 3 (see, for example, Figure 1C). In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising two monomers by exemplary monomer 4 (see, for example, Figure 1D).

[0061] In some embodiments, the present disclosure provides a CD20-PD1 binding molecule comprising two monomers, represented by exemplary monomer 5 (see, for example, Figure 1E). In the CD20-PD1 binding molecules of this disclosure, if the CD20 targeting moiety is the antigen-binding domain ("ABD") of an antibody, each monomer may consist of two or more polypeptide chains, including one polypeptide chain having a heavy chain variable region and other polypeptide chains having light chain variable regions. The CD20 targeting moiety may include heavy and light chain variable domains on separate polypeptide chains. For example, the monomer may consist of polypeptide A and polypeptide B. Polypeptide A may include, for example, the heavy chain variable domain of the CD20 targeting moiety - an optional linker - a PD1 agonist moiety - an optional linker - a dimerization moiety from the N-terminus to the C-terminus, and polypeptide B may include the light chain variable domain of the CD20 targeting moiety. If the monomer is divalent with respect to the CD20 targeting moiety, the monomer may include a third polypeptide chain (polypeptide C) containing another light chain variable domain of the CD20 targeting moiety.

[0062] Alternatively, the CD20-targeting portion may be in the form of an scFv, where the heavy and light chain variable regions of the CD20-targeting portion are fused together in a single polypeptide.

[0063] Further details of the components of the CD20-PD1 binding molecule of this disclosure are shown below. 6.3.CD20 targeting moiety: The incorporation of the CD20-targeting moiety in the CD20-PD1 binding molecule of this disclosure provides, in some embodiments, the delivery of high concentrations of a topical PD1 agonist moiety for the activation of the immune system and the treatment of autoimmune disorders including, but not limited to, type 1 diabetes, systemic lupus erythematosus, and Crohn's disease, as well as graft-versus-host disease (GVHD). In some embodiments, in addition to facilitating the topical delivery of the PD1 agonist moiety, the anti-CD20 moiety provides an additional therapeutic pathway for such autoimmune diseases.

[0064] In certain embodiments of this disclosure, each CD20-targeting moiety of the CD20-PD1 binding molecule contains an antigen-binding domain of an anti-CD20 antibody. In some embodiments, the CD20-PD1 binding molecule of this disclosure comprises a single CD20-targeting moiety (e.g., in embodiments where the CD20-PD1 binding molecule is monovalent with respect to the CD20-targeting moiety, the CD20-targeting moiety is on a first monomer or a second monomer). In some embodiments, the CD20-PD1 binding molecule of this disclosure comprises two CD20-targeting moieties (e.g., in embodiments where the CD20-PD1 binding molecule is divalent with respect to the CD20-targeting moiety, the first CD20-targeting moiety is on a first monomer and the second CD20-targeting moiety is on a second monomer, or both the first and second CD20-targeting moieties are on either the first or second monomer). In such embodiments, the two CD20-targeting moieties may be identical or different. If different, the two CD20 targeting regions may be orthogonal, may bind to separate epitopes of CD20, and / or may be non-competitive.

[0065] In some embodiments, the CD20-targeting moiety includes 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, ubrituximab, okalatuzumab, TRU-015, and bertuzumab (each a “reference CD20 antibody”). Additional exemplary reference anti-CD20 antibodies are provided in Table T below.

[0066] [Table 1]

[0067] In further embodiments, the CD20-targeting moiety includes a CDR having the CDR sequence of a reference anti-CD20 antibody (e.g., the anti-CD20 antibody shown in Table T). In some embodiments, the CD20-targeting moiety includes all six CDR sequences of the reference anti-CD20 antibody. In other embodiments, the targeting moiety includes at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the reference anti-CD20 antibody and the light chain CDR sequence of the universal light chain. In further embodiments, the CD20-targeting moiety includes a VH having the amino acid sequence of the VH of the reference anti-CD20 antibody. In some embodiments, the CD20-targeting moiety further includes a VL having the amino acid sequence of the VL of the reference anti-CD20 antibody. In other embodiments, the targeting moiety further includes the universal light chain VL sequence.

[0068] In other embodiments, the CD20-targeting moiety includes an antigen-binding domain that binds to the same region of CD20 as rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or vertuzumab, 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 conjugated to a solid support. A first antibody and a second antibody are then added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies bind to separate sites on CD20, the labeled antibody will bind at the same level regardless of the presence of the unlabeled antibody. 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 decrease. If an excess of unlabeled antibody is present, the labeled antibody will bind, if any, only in very small amounts. In some embodiments, the competing antibody is one that reduces the binding of another antibody to CD20 by about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. Details of the procedures for performing such a competing assay 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 performed quantitatively 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 compared to the concentrations required to achieve the desired degree of binding inhibition.In some embodiments, competition for binding to the target molecule can be determined, for example, using a real-time, label-free biolayer interference assay on the Octet HTX biosensor platform (Pall ForteBio Corp.).

[0069] A preferred format for the CD20-targeting moiety is 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.

[0070] 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 typically consists of multiple polypeptide chains, as described in Section 6.2.

[0071] 6.3.1. Format of the CD20 Targeted Section In certain embodiments, the CD20-targeting moiety can be any type of antibody or fragment thereof that retains specific binding to CD20. In some embodiments, the antigen-binding moiety is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more specifically an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments can be, but are not limited to, VH (or V H ) Fragment, VL (or V L Examples include the ) fragment, Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, minibody, diabody, triabody, and tetrabody.

[0072] 6.3.1.1.Fab Traditionally, Fab domains have been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the CD20-PD1 binding molecules of this disclosure, the Fab domain is typically expressed recombinantly as part of the CD20-PD1 binding molecule.

[0073] The Fab domain may contain constant domain sequences and variable region sequences from any suitable species, and may therefore be mouse, chimeric, human, or humanized. In some embodiments, the variable region sequence and / or constant domain region sequence are derived from known anti-CD20 antibodies. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, and beltuzumab.

[0074] In some embodiments, the CD20-targeting moiety includes Fabs that bind to the same CD20 epitope as the Fabs of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or bertuzumab (each a "reference CD20 antibody"), and / or compete with these Fabs for binding to CD20. In further embodiments, the CD20-targeting moiety includes a CDR having the CDR sequence of the reference CD20 antibody. In some embodiments, the CD20-targeting moiety includes all six CDR sequences of the reference CD20 antibody. In other embodiments, the targeting moiety includes at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the reference CD20 antibody and the light chain CDR sequence of the universal light chain. In further embodiments, the CD20 targeting moiety includes VH, which comprises the amino acid sequence of VH of a reference CD20 antibody. In some embodiments, the CD20 targeting moiety further includes VL, which comprises the amino acid sequence of VL of a reference CD20 antibody. In other embodiments, the targeting moiety further includes the universal light chain VL sequence.

[0075] The Fab domain typically contains a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulin, 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.

[0076] For the CD20-PD1 binding molecules of this disclosure, particularly when the light chains are not common or universal light chains, it is advantageous to use Fab heterodimerization strategies to enable precise association of Fab domains belonging to the same ABD and to minimize anomalous pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategies shown in Table 1 below can be used:

[0077] [Table 2]

[0078] Therefore, in certain embodiments, the correct association between two polypeptides of Fab is facilitated, for example, by exchanging the VL and VH domains of Fab with each other, or by exchanging the CH1 and CL domains with each other, as described in WO2009 / 080251.

[0079] Correct Fab pairing can also be facilitated by introducing one or more amino acid modifications to the CH1 domain and one or more amino acid modifications to the CL domain of the Fab, and / or by introducing one or more amino acid modifications to the VH domain and one or more amino acid modifications to the VL domain. The amino acids to be modified are typically those that are part of the VH:VL and CH1:CL interfaces so that the Fab components pair preferentially with each other rather than with other Fab components.

[0080] In one embodiment, one or more amino acid modifications are limited to conserved framework residues in 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 Kabat, Chothia, and IMGT numbering schemes.

[0081] In one embodiment, modifications introduced in the VH and CH1 domains and / or VL and CL domains are complementary to each other. Complementarity at the heavy-light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or various combinations of 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, all of which suggest a structural and chemical correspondence between two interacting surfaces.

[0082] In one embodiment, one or more introduced modifications introduce new hydrogen bonds across the interfaces of Fab components. In one embodiment, one or more introduced modifications introduce new salt bridges across the interfaces of Fab components. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, which are incorporated herein by reference.

[0083] In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, thereby introducing a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0084] In some embodiments, the Fab domain includes 143Q and 188V substitutions within the CH1 domain, as well as 113T and 176V substitutions within the CL domain, which helps to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0085] In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that facilitate the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, the 39K, 62E modifications are introduced into the VH domain, the H172A, F174G modifications into the CH1 domain, the 1R, 38D, (36F) modifications into the VL domain, and the L135Y, S176W modifications into the CL domain. In another embodiment, the 39Y modification is introduced into the VH domain and the 38R modification is introduced into the VL domain.

[0086] The Fab domain can also be modified to replace the natural CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of Fab components. For example, the engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, for example, Mazor et al., 2015, MAbs 7:377-89).

[0087] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that facilitate correct assembly. For example, Wu et al., 2015, MAbs 7:364-76 describe replacing the CH1 domain with the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, and pairing these domain substitutions with additional charge-charge interactions between the VL and VH domains by introducing 38D modification to the VL domain and 39K modification to the VH domain.

[0088] Instead of, or in addition to, the use of 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 molecule of this disclosure. In various embodiments, using the common light chain described herein reduces the number of inappropriate species of the CD20-PD1 binding molecule compared to using the original congeneral VL. In various embodiments, the VL domain of the CD20-PD1 binding molecule is identified from a monospecific antibody containing the common light chain. In various embodiments, the VH region of the CD20-PD1 binding molecule contains a limited human light chain repertoire, or a human heavy chain variable gene segment rearranged in vivo in mouse B cells pre-engineered to express a single human light chain, which is congeneral to the human heavy chain and generates an antibody repertoire containing one or more human VHs congenerate to one of two possible human VLs in response to exposure to the antigen of interest, and this antibody repertoire is specific to the antigen of interest. The common light chain is derived from a reconstituted human Vκ1-39Jκ5 sequence or a reconstituted human Vκ3-20Jκ1 sequence, including somatic mutants (e.g., affinity matured mutants). See, for example, U.S. Patent No. 10,412,940.

[0089] 6.3.1.2.scFv Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain and can be expressed as single-chain polypeptides, retaining the specificity of the intact antibody from which they originate. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for target binding. Examples of suitable linkers for linking the VH and VL chains of scFv are the linkers identified in Section 6.8.

[0090] As used herein, unless otherwise specified, scFv may have a VL variable region and a VH variable region in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and scFv may contain a VL-linker-VH or a VH-linker-VL.

[0091] The scFv may include VH and VL sequences from any preferred species, such as mouse, human, or humanized VH and VL sequences. In some embodiments, the scFv may include VH and VL sequences from known anti-CD20 antibodies. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, and beltuzumab.

[0092] In some embodiments, the CD20-targeting moiety includes scFvs that bind to the same CD20 epitope as scFvs derived from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or vertuzumab, and / or compete for binding to CD20.

[0093] To create nucleic acids encoding scFv, DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding one of the linkers described in Section 6.8 (typically a repeat of a sequence containing amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 50)), thereby allowing the VH and VL sequences to be expressed as a continuous single-stranded protein having VL and VH regions linked 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).

[0094] 6.4. PD1 Agonist Section In certain embodiments of the present disclosure, the PD1 agonist moiety of the CD20-PD1 binding molecule includes a wild-type or variant PD1 binding domain of programmed dead ligand 1 (PDL1) or programmed dead ligand 2 (PDL2). In some embodiments, the CD20-PD1 binding molecule of the present disclosure includes a single PD1 agonist moiety (e.g., a PD1 agonist moiety on a first monomer or a second monomer in embodiments where the CD20-PD1 binding molecule is monovalent to the PD1 agonist moiety). In some embodiments, the CD20-PD1 binding molecule of the present disclosure includes two PD1 agonist moieties (e.g., a first PD1 agonist moiety on a first monomer and a second PD1 agonist moiety on a second monomer, or both a first PD1 agonist moiety and a second PD1 agonist moiety on either the first or second monomer). In such embodiments, the two PD1 agonist moieties may be identical or different. If different, the two PD1 agonist moieties can interact with PD1 in different ways (for example, with different affinities).

[0095] 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 typically consists of multiple polypeptide chains, as represented, for example, 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 1, 2, 3, 4, and 5. Exemplary CD20-PD1 binding molecules incorporating one or more of the exemplary monomers 1, 2, 3, 4, and 5 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 PD1 agonist moiety is a PDL2-based agonist moiety.

[0096] 6.4.1. PD1 agonist section based on PDL1 PDL1 plays a crucial role in inducing and maintaining immune tolerance to self. As a ligand for the inhibitory receptor PD1, PDL1 modulates the activation threshold of T cells and limits T cell effector responses. This disclosure provides CD20-PD1 binding molecules in which at least one PD1 agonist moiety comprises an amino acid sequence comprising the PDL1 amino acid sequence described herein or an amino acid sequence homologous thereto. Such PD1 agonist moieties are referred herein as “PDL1-based PD1 agonist moieties” or similar terms.

[0097] The human PDL1 protein is synthesized as a 290-amino acid precursor polypeptide, from which 18 amino acids are removed to produce mature hPDL1, with amino acids 19-238 (numbered based on the precursor protein) forming the hPDL1 extracellular domain or ectodomain. The sequence of human PDL1 has the Uniprot identifier Q9NZQ7 (uniprot.org / uniprot / Q9NZQ7). The sequence of mouse PDL1 has the Uniprot identifier Q9EP73 (uniprot.org / uniprot / Q9EP73).

[0098] The precursor human PDL1 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold):

[0099] [ka]

[0100] The IgV domain of human PDL1 (sometimes referred to as the "IgV ectodomain" in this specification) has the following amino acid sequence: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY(Sequence ID 2) In some embodiments, the PD1 agonist moiety is a PDL1-based agonist moiety comprising or consisting of an amino acid sequence having at least 70% sequence identity to the human PDL1 IgV domain, for example, 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%, or 100% sequence identity. In some embodiments, the PD1 agonist moiety is a PDL1-based agonist moiety comprising, or consisting of, an amino acid sequence corresponding to the IgV domain of human PDL1, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional amino acids at the N-terminus of the IgV domain, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional amino acids at the C-terminus of the IgV domain.

[0101] In certain embodiments, the PD1 agonist moiety comprises or consists of the IgV domain of human PDL1 (SEQ ID NO: 2), or a variant thereof having one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type human PDL1 sequence. In some embodiments, the PD1 agonist moiety comprises or consists of amino acids 19-134 of human PDL1, and in some cases has one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type human PDL1 sequence. For example, in certain embodiments, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0102] In some embodiments, the PD1 agonist moiety does not contain an amino acid sequence corresponding to amino acids 150–238 of human PDL1. In certain embodiments, the PD1 agonist moiety does not contain an amino acid sequence corresponding to any subsequence of amino acids 150–238 of human PDL1 for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths.Therefore, for example, in some embodiments, the PD1 agonist moiety corresponds to human PDL1 residues 150-161, 151-162, 152-163, 153-164, 154-165, 155-166, 156-167, 157-168, 158-169, 159-170, 160-171, 161-172, 162-173, 163-174, 164-175, 165-176, and 166-177. , residues 167-178, residues 168-179, residues 169-180, residues 170-181, residues 171-182, residues 172-183, residues 173-184, residues 174-185, residues 175-186, residues 176-187, residues 177-188, residues 178-189, residues 179-190, residues 180-191, residues 181-192, residues 182-193, residues 183-194, residues 184-195, residues 185-196, residues 186-197, residues 187-198 , residues 188-199, residues 189-200, residues 190-201, residues 191-202, residues 192-203, residues 193-204, residues 194-205, residues 195-206, residues 196-207, residues 197-208, residues 198-209, residues 199-210, residues 200-211, residues 201-212, residues 202-213, residues 203-214, residues 204-215, residues 205-216, residues 206-217, residues 207-218, residues 208-219 It does not contain the amino acid sequence corresponding to residues 209-220, 210-221, 211-222, 212-223, 213-224, 214-225, 215-226, 216-227, 217-228, 218-229, 219-230, 220-231, 221-232, 222-233, 223-234, 224-235, 225-236, 226-237, or 227-238.In some embodiments, the PD1 agonist moiety consists of residues 1-11, 2-12, 3-13, 4-14, 5-15, 6-16, 7-17, 8-18, 9-19, 10-20, 11-21, 12-22, 13-23, 14-24, 15-25, 16-26, 17-27, 18-28, and the remaining residues. Residues 19-29, 20-30, 21-31, 22-32, 23-33, 24-34, 25-35, 26-36, 27-37, 28-38, 29-39, 30-40, 31-41, 32-42, 33-43, 34-44, 35-45, 36-46, 37-47, 38-48, 39-49 , residues 40-50, residues 41-51, residues 42-52, residues 43-53, residues 44-54, residues 45-55, residues 46-56, residues 47-57, residues 48-58, residues 49-59, residues 50-60, residues 51-61, residues 52-62, residues 53-63, residues 54-64, residues 55-65, residues 56-66, residues 57-67, residues 58-68, residues 59-69, residues 60- It does not contain amino acid sequences corresponding to residues 70, 61-71, 62-72, 63-73, 64-74, 65-75, 66-76, 67-77, 68-78, 69-79, 70-80, 71-81, 72-82, 73-83, 74-84, 75-85, 76-86, 77-87, or 78-88.

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

[0104] [ka]

[0105] The IgV domain of mouse PDL1 (sometimes referred to as the "IgV ectodomain" in this specification) has the following amino acid sequence. FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPY(Sequence ID 8) In some embodiments, the PD1 agonist moiety is a PDL1-based agonist moiety comprising an amino acid sequence having at least 70% sequence identity to the IgV domain of mouse PDL1, for example, 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%, or 100% sequence identity.

[0106] In certain embodiments, the PD1 agonist moiety comprises or consists of the IgV domain of mouse PDL1 (SEQ ID NO: 8), or a variant thereof having one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type mouse PDL1 sequence. In some embodiments, the PD1 agonist moiety comprises or consists of amino acids 19-134 of mouse PDL1, and in some cases has one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type mouse PDL1 sequence. For example, in certain embodiments, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0107] In some embodiments, the PD1 agonist moiety does not contain amino acids 150–239 of mouse PDL1. In certain embodiments, the PD1 agonist moiety does not contain an amino acid sequence corresponding to any subsequence of amino acids 150–239 of mouse PDL1 for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths.Therefore, for example, in some embodiments, the PD1 agonist moiety is derived from residues 150-161, 151-162, 152-163, 153-164, 154-165, 155-166, 156-167, 157-168, 158-169, 159-170, 160-171, 161-172, 162-173, 163-174, 164-175, 165-176, 166-177 of mouse PDL1. Residues 167-178, 168-179, 169-180, 170-181, 171-182, 172-183, 173-184, 174-185, 175-186, 176-187, 177-188, 178-189, 179-190, 180-191, 181-192, 182-193, 183-194, 184-195, 185-196, 186-197, 187-198, 1 88-199, residues 189-200, residues 190-201, residues 191-202, residues 192-203, residues 193-204, residues 194-205, residues 195-206, residues 196-207, residues 197-208, residues 198-209, residues 199-210, residues 200-211, residues 201-212, residues 202-213, residues 203-214, residues 204-215, residues 205-216, residues 206-217, residues 207-218, residues 208-219, residues 209- It does not contain the amino acid sequence corresponding to residues 220, 210-221, 211-222, 212-223, 213-224, 214-225, 215-226, 216-227, 217-228, 218-229, 219-230, 220-231, 221-232, 222-233, 223-234, 224-235, 225-236, 226-237, 227-238, or 228-239.In a mixed embodiment, the PD1 agonist moiety consists of residues 1-11, 2-12, 3-13, 4-14, 5-15, 6-16, 7-17, 8-18, 9-19, 10-20, 11-21, 12-22, 13-23, 14-24, 15-25, 16-26, 17-27, 18-28, 19-29, and 20 of SEQ ID NO: 11. ~30, residues 21~31, residues 22~32, residues 23~33, residues 24~34, residues 25~35, residues 26~36, residues 27~37, residues 28~38, residues 29~39, residues 30~40, residues 31~41, residues 32~42, residues 33~43, residues 34~44, residues 35~45, residues 36~46, residues 37~47, residues 38~48, residues 39~49, residues 40~50, residues 41~51, residues 42~5 2. Residues 43-53, 44-54, 45-55, 46-56, 47-57, 48-58, 49-59, 50-60, 51-61, 52-62, 53-63, 54-64, 55-65, 56-66, 57-67, 58-68, 59-69, 60-70, 61-71, 62-72, 63-73, 64-74, It does not contain amino acid sequences corresponding to residues 65-75, 66-76, 67-77, 68-78, 69-79, 70-80, 71-81, 72-82, 73-83, 74-84, 75-85, 76-86, 77-87, 78-88, 79-84, 80-85, 81-86, 82-87, 83-88, or 84-89.

