CD20-PD1 binding molecules and methods of use thereof

Abstract

The present disclosure relates to molecules capable of binding to both CD20 and PD1, as well as nucleic acids encoding such molecules, pharmaceutical compositions comprising such molecules, and methods of use thereof. In certain aspects, disclosed are CD20-PD1 binding molecules comprising at least one CD20 targeting moiety and at least one PD1 agonist moiety wherein in certain aspects the PD1 agonist moiety is a PDL1 or PDL2 extracellular domain or a PD1 binding moiety thereof.

Description

[0001] 1. Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 501,252, filed May 10, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence List

[0004] This application contains a sequence list that has been electronically submitted in XML format, the contents of which are incorporated herein by reference in their entirety. The XML sequence list, created on April 25, 2024, is named RGN-030WO_SL and has a size of 139,033 bytes. 3. Background Technology

[0006] Autoimmune diseases occur when an organism mounts an abnormal immune response to its own cells and tissues. For decades, researchers have strived to understand autoimmunity. During this time, it has become clear that the immune system has evolved a variety of mechanisms to control its own responses. Defects in one or more of these mechanisms can lead to compromised tolerance and, consequently, autoimmune diseases.

[0007] The initial triggers for systemic and organ-specific autoimmune disorders may involve the recognition of self or foreign molecules by innate sensors. This recognition triggers inflammatory responses as well as the involvement of previously dormant autoreactive T and B cells (Theofilopolous, Kono and Baccala, 2017, Nat Immunol, 18(17):716-724). Autoreactivity ranges from low “physiological” levels of autoreactivity that are crucial for lymphocyte selection and immune system homeostasis, to intermediate levels of autoimmunity characterized by circulating autoantibodies and small tissue infiltration without clinical consequences, to pathogenic autoimmunity associated with immune-mediated organ damage (Theofilopolous, Kono and Baccala, 2017, Nat Immunol, 18(17):716-724). Autoimmune diseases are classified into organ-specific diseases (e.g., type 1 diabetes (T1D), multiple sclerosis (MS), inflammatory bowel disease (IBD), myasthenia gravis) and systemic diseases (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome), and can be mediated by autoantibodies or cytotoxic T cells, but helper T cells are required in all cases (Theofilopolous, Kono and Baccala, 2017, NatImmunol, 18(17):716-724).

[0008] Most autoimmune diseases exhibit clinical heterogeneity, a polygenic nature, and a multifactorial effect, typically requiring both genetic and environmental factors. Four mechanisms contribute to the control of escaped self-reactive T and B cells: inhibitory molecules, disabling, neglect, and active suppression (Kono and Theofilopolous, Kono and Baccala, 2017, NatImmunol, 18(17):716-724). A variety of inhibitory molecules (e.g., CTLA-4, PD-1, LAG-3, TIM3, VISTA, TIGIT, FcγRIIb, and certain Siglecs) are expressed on the surface of both T and B cells to suppress excessive immune responses, both normal and anti-self. The absence of some of these molecules leads to autoimmunity, providing strong evidence that self-reactive lymphocytes exist in a peripheral cell pool but are generally controlled. See Paterson and Sharpe, 2010, Nat Ummunol, 11:109-111; Okazaki et al., 2013, Nat Immunol, 14:1212-1218; Pincetic et al., 2014, Nat Immunol, 15:707-716; Macauley, Crocker and Paulson, 2914, Nat Rev Immunol, 14:653-666; Ceeraz et al., 2016, ArthritisRheumatol 69(4):814-825; and Schmitt et al., 2016, J Exp Med, 213:1627-1644. Widespread immune-related adverse events often occur due to unexamined autoreactivity (Michot et al., 2016, Eur J Cancer, 54:139-148).

[0009] Existing treatments for autoimmune diseases have achieved only limited success. For example, organ-specific autoimmune diseases can often be corrected through metabolic control. If function is lost and cannot be restored, mechanical replacements or tissue transplantation may be appropriate. While this approach may alleviate some symptoms, there is no effective long-term cure for some of the most disabling autoimmune diseases. Although many compounds, including insulin, corticosteroids, and modified beta-interferon, can improve some symptoms of autoimmune diseases, they can produce serious side effects and / or require long-term use. Conventional immunosuppressive drug therapies, such as chronic treatment with cyclosporine A, FK506, and rapamycin, do not cure these diseases, and their use is accompanied by many harmful side effects. These effects include nephrotoxicity, increased susceptibility to infectious diseases, and increased incidence of tumors.

[0010] Therefore, there is a need to seek novel therapeutic compositions and regimens that can be used to suppress immune responses and treat autoimmune diseases. 4. Summary of the Invention

[0012] This disclosure provides novel CD20-PD1 binding molecules. The CD20-PD1 binding molecules of this disclosure generally comprise or consist of a CD20-PD1 monomer, which includes one or more CD20 targeting moieties and / or one or more PD1 agonist moieties. In some embodiments, the CD20-PD1 monomer further includes one or more antigen-binding fragments of an agonist anti-PD1 antibody.

[0013] The CD20-PD1 binding molecule disclosed herein is typically a protein comprising at least one CD20 targeting moiety, at least one PD1 agonist moiety, at least one dimerizing moiety, and optionally a linker moiety of one or more portions of the separated protein. In some embodiments, the protein further comprises at least one antigen-binding fragment of an agonist anti-PD1 antibody. In specific embodiments, the CD20 targeting moiety is anti-CD20 Fab or scFv, the PD1 agonist moiety is an IgV domain of PDL1 (or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions), and the dimerizing moiety is an Fc domain. Thus, the CD20-PD1 binding domain is typically a dimer composed of two polypeptide chains, each polypeptide chain 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) a linker moiety of one or more portions of the separated polypeptide chains.

[0014] Exemplary CD20-PD1 binding molecules are disclosed in Section 6.2 below and in Examples 1 through 159. Exemplary CD20 targeting portions are disclosed in Section 6.3. Exemplary PD1 agonist portions are disclosed in Section 6.4. Exemplary antigen-binding fragments of agonist anti-PD1 antibodies are disclosed in Section 6.5.

[0015] This disclosure further provides nucleic acids encoding CD20-PD1 binding molecules, CD20-PD1 monomers, and CD20 targeting and PD1 agonist moieties. The nucleic acids encoding CD20-PD1 binding molecules and CD20-PD1 monomers consisting 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). This disclosure further provides host cells and cell lines engineered to express the nucleic acids of this disclosure and the CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting and PD1 agonist moieties. This disclosure further provides methods for generating the CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting and PD1 agonist moieties of this disclosure. Exemplary nucleic acids, host cells, cell lines, and methods for generating CD20-PD1 binding molecules, CD20-PD1 monomers, CD20 targeting and PD1 agonist moieties are described below in Section 6.9 and in Examples 160 to 162.

[0016] This disclosure further provides pharmaceutical compositions comprising the CD20-PD1 binding molecule, CD20-PD1 monomer, CD20 targeting moiety, and PD1 agonist moiety of this disclosure. Exemplary pharmaceutical compositions are described in Section 6.10 below and in Example 163.

[0017] This document further provides methods for using the CD20-PD1 binding molecule, CD20-PD1 monomer, CD20 targeting moiety, PD1 agonist moiety, and pharmaceutical composition disclosed herein, for example, for treating autoimmune diseases, suppressing cellular autoimmune responses, or suppressing the immune system of a subject. Exemplary methods are described in Section 6.11 below and in Examples 164 through 186. 5. Description of the attached drawings

[0019] Figures 1A to 1E This is a series of cartoons representing an exemplary format of a CD20-PD1 binding molecule according to certain embodiments. The heavy chain variable domain of the CD20 targeting moiety is shown in a dotted pattern, the VH domain of the agonist anti-PD1 antibody is shown in a striped pattern, and the IgV domain of the extracellular domain of PDL1 or PDL2 is shown in a semicircle. Although the CD20 and PD1 targeting moiety are shown as Fab for convenience, Figures 1A to 1E One or more Fabs described herein may be replaced with any targeted portion described herein, such as an Fv fragment or scFv.

[0020] Figure 2A and Figure 2BTraces from flow cytometry binding studies are depicted, and mPDL1 binding (top panel) or anti-mCD20 binding (bottom panel) of the specified CD20-PD1 binding molecule (anti-mCD20 x mPDL1 extracellular domain) is represented on Jurkat / mPD1 and MC38 / mCD20 or HEK293 / mCD20 cells, respectively.

[0021] Figures 3A to 3C The luciferase assay protocol is described. Figure 3A This is a schematic diagram of luciferase reporter gene assay, and Figure 3B and Figure 3C It is a description Figure 3A Cartoon representations of the interactions between key participants described in the text, showing molecules in alternative formats of 2+1 or 2+2.

[0022] Figure 4A Figure 4E depicts the test molecule ( Figure 4A The study used a series of traces (Figures 4B to 4E) using the same test molecules. These traces depict the mPD1 agonist activity measured using the bioassays depicted in Figure 3. Cells and molecules were used according to the instructions for each individual trace.

[0023] Figure 5 An experimental design for dose titration efficacy testing in prediabetic non-obese diabetic (NOD) mice was described.

[0024] Figures 6A to 6I A series of traces depicting individual animal data are presented, demonstrating the onset of spontaneous diabetes in the presence of the indicated control or CD20-PD1 binding molecule (anti-mCD20 x mPDL1 extracellular domain).

[0025] Figure 7A and Figure 7B The image depicts the CD20-PD1 binding molecule (anti-mCD20 x mPDL1 extracellular domain) (top: Figure 2A The molecule L; bottom: Figure 2B A diagram illustrating the ability of the molecule G to regulate the onset of diabetes in NOD mice.

[0026] Figures 8A to 8C This is a box plot depicting the reduction in pancreatic infiltration of activated, autoreactive islet-specific CD8+ T cells in NOD mice after treatment with CD20-PD1 binding molecules (anti-mCD20 x mPDL1 extracellular domain). * p < 0.05.

[0027] Figures 9A to 9CThis is a box plot depicting the reduction in infiltration of activated autoreactive CD3+, CD4+, and CD8+ T cells into the spinal cord of EAE-MS mice after treatment with CD20-PD1 binding molecules (anti-mCD20 x mPDL1 extracellular domain). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0028] Figures 10A to 10C are cartoons illustrating exemplary constructs of CD20-PD1 binding molecules containing the mouse PDL1-IgV extracellular domain. Figure 10A depicts the anti-mCD20 x mPDL1 IgV extracellular domain construct AF-12.1, which has the conformation anti-mCD20Fab – 10 amino acid linker – mPDL1 IgV extracellular domain – 5 amino acid linker – mIgG1. Figure 10B depicts the anti-mCD20 x mPDL1 IgV extracellular domain construct AF-12.2, which has the conformation anti-mCD20 Fab – 10 amino acid linker – mPDL1 IgV extracellular domain – 5 amino acid linker – hIgG1. Figure 10C depicts the control mPDL1 IgV extracellular domain construct AF-17.1, which has the conformation mPDL1 IgV extracellular domain – 5 amino acid 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 is shown as a broken wave pattern, and the mouse PDL1 extracellular domain is shown as a dotted semicircle.

[0029] Figures 11A to 11E are cartoons illustrating exemplary constructs of CD20-PD1 binding molecules containing the human PDL1-IgV extracellular domain. Figure 11A depicts the anti-hCD20 x hPDL1 IgV extracellular domain construct AF-16.1, which has the conformation anti-hCD20Fab – 10 amino acid linker – hPDL1 IgV extracellular domain – 5 amino acid linker – hIgG1. Figure 11B depicts the anti-hCD20 x hPDL1 IgV extracellular domain construct AF-16.2, which has the conformation anti-hCD20 Fab – 10 amino acid linker – hPDL1 IgV extracellular domain – 5 amino acid linker – hIgG1. Figure 11C depicts the hPDL1 IgV extracellular domain construct AF-24.1, which has the conformation hPDL1 IgV extracellular domain – 5 amino acid linker – hIgG1. Figure 11D depicts the hPDL1 IgV–anti-hPD1–anti-hCD20 construct AF-69, which has the conformation hPDL1 IgV extracellular domain–18 amino acid linker–anti-hPD1 Fab–15 amino acid linker–anti-hCD20 Fab–hIgG1. Figure 11E depicts the hPDL1 IgV–anti-hPD1–anti-hCD20 construct AF-70, which has the conformation anti-hPD1 Fab–10 amino acid linker–hPDL1 IgV extracellular domain–10 amino acid linker–anti-hCD20 Fab–hIgG1. The heavy chain variable domain of the anti-hCD20 Fab domain is shown in a diamond pattern, the heavy chain variable domain of the anti-hPD1 Fab domain is shown in a striped pattern, and the human PDL1 extracellular domain is shown in a semicircle with an open-loop pattern.

[0030] Figures 12A to 12D A diagram depicting the binding efficacy of mPDL1 IgV and full-length mPDL1 molecules containing extracellular domains is shown. Figure 12A and Figure 12B The binding efficacy of the evaluated construct with Jurkat cells overexpressing mPD1 and MC38 cells overexpressing mCD20 was demonstrated, respectively. Figure 12C and 12D The binding efficacy of the same construct with parental Jurkat and MC38 cells was demonstrated, respectively.

[0031] Figure 13 A diagram depicting the PD1 agonist activity of mPDL1 IgV and full-length mPDL1 molecules containing extracellular domains, as measured using the bioassays depicted in Figure 3, is presented. 6. Detailed Implementation Methods

[0033] 6.1. Definition

[0034] ApproximatelyThroughout the specification, the terms “about,” “approximately,” etc., are used before numbers to indicate that the number is not necessarily precise (e.g., considering variations in fractions, measurement accuracy and / or precision, timing, etc.). It should be understood that disclosures of “about X” or “approximately X,” where X is a number, are also disclosures of “X.” Thus, for example, a disclosure of an embodiment in which one sequence has “about X% sequence identity” with another sequence is also a disclosure of an embodiment in which that sequence has “X% sequence identity” with another sequence.

[0035] and or Unless otherwise stated, the conjunction “or” is intended to be used in its proper sense as a Boolean logic operator, including feature selection in alternatives (A or B, where selection of A is mutually exclusive with selection of B) and feature selection in conjunctions (A or B, where both A and B are selected). In some places in the text, the terms “and / or” are used for the same purpose, which should not be interpreted as implying that “or” is used to refer to mutually exclusive alternatives.

[0036] 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 the portion of the targeting region that can specifically, non-covalently, and reversibly bind to the target molecule.

[0037] antigen-binding fragment of agonist anti-PD1 antibody The term "antigen-binding fragment of an agonist anti-PD1 antibody" refers to a binding portion of an immunoglobulin or its antigen-binding fragment that can bind to and activate PD1 (i.e., activate PD1 signaling when bound to PD1 expressed on the surface of immune cells). The antigen-binding fragment of an agonist anti-PD1 antibody can be in the form of Fab, Fv, or scFv. The antigen-binding fragment of an agonist anti-PD1 antibody is further described in Section 6.5.

[0038] Association In the context of CD20-PD1 binding molecules or their components (e.g., CD20 targeting moiety; PD1 agonist moiety; dimerizing moiety), the term "association" refers to a functional relationship between two or more polypeptide chains or portions thereof. Specifically, the term "association" means that two or more polypeptides associate with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinks, to produce a functional CD20-PD1 binding molecule. Examples of possible associations in the CD20-PD1 binding molecules of this disclosure include (but are not limited to) association between homodimeric or heterodimeric Fc domains in the Fc region, association between VH and VL regions in Fab or scFv, association between CH1 and CL in Fab, and association between CH3 and CH3 in domain-substituted Fab.

[0039] BivalentAs used herein, the term "bivalent" in relation to a CD20-PD1 binding molecule relative to its CD20 targeting moiety and / or PD1 agonist moiety means that the CD20-PD1 binding molecule has two CD20 targeting moieties (e.g., two antigen-binding fragments of an anti-CD20 antibody) and / or two PD1 agonist moieties (e.g., two PDL1 agonist moieties, two PDL2 agonist moieties, or a combination thereof). A CD20-PD1 binding molecule may be bivalent for one type of moieties (e.g., the CD20 targeting moiety) and monovalent for another type of moieties (e.g., the PD1 agonist moiety).

[0040] CD20-PD1 binding molecules 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. Typically, a CD20-PD1 binding molecule is a molecule composed of one or more polypeptide chains (e.g., one, two, three, or four polypeptide chains) that together contain at least one CD20 targeting moiety and at least one PD1 agonist moiety.

[0041] In the context of the CD20-PD1 binding molecules disclosed herein, the term "CD20-PD1 binding molecule" sometimes refers to the core components of the molecule, namely the CD20 targeting moiety and the PD1 agonist moiety, and sometimes also refers to the dimerizing moiety, such as the Fc domain and / or the associated linker moiety. It should be understood that, unless the context otherwise indicates, the term "CD20-PD1 binding molecule" also extends to molecules containing additional features, such as one or more stable moieties, one or more dimerizing moieties, one or more linker moieties, and any combination thereof.

[0042] CD20-targeted portion The term "CD20 targeting moiety" refers to any molecule or its binding portion (e.g., an immunoglobulin or its antigen-binding fragment) that can bind to CD20. In some embodiments, the CD20 targeting moiety comprises an antigen-binding fragment of an anti-CD20 antibody. The CD20 binding fragment of an anti-CD20 antibody may be in the form of Fab, Fv, or scFv. The term "CD20 targeting moiety" includes molecules that can bind to any domain or region of CD20, including topological domains or transmembrane domains. In some embodiments, the CD20 targeting moiety is a molecule that can bind to a region of CD20 displayed extracellularly on the surface of a cell (e.g., B cell). The CD20 targeting moiety is further described in Section 6.3.

[0043] Complementary Determinant Region or CDRAs used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. Typically, three CDRs (CDR-H1, CDR-H2, CDR-H3) are present in each heavy chain variable region, and three CDRs (CDR1-L1, CDR-L2, CDR-L3) are present in each light chain variable region. Exemplary specifications that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. See, for example, Kabat, 1991, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J.Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABM numbering scheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). Public databases can also be used to identify CDR sequences within antibodies.

[0044] Dimerization portion The term "dimerization moiety" refers to a polypeptide chain or amino acid sequence capable of promoting association between two polypeptide chains to form a dimer. A first dimerization moiety may associate with the same second dimerization moiety or with a second dimerization moiety different from the first dimerization moiety. In some embodiments, the dimerization moiety is an Fc domain, and the association of two Fc domains forms an Fc region. Therefore, the Fc region can be a VH homodimer or a heterodimer.

[0045] EC50 The term "EC50" refers to the half-maximal effective concentration of a molecule (such as a CD20-PD1 binding molecule) that elicits half the response between baseline and maximum after a specific exposure time. EC50 essentially represents the concentration of antibody or CD20-PD1 binding molecule at which 50% of the maximum effect is observed. In some embodiments, the EC50 value is equal to the concentration of CD20-PD1 binding molecule that produces half-maximal activation in a luciferase reporter assay.

[0046] extracellular domainAs used herein, the term "extracellular domain" refers to a region of a transmembrane protein (e.g., PDL1 or PDL2) that, when expressed on the cell surface, is located on the outer side of the membrane. An extracellular domain typically contains a binding domain that specifically binds to a ligand or cell surface receptor. The term "extracellular domain" is used conveniently in this specification not only to the extracellular domain of a transmembrane protein but also to fragments and variant sequences possessing target-binding activity.

[0047] Epitope An epitope or antigenic determinant is a portion of an antigen (e.g., CD20) recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational.

[0048] Fab In the context of the CD20 targeting portion of this disclosure, the term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain containing a variable heavy chain (VH) domain of the antibody at the N-terminus of a first constant domain (referred to herein as C1), and the second polypeptide chain containing a variable light chain (VL) domain of the antibody at the N-terminus of a second constant domain (referred to herein as C2), which is capable of pairing with the first constant domain. In native 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 of the light chain (CL). The Fab of this disclosure can be aligned according to the native orientation or include domain substitutions or exchanges that facilitate correct VH and VL pairing. For example, the CH1 and CL domain pairs in Fab can be replaced with CH3 domain pairs to facilitate correct modified Fab chain pairing in the heterodimer molecule. CH1 and CL can also be reversed, such that CH1 is attached to VL and CL is attached to VH; this configuration is commonly referred to as a Crossmab, a type of "domain exchange".

[0049] Fc structural domain and Fc region 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 different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, for example via knock-in-hole interactions, and / or to allow purification, for example via star mutations.

[0050] Host cells or recombinant host cellsAs used herein, the terms "host cell" and "recombinant host cell" refer to cells that have been genetically engineered, for example, by introducing heterologous nucleic acids. It should be understood that such terms refer not only to specific subject cells but also to the progeny of such cells. Because certain modifications may occur in offspring due to mutations or environmental influences, these progeny may actually differ from the parent cells but are still included within the scope of the term "host cell" as used herein. Host cells may, for example, transiently carry heterologous nucleic acids on extrachromosomal heterologous expression vectors, or stably carry heterologous nucleic acids, for example, by integrating them into the host cell genome. For the purpose of expressing the CD20-PD1 binding molecule, the host cell can be a mammalian-derived or mammalian-like cell line, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, juvenile hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives thereof and / or engineered variants. Engineered variants include, for example, glycan-modified and / or site-specific integration site derivatives.

[0051] Monomer and CD20-PD1 monomer As used herein, the terms "monomer" and "CD20-PD1 monomer" refer to a molecule comprising a first polypeptide chain that (a) contains at least one CD20 targeting moiety and is capable of associating with a second polypeptide chain; (b) contains at least one PD1 agonist moiety and is capable of associating with a second polypeptide chain; (c) contains a dimerizing moiety (e.g., an Fc domain) and is capable of associating with a corresponding dimerizing moiety (e.g., another Fc domain) on a second polypeptide chain; or (d) any combination of (a), (b), and (c) above. A monomer can associate with other monomers by pairing through a dimerizing moiety (e.g., an Fc domain). In some embodiments, one or more associations between monomers are stabilized by a hinge sequence or other portions of the Fc domain. Thus, a monomer of this disclosure can associate with another monomer to form a dimer. The dimer can be a homodimer (where each constituent monomer is identical) or a heterodimer (in which case each constituent monomer is different). As used herein, the term "monomer" is used for convenience and does not exclude the presence of one or more additional polypeptide chains, such as one or more light chains of one or more Fab domains. Therefore, a "dimer" of two monomers can include more than two polypeptide chains, for example, it can include three, four or more polypeptide chains, and the reference to monomer or dimer is not intended to indicate any temporal order of association between polypeptide chains.

[0052] unit priceAs used herein, the term "monovalent" in relation to a CD20-PD1 binding molecule relative to its CD20 targeting moiety and / or PD1 agonist moiety means that the CD20-PD1 binding molecule has one CD20 targeting moiety (e.g., an antigen-binding domain of an anti-CD20 antibody) and / or one PD1 agonist moiety (e.g., a PDL1 agonist moiety or a PDL2 agonist moiety). A CD20-PD1 binding molecule may be monovalent for one type of moiety (e.g., the PD1 agonist moiety) and divalent for another type of moiety (e.g., the CD20 targeting moiety).

