Multiple specific binding agents and their use

Abstract

The present disclosure provides multispecific binding agents (e.g., antibodies, such as bispecific antibodies) having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47, such as PD-L1, and uses thereof. The present disclosure also provides compositions comprising the multispecific binding agents described herein. The present disclosure also provides methods of treating, preventing, or alleviating an immune cell dysfunction disease, disorder, or condition (e.g., a phagocyte dysfunction disease, disorder, or condition, or a T cell dysfunction disease, disorder, or condition), including one or more symptoms of the immune cell dysfunction disease, disorder, or condition, using such compositions.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 061,112 filed 4 August 2020, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence List This application incorporates, by reference, a sequence listing file submitted with this application as a text file titled 14529-008-228_SEQ_LISTING.txt, created on July 30, 2021, with a size of 172,431 bytes.

[0003] field This disclosure generally relates to multispecific binding agonists, such as bispecific antibodies, having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47, such as PD-L1, and methods of using them. [Background technology]

[0004] background Tumor cells have been shown to utilize both innate and adaptive checkpoints to evade anti-tumor immune responses. CD47 and PD-L1 are two targets widely expressed on the surface of tumor cells that may synergistically suppress innate and adaptive sensing, respectively, to evade immune regulation.

[0005] background CD47 is a cell surface glycoprotein that functions as a regulator of phagocytosis mediated by cells of the innate immune system. CD47 interacts with numerous ligands, including integrins, signal regulatory protein alpha (SIRPα), signal regulatory protein gamma (SIRPγ), and thrombospondin. CD47 inhibits phagocytosis by interacting with SIRPα on the surface of macrophages and dendritic cells, thereby inducing a "don't eat me" signal.

[0006] CD47 expression allows tumor cells to evade phagocytosis and escape innate immune surveillance. Therefore, CD47 is a potential target for therapeutic agents. However, CD47 is widely expressed in normal cells such as hematopoietic cells, red blood cells (RBCs), and platelets. This widespread expression of CD47 in healthy cells raises safety and efficacy concerns, as targeting CD47 with neutralizing antibodies could affect healthy cells, potentially leading to toxicity. Furthermore, widespread CD47 expression could lead to the rapid elimination of CD47-binding agonists, potentially resulting in poor pharmacokinetics and reduced efficacy.

[0007] Furthermore, activation or loss of CD47 may lead to enhanced proliferation in a cell type-dependent manner. For example, increased proliferation of astrocytoma cells has been shown after activation of CD47 and TSP-1, whereas no such increase has been observed in normal astrocytes. It has also been proposed that CD47 may promote cancer cell proliferation through the PI3K / Akt pathway.

[0008] Many reported anti-CD47 antibodies are known to cause RBC aggregation when the binding of CD47 to SIRPα is blocked, which significantly reduces the therapeutic effect of such antibodies.

[0009] Programmed death ligand 1 (PD-L1) is a cell surface glycoprotein ligand that specifically binds to programmed death receptor 1 (PD-1), an important immune checkpoint receptor. PD-1 is upregulated on activated T cells, B cells, and monocytes, mediating immunosuppression. While PD-L2, the other PD-1 ligand, is primarily expressed on activated antigen-presenting cells (APCs), PD-L1 is widely expressed in hematopoietic cells such as activated T cells, B cells, monocytes, dendritic cells, and macrophages, as well as in peripheral tissues including the heart, skeletal muscle, placenta, lungs, kidneys, and liver.

[0010] The binding of PD-L1 to PD-1 is a negative checkpoint that can activate downstream signaling of the PD-1 receptor in T cells, thereby inhibiting T cell proliferation, cytokine production and release, and cytotoxicity. This inhibition of T cell activation and effector cytokine secretion can prevent autoimmune and chronic infections.

[0011] However, many tumor cells use this mechanism to protect themselves from immune attack, resulting in tumor immune evasion. Many cancers overexpress PD-L1, and this overexpression is often associated with a poor prognosis. In cancer, the PD-1 / PD-L1 interaction stimulates downstream signaling, suppressing T cell activation and leading to tumor cell survival.

[0012] Blocking the PD-1 interaction with its ligand has been proposed as an immunotherapy method to enhance the T-cell immune response against tumor cells. While current strategies for PD-1 / PD-L1-based immunotherapy have shown efficacy in treating some advanced cancers, their effects on many solid tumors and certain PD-L1 functions are limited. Therefore, there remains an urgent need in the art for agonists that can block or prevent PD-1 / PD-L1 interactions. [Overview of the Initiative] [Means for solving the problem]

[0013] Therefore, in order to treat, prevent or alleviate immunodeficiency diseases, disorders, or conditions, including those involving tumor cells expressing CD47 and / or PD-L1, there remains a need for agonists that overcome these concerns and target CD47 and other targets, such as PD-L1. The multispecific binding agonists, compositions, and methods provided herein satisfy this need and offer relevant advantages.

[0014] Abstract This disclosure provides a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47 (e.g., PD-L1). Such agonists include multispecific antibodies (e.g., antibodies such as bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1), for example, a multispecific antibody having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47 (e.g., PD-L1). In some embodiments, such activators include multispecific antibodies (e.g., bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1), for example, antibodies having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47 (e.g., PD-L1), and having heavy chain variable regions and light chain variable regions as described herein (e.g., Tables 1-3), which compete for binding to human CD47.

[0015] This disclosure also provides compositions comprising the multispecific binding agents described herein. In some embodiments, such compositions include multispecific antibodies (e.g., bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1), for example, a multispecific antibody having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47 (e.g., PD-L1). In some embodiments, such compositions include multispecific antibodies that compete for binding to human CD47 with antibodies having a heavy chain variable region and a light chain variable region as described herein (e.g., Tables 1-3).

[0016] This disclosure also provides methods for treating, preventing or mitigating one or more symptoms of an immune cell dysfunction disorder, disorder, or condition (e.g., phagocyte dysfunction disorder, disorder, or condition, or T-cell dysfunction disorder, disorder, or condition) using a multispecific binding agonist or a composition comprising a bispecific antibody or a composition comprising a bispecific antibody as described herein. Such compositions include multispecific antibodies (e.g., antibodies such as bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1), for example, a multispecific antibody having a first binding domain that binds to CD47, including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47 (e.g., PD-L1). In some embodiments, such compositions include multispecific antibodies (e.g., bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1), for example, antibodies having a first binding domain that binds to CD47 including human CD47, and one or more additional binding domains that bind to one or more targets other than CD47 (e.g., PD-L1), and having heavy chain variable regions and light chain variable regions as described herein (e.g., Tables 1-3), which compete for binding to human CD47. [Brief explanation of the drawing]

[0017] [Figure 1-1] Figures 1A–1D show exemplary results from the cell binding assay, which are further described in Example 3. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above.

[0018] [Figure 2-1] Figures 2A–2E show exemplary results from additional cell binding assays, which are further described in Example 3. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above.

[0019] [Figure 3] Figure 3 shows exemplary results from the CD47 / SIRPα inhibition assay, which is further described in Example 4.

[0020] [Figure 4] Figure 4 shows exemplary results from the PD-L1 / PD-1 inhibition assay, which is further described in Example 4.

[0021] [Figure 5-1] Figures 5A–5G show exemplary results from the phagocytosis assay, which are further described in Example 5. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 5-6] Same as above. [Figure 5-7] Same as above.

[0022] [Figure 6-1] Figures 6A–6G show exemplary results from additional phagocytosis assays, which are further described in Example 5. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above. [Figure 6-6] Same as above. [Figure 6-7] Same as above.

[0023] [Figure 7]Figure 7 shows exemplary results from in vivo animal studies regarding tumor volume, which are further described in Example 7.

[0024] [Figure 8] Figure 8 shows exemplary results from in vivo animal studies on body weight, which are further described in Example 7.

[0025] [Figure 9-1] Figures 9A–9E show exemplary results from in vivo animal studies on blood parameters, which are further described in Example 7. [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0026] [Figure 10-1] Figures 10A–10G show exemplary results from in vivo animal studies on additional blood parameters, which are further described in Example 7. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above.

[0027] [Figure 11] Figure 11 shows exemplary results from in vivo animal studies on antibody concentrations in the blood, which are further described in Example 7.

[0028] [Figure 12-1] Figures 12A–12E show exemplary results from additional in vivo animal studies regarding tumor volume and body weight, which are further described in Example 7. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above.

[0029] [Figure 13-1]Figures 13A–13D show exemplary results from additional in vivo animal studies on tumor-infiltrating leukocytes, which are further described in Example 7. [Figure 13-2] Same as above.

[0030] [Figure 14] Figure 14 shows exemplary results from additional in vivo animal studies on antibody concentrations in the blood, which are further described in Example 7.

[0031] [Figure 15-1] Figures 15A–15H show exemplary results from additional in vivo animal studies regarding tumor volume and body weight, which are further described in Example 7. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. [Figure 15-5] Same as above. [Figure 15-6] Same as above. [Figure 15-7] Same as above. [Figure 15-8] Same as above.

[0032] [Figure 16-1] Figures 16A–16B show exemplary results from additional in vivo animal studies regarding tumor volume and body weight, which are further described in Example 7. [Figure 16-2] Same as above.