[0108] In certain embodiments, the PDL1-based PD1 agonist moiety contains or comprises an amino acid sequence having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) sequence identity with respect to the IgV domain of one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions of PDL1 compared to wild-type PDL1. In some embodiments, one or more amino acid substitutions increase the stability of the PDL1-based PD1 agonist moiety. For example, in some embodiments, the PDL1-based PD1 agonist moiety contains the amino acid substitution C113S (numbered based on the precursor PDL1 protein).

[0109] In some embodiments, the PDL1-based PD1 agonist moiety is optionally fused to the CD20-targeting moiety directly or indirectly via a linker (e.g., as described in Section 6.8). When present on the same monomer, the PDL1-based PD1 agonist moiety may be located at the N-terminus or C-terminus of the CD20-targeting moiety. When the PDL1-based PD1 agonist moiety is fused "directly" to the CD20-targeting moiety, the PDL1-based PD1 agonist moiety and the CD20-targeting moiety are located adjacent to each other on the same monomer and separated only by a linker, if present. When the PDL1-based PD1 agonist moiety is fused "indirectly" to the CD20-targeting moiety, the PDL1-based PD1 agonist moiety and the CD20-targeting moiety are separated by one or more other domains on the same monomer (e.g., a dimerization moiety, an antigen-binding fragment of the agonist anti-PD1 antibody) or located on separate monomers.

[0110] 6.4.2. PD1 agonist section based on PDL2 The interaction between PDL2 and PD1 inhibits T cell proliferation by blocking cell cycle progression and cytokine production. This disclosure provides a CD20-PD1 binding molecule in which at least one PD1 agonist moiety comprises an amino acid sequence homologous to or containing the PDL2 amino acid sequence described herein. Such a PD1 agonist moiety is referred herein to as a “PDL2-based PD1 agonist moiety” or similar terminology.

[0111] The human PDL2 protein is synthesized as a 273-amino acid precursor polypeptide, from which 19 amino acids are removed to produce mature hPDL2, with amino acids 20-220 (numbered based on the precursor protein) forming the hPDL2 extracellular domain or ectodomain. The human PDL2 sequence has the Uniprot identifier Q9BQ51 (uniprot.org / uniprot / Q9BQ51). The mouse PDL2 sequence has the Uniprot identifier Q9WUL5 (uniprot.org / uniprot / Q9WUL5).

[0112] The precursor human PDL2 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold).

[0113] [ka]

[0114] The IgV domain of human PDL2 (sometimes referred to as the "IgV ectodomain" in this specification) has the following amino acid sequence: LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK(Sequence ID 14) In some embodiments, the PD1 agonist moiety is a PDL2-based agonist moiety comprising an amino acid sequence having at least 70% sequence identity to the human PDL2 IgV domain, for example, 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.

[0115] In certain embodiments, the PD1 agonist moiety comprises or consists of the IgV domain of human PDL2 (SEQ ID NO: 14), or a variant thereof having one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type human PDL2 sequence. In some embodiments, the PD1 agonist moiety comprises or consists of amino acids 20-121 of human PDL2, and in some cases has one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type human PDL2 sequence. For example, in certain embodiments, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0116] In some embodiments, the PD1 agonist moiety does not contain amino acids 150–221 of human PDL2. In certain embodiments, the PD1 agonist moiety does not contain an amino acid sequence corresponding to any subsequence of amino acids 150–221 of human PDL2 for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths. Therefore, for example, in some embodiments, the PD1 agonist moiety is derived from human PDL2 residues 150-161, 151-162, 152-163, 153-164, 154-165, 155-166, 156-167, 157-168, 158-169, 159-170, 160-171, 161-172, 162- 173, residues 163-174, residues 164-175, residues 165-176, residues 166-177, residues 167-178, residues 168-179, residues 169-180, residues 170-181, residues 171-182, residues 172-183, residues 173-184, residues 174-185, residues 175-186, residues 176-187, residues 177-188, residues 178-189, residue 17 9-190, residues 180-191, residues 181-192, residues 182-193, residues 183-194, residues 184-195, residues 185-196, residues 186-197, residues 187-198, residues 188-199, residues 189-200, residues 190-201, residues 191-202, residues 192-203, residues 193-204, residues 194-205, residues 195-206, remaining It does not contain amino acid sequences corresponding to groups 196-207, residues 197-208, 198-209, 199-210, 200-211, 201-212, 202-213, 203-214, 204-215, 205-216, 206-217, 207-218, 208-219, 209-220, or residues 210-221.In the mixed embodiment, the PD1 agonist portion consists of residues 1-11, 2-12, 3-13, 4-14, 5-15, 6-16, 7-17, 8-18, 9-19, 10-20, 11-21, 12-22, 13-23, 14-24, 15-25, 16-26, 17-27, 18-28, 19-29, 20-30, 21-31, 22-32, 23-33, 24-34, 25-35, 26-36, 27-37, 28-38, 29-39, 30-40, residues of SEQ ID NO: 17 It does not contain amino acid sequences corresponding to residues 31-41, residues 32-42, residues 33-43, residues 34-44, residues 35-45, residues 36-46, residues 37-47, residues 38-48, residues 39-49, residues 40-50, residues 41-51, residues 42-52, residues 43-53, residues 44-54, residues 45-55, residues 46-56, residues 47-57, residues 48-58, residues 49-59, residues 50-60, residues 51-61, residues 52-62, residues 53-63, residues 54-64, residues 55-65, residues 56-66, residues 57-67, residues 58-68, residues 59-69, residues 60-70, or residues 61-71.

[0117] The mouse PDL2 polypeptide is synthesized as a 247-amino acid precursor polypeptide, from which 19 amino acids are removed to produce mature mPDL2. Amino acids 20-221 (numbered based on the precursor protein) form the mPDL2 extracellular domain, or ectodomain. The precursor mouse PDL2 polypeptide has the following amino acid sequence (signal sequence = underlined; extracellular domain = bold).

[0118] [ka]

[0119] The IgV domain of mouse PDL2 (sometimes referred to as the "IgV ectodomain" in this specification) has the following amino acid sequence. LFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQKVENDTSLQSERATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVICGAAWDYKYLTVKVK(Sequence ID 20) In some embodiments, the PD1 agonist moiety is a PDL2-based agonist moiety comprising an amino acid sequence having at least 70% sequence identity to the IgV domain of mouse PDL2, for example, 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.

[0120] In certain embodiments, the PD1 agonist moiety comprises or consists of the IgV domain of mouse PDL2 (SEQ ID NO: 20), or a variant thereof having one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type mouse PDL2 sequence. In some embodiments, the PD1 agonist moiety comprises or consists of amino acids 20-121 of mouse PDL2, and in some cases has one or more (e.g., 1, 2, 3, 4, or 5) amino acid variants or substitutions relative to the wild-type mouse PDL2 sequence. For example, in certain embodiments, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0121] In some embodiments, the PD1 agonist moiety does not contain amino acids 150–222 of mouse PDL2. In certain embodiments, the PD1 agonist moiety does not contain an amino acid sequence corresponding to any subsequence of amino acids 150–222 of mouse PDL2 for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths.Therefore, for example, in some embodiments, the PD1 agonist moiety corresponds to residues 150-161, 151-162, 152-163, 153-164, 154-165, 155-166, 156-167, 157-168, 158-169, 159-170, 160-171, 161-172, and 162-17 3. Residues 163-174, 164-175, 165-176, 166-177, 167-178, 168-179, 169-180, 170-181, 171-182, 172-183, 173-184, 174-185, 175-186, 176-187, 177-188, 178-189, 179-19 0, residues 180-191, residues 181-192, residues 182-193, residues 183-194, residues 184-195, residues 185-196, residues 186-197, residues 187-198, residues 188-199, residues 189-200, residues 190-201, residues 191-202, residues 192-203, residues 193-204, residues 194-205, residues 195-206, residues 196-20 7. Does not contain amino acid sequences corresponding to residues 197-208, 198-209, 199-210, 200-211, 201-212, 202-213, 203-214, 204-215, 205-216, 206-217, 207-218, 208-219, 209-220, 210-221, or 211-222.In the mixed embodiment, the PD1 agonist portion consists of residues 1-11, 2-12, 3-13, 4-14, 5-15, 6-16, 7-17, 8-18, 9-19, 10-20, 11-21, 12-22, 13-23, 14-24, 15-25, 16-26, 17-27, 18-28, 19-29, 20-30, 21-31, 22-32, 23-33, 24-34, 25-35, 26-36, 27-37, 28-38, 29-39, 30-40, and 31-4 1. Does not contain amino acid sequences corresponding to residues 32-42, 33-43, 34-44, 35-45, 36-46, 37-47, 38-48, 39-49, 40-50, 41-51, 42-52, 43-53, 44-54, 45-55, 46-56, 47-57, 48-58, 49-59, 50-60, 51-61, 52-62, 53-63, 54-64, 55-65, 56-66, 57-67, 58-68, 59-69, 60-70, 61-71, or 62-72.

[0122] In some embodiments, the PDL2-based PD1 agonist moiety is optionally fused to the CD20-targeting moiety directly or indirectly via a linker (e.g., as described in Section 6.8). When present on the same monomer, the PDL2-based PD1 agonist moiety may be located at the N-terminus or C-terminus of the CD20-targeting moiety. When the PDL2-based PD1 agonist moiety is fused "directly" to the CD20-targeting moiety, the PDL2-based PD1 agonist moiety and the CD20-targeting moiety are located adjacent to each other on the same monomer and separated only by a linker, if present. When the PDL2-based PD1 agonist moiety is fused "indirectly" to the CD20-targeting moiety, the PDL2-based PD1 agonist moiety and the CD20-targeting moiety are separated by one or more other domains on the same monomer (e.g., a dimerization moiety, an antigen-binding fragment of the agonist anti-PD1 antibody) or are located on separate monomers.

[0123] 6.5. Antigen-binding fragments of agonist anti-PD1 antibodies In certain embodiments, the CD20-PD1 conjugated molecule of the Disclosure comprises an antigen-binding fragment of an agonist anti-PD1 antibody in addition to the CD20 targeting moiety and the PD1 agonist moiety. In some embodiments, the CD20-PD1 conjugated molecule of the Disclosure comprises a single antigen-binding fragment of an agonist anti-PD1 antibody (for example, in embodiments where the CD20-PD1 conjugated molecule is monovalent to the antigen-binding fragment of the agonist anti-PD1 antibody, the antigen-binding fragment of the agonist anti-PD1 antibody on a first monomer or a second monomer). In some embodiments, the CD20-PD1 binding molecule of the present disclosure comprises two antigen-binding fragments of an agonist anti-PD1 antibody (for example, in embodiments where the CD20-PD1 binding molecule is bivalent to the antigen-binding fragments of the agonist anti-PD1 antibody, a first antigen-binding fragment of the agonist anti-PD1 antibody on a first monomer and a second antigen-binding fragment of the agonist anti-PD1 antibody on a second monomer, or both the first and second antigen-binding fragments of the agonist anti-PD1 antibody may be on either the first or second monomer). In such embodiments, the two antigen-binding fragments of the agonist anti-PD1 antibody may be identical or they may be different. If they are different, the two antigen-binding fragments of the agonist anti-PD1 antibody may be orthogonal, may bind to distinct epitopes of CD20, and / or may be non-competitive.

[0124] In some embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody comprises the antigen-binding domain (e.g., VH / VL) of any known anti-PD1 agonist antibody. Examples of known anti-PD1 agonist antibodies include, but are not limited to, rosnilimaibu (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 described in WO2016 / 020856; clones 2, 10, and 19 described in WO / 2013 / 022091; and hi Examples include PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 949;WO / 2017 / 058859; clones 2 and 19, described in WO / 2010 / 029434; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35, described in WO / 2004 / 056875.

[0125] In other embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody is rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 described in WO2016 / 020856; clones 2, 10, and 19 described in WO / 2013 / 022091; humanized antibody 949 described in WO / 2011 / 110621; humanized antibody 949 described in WO / 2017 / 058859 These antibodies contain antigen-binding domains that bind to the same PD1 epitopes as antibodies PD1-17, PD1-28, PD1-33, and PD1-35, as described in WO / 2010 / 029434, and antibodies PD1-17, PD1-28, PD1-33, and PD1-35, and / or compete with them for binding to PD1. Assays for measuring antibody competition are known in the art. For example, a sample of PD1 can be conjugated to a solid support. A first antibody and a second antibody are then added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies bind to separate sites on PD1, the labeled antibody will bind at the same level regardless of the presence of the unlabeled antibody. 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 decrease. If an excess of unlabeled antibody is present, the labeled antibody will only bind, if any, to the PD1 antibody. In some embodiments, the competing antibody is one that reduces the binding of another antibody to PD1 by approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. Details of the procedures for performing such a competing assay 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 performed quantitatively 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 the target molecule can be determined using, for example, a real-time, label-free biolayer interference assay on the Octet HTX biosensor platform (Pall ForteBio Corp.).

[0126] Preferred formats for the antigen-binding fragment of the agonist anti-PD1 antibody are described in Section 6.5.1. The antigen-binding fragment of the agonist anti-PD1 antibody is preferably a PD1-binding fragment of an anti-PD1 agonist antibody, such as a Fab, Fv fragment as described in Section 6.5.1.1, or an scFv as described in Section 6.5.1.2.

[0127] The antigen-binding fragment of an agonist anti-PD1 antibody can be incorporated into a CD20-PD1 binding molecule having one of the configurations described herein. A CD20-PD1 binding molecule typically consists of multiple polypeptide chains, as represented, for example, by the exemplary monomers described in Section 6.2. As described in Section 6.2, the antigen-binding fragment of an agonist anti-PD1 antibody can be incorporated into one of the exemplary monomers 2, 3, 4, and 5. Exemplary CD20-PD1 binding molecules incorporating one or more of the exemplary monomers 2, 3, 4, and 5 are described in detail in Section 6.2.

[0128] 6.5.1. Antigen-binding fragment format In certain embodiments, the antigen-binding fragment of an agonist anti-PD1 antibody can be any type of antibody fragment that retains specific binding to PD1. In some embodiments, the antigen-binding fragment is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more specifically an IgG1 or IgG4 immunoglobulin molecule. The antibody fragment can be, but is not limited to, VH (or V H ) Fragment, VL (or V L Examples include the ) fragment, Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, minibody, diabody, triabody, and tetrabody.

[0129] 6.5.1.1.Fab Traditionally, Fab domains have been generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the CD20-PD1 binding molecules of this disclosure, the Fab domain is typically expressed recombinantly as part of the CD20-PD1 binding molecule.

[0130] In certain embodiments, the variable region sequence and / or constant domain region sequence are derived from a known anti-PD1 agonist antibody. Examples of known anti-PD1 agonist antibodies include, but are not limited to, rosnilimaibu (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 described in WO2016 / 020856; clones 2, 10, and 19 described in WO / 2013 / 022091; and hi Examples include PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 949;WO / 2017 / 058859; clones 2 and 19, described in WO / 2010 / 029434; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35, described in WO / 2004 / 056875.

[0131] In some embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody is one of clones C8-1 and G10-2 described in rosnilimab (ANB030);LY3462817;CC-90006;PT627;PT001;WO2016 / 020856;one of clones 2, 10, and 19 described in WO / 2013 / 022091;humanized antibody 949 described in WO / 2011 / 110621;WO / 2017 / 058859 The Fab includes one of PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6; one of clones 2 and 19 described in WO / 2010 / 029434; or one of the Fabs of antibodies PD1-17, PD1-28, PD1-33, and PD1-35 described in WO / 2004 / 056875 that bind to the same PD1 epitope as and / or compete with them for binding to PD1.

[0132] The Fab domain typically contains a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulin, 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.

[0133] For the CD20-PD1 binding molecules of this disclosure, particularly when the light chains are not common or universal light chains, it is advantageous to use Fab heterodimerization strategies to enable precise association of Fab domains belonging to the same ABD and to minimize abnormal pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategies shown in Table 1 above can be used.

[0134] Therefore, in certain embodiments, the correct association between two polypeptides of Fab is facilitated, for example, by exchanging the VL and VH domains of Fab with each other, or by exchanging the CH1 and CL domains with each other, as described in WO2009 / 080251.

[0135] Correct Fab pairing can also be facilitated by introducing one or more amino acid modifications to the CH1 domain and one or more amino acid modifications to the CL domain of the Fab, and / or by introducing one or more amino acid modifications to the VH domain and one or more amino acid modifications to the VL domain. The amino acids to be modified are typically those that are part of the VH:VL and CH1:CL interfaces so that the Fab components pair preferentially with each other rather than with other Fab components.

[0136] In one embodiment, one or more amino acid modifications are limited to conserved framework residues in 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 Kabat, Chothia, and IMGT numbering schemes.

[0137] In one embodiment, modifications introduced in the VH and CH1 domains and / or VL and CL domains are complementary to each other. Complementarity at the heavy-light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or various combinations of 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, all of which suggest a structural and chemical correspondence between two interacting surfaces.

[0138] In one embodiment, one or more introduced modifications introduce new hydrogen bonds across the interfaces of Fab components. In one embodiment, one or more introduced modifications introduce new salt bridges across the interfaces of Fab components. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, which are incorporated herein by reference.

[0139] In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, thereby introducing a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0140] In some embodiments, the Fab domain includes 143Q and 188V substitutions within the CH1 domain, as well as 113T and 176V substitutions within the CL domain, which helps to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0141] In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that facilitate the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, the 39K, 62E modifications are introduced into the VH domain, the H172A, F174G modifications into the CH1 domain, the 1R, 38D, (36F) modifications into the VL domain, and the L135Y, S176W modifications into the CL domain. In another embodiment, the 39Y modification is introduced into the VH domain and the 38R modification is introduced into the VL domain.

[0142] The Fab domain can also be modified to replace the natural CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of Fab components. For example, the engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, for example, Mazor et al., 2015, MAbs 7:377-89).