[0053] Multi-price As used herein, the term "multivalent" in relation to CD20-PD1 binding molecules relative to the CD20 targeting moiety and / or the PD1 agonist moiety means that a 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 combinations thereof). A CD20-PD1 binding molecule may be multivalent for one type of moieties (e.g., the CD20 targeting moiety) and monovalent for another type of moieties (e.g., the PD1 agonist moiety).

[0054] Operable connection As used herein, the term “operably linked” refers to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are linked to produce a functional polypeptide, or two or more nucleic acid sequences are linked to produce, for example, an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0055] PD1 agonist portionThe term “PD1 agonist moiety” refers to a molecule or portion thereof capable of binding to and activating PD1, which: (1) contains an amino acid sequence having at least 70% sequence identity with 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 with the membrane-adjacent portion of human or mouse PDL1 or human-mouse PDL1. As described herein, the “membrane-adjacent portion” of PDL1 or PDL2 refers to the C-terminal region of the extracellular domain of PDL1 or PDL2 immediately following the IgV domain. In some embodiments, the membrane-adjacent portion of PDL1 or PDL2 is 20, 40, 60, or 80 amino acids from the C-terminus of the extracellular domain of PDL1 or PDL2. Therefore, in specific embodiments, the PD1 agonist moiety is a molecule that: (1) contains an amino acid sequence having at least 70% sequence identity with 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 with the C-terminal 20, 40, 60, or 80 amino acids 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 with the IgV domain of mammalian PDL1 (e.g., human or mouse PDL1). In other embodiments, the PD1 agonist moiety contains an amino acid sequence having at least 70% sequence identity with the IgV domain of mammalian PDL2 (e.g., human or mouse PDL2). The extracellular domains of PDL1 and PDL2 are sometimes referred to as the “PDL1 extracellular domain” and the “PDL2 extracellular domain”, respectively. The terms “PDL1 extracellular domain” and “PDL2 extracellular domain” are used conveniently in this specification to refer not only to the PDL1 and PDL2 extracellular domains, but also to fragments and variant sequences having PD1 binding activity. Therefore, the terms “PDL1 extracellular domain” and “PDL2 extracellular domain” as used in this specification are intended to cover the PD1-binding portions of the PDL1 and PDL2 extracellular domains, as well as their variants having PD1-binding function.

[0056] The extracellular domains of PDL1 and PDL2 consist of IgC and IgV domains. Without being bound by theory, PDL1 and PDL2 are understood to interact with PD1 via their IgV domains. In some embodiments, the PD1 agonist moiety of this disclosure is a PDL1 extracellular domain comprising or consisting of a 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 extracellular domain comprising a PDL2 IgV domain or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions.

[0057] CD20-PD1 binding molecules may include a PD1 agonist moiety with 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-substituted domain.

[0058] Single-chain Fv or scFv As used herein, the terms “single-chain Fv” or “scFv” refer to a polypeptide chain containing the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0059] Subjects The term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably in this document.

[0060] Treatment (Treatment, Treating) As used herein, the terms "treat," "treatment," and "treating" refer to reducing or improving the progression, severity, and / or duration of a disease described herein, improving one or more symptoms (preferably one or more identifiable symptoms) of a disease or condition described herein, or preventing a disease or condition described herein, such as an autoimmune or inflammatory disease or condition, by applying a molecule or composition (e.g., one or more CD20-PD1 binding molecules disclosed herein). In specific embodiments, the terms "treat," "treatment," and "treating" refer to improving at least one measurable physical parameter of a disease (e.g., an autoimmune disease) that may not necessarily be identifiable by the patient. In other embodiments, the term "treatment" refers to suppressing the progression or onset of a disease physically by, for example, stabilizing identifiable symptoms, or physiologically by, for example, stabilizing physical parameters, or both.

[0061] General Light Chain As used herein, in the context of the target moiety, the term "universal light chain" refers to a light chain polypeptide that can pair with the heavy chain region of the target moiety and also with other heavy chain regions. Universal light chains are also referred to as "shared light chains".

[0062] VH The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, including the heavy chain of scFv or Fab.

[0063] VL The term "VL" refers to the variable region of the immunoglobulin light chain, including the light chain of scFv or Fab.

[0064] 6.2. CD20-PD1 binding molecules

[0065] 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 dimerizing 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 molecule of this disclosure generally comprises or is composed of a CD20-PD1 binding molecule monomer, which includes one or more CD20 targeting moieties and / or one or more PD1 agonist moieties.

[0066] In some embodiments, the PD1 agonist moiety is located between the CD20 targeting moiety and the dimerizing moiety of the CD20-PD1 binding monomer. In such embodiments, the CD20-PD1 binding monomer thus has an N-terminal to C-terminal orientation of the CD20 targeting moiety – PD1 agonist moiety – dimerizing moiety. The CD20-PD1 binding molecule may further comprise an antigen-binding fragment of an agonist anti-PD1 antibody located at the N-terminus of the PD1 agonist moiety.

[0067] In other embodiments, the CD20 targeting portion is located between the PD1 agonist portion and the dimerizing portion of the CD20-PD1 binding molecule monomer. In such embodiments, the CD20-PD1 binding molecule monomer thus has an N-terminal to C-terminal orientation of the PD1 agonist portion – CD20 targeting portion – dimerizing portion. The CD20-PD1 binding molecule may further comprise an antigen-binding fragment of an agonist anti-PD1 antibody located between the PD1 agonist portion and the CD20 targeting portion.

[0068] Exemplary dimerization portions are described in Section 6.6 and include Fc domains that endow CD20-PD1 binding molecules with the ability to homodimerize or heterodimerize.

[0069] CD20-PD1 binding molecules may consist of one or more polypeptides. In some embodiments, CD20-PD1 binding molecules consist of a plurality of (e.g., two) monomers comprising at least one CD20 targeting moiety and / or at least one PD1 agonist moiety, and in some embodiments, also comprising a dimerizing moiety. In some embodiments, the CD20-PD1 binding molecule of this disclosure consists of two monomers optionally associated with one or more additional polypeptide chains (e.g., a polypeptide chain comprising a light chain containing an anti-CD20 Fab moiety). The monomers may be identical, thus forming a homodimer, or different, thus forming a heterodimer. The dimerizing moiety of each monomer of the CD20-PD1 binding molecule may be configured to dimerize together. Exemplary dimerizing moieties are described in Section 6.6.

[0070] One or more CD20-targeting moieties and one or more PD1-agonist moieties may be located on the same arm of a CD20-PD1 binding molecule (e.g., where the CD20-targeting moieties comprise anti-CD20 Fab and the PD1-agonist moieties comprise a PDL1-based PD1-agonist moieties, with the anti-CD20 Fab variable heavy or light chain and the PDL1-based PD1-agonist moieties located on the same polypeptide chain), or may be located on different arms of a bispecific CD20-PD1 agonist (e.g., where the CD20-targeting moieties comprise anti-CD20 Fab and the PD1-agonist moieties comprise a PDL1-based PD1-agonist moieties, with the anti-CD20 Fab variable heavy or light chain and the PDL1-based PD1-agonist moieties located on different polypeptide chains).

[0071] The CD20-PD1 binding molecule can be monovalent (i.e., having a single CD20 targeting portion) or polyvalent (i.e., having multiple CD20 targeting portions) with respect to the CD20 targeting portion. Similarly, the CD20-PD1 binding molecule can be monovalent (i.e., having a single PD1 agonist portion) or polyvalent (i.e., having multiple PD1 agonist portions) with respect to the PD1 agonist portion. In some embodiments, the CD20-PD1 binding molecule is bivalent (i.e., having two CD20 targeting portions) with respect to the CD20 targeting portion. In some embodiments, the CD20-PD1 binding molecule is bivalent (i.e., having two PD1 agonist portions) with respect to the PD1 agonist portion. When the CD20-PD1 binding molecule is polyvalent with respect to the CD20 targeting portion and / or the PD1 agonist portion, the multiple CD20 targeting portions may be the same or different from each other, and / or the multiple PD1 agonist portions may be the same or different from each other.

[0072] In some embodiments, a CD20-PD1 binding molecule may include one or more linker sequences connecting various components of one or more of its polypeptide chains, such as (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), when present on the same polypeptide chain; (2) a CD20 targeting moiety and a dimerizing domain (e.g., an Fc domain); (3) a PD1 agonist moiety and a dimerizing domain (e.g., an 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 a CD20 agonist moiety; or (6) any combination thereof. Exemplary linkers are described in Section 6.8.

[0073] 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 associating via dimerizing moieties and others via VH-VL interactions. For convenience and descriptive purposes only, this disclosure generally refers to polypeptides containing a CD20 targeting moiety, a PD1 agonist moiety, and / or a dimerizing moiety (e.g., a first Fc domain) capable of associating with another polypeptide chain containing a CD20 targeting moiety, a PD1 agonist moiety, and / or a corresponding dimerizing moiety (e.g., a second Fc domain). Monomers may comprise one, two, three, or more polypeptide chains. For example, in one embodiment, a monomer may consist of (a) a first polypeptide chain containing an anti-CD20 VH, a PD1 agonist moiety, and an Fc domain, and (b) a second polypeptide chain containing a VL capable of pairing with the anti-CD20 VH. In another embodiment, the monomer may consist of (a) a first polypeptide chain containing a first anti-CD20 VH, a second anti-CD20 VH, and an Fc domain, (b) a second polypeptide chain containing a first VL capable of pairing with the first anti-CD20 VH, and (c) a third polypeptide chain containing a second VL capable of pairing with the second anti-CD20 VH.

[0074] The following are some exemplary examples of monomers of this disclosure described in terms of N-terminal to C-terminal orientation. The CD20-PD1 binding molecule of this disclosure may comprise any two of the exemplary monomers referenced below, whether two identical monomers or two different monomers. The various elements of each monomer are described in detail herein, as in the following subsections and numbered examples.

[0075] (1) Exemplary Monomer 1 CD20 targeting portion – optional linker – PD1 agonist portion – optional linker – dimerization portion (see example, Figure 1A (All are monomers).

[0076] (2) Exemplary monomer 2 PD1 agonist portion – optional linker – antigen-binding fragment of agonist anti-PD1 antibody – optional linker – CD20 targeting portion – optional linker – dimerization portion (see, for example, Figure 1B (All are monomers).

[0077] (3) Exemplary monomer 3 PD1 agonist portion – optional linker – CD20 targeting portion – optional linker – antigen-binding fragment of agonist anti-PD1 antibody – optional linker – dimerization portion (see, for example, Figure 1C (All are monomers).

[0078] (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 (All are monomers).

[0079] (5) Exemplary Monomer 5 CD20 targeting portion – optional linker – PD1 agonist portion – optional linker – antigen-binding fragment of agonist anti-PD1 antibody – optional linker – dimerization portion (see, for example, Figure 1E (All are monomers).

[0080] In some embodiments, this disclosure provides two components according to an exemplary monomer 1 (see, for example, Figure 1A The monomer of CD20-PD1 bound molecules.

[0081] In some embodiments, this disclosure provides two components according to an exemplary monomer 2 (see, for example, Figure 1B The monomer of CD20-PD1 bound molecules.

[0082] In some embodiments, this disclosure provides two components according to an exemplary monomer 3 (see, for example, Figure 1C The monomer of CD20-PD1 bound molecules.

[0083] In some embodiments, this disclosure provides two components according to an exemplary monomer 4 (see, for example, Figure 1D The monomer of CD20-PD1 bound molecules.

[0084] In some embodiments, this disclosure provides two components according to an exemplary monomer 5 (see, for example, Figure 1E The monomer of CD20-PD1 bound molecules.

[0085] In the CD20-PD1 binding molecule disclosed herein, when the CD20 targeting moiety is an antigen-binding domain (“ABD”) of an antibody, each monomer may consist of two or more polypeptide chains (one polypeptide chain having a heavy chain variable region and another polypeptide chain having a light chain variable region). The CD20 targeting moiety may contain both the heavy chain variable domain and the light chain variable domain on separate polypeptide chains. For example, the monomer may consist of polypeptide A and polypeptide B. Polypeptide A may include, for example, from the N-terminus to the C-terminus: the heavy chain variable domain of the CD20 targeting moiety – optional linker – PD1 agonist moiety – optional linker – dimerization moiety; and polypeptide B may contain the light chain variable domain of the CD20 targeting moiety. When 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.

[0086] Alternatively, the CD20 targeting moiety may be in the form of scFv, wherein the heavy chain variable region and the light chain variable region of the CD20 targeting moiety are fused to each other in a single peptide.

[0087] Further details of the composition of the CD20-PD1 binding molecule disclosed herein are presented below.

[0088] 6.3. CD20 Targeting Component

[0089] In some embodiments, incorporating the CD20-targeting portion into the CD20-PD1 binding molecule of this disclosure provides delivery of high concentrations of the targeted PD1 agonist portion for purposes such as immune system activation and treatment of autoimmune diseases, including but not limited to type 1 diabetes, systemic lupus erythematosus, and Crohn's disease, as well as for the treatment of graft-versus-host disease (GVHD). In some embodiments, in addition to promoting the targeted delivery of the PD1 agonist portion, the anti-CD20 portion also provides an additional therapeutic pathway for such autoimmune diseases.

[0090] In some embodiments of this disclosure, each CD20-targeting portion of the CD20-PD1 binding molecule includes an antigen-binding domain of an anti-CD20 antibody. In some embodiments, the CD20-PD1 binding molecule of this disclosure includes a single CD20-targeting portion (e.g., in embodiments where the CD20-PD1 binding molecule is monovalent for the CD20-targeting portion, a CD20-targeting portion on a first monomer or on a second monomer). In some embodiments, the CD20-PD1 binding molecule of this disclosure includes two CD20-targeting portions (e.g., in embodiments where the CD20-PD1 binding molecule is bivalent for the CD20-targeting portion, a first CD20-targeting portion on a first monomer and a second CD20-targeting portion on a second monomer; or both the first and second CD20-targeting portions may be on either the first or second monomer). In such embodiments, the two CD20-targeting portions may be identical or different. When different, the two CD20-targeting portions may be orthogonal, bind to different epitopes of CD20, and / or not compete.

[0091] In some embodiments, the CD20 targeting portion includes an antigen-binding domain of a known anti-CD20 antibody. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, ozoglucomancil, oxautumumab, oflamumab, teimomab, tosimob, utuximab, oxcartuzumab, TRU-015, and vertuzumab (each a “reference anti-CD20 antibody”). Further examples of reference anti-CD20 antibodies are provided in Table T below.

[0092]

[0093] In a further embodiment, the CD20 targeting portion comprises a CDR having a CDR sequence of a reference anti-CD20 antibody (e.g., an anti-CD20 antibody listed in Table T). In some embodiments, the CD20 targeting portion comprises all six CDR sequences of the reference anti-CD20 antibody. In other embodiments, the targeting portion comprises 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 a universal light chain. In a further aspect, the CD20 targeting portion comprises a VH containing the amino acid sequence of the VH of the reference anti-CD20 antibody. In some embodiments, the CD20 targeting portion further comprises a VL containing the amino acid sequence of the VL of the reference anti-CD20 antibody. In other embodiments, the targeting portion further comprises a universal light chain VL sequence.

[0094] In other embodiments, the CD20 targeting portion includes an antigen-binding domain that binds to and / or competes with the same CD20 region as the following: rituximab, olizumab, oxatuzumab, oflavumab, teimozumab, tosimomumab, utuximab, oxcartuzumab, TRU-015, or vetozumab. Assays for measuring antibody competition are known in the art. For example, a CD20 sample can be bound to a solid support. A first antibody and a second antibody are then added. One of the two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to separate and discrete 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 binding to the antigen will be reduced. If an excess of unlabeled antibody is present, very little labeled antibody will bind (if any). In some embodiments, the competitive antibody is an antibody that reduces the binding of another antibody to CD20 by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Details of the procedures for performing such competitive assays are well known in the art and can be found, for example, in Greenfield, ed., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2014. Such assays can be quantified using purified antibodies. A standard curve can be established by titrating the antibody against itself, i.e., the same antibody used for both the label and the competitor. The ability of the unlabeled competitive 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 level of binding inhibition can be compared. In some embodiments, the competitiveness for binding to a target molecule can be determined, for example, using a real-time, label-free biolayer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.).

[0095] A suitable CD20 targeting moiety format is described in Section 6.3.1. The CD20 targeting moiety is preferably a CD20 binding fragment of an anti-CD20 antibody, such as Fab, as described in Section 6.3.1.1, an Fv fragment, or scFv, as described in Section 6.3.1.2.

[0096] The CD20 targeting moiety can be incorporated into CD20-PD1 binding molecules having any of the conformations described herein. CD20-PD1 binding molecules typically consist of multiple polypeptide chains, as described in Section 6.2.

[0097] 6.3.1. CD20 Target Format

[0098] In some respects, the CD20-targeting portion can be any type of antibody or fragment thereof that retains specific binding to CD20. In some embodiments, the antigen-binding portion is an immunoglobulin molecule, particularly an IgG-like immunoglobulin molecule, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include, but are not limited to, VH (or V H ) fragment, VL (or V L Fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, microantibodies, biantibodies, triantibodies, and tetraantibodies.

[0099] 6.3.1.1.Fab

[0100] Traditionally, Fab domains are generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the CD20-PD1 binding molecule disclosed herein, the Fab domain is typically recombinantly expressed as part of the CD20-PD1 binding molecule.

[0101] The Fab domain can contain constant domain and variable region sequences from any suitable species, and therefore can be mouse, chimeric, human, or humanized. In some embodiments, the variable region sequence and / or constant domain sequence is derived from a known anti-CD20 antibody. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, olizumab, oxautumumab, oflamumab, teimomab, tosimob, utuximab, oxcartuzumab, TRU-015, and vetuzumab.

[0102] In some embodiments, the CD20 targeting portion comprises a Fab that binds to and / or competes with the Fab of the following CD20 epitopes: rituximab, ozoglucomancil, ozoglucomancil, ozoglucomancil, tivamomumab, tosimomumab, utuximab, oxcartuzumab, TRU-015, or vertuzumab (each a “reference CD20 antibody”). In a further embodiment, the CD20 targeting portion comprises a CDR having the CDR sequence of the reference CD20 antibody. In some embodiments, the CD20 targeting portion comprises all six CDR sequences of the reference CD20 antibody. In other embodiments, the targeting portion comprises at least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of the reference CD20 antibody and the light chain CDR sequence of the universal light chain. In a further aspect, the CD20 targeting portion comprises a VH that comprises the amino acid sequence of the VH of the reference CD20 antibody. In some embodiments, the CD20 targeting portion further includes a VL containing the amino acid sequence of a VL referencing a CD20 antibody. In other embodiments, the targeting portion further includes a universal light chain VL sequence.

[0103] 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 immunoglobulins, the VH and VL domains pair to form the Fv region, and the CH1 and CL domains pair to further stabilize the bound molecule. The disulfide bond between the two constant domains further stabilizes the Fab domain.

[0104] 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 a Fab heterodimerization strategy to allow the correct 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:

[0105]

[0106] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0107] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain of the Fab, one or more amino acid modifications into the CL domain of the Fab, and / or one or more amino acid modifications into the VH domain, and one or more amino acid modifications into the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, causing Fab components to preferentially pair with each other rather than with components from other Fabs.

[0108] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0109] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be based on spatial and hydrophobic contacts, electrostatic / charge interactions, or combinations of various interactions. Complementarity between protein surfaces is widely described in the literature as lock-and-key mating, pestle-and-mortar, protrusion-and-cavity, donor-and-acceptor, etc., all of which imply a structural and chemical matching property between two interacting surfaces.

[0110] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds at the interface of the Fab components. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary alternatives are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0111] In some embodiments, the Fab domain includes 192E substitution in the CH1 domain and 114A and 137K substitution in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, JImmunol 196:3199-211).

[0112] In some embodiments, the Fab domain includes 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which are used to exchange the hydrophobic and polar regions of the contact between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0113] In some embodiments, the Fab domains may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that promote proper assembly of the Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modification is introduced in the VH domain and 38R modification is introduced in the VL domain.

[0114] The Fab domains can also be modified to replace the natural CH1:CL disulfide bonds with engineered disulfide bonds, thereby improving the efficiency of Fab component pairing. For example, engineered disulfide bonds can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, for example, Mazor et al., 2015, MAbs 7:377-89).

[0115] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, described 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 replacing these domains with additional charge-charge interaction pairings between the VL and VH domains by introducing 38D modification into the VL domain and 39K modification into the VH domain.

[0116] Instead of using a Fab heterodimerization strategy to facilitate proper VH-VL pairing, or in addition to using a common light chain (also known as a universal light chain), a VL of the common light chain can be used in each Fab VL region of the CD20-PD1 binding molecule of this disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species in the CD20-PD1 binding molecule compared to employing a pristine homologous VL. In various embodiments, the VL domain of the CD20-PD1 binding molecule is recognized from a monospecific antibody containing the common light chain. In various embodiments, the VH region of the CD20-PD1 binding molecule contains human heavy chain variable gene segments rearranged in vivo within mouse B cells previously engineered to express a limited human light chain library, or a single human light chain homologous to the human heavy chain, and in response to exposure to the antigen of interest, generates an antibody library containing multiple human VHs homologous to one or both of two possible human VLs, wherein the antibody library is specific to the antigen of interest. Common light chains are derived from rearranged human Vκ1-39Jκ5 sequences or rearranged human Vκ3-20Jκ1 sequences, and include somatic mutant (e.g., affinity maturation) versions. See, for example, U.S. Patent No. 10,412,940.

[0117] 6.3.1.2.scFv

[0118] Single-chain Fv or “scFv” antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain, enabling expression as a single polypeptide chain while retaining the specificity of the complete antibody from which they originated. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of scFv are identified in Section 6.8.

[0119] Unless otherwise stated, scFv as used herein may have VL and VH variable regions in either order. For example, scFv may contain VL-connector-VH or VH-connector-VL relative to the N-terminus and C-terminus of the polypeptide.

[0120] The scFv may contain VH and VL sequences from any suitable species, such as mouse, human, or humanized VH and VL sequences. In some embodiments, the scFv may contain VH and VL sequences from a known anti-CD20 antibody. Examples of known anti-CD20 antibodies include, but are not limited to, rituximab, olizumab, oxautumumab, oflamumab, teimomab, tosimob, utuximab, oxcartuzumab, TRU-015, and vetuzumab.

[0121] In some embodiments, the CD20-targeting portion comprises an scFv that binds to the same CD20 epitope as and / or competes with scFv derived from the following: rituximab, ozoglucomannab, ozoglucomannab, ozoglucomannab, ozoglucomannab, tosimomumab, tosimocomannab, ozoglucomannab, ozoglucomannab, TRU-015, or vertutuzumab.

[0122] To generate nucleic acids encoding scFv, DNA fragments encoding VH and VL are operatively ligated to another fragment encoding a linker, for example, any linker encoding a linker described in Section 6.8 (typically a repeating sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO:50), such that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by a flexible linker (see, for example, 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).