[0033] [Figure 17] Figure 17 shows exemplary results from additional in vivo animal studies on macrophage infiltration into tumors, which are further described in Example 7.

[0034] [Figure 18-1] Figures 18A–18B show exemplary results from additional in vivo animal studies regarding tumor volume and body weight, which are further described in Example 7. [Figure 18-2] Same as above.

[0035] [Figure 19-1] Figures 19A–19C show exemplary results from SEC chromatography, which are further described in Example 8. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0036] [Figure 20-1] Figures 20A–20D show exemplary results from HIC chromatography, which are further described in Example 8. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above.

[0037] [Figure 21-1] Figures 21A–21D show exemplary results from SMAC chromatography, which are further described in Example 8. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 21-4] Same as above.

[0038] [Figure 22-1] Figures 22A–22B show the sequence alignments of the heavy-chain and light-chain variable regions of C40, C56, and C59, including the consensus sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The CDR boundaries are indicated by Kabat, AbM, Chothia, Contact, IMGT, and AHon numbering. [Figure 22-2] Same as above.

[0039] [Figure 23-1]Figures 23A–23B show the sequence alignments of the heavy-chain and light-chain variable regions of P22, P24, and P31.2, including the consensus sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The CDR boundaries are indicated by Kabat, AbM, Chothia, Contact, and IMGT numbering. [Figure 23-2] Same as above. [Modes for carrying out the invention]

[0040] Detailed explanation This disclosure provides multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1). Such multispecific binding agonists include CD47-binding antibodies (e.g., antibodies, e.g., bispecific antibodies) including antibodies that bind to human CD47 and one or more additional targets other than CD47 (e.g., PD-L1). Such multispecific binding agonists are useful in compositions and methods for treating, preventing or mitigating immune cell dysfunction disorders, disorders or conditions (e.g., phagocyte dysfunction disorders, disorders or conditions or T cell dysfunction disorders, disorders or conditions) including one or more symptoms of the disease, disorder or condition. Phagocyte dysfunction disorders and conditions include, but are not limited to, any cancers in which tumor cells express or overexpress CD47, as well as tumor immunology and related cancers. T cell dysfunction disorders and conditions include, but are not limited to, any cancers in which tumor cells express or overexpress PD-L1, as well as tumor immunology and related cancers. Such CD47 and / or PD-L1 expressing tumor cells may help tumor cells evade immune surveillance and clearance (e.g., tumor immunity). Furthermore, multispecific binding agonists described herein, e.g., multispecific antibodies (e.g., bispecific antibodies) that bind to CD47 and one or more additional targets other than CD47 (e.g., PD-L1), are useful for inhibiting SIRPα signaling and / or PD-1 signaling, for enhancing phagocytic function and / or immune surveillance, and for enhancing the removal of tumor cells. Multispecific binding agonists (e.g., antibodies, e.g. bispecific antibodies), e.g., multispecific binding antibodies (e.g. bispecific antibodies) described herein are useful in compositions and methods for enhancing phagocytic function and T cell function, including upmodulation of cell-mediated immune responses.

[0041] The terms “CD47,” “Surface Antigen Classification 47,” or “CD47 polypeptide,” and similar terms, unless otherwise specified, refer to polypeptides (“polypeptide” and “protein” are used interchangeably herein) or any native CD47 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). CD47 is also known in the art as an integrin-associated protein (IAP) and has an extracellular N-terminal IgV domain, a five-transmembrane domain, and a short C-terminal intracellular tail. The term CD47 encompasses any form of CD47 or any fragment thereof resulting from cellular processing, including “full-length” unprocessed CD47 and isoforms of four known CD47 that have undergone alternative splicing with different intracellular tail lengths. The term CD47 also encompasses naturally occurring variants of CD47, such as SNP variants, splice variants, and allelic variants. CD47 is known in the art to interact with SIRPα, and this interaction leads to cellular signaling, including, in particular, the inhibition of macrophage-mediated phagocytosis. The full amino acid sequence of human CD47 is shown below (exemplary extracellular domain = underlined text): [ka]

[0042] Other related CD47 polypeptides, also encompassed by the term CD47, include fragments, derivatives (e.g., substitution, deletion, shortening, and insertion variants), fusion polypeptides, and interspecific homologs sufficient to retain CD47 activity and / or produce an anti-CD47 immune response. As those skilled in the art will understand, the multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) described herein may bind to CD47 polypeptides, CD47 polypeptide fragments, CD47 antigens, and / or CD47 epitopes. Epitopes may, in turn, be part of a larger CD47 antigen, which may be part of a larger CD47 polypeptide fragment, which may be part of a larger CD47 polypeptide. CD47 may exist in native or denatured forms. The CD47 polypeptides described herein may be isolated from various sources, e.g., human tissue types or other sources, or prepared by recombinant or synthetic methods. CD47 polypeptides may include polypeptides having the same amino acid sequence as the corresponding CD47 polypeptides of natural origin. Orthologues to CD47 polypeptides are also well known in the art.

[0043] The terms “SIRPα,” “Signal Regulatory Protein Alpha,” or “Signal Regulatory Protein α,” and similar terms, unless otherwise specified, refer to any native SIRPα derived from any vertebrate source, including polypeptides ("polypeptide" and "protein" are used interchangeably herein) or mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). SIRPα has three immunoglobulin superfamily domains—an extracellular domain containing a single V set and two C1 set IgSF domains, a transmembrane domain, and an intracellular domain containing an immunoreceptor-suppressive tyrosine motif (ITIM). The term SIRPα also encompasses naturally occurring variants of SIRPα, such as SNP variants, splice variants, and allele variants. It is known in the art that SIRPα interacts with CD47, thereby leading to phosphorylation of ITIM, which in turn mediates association with the phosphatase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2). The full amino acid sequence of human SIRPα is presented below: [ka]

[0044] The terms “Programmed Cell Death Ligand-1 (PD-L1),” “Programmed Cell Death Ligand-1,” “PD-1 Ligand 1,” or similar terms refer, unless otherwise indicated, to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native PD-L1 derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). PD-L1, also known as surface antigen classification 274 (CD274) or B7 homolog 1 (B7-H1), is a human protein encoded by the CD274 gene. PD-L1 is one of two naturally occurring cell surface glycoprotein ligands for PD-1 (the other being PD-L2). Like PD-1, PD-L1 belongs to the immunoglobulin superfamily and consists of two extracellular Ig domains, an N-terminal V domain, and a C-terminal constant domain. It is known in the field that PD-L1, upon binding to PD-1, downregulates T cell activation and cytokine secretion. The term PD-L1 encompasses "full-length" PD-L1, as well as any form of PD-L1 or any fragment thereof resulting from cellular processing. The term PD-L1 also encompasses naturally occurring variants of PD-L1, such as SNP variants, splice variants, and allelic variants. The full-length amino acid sequence of human PD-L1 is provided below (exemplary extracellular domain = underlined string): [ka] [ka]

[0045] Other related PD-L1 polypeptides, also encompassed by the term PD-L1, include fragments, derivatives (e.g., substitution, deletion, shortening, and insertion variants), fusion polypeptides, and interspecies homologs sufficient to retain PD-L1 activity and / or produce an anti-PD-L1 immune response. As those skilled in the art will understand, the PD-L1 binding agonists (e.g., antibodies) described herein may bind to PD-L1 polypeptides, PD-L1 polypeptide fragments, PD-L1 antigens, and / or PD-L1 epitopes. Epitopes may, in turn, be part of a larger PD-L1 polypeptide, part of a larger PD-L1 polypeptide fragment, or part of a larger PD-L1 antigen. PD-L1 may exist in native or denatured forms. The PD-L1 polypeptides described herein may be isolated from various sources, e.g., human tissue types or other sources, or prepared by recombinant or synthetic methods. PD-L1 polypeptides may include polypeptides having the same amino acid sequence as the corresponding PD-L1 polypeptides derived in nature. Orthologs for PD-L1 polypeptides are also well-known in this field.

[0046] The terms “Programmed Cell Death-1 (PD-1),” “Programmed Cell Death-1,” “PD-1 Receptor,” or similar terms refer, unless otherwise indicated, to polypeptides derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats) (the terms “polypeptide” and “protein” are used interchangeably herein) or any native PD-1. Also known as CD279 (surface antigen classification 279), PD-1 is an immunosuppressive receptor belonging to the CD28 family. PD-1 is predominantly expressed in vivo on pre-activated T cells and binds to two ligands, PD-L1 and PD-L2. PD-1 belongs to the immunoglobulin superfamily and consists of two extracellular Ig domains, an N-terminal V domain, and a C-terminal constant domain. PD-1 contains two cytoplasmic tyrosine-dependent signaling motifs: the immunoreceptor suppressive tyrosine motif (ITIM) and the immunoreceptor tyrosine-dependent switch motif (ITSM). The term PD-1 encompasses "full-length" PD-1, as well as any form of PD-1 or any fragment thereof resulting from processing in cells. The term PD-1 also encompasses naturally occurring variants of PD-1, such as SNP variants, splice variants, and allelic variants. It is well known in the art that, after T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif in its cytoplasmic tail, resulting in the dephosphorylation of effector molecules involved in the CD3 T cell signaling cascade, such as CD3 zeta, PKC theta, and ZAP70 (Carter et al. (2002) Eur J Immunol 32:634-43). The full-length amino acid sequence of human PD-1 is provided below: [ka]

[0047] As used herein, the term “binding agonist” or its grammatical equivalent refers to a molecule having one or more antigen-binding sites that bind to an antigen (e.g., an antibody, e.g., a bispecific antibody). In some embodiments, the bispecific binding agonists described herein are antibodies, antibody fragments, or other peptide-based molecules that bind to CD47, e.g., human CD47.