[0143] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that facilitate correct assembly. For example, Wu et al., 2015, MAbs 7:364-76 describe replacing the CH1 domain with the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, and pairing these domain substitutions with additional charge-charge interactions between the VL and VH domains by introducing 38D modification to the VL domain and 39K modification to the VH domain.

[0144] Instead of, or in addition to, the use of 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 molecule of this disclosure. In various embodiments, using the common light chain described herein reduces the number of inappropriate species of the CD20-PD1 binding molecule compared to using the original congeneral VL. In various embodiments, the VL domain of the CD20-PD1 binding molecule is identified from a monospecific antibody containing the common light chain. In various embodiments, the VH region of the CD20-PD1 binding molecule contains a limited human light chain repertoire, or a human heavy chain variable gene segment rearranged in vivo in mouse B cells pre-engineered to express a single human light chain, which is congeneral to the human heavy chain and generates an antibody repertoire containing one or more human VHs congenerate to one of two possible human VLs in response to exposure to the antigen of interest, and this antibody repertoire is specific to the antigen of interest. The common light chain is derived from a reconstituted human Vκ1-39Jκ5 sequence or a reconstituted human Vκ3-20Jκ1 sequence, including somatic mutants (e.g., affinity matured mutants). See, for example, U.S. Patent No. 10,412,940.

[0145] 6.5.1.2.scFv Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain and can be expressed as single-chain polypeptides, retaining the specificity of the intact antibody from which they originate. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for target binding. Examples of suitable linkers for linking the VH and VL chains of scFv are the linkers identified in Section 6.8.

[0146] As used herein, unless otherwise specified, scFv may have a VL variable region and a VH variable region in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and scFv may contain a VL-linker-VH or a VH-linker-VL.

[0147] In certain embodiments, scFv may include VH and VL sequences from known anti-PD1 agonist antibodies. Examples of known anti-PD1 agonist antibodies include, but are not limited to, rosnilimaibu (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 described in WO2016 / 020856; clones 2, 10, and 19 described in WO / 2013 / 022091; and hi Examples include PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 949;WO / 2017 / 058859; clones 2 and 19, described in WO / 2010 / 029434; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35, described in WO / 2004 / 056875.

[0148] In some embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody is one of clones C8-1 and G10-2 described in rosnilimab (ANB030);LY3462817;CC-90006;PT627;PT001;WO2016 / 020856;one of clones 2, 10, and 19 described in WO / 2013 / 022091;humanized antibody 949 described in WO / 2011 / 110621;WO / 2017 / 058859 The following are included: one of PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6; one of clones 2 and 19 as described in WO / 2010 / 029434; or am scFv that bind to the same PD1 epitope as scFv derived from one of antibodies PD1-17, PD1-28, PD1-33, and PD1-35 as described in WO / 2004 / 056875, and / or compete with them for binding to PD1.

[0149] To create nucleic acids encoding scFv, DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding one of the linkers described in Section 6.8 (typically a repeat of a sequence containing amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 50)), thereby allowing the VH and VL sequences to be expressed as a continuous single-stranded protein having VL and VH regions linked 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).

[0150] 6.6. Dimerization moiety 6.6.1. Fc Domain In some embodiments, the CD20-PD1 binding molecule and CD20-PD1 monomer of the present disclosure include one or more dimerizable moieties, e.g., an Fc domain, or one or more dimerizable moieties containing such moieties. In certain embodiments, the CD20-PD1 monomer of the present disclosure includes a single dimerizable moiety (e.g., a single Fc domain), and / or the CD20-PD1 binding molecule of the present disclosure includes two dimerizable moieties (e.g., two Fc domains that can associate to form an Fc region).

[0151] The CD20-PD1 binding molecules and CD20-PD1 monomers of this disclosure may be derived from any preferred species and may include an Fc domain, or a pair of Fc domains that associate to form an Fc region, operably linked to the CD20 targeting moiety and / or the PD1 agonist moiety. In one embodiment, the Fc domain is derived from a human Fc domain. In a particular embodiment, the Fc domain is derived from a human IgG Fc domain.

[0152] The CD20 targeting moiety and / or PD1 agonist moiety may be fused to the N-terminus or C-terminus of the IgGFc domain. One embodiment of the present disclosure relates to a dimer comprising two Fc-fusion polypeptides, which are created by fusing one or more CD20-targeting moieties and / or PD1 agonist moieties to an Fc domain, for example, by fusing both a CD20-targeting moiety and a PD1 agonist moiety to an Fc domain that can form a CD20-PD1 monomer that can homodimerize at 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 that forms two different CD20-PD1 monomers that can heterodimerize at expression. Dimers can be produced, for example, by inserting a gene fusion product encoding a fusion protein(s) into a suitable expression vector, expressing the gene fusion product(s) in host cells transformed with a recombinant expression vector, and then assembling the expressed fusion protein(s) in a similar manner to antibody molecules, thereby immediately forming interchain bonds between the Fc portions and generating a dimer.

[0153] The Fc domain that can be incorporated into the CD20-PD1 monomer may be derived from any preferred 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.

[0154] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but for the purpose of producing multispecific binding molecules, such as the CD20-PD1 binding molecule of this disclosure, the Fc domains may be advantageously different to enable heterodimerization, as described in Section 6.6.1.2 below.

[0155] In natural antibodies, the heavy chain Fc domains of IgA, IgD, and IgG consist of two heavy chain constant domains (CH2 and CH3), while the domains of IgE and IgM consist of three heavy chain constant domains (CH2, CH3, and CH4). These dimerize to form the Fc region.

[0156] In the CD20-PD1 binding molecule of this disclosure, the Fc region, and / or the Fc domain within it, may contain heavy chain constant domains derived from one or more different classes of antibodies, e.g., one, two, or three different classes.

[0157] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG1. In one embodiment, the Fc region includes a CH2 domain and a CH3 domain derived from IgG2.

[0158] In one embodiment, the Fc region includes a CH2 domain and a CH3 domain derived from IgG3. In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG4.

[0159] In one embodiment, the Fc region includes an IgM-derived CH4 domain. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain. In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG, as well as a CH4 domain derived from IgM.

[0160] It will be understood that the heavy chain constant domains for use in the production of the Fc region of the CD20-PD1 binding molecule of this disclosure may include variants of the naturally occurring constant domains described above. Such variants may contain one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of this disclosure includes at least one constant domain whose sequence differs from that of 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 yet another example, the variant constant domain is at least 80% identical or similar. In yet another example, the variant constant domain is at least 90% identical or similar. In yet another example, the variant constant domain is at least 95% identical or similar.

[0161] IgM and IgA are naturally present in humans as covalent polymers of a common H2L2 antibody unit. IgM exists as a pentamer when a J chain is incorporated, and as a hexamer when the J chain is absent. IgA exists in monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to a C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains in the polymer and is thought to play a crucial role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the CD20-PD1 binding molecules of this disclosure do not contain a tail.

[0162] The Fc domain incorporated into the CD20-PD1 binding molecule of this disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, a modified disulfide bond structure, or an altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.6.1.1.

[0163] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric CD20-PD1 binding molecules, for example, by enabling 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 linked together by Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.6.1.2.

[0164] It will be understood that any of the above modifications can be combined in any preferred manner to achieve desired functional properties, and / or combined with other modifications to alter the properties of the CD20-PD1 binding molecule.

[0165] 6.6.1.1. Changes to the effector function of the Fc domain In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0166] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0167] 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) includes an amino acid substitution at a position selected from the group E233, L234, L235, G237, N297, A330, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region includes an amino acid substitution at a position selected from the group L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain or Fc region includes the amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, in particular a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbered according to the Kabat EU index). In one embodiment, the Fc domain or Fc region includes an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region includes amino acid substitutions at positions P329, L234 and L235 (numbered according to the Kabat EU index). In more specific embodiments, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "LALAPG").

[0168] In some embodiments, the Fc domain or Fc region includes amino acid substitutions at positions L234, L235, G237, A330, and P331 (numbered according to the Kabat EU index). In more specific embodiments, the amino acid substitutions are L234A, L235E, G237A, A330S, and P331S (numbered according to the Kabat EU index).

[0169] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region contains amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), namely, 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), and the proline residue at position 329 is substituted with a glycine residue (P329G) (Kabat EU index numbering). In another specific embodiment, each Fc domain of the Fc region includes amino acid substitutions L234A, L235E, G237A, A330S, and P331S (numbered according to the Kabat EU index), namely, 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) (numbered according to the Kabat EU index).

[0170] 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 containing D265A and N297A mutations (EU numbering) to reduce effector function. In other embodiments, the IgG1 Fc domain is a variant IgG1 containing L234A, L235E, G237A, A330S, and P331S mutations (numbered by the 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.

[0171] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to the Fc receptor. Exemplary IgG4 Fc domains with reduced binding to the Fc receptor may include amino acid sequences selected from Table 2 below. In some embodiments, the Fc domain includes only the bolded portion of the sequence shown below:

[0172] [Table 3-1]

[0173] [Table 3-2]

[0174] [Table 3-3]

[0175] In certain embodiments, IgG4 having reduced effector function comprises the bolded portion of the amino acid sequence of Sequence ID No. 31 of WO2014 / 121087, which may be referred to herein as IgG4s or hIgG4s.

[0176] Regarding the heterodimer Fc region, it is possible to incorporate the above-mentioned combinations of variant IgG4 Fc sequences, for example, an Fc region containing an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 30 of WO2014 / 121087 and an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 37 of WO2014 / 121087, or an Fc region containing an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 31 of WO2014 / 121087 and an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 38 of WO2014 / 121087.

[0177] 6.6.1.2. Fc Heterodimerized Variants Certain CD20-PD1 binding molecules, unlike innate immunoglobulins, involve dimerization between two Fc domains operably linked to non-identical N-terminal regions (e.g., one Fc domain is linked to Fab, and the other to the PD1 agonist moiety). Insufficient heterodimerization of the two Fc domains to form the Fc region can hinder increasing the yield of the desired heterodimer molecule and present a purification challenge. For example, as disclosed in EP1870459A1; 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 Publication No. 2006204493A1; and PCT Publication No. WO2009 / 089004A1, various approaches available in the art can be used to enhance the dimerization of the Fc domain that may be present in the CD20-PD1 binding molecule of this disclosure.

[0178] In some embodiments, this disclosure provides CD20-PD1 binding molecules comprising Fc heterodimers, i.e., Fc regions containing heterogeneous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer contains a CH3 domain of an antibody. The CH3 domains are derived from the constant region of an antibody of any isotype, class, or subclass, preferably an IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the preceding section.

[0179] Heterodimerization of two different heavy chains in the CH3 domain yields the desired CD20-PD1 binding molecule, while homodimerization of the same heavy chain would reduce the yield of the desired CD20-PD1 binding molecule. Therefore, in a preferred embodiment, the polypeptide that associates to form the heterodimer CD20-PD1 binding molecule of this disclosure would contain a CH3 domain having modifications that favor heterodimer association compared to an unmodified Fc domain.

[0180] In certain embodiments, the modification that promotes the formation of an Fc heterodimer is a so-called "knob-into-hole" or "knob-in-hole" modification, which includes a "knob" modification in one Fc domain and a "hole" modification in the other Fc domain. Knob-into-hole techniques are described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide to allow the projection to be positioned within the cavity, in order to promote heterodimer formation and prevent homodimer formation. The protrusions are constructed by replacing smaller amino acid side chains (e.g., tyrosine or tryptophan) from the interface of the first polypeptide. Compensatory cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing larger amino acid side chains (e.g., alanine or threonine) with smaller amino acid side chains.

[0181] Accordingly, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side-chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be placed in a cavity in the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side-chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can be placed therein. Preferably, the amino acid residue having a larger side-chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side-chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity 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.

[0182] In certain such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to the Kabat EU index). In further embodiments, in the first Fc domain, the serine residue at position 354 is additionally replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue). In the second Fc domain, the tyrosine residue at position 349 is additionally replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In certain embodiments, the first Fc domain includes amino acid substitutions S354C and T366W, and the second Fc domain includes amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index).

[0183] In some embodiments, electrostatic steering (e.g., Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to facilitate the association of the first and second Fc domains of the Fc region.

[0184] As an alternative to, or in addition to, the use of Fc domains modified to promote heterodimerization, Fc domains can be modified to enable purification strategies that allow for the selection of Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that inhibits its binding to protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Such a CD20-PD1 binding molecule comprises 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, and the difference of at least one amino acid reduces the 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 to protein A, and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further contain the Y96F modification (according to IMGT; Y436F according to EU). This class of modifications is referred to herein as “star” mutations.

[0185] In some embodiments, Fc may include one or more mutations (e.g., knob and hole mutations) to promote heterodimerization, as well as star mutations to promote purification.

[0186] 6.7. Stabilization part The CD20-PD1 binding molecules of this disclosure may include a stabilizing moiety that can extend the serum half-life of the molecule in vivo. The serum half-life is often divided into alpha and beta phases. Either or both phases may 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 the purposes of this disclosure, serum half-life may refer to the half-life in humans or other mammals (e.g., mice or non-human primates).

[0187] Examples of stabilizing components include polyoxyalkylene moieties (e.g., polyethylene glycol), sugars (e.g., sialic acid), and highly tolerable protein moieties (e.g., Fc and its fragments and variants, transferrin, or serum albumin).

[0188] Other stabilizing moieties that can be used in the CD20-PD1 binding molecule of this 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., adnectin 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.

[0189] Therefore, in some embodiments, the present disclosure provides CD20-PD1 bound molecules comprising a stabilizing moiety that is a high molecular weight sugar. Serum albumin can also be involved in half-life extension via modules that have the ability to interact with albumin non-covalently. Therefore, the CD20-PD1 binding molecule of this disclosure may include an albumin-binding protein as a stabilizing portion. The albumin-binding protein may be conjugated or genetically fused to one or more other components of the CD20-PD1 binding molecule of this disclosure. Proteins with albumin-binding activity are known from certain bacteria. For example, Streptococcal protein G contains several small albumin-binding domains consisting of approximately 50 amino acid residues (6 kDa). Examples of additional serum albumin-binding proteins include those described in U.S. Publications 2007 / 0178082 and 2007 / 0269422. Fusion of albumin-binding domains to proteins results in a significantly extended half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76).

[0190] In other embodiments, the stabilizing portion is human serum albumin. In other embodiments, the stabilizing portion is transferrin. In some embodiments, the stabilizing portion is an Fc domain, for example, one of the Fc domains described in Section 6.6.1 and its subsections, which are incorporated herein by reference. The Fc domains described in Section 6.6.1 can generally be dimerized. However, for stabilization purposes, the Fc domain can be a soluble monomeric Fc domain with reduced self-associating ability. 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 is described in U.S. Patent Publication 2019 / 0367611, which includes amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3. The monomeric Fc domain may be any Ig subtype and may include additional substitutions that reduce effector function, as described in Section 6.6.1 and its subsections.

[0191] In yet another embodiment, the stabilizing portion is a polyethylene glycol portion or another polymer, as described in Section 6.7.1 below. The stabilizing portion can be connected to one or more other components of the CD20-PD1 binding molecule of this disclosure via a linker, for example, as described in Section 6.8 below.

[0192] 6.7.1. Polyethylene glycol In some embodiments, the CD20-PD1 bonded molecule includes polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing moiety, e.g., ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymer or random copolymer), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and copolymers of mixtures thereof. The polymer can have any molecular weight and can be branched or unbranched.

[0193] 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 widely used to encompass any polyethylene glycol molecule, regardless of size or modification at the PEG terminus, and can be represented by the following formula: X--O(CH2CH2O) n -1CH2CH2OH (wherein n is between 20 and 2300, and X is H or terminal modification, e.g., C) 1-4 (It is alkyl). PEG may contain additional chemical groups that are necessary for bonding reactions resulting from the chemical synthesis of the molecule, or that function as spacers for optimal distances between molecular parts. In addition, such PEG may consist of one or more PEG side chains linked together. PEG having multiple PEG chains is called multi-armed or branched PEG. Branched PEG is described, for example, in European Patent Application No. 473084A and U.S. Patent No. 5,932,462.

[0194] One or more PEG molecules can be attached to the CD20-PD1 binding molecule at different positions, and such attachment can be achieved 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 its components), or an amine group present in an amino acid such as lysine or arginine.

[0195] PEGylation can be achieved by site-directed PEGylation, in which a suitable reactive group is introduced into the protein to create a site where PEGylation preferentially occurs. In some embodiments, the CD20-PD1 binding molecule is modified to introduce a cysteine ​​residue at a desired position, enabling site-directed PEGylation on cysteine. A cysteine ​​residue can be generated by introducing a mutation into the coding sequence of the CD20-PD1 binding molecule of this disclosure. This can be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutation into cysteine ​​residues include serine, threonine, alanine, and other hydrophilic residues. Preferably, the residue to be mutated into cysteine ​​is a surface-exposed residue. Algorithms for predicting the surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. PEGylation of cysteine ​​residues can be carried out using, for example, PEG-maleimide, PEG-vinyl sulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

[0196] PEGs are typically activated with suitable activating groups appropriate for coupling to a desired site on a polypeptide. Methods for PEGylation 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.

[0197] The PEG portion can vary considerably in molecular weight and can be branched or linear. Typically, the weight-average molecular weight of PEG ranges from 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 aforementioned total molecular weights can also be used. In some embodiments, PEG has two branches. In other embodiments, PEG has four branches. In yet another embodiment, PEG is bis-PEG (NOF Corporation, DE-200MA).

[0198] Using conventional separation and purification techniques known in the art, PEGylated CD20-PD1 binding molecules such as size exclusion (e.g., gel filtration) and ion exchange chromatography can be purified. The product 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 a wider fraction near the elution peak to increase the percentage of mono-PEG in the composition. Approximately 90% mono-PEG conjugate represents a good balance of yield and activity.

[0199] In some embodiments, the PEGylated CD20-PD1 binding molecule will preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the biological activity associated with the unmodified CD20-PD1 binding molecule. In some embodiments, the biological activity is the ability of the CD20-PD1 binding molecule to bind to CD20, PD1, or both CD20 and PD1, as evaluated by K D , k on , or k off .

[0200] 6.8. Linker In certain embodiments, the Disclosure provides a CD20-PD1 binding molecule in which two or more components of the CD20-PD1 binding molecule are linked together by a peptide linker (also known as the "linker"). By example, but not limited to, a linker may be used to link (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, (d) a CD20 targeting moiety and an antigen-binding fragment of an agonist anti-PD1 antibody, (e) a PD1 agonist moiety and an antigen-binding fragment of an agonist anti-PD1 antibody, (f) different domains within the CD20 targeting moiety (e.g., the VH domain and VL domain in the scFv), or (g) different domains within the antigen-binding fragment of an agonist anti-PD1 antibody (e.g., the VH domain and VL domain in the scFv).

[0201] The peptide linker can be in the range of 2 amino acids to 60 amino acids or more, 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.

[0202] In certain embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids long, and optionally up to 30 amino acids, at least 40 amino acids, at least 50 amino acids, or at least 60 amino acids long.

[0203] In some of the embodiments described above, the linker is in the range of 5 to 50 amino acid lengths, for example, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acid lengths. In other embodiments described above, the linker is in the range of 6 to 50 amino acid lengths, for example, 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acid lengths. In yet another embodiment described above, the linker is in the range of 7 to 50 amino acid lengths, for example, 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acid lengths.

[0204] Charged (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that can be used in the CD20-PD1 binding molecule of this disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers are glycine and serine repeats, for example, G n S (Sequence ID 57) or SG n The monomer or polymer of (SEQ ID NO: 58), or containing thereof, 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 G4S (SEQ ID NO: 33), e.g., (GGGGS) n It is a monomer or polymer of the repeat of (SEQ ID NO: 33), or contains one.