[0123] 6.4. PD1 agonist section

[0124] In some embodiments of this disclosure, the PD1 agonist portion of the CD20-PD1 binding molecule comprises a wild-type or variant PD1-binding domain of programmed death ligand 1 (PDL1) or programmed death ligand 2 (PDL2). In some embodiments, the CD20-PD1 binding molecule of this disclosure comprises a single PD1 agonist portion (e.g., in embodiments where the CD20-PD1 binding molecule is monovalent for the PD1 agonist portion, a PD1 agonist portion on a first monomer or on a second monomer). In some embodiments, the CD20-PD1 binding molecule of this disclosure comprises two PD1 agonist portions (e.g., a first PD1 agonist portion on a first monomer and a second PD1 agonist portion on a second monomer, or both first and second PD1 agonist portions on either the first or second monomer). In such embodiments, the two PD1 agonist portions may be identical or they may be different. When different, the two PD1 agonist portions may interact differently with PD1 (e.g., with different affinities).

[0125] The PD1 agonist moiety can be incorporated into a CD20-PD1 binding molecule having any of the configurations described herein. The CD20-PD1 binding molecule typically consists of multiple polypeptide chains, such as the exemplary monomers described in Section 6.2. As described in Section 6.2, the PD1 agonist moiety can be incorporated into any of the exemplary monomers 1, 2, 3, 4, and 5. Exemplary CD20-PD1 binding molecules incorporated into one or more of the exemplary monomers 1, 2, 3, 4, and 5 are detailed in Section 6.2. In some embodiments, the PD1 agonist moiety is a PDL1-based agonist moiety. In other embodiments, the PD1 agonist is a PDL2-based agonist moiety.

[0126] 6.4.1. PD1 agonist moiety based on PDL1

[0127] PDL1 plays a crucial role in inducing and maintaining autoimmune tolerance. As a ligand for the inhibitory receptor PD1, PDL1 regulates the activation threshold of T cells and limits T cell effector responses. This disclosure provides a CD20-PD1 binding molecule wherein at least one PD1 agonist moiety comprises an amino acid sequence that comprises or is homologous to the PDL1 amino acid sequence described herein. Such PD1 agonist moieties are referred to herein as “PDL1-based PD1 agonist moieties” or similar terms.

[0128] Human PDL1 protein is synthesized into a 290-amino acid precursor polypeptide, from which 18 amino acids are removed to generate mature hPDL1. Amino acids 19 through 238 (according to the precursor protein number) form the hPDL1 extracellular domain. 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).

[0129] The precursor human PDL1 polypeptide has the following amino acid sequence (signal sequence = underline (Extracellular domain = bold):

[0130] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELT CQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO:1)

[0131] The IgV domain of human PDL1 (sometimes referred to as the "IgV extracellular domain" in this paper) has the following amino acid sequence:

[0132] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY (SEQ ID NO:2)

[0133] In some embodiments, the PD1 agonist portion is a PDL1-based agonist portion comprising or consisting of an amino acid sequence having at least 70% sequence identity with the IgV domain of human 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% sequence identity or 100% sequence identity. In some embodiments, the PD1 agonist portion is a PDL1-based agonist portion comprising an amino acid sequence corresponding to the IgV domain of human PDL1 plus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additional amino acids located 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 located at the C-terminus of the IgV domain, or composed thereof.

[0134] In some aspects, 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 aspects, the PD1 agonist moiety comprises or consists of amino acids 19 to 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 a specific embodiment, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0135] In some embodiments, the PD1 agonist portion does not contain an amino acid sequence corresponding to amino acids 150 to 238 of human PDL1. In specific embodiments, the PD1 agonist portion does not contain an amino acid sequence corresponding to any subsequence of length of 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 acids in amino acids 150 to 238 of human PDL1.Therefore, for example, in some embodiments, the PD1 agonist moiety does not contain 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, and 167-178 of human PDL1. Residues 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 Residues 188-199, 189-200, 190-201, 191-202, 192-203, 193-204, 194-205, 195-206, 196-207, 197-208, 198-209, 199-210, 200-211, 201-212, 202-213, 203-214, 204-215, 205-216, 206-217, 207-218, 208 The amino acid sequences corresponding to residues 219, 209 to 220, 210 to 221, 211 to 222, 212 to 223, 213 to 224, 214 to 225, 215 to 226, 216 to 227, 217 to 228, 218 to 229, 219 to 230, 220 to 231, 221 to 232, 222 to 233, 223 to 234, 224 to 235, 225 to 236, 226 to 237, or 227 to 238.In some embodiments, the PD1 agonist moiety does not contain residues 1 to 11, 2 to 12, 3 to 13, 4 to 14, 5 to 15, 6 to 16, 7 to 17, 8 to 18, 9 to 19, 10 to 20, 11 to 21, 12 to 22, 13 to 23, 14 to 24, 15 to 25, 16 to 26, 17 to 27, 18 to 28, 19 to 29, and 20 to 30 corresponding to SEQ ID NO:5. Residues 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, 40- Residues 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 The amino acid sequence of residues 60 to 70, 61 to 71, 62 to 72, 63 to 73, 64 to 74, 65 to 75, 66 to 76, 67 to 77, 68 to 78, 69 to 79, 70 to 80, 71 to 81, 72 to 82, 73 to 83, 74 to 84, 75 to 85, 76 to 86, 77 to 87, or 78 to 88.

[0136] The mouse PDL1 polypeptide is synthesized into a 290-amino acid precursor polypeptide, from which 18 amino acids are removed to generate mature mPDL1. Amino acids 19 to 239 (according to the precursor protein number) form the extracellular domain of mPDL1. The precursor mouse PDL1 polypeptide has the following amino acid sequence (signal sequence = underline (Extracellular domain = bold):

[0137] MRIFAGIIFTACCHLLRA FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELIC QAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET (SEQ ID NO:7)

[0138] The IgV domain of mouse PDL1 (sometimes referred to as the "IgV extracellular domain" in this paper) has the following amino acid sequence:

[0139] FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPY (SEQ ID NO:8)

[0140] In some embodiments, the PD1 agonist portion is a PDL1-based agonist portion comprising an amino acid sequence having at least 70% sequence identity with 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% sequence identity or 100% sequence identity.

[0141] In some aspects, 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 aspects, the PD1 agonist moiety comprises or consists of amino acids 19 to 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 a specific embodiment, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0142] In some embodiments, the PD1 agonist portion does not contain amino acids 150 to 239 of mouse PDL1. In specific embodiments, the PD1 agonist portion does not contain an amino acid sequence corresponding to any subsequence of 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 acids in the PD1 amino acid sequence 150 to 239 of mouse PDL1.Therefore, for example, in some embodiments, the PD1 agonist moiety does not contain 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, and 167-178 of mouse PDL1. Residues 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, 188-1... Residue 99, 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, and so on. The amino acid sequence corresponding to residues 209 to 220, 210 to 221, 211 to 222, 212 to 223, 213 to 224, 214 to 225, 215 to 226, 216 to 227, 217 to 228, 218 to 229, 219 to 230, 220 to 231, 221 to 232, 222 to 233, 223 to 234, 224 to 235, 225 to 236, 226 to 237, 227 to 238, or 228 to 239.In some embodiments, the PD1 agonist portion does not contain residues 1 to 11, 2 to 12, 3 to 13, 4 to 14, 5 to 15, 6 to 16, 7 to 17, 8 to 18, 9 to 19, 10 to 20, 11 to 21, 12 to 22, 13 to 23, 14 to 24, 15 to 25, 16 to 26, 17 to 27, 18 to 28, 19 to 29, 20 to 30, 21 to 31, and 2... corresponding to SEQ ID NO: 11. Residues 2 to 32, residues 23 to 33, residues 24 to 34, residues 25 to 35, residues 26 to 36, residues 27 to 37, residues 28 to 38, residues 29 to 39, residues 30 to 40, residues 31 to 41, residues 32 to 42, residues 33 to 43, residues 34 to 44, residues 35 to 45, residues 36 to 46, residues 37 to 47, residues 38 to 48, residues 39 to 49, residues 40 to 50, residues 41 to 51, residues 42 to 52, residues 43 Residues 44 to 54, 45 to 55, 46 to 56, 47 to 57, 48 to 58, 49 to 59, 50 to 60, 51 to 61, 52 to 62, 53 to 63, 54 to 64, 55 to 65, 56 to 66, 57 to 67, 58 to 68, 59 to 69, 60 to 70, 61 to 71, 62 to 72, 63 to 73, 64 to 55, 56 to 66, 57 to 67, 58 to 68, 59 to 69, 60 to 70, 61 to 71, 62 to 72, 63 to 73, 64 to 55, 56 to 67, 58 to 68, 59 to 69, 64 to 65, 56 to 67, 58 to 68, 59 to 69, 60 to 70, 61 to 71, 62 to 72, 63 to 73, 64 to 6 ...7, 58 to 67, 58 to 67, 58 to 67, 68 to 67, 68 to 67, 68 to 67, 68 to 67, 68 The amino acid sequence of residues 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, 78-88, 79-84, 80-85, 81-86, 82-87, 83-88, or 84-89.

[0143] In some embodiments, the PDL1-based PD1 agonist moiety comprises, or is composed of, 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 the IgV domain of PDL1 and having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions compared to wild-type PDL1. In some embodiments, these 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 comprises the amino acid substitution C113S (based on the precursor PDL1 protein number).

[0144] In some embodiments, the PDL1-based PD1 agonist moiety is optionally fused directly or indirectly to the CD20 targeting moiety 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” with the CD20 targeting moiety, the PDL1-based PD1 agonist moiety and the CD20 targeting moiety are adjacent on the same monomer and separated only by a linker (if present). When the PDL1-based PD1 agonist moiety is fused “indirectly” with 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., dimerization moieties, antigen-binding fragments of agonist anti-PD1 antibodies), or are located on separate monomers.

[0145] 6.4.2. PD1 agonist moiety based on PDL2

[0146] 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 wherein at least one PD1 agonist moiety comprises an amino acid sequence comprising or homologous to the PDL2 amino acid sequence described herein. Such PD1 agonist moiety is referred to herein as a "PDL2-based PD1 agonist moiety" or similar terminology.

[0147] Human PDL2 protein is synthesized into a 273-amino acid precursor polypeptide, from which 19 amino acids are removed to generate mature hPDL2. Amino acids 20 through 220 (according to the precursor protein number) form the hPDL2 extracellular domain. The sequence of human PDL2 has the Uniprot identifier Q9BQ51 (uniprot.org / uniprot / Q9BQ51). The sequence of mouse PDL2 has the Uniprot identifier Q9WUL5 (uniprot.org / uniprot / Q9WUL5).

[0148] The precursor human PDL2 polypeptide has the following amino acid sequence (signal sequence = underline (Extracellular domain = bold):

[0149] MIFLLLMLSLELQLHQIAA LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQAT GYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI (SEQ ID NO:13)

[0150] The IgV domain of human PDL2 (sometimes referred to as the "IgV extracellular domain" in this paper) has the following amino acid sequence:

[0151] LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK (SEQ ID NO:14)

[0152] In some embodiments, the PD1 agonist portion is a PDL2-based agonist portion comprising an amino acid sequence having at least 70% sequence identity with the IgV domain of human 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.

[0153] In some aspects, 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 aspects, the PD1 agonist moiety comprises or consists of amino acids 20 to 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 a specific embodiment, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO:14.

[0154] In some embodiments, the PD1 agonist portion does not contain amino acids 150 to 221 of human PDL2. In specific embodiments, the PD1 agonist portion does not contain an amino acid sequence corresponding to any subsequence of 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 acids in the amino acid sequence corresponding to the amino acid sequence 150 to 221 of human PDL2. Therefore, for example, in some embodiments, the PD1 agonist moiety does not contain 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, and 163-164 of human PDL2. Residue 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, residues 179-1 Residue 90, 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 195-20 The amino acid sequence corresponding to residues 6, 196 to 207, 197 to 208, 198 to 209, 199 to 210, 200 to 211, 201 to 212, 202 to 213, 203 to 214, 204 to 215, 205 to 216, 206 to 217, 207 to 218, 208 to 219, 209 to 220, or 210 to 221.In some embodiments, the PD1 agonist moiety does not contain residues 1 to 11, 2 to 12, 3 to 13, 4 to 14, 5 to 15, 6 to 16, 7 to 17, 8 to 18, 9 to 19, 10 to 20, 11 to 21, 12 to 22, 13 to 23, 14 to 24, 15 to 25, and 16 to 17 corresponding to SEQ ID NO: 17. Residues 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, 31-41 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, 4 The amino acid sequence of residues 7 to 57, 48 to 58, 49 to 59, 50 to 60, 51 to 61, 52 to 62, 53 to 63, 54 to 64, 55 to 65, 56 to 66, 57 to 67, 58 to 68, 59 to 69, 60 to 70, or 61 to 71.

[0155] The mouse PDL2 polypeptide is synthesized into a 247-amino acid precursor polypeptide, from which 19 amino acids are removed to generate mature mPDL2. Amino acids 20 to 221 (according to the precursor protein number) form the extracellular domain of mPDL2. The precursor mouse PDL2 polypeptide has the following amino acid sequence (signal sequence = underline (Extracellular domain = bold):

[0156] MLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQKVENDTSLQSERATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVICGAAWDYKYLTVKVKASYMRIDTRILEV PGTGEVQLTCQARGYPLAEVSWQNVSVPANTSHIRTPEGLYQVTSVLRLKPQPSRNFSCMFWNAHMKELTSAIIDPLSRMEPKVPRTWPLHVFIPACTIALIFLAIVIIQRKRI (SEQ ID NO:19)

[0157] The IgV domain of mouse PDL2 (sometimes referred to as the "IgV extracellular domain" in this paper) has the following amino acid sequence:

[0158] LFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQKVENDTSLQSERATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVICGAAWDYKYLTVKVK (SEQ ID NO:20)

[0159] In some embodiments, the PD1 agonist portion is a PDL2-based agonist portion comprising an amino acid sequence having at least 70% sequence identity with 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.

[0160] In some aspects, 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 aspects, the PD1 agonist moiety comprises or consists of amino acids 20 to 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 a specific embodiment, the PD1 agonist moiety comprises or consists of the amino acid sequence of SEQ ID NO:20.

[0161] In some embodiments, the PD1 agonist moiety does not contain amino acids 150 to 222 of mouse PDL2. In specific embodiments, the PD1 agonist moiety does not contain an amino acid sequence corresponding to any subsequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in the 150th to 222nd amino acids of mouse PDL2.Therefore, for example, in some embodiments, the PD1 agonist moiety does not contain 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, and 163-174 of human PDL2. Residues 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, 179-190 Residues 180-191, 181-192, 182-193, 183-194, 184-195, 185-196, 186-197, 187-198, 188-199, 189-200, 190-201, 191-202, 192-203, 193-204, 194-205, 195-206, 196 The amino acid sequences corresponding to residues 207, 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 some embodiments, the PD1 agonist portion does not contain residues 1 to 11, 2 to 12, 3 to 13, 4 to 14, 5 to 15, 6 to 16, 7 to 17, 8 to 18, 9 to 19, 10 to 20, 11 to 21, 12 to 22, 13 to 23, 14 to 24, 15 to 25, and 16 to 26 corresponding to SEQ ID NO:23. Residues, residues 17-27, residues 18-28, residues 19-29, residues 20-30, 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, residue 32 Residues 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 The amino acid sequence of residues 48 to 58, 49 to 59, 50 to 60, 51 to 61, 52 to 62, 53 to 63, 54 to 64, 55 to 65, 56 to 66, 57 to 67, 58 to 68, 59 to 69, 60 to 70, 61 to 71, or 62 to 72.

[0162] In some embodiments, the PD1 agonist moiety based on PDL2 is optionally fused directly or indirectly to the CD20 targeting moiety via a linker (e.g., as described in Section 6.8). When present on the same monomer, the PD1 agonist moiety based on PDL2 may be located at the N-terminus or C-terminus of the CD20 targeting moiety. When the PD1 agonist moiety based on PDL2 is fused “directly” with the CD20 targeting moiety, the PD1 agonist moiety based on PDL2 and the CD20 targeting moiety are adjacent on the same monomer and separated only by a linker (if present). When the PD1 agonist moiety based on PDL2 is fused “indirectly” with the CD20 targeting moiety, the PD1 agonist moiety based on PDL2 and the CD20 targeting moiety are separated by one or more other domains on the same monomer (e.g., dimerization moieties, antigen-binding fragments of agonist anti-PD1 antibodies) or located on separate monomers.

[0163] 6.5. Antigen-binding fragments of agonist anti-PD1 antibodies

[0164] In some embodiments, the CD20-PD1 binding molecule of this disclosure includes, in addition to the CD20 targeting portion and the PD1 agonist portion, an antigen-binding fragment of an agonist anti-PD1 antibody. In some embodiments, the CD20-PD1 binding molecule of this disclosure includes a single antigen-binding fragment of an agonist anti-PD1 antibody (e.g., in embodiments where the CD20-PD1 binding molecule is monovalent for the antigen-binding fragment of the agonist anti-PD1 antibody, an 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 this disclosure includes two antigen-binding fragments of an agonist anti-PD1 antibody (e.g., in embodiments where the CD20-PD1 binding molecule is bivalent for the antigen-binding fragment 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 the same or they may be different. When not in use, the two antigen-binding fragments of an agonist anti-PD1 antibody can be orthogonal, binding to different epitopes of CD20, and / or not competing.

[0165] In some embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody includes 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, 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; and humanized antibody 949, described in WO / 2011 / 11. In 0621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in 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.

[0166] In other embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody includes an antigen-binding domain that binds to and / or competes with the same PD1 epitope as the following: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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. Assays for measuring antibody competition are known in the art. For example, a PD1 sample can be bound to a solid support. Then, a first antibody and a second antibody are added. One of the two antibodies is labeled. If labeled and unlabeled antibodies bind to individual and discrete sites on PD1, the labeled antibody will bind at the same level regardless of the presence of unlabeled antibody. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete, and the amount of labeled antibody binding to the antigen will be reduced. If an excess of unlabeled antibody is present, very little labeled antibody will bind (if any). In some embodiments, the competing antibody is an antibody that reduces the binding of another antibody to PD1 by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Details of the procedure for performing such competitive assays are well known in the art and can be found, for example, in Greenfield, ed., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2014. Such assays can be quantified using purified antibodies. A standard curve can be established by titrating the antibody against itself, i.e., using the same antibody for both the labeler and the competitor. The ability of an unlabeled competitive antibody to inhibit the binding of a labeled antibody to a plate is titrated. Results can be plotted and compared to concentrations required to achieve the desired level of binding inhibition. In some embodiments, the competitiveness for binding to a target molecule can be determined, for example, using real-time, label-free biolayer interferometry on the Octet HTX biosensor platform (Pall ForteBio Corp.).

[0167] A suitable format for the antigen-binding fragment of an agonist anti-PD1 antibody is described in Section 6.5.1. The antigen-binding fragment of an agonist anti-PD1 antibody is preferably a PD1-binding fragment of an anti-PD1 agonist antibody, such as Fab, as described in Section 6.5.1.1, an Fv fragment, or scFv, as described in Section 6.5.1.2.

[0168] The antigen-binding fragment of an agonist anti-PD1 antibody can be incorporated into a CD20-PD1 binding molecule having any of the conformations described herein. A CD20-PD1 binding molecule typically consists of multiple polypeptide chains, such as those shown in 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 any of the exemplary monomers 2, 3, 4, and 5. Exemplary CD20-PD1 binding molecules incorporated into one or more of the exemplary monomers 2, 3, 4, and 5 are detailed in Section 6.2.

[0169] 6.5.1. Antigen-binding fragment format

[0170] In some respects, 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 particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include, but are not limited to, VH (or V... H ) fragment, VL (or V L Fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, microantibodies, biantibodies, triantibodies, and tetraantibodies.

[0171] 6.5.1.1.Fab

[0172] Traditionally, Fab domains are generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the CD20-PD1 binding molecule disclosed herein, the Fab domain is typically recombinantly expressed as part of the CD20-PD1 binding molecule.

[0173] In some embodiments, the variable region sequence and / or constant domain sequence are derived from known anti-PD1 agonist antibodies. Examples of known anti-PD1 agonist antibodies include, but are not limited to, rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10 and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in 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.

[0174] In some embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody comprises a Fab that binds to and / or competes with the Fab of the following Fabs for binding to PD1: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; one of clones C8-1 and G10-2, as described in WO In 2016 / 020856; one of clones 2, 10 and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; one of PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in WO / 2017 / 058859; one of clones 2 and 19, described in WO / 2010 / 029434; or one of antibodies PD1-17, PD1-28, PD1-33 and PD1-35, described in WO / 2004 / 056875.

[0175] 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 immunoglobulins, the VH and VL domains pair to form the Fv region, and the CH1 and CL domains pair to further stabilize the bound molecule. The disulfide bond between the two constant domains further stabilizes the Fab domain.

[0176] 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 a Fab heterodimerization strategy to allow the correct 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 above can be used.

[0177] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0178] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain of the Fab, one or more amino acid modifications into the CL domain of the Fab, and / or one or more amino acid modifications into the VH domain, and one or more amino acid modifications into the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, causing Fab components to preferentially pair with each other rather than with components from other Fabs.

[0179] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0180] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be based on spatial and hydrophobic contacts, electrostatic / charge interactions, or combinations of various interactions. Complementarity between protein surfaces is widely described in the literature as lock-and-key mating, pestle-and-mortar, protrusion-and-cavity, donor-and-acceptor, etc., all of which imply a structural and chemical matching property between two interacting surfaces.

[0181] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds at the interface of the Fab components. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary alternatives are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0182] In some embodiments, the Fab domain includes 192E substitution in the CH1 domain and 114A and 137K substitution in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, JImmunol 196:3199-211).

[0183] In some embodiments, the Fab domain includes 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which are used to exchange the hydrophobic and polar regions of the contact between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0184] In some embodiments, the Fab domains may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that promote proper assembly of the Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modification is introduced in the VH domain and 38R modification is introduced in the VL domain.

[0185] The Fab domains can also be modified to replace the natural CH1:CL disulfide bonds with engineered disulfide bonds, thereby improving the efficiency of Fab component pairing. For example, engineered disulfide bonds can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, for example, Mazor et al., 2015, MAbs 7:377-89).

[0186] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, described 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 replacing these domains with additional charge-charge interaction pairings between the VL and VH domains by introducing 38D modification into the VL domain and 39K modification into the VH domain.

[0187] Instead of using a Fab heterodimerization strategy to facilitate proper VH-VL pairing, or in addition to using a common light chain (also known as a universal light chain), a VL of the common light chain can be used in each Fab VL region of the CD20-PD1 binding molecule of this disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species in the CD20-PD1 binding molecule compared to employing a pristine homologous VL. In various embodiments, the VL domain of the CD20-PD1 binding molecule is recognized from a monospecific antibody containing the common light chain. In various embodiments, the VH region of the CD20-PD1 binding molecule contains human heavy chain variable gene segments rearranged in vivo within mouse B cells previously engineered to express a limited human light chain library, or a single human light chain homologous to the human heavy chain, and in response to exposure to the antigen of interest, generates an antibody library containing multiple human VHs homologous to one or both of two possible human VLs, wherein the antibody library is specific to the antigen of interest. Common light chains are derived from rearranged human Vκ1-39Jκ5 sequences or rearranged human Vκ3-20Jκ1 sequences, and include somatic mutant (e.g., affinity maturation) versions. See, for example, U.S. Patent No. 10,412,940.

[0188] 6.5.1.2.scFv

[0189] Single-chain Fv or “scFv” antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain, enabling expression as a single polypeptide chain while retaining the specificity of the complete antibody from which they originated. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of scFv are identified in Section 6.8.