[0048] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in their broadest sense, specifically encompassing, for example, polyclonal antibodies having full-length heavy and / or light chains, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multi-epitope specificity or single-epitope specificity, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies. This disclosure also includes antibody fragments (and / or polypeptides containing antibody fragments) that retain the characteristic of binding to CD47. Non-limiting examples of antibody fragments include the antigen-binding region and / or effector region of an antibody, e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate region antibodies, single variable region antibodies, linear antibodies, V region, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fv (dsFv), single-domain antibodies (e.g., nanobody) or other fragments (e.g., fragments consisting of a heavy-chain variable region and a light-chain variable region coupled by non-covalent bonds). Generally speaking, the variable (V) region domain can be an immunoglobulin heavy-chain (VH) and / or light-chain (VL) variable domain in any suitable arrangement. For example, this disclosure also includes two heavy-chain molecules and two light-chain molecules, antibody light-chain monomers, and tetrameric antibodies containing an antibody heavy-chain monomer. Therefore, for example, the V region domain may be a dimer and may contain a VH-VH, VH-VL, or VL-VL dimer that binds to CD47. If desired, the VH and VL chains may be covalently coupled directly or through a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein in the category of “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody.CDRs (also known as “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode the desired CDR. Such polynucleotides are prepared, for example, by using mRNA from antibody-producing cells as a template and synthesizing the variable region using polymerase chain reaction (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments can be incorporated into single-domain antibodies, maxibodies, minibodies, intracellular antibodies, diabodies, triabodies, tetrabodies, variable domains (v-NARs) of novel antigen receptors, and bis-single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some embodiments, the binding agonist contains one or more constant regions including light chain and / or heavy chain constant regions, e.g., one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, the antibody may contain any of the above epitope-binding fragments.The antibodies described herein may be any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies may be agonist antibodies or antagonist antibodies.

[0049] As used herein, the term "single-specific" refers, when used in reference to a binding agonist (e.g., an antibody), to a binding agonist having one or more binding sites, each binding to the same epitope of the same antigen.

[0050] When the term "multispecificity" is used in reference to a binding agonist (e.g., an antibody), it means that the binding agonist has binding specificity to at least two different antigens or at least two different epitopes on the same antigen (e.g., a bispecific antibody against CD47 having a first binding site for a first epitope of CD47 and a second binding site for a second epitope of CD47).

[0051] When the term "bispecificity" is used in reference to a binding agent (e.g., an antibody), it means that the binding agent is capable of specifically binding to two distinct antigenic determinants, for example, two binding sites formed by pairs of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), which bind to different antigens or different epitopes on the same antigen. Such a bispecific binding agent may have a 1+1 format. Other bispecific binding agent (e.g., an antibody) formats may be a 2+1 or 1+2 format (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). If a bispecific binding agent (e.g., an antibody) contains two antigen-binding sites, each may bind to a different antigenic determinant. Such bispecific binding agents (e.g., antibodies) can bind to two different epitopes on the same antigen (e.g., epitopes on CD47).

[0052] The terms "identical" or "percent "identical" refer to two or more sequences or subsequences that, in the context of two or more nucleic acids or polypeptides, are identical or have a specific percentage of the same nucleotide or amino acid residues when compared and aligned (with gaps introduced if necessary) to maximize correspondence, without considering any conserved amino acid substitutions as part of sequence identity. Percent identical can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variations. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that when compared and aligned to maximize correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, and 99% nucleotide or amino acid residue identity, as measured by using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40–60 residues, at least about 60–80 residues, or any integer value in between. In some embodiments, the identity exists over a region longer than 60–80 residues, e.g., at least about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequence being compared, e.g., the coding region of a target protein or antibody. In some embodiments, identity exists over a region of nucleotide sequence having a length of at least about 10 bases, at least about 20 bases, at least about 40–60 bases, at least about 60–80 bases, or any integer value in between.In some embodiments, the identity exists over a region longer than 60–80 bases, for example, at least about 80–1000 bases or longer, and in some embodiments, the sequence is substantially identical over the entire length of the sequence being compared, for example, the nucleotide sequence encoding the protein of interest.

[0053] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain with similar chemical characteristics. Families of amino acid residues with similar side chains are commonly defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine in phenylalanine is a conservative substitution. Generally, conserved substitutions in the sequences of polypeptides, soluble proteins, and / or antibodies of this disclosure do not prevent binding of the polypeptide, soluble protein, or antibody containing that amino acid sequence to a target binding site. Methods for identifying conserved amino acid substitutions that do not preclude binding are well known in the art.

[0054] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, or may contain non-amino acids (e.g., may be interrupted by non-amino acids). The term also includes amino acid polymers that have been modified naturally or by intervention; for example, by the formation of disulfide bonds, glycosylation, lipid addition, acetylation, phosphorylation, or any other manipulation or modification, such as linkage or conjugation (directly or indirectly) with parts such as labeling components. For example, polypeptides containing one or more analogues of amino acids (e.g., non-natural amino acids) and other modifications known in the art are also included in this definition. Since the polypeptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it will be understood that in some embodiments, polypeptides may exist as single chains.

[0055] As used herein, “antigen” is a portion or molecule containing an epitope to which a binding agent (e.g., an antibody, e.g., a bispecific antibody) can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen to which the binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein binds is CD47 (e.g., human CD47) or a fragment thereof.

[0056] As used herein, “epitope” is a term used in the art and refers to a localized region of an antigen to which an antibody can bind. Epitopes can be linear epitopes or structural, nonlinear, or discontinuous epitopes. In the case of polypeptide antigens, for example, an epitope may be a sequence of amino acids in a polypeptide (a “linear” epitope), or it may consist of amino acids from two or more discontinuous regions of a polypeptide, such as human CD47 (a “structural,” “nonlinear,” or “discontinuous” epitope). Generally, it will be understood by those skilled in the art that linear epitopes may or may not depend on the secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids, whether folded into a native three-dimensional protein structure or not. In other embodiments, for an antibody to recognize and bind to an epitope, the amino acid residues must constitute the epitope in such a way that it exhibits a specific conformation (e.g., bending, twisting, turning, or folding).

[0057] An antibody binds to an "epitope," or to an "essentially identical epitope" or "same epitope" as a reference antibody, when the two antibodies recognize epitopes that are identical, overlapping, or adjacent in three-dimensional space. The most widely used and rapid method for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in three-dimensional space is a competitive assay, which can be constructed in several different formats, for example, by using either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized in a 96-well plate or expressed on the cell surface, and the ability of an unlabeled antibody to block the binding of a labeled antibody is measured using radiolabeling, fluorescent labeling, or enzymatic labeling.

[0058] Epitope binning is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition characteristics of different antibodies by combining competitive assays with computer processes to cluster antibodies based on their epitope recognition characteristics and identify antibodies with distinct binding specificities.

[0059] As used herein, the terms “specifically bind,” “specifically recognize,” “immunospecifically bind,” “selectively bind,” “immunospecifically recognize,” and “immunospecific” are synonymous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope), and such binding is understood by those skilled in the art. In some embodiments, “specifically bind” means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with higher affinity, or in some combination of the above, than with alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity, as determined by immunoassays, Biacore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds to or specifically binds to an antigen if, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), it binds to the antigen with a higher affinity than any cross-reactive antigen. Typically, specific or selective reactions are at least twice the background signal or noise, and may exceed 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In some embodiments, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA, the degree of binding of an antibody or antigen-binding domain to a “non-target” protein is less than about 10% of the binding of the antibody or antigen-binding domain to its particular target antigen. In some embodiments, a molecule that specifically binds to an antigen binds to that antigen with a Ka of at least 2log, 2.5log, 3log, 4log, or greater than the Ka of the molecule when it binds to another antigen. In some embodiments, molecules that specifically bind to the antigen do not cross-react with other proteins.In other specific embodiments, molecules that specifically bind to an antigen do not cross-react with other non-CD47 proteins. In some embodiments, “specifically binding” means, for example, that a polypeptide or molecule binds to a protein or target with a KD of about 0.1 mM or less, but typically less than 1 μM. In some embodiments, “specifically binding” means that a polypeptide or molecule binds to a target with a KD of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. Due to sequence identity between homologous proteins in different species, specific binding may include polypeptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of polypeptide sequences of different proteins, specific binding may include polypeptides or molecules that recognize more than one protein or target. In some embodiments, it is understood that a polypeptide or molecule that specifically binds to a first target may also specifically bind to a second target, or it may not specifically bind to the second target. Therefore, “specific binding” does not necessarily require exclusive binding, e.g., binding to a single target (although it may include such binding). Thus, a polypeptide or molecule may, in some embodiments, specifically bind to more than one target. In some embodiments, the same antigen-binding site of a polypeptide or molecule may bind to multiple targets. For example, an antibody may, in certain cases, contain two identical antigen-binding sites, each specifically binding to the same epitope of two or more proteins. In certain alternative embodiments, an antibody may be bispecific, containing at least two antigen-binding sites with different specificities. Generally, though not always, references to “binding” mean “specific binding.”