[0205] Polyglycine linkers can be suitably used with the CD20-PD1 binding molecules of this disclosure. In some embodiments, the peptide linkers include two consecutive glycine molecules (2Gly), three consecutive glycine molecules (3Gly), four consecutive glycine molecules (4Gly) (SEQ ID NO: 59), five consecutive glycine molecules (5Gly) (SEQ ID NO: 60), six consecutive glycine molecules (6Gly) (SEQ ID NO: 61), seven consecutive glycine molecules (7Gly) (SEQ ID NO: 62), eight consecutive glycine molecules (8Gly) (SEQ ID NO: 63), or nine consecutive glycine molecules (9Gly) (SEQ ID NO: 64).

[0206] Exemplary linker sequences are shown in Table L below. The CD20-PD1 binding molecules of this disclosure may contain one or more linkers from Table L.

[0207] [Table 4-1]

[0208] [Table 4-2]

[0209] In some embodiments, the CD20-PD1 binding molecule includes linker L1. In some embodiments, the CD20-PD1 binding molecule includes linker L2. In some embodiments, the CD20-PD1 binding molecule includes linker L3. In some embodiments, the CD20-PD1 binding molecule includes linker L4. In some embodiments, the CD20-PD1 binding molecule includes linker L5. In some embodiments, the CD20-PD1 binding molecule includes linker L6. In some embodiments, the CD20-PD1 binding molecule includes linker L7. In some embodiments, the CD20-PD1 binding molecule includes linker L8. In some embodiments, the CD20-PD1 binding molecule includes linker L9. In some embodiments, the CD20-PD1 binding molecule includes linker L10. In some embodiments, the CD20-PD1 binding molecule includes linker L11. In some embodiments, the CD20-PD1 binding molecule includes linker L12. In some embodiments, the CD20-PD1 binding molecule includes linker L13. In some embodiments, the CD20-PD1 binding molecule includes linker L14. In some embodiments, the CD20-PD1 binding molecule includes linker L15. In some embodiments, the CD20-PD1 binding molecule includes linker L16. In some embodiments, the CD20-PD1 binding molecule includes linker L17. In some embodiments, the CD20-PD1 binding molecule includes linker L18. In some embodiments, the CD20-PD1 binding molecule includes linker L19. In some embodiments, the CD20-PD1 binding molecule includes linker L20. In some embodiments, the CD20-PD1 binding molecule includes linker L21. In some embodiments, the CD20-PD1 binding molecule includes linker L22. In some embodiments, the CD20-PD1 binding molecule includes linker L23. In some embodiments, the CD20-PD1 binding molecule includes linker L24. In some embodiments, the CD20-PD1 binding molecule includes linker L25. In some embodiments, the CD20-PD1 binding molecule includes linker L26. In some embodiments, the CD20-PD1 binding molecule includes linker L27.In some embodiments, the CD20-PD1 binding molecule includes linker L28. In some embodiments, the CD20-PD1 binding molecule includes linker L29. In some embodiments, the CD20-PD1 binding molecule includes linker L30. In some embodiments, the CD20-PD1 binding molecule includes linker L31. In some embodiments, the CD20-PD1 binding molecule includes linker L32. In some embodiments, the CD20-PD1 binding molecule includes linker L33. In some embodiments, the CD20-PD1 binding molecule includes linker L34. In some embodiments, the CD20-PD1 binding molecule includes linker L35. In some embodiments, the CD20-PD1 binding molecule includes linker L36. In some embodiments, the CD20-PD1 binding molecule includes linker L37. In some embodiments, the CD20-PD1 binding molecule includes linker L38. In some embodiments, the CD20-PD1 binding molecule includes linker L39. In some embodiments, the CD20-PD1 binding molecule includes linker L40. In some embodiments, the CD20-PD1 binding molecule includes linker L41. In some embodiments, the CD20-PD1 binding molecule includes linker L42. In some embodiments, the CD20-PD1 binding molecule includes linker L43. In some embodiments, the CD20-PD1 binding molecule includes linker L44. In some embodiments, the CD20-PD1 binding molecule includes linker L45. In some embodiments, the CD20-PD1 binding molecule includes linker L46. In some embodiments, the CD20-PD1 binding molecule includes linker L47. In some embodiments, the CD20-PD1 binding molecule includes linker L48. In some embodiments, the CD20-PD1 binding molecule includes linker L49. In some embodiments, the CD20-PD1 binding molecule includes linker L50. In some embodiments, the CD20-PD1 binding molecule includes linker L51. In some embodiments, the CD20-PD1 binding molecule includes linker L52. In some embodiments, the CD20-PD1 binding molecule includes linker L53. In some embodiments, the CD20-PD1 binding molecule includes linker L54.In some embodiments, the CD20-PD1 binding molecule includes linker L55. In some embodiments, the CD20-PD1 binding molecule includes linker L56. In some embodiments, the CD20-PD1 binding molecule includes linker L57. In some embodiments, the CD20-PD1 binding molecule includes linker L58. In some embodiments, the CD20-PD1 binding molecule includes linker L59. In some embodiments, the CD20-PD1 binding molecule includes linker L60. In some embodiments, the CD20-PD1 binding molecule includes linker L61. In some embodiments, the CD20-PD1 binding molecule includes linker L62. In some embodiments, the CD20-PD1 binding molecule includes linker L63. In some embodiments, the CD20-PD1 binding molecule includes linker L64. In some embodiments, the CD20-PD1 binding molecule includes linker L65. In some embodiments, the CD20-PD1 binding molecule includes linker L66. In some embodiments, the CD20-PD1 binding molecule includes linker L67. In some embodiments, the CD20-PD1 binding molecule includes linker L68. In some embodiments, the CD20-PD1 binding molecule includes linker L69. In some embodiments, the CD20-PD1 binding molecule includes linker L70. In some embodiments, the CD20-PD1 binding molecule includes linker L71. In some embodiments, the CD20-PD1 binding molecule includes linker L72. In some embodiments, the CD20-PD1 binding molecule includes linker L73. In some embodiments, the CD20-PD1 binding molecule includes linker L74. In some embodiments, the CD20-PD1 binding molecule includes linker L75. In some embodiments, the CD20-PD1 binding molecule includes linker L76. In some embodiments, the CD20-PD1 binding molecule includes linker L77. In some embodiments, the CD20-PD1 binding molecule includes linker L78. In some embodiments, the CD20-PD1 binding molecule includes the linker L79.

[0210] 6.8.1. Hinge Arrangement In some embodiments, the CD20-PD1 binding molecules of this disclosure include a hinge region or a linker containing a hinge region. In particular, the hinge can be used to connect a CD20 targeting moiety, e.g., a Fab domain, to a dimerizing domain, e.g., an Fc domain. The hinge region can be a native hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of an Fc domain. The term “hinge region” refers, with respect to a dimerized polypeptide (e.g., a homodimer or heterodimer CD20-PD1 binding molecule formed by the association of two Fc domains), to two associated hinge sequences on separate polypeptide chains.

[0211] A natural hinge region is typically the hinge region found between the Fab domain and the Fc domain in naturally occurring antibodies. A modified hinge region is any hinge that differs from a natural hinge region in length and / or composition. Such hinges may include hinge regions from other species, such as those from humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, or goats. Other modified hinge regions may include complete hinge regions derived from antibodies of a different class or subclass than those of the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may include a portion or repeating unit of a natural hinge, where each repeating unit is derived from a natural hinge region. In yet another way, a natural hinge region may be modified by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting suitably positioned 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.

[0212] Several modified hinge regions are already described, for example, in U.S. Patent Nos. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0213] In one embodiment, the CD20-PD1 binding molecule of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge region at its N-terminus. In various embodiments, positions 233–236 within the hinge region may be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, and the positions are numbered by EU numbering.

[0214] In some embodiments, the CD20-PD1 binding molecules of this disclosure include a modified hinge region that reduces binding affinity to the Fcγ receptor compared to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0215] In one embodiment, the CD20-PD1 binding molecule of the present disclosure comprises an Fc domain in which each Fc domain has an intact hinge region at its N-terminus, each Fc domain and hinge region is derived from IgG4, and each hinge region contains the modified sequence CPPC (SEQ ID NO: 137). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 138) compared to IgG1, which contains the sequence CPPC (SEQ ID NO: 137). The serine residue present in the IgG4 sequence results in increased flexibility in this region, and therefore a certain percentage of molecules form disulfide bonds (intrachain disulfides) within the same protein chain 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). By replacing serine residues with proline residues to obtain the same core sequence as IgG1, complete interchain disulfide formation becomes possible within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0216] 6.8.1.1. Chimera Hinge Array The hinge region can be a chimeric hinge region. For example, a chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region, combined with an "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0217] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (sequence number 139, previously disclosed as sequence number 8 in WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (sequence number 140, previously disclosed as sequence number 9 in WO2014 / 121087). Such a chimeric hinge sequence can be suitably linked to the IgG4 CH2 region (for example, by incorporating it into the IgG4 Fc domain, e.g., a human or mouse Fc domain, and can be further modified with CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.6.1.1).

[0218] 6.8.1.2. Hinge arrangement with reduced effector functionality In further embodiments, the hinge region may be modified to reduce the effector function, for example, as described in WO2016161010A2, which is incorporated herein in its entirety by reference. In various embodiments, the modified hinge region positions 233–236 are G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with the positions numbered by EU numbering (as shown in Figure 1 of WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" indicates an empty position.

[0219] Position 236 is empty in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233–235 are occupied by non-G residues in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0220] Hinge modifications within positions 233-236 can be combined with position 228, which is occupied by P. Position 228 is originally 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 for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0221] The exemplary hinge region has residues 226–236, sometimes referred to as the intermediate (or core) and lower hinge, and is occupied by modified hinge sequences called GGG-(233–236), GG--(233–236), G---(233–236), and G-less(233–236). Optionally, the hinge domain amino acid sequence may include CPPPAPGGG-GPSVF (SEQ ID NO: 141, previously disclosed as SEQ ID NO: 1 in WO2016161010A2), CPPPPAPGG--GPSVF (SEQ ID NO: 142, previously disclosed as SEQ ID NO: 2 in WO2016161010A2), CPPPAPG---GPSVF (SEQ ID NO: 143, previously disclosed as SEQ ID NO: 3 in WO2016161010A2), or CPPPPAP----GPSVF (SEQ ID NO: 144, previously disclosed as SEQ ID NO: 4 in WO2016161010A2).

[0222] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically includes the CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinge) adjacent to the specified region. Such additional constant region segments that are present are typically of the same isotype, preferably a human isotype, but may 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 with different domains. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016161010A2.

[0223] In certain embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., incorporated into an IgG4 Fc domain, e.g., a human or mouse Fc domain, and further modified in the CH2 and / or CH3 domains to reduce effector function, as described in Section 6.6.1.1).

[0224] 6.9. Nucleic Acids and Host Cells In another aspect, the disclosure provides a nucleic acid encoding a CD20-PD1 binding molecule of the disclosure. In some embodiments, the CD20-PD1 binding molecule is encoded by a single nucleic acid. In other embodiments, e.g., in the case of a heterodimeric molecule or a molecule comprising a CD20 targeting moiety composed of two or more polypeptide chains, the CD20-PD1 binding molecule can be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0225] A single nucleic acid can encode a CD20-PD1 binding molecule containing a single polypeptide chain, a CD20-PD1 binding molecule containing two or more polypeptide chains, or a portion of a CD20-PD1 binding molecule containing three or more polypeptide chains (for example, a single nucleic acid can encode two polypeptide chains of a CD20-PD1 binding molecule containing three, four or more polypeptide chains, or three polypeptide chains of a CD20-PD1 binding molecule containing four or more polypeptide chains). To control expression separately, 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 element, separated by an internal ribosome entry site (IRES) sequence, and allowed to be translated into separate polypeptides.

[0226] In some embodiments, a CD20-PD1 binding molecule containing two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the CD20-PD1 binding molecule may be less than or equal to the number of polypeptide chains in the CD20-PD1 binding molecule (for example, when two or more polypeptide chains are encoded by a single nucleic acid).

[0227] The nucleic acids in this disclosure may be DNA or RNA (e.g., 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 below herein.

[0228] 6.9.1. Vectors This disclosure provides vectors comprising a nucleotide sequence encoding one or two polypeptide chains of the CD20-PD1 monomer, such as a CD20-PD1 binding molecule or a CD20-PD1 binding molecule component as described herein. The vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Vectors encoding the CD20-PD1 binding molecule (or its components) as described herein may be useful for the expression and / or delivery of the CD20-PD1 binding molecule.

[0229] A variety of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, 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 Semlik Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0230] Additionally, cells in which DNA has been stably incorporated into the chromosome can be selected by introducing one or more markers that enable selection of the transfected host cell. These markers may provide, for example, prototropy to a trophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly ligated to the expressed DNA sequence or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0231] Once a DNA sequence containing an 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 may be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and may be modified or optimized according to the specific expression vector and mammalian host cell used, as described herein.

[0232] 6.9.2.Host cells This disclosure also provides host cells containing the nucleic acids of this disclosure. In one embodiment, a host cell is genetically engineered to contain one or more nucleic acids described herein.

[0233] In one embodiment, host cells are genetically engineered by using an expression cassette. The term “expression cassette” refers to a nucleotide sequence that can influence gene expression in a host that is compatible with such a sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in bringing about expression, such as an inducible promoter, may also be used.

[0234] This disclosure also provides host cells containing the vectors described herein. The cells can be eukaryotic cells, bacterial cells, insect cells, or human cells, but are not limited to these. Preferred eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Preferred insect cells include, but are not limited to, Sf9 cells.

[0235] 6.10. Pharmaceutical Compositions 6.10.1. Pharmaceutical compositions containing CD20-PD1 binding molecules The CD20-PD1 conjugated molecules of this disclosure may be in the form of a composition comprising the CD20-PD1 conjugated molecule and one or more carriers, excipients, and / or diluents. The composition may be formulated for specific uses, 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 conjugated molecule and, in the case of therapeutic use, the mode of administration.

[0236] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any preferred form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by various routes, including orally, percutaneously, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or locally. The most preferred route of administration in any given case will depend on the specific antibody, the target, as well as the nature and severity of the disease, and the physical condition of the target. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0237] Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of the CD20-PD1 conjugated molecules of this disclosure per dose. The amount of CD20-PD1 conjugated molecules 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 may be in the form of a lyophilized dry powder containing an amount of CD20-PD1 conjugated molecules suitable for a single dose, or in liquid form. Dry powder unit dosage forms can be packaged in a kit with a syringe, an appropriate amount of diluent, and / or other components useful for administration. Unit dosages in liquid form may be conveniently supplied in the form of a syringe pre-filled with an amount of CD20-PD1 conjugated molecules suitable for a single dose.

[0238] The pharmaceutical composition may also be supplied in bulk, as it contains an amount of CD20-PD1 binding molecule suitable for multiple doses. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing CD20-PD1 bound molecules of desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”) typically used in the art, namely buffers, stabilizers, preservatives, isotonic agents, nonionic 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 the recipient at the dosage and concentration used.

[0239] Buffers help maintain pH within a range close to physiological conditions. They can exist at a wide variety of concentrations, but typically they will be present in the range of approximately 2 mM to 50 mM. Suitable buffers for use in this disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.), and gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.). Examples include (glucate) mixtures, oxalate buffers (e.g., oxalate-sodium oxalate mixture, oxalate-sodium hydroxide mixture, oxalate-potassium oxalate mixture, etc.), lactate buffers (e.g., lactate-sodium lactate mixture, lactate-sodium hydroxide mixture, lactate-potassium lactate mixture, etc.), and acetate buffers (e.g., acetate-sodium acetate mixture, acetate-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0240] Preservatives may be added to delay microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in this disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this 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 functionally from volume extenders to additives, and help to solubilize therapeutic drugs or prevent them from denatured or adhering to the container walls.Typical stabilizers include polyhydric sugar alcohols (listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, and sulfur-containing reducing agents (e.g., urea, glutathione, thio). These can be stolic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate, etc., low molecular weight polypeptides (e.g., peptides with 10 or fewer residues), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins, etc.), 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 CD20-PD1 bound molecule.

[0241] Nonionic surfactants or detergents (also known as "wetting agents") can be added to aid in the solubilization of glycoproteins and protect them from agitation-induced aggregation, thereby allowing the formulation to be exposed to a stressful shear surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and pluronic polyols. Nonionic surfactants may be present in concentrations ranging from approximately 0.05 mg / mL to approximately 1.0 mg / mL (e.g., approximately 0.07 mg / mL to approximately 0.2 mg / mL).

[0242] Examples of various additional excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and co-solvents.

[0243] 6.10.2. Pharmaceutical compositions for nucleic acid delivery encoding CD20-PD1 binding molecules The CD20-PD1 binding molecules of the present 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.

[0244] Exemplary gene therapy vectors include adenovirus or AAV-based therapeutics.Non-limiting examples of adenovirus-based or AAV-based therapeutic agents for use in the methods, uses, or compositions herein include, for example, rAd-p53 (also known as Gendicine® or Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297), a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53 for use in the treatment of cancer; Ad5 d11520 (also known as H101 or ONYX-015, e.g., Russell et al., 2012, Nature Biotechnology 30:658-670), an adenovirus lacking the E1B gene for inactivating host p53; and, for example, AD5-D24-GM-CSF (Cerullo et al., 2010, Cancer), an adenovirus containing the cytokine GM-CSF for use in the treatment of cancer. Res.70:4297); For example, rAd-HSVtk (Cerepro®, developed by Ark Therapeutics, see, for example, U.S. Patent No. 6,579,855; developed by Advantagene as ProstAtak®; International PCT Application No. WO2005 / 049094), a replication-deficient adenovirus vector expressing human tumor necrosis factor alpha (TNFα) under the control of a chemoradiation-inducible EGR-1 promoter for use in cancer treatment; For example, rAd-TNFα (TNFerade®, GenVec; Rasmussen et al., 2002, Cancer Gene For example, Ad-IFNβ (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol.Ther.18:852-860) is an adenovirus serotype 5 vector in which the E1 and E3 genes, which express the human interferon-beta gene under the direction of the cytomegalovirus (CMV) pre-initial promoter, are deleted, for the treatment of cancer.Additional vectors known in the art include, for example, lentiviral vectors (e.g., VSV), retroviral vectors, and other vectors.

[0245] Any currently known or future-developed delivery vector, whether natural or engineered, can be used to deliver the CD20-PD1 binding molecule of this disclosure. In some embodiments, the delivery vector is a viral vector, including, for example, a virus, a viral capsid, or a viral genome. In some embodiments, the delivery vector is a naked nucleic acid, such as an episome. In some embodiments, the delivery vector includes a nucleic acid complex. Exemplary, non-limiting nucleic acid complexes for use as delivery vectors include lipoplexes, polymerosomes, polypexes, dendrimers, and inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, calcium phosphate, etc.). In some embodiments, the delivery vectors described herein include combinations of viral vectors, naked nucleic acids, and nucleic acid complexes.

[0246] In one embodiment, the delivery vector is a virus including retroviruses, adenoviruses, herpes simplex viruses, poxviruses, vaccinia viruses, lentiviruses, or adeno-associated viruses. In one embodiment, the delivery vector is adeno-associated viruses (AAVs) including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof.