[0190] Unless otherwise stated, scFv as used herein may have VL and VH variable regions in either order. For example, scFv may contain VL-connector-VH or VH-connector-VL relative to the N-terminus and C-terminus of the polypeptide.

[0191] In some embodiments, the scFv may comprise VH and VL sequences from known anti-PD1 agonist antibodies. Examples of known anti-PD1 agonist antibodies include, but are not limited to, rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10 and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in 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.

[0192] In some embodiments, the antigen-binding fragment of the agonist anti-PD1 antibody comprises an scFv that binds to and / or competes with scFv derived from the same PD1 epitope as and / or with scFv that binds to PD1: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; one of clones C8-1 and G10-2, as described in WO In 2016 / 020856; one of clones 2, 10 and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; one of PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in WO / 2017 / 058859; one of clones 2 and 19, described in WO / 2010 / 029434; or one of antibodies PD1-17, PD1-28, PD1-33 and PD1-35, described in WO / 2004 / 056875.

[0193] To generate nucleic acids encoding scFv, DNA fragments encoding VH and VL are operatively ligated to another fragment encoding a linker, for example, any linker encoding a linker described in Section 6.8 (typically a repeating sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO:50), such that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by a flexible linker (see, for example, 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).

[0194] 6.6. Dimerization Section

[0195] 6.6.1. Fc structural domain

[0196] In some embodiments, the CD20-PD1 binding molecule and CD20-PD1 monomer of this disclosure comprise one or more multimerizing moieties, such as one or more being Fc domains or dimerizing moieties comprising Fc domains. In some embodiments, the CD20-PD1 monomer of this disclosure comprises a single dimerizing moiety (e.g., a single Fc domain) and / or the CD20-PD1 binding molecule of this disclosure comprises two dimerizing moieties (e.g., two Fc domains that can associate to form an Fc region).

[0197] The CD20-PD1 binding molecule and CD20-PD1 monomer disclosed herein may include an Fc domain or a pair of Fc domains associated to form an Fc region, which are derived from any suitable species and operatively 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 some embodiments, the Fc domain is derived from a human IgG Fc domain.

[0198] The CD20 targeting portion and / or PD1 agonist portion can be fused to the N-terminus or C-terminus of the IgG Fc domain.

[0199] One embodiment of this disclosure relates to a dimer comprising two Fc fusion polypeptides formed by fusing one or more CD20 targeting moieties and / or PD1 agonist moieties to an Fc domain. For example, by fusing both the CD20 targeting moieties and the PD1 agonist moieties to an Fc domain, it can form homodimerizable CD20-PD1 monomers upon expression. Alternatively, by fusing one or more CD20 targeting moieties and / or one or more PD1 agonist moieties to a first Fc domain and fusing one or more CD20 targeting moieties and / or one or more PD1 agonist moieties to a second Fc domain, it can form two distinct CD20-PD1 monomers upon expression. The dimer can be manufactured, for example, by inserting a gene fusion encoding one or more fusion proteins into a suitable expression vector, expressing the one or more gene fusion proteins in host cells transformed with a recombinant expression vector, and assembling the expressed one or more fusion proteins like antibody molecules, and then forming interchain bonds between the Fc moieties to produce a dimer.

[0200] The Fc domain that can be incorporated into the CD20-PD1 monomer can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain is derived from IgG1. In some embodiments, the Fc domain is derived from IgG4.

[0201] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are usually identical, but for the purpose of generating multispecific binding molecules, such as the CD20-PD1 binding molecule of this disclosure, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.6.1.2 below.

[0202] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These dimers form the Fc region.

[0203] In the CD20-PD1 binding molecule disclosed herein, the Fc region and / or the Fc domain within it may contain one or more different types of antibodies, such as one, two or three different types of heavy chain constant domains.

[0204] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG1.

[0205] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG2.

[0206] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG3.

[0207] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG4.

[0208] In one embodiment, the Fc region contains a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0209] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0210] It should be understood that the heavy chain constant domain of the Fc region used to generate the CD20-PD1 binding molecule of this disclosure may include variants of the aforementioned naturally occurring constant domains. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one instance, the Fc region of this disclosure contains at least one constant domain that is sequence-different from the wild-type constant domain. It should be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another instance, the variant constant domain is at least 70% identical or similar. In another instance, the variant constant domain is at least 80% identical or similar. In another instance, the variant constant domain is at least 90% identical or similar. In yet another instance, the variant constant domain is at least 95% identical or similar.

[0211] IgM and IgA are naturally occurring covalent polymers of common H2L2 antibody units in the human body. When IgM is incorporated into the J chain, it appears as a pentamer; or when the J chain is absent, it appears as a hexamer. IgA appears as both a monomer and a dimer. The heavy chains of IgM and IgA have 18 amino acids extending to a constant C-terminal domain, called a tail. The tail contains cysteine ​​residues, which form disulfide bonds between the heavy chains of the polymer and are considered to play an important role in polymerization. The tail also contains glycosylation sites. In some embodiments, the CD20-PD1 binding molecule of this disclosure does not contain a tail.

[0212] The Fc domain incorporated into the CD20-PD1 binding molecule of this disclosure may contain one or more modifications that alter protein functional properties, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.6.1.1.

[0213] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric CD20-PD1 binding molecules, for example, by allowing heterodimerization, which is the preferential pairing of different Fc domains relative to the same Fc domain. Heterodimerization allows the generation of CD20-PD1 binding molecules in which different polypeptide components are linked to each other through Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.6.1.2.

[0214] It should be understood that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or combined with other modifications to alter the properties of the CD20-PD1 binding molecule.

[0215] 6.6.1.1. Fc domain with altered effector function

[0216] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptor and / or effector function.

[0217] In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is selected from one or more of the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0218] In one embodiment, the Fc domain (e.g., the Fc domain of a CD20-PD1 monomer) or the Fc region (e.g., one or both Fc domains of a CD20-PD1 binding molecule that can associate to form an Fc region) contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, G237, N297, A330, P331, and P329 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain or Fc region contains an amino acid substitution at a position selected from L234, L235, and P329 (according to Kabat EU index numbers). In some embodiments, the Fc domain or Fc region contains amino acid substitutions L234A and L235A (according to Kabat EU index numbers). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (according to the Kabat EU index number). In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (according to the Kabat EU index number). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain or Fc region contains amino acid substitutions at positions P329, L234, and L235 (according to the Kabat EU index number). In a more specific embodiment, the Fc domain contains amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0219] In some embodiments, the Fc domain or Fc region contains amino acid substitutions at positions L234, L235, G237, A330, and P331 (according to Kabat EU index numbers). In more specific embodiments, the amino acid substitutions are L234A, L235E, G237A, A330S, and P331S (according to Kabat EU index numbers).

[0220] Typically, each of the two Fc domains in the Fc region contains the same one or more amino acid substitutions. Therefore, in a specific embodiment, each Fc domain in the Fc region contains amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbers), meaning that 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) (according to the Kabat EU index number). In another specific embodiment, each Fc domain of the Fc region contains amino acid substitutions L234A, L235E, G237A, A330S, and P331S (according to Kabat EU index numbers), that is, 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) (according to Kabat EU index numbers).

[0221] In one embodiment, the Fc domain is the IgG1 Fc domain, such as the human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A and N297A mutations (EU numbers) to reduce effector function. In other embodiments, the IgG1 Fc domain is a variant IgG1 containing L234A, L235E, G237A, A330S, and P331S mutations (according to Kabat EU index numbers) to provide effector-ineffective IgG1 (IgG1EN). Amino acid substitutions for L234A, L235E, and G237A reduce binding to FcγRI, FcγRIIa, and FcγRIII, while substitutions for A330S and P331S reduce C1q-mediated complement fixation.

[0222] In another embodiment, the Fc domain is an IgG4 Fc domain that reduces binding to the Fc receptor. An exemplary IgG4 Fc domain with reduced binding to the Fc receptor may comprise an amino acid sequence selected from Table 2 below. In some embodiments, the Fc domain includes only the bolded portions of the sequences shown below:

[0223]

[0224] In a specific embodiment, the reduced effector function IgG4 comprises the bold portion of the amino acid sequence of SEQ ID NO:31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4.

[0225] For the heterodimer Fc region, combinations of the above-mentioned variant IgG4 Fc sequences can be incorporated, such as an Fc region containing an Fc domain of the amino acid sequence (or its bolded portion) of SEQ ID NO:30 of WO2014 / 121087 and an Fc domain containing an amino acid sequence (or its bolded portion) of SEQ ID NO:37 of WO2014 / 121087, or an Fc region containing an Fc domain of the amino acid sequence (or its bolded portion) of SEQ ID NO:31 of WO2014 / 121087 and an Fc domain containing an amino acid sequence (or its bolded portion) of SEQ ID NO:38 of WO2014 / 121087.

[0226] 6.6.1.2. Fc heterodimer variants

[0227] Some CD20-PD1 binding molecules require dimerization between two Fc domains. Unlike native immunoglobulins, these two Fc domains can be operatively linked to different N-terminal regions; for example, one Fc domain may be linked to the Fab region, while the other is linked to the PD1 agonist moiety. Insufficient heterodimerization of the two Fc domains to form the Fc region can be a barrier to increasing the yield of the desired heterodimer molecule and poses challenges to purification. Various methods 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 disclosed herein, for example, as disclosed in: EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0228] In some aspects, this disclosure provides CD20-PD1 binding molecules comprising Fc heterodimers (i.e., Fc regions containing heterologous, non-identical Fc domains). Typically, each Fc domain in the Fc heterodimer contains the CH3 domain of an antibody. The CH3 domain is derived from a constant region of any isotype, class, or subclass of antibody, and preferably from a constant region of antibodies of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0229] Heterodimerization of two different heavy chains at the CH3 domain produces the desired CD20-PD1 binding molecule, while homodimerization of the same heavy chain reduces the yield of the desired CD20-PD1 binding molecule. Therefore, in a preferred embodiment, the polypeptide associated to form the heterodimeric CD20-PD1 binding molecule of this disclosure will contain a modified CH3 domain that is favorable for heterodimer association compared to the unmodified Fc domain.

[0230] In specific embodiments, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "mortar" modification, comprising a "knob" modification in one Fc domain and a "mortar" modification in the other Fc domain. Knob-into-hole techniques are described, for example, in U.S. Patent Nos. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621; and Carter, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("mortar") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity, thereby promoting heterodimer formation and inhibiting homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the first polypeptide interface with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

[0231] Therefore, in some embodiments, amino acid residues in the CH3 domain of the first subunit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit. This protrusion can be positioned in a cavity within the CH3 domain of the second subunit. Conversely, amino acid residues in the CH3 domain of the second subunit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit. The protrusion within the CH3 domain of the first subunit can be positioned within this cavity. Preferably, the amino acid residues with a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues with a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. An exemplary substitution is Y470T.

[0232] In a specific embodiment of this kind, in the first Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V). Optionally, the threonine residue at position 366 is substituted with a serine residue (T366S), and the leucine residue at position 368 is substituted with an alanine residue (L368A) (according to the Kabat EU index number). In a further embodiment, in the first Fc domain, the serine residue at position 354 is additionally substituted with a cysteine ​​residue (S354C), or the glutamate residue at position 356 is substituted with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is substituted with a cysteine ​​residue), and in the second Fc domain, the tyrosine residue at position 349 is additionally substituted with a cysteine ​​residue (Y349C) (according to the Kabat EU index number). In a specific embodiment, the first Fc domain contains amino acid substitutions S354C and T366W, and the second Fc domain contains amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Kabat EU index numbers).

[0233] In some embodiments, electrostatic redirection (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to facilitate the bonding of the first and second Fc domains in the Fc region.

[0234] As an alternative or addition to using a modified Fc domain to promote heterodimerization, the Fc domain can be modified to allow for selective purification strategies for Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that eliminates its binding to protein A, thereby enabling purification methods that produce heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Thus, a CD20-PD1 binding molecule comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein at least one amino acid difference reduces the binding of the CD20-PD1 binding molecule to protein A compared to a corresponding CD20-PD1 binding molecule lacking the amino acid difference. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as an H95R modification (via IMGT exon numbering; H435R via EU numbering). The second CH3 may further comprise a Y96F modification (via IMGT; Y436F via EU numbering). This type of modification is referred to as the "star" mutation in this paper.

[0235] In some embodiments, Fc may contain one or more mutations (e.g., pestle and mortar mutations) to promote heterodimerization and star mutations to promote purification.

[0236] 6.7. Stable Part

[0237] The CD20-PD1 binding molecule of this disclosure may include a stabilizing moiety that prolongs the serum half-life of the molecule in vivo. Serum half-life is typically divided into α and β phases. By adding an appropriate stabilizing moiety, either or both phases can be significantly improved. For example, relative to the corresponding CD20-PD1 binding molecule without the stabilizing moiety, the stabilizing moiety can increase the serum half-life of the CD20-PD1 binding molecule by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 400%, 600%, 800%, 1000%, or more. For the purposes of this disclosure, serum half-life may refer to the half-life in humans or other mammals (e.g., mice or non-human primates).

[0238] The stable portion includes the polyoxyethylene portion (e.g., polyethylene glycol), sugars (e.g., sialic acid), and well-tolerated protein portions (e.g., Fc and its fragments and variants, transferrin, or serum albumin).

[0239] 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 conjugates (e.g., AdnectinPKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimics based on three amino acids (proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

[0240] Therefore, in some embodiments, this disclosure provides a CD20-PD1 binding molecule comprising a polymeric sugar as a stabilizing component.

[0241] Serum albumin can also participate in half-life extension through modules with the ability to interact non-covalently with albumin. Therefore, the CD20-PD1 binding molecule of this disclosure may include an albumin-binding protein as a stable component. 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 to exist in certain bacteria. For example, Streptococcus protein G contains several small albumin-binding domains consisting of approximately 50 amino acid residues (6 kDa). Further examples of serum albumin-binding proteins include those described in U.S. Publications 2007 / 0178082 and 2007 / 0269422. Fusion of the albumin-binding domain to the protein results in a significantly extended half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76).

[0242] In other embodiments, the stabilizing component is human serum albumin. In other embodiments, the stabilizing component is transferrin.

[0243] In some embodiments, the stabilizing portion is an Fc domain, such as any Fc domain described in Section 6.6.1 and its subsections, which are incorporated herein by reference. The Fc domains described in Section 6.6.1 are generally capable of dimerization. However, for stabilization purposes, the Fc domain may be a soluble monomeric Fc domain with reduced self-association ability. See, for example, Helm et al., 1996, J. Biol. Chem. 271: 7494-7500 and Ying et al., 2012, J BiolChem. 287(23):19399–19408. Examples of soluble monomeric Fc domains include amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3, as described in U.S. Patent Publication No. 2019 / 0367611. The monomeric Fc domain may be any Ig isotype and may include additional substitutions that reduce effector function, as described in Section 6.6.1 and its subsections.

[0244] In other embodiments, the stabilizing portion is a polyethylene glycol portion or another polymer, as described in Section 6.7.1 below.

[0245] The stabilizing portion may be connected via a connector to one or more other components of the CD20-PD1 binding molecule of this disclosure, such as as described in Section 6.8 below.

[0246] 6.7.1. Polyethylene glycol

[0247] In some embodiments, the CD20-PD1 binding molecule comprises polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing portion, such as a copolymer of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer can have any molecular weight and can be branched or unbranched.

[0248] 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 known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pp. 138-161). The term "PEG" is broadly used to cover any polyethylene glycol molecule, regardless of the size or modification of the PEG terminus, and can be represented by the following formula: X--O(CH2CH2O) n-1CH2CH2OH, where n is 20 to 2300, and X is H or a terminal modification, for example, C 1-4 Alkyl groups. PEG may contain other chemical groups required for the bonding reaction, which are generated from the chemical synthesis of the molecule; or they may act as spacers to achieve optimal spacing between the parts of the molecule. Additionally, such PEGs may consist of one or more PEG side chains linked together. PEGs having more than one PEG chain are called multi-armed or branched PEGs. Branched PEGs are described, for example, in European Application No. 473084A and US Patent No. 5,932,462.

[0249] One or more PEG molecules can attach to different positions on the CD20-PD1 binding molecule, and this attachment can be achieved by reacting with an amine, thiol, or other suitable reactive group. The amine moiety can be, for example, a primary amine found at the N-terminus of the CD20-PD1 binding molecule (or a component thereof) or an amine group present in an amino acid such as lysine or arginine.

[0250] Polyglycolization can be achieved through site-directed PEGylation, in which suitable reactive groups are introduced into the protein to create sites where PEGylation preferentially occurs. In some embodiments, the CD20-PD1 binding molecule is modified to introduce a cysteine ​​residue at the desired site, thereby allowing site-directed PEGylation of the cysteine. Mutations can be introduced into the coding sequence of the CD20-PD1 binding molecule of this disclosure to produce a cysteine ​​residue. For example, this can be achieved by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutation to cysteine ​​residues include serine, threonine, alanine, and other hydrophilic residues. Preferably, the residue to be mutated to cysteine ​​is a surface-exposed residue. Algorithms for predicting the surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. PEGylation of cysteine ​​residues can be performed using, for example, PEG-maleimide, PEG-vinyl sulfone, PEG-iodoacetamide, or PEG-o-pyridine disulfide.

[0251] PEG is typically activated with a suitable activating group appropriate for coupling to the desired site on the peptide. Polyethylene glycolation is a well-known method in the art and is further described below: Zalipsky et al., “Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides”, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, JM Harris, Plenus Press, New York (1992), and Zalipsky, 1995, Advanced Drug Reviews 16: 157-182.

[0252] The molecular weight of the PEG moiety can vary considerably and can be branched or linear. Typically, the weight-average molecular weight of PEG is 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 some embodiments, the molecular weight of PEG is 40,000 Daltons. Branched forms of PEG having any of the above 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 (NOFCorporation, DE-200MA).

[0253] Conventional separation and purification techniques known in the art can be used to purify PEGylated CD20-PD1 bound molecules, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. SDS-PAGE can also be used to separate the products. Separable products include mono-PEGylated, dimerized, trimerized, poly-PEGylated, and unPEGylated CD20-PD1 bound molecules, as well as free PEG. The percentage of monoPEG conjugates can be controlled by merging broader fractions around the elution peak to increase the percentage of monoPEG in the composition. Approximately 90% monoPEG conjugates indicate a good balance between yield and activity.

[0254] In some embodiments, the polyethylene glycol-modified CD20-PD1 binding molecule will preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the bioactivity associated with the unmodified CD20-PD1 binding molecule. In some embodiments, bioactivity refers to its ability to bind to CD20, PD1, or both CD20 and PD1, such as by K D k on or k off Evaluate.

[0255] 6.8. Connector

[0256] In some aspects, this disclosure provides a CD20-PD1 binding molecule in which two or more components of a CD20-PD1 binding molecule are linked to each other via peptide linkers (also referred to as “linkers”). By way of example and not limitation, linkers may be used to link (a) a CD20 targeting moiety and a dimerizing moiety; (b) a CD20 targeting moiety and a PD1 agonist moiety; (c) a PD1 agonist moiety and a dimerizing moiety; (d) a CD20 targeting moiety and an antigen-binding fragment of an agonist antiPD1 antibody; (e) a PD1 agonist moiety and an antigen-binding fragment of an agonist antiPD1 antibody; (f) different domains within the CD20 targeting moiety (e.g., VH and VL domains in scFv); or (g) different domains within the antigen-binding fragment of an agonist antiPD1 antibody (e.g., VH and VL domains in scFv).

[0257] The length of the peptide linker can range from 2 amino acids to 60 or more amino acids, and in some respects, the length of the peptide linker ranges from 3 amino acids to 50 amino acids, 4 amino acids to 30 amino acids, 5 amino acids to 25 amino acids, 10 amino acids to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids.

[0258] In specific respects, the length of the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids, and optionally, the length is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids.

[0259] In some of the foregoing embodiments, the length of the connector ranges from 5 to 50 amino acids, for example, from 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other foregoing embodiments, the length of the connector ranges from 6 to 50 amino acids, for example, from 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In some of the foregoing embodiments, the length of the connector ranges from 7 to 50 amino acids, for example, from 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0260] Electrically charged (e.g., electrically charged hydrophilic connectors) and / or flexible connectors are particularly preferred.

[0261] Examples of flexible linkers that can be used for the CD20-PD1 binding molecules disclosed herein 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 or contain repetitive sequences of glycine and serine, such as G... n S (SEQ ID NO:57) or SG n The monomer or polymer of (SEQ ID NO:58), where n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the connector is or comprises a monomer or polymer of G4S (SEQ ID NO:33) repeating, for example, (GGGGS). n (SEQ ID NO:33).

[0262] Polyglycine linkers can be suitably used in the CD20-PD1 binding molecules of this disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly) (SEQ ID NO:59), five consecutive glycines (5 Gly) (SEQ ID NO:60), six consecutive glycines (6 Gly) (SEQ ID NO:61), seven consecutive glycines (7 Gly) (SEQ ID NO:62), eight consecutive glycines (8 Gly) (SEQ ID NO:63), or nine consecutive glycines (9 Gly) (SEQ ID NO:64).

[0263] Exemplary linker sequences are listed in Table L below. The CD20-PD1 binding molecule of this disclosure may contain one or more linkers from Table L.