[0060] "Binding affinity" generally refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., a binding protein, e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is given by the dissociation constant (K). D ) can be generally shown by. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally tend to bind slowly to antigens and dissociate rapidly, while high affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them may be used for the purposes of this disclosure. In one embodiment, "K D " or "K D The value can be measured by assays known in the art, for example, by binding assays. D The values ​​were determined, for example, by biolayer interferometry (BLI) using the OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, K D This can also be measured, for example, in a radiolabeled antigen-binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881), or by using a surface plasmon resonance (SPR) assay with Biacore, for example, using BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). The term "on-rate" or "rate of association" or "association rate" or "k" can also be used. on " and "off-rate" or "dissociation rate" or "dissociation rate" or "k offThis can also be determined, for example, using the OctetQK384 system (ForteBio, Menlo Park, CA) or BIAcore(trademark)-2000 or BIAcore(trademark)-3000 (BIAcore, Inc., Piscataway, NJ) using the same SPR or BLI techniques described above.

[0061] When the term “competing” is used in the context of multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), it means binding agonists competing for the same epitope or binding site on a target, including competition among such binding agonists when it is determined by assays that prevent or inhibit the specific binding of a reference molecule (e.g., a reference ligand, or a reference antigen-binding protein, e.g., a reference antibody) to a common antigen (e.g., CD47). Numerous types of competitive binding assays can be used to determine whether a test binding agonist competes for binding to a reference molecule and to CD47 (e.g., human CD47). Examples of assays that may be used include solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., See (1990) Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82).Typically, such assays involve the use of purified antigen (e.g., CD47, e.g., human CD47) bound to a solid surface or cell possessing either an unlabeled test antigen-binding protein (e.g., test CD47 antibody) or a labeled reference antigen-binding protein (e.g., reference CD47 antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and / or antibodies that bind to an adjacent epitope (e.g., a similar or overlapping epitope) that is sufficiently proximal to the epitope to which the reference antibody binds to cause steric hindrance. Typically, when a competing antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 20%, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, or 97%, 98%, 99%, or more.

[0062] As used herein, the terms “constant region” or “constant domain” are well-known antibody terminology and refer to a portion of an antibody, for example, the carboxyl-terminal portion of the light and / or heavy chain, which is not directly involved in the antibody’s binding to an antigen but may exhibit various effector functions, such as interaction with Fc receptors. This term generally includes portions of immunoglobulin molecules that have a more conserved amino acid sequence compared to immunoglobulin variable domains.

[0063] Antibody "effector function" refers to the biological activity resulting from the antibody's Fc region (e.g., the Fc region of the native sequence or the Fc region of an amino acid sequence variant), and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated injury (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0064] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, the native sequence Fc region, recombinant Fc region, and variant Fc region. While the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is often defined as a continuum from the amino acid residue at position Cys226 (according to the EU numbering scheme) or from Pro230 (according to the EU numbering scheme) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering scheme) may be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are shown below (CH2 domain = bold; CH3 domain = underlined): [ka]

[0065] A "functional Fc region" possesses the "effector function" of the native sequence's Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated injury (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various disclosed assays.

[0066] A “native sequence Fc region” contains the same amino acid sequence as a naturally occurring Fc region and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, included in or combined with other sequences such as variable region sequences). Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A allotype and A allotype); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as their naturally occurring variants.

[0067] A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the Fc region of the native sequence by a modification (e.g., substitution, addition, or deletion) of at least one amino acid, preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, within the Fc region of the native sequence or the Fc region of the parent polypeptide, compared to the Fc region of the native sequence or the Fc region of the parent polypeptide. The variant Fc regions described herein may have at least about 80% homology with the Fc region of the native sequence and / or the Fc region of the parent polypeptide, or at least about 90% homology with them, for example, at least about 95% homology with them. The variant Fc regions described herein may be loss of effector function (e.g., silent Fc). An example variant Fc region ("silent Fc") sequence is shown below (CH2 domain = bold, amino acid changes are underlined; CH3 domain = underlined text): [ka] Exemplary variant CH2 domain (e.g., silent CH2) sequences useful for multispecific (e.g., bispecific) binding agents described herein are provided below (amino acid changes are underlined): [ka]

[0068] As used herein, the term “heavy chain,” when used in reference to antibodies, refers to a polypeptide chain of about 50–70 kDa, where the amino-terminal portion comprises a variable region of about 120–130 or more amino acids, and the carboxy-terminal portion comprises one or more constant regions. “Heavy chain” may refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG (including subclasses, e.g., IgG1, IgG2, IgG3, and IgG4) and IgM classes, respectively.

[0069] As used herein, the term “light chain,” when used in reference to antibodies, may refer to a polypeptide chain of approximately 25 kDa, where the amino-terminal portion contains a variable region of approximately 100 to 110 or more amino acids, and the carboxy-terminal portion contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, two distinct types exist, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0070] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to a portion of an antibody that interacts with an antigen and contains amino acid residues (e.g., CDRs) that confer specificity and affinity to the antigen to the binding fragment, domain, or region. “Antigen-binding fragment,” as used herein, includes an “antibody fragment” that contains a portion of an antibody, such as an antigen-binding region or variable region of the antibody, which contains one or more CDRs.

[0071] The antibodies described herein are not limited to, but include synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv(scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies, and any of the epitope-binding fragments described above.

[0072] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunoactive moieties of immunoglobulin molecules, including molecules containing one or more antigen-binding domains that bind to the CD47 antigen and one or more antigen-binding domains that bind to one or more targets other than CD47 (e.g., PD-L1).

[0073] The antibody may be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a, or IgG2b). In some embodiments, the antibody described herein is an IgG antibody (e.g., human IgG), or a class thereof (e.g., human IgG1, IgG2, IgG3, or IgG4), or a subclass thereof.

[0074] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, where the amino acid sequences of the H chains and L chains are identical. In some embodiments, the H chains and L chains include a constant region, e.g., a human constant region. In some embodiments, the L chain constant region of such an antibody is a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such an antibody includes a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody includes an IgG constant region, e.g., a human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions).

[0075] An antibody or fragment thereof may preferentially bind to CD47, for example, human CD47, meaning that the antibody or fragment binds to CD47 with a higher affinity than when it binds to an unrelated control protein, and / or to human CD47 with a higher affinity than when it binds to an unrelated control protein. For example, an antibody or fragment thereof may specifically recognize and bind to CD47 or a portion thereof. "Specific binding" means that the antibody or fragment binds to CD47 with an affinity at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times higher than its affinity to an unrelated control protein (e.g., chicken egg white lysozyme). In some embodiments, an antibody or fragment thereof may bind to CD47 substantially exclusively (for example, CD47 can be distinguished from other known polypeptides by a measurable difference in binding affinity). In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) may react with CD47 sequences other than human CD47 sequences (e.g., cynomolgus monkey CD47 sequences).

[0076] The term "variable region" or "variable domain" generally refers to a portion of an antibody's light or heavy chain located at the amino terminus of the light or heavy chain, with a heavy chain length of approximately 120–130 amino acids and a light chain length of approximately 100–110 amino acids, used for the binding and specificity of each particular antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that the sequence of a particular segment of the variable region varies widely among antibodies. The V region mediates binding to the antigen and determines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed across the 110-amino acid length of the variable region. Instead, the V region consists of a stretch of approximately 15–30 amino acids called the framework region (FR), which is less variable (e.g., relatively invariant), separated by a shorter region called the "hypervariable region" or "complementarity-determining region," which is more variable (e.g., extremely variable). The variable regions of the heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4), which primarily adopt a β-sheet configuration and are connected by three hypervariable regions that form loops connecting the β-sheet structure, and in some cases forming part of it. The hypervariable regions within each chain are held together in very close proximity by the FRs, and hypervariable regions from other chains contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant region does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). The sequences of the variable regions vary widely among different antibodies. Sequence variability is concentrated in the CDR, while less variable regions within the variable region are called framework regions (FRs).The CDRs of the light and heavy chains are primarily responsible for the antibody's interaction with the antigen. In certain embodiments, the variable region is a human variable region.

[0077] The terms “hypervariable region,” “HVR,” “HV,” “complementarity-determining region,” or “CDR,” as used herein, refer to regions of antibody variable regions where the sequence is hypervariable and / or forms structurally defined loops. Generally, antibodies contain six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several descriptions of hypervariable regions have been used and are included in the present invention. Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia, on the other hand, refers to the location of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length (this is because insertions are made at H35A and H35B in the Kabat numbering scheme; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region is a compromise between Kabat CDRs and Chothia structural loops and is used in Oxford Molecular's AbM antibody modeling software (see, for example, Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable region is based on the analysis of available complex crystal structures. The residues from each of these hypervariable regions or CDRs are listed below.