[0247] In one embodiment, the nucleic acid encoding the CD20-PD1 binding molecule (or its components) also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the vector is a chimeric adeno-associated virus containing gene elements from two or more serotypes. For example, an AAV vector (designated as AAV1 / 2 or AAV RC1 / 2) having a rep gene from AAV1 and a cap gene from AAV2 may be used as a delivery vector to deliver the CD20-PD1 binding molecule expressing the nucleic acid to cells or the cells of a patient in need.In one embodiment, the delivery vectors are AAV1 / 2, AAV1 / 3, AAV1 / 4, AAV1 / 5, AAV1 / 6, AAV1 / 7, AAV1 / 8, AAV1 / 9, AAV1 / 10, AAV1 / 11, AAV2 / 1, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV3 / 1, AAV3 / 2, AAV3 / 4, AAV3 / 5, AAV3 / 6, AAV3 / 7, AAV3 / 8, AAV3 / 9, AAV3 / 10, AAV3 / 10, AAV4 / 1, AAV4 / 2, AAV4 / 3, AAV4 / 5, AAV4 / 6, AAV4 / 7, AAV4 / 8, AAV4 / 9, AAV4 / 10, AAV4 / 11, AAV5 / 1, AAV5 / 2, A AV5 / 3, AAV5 / 4, AAV5 / 6, AAV5 / 7, AAV5 / 8, AAV5 / 9, AAV5 / 10, AAV5 / 11, AAV6 / 1, AAV6 / 2, AAV6 / 3, AAV6 / 4, AAV6 / 5, AAV6 / 7, AAV6 / 8, A AV6 / 9, AAV6 / 10, AAV6 / 10, AAV7 / 1, AAV7 / 2, AAV7 / 3, AAV7 / 4, AAV7 / 5, AAV7 / 6, AAV7 / 8, AAV7 / 9, AAV7 / 10, AAV7 / 11, AAV8 / 1, AAV8 / 2, AAV8 / 3, AAV8 / 4, AAV8 / 5, AAV8 / 6, AAV8 / 7, AAV8 / 9, AAV8 / 10, AAV8 / 11, AAV9 / 1, AAV9 / 2, AAV9 / 3, AAV9 / 4, AAV9 / 5, AAV9 / 6, AAV9 / 7. AAV9 / 8, AAV9 / 10, AAV9 / 11, AAV10 / 1, AAV10 / 2, AAV10 / 3, AAV10 / 4, AAV10 / 5, AAV10 / 6, AAV10 / 7, AAV10 / 8, AAV10 / 9, AAV10 / 11, AAV11 / 1, AAV11 / 2, AAV11 / 3, AAV11 / 4, AAV11 / 5, AAV11 / 6, AAV11 / 7, AAV11 / 8, AAV11 / 9, AAV11 / 10, chimeric virus vectors, or derivatives thereof.Gao et al., “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy,” PNAS 99(18):11854-11859, Sep.3, 2002 is incorporated herein by reference to AAV vectors and chimeric virus vectors useful as delivery vectors, as well as their construction and use.

[0248] Nucleic acid molecules (e.g., mRNA) or viruses can be formulated as the sole pharmaceutically active ingredient in a pharmaceutical composition, or in combination with other activators for a specific disease to be treated. Optionally, other drugs, pharmaceuticals, carriers, adjuvants, and diluents may be included in the compositions provided herein. For example, wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as one or more of colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Other exemplary agents and excipients that may be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium bisulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0249] When used as an adjunct therapy for adoptive cell transplantation, for example, CAR-expressing cell therapy as described in Section 6.11.2.1, the cell therapy, for example, CAR-expressing cells, can be manipulated to express the CD20-PD1 binding molecule of this disclosure. The CD20-PD1 binding molecule can target a specific genomic locus, for example, a locus that is active in activated or dysfunctional lymphocytes, such as the PD-1 locus, or it can be inserted into a non-specific genomic locus. Targeting a specific genomic locus can be achieved, for example, by gene editing using zinc finger proteins, CRISPR / Cas9 systems, etc.

[0250] 6.11. Indications for treatment and method of use The CD20-PD1 conjugated molecules of this disclosure are useful for treating disease conditions in which modulation of the host immune system is beneficial, particularly conditions in which suppression of the cellular immune response is desirable. Therefore, the CD20-PD1 conjugated molecules of this disclosure can be used to suppress immune responses in a variety of applications.

[0251] Conditions in which suppression of cellular immune responses is desirable include disease states resulting from autoimmune responses. Disease states in which the CD20-PD1 conjugated molecule of this disclosure can be administered include, for example, autoimmune diseases in which suppression of cellular autoimmune responses is an important mechanism. Specific disease states in which the CD20-PD1 conjugated molecule of this disclosure can be used include type 1 diabetes (T1D), systemic lupus erythematosus, Crohn's disease, and graft-versus-host disease (GVHD). The CD20-PD1 conjugated molecule of this disclosure may be administered on its own or in any suitable pharmaceutical composition.

[0252] In one embodiment, a CD20-PD1 conjugated molecule of the present disclosure is provided for use as a pharmaceutical. In a further embodiment, a CD20-PD1 conjugated molecule of the present disclosure is provided for use in the treatment of a disease. In a particular embodiment, a CD20-PD1 conjugated molecule of the present disclosure is provided for use in a therapeutic method. In one embodiment, the present disclosure provides a CD20-PD1 conjugated molecule described herein for use in the treatment of a disease in a subject requiring treatment of the disease. In a particular embodiment, the present disclosure provides a CD20-PD1 conjugated molecule for use in a method of treating a subject having an autoimmune disease, comprising administering a therapeutically effective amount of the CD20-PD1 conjugated molecule to the individual. In a particular embodiment, the disease to be treated is an autoimmune disease. In a particular embodiment, the disease is T1D. In another embodiment, the disease is systemic lupus erythematosus. In another embodiment, the disease is Crohn's disease. In yet another embodiment, the disease is GVHD. In a particular embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual. In further embodiments, the Disclosure provides CD20-PD1 conjugated molecules for use in suppressing the immune system. In certain embodiments, the Disclosure provides CD20-PD1 conjugated molecules for use in a method of suppressing the immune system in a subject, comprising administering an effective amount of the CD20-PD1 conjugated molecule to an individual for suppressing the immune system. The “individual” in any of the above embodiments is a mammal, e.g., a human. The “suppression of the immune system” in any of the above embodiments may include one or more of the following: a general reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell responsiveness, etc.

[0253] This disclosure further provides methods of topical PD1 agonism, including administering CD20-PD1 binding molecules or pharmaceutical compositions described herein. As used herein, the term “topically delivered” indicates that topical administration is not required, but rather that the CD20-PD1 binding molecule selectively or preferentially localizes to the intended site of immunomodulation, e.g., the site of autoimmune activity.

[0254] This disclosure further provides a method for administering PD1 agonist therapy to a subject with reduced systemic exposure and / or reduced systemic toxicity, which includes, for example, administering PD1 agonist therapy to a subject in the form of a CD20-PD1 conjugated molecule or pharmaceutical composition described herein, if CD20 is expressed in the tissue to which PD1 agonist therapy is desired and / or intended.

[0255] Therefore, the aforementioned method enables PD1 agonist therapy with reduced off-target side effects by preferentially delivering the CD20-PD1 binding molecule to the site intended for PD1 agonist treatment.

[0256] This disclosure further provides a method for locally modulating (e.g., inhibiting) an immune response in a target tissue expressing CD20, comprising administering a CD20-PD1 binding molecule or one of the pharmaceutical compositions described herein.

[0257] In some embodiments, the administration is not local to the tissue. For example, if the target tissue is cancerous tissue, the administration may be systemic or subcutaneous. In further embodiments, the Disclosure provides the use of the CD20-PD1 binding molecule of the Disclosure in the manufacture or preparation of a pharmacopoeia for the treatment of a disease in a subject requiring treatment of the disease. In one embodiment, the pharmacopoeia is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the pharmacopoeia to a subject having the disease. In one particular embodiment, the disease to be treated is an autoimmune disease. In one particular embodiment, the disease is T1D. In another embodiment, the disease is systemic lupus erythematosus. In yet another embodiment, the disease is Crohn's disease. In yet another embodiment, the disease is GVHD. In one particular embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the subject. In further embodiments, the pharmacopoeia is for suppressing the immune system. In further embodiments, the pharmacopoeia is for use in a method of suppressing the immune system in a subject, comprising administering to the subject an amount of the agent effective in suppressing the immune system. The “subject” in any of the above embodiments may be a mammal, for example, a human. "Suppression of the immune system" in any of the embodiments described above may include one or more of the following: a general decrease in immune function, a decrease in T cell function, a decrease in B cell function, a decrease in T cell responsiveness, etc.

[0258] In a further embodiment, the Disclosure provides a method for clustering PD1 and / or enhancing PD1 activity in a subject, comprising administering an effective amount of the CD20-PD1 binding molecule of the Disclosure to the subject. The CD20-PD1 binding molecule of the Disclosure can induce PD1 clustering at the interface between CD20-presenting cells and T cells. This results in targeted immunosuppression, protecting CD20-presenting cells and surrounding cells and tissues from T cell death. High levels of CD20 are found on B cells, which are abundant in afferent lymph nodes and autoimmune tissues (e.g., the pancreas in type 1 diabetes (T1D)). The CD20-PD1 binding molecule of the Disclosure can stimulate PD1 in a cell and / or tissue-specific manner and inhibit autoreactive T cell activation. In T1D, abundant CD20+ B cells result in PD1 clustering on autoreactive T cells, inhibiting autoreactive cytotoxic T cells from killing pancreatic islet cells. In one embodiment, a composition comprising the CD20-PD1 binding molecule of the present disclosure in a pharmaceutically acceptable form is administered to the subject.

[0259] In a further embodiment, the Disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the CD20-PD1 binding molecule of the Disclosure to the subject. In one embodiment, a composition comprising the CD20-PD1 binding molecule of the Disclosure in a pharmaceutically acceptable form is administered to the subject. In a particular embodiment, the disease to be treated is an autoimmune disease. Autoimmune diseases treatable with the CD20-PD1 binding molecule disclosed herein include type 1 diabetes, primary biliary cholangitis (PBC), Goodpasture syndrome, amyloidosis, ankylosing spondylitis, anti-glomerular basement nephritis, anti-tubulobasement 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 syndrome, fibrotic alveolitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, and IgA nephropathy. These include IgG4-related sclerosing diseases, immune thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), multiple sclerosis, polyneuropathy, organomegaly, endocrine disorders, monoclonal syndromes (POEMS), polyarteritis nodosa, rheumatoid arthritis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm or testicular autoimmunity, generalized rigidus syndrome (SPS), Takayasu's arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), and vasculitis.

[0260] 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 another embodiment, 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 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 for suppressing the immune system. The “individual” in any of the above embodiments may be a mammal, for example, a human. The “suppression of the immune system” in any of the above embodiments may include one or more of the following: a general reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell responsiveness, etc.

[0261] In certain embodiments, the disease being treated is an autoimmune disease. CD20-PD1 conjugated 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 CD20-PD1 conjugated molecules often do not provide a cure and may only provide partial benefits. In some embodiments, physiological changes that provide some benefit are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of CD20-PD1 conjugated molecule that produces a physiological change is considered an "effective dose" or "therapeutic effective dose." The subject, patient, or individual requiring treatment is typically a mammal, more specifically a human.

[0262] For the prevention or treatment of disease, the appropriate dosage of the CD20-PD1 conjugated molecule of this disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the route of administration, the patient's weight, the specific CD20-PD1 conjugated 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 conjugated molecule, and the judgment of the attending physician. In any case, the healthcare professional responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject. Various administration schedules, including but not limited to single or multiple doses, bolus administration, and pulse infusion, are contemplated herein.

[0263] CD20-PD1 conjugated molecules are preferably administered to patients in a single dose or over a series of treatments. For example, depending on the type and severity of the disease, a CD20-PD1 conjugated molecule of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to a patient, whether by one or more separate doses or by continuous infusions. A typical daily dose will range from approximately 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, treatment will generally be continued until the desired suppression of symptoms occurs, depending on the condition. One exemplary dose of CD20-PD1 conjugated molecules may range from approximately 0.005 mg / kg to approximately 10 mg / kg. In other non-limiting examples, doses may also include approximately 1 μg / kg / body weight, approximately 5 μg / kg / body weight, approximately 10 μg / kg / body weight, approximately 50 μg / kg / body weight, approximately 100 μg / kg / body weight, approximately 200 μg / kg / body weight, approximately 350 μg / kg / body weight, approximately 500 μg / kg / body weight, approximately 1 mg / kg / body weight, approximately 5 mg / kg / body weight, approximately 10 mg / kg / body weight, approximately 50 mg / kg / body weight, approximately 100 mg / kg / body weight, approximately 200 mg / kg / body weight, approximately 350 mg / kg / body weight, approximately 500 mg / kg / body weight, up to approximately 1000 mg / kg / body weight or more, and any range from there. In non-limiting examples of the derivable range from the numerical values ​​enumerated herein, doses can be administered in ranges such as approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, or approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight, based on the above numerical values. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives approximately 2 to approximately 20 doses, or for example, approximately 6 doses of the CD20-PD1 binding molecule). A higher loading dose may be administered first, followed by one or more lower doses. However, other dosing regimens may also be useful. The progress of this therapy is readily monitored by conventional techniques and assays.

[0264] The CD20-PD1 conjugated molecules of this disclosure will generally be used in amounts effective to achieve the intended purpose. For use in treating or preventing a disease condition, the CD20-PD1 conjugated molecules or their pharmaceutically effective compositions of this disclosure will be administered or applied in therapeutically effective doses. Determining therapeutically effective doses is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosures provided herein.

[0265] For systemic administration, the therapeutically effective dose can first be estimated from in vitro assays such as cell culture assays. Subsequently, the EC is determined by cell culture. 50 To achieve the circulating concentration range, including [specific concentration range], doses are formulated in animal models. Such information can be used to more accurately determine useful doses in humans.

[0266] The initial dose can also be estimated from in vivo data, such as animal models, using techniques known in the art. Those skilled in the art will be able to easily optimize the human dose based on the animal data.

[0267] The dosage and interval of administration may be individually adjusted to provide sufficient plasma levels of CD20-PD1 binding molecules to maintain therapeutic effect. Typical patient doses for administration by injection range from approximately 0.1 to 50 mg / kg / day, typically from approximately 0.5 to 1 mg / kg / day. Therapeutably effective plasma levels may be achieved by administering the drug multiple times daily. Plasma levels may be measured, for example, by ELISA HPLC.

[0268] In the case of local administration or selective uptake, the effective local concentration of the CD20-PD1 binding molecule may not be related to the plasma concentration. Those skilled in the art will be able to optimize the therapeutically effective local dosage without excessive experimentation.

[0269] The therapeutically effective doses of the CD20-PD1 binding molecules described herein will generally provide therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic efficacy of the CD20-PD1 binding molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal studies, LD 50 (A dose that causes death in 50% of the population) and ED 50 (The therapeutically effective dose in 50% of the population) can be determined. The dose ratio between the toxic effect and the therapeutic effect is the therapeutic indicator, and it is the LD 50 / ED 50 It can be expressed as a ratio. CD20-PD1 binding molecules that exhibit a large therapeutic index are preferred. In one embodiment, the CD20-PD1 binding molecule according to this disclosure exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a range of suitable dosages for use in humans. The dosage is preferably ED with little or no toxicity. 50 The circulating concentration range includes [specific concentration range]. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, and the patient's condition. The exact prescription, route of administration, and dosage can be selected by the individual physician, taking into account the patient's condition. (See, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch.1, p.1, which is incorporated herein by reference in its entirety).

[0270] The attending physician of a patient treated with the CD20-PD1 binding molecule of this disclosure will be aware of the methods and timing for discontinuing, interrupting, or adjusting the administration in the event of toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also be aware of how to adjust the treatment to a higher level. The size of the dose administered in managing the desired disorder will vary depending on the severity of the condition being treated and the route of administration, etc. The severity of the condition can be assessed, for example, in part by standard prognostic assessment methods. Furthermore, the dose and possibly the frequency of administration will also vary depending on the age, weight, and response of the individual patient.

[0271] 6.11.1.1 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. Therefore, in some embodiments, the CD20-PD1 binding molecules, nucleic acids, and / or pharmaceutical compositions of the present disclosure can be administered to subjects who have T1D or are at risk of developing T1D. Risk factors for developing T1D include, but are not limited to, genetic markers (e.g., human leukocyte antigen (HLA) complexes; 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, Caucasians are more susceptible to type 1 diabetes), family history, early diet, and 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 individuals at risk of developing T1D is within the scope of the art.

[0272] 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 in accordance with the methods of the Disclosure.

[0273] 6.11.2. Combination Therapy The CD20-PD1 conjugated molecules of this disclosure may be administered in combination with one or more other agents in therapeutic settings. For example, the CD20-PD1 conjugated molecules of this disclosure may be administered concurrently with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat symptoms or diseases in a subject requiring such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other.

[0274] In certain embodiments, additional therapeutic agents are immunosuppressants, but are not limited to, mycophenolate mofetil (MMF), mycophenolic acid (MPA), cyclosporine A, FK506-like compounds (e.g., FK506, FK506 derivatives, and FK506 analogs), rapamycin compounds (including rapamycin, rapamycin derivatives, and rapamycin analogs), corticosteroids (e.g., hydrocortisone, hydroxyl-triamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropio Hydrocortisone acetate, clobetasol valerate, desonide, desoxymethasone, deoxycorticosterone acetate, dexamethasone, dichlorizone, diflorasone acetate, diflucortolone valerate, fluadrenolone, fluchlorone acetonide, fludrocortisone, flumetasone pivalate, fluocinolone acetonide, fluocinonide, flucortin butyl ester, fluocortolone, fluprednilidene acetate, fludrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone Triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone acetate, fluradrenolon, fludrocortisone, diflurozone diacetate, fluradrenolon acetonide, medrizone, amsinafel, amsinafid, betamethasone and its ester balance, chloroprednisone, chloroprednisone acetate, crocorterone, cresinolone, dichlorizone, difluprednate, fluchloronide, flunisolide, fluoromethalone, fluperolon, Fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocoltamate, meprednisone, paramethasone, prednisolone, prednisone, betamethasone dipropionate, triamcinolone, and mixtures thereof), nonsteroidal anti-inflammatory drugs (e.g., oxicam, e.g., piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, e.g., aspirin, disalside, benolilate, trilysate, sapphin, sorprin, diflunisal, and fendosal;Acetic acid derivatives, e.g., diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, phlofenac, thiopinac, didomethacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenametes, e.g., mefenamic acid, meclofenamic acid, flufenamic acid, diflumic acid, and tolfenamic acid; propionic acid derivatives, e.g., ibuprofen, naproxen, benoxaprofen, flurbiprofen, ke Toprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, thioxaprofen, suprofen, aluminoprofen, and thiaprofen; pyrazoles (e.g., phenylbutazone, oxyfenbutazone, feprazone, azapropazone, and trimethazone), and anti-inflammatory cytokines or chemokines (e.g., IL-4, IL-6, IL-10, IL-11, and IL-13);

[0275] Such other agents are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of CD20-PD1 binding molecule used, the type of disorder or treatment, and other factors discussed above. CD20-PD1 binding molecules are generally used in the same dosages and routes of administration described herein, or in about 1–99% of the dosages described herein, or in any dosage and route that is deemed empirically / clinically appropriate.

[0276] Such combination therapies described above include concomitant administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case the CD20-PD1 binding molecule of this disclosure may be administered before, concurrently with, and / or after the administration of additional therapeutic agents and / or adjuvants.