[0264]

[0265] In some embodiments, the CD20-PD1 binding molecule includes a connector L1. In some embodiments, the CD20-PD1 binding molecule includes a connector L2. In some embodiments, the CD20-PD1 binding molecule includes a connector L3. In some embodiments, the CD20-PD1 binding molecule includes a connector L4. In some embodiments, the CD20-PD1 binding molecule includes a connector L5. In some embodiments, the CD20-PD1 binding molecule includes a connector L6. In some embodiments, the CD20-PD1 binding molecule includes a connector L7. In some embodiments, the CD20-PD1 binding molecule includes a connector L8. In some embodiments, the CD20-PD1 binding molecule includes a connector L9. In some embodiments, the CD20-PD1 binding molecule includes a connector L10. In some embodiments, the CD20-PD1 binding molecule includes a connector L11. In some embodiments, the CD20-PD1 binding molecule includes a connector L12. In some embodiments, the CD20-PD1 binding molecule includes a connector L13. In some embodiments, the CD20-PD1 binding molecule includes a connector L14. In some embodiments, the CD20-PD1 binding molecule includes a connector L15. In some embodiments, the CD20-PD1 binding molecule includes a connector L16. In some embodiments, the CD20-PD1 binding molecule includes a connector L17. In some embodiments, the CD20-PD1 binding molecule includes a connector L18. In some embodiments, the CD20-PD1 binding molecule includes a connector L19. In some embodiments, the CD20-PD1 binding molecule includes a connector L20. In some embodiments, the CD20-PD1 binding molecule includes a connector L21. In some embodiments, the CD20-PD1 binding molecule includes a connector L22. In some embodiments, the CD20-PD1 binding molecule includes a connector L23. In some embodiments, the CD20-PD1 binding molecule includes a connector L24. In some embodiments, the CD20-PD1 binding molecule includes a connector L25. In some embodiments, the CD20-PD1 binding molecule includes a connector L26. In some embodiments, the CD20-PD1 binding molecule includes a connector L27. In some embodiments, the CD20-PD1 binding molecule includes a connector L28. In some embodiments, the CD20-PD1 binding molecule includes a connector L29. In some embodiments, the CD20-PD1 binding molecule includes a connector L30. In some embodiments, the CD20-PD1 binding molecule includes a connector L31. In some embodiments, the CD20-PD1 binding molecule includes a connector L32. In some embodiments, the CD20-PD1 binding molecule includes a connector L33. In some embodiments, the CD20-PD1 binding molecule includes a connector L34. In some embodiments, the CD20-PD1 binding molecule includes a connector L35. In some embodiments, the CD20-PD1 binding molecule includes a connector L36.In some embodiments, the CD20-PD1 binding molecule includes a connector L37. In some embodiments, the CD20-PD1 binding molecule includes a connector L38. In some embodiments, the CD20-PD1 binding molecule includes a connector L39. In some embodiments, the CD20-PD1 binding molecule includes a connector L40. In some embodiments, the CD20-PD1 binding molecule includes a connector L41. In some embodiments, the CD20-PD1 binding molecule includes a connector L42. In some embodiments, the CD20-PD1 binding molecule includes a connector L43. In some embodiments, the CD20-PD1 binding molecule includes a connector L44. In some embodiments, the CD20-PD1 binding molecule includes a connector L45. In some embodiments, the CD20-PD1 binding molecule includes a connector L46. In some embodiments, the CD20-PD1 binding molecule includes a connector L47. In some embodiments, the CD20-PD1 binding molecule includes a connector L48. In some embodiments, the CD20-PD1 binding molecule includes a connector L49. In some embodiments, the CD20-PD1 binding molecule includes a connector L50. In some embodiments, the CD20-PD1 binding molecule includes a connector L51. In some embodiments, the CD20-PD1 binding molecule includes a connector L52. In some embodiments, the CD20-PD1 binding molecule includes a connector L53. In some embodiments, the CD20-PD1 binding molecule includes a connector L54. In some embodiments, the CD20-PD1 binding molecule includes a connector L55. In some embodiments, the CD20-PD1 binding molecule includes a connector L56. In some embodiments, the CD20-PD1 binding molecule includes a connector L57. In some embodiments, the CD20-PD1 binding molecule includes a connector L58. In some embodiments, the CD20-PD1 binding molecule includes a connector L59. In some embodiments, the CD20-PD1 binding molecule includes a connector L60. In some embodiments, the CD20-PD1 binding molecule includes a connector L61. In some embodiments, the CD20-PD1 binding molecule includes a connector L62. In some embodiments, the CD20-PD1 binding molecule includes a connector L63. In some embodiments, the CD20-PD1 binding molecule includes a connector L64. In some embodiments, the CD20-PD1 binding molecule includes a connector L65. In some embodiments, the CD20-PD1 binding molecule includes a connector L66. In some embodiments, the CD20-PD1 binding molecule includes a connector L67. In some embodiments, the CD20-PD1 binding molecule includes a connector L68. In some embodiments, the CD20-PD1 binding molecule includes a connector L69. In some embodiments, the CD20-PD1 binding molecule includes a connector L70. In some embodiments, the CD20-PD1 binding molecule includes a connector L71.In some embodiments, the CD20-PD1 binding molecule includes a connector L72. In some embodiments, the CD20-PD1 binding molecule includes a connector L73. In some embodiments, the CD20-PD1 binding molecule includes a connector L74. In some embodiments, the CD20-PD1 binding molecule includes a connector L75. In some embodiments, the CD20-PD1 binding molecule includes a connector L76. In some embodiments, the CD20-PD1 binding molecule includes a connector L77. In some embodiments, the CD20-PD1 binding molecule includes a connector L78. In some embodiments, the CD20-PD1 binding molecule includes a connector L79.

[0266] 6.8.1. Hinge Sequence

[0267] In some embodiments, the CD20-PD1 binding molecule of this disclosure includes a connector that is or includes a hinge region. Specifically, the hinge may be used to connect a CD20 targeting portion (e.g., a Fab domain) to a dimerizing domain (e.g., an Fc domain). The hinge region may be a natural or modified hinge region. The hinge region is typically located at the N-terminus of the Fc region. In the context of a dimer polypeptide (e.g., a homodimer or heterodimer CD20-PD1 binding molecule formed by the association of two Fc domains), the term "hinge region" refers to two associated hinge sequences on a single polypeptide chain.

[0268] A natural hinge region is a hinge region typically found between the Fab and Fc domains of a naturally occurring antibody. A modified hinge region is any hinge that differs from the natural hinge region in length and / or composition. Such hinges can include hinge regions derived from other species, such as those of humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, or goats. Other modified hinge regions may comprise complete hinge regions derived from antibodies of a different class or subclass than the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may comprise a portion of a natural hinge or repeating unit, where each unit in the repeat originates from the natural hinge region. In another alternative, the natural hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. In this way, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions can be fully synthetic and can be designed to have desired properties, such as length, cysteine ​​composition, and flexibility.

[0269] Many modified hinge areas have been described in, for example, U.S. Patent Nos. 5,677,425, WO 99 / 15549, WO2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971 and WO 2005 / 003171, and these contents are incorporated herein by reference.

[0270] In one embodiment, the CD20-PD1 binding molecule of this disclosure includes an Fc region, wherein one or both Fc domains have a fully hinged region at their N-terminus.

[0271] In various embodiments, positions 233-236 within the hinge area can be G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, wherein the positions are numbered by EU numbering.

[0272] In some embodiments, the CD20-PD1 binding molecule of this disclosure includes a modified hinge region that reduces the binding affinity to the Fcγ receptor relative to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0273] In the embodiments, the CD20-PD1 binding molecule of this disclosure comprises an Fc region, wherein each Fc domain has a fully hinged region at its N-terminus, wherein each Fc domain and hinged region is derived from IgG4 and each hinged region contains the modified sequence CPCC (SEQ ID NO:137). Compared to IgG1 containing the sequence CPCC (SEQ ID NO:137), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO:138). The presence of serine residues in the IgG4 sequence increases the flexibility of this region, thus allowing a portion of the molecule to form disulfide bonds within the same protein chain (intra-chain disulfide bonds) rather than bridging to other heavy chains in the IgG molecule to form inter-chain disulfide bonds (Angel et al., 1993, Mol Immunol 30(1):105-108). Replacing serine residues with proline to obtain the same core sequence as IgG1 allows for complete inter-chain disulfide bond formation in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This modified isotype is referred to as IgG4P.

[0274] 6.8.1.1. Interlocking Hinge Sequence

[0275] The hinge area can be a mating hinge area.

[0276] For example, the chimeric hinge may include an “upper hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, which is combined with a “lower hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4.

[0277] In a specific embodiment, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 139; previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 140; previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences may suitably be linked to the IgG4 CH2 region (e.g., by incorporating an IgG4 Fc domain, such as a human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, for example as described in Section 6.6.1.1).

[0278] 6.8.1.2. Hinge sequences with reduced effector functionality

[0279] In further embodiments, the hinge region may be modified to reduce the effector function, for example as described in WO2016161010A2, the entirety of which is incorporated herein by reference. In various embodiments, the modified hinge region positions 233-236 are G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, wherein the positions are numbered by EU numbering (as shown in Figure 1 of WO2016161010A2). These segments may be represented as GGG-, GG--, G---, or ----, where "-" indicates an unoccupied position.

[0280] Position 236 is not occupied in canonical human IgG2, but is occupied in other canonical human IgG isoforms. In all four human isoforms, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

[0281] The hinge modification at positions 233-236 can be combined with position 228, which is occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, by S in human IgG4, and by R in human IgG3. The S228P mutation in IgG4 antibodies is beneficial for stabilizing IgG4 antibodies and reducing the exchange of heavy and light chain pairs between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0282] The exemplary hinge region has residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated as GGG-(233-236), GG--(233-236), G---(233-236), and without G(233-236). Optionally, the hinge domain amino acid sequence comprises CPCPAPGGG-GPSVF (SEQ ID NO:141; previously disclosed as WO2016161010A2, SEQ ID NO:1), CPCPAPGG--GPSVF (SEQ ID NO:142; previously disclosed as WO2016161010A2, SEQ ID NO:2), CPCPAPG---GPSVF (SEQ ID NO:143; previously disclosed as WO2016161010A2, SEQ ID NO:3) or CPCPAPG----GPSVF (SEQ ID NO:144; previously disclosed as WO2016161010A2, SEQ ID NO:4).

[0283] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically includes CH2 and CH3 domains, and may have additional hinge segments (e.g., upper hinges) located on the flanks of the designated region. Such additional constant region fragments generally have the same isotype, preferably the human isotype, although they may be hybrids of different isotypes. The isotype of such additional human constant region fragments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3, or hybrids of their domains with different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 through 4 of WO2016161010A2.

[0284] In a specific embodiment, the modified hinge sequence may be linked to the IgG4 CH2 region (e.g., by incorporating the IgG4 Fc domain, such as the human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, for example as described in Section 6.6.1.1).

[0285] 6.9. Nucleic Acids and Host Cells

[0286] In another aspect, this disclosure provides nucleic acids encoding the CD20-PD1 binding molecule of this disclosure. In some embodiments, the CD20-PD1 binding molecule is encoded by a single nucleic acid. In other embodiments, for example, in the case of a heterodimeric molecule or a molecule comprising a CD20 targeting portion consisting of more than one polypeptide chain, the CD20-PD1 binding molecule may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0287] 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 two or more polypeptide chains (e.g., 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). For a single control of expression, open reading frames encoding two or more polypeptide chains can be under the control of individual 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 and separated by internal ribosome entry site (IRES) sequences, thereby allowing translation into individual polypeptides.

[0288] In some embodiments, a CD20-PD1 binding molecule comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding a CD20-PD1 binding molecule may be equal to or less than the number of polypeptide chains in the CD20-PD1 binding molecule (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0289] The nucleic acid disclosed herein may be DNA or RNA (e.g., mRNA).

[0290] In another aspect, this disclosure provides host cells and vectors containing the nucleic acids of this disclosure. The nucleic acids may be present in a single vector or in a single vector within the same host cell or a separate host cell, as described in more detail below.

[0291] 6.9.1. Carrier

[0292] This disclosure provides vectors comprising nucleotide sequences encoding the CD20-PD1 binding molecule or a component thereof (e.g., one or two polypeptide chains of a CD20-PD1 monomer) described herein. Vectors include, but are not limited to, viruses, plasmids, viscera, λ phages, or yeast artificial chromosomes (YACs). Vectors encoding the CD20-PD1 binding molecule (or a component thereof) of this disclosure can be used for the expression and / or delivery of the CD20-PD1 binding molecule.

[0293] Various vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rouse sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semleeki Forest virus, Eastern Equine Encephalitis Virus, and Flavivirosis.

[0294] Additionally, cells that have stably integrated their DNA into their chromosomes can be selected by introducing one or more markers that allow selective transfection of host cells. Markers can provide, for example, tropism against auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals (e.g., copper). The selection marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal mRNA synthesis may also require other elements. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0295] Once the expression vector or DNA sequence containing the construct is prepared, it can be transfected or introduced into a suitable host cell. This can be achieved using various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions used to culture the resulting transfected cells and to recover the expressed peptide are known to those skilled in the art and can be varied or optimized based on the specific expression vector and mammalian host cells used, according to this specification.

[0296] 6.9.2. Host Cell

[0297] This disclosure also provides host cells containing the nucleic acids contained in this disclosure.

[0298] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids as described herein.

[0299] In one embodiment, host cells are genetically engineered using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of influencing gene expression in a host 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 influencing expression, such as, for example, inducible promoters, may also be used.

[0300] This disclosure also provides host cells containing the vectors described herein.

[0301] The cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0302] 6.10. Pharmaceutical Compositions

[0303] 6.10.1. Pharmaceutical compositions comprising CD20-PD1 binding molecules

[0304] The CD20-PD1 binding molecule disclosed herein may be in the form of a composition comprising a CD20-PD1 binding molecule and one or more carriers, excipients, and / or diluents. The composition may be formulated for a specific purpose, such as for veterinary or human pharmaceutical use. The form of the composition used (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers will depend on the intended use of the CD20-PD1 binding molecule and the mode of administration for therapeutic purposes.

[0305] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the method required for its administration to the patient). The pharmaceutical composition may be administered to the patient via a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most appropriate route of administration in any given situation will depend on the specific antibody, the subject, the nature and severity of the disease, and the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0306] The pharmaceutical composition can be conveniently available in unit dosage forms containing a predetermined amount of the CD20-PD1 binding molecule of this disclosure per dose. The amount of CD20-PD1 binding molecule contained in a unit dose will depend on the disease being treated and other factors known in the art. Such unit doses can be in the form of a lyophilized powder containing a certain amount of CD20-PD1 binding molecule suitable for a single administration, or in a liquid form. The powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components for administration. The liquid unit dose can be conveniently supplied in the form of a syringe pre-filled with a certain amount of CD20-PD1 binding molecule suitable for a single administration.

[0307] The pharmaceutical composition can also be supplied in bulk from a certain amount of CD20-PD1 binding molecules suitable for multiple administrations.

[0308] Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing a CD20-PD1 binding molecule of desired purity with an optional, pharmaceutically acceptable carrier, excipient, or stabilizer (i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives) commonly used in the art (all of which are referred to herein as “carriers”). See Remington, The Science and Practice of Pharmacy, 23rd edition (Adejare, editor 2020). Such additives should be non-toxic to the recipient at the dosage and concentration used.

[0309] Buffers help maintain pH values ​​within a range close to physiological conditions. They can be present in a variety of concentrations, but are typically found in concentrations ranging from about 2 mM to about 50 mM. Suitable buffers for use with this invention include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, trisodium citrate mixtures, monosodium citrate mixtures, etc.), succinate buffers (e.g., succinate-monosodium succinate mixtures, succinate-sodium hydroxide mixtures, succinate-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), and fumarate buffers (e.g., fumarate-monosodium fumarate mixtures). Buffers include fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.; gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.); oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.); lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.); and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can also be used.

[0310] Preservatives may be added to slow microbial growth, and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use with this invention include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzyl ammonium halides (e.g., chlorides, bromides, and iodides), hexamethyl ammonium chloride, and alkyl esters of parabens, such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents (sometimes referred to as “stabilizers”) may be added to ensure the isotonicity of the liquid compositions of this disclosure, and isotonic agents include polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol. Stabilizers are a broad class of excipients whose functions range from fillers to additives. They can dissolve therapeutic agents or help prevent denaturation or adhesion to container walls. Typical stabilizers include polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc.; including cyclic polyols such as inositol; polyethylene glycol; amino acid polymers; and sulfur-containing reducing agents such as urea. Glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in amounts ranging from 0.5 wt% to 10 wt% per wt of CD20-PD1 binding molecules.

[0311] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve glycoproteins and protect them from agitation-induced aggregation. This also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and Pranic polyols. Nonionic surfactants can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0312] Additional miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0313] 6.10.2. Pharmaceutical compositions for delivering nucleic acids encoded by CD20-PD1 binding molecules.

[0314] The CD20-PD1 binding molecule disclosed herein can be delivered by any method that can be used for gene therapy, such as as mRNA or by a viral vector encoding a CD20-PD1 receptor agonist under the control of a suitable promoter.

[0315] Exemplary gene therapy vectors include adenovirus- or AAV-based therapeutic agents. Non-limiting examples of adenovirus- or AAV-based therapeutic agents used in the methods, uses, or compositions described herein include, but are not limited to: rAd-p53, a recombinant adenovirus vector encoding the wild-type human tumor suppressor protein p53, for example, for the treatment of cancer (also known as Gendicine®, Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297); Ad5_d11520, an adenovirus lacking the E1B gene for inactivating host p53 (also known as H101 or ONYX-015; see, for example, Russell et al., 2012, Nature Biotechnology 30:658-670); AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF, for example, for the treatment of cancer (Cerullo et al., 2010, Cancer). Res. 70:4297); rAd-HSVtk, a replication-deficient adenovirus with the HSV thymidine kinase gene, for example, for the treatment of cancer (developed as Cerepro®, Ark Therapeutics, see, for example, US Patent No. 6,579,855; developed by Advantagene as ProstAtak™; International PCT Application No. WO2005 / 049094); rAd-TNFα, a replication-deficient adenovirus vector expressing human tumor necrosis factor α (TNFα) under the control of the chemiradioactive EGR-1 promoter, for example, for the treatment of cancer (TNFerade™, GenVec; Rasmussen et al., 2002, Cancer Gene). Ther. 9:951-7; Ad-IFNβ, an adenovirus serotype 5 vector in which the E1 and E3 genes have been deleted, expresses the human interferon β gene under the guidance of the cytomegalovirus (CMV) immediate early promoter, for example for the treatment of cancer (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther. 18:852-860). Other vectors recognized in the art include, for example, lentiviral vectors (e.g., VSV), retroviral vectors, etc.

[0316] Any delivery vector now known or to be developed in the future, whether natural or engineered, can be used to deliver the CD20-PD1 binding molecule disclosed herein. In some embodiments, the delivery vector is a viral vector, for example, comprising a virus, a viral capsid, a viral genome, etc. In some embodiments, the delivery vector is a naked nucleic acid, for example, an episome. In some embodiments, the delivery vector comprises a nucleic acid complex. Exemplary non-limiting nucleic acid complexes used as delivery vectors include liposomes, polymeric vesicles, polymers, dendritic polymers, and inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, calcium phosphate, etc.). In some embodiments, the delivery vectors described herein comprise a combination of a viral vector, a naked nucleic acid, and a nucleic acid complex.

[0317] In one embodiment, the delivery vector is a virus, including a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. In one embodiment, the delivery vector is adeno-associated virus (AAV), including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof.

[0318] In one embodiment, the nucleic acid encoding the CD20-PD1 binding molecule (or a component thereof) also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the vector is a chimeric adeno-associated virus containing genetic elements from two or more serotypes. For example, an AAV vector having a rep gene from AAV1 and a cap gene from AAV2 (referred to as AAV1 / 2 or AAV RC1 / 2) can be used as a delivery vector to deliver nucleic acids expressing the CD20-PD1 binding molecule to cells in need or to the patient's cells.In one embodiment, the delivery carrier is 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, AA V3 / 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, AAV5 / 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, AA V6 / 8, AAV6 / 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, AA V9 / 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 viral vectors or their derivatives. Gao et al., “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy,” PNAS 99(18):11854-11859, September 3, 2002, incorporated herein by reference, relates to AAV vectors and chimeric viral vectors as delivery vectors, their construction and uses.

[0319] Nucleic acid molecules (e.g., mRNA) or viruses may be formulated as the sole active pharmaceutical ingredient in a pharmaceutical composition, or may be combined with other active agents for the specific disease to be treated. Optionally, the compositions provided herein may contain other pharmaceutical agents, drug agents, carriers, adjuvants, and diluents. For example, wetting agents, emulsifiers, and lubricants (such as sodium dodecyl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Exemplary other pharmaceutical agents and excipients that may be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0320] When used as adjunctive therapy to adoptive cell transfer therapy, for example, CAR-expressing cell therapies as described in Section 6.11.2.1, cell therapies, such as CAR-expressing cells, can be engineered to express the CD20-PD1 binding molecule of this disclosure. The CD20-PD1 binding molecule can target specific genomic sites, such as sites active in activated or dysfunctional lymphocytes, such as PD-1 sites, or insert into non-specific genomic sites. Targeting of specific genomic sites can be achieved through gene editing, for example, using zinc finger proteins, CRISPR / Cas9 systems, etc.

[0321] 6.11. Indications and Methods of Use

[0322] The CD20-PD1 binding molecule disclosed herein can be used to treat disease states that are beneficial to stimulating the host immune system, specifically conditions requiring suppression of cellular immune responses. Therefore, the CD20-PD1 binding molecule disclosed herein can be used to suppress immune responses in a variety of applications.

[0323] Conditions requiring suppression of cellular immune responses can include disease states caused by autoimmune responses. Disease states for which the CD20-PD1 binding molecule of this disclosure can be administered include, for example, autoimmune diseases, where suppression of cellular autoimmune responses is an important mechanism. Specific disease states for which the CD20-PD1 binding molecule of this disclosure can be used for treatment include type 1 diabetes (T1D), systemic lupus erythematosus, Crohn's disease, and graft-versus-host disease (GVHD). The CD20-PD1 binding molecule of this disclosure can be administered alone or in any suitable pharmaceutical composition.

[0324] In one aspect, the present disclosure provides a CD20-PD1 binding molecule for use as a medicament. In a further aspect, the present disclosure provides a CD20-PD1 binding molecule for treating a disease. In some embodiments, the present disclosure provides a CD20-PD1 binding molecule for use in a treatment method. In one embodiment, the present disclosure provides a CD20-PD1 binding molecule as described herein for treating a disease in a subject of need. In some embodiments, the present disclosure provides a CD20-PD1 binding molecule for use in a method of treating a subject with an autoimmune disease, the method comprising administering a therapeutically effective amount of the CD20-PD1 binding molecule to the individual. In some embodiments, the disease to be treated is an autoimmune disease. In a specific embodiment, the disease is T1D. In other embodiments, the disease is systemic lupus erythematosus. In other embodiments, the disease is Crohn's disease. In still other embodiments, the disease is GVHD. In some embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual. In a further embodiment, the present disclosure provides a CD20-PD1 binding molecule for suppressing the immune system. In some embodiments, this disclosure provides a CD20-PD1 binding molecule for use in a method of suppressing the immune system of a subject, the method comprising administering an effective amount of the CD20-PD1 binding molecule to an individual to suppress the immune system. According to any of the above embodiments, "individual" is a mammal, such as a human. According to any of the above embodiments, "suppression of the immune system" may include any and more of a generalized reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell responsiveness, etc.

[0325] This disclosure further provides a method for targeting PD1 agonist activity, the method comprising administering the CD20-PD1 binding molecule or pharmaceutical composition described herein to a subject. As used herein, the term “local delivery” does not require local application, but rather indicates that the CD20-PD1 binding molecule selectively or preferentially targets an intended site of immune regulation, such as an autoimmune active site.

[0326] This disclosure further provides a method of administering a PD1 agonist therapy to a subject with reduced systemic exposure and / or reduced systemic toxicity, the method comprising administering to the subject a PD1 agonist therapy in the form of a CD20-PD1 binding molecule or pharmaceutical composition as described herein, for example wherein CD20 is expressed by tissues requiring and / or anticipating PD1 agonist therapy.

[0327] Therefore, the above method allows PD1 agonist therapy to have reduced off-target side effects by preferentially delivering CD20-PD1 binding molecules at the site of PD1 agonist treatment.

[0328] This disclosure further provides a method for locally modulating (e.g., inhibiting) an immune response in a target tissue expressing CD20, the method comprising administering to a subject the CD20-PD1 binding molecule or pharmaceutical composition described herein.

[0329] In some embodiments, application is not localized to the tissue. For example, when the target tissue is cancerous tissue, application may be systemic or subcutaneous.