[0078] A universal numbering scheme, ImMunoGeneTics (IMGT) Information System (registered trademark) (Lefranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)), has been developed and is widely adopted. IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC) of human and other vertebrates. Herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. The “location” of CDRs within the structure of immunoglobulin variable domains is conserved across species and can be easily identified by a structure referred to as a loop, indicated by a numbering scheme that aligns the variable domain sequence according to structural features, CDRs, and framework residues. This information can be used for grafting and substitution of CDR residues from immunoglobulins of one species to acceptor frameworks, typically derived from human antibodies. An additional numbering scheme (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309: 657-670 (2001). For example, correspondences between numbering schemes, including Kabat numbering and the IMGT unique numbering scheme, are well known to those skilled in the art (see, e.g., Kabat, above; Chothia and Lesk, above; Martin, above; Lefranc et al., above), and are also illustrated below. The exemplary scheme shown herein combines Kabat and Chothia. [Table A]

[0079] The hypervariable region may include the "hypervariable region extensions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "hypervariable region," "HVR," "HV," "complementarity determination region," or "CDR" are used interchangeably.

[0080] The term "vector" refers to a substance used to carry or contain nucleic acid sequences, including, for example, those intended to introduce nucleic acid sequences into host cells. Applicable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers that can be activated for stable integration into the chromosomes of host cells. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes that may be included may, for example, confer resistance to antibiotics or toxins, compensate for nutritional requirement deficiencies, or supply critical nutrients that are not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcription enhancers, transcription terminators, etc., which are well known in the art. When co-expressing two or more nucleic acid molecules (e.g., both antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector, or into separate expression vectors. For expression by a single vector, the encoding nucleic acid can be operationally ligated to one common expression regulatory sequence, or to different expression regulatory sequences, such as one inductive promoter and one constitutive promoter. The introduction of the nucleic acid molecule into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for the expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., the multispecific binding agonists described herein), and further, that the expression level can be optimized using methods well known in the art to obtain sufficient expression.

[0081] "Immune cell dysfunction disorder," "immune cell dysfunction disorder," and "immune cell dysfunction state" are interchangeable terms and refer to any disease, disorder, or state that is entirely or partially caused by or a result of improper signaling to immune cells, and / or any disease, disorder, or state in which it is desirable to inhibit the in vivo effects of the interaction between immune cell receptors (e.g., SIRPα or PD-1) and their ligands (e.g., CD47 or PD-L1). Immune cell dysfunction disorders include phagocyte dysfunction disorders and T cell dysfunction disorders.

[0082] "Phasophageal dysfunction disorder," "phagocyte dysfunction disorder," and "phagocyte dysfunction state" are interchangeable terms and refer to any disease, disorder, or state that is entirely or partially caused by or a result of CD47 or the interaction between CD47 and SIRPα, and / or any disease, disorder, or state in which it is desirable to inhibit the in vivo effects of the interaction between CD47 and SIRPα instead. Phagocyte dysfunction disorders include diseases, disorders, or states characterized by or associated with reduced activity of phagocytic immune cells (e.g., neutrophils, macrophages, dendritic cells, B lymphocytes). In some embodiments, a phagocyte dysfunction disorder is a disease, disorder, or state specifically associated with an inappropriate increase in signaling through SIRPα. In some embodiments, a phagocyte dysfunction disorder is a reduced ability of phagocytes (e.g., macrophages) to take in or swallow other cells (e.g., tumor cells) or particles. In some embodiments, a reduced ability to take in or swallow other cells or particles results in impaired control of pathogens or tumors, including, but not limited to, tumors expressing CD47. Examples of phagocytic dysfunction disorders characterized by phagocytic dysfunction include unresolved acute infections, chronic infections, and tumor immunity (e.g., any cancer, including, but not limited to, cancers expressing or overexpressing CD47).

[0083] "T-cell dysfunction," "T-cell dysfunction disorder," and "T-cell dysfunction state" are interchangeable terms and refer to any disease, disorder, or state of T cells characterized by reduced responsiveness to antigenic stimulation. T-cell dysfunction disorders include diseases, disorders, or states that are entirely or partially caused by or result of PD-L1 or the interaction between PD-L1 and PD-1, and / or any disease, disorder, or state in which it is desirable to inhibit the in vivo effects of the interaction between PD-L1 and PD-1 instead. In some embodiments, a T-cell dysfunction disorder is a disease, disorder, or state specifically associated with an inappropriate increase in PD-1-mediated signaling. In some embodiments, a T-cell dysfunction disorder is a disease in which T cells are anergistic or have reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In some embodiments, reduced responsiveness results in uncontrolled effects of pathogens or tumors, including but not limited to tumors expressing PD-L1. Examples of T-cell dysfunction disorders characterized by T-cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity (e.g., from any cancer that expresses or overexpresses PD-L1, including but not limited to PD-L1).

[0084] "Tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when such evasion is weakened and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0085] "Enhancing T cell function" means inducing T cells to have sustained or increased biological function, causing T cells to have sustained or increased biological function, stimulating T cells to have sustained or increased biological function, or regenerating or reactivating depleted or inactive T cells. Examples of enhancing T cell function include: CD8 compared to such levels before intervention. + Increased secretion of cytokines (e.g., TNFα, IFNγ) from T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor cell elimination). In some embodiments, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Methods for measuring this enhancement are known to those skilled in the art.

[0086] An "effective dose" is generally a quantity sufficient to reduce the severity and / or frequency of symptoms resulting from or associated with a disease, disorder, or condition; to eliminate symptoms and / or their underlying causes; to prevent the appearance of symptoms and / or their underlying causes; and / or to improve or correct damage. In some embodiments, the effective dose is a therapeutic effective dose or a preventive effective dose.

[0087] When used herein, the term “therapeutic dose” refers to the amount of an agonist (e.g., the antibody described herein or any other agonist described herein) sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder, or condition and / or symptoms thereof. The therapeutic dose of an agonist, including an agonist, may be the amount necessary to (i) reduce or improve the progression or exacerbation of a given disease, disorder, or condition; (ii) reduce or improve the recurrence, occurrence, or onset of a given disease, disorder, or condition; and / or (iii) improve or enhance the preventive or therapeutic effect of another treatment (e.g., a treatment other than the administration of the antibody described herein). The “therapeutic dose” of a substance / molecule / agonist in this disclosure (e.g., CD47 antibody) may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the substance / molecule / agonist’s ability to elicit a desired response in the individual. A therapeutically effective dose encompasses the amount of a substance / molecule / agent in which the therapeutically beneficial effects outweigh any toxic or adverse effects. In certain embodiments, the term “therapeutically effective dose” refers to the amount of an antibody or other agonist (e.g., drug) that is effective in “treating” a disease, disorder, or condition in a subject or mammal.

[0088] The "prophylactic effective dose" is the amount of a pharmaceutical composition that, when administered to a subject, has the intended preventive effect, for example, preventing or delaying the onset (or recurrence) of a disease, disorder, or condition, or reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms(s). The complete therapeutic or prophylactic effect is not necessarily achieved by a single dose, but may only occur after a series of doses. Therefore, the therapeutic or prophylactic effective dose may be administered in one or more doses.

[0089] When used herein, the term "pharmaceutically acceptable" means that a product is approved by a federal or state regulatory authority for use in animals, more specifically in humans, or is listed in the United States Pharmacopeia, the European Pharmacopeia, or any other generally accepted pharmacopoeia.

[0090] When used herein, “carrier” includes carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the dosage and concentration used. Often, the carrier is an aqueous pH buffer. Examples of carriers include buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and / or nonionic surfactants, e.g., TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term "carrier" may also refer to diluents, adjuvants (e.g., Freund's adjuvants (complete or incomplete)), excipients, or vehicles with which the therapeutic agent is administered. Such carriers may be sterile liquids, such as water, and oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a typical carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly as injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, grain flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. The composition may also contain, if desired, trace amounts of wetting agents, emulsifiers, or pH buffers. The composition may take the form of a liquid, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like.Oral compositions containing the formulation may include, for example, standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions containing the pharmaceutical compound may contain, together with an appropriate amount of carrier, a prophylactic or therapeutically effective dose of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody), for example, in an isolated or purified form, to provide a form for appropriate administration to the target (e.g., a patient). The formulation should be suitable for the mode of administration.

[0091] In some embodiments, this disclosure provides multispecific binding agonists that can be used herein as therapeutic agents. Such agonists include multispecific antibodies (e.g., antibodies, e.g., bispecific antibodies) comprising a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more additional targets other than CD47 (e.g., PD-L1). Exemplary antibodies include humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0092] In some embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) comprising a first binding domain that binds to CD47, including CD47 polypeptide, CD47 polypeptide fragments, CD47 peptides, or CD47 epitopes, are described herein. In some embodiments, the multispecific binding agonist is a human antibody or humanized antibody (e.g., including a human constant region) comprising a first binding domain that binds to CD47, including CD47 polypeptide, CD47 polypeptide fragments, CD47 peptides, or CD27 epitopes. In some embodiments, the multispecific binding agonist (e.g., antibodies, e.g., bispecific antibodies) may bind to CD47 expressed on the surface of mammalian (e.g., human) cells, including CD47-expressing tumor cells. In some embodiments, the multispecific binding agonist (e.g., antibodies, e.g., bispecific antibodies) binds to CD47 extracellular epitopes (e.g., CD47 epitopes) exposed on cells such as tumor cells. In some embodiments, a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) that binds to CD47, for example, human CD47 or a portion thereof, is described herein. In some embodiments, CD47 is human CD47. In some embodiments, the multispecific binding agonist is a multispecific binding agonist that binds to CD47 (e.g., an antibody that binds to human CD47). An exemplary amino acid sequence of human CD47 is described herein.