[0277] 6.11.2.1. Combination therapy using CD20-PD1 conjugate molecule therapy and immunotherapy The CD20-PD1 binding molecules of this disclosure can be advantageously used in combination with chimeric antigen receptor ("CAR") expressing cells, such as CAR-expressing Treg ("CAR-Treg") cells, e.g., CAR-Treg, in the treatment of autoimmune diseases. In some embodiments, CAR-Treg cells are recognized by the CD20-targeting moiety in the CD20-PD1 binding molecule. The CD20-targeting moiety can recognize Treg cell receptors or other cell surface molecules on CAR-Treg cells. In some embodiments, the CD20-targeting moiety in the CD20-PD1 binding molecule can bind to the extracellular domain of the CAR, e.g., the antigen-binding domain. CAR-Treg cells are described in Fritsche et al., 2020, Trends Biotechnol, 38(10):1099-1112, Zhang et al., 2018, Front Immunol, 9:2359, and Mohseni et al. Front Immunol, 11:1608, each of which is incorporated herein by reference in its entirety.

[0278] 7. Array Certain sequences of this disclosure are provided in Table S below.

[0279] [Table 5-1]

[0280] [Table 5-2]

[0281] [Table 5-3]

[0282] [Table 5-4]

[0283] [Table 5-5]

[0284] [Table 5-6]

[0285] [Table 5-7]

[0286] [Table 5-8]

[0287] [Table 5-9]

[0288] [Table 5-10]

[0289] [Table 5-11]

[0290] 8. Specific embodiments and citation of references While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments described below.

[0291] 1. It is a protein, (a) CD20 targeting portion, (b) The PD1 agonist portion, (i) an amino acid sequence having at least approximately 70% sequence identity with SEQ ID NO: 2, or (ii) A PD1 agonist moiety comprising an amino acid sequence having at least approximately 70% sequence identity with SEQ ID NO: 14, (c) A protein containing a dimerized moiety.

[0292] 2. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO: 2. 3. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 2.

[0293] 4. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO: 2. 5. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 2.

[0294] 6. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 2. 7. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO: 2.

[0295] 8. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 2. 9. The protein according to any one of Embodiments 1 to 8, wherein the PD1 agonist portion contains or consists of the amino acid sequence of SEQ ID NO: 2.

[0296] 10. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO: 8. 11. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 8.

[0297] 12. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO: 8. 13. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 8.

[0298] 14. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 8. 15. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO: 8.

[0299] 16. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 8. 17. The protein according to any one of embodiments 10 to 16, wherein the PD1 agonist portion contains or consists of the amino acid sequence of SEQ ID NO: 8.

[0300] 18. The protein according to any one of embodiments 10 to 16, wherein the PD1 agonist portion contains or consists of the amino acid sequence of SEQ ID NO: 9. 19. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO: 14.

[0301] 20. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 14. 21. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO: 14.

[0302] 22. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 14. 23. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 14.

[0303] 24. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO: 14. 25. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 14.

[0304] 26. The protein according to any one of embodiments 19 to 25, wherein the PD1 agonist portion contains or consists of the amino acid sequence of SEQ ID NO: 14. 27. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO: 20.

[0305] 28. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 20. 29. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO: 20.

[0306] 30. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 20. 31. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 20.

[0307] 32. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO: 20. 33. The protein according to Embodiment 1, wherein the PD1 agonist comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 20.

[0308] 34. The protein according to any one of embodiments 27 to 33, wherein the PD1 agonist portion contains or consists of the amino acid sequence of SEQ ID NO: 20. 35. The protein according to any one of Embodiments 1 to 34, wherein the protein does not contain the PDL1 transmembrane domain.

[0309] 36. The protein according to any one of Embodiments 1 to 35, wherein the protein does not contain the PDL1 intracellular domain. 37. The protein according to any one of Embodiments 1 to 36, wherein the protein does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 6.

[0310] 38. The protein according to any one of Embodiments 1 to 36, wherein the protein does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 13. 39. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 5.

[0311] 40. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to Sequence ID No. 5. 41. The protein according to any one of Embodiments 1 to 40, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 5 having a length of at least 20 amino acids.

[0312] 42. The protein according to any one of Embodiments 1 to 40, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 5 having a length of at least 10 amino acids.

[0313] 43. The protein according to any one of Embodiments 1 to 40, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 5 having a length of at least 5 amino acids. 44. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 11.

[0314] 45. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to Sequence ID No. 11. 46. ​​The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 11 having a length of at least 20 amino acids.

[0315] 47. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 11 having a length of at least 10 amino acids.

[0316] 48. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 11 having a length of at least 5 amino acids.

[0317] 49. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 17. 50. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to Sequence ID No. 17.

[0318] 51. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 17 having a length of at least 20 amino acids.

[0319] 52. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 17 having a length of at least 10 amino acids.

[0320] 53. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 17 having a length of at least 5 amino acids.

[0321] 54. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 23. 55. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to Sequence ID No. 23.

[0322] 56. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 23 having a length of at least 20 amino acids.

[0323] 57. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 23 having a length of at least 10 amino acids.

[0324] 58. The protein according to any one of Embodiments 1 to 38, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 23 having a length of at least 5 amino acids.

[0325] 59. The protein according to any one of embodiments 1 to 58, wherein the PD1 agonist portion is not operably linked to the membrane-adjacent portion of PDL1. 60. The protein according to any one of embodiments 1 to 58, wherein the PD1 agonist portion is not operably linked to the membrane-adjacent portion of PDL2.

[0326] 61. The protein according to any one of Embodiments 1 to 60, wherein the PD1 agonist portion has an amino acid length of 120 or less. 62. The protein according to any one of Embodiments 1 to 61, wherein the portion of the protein is arranged in the order of CD20 targeting portion - PD1 agonist portion - dimerizing portion from the N-terminus to the C-terminus.

[0327] 63. The protein according to any one of Embodiments 1 to 61, wherein the portion of the protein is arranged from the N-terminus to the C-terminus in the order of PD1 agonist portion - CD20 targeting portion - dimerization portion.

[0328] 64. The protein according to any one of Embodiments 1 to 61, wherein the CD20 targeting portion is an anti-CD20 Fab, the dimerizing portion is an Fc domain, and the light chain of the Fab is not fused to the PD1 agonist portion.

[0329] 65. The protein according to any one of Embodiments 1 to 61, wherein the dimerized portion is an Fc domain and the PD1 agonist portion is not at the C-terminus of the Fc domain. 66. The protein according to any one of Embodiments 1 to 65, further comprising an antigen-binding fragment of an agonist anti-PD1 antibody.

[0330] 67. The protein according to Embodiment 66, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is N-terminus with respect to the PD1 agonist moiety. 68. The protein according to Embodiment 66, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is C-terminus with respect to the PD1 agonist portion.

[0331] 69. The protein according to Embodiment 66, wherein the portion of the protein is arranged from the N-terminus to the C-terminus in the order of PD1 agonist portion - antigen-binding fragment of the agonist anti-PD1 antibody - CD20 targeting portion - dimerization portion.

[0332] 70. The protein according to Embodiment 66, wherein the portion of the protein is arranged from the N-terminus to the C-terminus in the order of antigen-binding fragment of the agonist anti-PD1 antibody - PD1 agonist portion - CD20 targeting portion - dimerization portion.

[0333] 71. The protein according to any one of embodiments 66 to 70, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is in the form of Fab, Fv, or scFv. 72. The antigen-binding fragment of the agonist anti-PD1 antibody is (a) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; as described in WO / 2017 / 058859 The antigen-binding fragment of an agonist PD1 antibody selected from PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6; clones 2 and 19 described in WO / 2010 / 029434; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35 described in WO / 2004 / 056875, (b) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; PD as described in WO / 2017 / 058859 Containing heavy and / or light chain CDRs of agonist PD1 antibodies selected from antibodies PD1-17, PD1-28, PD1-33, and PD1-35, as described in WO / 2010 / 029434; clones 2 and 19; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35, as described in WO / 2004 / 056875, (c) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; PD as described in WO / 2017 / 058859 This includes VH and / or VL of agonist PD1 antibodies selected from antibodies PD1-17, PD1-28, PD1-33, and PD1-35, as described in WO / 2010 / 029434; clones 2 and 19; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35, as described in WO / 2004 / 056875, or (d) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1A as described in WO / 2017 / 058859 A protein according to any one of embodiments 66 to 71, which competes with agonist PD1 antibodies selected from antibodies PD1-17, PD1-28, PD1-33, and PD1-35 as described in WO / 2010 / 029434 for binding to PD1, and / or binds to the same epitope as the agonist PD1 antibody.

[0334] 73. The protein according to any one of embodiments 66 to 72, wherein the antigen-binding fragment of the agonist anti-PD1 antibody stimulates human PD1. 74. The protein according to any one of embodiments 66 to 72, wherein the antigen-binding fragment of the agonist anti-PD1 antibody stimulates mouse PD1.

[0335] 75. The protein according to any one of Embodiments 1 to 74, wherein the CD20 targeting portion binds to the extracellular domain of human CD20. 76. The protein according to any one of Embodiments 1 to 74, wherein the CD20 targeting portion binds to the extracellular domain of mouse CD20.

[0336] 77. The protein according to any one of Embodiments 1 to 76, wherein the CD20 targeting portion is an antigen-binding fragment of an anti-CD20 antibody. 78. The protein according to Embodiment 77, wherein the antigen-binding fragment of the anti-CD20 antibody is in the form of Fab, Fv, or scFv.

[0337] 79. The anti-CD20 antibody is (a) Topological domain of CD20, (b) Transmembrane domain of CD20, or (c) The protein according to Embodiment 77 or 78, which binds to an extracellularly displayed region of CD20 on the surface of a cell (e.g., a B cell).

[0338] 80. The anti-CD20 antibody is (a) Complementarity-determining regions ("CDR") having the CDR sequence of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or vertuzumab, (b) All six CDR sequences of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or vertuzumab. (c) At least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or bertuzumab, and the light chain CDR sequence of the universal light chain. (d) VH containing the amino acid sequence of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or vertuzumab, and VL containing the amino acid sequence of the same antibody, or (e) The protein according to any one of Embodiments 77 to 79, comprising VH, which includes the amino acid sequence of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, or vertuzumab, and VL, which includes the universal light chain VL sequence.

[0339] 81. The anti-CD20 antibody is (a) Selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, and vertuzumab, or (b) A protein according to any one of Embodiments 77 to 80, which competes with an anti-CD20 antibody selected from the group consisting of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, and bertuzumab for binding to CD20, and / or binds to the same epitope as the anti-CD20 antibody.

[0340] 82. A protein according to any one of Embodiments 1 to 81, comprising one or more linker moieties. 83. The protein according to embodiment 82, wherein the CD20 targeting portion and the PD1 agonist portion are separated by a linker portion.

[0341] 84. The protein according to embodiment 82 or 83, wherein the PD1 agonist portion and the dimerized portion are separated by a linker portion. 85. The protein according to any one of embodiments 82 to 84, wherein each linker portion is (a) at least 5 or at least 10 amino acids long, (b) up to 20, up to 25, or up to 30 amino acids long, and / or (c) 5 to 15 amino acids or 5 to 20 amino acids long.

[0342] 86. The protein according to any one of embodiments 82 to 85, wherein the protein comprises a glycine-serine linker. 87. The protein according to Embodiment 86, wherein the glycine-serine linker comprises sequence G4S (SEQ ID NO: 33) or a polymer thereof.

[0343] 88. The protein according to Embodiment 87, wherein the glycine-serine linker comprises a polymer containing 2, 3, 4, 5 or more repeats of the amino acid sequence G4S (SEQ ID NO: 33).

[0344] 89. The protein according to any one of Embodiments 1 to 88, further comprising (1) an additional CD20 targeting moiety, (2) an additional PD1 agonist moiety of 120 amino acids or less including (i) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO: 2, or (ii) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO: 14, and (3) an additional dimerizing moiety.

[0345] 90. The protein according to Embodiment 89, wherein the additional CD20 targeting portion and the additional PD1 agonist portion are separated by a linker portion. 91. The protein according to embodiment 89 or 90, wherein the PD1 agonist portion and the dimerized portion are separated by a linker portion.

[0346] 92. The protein according to any one of embodiments 89 to 91, wherein the linker portion is (a) at least 5 or at least 10 amino acids long, (b) up to 20, up to 25, or up to 30 amino acids long, and / or (c) 5 to 15 amino acids long or 5 to 20 amino acids long.

[0347] 93. The protein according to any one of embodiments 89 to 92, wherein the linker portion is a glycine-serine linker. 94. The protein according to Embodiment 93, wherein the glycine-serine linker comprises sequence G4S (SEQ ID NO: 33) or a polymer thereof.

[0348] 95. The protein according to Embodiment 93, wherein the glycine-serine linker comprises a polymer containing 2, 3, 4, 5 or more repeats of the amino acid sequence G4S (SEQ ID NO: 33).

[0349] 96. A protein according to any one of Embodiments 1 to 95, comprising two monomers by exemplary monomer 1. 97. The protein according to Embodiment 96, wherein exemplary monomer 1 comprises, or comprises, in an N-terminal to C-terminal orientation, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, and the dimerization moiety.

[0350] 98. The protein according to Embodiment 96 or 97, wherein exemplary monomer 1 consists of a single polypeptide chain. 99. The protein according to embodiment 96 or 97, wherein exemplary monomer 1 is composed of two polypeptide chains.

[0351] 100. A protein according to any one of embodiments 96 to 99, wherein exemplary monomer 1 comprises one or more universal light chains. 101. A protein according to any one of embodiments 96 to 100, having the configuration shown in Figure 1A.

[0352] 102. A protein according to any one of Embodiments 1 to 88, comprising two monomers by exemplary monomer 2. 103. The protein according to Embodiment 102, wherein exemplary monomer 2 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, and the dimerization moiety, or comprising the same.

[0353] 104. The protein according to embodiment 102 or 103, wherein exemplary monomer 2 consists of a single polypeptide chain. 105. The protein according to embodiment 102 or 103, wherein exemplary monomer 2 is composed of two polypeptide chains.

[0354] 106. The protein according to embodiment 102 or 103, wherein exemplary monomer 2 is composed of three polypeptide chains. 107. A protein according to any one of embodiments 102 to 106, wherein exemplary monomer 2 comprises one or more universal light chains.

[0355] 108. A protein according to any one of embodiments 102 to 107, having the configuration shown in Figure 1B. 109. A protein according to any one of Embodiments 1 to 88, comprising two monomers by exemplary monomer 3.

[0356] 110. The protein according to Embodiment 109, wherein exemplary monomer 3 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, and the dimerization moiety, or comprising the same.

[0357] 111. The protein according to Embodiment 109 or 110, wherein exemplary monomer 3 consists of a single polypeptide chain. 112. The protein according to Embodiment 109 or 110, wherein exemplary monomer 3 is composed of two polypeptide chains.

[0358] 113. The protein according to Embodiment 109 or 110, wherein exemplary monomer 3 is composed of three polypeptide chains. 114. A protein according to any one of embodiments 109 to 113, wherein exemplary monomer 3 comprises one or more universal light chains.

[0359] 115. A protein according to any one of embodiments 109 to 114, having the configuration shown in Figure 1C. 116. A protein according to any one of Embodiments 1 to 88, comprising two monomers by exemplary monomer 4.

[0360] 117. The protein according to Embodiment 116, wherein exemplary monomer 4 comprises, in an N-terminal to C-terminal orientation, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, and the dimerization moiety.

[0361] 118. The protein according to embodiment 116 or 117, wherein exemplary monomer 4 consists of a single polypeptide chain. 119. The protein according to embodiment 116 or 117, wherein exemplary monomer 4 is composed of two polypeptide chains.

[0362] 120. The protein according to embodiment 116 or 117, wherein exemplary monomer 4 is composed of three polypeptide chains. 121. A protein according to any one of embodiments 116 to 120, wherein the exemplary monomer 4 comprises one or more universal light chains.

[0363] 122. A protein according to any one of embodiments 59 to 121, having the configuration shown in Figure 1D. 123. A protein according to any one of Embodiments 1 to 88, comprising two monomers by exemplary monomer 5.

[0364] 124. The protein according to Embodiment 123, wherein exemplary monomer 5 comprises, in an N-terminal to C-terminal orientation, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, an optional linker moiety, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, and the dimerization moiety.

[0365] 125. The protein according to Embodiment 123 or 124, wherein exemplary monomer 5 consists of a single polypeptide chain. 126. The protein according to Embodiment 123 or 124, wherein exemplary monomer 5 is composed of two polypeptide chains.

[0366] 127. The protein according to Embodiment 123 or 124, wherein exemplary monomer 5 is composed of three polypeptide chains. 128. A protein according to any one of embodiments 123 to 127, wherein exemplary monomer 5 comprises one or more universal light chains.

[0367] 129. A protein according to any one of embodiments 123 to 128, having the configuration shown in Figure 1E. 130. A protein according to any one of embodiments 1 to 129, comprising two CD20 targeting moieties.

[0368] 131. The protein according to embodiment 130, wherein the two CD20 targeting regions are identical. 132. A protein according to any one of embodiments 1 to 131, comprising two PD1 agonist moieties.

[0369] 133. The protein according to embodiment 132, wherein the two PD1 agonist portions are identical. 134. A protein according to any one of embodiments 1 to 133, comprising two antigen-binding fragments of an agonist anti-PD1 antibody.

[0370] 135. The protein according to Embodiment 134, wherein the two antigen-binding fragments of the agonist anti-PD1 antibody are identical. 136. The protein according to any one of Embodiments 1 to 135, wherein the dimerized portion is an Fc domain.

[0371] 137. The protein according to Embodiment 136, wherein the Fc domain is a human Fc domain. 138. The protein according to embodiment 136 or 137, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0372] 139. The protein according to Embodiment 138, wherein the Fc domain is an IgG4 Fc domain. 140. The protein according to any one of Embodiments 136 to 139, wherein the Fc domain comprises the amino acid sequence ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 27) or a portion thereof.

[0373] 141. The protein according to Embodiment 138, wherein the Fc domain is an IgG1 Fc domain. 142. A protein according to any one of embodiments 136 to 141, comprising an Fc dimer.

[0374] 143. The protein according to Embodiment 142, wherein the Fc dimer is an Fc homodimer. 144. The protein according to Embodiment 142, wherein the Fc dimer is an Fc heterodimer.

[0375] 145. The protein according to Embodiment 144, wherein the Fc heterodimer contains a knob-in-hole mutation. 146. The protein according to Embodiment 144 or 145, wherein the Fc heterodimer contains a star mutation.

[0376] 147. Proteins, (a) Means for coupling to CD20, (b) PD1 agonist moieties of 120 or fewer amino acids, (i) an amino acid sequence having at least approximately 70% sequence identity with SEQ ID NO: 2, or (ii) A PD1 agonist moiety comprising an amino acid sequence having at least approximately 70% sequence identity with SEQ ID NO: 14, (c) A protein containing a dimerized moiety.

[0377] 148. A protein, which has a length from the N terminus to the C terminus. (a) CD20 targeting portion, (b) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) A protein comprising a polypeptide chain containing an Fc domain.

[0378] 149. The protein is shaped from the N-terminus to the C-terminus. (a) Additional CD20 targeting portion, (b) An additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) The protein according to Embodiment 148, further comprising an additional polypeptide chain containing an additional Fc domain.

[0379] 150. It is a protein, (a) CD20 targeting portion, (b) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 9, (c) A protein containing an Fc domain.