[0330] In a further aspect, this disclosure provides the use of the CD20-PD1 binding molecule of this disclosure in the manufacture or preparation of a medicament for treating a disease in a subject of need. In one embodiment, the medicament is provided for use in a method of treating a disease, the method comprising administering a therapeutically effective amount of the medicament to a subject suffering from the disease. In some embodiments, the disease to be treated is an autoimmune disease. In a specific embodiment, the disease is T1D. In other embodiments, the disease is systemic lupus erythematosus. In other embodiments, the disease is Crohn's disease. In still other embodiments, the disease is GVHD. In some embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual. In a further embodiment, the medicament is used to suppress the immune system. In a further embodiment, the medicament is provided for use in a method of suppressing the immune system of a subject, the method comprising administering an effective amount of the medicament to the individual to suppress the immune system. According to any of the above embodiments, the "individual" can be a mammal, such as a human. According to any of the above embodiments, "suppression of the immune system" can include any one or more of a generalized reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell reactivity, etc.

[0331] In a further aspect, this disclosure provides a method for accumulating PD1 and / or enhancing PD1 activity in a subject, the method comprising administering to the subject an effective amount of the disclosed CD20-PD1 binding molecule. The disclosed CD20-PD1 binding molecule can induce PD1 accumulation at the interface between CD20-presenting cells and T cells. This provides targeted immunosuppression, wherein CD20-presenting cells, as well as surrounding cells and tissues, are protected from T cell killing. High levels of CD20 are found on B cells, which are abundant in draining lymph nodes and autoimmune tissues (e.g., the pancreas in type 1 diabetes (T1D)). The CD20-PD1 binding molecule of the present invention can stimulate PD1 in a cell- and / or tissue-specific manner, thereby inhibiting the activation of autoreactive T cells. In T1D, a large number of CD20+ B cells cause PD1 to accumulate on autoreactive T cells, thereby inhibiting the killing of pancreatic islet cells by autoreactive cytotoxic T cells. In one embodiment, a composition comprising the disclosed CD20-PD1 binding molecule in a pharmaceutically acceptable form is administered to the subject.

[0332] In a further aspect, this disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering to the individual a therapeutically effective amount of the CD20-PD1 binding molecule of this disclosure. In one embodiment, the individual is administered a composition comprising the CD20-PD1 binding molecule of this disclosure in a pharmaceutically acceptable form. In some embodiments, the disease to be treated is an autoimmune disease. Autoimmune diseases treatable with the CD20-PD1 binding molecule of this disclosure may include type 1 diabetes, primary biliary cholangitis (PBC), Goodpasture syndrome, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane nephritis, antitubular basement membrane nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune oophoritis, graft-versus-host disease (GVHD), autoimmune pancreatitis, autoimmune retinopathy, Behçet's disease, Crohn's disease, Dervéd's disease, systemic lupus erythematosus (SLE), Dressler's syndrome, fibrotic alveolitis, glomerulonephritis, Graves' disease, Guillain-Ray syndrome, etc. Barley syndrome, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), multiple sclerosis, polyneuropathy, organ enlargement, endocrine disorders, monoclonal syndrome (POEMS), polyarteritis nodosa, rheumatoid arthritis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm or testicular autoimmunity, stiff-person syndrome (SPS), aortitis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), and vasculitis.

[0333] In a specific embodiment, the disease is T1D. In other embodiments, the disease is systemic lupus erythematosus. In other embodiments, the disease is Crohn's disease. In still other embodiments, the disease is GVHD. In some embodiments, the method further includes administering a therapeutically effective amount of at least one additional therapeutic agent to the individual. In a further aspect, this disclosure provides a method for suppressing the immune system of a subject, the method comprising administering an effective amount of a CD20-PD1 binding molecule to the individual to suppress the immune system. According to any of the above embodiments, the "individual" can be a mammal, such as a human. According to any of the above embodiments, "suppression of the immune system" can include any and more of a generalized reduction in immune function, a reduction in T cell function, a reduction in B cell function, a reduction in T cell reactivity, etc.

[0334] In some embodiments, the disease to be treated is an autoimmune disease. CD20-PD1 binding molecules can be used to eliminate cells involved in immune cell-mediated disorders, autoimmunity, transplant rejection, and graft-versus-host disease. Those skilled in the art will readily recognize that in many cases, CD20-PD1 binding molecules may not cure the disease and may only provide partial benefit. In some embodiments, physiological changes that provide some benefit are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of CD20-PD1 binding molecules providing physiological changes is considered an “effective amount” or a “therapeuticly effective amount.” The subject, patient, or individual requiring treatment is typically a mammal, more specifically a human.

[0335] For the prevention or treatment of disease, the appropriate dose of the CD20-PD1 binding molecule disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the specific CD20-PD1 binding molecule, the severity and duration of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the CD20-PD1 binding molecule, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject. This document covers a variety of dosing regimens, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsatile infusion.

[0336] CD20-PD1 binding molecules are suitable for single-dose administration to patients or as part of a series of treatments. Depending on the type and severity of the disease, a CD20-PD1 binding molecule at doses ranging from about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be an initial candidate dose for administration to a patient, whether, for example, by single or multiple administrations alone or by continuous infusion. Based on the factors described above, a typical daily dose range can be from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms is achieved. An exemplary dose range for CD20-PD1 binding molecules is from about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, the dosage may also include administration of about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight to about 1000 mg / kg / body weight or more, and any range derived therefrom. In non-limiting examples of ranges derived from the figures listed herein, based on the above figures, ranges such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc., may be administered. Therefore, patients can be administered 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). Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives approximately two to approximately twenty, or for example, approximately six, doses of the CD20-PD1 binding molecule). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may also be useful. Progression to this therapy is easily monitored using routine techniques and assays.

[0337] The CD20-PD1 binding molecules disclosed herein are generally used in amounts that are effective in achieving the intended purpose. For the treatment or prevention of disease symptoms, the CD20-PD1 binding molecules of this disclosure or pharmaceutical compositions thereof are administered or applied in therapeutically effective amounts. The determination of therapeutically effective amounts is entirely within the competence of those skilled in the art, especially based on the detailed disclosure provided herein.

[0338] For systemic administration, the effective therapeutic dose can be initially estimated through in vitro assays (such as cell culture assays). The dose can then be formulated in animal models to achieve the EC50 levels determined in cell culture. 50 The circulating concentration range, including [specific concentration range]. This information can be used to more accurately determine the useful dose in the human body.

[0339] The initial dose can also be estimated from in vivo data, such as animal models, using techniques well known in the art. Those skilled in the art can easily optimize human administration based on animal data.

[0340] The dosage and interval can be individually adjusted to provide sufficient plasma levels of CD20-PD1 binding molecules to maintain a therapeutic effect. Typically, the dose administered to patients by injection ranges from approximately 0.1 to 50 mg / kg / day, usually from approximately 0.5 to 1 mg / kg / day. Therapeuticly effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by ELISA or HPLC.

[0341] In cases of topical application or selective uptake, the effective local concentration of CD20-PD1 binding molecules may be independent of plasma concentration. Those skilled in the art will be able to optimize the therapeutically effective local dose without excessive experimentation.

[0342] The therapeutically effective doses of the CD20-PD1 binding molecules described herein will generally provide therapeutic benefit without significant toxicity. The toxicity and therapeutic efficacy of the CD20-PD1 binding molecules can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals. Cell culture assays and animal studies can be used to determine the LD50. 50（ (the dose that would be fatal to 50% of the population) and ED 50 (The dose effective for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 CD20-PD1 binding molecules exhibiting 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 to determine a dosage range suitable for human use. The dosage is preferably within the range including ED. 50Within a range of circulating concentrations with very low or no toxicity. Dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration employed, and the patient's condition. Precise preparation, route of administration, and dosage can be selected by an individual physician based on the patient's condition. (See, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Chapter 1, page 1, incorporated herein by reference in its entirety).

[0343] The attending physician for patients receiving CD20-PD1 combination molecular therapy according to this disclosure will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. In the treatment of the disease of interest, the dosage will vary depending on the severity of the disease being treated, the route of administration, etc. For example, the severity of the disease can be assessed in part using standard prognostic assessment methods. Furthermore, the dosage and possible dosing frequency will also vary based on the individual patient's age, weight, and response.

[0344] 6.11.1.1 Type 1 diabetes

[0345] In some embodiments, the CD20-PD1 binding molecule according to this disclosure can prevent or slow the onset or progression of type 1 diabetes. Therefore, in some embodiments, the CD20-PD1 binding molecule, nucleic acid, and / or pharmaceutical composition of this disclosure can be administered to subjects with T1D or at risk of developing T1D. Risk factors for 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., white people are more likely to develop type 1 diabetes in the United States), family history, early diet, and the presence of other autoimmune conditions (e.g., Graves' disease, multiple sclerosis, pernicious anemia). Cancer patients receiving immune checkpoint inhibitor therapy are also at risk of developing T1D. See de Filette et al., 2019, Eur J Endocrinol, 181(3):363-374. Identifying and selecting individuals at risk of developing T1D is within the capabilities of those skilled in the art.

[0346] 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 present disclosure according to the methods of the present disclosure.

[0347] 6.11.2. Combination Therapy

[0348] The CD20-PD1 binding molecule according to this disclosure can be administered in combination with one or more other pharmaceutical agents in a therapy. For example, the CD20-PD1 binding molecule of this disclosure can be administered co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any pharmaceutical agent administered to treat symptoms or diseases in a subject requiring such treatment. Such additional therapeutic agents may contain any active ingredient suitable for a particular indication being treated, preferably those having complementary activities that do not adversely affect each other.

[0349] In some embodiments, additional therapeutic agents are immunosuppressants, including but 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), and corticosteroids (e.g., hydrocortisone, hydroxytriamcione, α-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, deoxymethasone acetate, dexamethasone, diclomethasone diacetate, diflubenzuron valerate, and fluadrenol). One), Flucrononide, Fludrocortisone, Flumethasone Pentate, Fluocinolone Acetate, Fluocinolone, Flucortin Butyl Acetate, Flucolone, Fluprednylidene Acetate, Fludrocortisone, Halcinonide, Hydrocortisone Acetate, Hydrocortisone Butyrate, Methylprednisolone, Triamcinolone, Cortisone, Todoxacin, Flucrononide, Flucetonide, Fludrocortisone, Difluorometholone Diacetate, Fludrocortisone, Fludrocortisone Difluorometholone Diacetate, Fludrocortisone, Methoxysone, Amcinafel, Ancifenofibrate, Betamethasone and the remainder of esters, Chlorprednisolone, Chlorprednisolone Acetate, Chlorprednisolone, Clescinolone Clomid, difluprednisolone, fluclofenone, flunisolone, flumethrin, fluperazine, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone hydrocortisone, methylprednisolone, peramisone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and mixtures thereof), nonsteroidal anti-inflammatory agents (e.g., oxacins, such as piroxicam, isoxacin, tenoxacin, suldoxicam; salicylates, such as aspirin, disalicylic acid, chlorpheniramine, choline magnesium trisalicylate, safapryn, solprin, diflunisal, and fendoxa; acetic acid derivatives, such as diclofenac, fenchloric acid, indomethacin, sulindac, tometidine, isocolic acid, furofenic acid, thiophene, zidomi Ciprofen, azimacin, fentimic acid, zolpidem, clindanac, oxipin, biphenylacetic acid, and ketorolac; fenamic acid, such as mefenamic acid, meclofenamic acid, flufenamic acid, niflunic acid, and tofenamic acid; propionic acid derivatives, such as ibuprofen, naproxen, phenoxprofen, flurbiprofen, ketoprofen, fenprofen, fenbufen, indobuprofen, pirprofen, carprofen, oxaprazin, pranoprofen, miprofen, thioprofen, sulprofen, amiprofen, and tiprofen; pyrazoles, such as phenylbutazone, hydroxyphenylbutazone, feprazolam, azaprofen, and trimethoprim), and anti-inflammatory cytokines or chemokines (e.g., IL-4, IL-6, IL-10, IL-11, and IL-13).

[0350] Other such agents are appropriately combined in amounts effective for the intended purpose. The effective dose of these other agents depends on the amount of CD20-PD1 binding molecules used, the type of disease or treatment, and other factors discussed above. CD20-PD1 binding molecules are generally used at the same doses and routes of administration as described herein, or approximately 1% to 99% of the doses described herein, or at any dose and route of administration as determined empirically / clinically.

[0351] Such combination therapies include combined administration (two or more therapeutic agents contained in the same or separate composition) and single administration, in which case the administration of the CD20-PD1 binding molecule of this disclosure may be performed before, simultaneously with and / or after the administration of additional therapeutic agents and / or adjuvants.

[0352] 6.11.2.1. Combination therapy using CD20-PD1 combined with molecular therapy and immunotherapy

[0353] The CD20-PD1 binding molecule disclosed herein can be advantageously used in combination with cells expressing chimeric antigen receptors (“CAR”), such as CAR-Treg cells expressing CAR (“CAR-Treg”), for example, for the treatment of autoimmune diseases. In some embodiments, CAR-Treg cells are recognized by the CD20-targeting portion of the CD20-PD1 binding molecule. The CD20-targeting portion may recognize a Treg cell receptor or another cell surface molecule on the CAR-Treg cells. In some embodiments, the CD20-targeting portion of the CD20-PD1 binding molecule is capable of binding to an extracellular domain of the CAR, such as an antigen-binding domain. CAR-Treg cells are described in the following references: 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.

[0354] 7. Sequence

[0355] Certain sequences disclosed herein are provided in Table S below.

[0356]

[0357] 8. Specific embodiments and references

[0358] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by way of examples with reference to the numbers set forth below.

[0359] 1. A protein comprising:

[0360] (a) CD20 targeting region;

[0361] (b) The PD1 agonist portion, which comprises:

[0362] (i) An amino acid sequence having at least about 70% sequence identity with SEQ ID NO:2; or

[0363] (ii) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO:14; and

[0364] (c) Dimerization portion.

[0365] 2. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO:2.

[0366] 3. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:2.

[0367] 4. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:2.

[0368] 5. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:2.

[0369] 6. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:2.

[0370] 7. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2.

[0371] 8. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:2.

[0372] 9. The protein according to any one of Examples 1 to 8, wherein the PD1 agonist portion comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0373] 10. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO:8.

[0374] 11. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:8.

[0375] 12. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:8.

[0376] 13. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:8.

[0377] 14. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:8.

[0378] 15. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:8.

[0379] 16. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:8.

[0380] 17. The protein according to any one of Examples 10 to 16, wherein the PD1 agonist portion comprises or consists of the amino acid sequence of SEQ ID NO:8.

[0381] 18. The protein according to any one of Examples 10 to 16, wherein the PD1 agonist portion comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0382] 19. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO:14.

[0383] 20. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:14.

[0384] 21. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:14.

[0385] 22. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:14.

[0386] 23. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:14.

[0387] 24. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:14.

[0388] 25. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:14.

[0389] 26. The protein according to any one of Examples 19 to 25, wherein the PD1 agonist portion comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0390] 27. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO:20.

[0391] 28. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:20.

[0392] 29. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:20.

[0393] 30. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:20.

[0394] 31. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:20.

[0395] 32. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:20.

[0396] 33. The protein according to Example 1, wherein the PD1 agonist protein comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:20.

[0397] 34. The protein according to any one of Examples 27 to 33, wherein the PD1 agonist portion comprises or is composed of the amino acid sequence of SEQ ID NO:20.

[0398] 35. The protein according to any one of Examples 1 to 34, wherein the protein does not contain the PDL1 transmembrane domain.

[0399] 36. The protein according to any one of Examples 1 to 35, wherein the protein does not contain the PDL1 intracellular domain.

[0400] 37. The protein according to any one of Examples 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.

[0401] 38. The protein according to any one of Examples 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.

[0402] 39. The protein according to any one of Examples 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.

[0403] 40. The protein according to any one of Examples 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to SEQ ID NO:5.

[0404] 41. The protein according to any one of Examples 1 to 40, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 20 amino acids in length corresponding to SEQ ID NO:5.

[0405] 42. The protein according to any one of Examples 1 to 40, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 10 amino acids in length corresponding to SEQ ID NO:5.

[0406] 43. The protein according to any one of Examples 1 to 40, wherein the protein does not contain any subsequence of at least 5 amino acids corresponding to SEQ ID NO:5.

[0407] 44. The protein according to any one of Examples 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.

[0408] 45. The protein according to any one of Examples 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to SEQ ID NO:11.

[0409] 46. ​​The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 20 amino acids in length corresponding to SEQ ID NO:11.

[0410] 47. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 10 amino acids in length corresponding to SEQ ID NO: 11.

[0411] 48. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 5 amino acids in length corresponding to SEQ ID NO:11.

[0412] 49. The protein according to any one of Examples 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.

[0413] 50. The protein according to any one of Examples 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to SEQ ID NO:17.

[0414] 51. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 20 amino acids in length corresponding to SEQ ID NO:17.

[0415] 52. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 10 amino acids in length corresponding to SEQ ID NO:17.

[0416] 53. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 5 amino acids in length corresponding to SEQ ID NO:17.

[0417] 54. The protein according to any one of Examples 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.

[0418] 55. The protein according to any one of Examples 1 to 38, wherein the protein does not contain the amino acid sequence corresponding to SEQ ID NO:23.

[0419] 56. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence of at least 20 amino acids corresponding to any subsequence of SEQ ID NO:23.

[0420] 57. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 10 amino acids in length corresponding to SEQ ID NO:23.

[0421] 58. The protein according to any one of Examples 1 to 38, wherein the protein does not contain any amino acid sequence corresponding to any subsequence of at least 5 amino acids in length corresponding to SEQ ID NO:23.

[0422] 59. The protein according to any one of Examples 1 to 58, wherein the PD1 agonist portion is not operatively attached to the membrane-adjacent portion of PDL1.

[0423] 60. The protein according to any one of Examples 1 to 58, wherein the PD1 agonist portion is not operatively attached to the membrane-adjacent portion of PDL2.

[0424] 61. The protein according to any one of Examples 1 to 60, wherein the length of the PD1 agonist moiety is 120 or fewer amino acids.

[0425] 62. The protein according to any one of Examples 1 to 61, wherein the portions of the protein are arranged from the N-terminus to the C-terminus in the order of CD20 targeting portion – PD1 agonist portion – dimerizing portion.

[0426] 63. The protein according to any one of Examples 1 to 61, wherein the portions of the protein are arranged from the N-terminus to the C-terminus in the order of PD1 agonist portion – CD20 targeting portion – dimerizing portion.

[0427] 64. The protein according to any one of Examples 1 to 61, wherein the CD20 targeting portion is anti-CD20Fab, the dimerizing portion is an Fc domain, and the light chain of the Fab is not fused with the PD1 agonist portion.

[0428] 65. The protein according to any one of Examples 1 to 61, wherein the dimerization portion is an Fc domain and the PD1 agonist portion is not located at the C-terminus of the Fc domain.

[0429] 66. The protein according to any one of Examples 1 to 65, further comprising an antigen-binding fragment of an agonist anti-PD1 antibody.

[0430] 67. The protein according to Example 66, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is located at the N-terminus of the PD1 agonist moiety.

[0431] 68. The protein according to Example 66, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is located at the C-terminus of the PD1 agonist moiety.

[0432] 69. The protein according to Example 66, wherein the portions of the protein are arranged from the N-terminus to the C-terminus in the following order: PD1 agonist portion – antigen-binding fragment of the agonist anti-PD1 antibody – CD20 targeting portion – dimerizing portion.

[0433] 70. The protein according to Example 66, wherein the portions of the protein are 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.

[0434] 71. The protein according to any one of Examples 66 to 70, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is in the form of Fab, Fv or scFv.

[0435] 72. The protein according to any one of Examples 66 to 71, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is:

[0436] (a) is an antigen-binding fragment of an agonist PD1 antibody selected from the following: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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;

[0437] (b) A heavy chain and / or light chain CDR comprising an agonist PD1 antibody selected from: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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;

[0438] (c) VH and VL containing PD1 agonist antibodies selected from the following: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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; or

[0439] (d) Competing with PD1 agonist antibodies selected from the following to bind to PD1 and / or to the same epitope: 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 In WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in 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.

[0440] 73. The protein according to any one of Examples 66 to 72, wherein the antigen-binding fragment of the agonist anti-PD1 antibody activates human PD1.

[0441] 74. The protein according to any one of Examples 66 to 72, wherein the antigen-binding fragment of the agonist anti-PD1 antibody agonizes mouse PD1.

[0442] 75. The protein according to any one of Examples 1 to 74, wherein the CD20 targeting portion binds to the extracellular domain of human CD20.

[0443] 76. The protein according to any one of Examples 1 to 74, wherein the CD20 targeting portion binds to the extracellular domain of mouse CD20.

[0444] 77. The protein according to any one of Examples 1 to 76, wherein the CD20 targeting portion is an antigen-binding fragment of an anti-CD20 antibody.

[0445] 78. The protein according to Example 77, wherein the antigen-binding fragment of the anti-CD20 antibody is in the form of Fab, Fv or scFv.

[0446] 79. The protein according to Example 77 or 78, wherein the anti-CD20 antibody binds to:

[0447] (a) The topological domain of CD20;

[0448] (b) The transmembrane domain of CD20; or

[0449] (c) Regions of CD20 displayed extracellularly on the surface of cells (e.g., B cells).

[0450] 80. The protein according to any one of Examples 77 to 79, wherein the anti-CD20 antibody comprises:

[0451] (a) Having a complementarity-determining region (“CDR”) of the CDR sequence of rituximab, ozoglucomannab, ozoglucomannab, ozoglucomannab, ozoglucomannab, ozoglucomannab, ozoglucomannab, TRU-015 or vertuzumab.

[0452] (b) All six CDR sequences of rituximab, ozoglucilimab, oxatuzumab, oxfamumab, tiimomab, tosimob, utuximab, oxcartuzumab, TRU-015 or vertuzumab.

[0453] (c) At least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of rituximab, ozoglucomab, oxautumumab, tivamomumab, tosimomumab, utuximab, oxcartuzumab, TRU-015 or vertuzumab, and the light chain CDR sequence of the universal light chain;

[0454] (d) A VH comprising the amino acid sequence of said VH of rituximab, olarelizumab, oxutuzumab, oflamumab, teimomab, tosimob, utuximab, oxcartuzumab, TRU-015, or vertuzumab, and a VL comprising the amino acid sequence of the same antibody; or

[0455] (e) A VH comprising the amino acid sequence of said VH of rituximab, ozoglucilimab, ozoglucilimab, ozoglucilimab, ozoglucilimab, tiemomumab, tosimomumab, ozoglucilimab, ozoglucilimab, TRU-015 or vertuzumab, and a VL comprising the universal light chain VL sequence.

[0456] 81. The protein according to any one of Examples 77 to 80, wherein the anti-CD20 antibody:

[0457] (a) Selected from rituximab, ozoglucilimab, oxatuzumab, oxfamumab, tiimozumab, tosimomumab, utuximab, oxcartuzumab, TRU-015, and vertuzumab; or

[0458] (b) Compete with anti-CD20 antibodies selected from the group consisting of: rituximab, ozoglucomancil, ozoglucomancil, ozoglucomancil, ozoglucomancil, ozoglucomancil, tosimumab, tosimocomancil, ozoglucomancil, ozoglucomancil, TRU-015 and ozoglucomancil.

[0459] 82. The protein according to any one of Examples 1 to 81, comprising one or more linker portions.

[0460] 83. The protein according to Example 82, wherein the CD20 targeting portion and the PD1 agonist portion are separated by a linker portion.