[0093] In some embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) comprising a second binding domain that binds to PD-L1, including a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 peptide, or a PD-L1 epitope are described herein. In some embodiments, the multispecific binding agonist is a humanized antibody (e.g., including a human constant region) comprising a second binding domain that binds to PD-L1, including a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 peptide, or a PD-L1 epitope. In some embodiments, the multispecific binding agonist (e.g., antibodies, e.g., bispecific antibodies) may bind to PD-L1 expressed on the surface of mammalian (e.g., human) cells, including PD-L1-expressing antigen-presenting cells and tumor cells. In some embodiments, the multispecific binding agonist (e.g., antibodies, e.g., bispecific antibodies) binds to an exposed PD-L1 extracellular epitope (e.g., a CD47 epitope) on cells such as tumor cells. In some embodiments, a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) comprising a second binding domain that binds to PD-L1, for example, human PD-L1 or a portion thereof, is described herein. In some embodiments, PD-L1 is human PD-L1. In some embodiments, the multispecific binding agonist is a multispecific binding agonist that binds to human PD-L1 (e.g., an antibody that binds to human PD-L1). An exemplary amino acid sequence of human PD-L1 is described herein.

[0094] In some embodiments, the multispecific binding agents described herein (e.g., antibodies, e.g., bispecific antibodies) compete for binding to CD47, e.g., human CD47, with any one of the antibodies described herein that contains the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, e.g., the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 shown in Tables 1-3. Therefore, in some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) compete with binding agonists (e.g., antibodies, e.g., bispecific antibodies) comprising one, two, and / or three VH CDRs and / or one, two, and / or three VL CDRs from the antibodies designated as (a) C40; (b) C56; or (c) C59 shown in Tables 1-3 for binding to CD47, e.g., human CD47. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) compete for binding to CD47, e.g., human CD47, with binding agonists (e.g., antibodies, e.g., bispecific antibodies) containing one, two, and / or three VH CDRs and one, two, and / or three VL CDRs from antibodies designated as (a) C40; (b) C56; or (c) C59, as shown in Tables 1-3.In some embodiments, the multispecific binding agents described herein (e.g., antibodies, e.g., bispecific antibodies) compete for binding to CD47, e.g., human CD47, with binding agents (e.g., antibodies, e.g., bispecific antibodies) comprising (a) a VH region containing the amino acid sequence of SEQ ID NO: 25 and a VL region containing the amino acid sequence of SEQ ID NO: 26; (b) a VH region containing the amino acid sequence of SEQ ID NO: 51 and a VL region containing the amino acid sequence of SEQ ID NO: 52; or (c) a VH region containing the amino acid sequence of SEQ ID NO: 77 and a VL region containing the amino acid sequence of SEQ ID NO: 78.

[0095] In some embodiments, the multispecific binding activators described herein (e.g., antibodies, e.g., bispecific antibodies) include the amino acid sequences of any one of the antibodies described herein, such as the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, for example, the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 shown in Tables 1 to 6. Accordingly, in some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from an antibody designated as (a) C40; (b) C56; or (c) C59, as shown in Tables 1-3. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from an antibody designated as (a) P22; (b) P24; or (c) P31.2, as shown in Tables 4-6. In some embodiments, the multispecific binding activators described herein (e.g., antibodies, e.g., bispecific antibodies) include a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from an antibody referred to as (a) C40; (b) C56; or (c) C59, as shown in Tables 1-3, and a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from an antibody referred to as (a) P22; (b) P24; or (c) P31.2, as shown in Tables 4-6.In some embodiments, the multispecific binding activators described herein (e.g., antibodies, e.g., bispecific antibodies) include a first binding domain comprising one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from an antibody referred to as (a) C40; (b) C56; or (c) C59, as shown in Tables 1-3, and a second binding domain comprising one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from an antibody referred to as (a) P22; (b) P24; or (c) P31.2, as shown in Tables 4-6.

[0096] In some embodiments, a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) includes a first binding domain that binds to CD47 and includes a VH region containing VH CDR1, VH CDR2, and / or VH CDR3 of any one of the binding agonists listed in Tables 1, 2, and 3, and a VL region containing VL CDR1, VL CDR2, and / or VL CDR3; and a second binding domain that binds to PD-L1 and includes a VH region containing VH CDR1, VH CDR2, and / or VH CDR3 of any one of the binding agonists listed in Tables 4, 5, and 6, and a VL region containing VL CDR1, VL CDR2, and / or VL CDR3. Therefore, in some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a first binding domain comprising one, two, and / or three heavy chain CDRs from Table 1 and / or one, two, and / or three light chain CDRs. In some embodiments, the multispecific binding agonists described herein include a first binding domain comprising one, two, and / or three heavy chain CDRs from Table 2 and / or one, two, and / or three light chain CDRs. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a first binding domain comprising one, two, and / or three heavy chain CDRs from Table 3 and / or one, two, and / or three light chain CDRs. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a second binding domain comprising one, two, and / or three heavy chain CDRs from Table 4 and / or one, two, and / or three light chain CDRs. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a second binding domain comprising one, two, and / or three heavy chain CDRs from Table 5 and / or one, two, and / or three light chain CDRs.In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) include a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from Table 6. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) are bispecific and include a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from Table 1, Table 2, or Table 3, and a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from a binding agonist that binds to a second target antigen other than CD47. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies, e.g., bispecific antibodies) are bispecific and comprise a first binding domain containing one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from Table 1, Table 2, or Table 3, and a second binding domain containing one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from Table 4, Table 5, or Table 6.

[0097] The antibody referred to as C40 contains the VH sequence, which is sequence number 25, and the VL sequence, which is sequence number 26.

[0098] The antibody referred to as C56 contains the VH sequence, which is sequence number 51, and the VL sequence, which is sequence number 52.

[0099] The antibody referred to as C59 contains the VH sequence, which is sequence number 77, and the VL sequence, which is sequence number 78.

[0100] The antibody referred to as P22 contains the VH sequence, which is sequence number 103, and the VL sequence, which is sequence number 104.

[0101] The antibody referred to as P24 contains the VH sequence, which is sequence number 129, and the VL sequence, which is sequence number 130.

[0102] The antibody referred to as P31.2 contains the VH sequence, which is sequence number 155, and the VL sequence, which is sequence number 156.

[0103] In some embodiments, a multispecific binding agonist (e.g., an antibody) that binds to CD47 includes (i) a VH domain containing a VH sequence such as SEQ ID NO: 26 (C40 VH), SEQ ID NO: 51 (C56 VH), or SEQ ID NO: 77 (C59 VH), and (ii) a VL domain containing a VL sequence such as SEQ ID NO: 26 (C40 VL), SEQ ID NO: 52 (C56 VL), or SEQ ID NO: 78 (C59 VL). In some embodiments, such VH and VL domains are used to construct a bispecific binding agonist (e.g., an antibody) each having a first binding domain that binds to CD47, where the first binding domain includes (i) a C40 VH domain and a C40 VL domain, (ii) a C56 VH domain and a C56 VL domain, or (iii) a C59 VH domain and a C59 VL domain.

[0104] In some embodiments, a multispecific binding agonist (e.g., an antibody) that binds to PD-L1 includes (i) a VH domain containing a VH sequence such as SEQ ID NO: 103 (P22 VH), SEQ ID NO: 129 (P24 VH), or SEQ ID NO: 155 (P31.2 VH), and (ii) a VL domain containing a VL sequence such as SEQ ID NO: 104 (P22 VL), SEQ ID NO: 130 (P24 VL), or SEQ ID NO: 156 (P31.2 VL). In some embodiments, such VH domains and VL domains are used to construct a bispecific binding agonist (e.g., an antibody) each having a first binding domain that binds to PD-L1, where the first binding domain includes (i) a P22 VH domain and a P22 VL domain, (ii) a P24 VH domain and a P24 VL domain, or (iii) a P31.2 VH domain and a P31.2 VL domain.

[0105] In some embodiments, such VH and VL domains were used to construct a bispecific antibody comprising four polypeptide chains, wherein (i) polypeptide chain 1 comprises a VL domain (e.g., C40 VL, C56 VL, or C59 VL), polypeptide chain 2 comprises a VH domain (e.g., C40 VH, C56 VH, or C59 VH), and the VL and VH domains form a first binding domain that binds to CD47, and (ii) polypeptide chain 3 comprises a VL domain (e.g., P22 VL, P24 VL, or P31.2 VL), polypeptide chain 4 comprises a VH domain (e.g., P22 VH, P24 VH, or P31.2 VH), and the VL and VH domains form a second binding domain that binds to PD-L1.