[0380] 151. The protein is shaped from the N-terminus to the C-terminus. (a) Additional CD20 targeting portion, (b) An additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 9, (c) The protein according to Embodiment 150, further comprising an additional polypeptide chain containing an additional Fc domain.

[0381] 152. A protein, which has a length from the N terminus to the C terminus. (a) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (b) Antigen-binding fragment of agonist anti-PD1 antibody, (c) CD20 targeting portion, (d) A protein comprising a polypeptide chain containing an Fc domain.

[0382] 153. The protein is shaped from the N-terminus to the C-terminus. (a) an additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (b) Additional antigen-binding fragments of the agonist anti-PD1 antibody, (c) Additional CD20 targeting portion, (d) The protein according to Embodiment 152, further comprising an additional polypeptide chain containing an additional Fc domain.

[0383] 154. A protein, which has a length from the N terminus to the C terminus. (a) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 9, (b) Antigen-binding fragment of agonist anti-PD1 antibody, (c) CD20 targeting portion, (d) A protein comprising a polypeptide chain containing an Fc domain.

[0384] 155. The protein is shaped from the N-terminus to the C-terminus. (a) an additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 9, (b) Additional antigen-binding fragments of the agonist anti-PD1 antibody, (c) Additional CD20 targeting portion, (d) The protein according to Embodiment 154, further comprising an additional polypeptide chain containing an additional Fc domain.

[0385] 156. A protein, which has a length from the N terminus to the C terminus. (a) Antigen-binding fragment of an agonist anti-PD1 antibody, (b) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) CD20 targeting portion, (d) A protein comprising a polypeptide chain containing an Fc domain.

[0386] 157. The protein is shaped from the N-terminus to the C-terminus. (a) Additional antigen-binding fragments of the agonist anti-PD1 antibody, (b) An additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) Additional CD20 targeting portion, (d) The protein according to Embodiment 156, further comprising an additional polypeptide chain containing an additional Fc domain.

[0387] 158. A protein, which has a length from the N terminus to the C terminus. (a) Antigen-binding fragment of an agonist anti-PD1 antibody, (b) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 9, (c) CD20 targeting portion, (d) A protein comprising a polypeptide chain containing an Fc domain.

[0388] 159. The protein is shaped from the N-terminus to the C-terminus. (a) Additional antigen-binding fragments of the agonist anti-PD1 antibody, (b) An additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 9, (c) Additional CD20 targeting portion, (d) The protein according to Embodiment 158, further comprising an additional polypeptide chain containing an additional Fc domain.

[0389] 160. A nucleic acid or a group of nucleic acids encoding a protein according to any one of Embodiments 1 to 159. 161. A host cell manipulated to express any one of the proteins described in Embodiments 1 to 159 or any nucleic acid(s) described in Embodiment 160.

[0390] 162. A method for producing a protein according to any one of Embodiments 1 to 159, comprising culturing a host cell according to Embodiment 161 and thereby recovering the expressed protein.

[0391] 163. A pharmaceutical composition comprising a protein according to any one of Embodiments 1 to 159 and an excipient. 164. 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 according to any one of Embodiments 1 to 159 or a pharmaceutical composition according to Embodiment 163.

[0392] 165. The aforementioned immune deficiency or condition is type 1 diabetes, primary biliary cholangitis (PBC), Goodpasture syndrome, amyloidosis, ankylosing spondylitis, anti-glomerular basement nephritis, anti-tubulobasement 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 syndrome, fibrotic alveolitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, IgA nephropathy, IgG4-related sclerosing disease, The method according to Embodiment 164, wherein the condition is immune thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), multiple sclerosis, polyneuropathy, organomegaly, endocrine disorders, monoclonal syndromes (POEMS), polyarteritis nodosa, rheumatoid arthritis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm or testicular autoimmunity, generalized rigidus syndrome (SPS), Takayasu's arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), or vasculitis.

[0393] 166. The method according to Embodiment 165, wherein the immune disorder or condition is type 1 diabetes. 167. The method according to Embodiment 165, wherein the type 1 diabetes is childhood-onset type 1 diabetes.

[0394] 168. The method according to Embodiment 165, wherein the type 1 diabetes is adult-onset type 1 diabetes. 169. The method according to any one of embodiments 165 to 168, wherein the subject is a pediatric patient.

[0395] 170. The method according to any one of embodiments 165 to 168, wherein the subject is an adult patient. 171. The method according to Embodiment 164, wherein the immune disorder or condition is systemic lupus erythematosus.

[0396] 172. The method according to Embodiment 164, wherein the immune disorder or condition is Crohn's disease. 173. The method according to Embodiment 164, wherein the immune disorder or condition is graft-versus-host disease (GVHD).

[0397] 174. A method for suppressing a cellular autoimmune response, comprising administering an effective amount of a protein according to any one of Embodiments 1 to 159 or a pharmaceutical composition according to Embodiment 163 to the target.

[0398] 175. The method according to Embodiment 174, wherein the method reduces T cell function in the subject. 176. The method according to Embodiment 174 or 175, wherein the method reduces B cell function in the subject.

[0399] 177. The method according to any one of embodiments 174 to 176, wherein the method reduces T cell responsiveness in the subject. 178. The method according to any one of embodiments 164 to 177, further comprising administering an additional therapeutic agent to the subject.

[0400] 179. The method according to Embodiment 178, wherein the additional therapeutic agent is an immunomodulator, a cell proliferation inhibitor, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or a drug that increases the sensitivity of cells to apoptosis inducers.

[0401] 180. The method according to Embodiment 178, wherein the additional therapeutic agent is or contains CAR-expressing cells. 181. The method according to Embodiment 180, wherein the CAR-expressing cells are CAR-regulating T cells.

[0402] 182. A method for local PD1 agonism, comprising administering an effective amount of the protein described in any one of Embodiments 1 to 159 or the pharmaceutical composition described in Embodiment 163 to the subject.

[0403] 183. The method according to Embodiment 182, wherein administration of the protein or the pharmaceutical composition localizes PD1 agonism to the target B cells. 184. A method for locally modulating an immune response in a target tissue or cell expressing CD20, comprising administering an effective amount of the protein described in any one of Embodiments 1 to 159 or the pharmaceutical composition described in Embodiment 163 to the subject.

[0404] 185. The method according to Embodiment 184, wherein administration of the protein or the pharmaceutical composition modulates the immune response in the target B cells. 186. The method according to any one of Embodiments 164 to 185, wherein the protein described in any one of Embodiments 1 to 159 or the pharmaceutical composition described in Embodiment 163 is administered as a single dose.

[0405] 187. The method according to any one of embodiments 164 to 186, wherein the administration of the protein described in any one of embodiments 1 to 159 or the pharmaceutical composition described in embodiment 163 is not repeated. [Examples]

[0406] 9. Examples 9.1. Materials and Methods 9.1.1. Design and Production of CD20-PD1 Binding Molecules Constructs encoding bispecific CD20-PD1 agonists and controls, as shown in Tables 3 and 4 below, were generated. The bispecific CD20-PD1 agonists contained different compositions of mouse anti-CD20 and modified mouse PDL1 ectodomains, IgG1 effector null (EN) (L234A, L235E, G237A, A330S, and P331S, EU numbered) domains, and linkers of different lengths from different repeats of G4S (SEQ ID NO: 33). 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 mature proteins.

[0407] Knob-forming mutation: T366W (EU numbering). Hole-forming mutations: T366S, L368A, and Y407V (EU numbered). Star mutations: H435R and Y436F (EU numbering).

[0408] [Table 6-1]

[0409] [Table 6-2]

[0410] The constructs were expressed in Expi293F® cells by transient transfection (Thermo Fisher Scientific). Proteins from the Expi293F supernatant were purified using a ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with either a HiTrap® Protein G HP or a MabSelect SuRe pcc column (Cytiva). After a single-step elution, the antibodies were neutralized, dialyzed to a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, divided, 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.

[0411] [Table 7-1]

[0412] [Table 7-2]

[0413] [Table 7-3]

[0414] [Table 7-4]

[0415] [Table 7-5]

[0416] [Table 7-6]

[0417] [Table 7-7]

[0418] [Table 7-8]

[0419] [Table 7-9]

[0420] [Table 7-10]

[0421] 9.1.2. Flow Cytometry Cells (MC38 overexpressing HEK293 and mCD20, or Jurkat overexpressing mPD1) were divided into 1 × 10⁶ cells. 6 Cells / mL were resuspended in FACS washing solution (PBS containing 1% FBS). Staining was performed at 1 × 10⁶ per well. 5 The experiment was performed on cells. The antibody was divided into 1.3 × 10⁶ units. -07 The antibody was diluted at a ratio of 1:5 from the initial concentration of M. The diluted antibody was then added to the wells containing cells. Cells were stained at 2–8°C for 30 minutes and washed twice with FACS wash buffer. APC conjugate goat anti-human IgG (Jackson Immuno Research, 109-607-003, 1:400) or AF647 conjugate goat anti-mFc Fab (Jackson Immuno Research, 115-607-185, 1:400) was added, and cells were stained at 2–8°C for 30 minutes. After washing, cells were fixed in 2% paraformaldehyde at 2–8°C for 30 minutes. After two washes, stained cells were analyzed using a BD LSR Fortessa® FACS instrument. Results were analyzed by FlowJo. Mononuclear cells were selected using an FSC / SSC gate.

[0422] For spinal cord T cell infiltration flow cytometry analysis, single-cell suspensions from the spinal cord were first prepared by collagenase D (Roche, 11088882001) digestion and Percoll (GE Healthcare, 17-0891-02) gradient separation. Cells were resuspended in FACS wash 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 using a BD FAC Symphony Cell Analyzer. Results were analyzed using OMIQ cytometry software.

[0423] 9.1.3. Luciferase Reporter Assay: Anti-mCD20 × mPDL1 Ectodomain Molecules The ability of the anti-mCD20×mPDL1 ectodomain molecule to stimulate mouse PD1 (mPD1) on Jurkat cells in the presence of mouse CD20 (mCD20) presented on HEK293 cells was evaluated using a luciferase-based reporter assay. The overall design of the reporter assay is shown in Figures 3A-3C. AP1 is a transcription factor involved in regulating gene expression during T cell activation (Samelson 2002, PMID:11861607). A bispecific antibody (bsAb) that binds to human CD3 and CD22, CD3 bsAb (REGN10551), was used to stimulate T cell activation via the binding of antigen on target cells to receptors on T cells, similar to the CD3×CD20 bsAb mentioned above (Smith et al. 2015, PMID:26659273). Engagement of mPD1 on Jurkat cells via mCD20-anchored mCD20×mPDL1 resulted in inhibition of PD1 agonism-driven luciferase signaling.

[0424] 9.1.3.1. Manipulation of Jurkat / AP1-luc / mPD1 cells Jurkat / AP1-luc / mPD1 cells were generated by sequentially transducing AP1 (activated protein 1)-luciferase reporter lentivirus (QIAGEN CLS-011L) followed by mPD1 ORF-containing lentivirus (mPD1 NM_008798) into Jurkat E6-1 cells (ATCC number TIB-152).

[0425] 9.1.3.2. Manipulation of HEK293 / hCD22 / mCD20 cells HEK293 / hCD22 / mCD20 cells were generated by sequential lentiviral transduction using human CD22 ORF-coded lentivirus (NP_001762.2) followed by mCD20 ORF-coded lentivirus (NP_031667.1).

[0426] 9.1.3.3. Luciferase assay setup For bioassays, HEK293 / CD22 / mCD20 target cells were seeded at 10,000, 15,000, or 20,000 cells / well in 96-well plates in assay medium (RPMI1640 (Irvine Scientific) supplemented with 10% fetal bovine serum and L-glutamine-penicillin-streptomycin (Invitrogen)) and incubated overnight at 37°C in 5% CO2. The following day, Jurkat / AP1-luc / mPD1 reporter cells were added to the wells containing the cultured target cells at 30,000, 40,000, or 50,000 cells / well. The molecules of the disclosed or control antibodies were then serially diluted in assay medium 1:3 to final concentrations ranging from 100 nM to 1 pM or 1.69 pM (with additional conditions excluding the test molecule) and added to the cells together with 1 nM or 2.5 nM anti-CD3 bsAb. To obtain a range of activation, CD3 bsAb was serially diluted 1:3 to final concentrations ranging from 100 nM to 1.69 pM (with additional conditions excluding bispecific mAbs) and added to cells. 37°C / 5% CO2 2After a 5-hour incubation, luciferase activity was detected using an Envision multi-label plate reader (PerkinElmer) following the addition of ONE-Glo® (Promega) reagent. All conditions were tested in two consecutive sets.

[0427] EC50 / IC50 values ​​were determined using GraphPad Prism™ software with nonlinear regression (4-parameter logistic regression). The percentage of inhibition was calculated based on the relative luminescence unit (RLU) value using the following formula.

[0428]

number

[0429] In this formula, "RLU ベースライン "RLU" is the luminescence value from cells treated with a fixed amount of CD3 bsAb without the test molecule. 阻害 "RLU" is the luminescence value at the highest concentration of a test molecule containing a certain amount of CD3 bsAb. バックグラウンド " is the luminescence value from cells without CD3 bsAb or the test molecule.

[0430] 9.1.4. Determination of the oligomerization state of anti-mCD20×mPDL1 ectodomain molecules by size exclusion chromatography Size exclusion ultrahigh performance liquid chromatography (SE-UPLC) was used to evaluate the size heterogeneity of anti-mCD20×mPDL1 ectodomain molecules. SE-UPLC analysis was performed using a Waters Acquity UPLC H-Class system, with 10 μg of each protein sample injected into an Acquity BEH SEC column (200 Å, 1.7 μm, 4.6 × 300 mm) at a flow rate of 0.3 mL / min. The mobile phase buffer contained 10 mM sodium phosphate, 500 mM NaCl, and pH 7.0. Eluted samples were detected by UV absorbance at 280 nm using a photodiode array module.

[0431] 9.1.5.Thermal stability The thermal stability of the anti-mCD20×mPDL1 ectodomain molecule was evaluated using differential scanning fluorescence (DSF). DSF analysis was performed using a ThermoFisher QuantStudio5 system. The stock solution of each sample was diluted to 0.2 mg / mL in 1×PBS-glycerol, pH 7.4 and transferred to a 96-well plate. An excess of (8×)Sypro Orange® fluorescent dye, which preferentially binds to embedded hydrophobic residues when the protein unfolds, was added to each well, and the thermal stability profile was determined by a linear temperature increase from 25°C to 95°C over 20 minutes.

[0432] 9.1.6. Percentage of Assembly The bifunctional fusion molecule assemblies were assayed by high-throughput analysis using a Clipper LabChip GX (Perkin Elmer, Waltham, MA) according to the manufacturer's protocol. Briefly, sample buffers were prepared by mixing 7 ml of HT protein expression sample buffer with either 240 μl of BME (reducing) or 25 mM iodoacetamide (IAM, for non-reducing assays). Samples were normalized to 0.5 mg / ml using the sample buffer and then heated at 70°C for 10 minutes. 70 μl of water was added to each sample before loading it into the instrument. Tips were prepared according to the manufacturer's instructions. Electrophoresis of the samples was analyzed using LabChip GX software. Peaks from the non-reducing electrophoresis indicate the percentage of intact antibody.

[0433] 9.1.7. Activity of anti-mCD20×mPDL1 ectodomain molecule in prediabetic NOD mice Ten-week-old pre-diabetic non-obese diabetic (NOD) mice (The Jackson Laboratory) were intraperitoneally treated twice weekly with selected anti-mCD20×mPDL1 ectodomain molecules at doses of 1, 0.1, or 0.01 mg / kg for the duration of the experiment (e.g., until 28 weeks of age). Blood glucose was monitored every other week, and body weight was monitored weekly. The overall experimental design is shown in Figure 5.

[0434] 9.1.8. Activity of the anti-mCD20×mPDL1 ectodomain molecule in an experimental autoimmune encephalomyelitis / multiple sclerosis mouse model. Myelin oligodendrocyte glycoprotein (MOG) in mice 35-55 Administration of immunodominant 35-55 epitopes of ) produces anti-MOG antibodies that cause demyelination and chronic experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis (MS).

[0435] EAE was found in wild-type C57BL / 6 mice (10-12 weeks old, male, Jackson Laboratory) at 200 mg of MOG in CFA on day 1. 35-55 Pertussis was induced by subcutaneous injection. Considering that pertussis toxin administration promotes the migration of T cells to the central nervous system by weakening the blood-brain barrier, mice were also intraperitoneally injected 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 deficiency of righting reflex, 3: partial hindlimb paralysis, 4: complete hindlimb paralysis, 5: complete hindlimb and partial forelimb paralysis or morbidity.

[0436] Starting on day 2, mice were administered intraperitoneally twice a week with a selected anti-mCD20×mPDL1 ectodomain molecule or an appropriate control molecule. Endpoint tissue collection was performed at the peak of disease on day 20. Spinal cord infiltrates were used for flow cytometry, and spleen MOG-specific T cell responses were evaluated using ELISPOT.

[0437] 9.2. Example 1: Production and Stability of Bispecific Anti-mCD20-mPDL1 Ectodomain Agonist 9.2.1.Summary Mammalian expression vectors for individual heavy and light chains were prepared by DNA synthesis and cloning using ready-to-use constructs in the pcDNA3.4 Topo expression system from Life Technologies (Carlsbad, CA). To express the molecules, the heavy and universal light chain DNAs were co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was collected and processed for purification via HiTrap Protein A FF or Mab Select SuRe column (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.

[0438] 9.2.2.Results Various anti-mCD20 × mPDL1 ectodomain molecules were expressed and purified from Expi293 Freestyle cells (Table 5) via a one-step Mab-Select SuRe column, with total yields ranging from 2.7 to 7.7 mg. In general, molecules with a 1:1 valence ratio (anti-mCD20:mPDL1 ectodomain) showed higher yields (4.1 to 7.7 mg) than those with a 2:1 or 2:2 ratio (Table 5).

[0439] [Table 8]

[0440] After a one-step affinity purification, high molecular weight (HMW)% and monomer% were examined by SE-UPLC, and thermal stability was monitored by differential scanning fluorescence (DSF) (Table 6). The majority of anti-mCD20×mPDL1 ectodomain fusion molecules showed more than 85% monomer species without additional size exclusion chromatography (SEC). In 2+2 m20_mPL_4(L), the monomer percentage increased to 99% after two column purifications including an SEC step (Table 6). All anti-mCD20×mPDL1 ectodomain fusions exhibited similar thermal stability as measured by DSF using Tm1 at approximately 60°C. Furthermore, all bifunctional fusions had excellent heavy chain assembly as determined by capillary electrophoresis SDS (CE-SDS) (Table 6).

[0441] [Table 9]

[0442] 9.3. Example 2: Evaluation of binding properties of anti-mCD20 × mPDL1 ectodomain molecule The ability of anti-mCD20×mPDL1 ectodomain molecules to bind to two targets on the cell surface was evaluated using a flow-coupled assay.

[0443] 9.3.1.Results Binding curves are shown in Figures 2A and 2B. For both mPD1 and mCD20 binding, higher potency and maximal MFI were observed in the divalent molecule compared to a monovalent molecule of similar format. In particular, 2+2 m20_mPL_4(L) shared similar binding to HEK293 / mCD20 cells as 2+1 m20_mPL_3(G) (Figures 2A and 2B, Table 7), 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 valence for mPD1 binding. Across both divalent and monovalent molecules, the binding signal appeared to be orientation-dependent, with orientation of the N-terminal anti-mCD20 and hPDL1 ectodomain relative to the Fc domain generally resulting in higher potency and maximal MFI. The anti-mCD20 or mPDL1 ectodomain showed reduced binding when positioned between the N-terminal portion and the hinge region preceding Fc (Figures 2A and 2B, Table 7).