[0461] 84. The protein according to Example 82 or 83, wherein the PD1 agonist portion and the dimerized portion are separated by a linker portion.

[0462] 85. The protein according to any one of Examples 82 to 84, wherein each linker portion (a) has a length of at least 5 or at least 10 amino acids, (b) has a length of up to 20, up to 25 or up to 30 amino acids and / or (c) has a length of 5 to 15 amino acids or 5 to 20 amino acids.

[0463] 86. The protein according to any one of Examples 82 to 85, wherein the protein comprises a glycine-serine linker.

[0464] 87. The protein according to Example 86, wherein the glycine-serine linker comprises the sequence G4S (SEQ ID NO:33) or a polymer thereof.

[0465] 88. The protein according to Example 87, wherein the glycine-serine linker comprises a polymer comprising repeating sequences 2, 3, 4, 5 or more of the amino acid sequence G4S (SEQ ID NO:33).

[0466] 89. The protein according to any one of Examples 1 to 88, further comprising: (1) an additional CD20 targeting moiety; (2) an additional PD1 agonist moiety of 120 or fewer amino acids, 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.

[0467] 90. The protein according to Example 89, wherein the additional CD20 targeting portion and the additional PD1 agonist portion are separated by a linker portion.

[0468] 91. The protein according to Example 89 or 90, wherein the PD1 agonist portion and the dimerized portion are separated by a linker portion.

[0469] 92. The protein according to any one of Examples 89 to 91, wherein the linker portion has (a) a length of at least 5 or at least 10 amino acids, (b) a length of up to 20, up to 25 or up to 30 amino acids and / or (c) a length of 5 to 15 amino acids or 5 to 20 amino acids.

[0470] 93. The protein according to any one of Examples 89 to 92, wherein the linker portion is a glycine-serine linker.

[0471] 94. The protein according to Example 93, wherein the glycine-serine linker comprises the sequence G4S (SEQ ID NO:33) or a polymer thereof.

[0472] 95. The protein according to Example 93, wherein the glycine-serine linker comprises a polymer comprising repeating sequences 2, 3, 4, 5 or more of the amino acid sequence G4S (SEQ ID NO:33).

[0473] 96. The protein according to any one of Examples 1 to 95, comprising two monomers according to Exemplary Monomer 1.

[0474] 97. The protein according to Example 96, wherein exemplary monomer 1 comprises, in an N-terminal to C-terminal orientation, the CD20 targeting portion (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker portion, the PD1 agonist portion (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:9), an optional linker portion, and the dimerizing portion or consisting thereof.

[0475] 98. The protein according to Example 96 or 97, wherein exemplary monomer 1 consists of a single polypeptide chain.

[0476] 99. The protein according to Example 96 or 97, wherein exemplary monomer 1 consists of two polypeptide chains.

[0477] 100. The protein according to any one of Examples 96 to 99, wherein exemplary monomer 1 comprises one or more universal light chains.

[0478] 101. The protein according to any one of Examples 96 to 100, which has Figure 1A The configuration described in the text.

[0479] 102. The protein according to any one of Examples 1 to 88, comprising two monomers according to Exemplary Monomer 2.

[0480] 103. The protein according to Example 102, wherein exemplary monomer 2 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:9), optional linker moiety, antigen-binding fragment of agonist antiPD1 antibody (e.g., Fab, Fv or scFv), optional linker moiety, the CD20 targeting moiety (e.g., antiCD20 Fab, Fv or scFV), optional linker moiety and the dimerizing moiety or consisting thereof.

[0481] 104. The protein according to Example 102 or 103, wherein exemplary monomer 2 consists of a single polypeptide chain.

[0482] 105. The protein according to Example 102 or 103, wherein exemplary monomer 2 consists of two polypeptide chains.

[0483] 106. The protein according to Example 102 or 103, wherein exemplary monomer 2 consists of three polypeptide chains.

[0484] 107. The protein according to any one of Examples 102 to 106, wherein the exemplary monomer 2 comprises one or more universal light chains.

[0485] 108. The protein according to any one of Examples 102 to 107, having Figure 1B The configuration described in the text.

[0486] 109. The protein according to any one of Examples 1 to 88, comprising two monomers according to Exemplary Monomer 3.

[0487] 110. The protein according to Example 109, wherein exemplary monomer 3 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:9), optional linker moiety, the CD20 targeting moiety (e.g., anti-CD20 Fab, Fv or scFV), optional linker moiety, antigen-binding fragment of agonist anti-PD1 antibody (e.g., Fab, Fv or scFv), optional linker moiety and the dimerizing moiety or consisting thereof.

[0488] 111. The protein according to Example 109 or 110, wherein exemplary monomer 3 consists of a single polypeptide chain.

[0489] 112. The protein according to Example 109 or 110, wherein exemplary monomer 3 consists of two polypeptide chains.

[0490] 113. The protein according to Example 109 or 110, wherein exemplary monomer 3 consists of three polypeptide chains.

[0491] 114. The protein according to any one of Examples 109 to 113, wherein the exemplary monomer 3 comprises one or more universal light chains.

[0492] 115. The protein according to any one of Examples 109 to 114, having Figure 1C The configuration described in the text.

[0493] 116. The protein according to any one of Examples 1 to 88, comprising two monomers according to Exemplary Monomer 4.

[0494] 117. The protein according to Example 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 portion, the PD1 agonist portion (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:9), an optional linker portion, the CD20 targeting portion (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker portion, and the dimerizing portion or consisting thereof.

[0495] 118. The protein according to Example 116 or 117, wherein exemplary monomer 4 consists of a single polypeptide chain.

[0496] 119. The protein according to Example 116 or 117, wherein exemplary monomer 4 consists of two polypeptide chains.

[0497] 120. The protein according to Example 116 or 117, wherein exemplary monomer 4 consists of three polypeptide chains.

[0498] 121. The protein according to any one of Examples 116 to 120, wherein the exemplary monomer 4 comprises one or more universal light chains.

[0499] 122. The protein according to any one of Examples 59 to 121, having Figure 1D The configuration described in the text.

[0500] 123. The protein according to any one of Examples 1 to 88, comprising two monomers according to Exemplary Monomer 5.

[0501] 124. The protein according to Example 123, wherein exemplary monomer 5 comprises, in an N-terminal to C-terminal orientation, the CD20 targeting portion (e.g., anti-CD20 Fab, Fv, or scFV), an optional linker portion, the PD1 agonist portion (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:9), an optional linker portion, an antigen-binding fragment of an agonist anti-PD1 antibody (e.g., Fab, Fv, or scFv), an optional linker portion, and the dimerizing portion or consisting thereof.

[0502] 125. The protein according to Example 123 or 124, wherein exemplary monomer 5 consists of a single polypeptide chain.

[0503] 126. The protein according to Example 123 or 124, wherein exemplary monomer 5 consists of two polypeptide chains.

[0504] 127. The protein according to Example 123 or 124, wherein exemplary monomer 5 consists of three polypeptide chains.

[0505] 128. The protein according to any one of Examples 123 to 127, wherein the exemplary monomer 5 comprises one or more universal light chains.

[0506] 129. The protein according to any one of Examples 123 to 128, having Figure 1E The configuration described in the text.

[0507] 130. The protein according to any one of Examples 1 to 129, comprising two CD20 targeting moieties.

[0508] 131. The protein according to Example 130, wherein the two CD20 targeting regions are identical.

[0509] 132. The protein according to any one of Examples 1 to 131, comprising two PD1 agonist moieties.

[0510] 133. The protein according to Example 132, wherein the two PD1 agonist moieties are identical.

[0511] 134. The protein according to any one of Examples 1 to 133, comprising two antigen-binding fragments of an agonist anti-PD1 antibody.

[0512] 135. The protein according to Example 134, wherein the two antigen-binding fragments of the agonist anti-PD1 antibody are identical.

[0513] 136. The protein according to any one of Examples 1 to 135, wherein the dimerized portion is an Fc domain.

[0514] 137. The protein according to Example 136, wherein the Fc domain is a human Fc domain.

[0515] 138. The protein according to Example 136 or 137, wherein the Fc domain is an IgG1, IgG2, IgG3 or IgG4 Fc domain.

[0516] 139. The protein according to Example 138, wherein the Fc domain is the IgG4 Fc domain.

[0517] 140. The protein according to any one of Examples 136 to 139, wherein the Fc domain comprises the amino acid sequence ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:27) or a portion thereof.

[0518] 141. The protein according to Example 138, wherein the Fc domain is the IgG1 Fc domain.

[0519] 142. The protein according to any one of Examples 136 to 141, comprising an Fc dimer.

[0520] 143. The protein according to Example 142, wherein the Fc dimer is an Fc homodimer.

[0521] 144. The protein according to Example 142, wherein the Fc dimer is an Fc heterodimer.

[0522] 145. The protein according to Example 144, wherein the Fc heterodimer contains a club-and-mortar mutation.

[0523] 146. The protein according to Example 144 or 145, wherein the Fc heterodimer comprises a star mutation.

[0524] 147. A protein comprising:

[0525] (a) A device for combining CD20;

[0526] (b) A PD1 agonist moiety of 120 or fewer amino acids, comprising:

[0527] (i) An amino acid sequence having at least about 70% sequence identity with SEQ ID NO:2; or

[0528] (ii) an amino acid sequence having at least about 70% sequence identity with SEQ ID NO:14; and

[0529] (c) Dimerization portion.

[0530] 148. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus:

[0531] (a) CD20 targeting region;

[0532] (b) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; and

[0533] (c) Fc structural domain.

[0534] 149. The protein according to Example 148, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus:

[0535] (a) Additional CD20 targeting regions;

[0536] (b) A further PD1 agonist moiety, comprising an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; and

[0537] (c) The other Fc structural domain.

[0538] 150. A protein comprising:

[0539] (a) CD20 targeting region;

[0540] (b) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:9; and

[0541] (c) Fc structural domain.

[0542] 151. The protein according to Example 150, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus:

[0543] (a) Additional CD20 targeting regions;

[0544] (b) A further PD1 agonist moiety, comprising an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:9; and

[0545] (c) The other Fc structural domain.

[0546] 152. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus:

[0547] (a) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2;

[0548] (b) The antigen-binding fragment of the agonist anti-PD1 antibody;

[0549] (c) CD20 targeting region; and

[0550] (d) Fc structural domain.

[0551] 153. The protein according to Example 152, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus:

[0552] (a) An additional PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2;

[0553] (b) Additional antigen-binding fragments of agonist anti-PD1 antibodies;

[0554] (c) Additional CD20-targeting regions; and

[0555] (d) The other Fc structural domain.

[0556] 154. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus:

[0557] (a) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:9;

[0558] (b) The antigen-binding fragment of the agonist anti-PD1 antibody;

[0559] (c) CD20 targeting region; and

[0560] (d) Fc structural domain.

[0561] 155. The protein according to Example 154, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus:

[0562] (a) An additional PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:9;

[0563] (b) Additional antigen-binding fragments of agonist anti-PD1 antibodies;

[0564] (c) Additional CD20-targeting regions; and

[0565] (d) The other Fc structural domain.

[0566] 156. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus:

[0567] (a) The antigen-binding fragment of an agonist anti-PD1 antibody;

[0568] (b) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2;

[0569] (c) CD20 targeting region; and

[0570] (d) Fc structural domain.

[0571] 157. The protein according to Example 156, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus:

[0572] (a) Additional antigen-binding fragments of agonist anti-PD1 antibodies;

[0573] (b) The additional PD1 agonist portion, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2;

[0574] (c) Additional CD20-targeting regions; and

[0575] (d) The other Fc structural domain.

[0576] 158. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus:

[0577] (a) Antigen-binding fragment of agonist anti-PD1 antibody;

[0578] (b) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:9;

[0579] (c) CD20 targeting region; and

[0580] (d) Fc structural domain.

[0581] 159. The protein according to Example 158, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus:

[0582] (a) Additional antigen-binding fragments of agonist anti-PD1 antibodies;

[0583] (b) The additional PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:9;

[0584] (c) Additional CD20-targeting regions; and

[0585] (d) The other Fc structural domain.

[0586] 160. One or more nucleic acids encoding a protein according to any one of Examples 1 to 159.

[0587] 161. A host cell engineered to express the protein according to any one of Examples 1 to 159 or the nucleic acid according to Example 160.

[0588] 162. A method for producing a protein according to any one of Examples 1 to 159, the method comprising culturing a host cell according to Example 161 and recovering the protein expressed therefrom.

[0589] 163. A pharmaceutical composition comprising a protein according to any one of Examples 1 to 159 and an excipient.

[0590] 164. A method of treating a subject suffering from an immune disorder or condition associated with T-cell dysregulation, the method comprising administering to the subject an effective amount of a protein according to any one of Examples 1 to 159 or a pharmaceutical composition according to Example 163.

[0591] 165. The method according to Example 164, wherein the immune disorder or condition is type 1 diabetes mellitus, primary biliary cholangitis (PBC), Goodpasture syndrome, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane nephritis, antitubular basement membrane nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune oophoritis, graft-versus-host disease (GVHD), autoimmune pancreatitis, autoimmune retinopathy, Behçet's disease, Crohn's disease, Dervéd's disease, systemic lupus erythematosus (SLE), Dressler's syndrome, fibrotic alveolitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, etc. Levi syndrome, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), multiple sclerosis, polyneuropathy, organ enlargement, endocrine disorders, monoclonal syndrome (POEMS), polyarteritis nodosa, rheumatoid arthritis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm or testicular autoimmunity, stiff-person syndrome (SPS), aortitis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), or vasculitis.

[0592] 166. The method according to Example 165, wherein the immune disorder or condition is type 1 diabetes.

[0593] 167. The method according to Example 165, wherein the type 1 diabetes is pediatric type 1 diabetes.

[0594] 168. The method according to Example 165, wherein the type 1 diabetes is type 1 diabetes that occurs in an adult.

[0595] 169. The method according to any one of Examples 165 to 168, wherein the subject is a pediatric patient.

[0596] 170. The method according to any one of Examples 165 to 168, wherein the subject is an adult patient.

[0597] 171. The method according to Example 164, wherein the immune disorder or condition is systemic lupus erythematosus.

[0598] 172. The method according to Example 164, wherein the immune disorder or condition is Crohn's disease.

[0599] 173. The method according to Example 164, wherein the immune disorder or condition is graft-versus-host disease (GVHD).

[0600] 174. A method for suppressing cellular autoimmune responses, the method comprising administering to the subject an effective amount of a protein according to any one of Examples 1 to 159 or a pharmaceutical composition according to Example 163.

[0601] 175. The method according to Example 174, wherein the method reduces the T cell function of the subject.

[0602] 176. The method according to Example 174 or 175, wherein the method reduces the B-cell function of the subject.

[0603] 177. The method according to any one of Examples 174 to 176, wherein the method reduces the T-cell reactivity of the subject.

[0604] 178. The method according to any one of Examples 164 to 177, further comprising administering an additional therapeutic agent to the subject.

[0605] 179. The method according to Example 178, wherein the additional therapeutic agent is or includes an immunomodulator, cell inhibitor, cell adhesion inhibitor, cytotoxic agent, apoptosis activator, or agent that increases the sensitivity of cells to apoptosis inducers.

[0606] 180. The method according to Example 178, wherein the additional therapeutic agent is or comprises cells expressing CAR.

[0607] 181. The method according to Example 180, wherein the CAR-expressing cell is a CAR-expressing regulatory T cell.

[0608] 182. A method for targeting PD1 agonist activity, the method comprising administering to the subject an effective amount of a protein according to any one of Examples 1 to 159 or a pharmaceutical composition according to Example 163.

[0609] 183. The method according to Example 182, wherein administration of the protein or pharmaceutical composition targets the PD1 agonist effect to the B cells of the subject.

[0610] 184. A method for locally modulating an immune response in a target tissue or cell expressing CD20, the method comprising administering to the subject an effective amount of a protein according to any one of Examples 1 to 159 or a pharmaceutical composition according to Example 163.

[0611] 185. The method according to Example 184, wherein administration of the protein or pharmaceutical composition modulates the immune response in the subject's B cells.

[0612] 186. The method according to any one of Examples 164 to 185, wherein the protein according to any one of Examples 1 to 159 or the pharmaceutical composition according to Example 163 is administered as a single dose.

[0613] 187. The method according to any one of Examples 164 to 186, wherein the administration of the protein according to any one of Examples 1 to 159 or the pharmaceutical composition according to Example 163 is not repeated.

[0614] 9. Example

[0615] 9.1. Materials and Methods

[0616] 9.1.1. Design and production of CD20-PD1 binding molecules

[0617] Constructs encoding bispecific CD20-PD1 agonists and controls were generated, as shown in Tables 3 and 4 below. The bispecific CD20-PD1 agonists comprised different conformations of mouse anti-CD20 and modified mouse PDL1 extracellular domains, IgG1 effector null (EN) domains (L234A, L235E, G237A, A330S, and P331S, EU numbers), and linkers of varying lengths from different repeat sequences derived from G4S (SEQ ID NO: 33). A 29-amino acid signal sequence from the mouse inactivated tyrosine protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the constructs. All bispecific CD20-PD1 agonists were expressed as preproteinogens containing the signal sequence. The signal sequence was cleaved via intracellular processing to produce mature proteins.

[0618] Mutation of pestle formation: T366W (EU number).

[0619] Mouth formation mutations: T366S, L368A, and Y407V (EU number).

[0620] Astral mutations: H435R and Y436F (EU number).

[0621]

[0622] Transfected via Thermo Fisher Scientific on Expi293F TM Expression constructs in cells. The ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) and HiTrap were used. TMProteins in the Expi 293F supernatant were purified using Protein GHP or a MabSelect SuRe pcc column (Cytiva). After a single-step elution, the antibody was neutralized, dialyzed into a final buffer of 5% glycerol phosphate-buffered saline (PBS), aliquoted, and stored at -80°C. For some constructs, an additional step of size exclusion chromatography using a HiPrep 26 / 60 Sephacryl S-200 column was employed.

[0623]

[0624] 9.1.2. Flow cytometry

[0625] Cells (HEK 293, MC38 overexpressing mCD20, or Jurkat overexpressing mPD1) were loaded at 1×10⁻⁶ cells / year. 6 Cells / mL were resuspended in FACS washing buffer (PBS containing 1% FBS). 1 × 10⁶ cells / mL were used per well. 5 Individual cells were stained. Antibodies were applied at a 1:5 ratio from 1.3 x 10⁻⁶ cells. -07Dilute the starting concentration of M. Then add the diluted antibody to the wells containing cells. Stain the cells at 2–8 °C for 30 min and wash twice with FACS wash buffer. Add APC-conjugated goat anti-human IgG (Jackson Immuno Research, 109-607-003, 1:400) or AF647-conjugated goat anti-mFc Fab (Jackson Immuno Research, 115-607-185, 1:400) and stain the cells at 2–8 °C for 30 min. After washing, fix the cells with 2% paraformaldehyde at 2–8 °C for 30 min. After two washes, use BD LSR Fortessa. TM The FACS instrument was used to analyze the stained cells. The results were analyzed using FlowJo. The FSC / SSC gate was used to select monocytes.

[0626] For flow cytometry analysis of spinal cord T-cell infiltration, a single-cell suspension of the spinal cord was first prepared by digestion with collagenase D (Roche, 11088882001) and gradient separation using Percoll (GE Healthcare, 17-0891-02). Cells were resuspended in FACS washing buffer and stained using the LIVE / DEAD™ fixable blue dead cell staining 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) as described above. Cells were analyzed using the BD FACSymphony cell analyzer. Results were analyzed using OMIQ cell counting software.

[0627] 9.1.3. Luciferase reporter gene assay: Anti-mCD20 x mPDL1 extracellular domain molecule

[0628] Luciferase-based reporter gene assays were used to evaluate the ability of anti-mCD20 x mPDL1 extracellular domain molecules to induce mouse PD1 (mPD1) activation in Jurkat cells in the presence of mouse CD20 (mCD20) in HEK293 cells. The overall design of the reporter gene assays was described in Figures 3A to 3CAP1 is a transcription factor involved in the regulation of gene expression during T cell activation (Samelson 2002, PMID: 11861607). A bispecific antibody (bsAb) binding to human CD3 and CD22, namely CD3 bsAb (REGN10551), is used to stimulate T cell activation via binding to antigens on target cells and receptors on T cells, similar to the previously described CD3 x CD20 bsAb (Smith et al. 2015, PMID: 26659273). mCD20xmPDL1, anchored via mCD20, binds to mPD1 on Jurkat cells, leading to inhibition of PD1 agonist-driven luciferase signaling.

[0629] 9.1.3.1. Engineering of Jurkat / AP1-luc / mPD1 cells

[0630] Jurkat E6-1 cells (ATCC#TIB-152) were generated by transducing them sequentially with AP1 (activator protein 1)-luciferase reporter gene lentivirus (QIAGEN CLS-011L) followed by transduction with lentivirus containing mPD1 ORF (mPD1 NM_008798).

[0631] 9.1.3.2. Engineering of HEK293 / hCD22 / mCD20 cells

[0632] HEK293 / hCD22 / mCD20 cells were generated by transducing with human CD22 ORF-encoded lentivirus (NP_001762.2) followed by mCD20 ORF-encoded lentivirus (NP_031667.1).

[0633] 9.1.3.3. Luciferase assay setup

[0634] For the bioassay, HEK293 / CD22 / mCD20 target cells were seeded at 10,000, 15,000, or 20,000 cells / well in 96-well plate assay medium (RPMI 1640 (Irvine Scientific), supplemented with 10% fetal bovine serum and L-glutamine-penicillin-streptomycin (Invitrogen)) and incubated overnight at 37°C and 5% CO2. The next 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 disclosed molecule or control antibody was then serially diluted 1:3 in assay medium to a final concentration in the range of 100 nM to 1 pM or 1.69 pM (under conditions without the additional testing molecule) and added to the cells along with 1 nM or 2.5 nM anti-CD3bsAb. To achieve a specific range of activation, CD3 bsAb was serially diluted 1:3 to a final concentration ranging from 100 nM to 1.69 pM (without the addition of bispecific mAb) and then added to cells. The cells were then incubated at 37°C / 5% CO2. 2 After incubation for 5 hours, ONE-Glo™ (Promega) reagent was added, and luciferase activity was detected on an Envision multilabel microplate reader (PerkinElmer). All conditions were tested in duplicate.

[0635] The EC50 / IC50 values ​​were determined using nonlinear regression (4-parameter logic) with GraphPad Prism™ software. The percentage of suppression was calculated based on the relative luminescence units (RLU) value using the following formula:

[0636]

[0637] In this formula, "RLU" 基线 "The luminescence value of cells treated with a constant amount of CD3 bsAb without testing the molecules," RLU 抑制 "This is the luminescence value at the highest concentration of the test molecule with a constant amount of CD3 bsAb," RLU 背景 "This is the luminescence value of cells without any CD3 bsAb or test molecules."

[0638] 9.1.4. Determination of the oligomeric state of anti-mCD20 x mPDL1 extracellular domain molecules by size exclusion chromatography.

[0639] Size heterogeneity of the anti-mCD20 x mPDL1 extracellular domain molecules was assessed using size exclusion ultra-high performance liquid chromatography (SE-UPLC). SE-UPLC analyses were performed on a Waters Acquity UPLC H-Class system, where 10 μg of each protein sample was injected onto an Acquity BEH SEC column (200 Å, 1.7 μm, 4.6 x 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.