[0106] In a first series of embodiments, the CD47×PD-L1 binding agonist (e.g., antibody) has first, second, third, and fourth polypeptide chains, (a) the first polypeptide chain comprises domain A, domain B, domain D, and domain E, which are aligned from the N-terminus to the C-terminus in an ABDE orientation, domain A having a first VL amino acid sequence (e.g., SEQ ID NO: 26 for C40 VL, SEQ ID NO: 52 for C56 VL, SEQ ID NO: 78 for C59 VL), domain B having a human IgG1 CH3 amino acid sequence having a T366K mutation and a C-terminal elongation incorporating a KSC tripeptide sequence followed by a DKTHT motif in the IgG1 hinge region, domain D having a human IgG1 CH2 amino acid sequence, and domain E having a human IgG1 having S354C and T366W mutations (b) The second polypeptide chain has a CH3 amino acid sequence; (c) The second polypeptide chain has domains F and G, which are aligned from the N-terminus to the C-terminus in an FG orientation, and domain F has a first VH amino acid sequence (e.g., SEQ ID NO: 25 for C40 VH, SEQ ID NO: 51 for C56 VH, SEQ ID NO: 77 for C59 VH), and domain G has a human IgG1 CH3 amino acid sequence with an L351D mutation and a C-terminal elongation incorporating a GEC amino acid disulfide motif; (d) The third polypeptide chain has domains H, I, J, and K, which are aligned from the N-terminus to the C-terminus in an HIJK orientation, and domain H has a second VL amino acid sequence (e.g., SEQ ID NO: 104 for P22 VL, SEQ ID NO: 130 for P24 VL, P31).(d) The fourth polypeptide chain has domains L and M, which are aligned from the N-terminus to the C-terminus in an LM orientation, and domain L has the second VH amino acid sequence (e.g., SEQ ID NO: 103 for P22 VH, SEQ ID NO: 129 for P24 VH, SEQ ID NO: 155 for P31.2 VH), and domain M has the human IgG1 CH3 amino acid sequence (e.g., SEQ ID NO: 103 for P22 VH, SEQ ID NO: 129 for P24 VH, SEQ ID NO: 155 for P31.2 VH), and domain M has the human IgG1 (e) The first polypeptide and the second polypeptide associate via interactions between the A domain and the F domain, and between the B domain and the G domain; (f) The third polypeptide and the fourth polypeptide associate via interactions between the H domain and the L domain, and between the I domain and the M domain; (g) The first polypeptide and the third polypeptide associate via interactions between the D domain and the J domain, and between the E domain and the K domain to form a bispecific binding agonist (e.g., an antibody); (h) Domain A and Domain F form a first binding domain that binds to CD47; and (i) Domain H and Domain L form a second binding domain that binds to PD-L1.

[0107] This four-polypeptide chain configuration is called the BC1 B-Body configuration: First polypeptide chain: Domain A = Target 1 (e.g., CD47) VL Domain B = CH3 (T366K; 445K, 446S, 447C tripeptide insertion) Domain D=CH2 Domain E=CH3(T366W, S354C) Second polypeptide chain: Domain F = Target 1 (e.g., CD47) VH Domain G=CH3 (L351D; 445G, 446E, 447C tripeptide insertion) The third polypeptide chain: Domain H = Target 2 (e.g., PD-L1) VL Domain I=CL Domain J=CH2 Domain K=CH3(Y349C, D356E, L358M, T366S, L368A, Y407V) The fourth polypeptide chain: Domain L = Target 2 (e.g., PD-L1) VH Domain M=CH1 >BC1 chain 1 Domain sorting: AB-Hinge-DE VL- CH3 -Hinge-CH2- CH3 (knob) Mutation in the first CH3 (domain B): T366K; 445K, 446S, 447C insertion Mutation in the second CH3 (domain E): S354C, T366W >BC1 chain 2 Domain sorting: FG VH- CH3 Mutations in CH3 (domain G): L351D; 445G, 446E, 447C insertion >BC1 chain 3 Domain sorting: HI-Hinge-JK VL- CL- Hinge-CH2- CH3 (Hall) Mutations in CH3 (domain K): Y349C, D356E, L358M, T366S, L368A, Y407V >BC1 chain 4 Domain sorting: LM VH- CH1

[0108] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 157, the second polypeptide chain has the sequence of SEQ ID NO: 158, the third polypeptide chain has the sequence of SEQ ID NO: 159, and the fourth polypeptide chain has the sequence of SEQ ID NO: 160. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 169, and the third polypeptide chain has the sequence of SEQ ID NO: 170.

[0109] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 157, the second polypeptide chain has the sequence of SEQ ID NO: 158, the third polypeptide chain has the sequence of SEQ ID NO: 161, and the fourth polypeptide chain has the sequence of SEQ ID NO: 162. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 169, and the third polypeptide chain has the sequence of SEQ ID NO: 171.

[0110] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 157, the second polypeptide chain has the sequence of SEQ ID NO: 158, the third polypeptide chain has the sequence of SEQ ID NO: 163, and the fourth polypeptide chain has the sequence of SEQ ID NO: 164. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 169, and the third polypeptide chain has the sequence of SEQ ID NO: 172.

[0111] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 165, the second polypeptide chain has the sequence of SEQ ID NO: 166, the third polypeptide chain has the sequence of SEQ ID NO: 159, and the fourth polypeptide chain has the sequence of SEQ ID NO: 160. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 173, and the third polypeptide chain has the sequence of SEQ ID NO: 170.

[0112] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 165, the second polypeptide chain has the sequence of SEQ ID NO: 166, the third polypeptide chain has the sequence of SEQ ID NO: 161, and the fourth polypeptide chain has the sequence of SEQ ID NO: 162. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 173, and the third polypeptide chain has the sequence of SEQ ID NO: 171.

[0113] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 165, the second polypeptide chain has the sequence of SEQ ID NO: 166, the third polypeptide chain has the sequence of SEQ ID NO: 163, and the fourth polypeptide chain has the sequence of SEQ ID NO: 164. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 173, and the third polypeptide chain has the sequence of SEQ ID NO: 172.

[0114] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 167, the second polypeptide chain has the sequence of SEQ ID NO: 168, the third polypeptide chain has the sequence of SEQ ID NO: 159, and the fourth polypeptide chain has the sequence of SEQ ID NO: 160. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 174, and the third polypeptide chain has the sequence of SEQ ID NO: 170.

[0115] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 167, the second polypeptide chain has the sequence of SEQ ID NO: 168, the third polypeptide chain has the sequence of SEQ ID NO: 161, and the fourth polypeptide chain has the sequence of SEQ ID NO: 162. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 174, and the third polypeptide chain has the sequence of SEQ ID NO: 171.

[0116] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 167, the second polypeptide chain has the sequence of SEQ ID NO: 168, the third polypeptide chain has the sequence of SEQ ID NO: 163, and the fourth polypeptide chain has the sequence of SEQ ID NO: 164. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 174, and the third polypeptide chain has the sequence of SEQ ID NO: 172.

[0117] In a second set of embodiments, the CD47×PD-L1 binding agonist (e.g., antibody) has first, second, third, and fourth polypeptide chains, (a) the first polypeptide chain comprises domain A, domain B, domain D, and domain E, the domains arranged in the ABDE direction from N-terminus to C-terminus, with domain A having a first VL amino acid sequence (e.g., SEQ ID NO: 26 for C40 VL, SEQ ID NO: 52 for C56 VL, SEQ ID NO: 78 for C59 VL), domain B having a human IgG1 CH3 amino acid sequence with a C-terminal extension incorporating a Y349C mutation, a P343V mutation, and a PGK tripeptide sequence followed by a DKTHT motif in the IgG1 hinge region, domain D having a human IgG1 CH2 amino acid sequence, and domain E having a human IgG1 having a S354C mutation and a T366W mutation (b) The second polypeptide chain has a CH3 amino acid sequence; (c) The second polypeptide chain has domains F and G, the domains are arranged from the N-terminus to the C-terminus in the FG direction, and domain F has the first VH amino acid sequence (e.g., SEQ ID NO: 25 for C40 VH, SEQ ID NO: 51 for C56 VH, SEQ ID NO: 77 for C59 VH), and domain G has the human IgG1 CH3 amino acid sequence with a C-terminal extension incorporating the S354C mutation and the PGK tripeptide sequence; (d) The third polypeptide chain has domains H, I, J, and K, the domains are arranged from the N-terminus to the C-terminus in the HIJK direction, domain H has the second VL amino acid sequence (e.g., SEQ ID NO: 104 for P22 VL, SEQ ID NO: 130 for P24 VL, SEQ ID NO: 156 for P31.2 VL), domain I has the human CL kappa amino acid sequence, and domain J has the human IgG1 (d) The CH2 amino acid sequence has a human IgG1 CH3 amino acid sequence having K as Y349C, T366S, L368A, and Y407V; (d) The fourth polypeptide chain has domain L and domain M, the domains are arranged from the N-terminus to the C-terminus in the LM direction, and domain L is the second VH amino acid sequence (e.g., SEQ ID NO: 103 for P22 VH, SEQ ID NO: 129 for P24 VH, P31).Regarding VH, it has Sequence ID No. 155), and domain M has a human IgG1 amino acid sequence; (e) the first and second polypeptides associate through interactions between domains A and F, and between domains B and G; (f) the third and fourth polypeptides associate through interactions between domains H and L, and between domains I and M; (g) the first and third polypeptides associate through interactions between domains D and J, and between domains E and K to form a bispecific binding agonist (e.g., an antibody); (h) domains A and F form a first binding domain that binds to CD47; (i) domains H and L form a second binding domain that binds to PD-L1.