[0444] 9.4. Example 3: mPDL1 agonism by anti-mCD20 × mPDL1 ectodomain molecule Using the bioassay shown in Figure 3 and described in Section 9.1.3, we studied mPD1 agonism induced by anti-mCD20×mPDL1 ectodomain molecules.

[0445] 9.4.1.Results The results of the luciferase assay are shown in Figure 4 and Table 7. The anti-mCD20×mPDL1 ectodomain molecules of this 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, the four molecules of this 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 7) showed the strongest PD1 agonism with maximum inhibition of 74% to 84% (Figures 4C and 4E). The remaining molecules showed weak inhibition or no inhibition, with maximum inhibition ranging from -10 to 40%. Isoform control antibodies did not show inhibition of signaling. CD3 bsAb showed activation of T cell signaling at EC50 values ​​of 627 pM and 1.15 nM.

[0446] 9.4.2. Summary of data from in vitro assays using anti-mCD20 × mPDL1 ectodomain molecules Table 7 provides an overview of in vitro data collected with various anti-mCD20×mPDL1 ectodomain molecules, including cell-based flow binding and in vitro bioassay results. Results from luciferase assays are shown in Figures 4A–4E. The molecules 2+2 m20_mPL_4 and 2+1 m20_mPL_3 (G and L in Table 6, respectively) showed the strongest PD1 agonism. Cell-based flow analysis revealed that 2+2 m20_mPL_4 exhibited the strongest binding to mPD1 and mCD20, while 2+1 m20_mPL_3 revealed only moderate binding to mPD1-expressing cells, suggesting that clustering of mPD1 via divalent binding of mCD20 is required in the presence of both APCs and effector cells. Overall, similar cell-binding affinities (mPD1 or mCD20) were not converted to similar PD1 agonisms, e.g., F vs. G and K vs. L (Figure 4A and Table 7), indicating that both the valence and structural configuration of the CD20 and mPDL1 ectodomain arms are important for conferring activity.

[0447] [Table 10-1]

[0448] [Table 10-2]

[0449] 9.5. Example 5: In vivo efficacy of anti-mCD20×mPDL1 ectodomain molecule The ability of selected 2+2 and 2+1 anti-mCD20×mPDL1 ectodomain molecules to prevent the development of type 1 diabetes (T1D) was evaluated in pre-diabetic NOD mice. The experimental design is shown in Figure 5 and described in Section 9.1.7.

[0450] 9.5.1.Results Individual animal data are shown in Figures 6A-6I. Figures 7A and 7B 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. While control NOD mice had a lower incidence of diabetes, higher doses of the (2+2) anti-mCD20×mPDL1 ectodomain molecule, 2+2m20_mPL_4 (molecule L in Figure 2A), showed a clear protective trend, but (2+1 anti-mCD20×mPDL1 ectodomain molecule, 2+1m20_mPL_3 (molecule G in Figure 2A) did not (Figures 7A and 7B).

[0451] 9.6. Example 6: Reduction of autoimmune T cell infiltration by induction of anti-mCD20×mPDL1 ectodomain molecule T cell infiltration is 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, against T cell infiltration was evaluated by flow cytometry as described in Section 9.1.2.

[0452] 9.6.1.Results In one evaluation, populations of proliferating (activated) and inactivated islet-specific CD8+ T cells were analyzed in NOD mice described in Section 9.1.7, administered 0.1 or 1 mg / kg of 2+2 m20_mPL_4 or a control molecule. Treatment with 1 mg / kg of 2+2 m20_mPL_4 was associated with a significant percentage increase in low-activation CD8+ T cell clusters (Figure 8A). While the percentage of proliferating cell clusters did not differ across conditions (Figure 8B), the percentage of low-activation and low-proliferation cell clusters was higher in pancreatic tissue isolated from NOD mice treated with 1 mg / kg of 2+2 m20_mPL_4 (Figure 8C), indicating that this treatment was able to reduce pancreatic infiltration of activated autoimmune T cells.

[0453] In another assessment, T cell infiltration of the spinal cord was evaluated in the mouse model of multiple sclerosis described in Section 9.1.8. The spinal cords of mice treated with 1 mg / kg of 2+2 m20_mPL_4 contained significantly fewer CD3+ (Figure 9A), CD4+ (Figure 9B), and CD8+ (Figure 9C) T cells compared to the spinal cords of control mice treated with the same dosage. Therefore, treatment with 2+2 m20_mPL_4 was able to reduce T cell infiltration of the spinal cord in the multiple sclerosis model.

[0454] 9.7. Example 7: Design and production of a bispecific anti-mCD20×h / mPDL1 IgV agonist CD20-PD1 agonist constructs corresponding to certain examples from those described in Examples 1-6 were designed, with the full-length PDL1 ectodomain replaced by the PDL1 IgV domain. The constructs were designed to include (1) different configurations of anti-CD20 (mouse or human) and PDL1 IgV domains (mouse or human), (2) an IgG1 domain, and (3) linkers of different lengths from different repeats of G4S (SEQ ID NO: 33) (Table 8; Figures 10A-10C; 11A-11E). A 29-amino acid signal sequence from the 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 mature proteins.

[0455] Mammalian expression vectors for individual heavy and light chains were prepared by DNA synthesis and cloning using ready-to-use constructs in the pcDNA3.4 Topo expression system from Life Technologies (Carlsbad, CA). To express the molecules, the heavy and universal light chain DNAs were co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was collected and processed for purification via HiTrap Protein A FF or Mab Select SuRe column (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.

[0456] [Table 11-1]

[0457] [Table 11-2]

[0458] 9.8. Example 8: mPDL1 IgV binding to mPD1 with higher efficacy than the full-length ectodomain molecule of mPDL1. As described in Section 9.1.1, seven binding molecules and control molecules were generated. To evaluate whether the binding properties of the full-length mPDL1 ectodomain and the mPDL1 IgV construct differed, a flow binding assay was performed using mPD1-overexpressing Jurkat cells as described in Section 9.1.2.

[0459] 9.8.1.Results Table 9 shows the structures produced and evaluated in this embodiment.

[0460] [Table 12]

[0461] The hPDL1 full-length ectodomain-containing molecule AF_MH and the mPDL1 full-length ectodomain-containing molecule AF-12 showed similar binding to mPD1-overexpressing Jurkat cells (Figure 12A). In contrast, the mPDL1 IgV-containing molecule AF-12.1 showed a higher maximum MFI to mPD1-overexpressing Jurkat cells than the mPDL1 full-length ectodomain molecule AF-12, indicating that AF-12.1 had higher efficacy than AF-12 (Figure 12A).

[0462] The mPDL1 IgV bivalent molecule AF-17.1 did not show binding, but the mPDL1 full-length ectodomain bivalent molecule AF-17 showed minimal binding (Figure 12A), indicating the requirements for the CD20 targeting moiety. Neither construct showed binding to parental Jurkat cells (Figure 12C).

[0463] Next, we evaluated mCD20 arm binding using mCD20-overexpressing MC38 cells. AF-12.1, which has the N-terminus of the mCD20 arm, showed superior efficacy against each mPDL1 IgV compared to the mCD20 × mPDL1 full-length ectodomain molecule, AF-12, but had a similar EC50 (Figure 12B). Again, neither construct showed binding to the parent MC38 cells (Figure 12D).

[0464] 9.9. Example 9: mPDL1 IgV and molecules containing the full-length ectodomain exhibit similar agonism. To evaluate the PD1 agonism and its regulation of T cell signaling by mPDL1 IgV and full-length ectodomain-containing molecules, luciferase reporter assays were performed using HEK293 / hCD22 / mCD20 and Jurkat / AP1-luc / mPD1 reporter cells with anti-CD3 bsAb, as described in Section 9.1.3.

[0465] 9.9.1.Results The seven constructs presented in Table 9 were evaluated for PD1 agonism. Three of the seven constructs, AF-12, AF-12.1, and AF_MH1, were ICs in the range of 182–270 pM. 50 The values ​​indicated inhibition of T cell signaling, with inhibition percentages ranging from 85 to 86% (Figure 13). Therefore, these three constructs were associated with relatively strong PD1 agonism. The remaining four constructs did not show inhibition of T cell signaling. Similarly, the unrelated control antibody BetV1_mIgG1_AA also did not show inhibition of T cell signaling. The control CD3 bsAb, REGN10551, showed inhibition of 696 pM EC 50 The value activated T cell signaling (Figure 13).

Claims

1. It is a protein, (a) CD20 targeting portion, (b) The PD1 agonist portion, (i) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO: 2, or (ii) A PD1 agonist moiety comprising an amino acid sequence having at least approximately 70% sequence identity with SEQ ID NO: 14, (c) A protein containing a dimerized portion.

2. The aforementioned PD1 agonist, (a) Sequence identity of at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% with respect to Sequence ID No. 2, (b) The protein according to claim 1, comprising an amino acid sequence having at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO:

14.

3. The protein according to claim 1 or 2, wherein the PD1 agonist portion includes or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

14.

4. The protein according to any one of claims 1 to 3, wherein the protein does not contain a PDL1 transmembrane domain and / or a PDL1 intracellular domain.

5. The aforementioned protein, (a) Sequence identity of at least approximately 95% for sequence number 6, (b) Sequence identity of at least approximately 95% with respect to sequence number 13, (c) At least approximately 95% sequence identity with respect to sequence number 5, or (d) The protein according to any one of claims 1 to 4, which does not contain an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:

17.

6. The protein according to any one of claims 1 to 5, wherein the protein does not contain an amino acid sequence corresponding to SEQ ID NO: 5 or SEQ ID NO:

17.

7. The protein according to any one of claims 1 to 6, wherein the protein does not contain an amino acid sequence corresponding to any subsequence of Sequence ID No. 5 or Sequence ID No. 17 having a length of at least 20, at least 10, or at least 5 amino acids.

8. The protein according to any one of claims 1 to 7, wherein the PD1 agonist portion is not operably linked to the membrane-adjacent portion of PDL1 or PDL2.

9. The protein according to any one of claims 1 to 8, wherein the PD1 agonist portion has an amino acid length of 120 or less.

10. The aforementioned portion of the protein moves from the N-terminus to the C-terminus. (a) CD20 targeting moiety - PD1 agonist moiety - dimerization moiety, (b) The protein according to any one of claims 1 to 9, wherein the PD1 agonist portion, the CD20 targeting portion, and the dimerizing portion are arranged in that order.

11. The protein according to any one of claims 1 to 9, wherein the CD20 targeting portion is an anti-CD20 Fab, the dimerization portion is an Fc domain, and the light chain of the Fab is not fused to the PD1 agonist portion.

12. The protein according to any one of claims 1 to 9, wherein the dimerized portion is an Fc domain, and the PD1 agonist portion is not at the C-terminus of the Fc domain.

13. The protein according to any one of claims 1 to 12, further comprising an antigen-binding fragment of an agonist anti-PD1 antibody, wherein optionally the antigen-binding fragment of the agonist anti-PD1 antibody is in the form of Fab, Fv, or scFv.

14. The protein according to claim 13, wherein the portion of the protein is arranged from the N-terminus to the C-terminus in the order of PD1 agonist portion - antigen-binding fragment of the agonist anti-PD1 antibody - CD20 targeting portion - dimerization portion.

15. The protein according to claim 13, wherein the portion of the protein is arranged from the N-terminus to the C-terminus in the order of antigen-binding fragment of the agonist anti-PD1 antibody - PD1 agonist portion - CD20 targeting portion - dimerization portion.

16. The antigen-binding fragment of the agonist anti-PD1 antibody is (a) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; as described in WO / 2017 / 058859 The antigen-binding fragment of an agonist PD1 antibody selected from PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6; clones 2 and 19 described in WO / 2010 / 029434; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35 described in WO / 2004 / 056875, (b) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; PD as described in WO / 2017 / 058859 1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6; clones 2 and 19 as described in WO / 2010 / 029434; and agonist PD1 antibodies selected from antibodies PD1-17, PD1-28, PD1-33, and PD1-35 as described in WO / 2004 / 056875, comprising heavy and / or light chain CDRs of the agonist PD1 antibody, (c) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; PD as described in WO / 2017 / 058859 This includes VH and / or VL of agonist PD1 antibodies selected from 1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6; clones 2 and 19 as described in WO / 2010 / 029434; and antibodies PD1-17, PD1-28, PD1-33, and PD1-35 as described in WO / 2004 / 056875, or (d) Rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2 as described in WO2016 / 020856; clones 2, 10, and 19 as described in WO / 2013 / 022091; humanized antibody 949 as described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1 as described in WO / 2017 / 058859 The protein according to any one of claims 13 to 15, which competes with and / or binds to the same epitope as the agonist PD1 antibody selected from antibodies PD1-17, PD1-28, PD1-33, and PD1-35 as described in WO / 2010 / 029434; and PD1-17, PD1-28, PD1-33, and PD1-35 as described in WO / 2004 / 056875 for binding to PD1.

17. The protein according to any one of claims 13 to 16, wherein the antigen-binding fragment of the agonist anti-PD1 antibody stimulates human PD1.

18. The protein according to any one of claims 1 to 17, wherein the CD20 targeting portion binds to the extracellular domain of human CD20.

19. The protein according to any one of claims 1 to 18, wherein the CD20 targeting portion is an antigen-binding fragment of an anti-CD20 antibody, and optionally the antigen-binding fragment of the anti-CD20 antibody is in the form of Fab, Fv, or scFv.

20. The aforementioned anti-CD20 antibody (a) Selected from rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, and vertzuzumab, or (b) The protein according to claim 19, which competes with an anti-CD20 antibody selected from the group consisting of rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab ituxetan, tositumomab, ubrituximab, okalatuzumab, TRU-015, and bertuzumab for binding to CD20, and / or binds to the same epitope as the anti-CD20 antibody.

21. A protein according to any one of claims 1 to 20, comprising one or more linker moieties.

22. The protein according to claim 21, wherein (a) the CD20 targeting portion and the PD1 agonist portion are separated by the linker portion, and (b) the PD1 agonist portion and the dimerized portion are separated by the linker portion.

23. The protein according to claim 21 or 22, wherein each linker portion is (a) at least 5 or at least 10 amino acids long, (b) up to 20, up to 25, or up to 30 amino acids long, and / or (c) 5 to 15 amino acids or 5 to 20 amino acids long.

24. The protein according to any one of claims 21 to 23, wherein the protein comprises a glycine-serine linker.

25. The protein according to any one of claims 1 to 24, further comprising: (1) an additional CD20 targeting moiety; (2) an additional PD1 agonist moiety of 120 amino acids or less, comprising (i) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO: 2, or (ii) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO: 14; and (3) an additional dimerizing moiety.

26. The protein according to claim 25, wherein (a) the additional CD20 targeting portion and the additional PD1 agonist portion are separated by the linker portion, and (b) the additional PD1 agonist portion and the additional dimerizing portion are separated by the linker portion.

27. The protein according to claim 25, wherein the linker portion (a) is at least 5 or at least 10 amino acids long, (b) is up to 20, up to 25, or up to 30 amino acids long, and / or (c) is 5 to 15 amino acids or 5 to 20 amino acids long.

28. The protein according to any one of claims 25 to 27, wherein the linker portion is a glycine-serine linker.

29. A protein according to any one of claims 1 to 28, comprising two monomers of exemplary monomer 1, wherein exemplary monomer 1 comprises, in an N-terminal to C-terminal orientation, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, and the dimerization moiety.

30. The protein according to claim 29, having the configuration shown in Figure 1A.

31. A protein according to any one of claims 1 to 24, comprising two monomers by exemplary monomer 2, wherein exemplary monomer 2 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, and the dimerization moiety.

32. The protein according to claim 31, having the configuration shown in Figure 1B.

33. The protein according to any one of claims 1 to 24, comprising two monomers by exemplary monomer 3, wherein exemplary monomer 3 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, and the dimerization moiety, or comprising or comprising the same.

34. The protein according to claim 33, having the configuration shown in Figure 1C.

35. A protein according to any one of claims 1 to 24, comprising two monomers by exemplary monomer 4, wherein exemplary monomer 4 comprises, in an N-terminal to C-terminal orientation, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, and the dimerization moiety.

36. The protein according to claim 35, having the configuration shown in Figure 1D.

37. A protein according to any one of claims 1 to 24, comprising two monomers comprising exemplary monomer 5, wherein exemplary monomer 5 comprises, in an N-terminal to C-terminal orientation, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker moiety, an optional linker moiety, the PD1 agonist moiety (e.g., comprising or comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9), an optional linker moiety, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker moiety, and the dimerization moiety.

38. The protein according to claim 37, having the configuration shown in Figure 1E.

39. The protein according to any one of claims 1 to 38, wherein the dimerized portion is an Fc domain.

40. It is a protein, (a) means for coupling to CD20, (b) PD1 agonist moieties of 120 or fewer amino acids, (i) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO: 2, or (ii) A PD1 agonist moiety comprising an amino acid sequence having at least approximately 70% sequence identity with SEQ ID NO: 14, (c) A protein containing a dimerized portion.

41. It is a protein, and from the N terminus to the C terminus, (a) CD20 targeting portion, (b) A PD1 agonist moiety comprising an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) A protein comprising a polypeptide chain containing an Fc domain.

42. The aforementioned protein is located from the N-terminus to the C-terminus. (a) Additional CD20 targeting portion, (b) An additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) The protein according to claim 41, further comprising an additional polypeptide chain containing an additional Fc domain.

43. It is a protein, and from the N terminus to the C terminus, (a) A PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (b) Antigen-binding fragment of agonist anti-PD1 antibody, (c) CD20 targeting portion, (d) A protein comprising a polypeptide chain containing an Fc domain.

44. The aforementioned protein is located from the N-terminus to the C-terminus. (a) an additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (b) Additional antigen-binding fragments of the agonist anti-PD1 antibody, (c) Additional CD20 targeting portion, (d) The protein according to claim 43, further comprising an additional polypeptide chain containing an additional Fc domain.

45. It is a protein, and from the N terminus to the C terminus, (a) Antigen-binding fragment of agonist anti-PD1 antibody, (b) A PD1 agonist moiety comprising an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) CD20 targeting portion, (d) A protein comprising a polypeptide chain containing an Fc domain.

46. The aforementioned protein is located from the N-terminus to the C-terminus. (a) Additional antigen-binding fragments of the agonist anti-PD1 antibody, (b) An additional PD1 agonist moiety consisting of an amino acid sequence having at least approximately 98% sequence identity with SEQ ID NO: 2, (c) Additional CD20 targeting portion, (d) The protein according to claim 45, further comprising an additional polypeptide chain containing an additional Fc domain.

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

48. A host cell manipulated to express the protein according to any one of claims 1 to 46 or the nucleic acid(s) according to claim 47.

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

50. A pharmaceutical composition comprising the protein and excipient described in any one of claims 1 to 46.

51. 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 according to any one of claims 1 to 46 or a pharmaceutical composition according to claim 50.

52. The method according to claim 51, wherein the immune disorder or condition is type 1 diabetes, Crohn's disease, or graft-versus-host disease (GVHD).

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

54. The method described above is (a) reducing T cell function in the subject, (b) reducing B cell function in the subject, (c) reducing T cell responsiveness in the subject, or The method according to claim 53, which is any combination of (d)(a) to (c).

55. A method for local PD1 agonism, comprising administering an effective amount of the protein described in any one of claims 1 to 46 or the pharmaceutical composition described in claim 50 to a target.

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