[0640] 9.1.5. Thermal stability

[0641] The thermostability of the anti-mCD20 x mPDL1 extracellular domain molecule was assessed using differential scanning fluorometry (DSF). DSF analysis was performed on a ThermoFisher QuantStudio 5 system. Stock solutions of each sample were diluted to 0.2 mg / mL in 1X PBS-glycerol pH 7.4 and transferred to 96-well plates. Excess (8X) of Sypro Orange™ fluorescent dye (which preferentially binds to buried hydrophobic residues when the protein unfolds) was added to each well, and thermostability profiles were determined on a linear thermal ramp from 25 °C to 95 °C over 20 minutes.

[0642] 9.1.6. Assembly Percentage

[0643] The assembly of bifunctional fusion molecules was determined using high-throughput analysis on the Cliper LabChip GX according to the manufacturer's protocol (Perkin Elmer, Waltham, MA). Briefly, the sample buffer was prepared by mixing 7 ml of HT protein expression sample buffer with 240 µl of BME (reducing) or 25 mM iodoacetamide (IAM, for non-reducing assays). Samples were normalized to 0.5 mg / ml with the sample buffer and then heated at 70 °C for 10 min. 70 µl of water was added to each sample before loading onto the instrument. The chip was prepared according to the manufacturer's instructions. Electrophoresis patterns of the samples were analyzed using LabChip GX software. Peaks from the non-reducing electrophoresis pattern represent intact antibody percentages.

[0644] 9.1.7. Activity of anti-mCD20 x mPDL1 extracellular domain molecules in prediabetic NOD mice

[0645] During the experimental period (e.g., until mice reached 28 weeks of age), 10-week-old prediabetic non-obese diabetic (NOD) mice (The Jackson Laboratory) received intraperitoneal treatment twice weekly with a selected anti-mCD20 x mPDL1 extracellular domain molecule at doses of 1 mg / kg, 0.1 mg / kg, or 0.01 mg / kg. Blood glucose levels were monitored every two weeks, and body weight was monitored weekly. The overall experimental design is described in Figure 5 middle.

[0646] 9.1.8. Activity of anti-mCD20 x mPDL1 extracellular domain molecules in an experimental autoimmune encephalomyelitis / multiple sclerosis mouse model

[0647] Administering myelin oligodendrocyte glycoprotein (MOG) to mice 35-55 The immunodominant 35-55 epitope of MOG produces anti-MOG antibodies, leading to demyelination and chronic experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis (MS).

[0648] 200 mg of MOG dissolved in CFA was delivered subcutaneously on day 1. 35-55 EAE was induced in wild-type C57BL / 6 mice (10-12 weeks old, male, The Jackson Laboratory). Given that pertussis toxin administration promotes T cell migration to the central nervous system by weakening the blood-brain barrier, mice were also given intraperitoneal injections of 200 ng of pertussis toxin on days 1 and 2. Body weight and EAE progression 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: weak tail droop; 2: gait abnormalities and / or righting reflex defects; 3: partial hind limb paralysis; 4: complete hind limb paralysis; and 5: complete hind limb paralysis with partial forelimb paralysis, or a dying state.

[0649] Starting on day 2, mice were intraperitoneally injected twice weekly with a selected anti-mCD20 x mPDL1 extracellular domain molecule or an appropriate control molecule. Endpoint tissue harvesting was performed at the disease peak on day 20. Spinal cord infiltrates were used for flow cytometry, and MOG-specific T cell responses in the spleen were assessed using ELISPOT.

[0650] 9.2. Example 1: Production and stability of a bispecific anti-mCD20-mPDL1 extracellular domain agonist

[0651] 9.2.1. Overview

[0652] Mammalian expression vectors for individual heavy and light chains were created via DNA synthesis and cloned into a ready-to-use construct from Life Technologies' (Carlsbad, CA) pcDNA3.4 Topo expression system. For the expression molecule, heavy and universal light chain DNA were co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was harvested and purified using a HiTrap Protein A FF or MabSelect SuRe column (GE Healthcare). For functional validation, the selected MBM was scaled up to 2 L and subjected to a series of purification procedures, including size exclusion chromatography as a final step.

[0653] 9.2.2. Results

[0654] Various anti-mCD20 x mPDL1 extracellular domain molecules were expressed and purified from Expi293 Freestyle cells via a one-step Mab-Select SuRe column (Table 5), with total yields ranging from 2.7 mg to 7.7 mg. Generally, molecules with a valence ratio of 1:1 (anti-mCD20:mPDL1 extracellular domain) showed higher yields (4.1 mg to 7.7 mg) than molecules with a valence ratio of 2:1 or 2:2 (Table 5).

[0655]

[0656] Following one-step affinity purification, the high molecular weight (HMW) % and monomer % were examined by SE-UPLC, while thermal stability was monitored by differential scanning fluorometry (DSF) (Table 6). Most anti-mCD20 x mPDL1 extracellular domain fusion molecules showed more than 85% monomer species without the need for additional size exclusion chromatography (SEC). For 2+2 m20_mPL_4(L), the monomer percentage increased to 99% after two-step column purification (including the SEC step) (Table 6). All anti-mCD20 x mPDL1 extracellular domain fusions exhibited similar thermal stability (measured by DSF), with a Tm1 of approximately 60 °C. Furthermore, all bifunctional fusions showed excellent assembly between heavy chains as determined by capillary electrophoresis SDS (CE-SDS) (Table 6).

[0657]

[0658] 9.3. Example 2: Binding characterization of anti-mCD20 x mPDL1 extracellular domain molecules

[0659] The ability of anti-mCD20 x mPDL1 extracellular domain molecules to bind to two targets on the cell surface was evaluated in a flow cytometry binding assay.

[0660] 9.3.1. Results

[0661] Combined with curves shown Figure 2A and Figure 2B In the study, for the combination of mPD1 and mCD20, the divalent molecule was observed to have higher potency and maximum MFI compared to the monovalent molecule of a similar configuration. Specifically, although 2+2 m20_mPL_4 (L) and 2+1 m20_mPL_3 (G) ( Figure 2A and Figure 2B (Table 7) Similar binding was shared for HEK293 / mCD20 cells, but due to the increased valence of mPD1 binding, 2+2 m20_mPL_4 (L) bound more strongly to Jurkat / mPD1 cells than 2+1 m20_mPL_3 (G). In both divalent and monovalent molecules, the binding signal appears to be orientation-dependent, with the N-terminal anti-mCD20 and hPDL1 extracellular domains generally exhibiting higher potency and maximum MFI relative to the Fc domain. When the anti-mCD20 or mPDL1 extracellular domain is located between the N-terminal portion and the pre-Fc hinge region, its binding is shown to be reduced (…). Figure 2A and Figure 2B (Table 7).

[0662] 9.4. Example 3: MPDL1 agonism by inhibiting the extracellular domain molecules of mCD20 x mPDL1

[0663] The agonistic effect of anti-mCD20 x mPDL1 extracellular domain molecules on mPD1 was investigated using the depiction in Figure 3 and the bioassays described in Section 9.1.3.

[0664] 9.4.1. Results

[0665] The results of the luciferase assay are depicted in Figure 4 and Table 7. The PD1 agonistic effect and T cell signaling regulation of the disclosed anti-mCD20 x mPDL1 extracellular domain molecules were tested using HEK293 / CD22 / mCD20 and Jurkat / AP1-luc / mPD1 reporter cells and 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 pM to 770 pM and 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 agonistic effect, with maximum inhibition rates of 74% to 84% (Figures 4C and 4E). The remaining molecules showed weak or no inhibition, with maximum inhibition ranging from -10% to 40%. The allotype control antibody did not show inhibition of signaling. CD3 bsAb showed activation of T cell signaling, with EC50 values ​​of 627 pM and 1.15 nM.

[0666] 9.4.2. Summary of data from in vitro assays of anti-mCD20 x mPDL1 extracellular domain molecules

[0667] Table 7 provides a summary of in vitro data collected using various anti-mCD20 x mPDL1 extracellular domain molecules, including cell-based flow cytometry binding and in vitro bioassay results. Results from the luciferase assay are plotted on... Figure 4A As shown in Figure 4E, molecules 2+2m20_mPL_4 and 2+1m20_mPL_3 (G and L, respectively, in Table 6) exhibited the strongest PD1 agonistic activity. Cell-based flow cytometry analysis revealed that 2+2m20_mPL_4 exhibited the strongest binding to both mPD1 and mCD20, while 2+1m20_mPL_3 showed only moderate binding to mPD1-expressing cells. This suggests that in the presence of both APCs and effector cells, mPD1 aggregation requires bivalent binding to mCD20. Overall, similar cell-binding affinities (mPD1 or mCD20) did not translate into similar PD1 agonistic activity, such as F with G and K with L (…). Figure 4A (as shown in Table 7), indicating that the valence state and structural arrangement of the extracellular domain arms of CD20 and mPDL1 are important for conferring activity.

[0668]

[0669] 9.5. Example 5: In vivo efficacy of anti-mCD20 x mPDL1 extracellular domain molecules

[0670] The ability of selected 2+2 and 2+1 anti-mCD20 x mPDL1 extracellular domain molecules to prevent the onset of type 1 diabetes (T1D) was evaluated in prediabetic NOD mice. The experimental design was described in Figure 5 This is described in Section 9.1.7.

[0671] 9.5.1. Results

[0672] Individual animal data shows that Figures 6A to 6I middle. Figure 7A and Figure 7B The percentage of diabetic-free mice at each specified time point was depicted. Typically, 80% to 90% of NOD mice develop diabetes around 25 weeks of age. However, in this experiment, only about 30% of the mice developed diabetes at 27 weeks. While the incidence of diabetes was lower in control NOD mice, higher doses of (2+2) anti-mCD20 x mPDL1 extracellular domain molecule 2+2 m20_mPL_4 ( Figure 2A The molecule L in the middle showed a clear protective trend, but the (2+1) anti-mCD20 x mPDL1 extracellular domain molecule 2+1 m20_mPL_3 ( Figure 2A The molecule G in the middle does not show a protective tendency. Figure 7A and Figure 7B ).

[0673] 9.6. Example 6: Decreased autoimmune T cell infiltration induced by anti-mCD20 x mPDL1 extracellular domain molecules

[0674] 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 effect of (2+2) anti-mCD20 x mPDL1 extracellular domain molecule 2+2 m20_mPL_4 against T-cell infiltration was assessed by flow cytometry as described in Section 9.1.2.

[0675] 9.6.1. Results

[0676] In one evaluation, the proliferation (activation) and hypoactivated islet-specific CD8+ T cell populations of NOD mice described in Section 9.1.7 were analyzed after administration of 0.1 mg / kg or 1 mg / kg 2+2 m20_mPL_4 or a control molecule. Treatment with 1 mg / kg 2+2 m20_mPL_4 was associated with a significant increase in the percentage of hypoactivated CD8+ T cell clusters. Figure 8A Although the percentage of proliferating cell clusters did not differ under different conditions (). Figure 8B However, in pancreatic tissue isolated from NOD mice treated with 1 mg / kg of 2+2 m20_mPL_4, the ratio of low-activation cell clusters to proliferating cell clusters was high. Figure 8C This indicates that the treatment can reduce pancreatic infiltration of activated autoimmune T cells.

[0677] In another evaluation, T-cell spinal cord infiltration was assessed in a mouse model of multiple sclerosis described in Section 9.1.8. Significantly fewer CD3+ cells were found in the spinal cord of mice treated with 1 mg / kg of 2+2 m20_mPL_4 compared to the spinal cord of mice treated with the same dose as the control group. Figure 9A ), CD4+ Figure 9B ) and CD8+ Figure 9C T cells. Therefore, in a multiple sclerosis model, treatment with 2+2 m20_mPL_4 reduced T cell infiltration into the spinal cord.

[0678] 9.7. Example 7: Design and production of a bispecific anti-mCD20xh / mPDL1 IgV agonist

[0679] CD20-PD1 agonist constructs corresponding to some of the constructs described in Examples 1 to 6 were designed, replacing the full-length PDL1 extracellular domain with a PDL1 IgV domain. These constructs were designed to include (1) anti-CD20 (mouse or human) and PDL1 IgV domains (mouse or human) in different conformations, (2) an IgG1 domain, and (3) ferritin, CRP, and D-dimer data for linkers of different lengths from different repeat sequences of G4S (SEQ ID NO: 33) (Table 8; Figures 10A to 10C; 11A-11E). A 29-amino acid signal sequence from the mouse inactivated tyrosine protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the constructs. All bispecific CD20-PD1 agonists were expressed as a preproteinogen containing the signal sequence. The signal sequence was cleaved by intracellular processing to produce the mature protein.

[0680] Mammalian expression vectors for individual heavy and light chains were created via DNA synthesis and cloned into a ready-to-use construct from Life Technologies' (Carlsbad, CA) pcDNA3.4 Topo expression system. For the expression molecule, heavy and universal light chain DNA were co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was harvested and purified using a HiTrap Protein A FF or MabSelect SuRe column (GE Healthcare). For functional validation, the selected MBM was scaled up to 2 L and subjected to a series of purification procedures, including size exclusion chromatography as a final step.

[0681]

[0682] 9.8. Example 8: mPDL1 IgV binding binds to mPD1 with higher efficacy than the full-length extracellular domain molecule of mPDL1.

[0683] Seven binding molecules and control molecules were generated, as described in Section 9.1.1. To evaluate whether the binding properties of the full-length mPDL1 extracellular domain and the mPDL1 IgV construct differed, flow cytometry binding assays were performed using Jurkat cells overexpressing mPD1, as described in Section 9.1.2.

[0684] 9.8.1. Results

[0685] The constructs generated and evaluated in this example are listed in Table 9.

[0686]

[0687] The molecule AF_MH, containing the full-length extracellular domain of hPDL1, and the molecule AF-12, containing the full-length extracellular domain of mPDL1, showed similar binding to Jurkat cells overexpressing mPD1. Figure 12A Conversely, for Jurkat cells overexpressing mPD1, the molecule AF-12.1, containing mPDL1 IgV, exhibited a higher maximum MFI than the full-length mPDL1 extracellular domain molecule AF-12, suggesting that AF-12.1 has higher efficacy than AF-12. Figure 12A ).

[0688] The mPDL1 IgV bivalent molecule AF-17.1 did not show binding, while the full-length mPDL1 extracellular domain bivalent molecule AF-17 showed minimal binding. Figure 12A This indicates a need for the CD20 targeting component. None of the constructs showed any binding with parental Jurkat cells. Figure 12C ).

[0689] Next, mCD20 arm binding was evaluated using MC38 cells overexpressing mCD20. AF-12.1, with an mCD20 arm located at the N-terminus of each mPDL1 IgV, showed better efficacy than the full-length mCD20 x mPDL1 extracellular domain molecule AF-12; however, it had a similar EC50 ( Figure 12B Similarly, none of the constructs showed any binding with parental MC38 cells. Figure 12D ).

[0690] 9.9. Example 9: mPDL1 IgV and full-length extracellular domain molecules exhibit similar agonistic effects.

[0691] To evaluate the PD1 agonistic effect of mPDL1 IgV and full-length extracellular domain-containing molecules and their regulation of T cell signaling, as described in Section 9.1.3, luciferase reporter assays were performed using HEK293 / hCD22 / mCD20 and Jurkat / AP1-luc / mPD1 reporter cells with anti-CD3bsAb.

[0692] 9.9.1. Results

[0693] The PD1 agonistic effects of the seven constructs listed in Table 9 were evaluated. Three of the seven constructs, AF-12, AF-12.1, and AF_MH1, showed inhibitory effects on T cell signaling, with IC50 being the most effective. 50 The values ​​range from 182 pM to 270 pM, and the inhibition percentage ranges from 85% to 86%. Figure 13 Therefore, these three constructs were associated with relatively strong PD1 agonist activity. 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 activated T cell signaling, in which EC10... 50 The value is 696pM ( Figure 13 ).

Claims

1. A protein comprising: (a) CD20 targeting region; (b) The PD1 agonist portion, which comprises: (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 (c) Dimerization portion.

2. The protein of claim 1, wherein the PD1 agonist comprises an amino acid sequence, said amino acid sequence: (a) Has 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 SEQ ID NO:2; or (b) Has 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 SEQ ID NO:

14.

3. The protein according to claim 1 or claim 2, wherein the PD1 agonist portion comprises or is composed 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 protein according to any one of claims 1 to 4, wherein the protein does not contain an amino acid sequence, wherein the amino acid sequence is: (a) It has at least approximately 95% sequence identity with SEQ ID NO: 6; (b) It has at least approximately 95% sequence identity with SEQ ID NO: 13; (c) Has at least approximately 95% sequence identity with SEQ ID NO:5; or (d) It has 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 the 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 any amino acid sequence corresponding to any subsequence of at least 20, at least 10, or at least 5 amino acids in length corresponding to SEQ ID NO:5 or SEQ ID NO:

17.

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

9. The protein according to any one of claims 1 to 8, wherein the length of the PD1 agonist moiety is 120 or fewer amino acids.

10. The protein according to any one of claims 1 to 9, wherein the portions of said protein are arranged in the following order from the N-terminus to the C-terminus: (a) CD20 targeting moiety – PD1 agonist moiety – dimerizing moiety; or (b) PD1 agonist moiety – CD20 targeting moiety – dimerization moiety.

11. The protein according to any one of claims 1 to 9, 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.

12. The protein according to any one of claims 1 to 9, wherein the dimerization portion is an Fc domain and the PD1 agonist portion is not located 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, optionally wherein the antigen-binding fragment of the agonist anti-PD1 antibody is in the form of Fab, Fv, or scFv.

14. The protein of claim 13, wherein the portions of the protein are arranged from the N-terminus to the C-terminus in the following order: PD1 agonist portion – antigen-binding fragment of the agonist anti-PD1 antibody – CD20 targeting portion – dimerizing portion.

15. The protein of claim 13, wherein the portions of the protein are 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 protein according to any one of claims 13 to 15, wherein the antigen-binding fragment of the agonist anti-PD1 antibody is: (a) is an antigen-binding fragment of an agonist PD1 antibody selected from the following: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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; (b) A heavy chain and / or light chain CDR comprising an agonist PD1 antibody selected from: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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; (c) VH and VL containing PD1 agonist antibodies selected from the following: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10, and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6, described in 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; or (d) Competing with PD1 agonist antibodies selected from the following to bind to PD1 and / or bind to the same epitope: rosnilimab (ANB030); LY3462817; CC-90006; PT627; PT001; clones C8-1 and G10-2, described in WO In WO / 2016 / 020856; clones 2, 10 and 19, described in WO / 2013 / 022091; humanized antibody 949, described in WO / 2011 / 110621; PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 and PD1AB-6, described in 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.

17. The protein according to any one of claims 13 to 16, wherein the antigen-binding fragment of the agonist anti-PD1 antibody activates 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, optionally wherein the antigen-binding fragment of the anti-CD20 antibody is in the form of Fab, Fv or scFv.

20. The protein of claim 19, wherein the anti-CD20 antibody: (a) Selected from rituximab, ozoglucilimab, oxatuzumab, oxfamumab, tiimozumab, tosimomumab, utuximab, oxcartuzumab, TRU-015, and vertuzumab; or (b) Compete with anti-CD20 antibodies selected from the group consisting of: rituximab, ozoglucomancil, ozoglucomancil, ozoglucomancil, ozoglucomancil, ozoglucomancil, tosimumab, tosimocomancil, ozoglucomancil, ozoglucomancil, TRU-015 and ozoglucomancil.

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

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

23. The protein according to any one of claims 21 or 22, wherein each linker portion (a) has a length of at least 5 or at least 10 amino acids, (b) has a length of at most 20, at most 25 or at most 30 amino acids and / or (c) has a length of 5 to 15 amino acids or 5 to 20 amino acids.

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 or fewer amino acids, 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 of claim 25, wherein (a) the additional CD20 targeting portion and the additional PD1 agonist portion are separated by a linker portion; and (b) the additional PD1 agonist portion and the additional dimerized portion are separated by a linker portion.

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

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

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

30. The protein of claim 29, having the conformation depicted in FIG1A.

31. The protein according to any one of claims 1 to 24, comprising two monomers according to exemplary monomer 2, wherein exemplary monomer 2 comprises, in an N-terminal to C-terminal orientation, the PD1 agonist moiety (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:9), optional linker moiety, antigen-binding fragment of agonist antiPD1 antibody (e.g., Fab, Fv or scFv), optional linker moiety, the CD20 targeting moiety (e.g., antiCD20 Fab, Fv or scFV), optional linker moiety and the dimerizing moiety or consisting thereof.

32. The protein of claim 31, having the conformation depicted in FIG1B.

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

34. The protein of claim 33, having the conformation depicted in FIG1C.

35. The protein according to any one of claims 1 to 24, comprising two monomers according to exemplary monomer 4, wherein exemplary monomer 4 comprises, in an N-terminal to C-terminal orientation, an antigen-binding fragment of an agonist antiPD1 antibody (e.g., Fab, Fv, or scFv), an optional linker portion, the PD1 agonist portion (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:9), an optional linker portion, the CD20 targeting portion (e.g., antiCD20 Fab, Fv, or scFV), an optional linker portion, and the dimerizing portion or consisting thereof.

36. The protein of claim 35, having the conformation depicted in FIG1D.

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

38. The protein of claim 37, having the conformation depicted in FIG1E.

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

40. A protein comprising: (a) A device for combining CD20; (b) A PD1 agonist moiety of 120 or fewer amino acids, 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 (c) Dimerization portion.

41. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus: (a) CD20 targeting region; (b) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; and (c) Fc structural domain.

42. The protein of claim 41, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus: (a) Additional CD20 targeting regions; (b) A further PD1 agonist moiety, comprising an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; and (c) The other Fc structural domain.

43. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus: (a) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; (b) The antigen-binding fragment of the agonist anti-PD1 antibody; (c) CD20 targeting region; and (d) Fc structural domain.

44. The protein of claim 43, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus: (a) An additional PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; (b) Additional antigen-binding fragments of agonist anti-PD1 antibodies; (c) Additional CD20-targeting regions; and (d) The other Fc structural domain.

45. A protein comprising a polypeptide chain, said polypeptide chain comprising, from the N-terminus to the C-terminus: (a) The antigen-binding fragment of an agonist anti-PD1 antibody; (b) The PD1 agonist moiety, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; (c) CD20 targeting region; and (d) Fc structural domain.

46. ​​The protein of claim 45, wherein the protein further comprises an additional polypeptide chain, the additional polypeptide chain comprising, from the N-terminus to the C-terminus: (a) Additional antigen-binding fragments of agonist anti-PD1 antibodies; (b) The additional PD1 agonist portion, which consists of an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2; (c) Additional CD20-targeting regions; and (d) The other Fc structural domain.

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

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

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

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

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

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

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

54. The method of claim 53, wherein the method: (a) Reduce the T cell function of the subjects; (b) Reduce the B-cell function of the subject; (c) Reduce the T-cell responsiveness of the subject; or Any combination of (d)(a) to (c).

55. A method of local PD1 agonism, the method comprising administering to the subject an effective amount of the protein according to any one of claims 1 to 46 or the pharmaceutical composition according to claim 50.

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