[0118] Such a four-laptop chain format is referred to as the BC44 B-body format. First polypeptide chain Domain A = Target 1 (e.g., CD47) VL Domain B = CH3 (P343V; Y349C; 445P, 446G, 447K insertions) Domain D=CH2 Domain E=CH3(S354C, T366W) Second polypeptide chain Domain F = Target 1 (e.g., CD47) VH Domain G=CH3(S354C; 445P, 446G, 447K insertion) Third polypeptide chain Domain H = Target 2 (e.g., PD-L1) VL Domain I = CL (Kappa) Domain J=CH2 Domain K=CH3(Y349C, D356E, L358M, T366S, L368A, Y407V) Fourth polypeptide chain Domain L = Target 2 (e.g., PD-L1) VH Domain M=CH1. >BC44 chain 1 Domain placement: AB-Hinge-DE VL- CH3 -Hinge-CH2- CH3 (knob) Mutation in the first CH3 (domain B): P343V;Y349C;445P, 446G, 447K inserted Mutation in the second CH3 (domain E): S354C, T366W >BC44 chain 2 Domain placement: FG VH- CH3 Mutations in CH3 (domain G): S354C; 445P, 446G, 447K insertion >BC44 chain 3 Domain placement: HI-Hinge-JK VL- CL -Hinge-CH2- CH3 (Hall) Mutations in CH3 (domain K): Y349C, D356E, L358M, T366S, L368A, Y407V >BC44 chain 4 Domain placement: LM VH- CH1

[0119] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 175, the second polypeptide chain has the sequence of SEQ ID NO: 176, the third polypeptide chain has the sequence of SEQ ID NO: 159, and the fourth polypeptide chain has the sequence of SEQ ID NO: 160. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 181, and the third polypeptide chain has the sequence of SEQ ID NO: 170.

[0120] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 175, the second polypeptide chain has the sequence of SEQ ID NO: 176, the third polypeptide chain has the sequence of SEQ ID NO: 161, and the fourth polypeptide chain has the sequence of SEQ ID NO: 162. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 181, and the third polypeptide chain has the sequence of SEQ ID NO: 171.

[0121] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 175, the second polypeptide chain has the sequence of SEQ ID NO: 176, the third polypeptide chain has the sequence of SEQ ID NO: 163, and the fourth polypeptide chain has the sequence of SEQ ID NO: 164. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 181, and the third polypeptide chain has the sequence of SEQ ID NO: 172.

[0122] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 177, the second polypeptide chain has the sequence of SEQ ID NO: 178, the third polypeptide chain has the sequence of SEQ ID NO: 159, and the fourth polypeptide chain has the sequence of SEQ ID NO: 160. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 182, and the third polypeptide chain has the sequence of SEQ ID NO: 170.

[0123] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 177, the second polypeptide chain has the sequence of SEQ ID NO: 178, the third polypeptide chain has the sequence of SEQ ID NO: 161, and the fourth polypeptide chain has the sequence of SEQ ID NO: 162. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 182, and the third polypeptide chain has the sequence of SEQ ID NO: 171.

[0124] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 177, the second polypeptide chain has the sequence of SEQ ID NO: 178, the third polypeptide chain has the sequence of SEQ ID NO: 163, and the fourth polypeptide chain has the sequence of SEQ ID NO: 164. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 182, and the third polypeptide chain has the sequence of SEQ ID NO: 172.

[0125] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 179, the second polypeptide chain has the sequence of SEQ ID NO: 180, the third polypeptide chain has the sequence of SEQ ID NO: 159, and the fourth polypeptide chain has the sequence of SEQ ID NO: 160. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 183, and the third polypeptide chain has the sequence of SEQ ID NO: 170.

[0126] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 179, the second polypeptide chain has the sequence of SEQ ID NO: 180, the third polypeptide chain has the sequence of SEQ ID NO: 161, and the fourth polypeptide chain has the sequence of SEQ ID NO: 162. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 183, and the third polypeptide chain has the sequence of SEQ ID NO: 171.

[0127] In exemplary embodiments of a bispecific binding agonist (e.g., an antibody), the first polypeptide chain has the sequence of SEQ ID NO: 179, the second polypeptide chain has the sequence of SEQ ID NO: 180, the third polypeptide chain has the sequence of SEQ ID NO: 163, and the fourth polypeptide chain has the sequence of SEQ ID NO: 164. Alternatively, the first polypeptide chain has the sequence of SEQ ID NO: 183, and the third polypeptide chain has the sequence of SEQ ID NO: 172. Table 1: Antibody C40 [Table 1] Table 2: Antibody C56 Table 2 Table 3: Antibody C59 Table 3 Table 4: Antibody P22 Table 4 Table 5: Antibody P24 Table 5 Table 6: Antibody P31.2 Table 6

[0128] In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain and / or the second binding domain include a VH region or VH domain. In other embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain and / or the second binding domain include a VL region or VL domain. In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain and / or the second binding domain have (i) a VH domain or VH region, and / or (ii) a VL domain or VL region.

[0129] In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) have a combination of (i) a VH domain or VH region and (ii) a VL domain or VL region. For example, an exemplary bispecific IgG antibody comprises (i) a heavy chain having a combination of a VH domain or VH region described herein and one or more heavy chain constant domains or constant regions (e.g., CH1, hinge, CH2, and CH3), and (ii) a light chain having a combination of a VL domain or VL region described herein and a light chain constant domain or constant region (CL). An exemplary IgG heavy chain has any VH domain described herein, as well as the following CH1, hinge, CH2, and CH3 amino acid sequences: [ka] Includes. Another exemplary IgG heavy chain is any VH domain described herein, as well as the following CH1, hinge, CH2, and CH3 amino acid sequences: [ka] Includes. An example light chain (for example, for pairing with an IgG heavy chain) is any VL domain described herein, and the following CL amino acid sequence: [ka] Includes.

[0130] In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain includes one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as identified in Table 1. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain includes one or more of six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as identified in Table 2. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain includes one or more of six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as identified in Table 3. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain includes one or more of six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as identified in Tables 1, 2 and / or 3.

[0131] In some embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist described herein that binds to one or more targets including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), includes a first binding domain comprising one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 1. In other embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist described herein that binds to one or more targets including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), includes a first binding domain comprising one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 1. In further embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) including a multispecific binding agonist described herein that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) comprises a first binding domain comprising one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 1, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 1.

[0132] In some embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist described herein that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1), includes a first binding domain comprising one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 2. In other embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist described herein that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1), includes a first binding domain comprising one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 2. In further embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) including a multispecific binding agonist described herein that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) comprises a first binding domain comprising one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 2, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 2.

[0133] In some embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist described herein that binds to one or more targets including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), includes a first binding domain comprising one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 3. In other embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist described herein that binds to one or more targets including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), includes a first binding domain comprising one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 3. In further embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) including a multispecific binding agonist described herein that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) comprises a first binding domain comprising one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 3, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 3.

[0134] In some embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises a second binding domain comprising one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 4. In other embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises a second binding domain comprising one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 4. In further embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 4, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 4.

[0135] In some embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises a second binding domain comprising one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 5. In other embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises a second binding domain comprising one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 5. In further embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 5, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 5.

[0136] In some embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises a second binding domain comprising one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 6. In other embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody), including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises a second binding domain comprising one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 6. In further embodiments, a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1, comprises one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 6, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 6.

[0137] In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47 and includes one or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47 and includes two or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47 and includes three or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain that binds to PD-L1 and includes one or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs. 79-102, 105-128, and 131-154. In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain that binds to PD-L1 and includes three or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs. 79-102, 105-128, and 131-154.

[0138] In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising a VH that binds to CD47 and has one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3. In other embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising a VL that binds to CD47 and has one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-3. In yet another embodiment, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain that binds to CD47 and comprises one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3, and one or more VL CDRs listed in Tables 1-3. Accordingly, in some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising VH CDR1 bound to CD47 and having one of the amino acid sequences of SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising VH CDR2 bound to CD47 and having one of the amino acid sequences of SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76. In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising a VH CDR3 that binds to CD47 and has one of the amino acid sequences of SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72.In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising VH CDR1 and / or VH CDR2 and / or VH CDR3, which are independently selected from VH CDR1, VH CDR2, and VH CDR3, which are represented by any one of the amino acid sequences shown in Tables 1 to 3, and which bind to CD47. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a first binding domain comprising VL CDR1, which binds to CD47 and has any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73. In another embodiment, the multispecific binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes a first binding domain comprising VL CDR2 bound to CD47 and having one of the amino acid sequences of SEQ ID NOs. 5, 11, 22, 31, 37, 48, 57, 63, and 74. In some embodiments, the multispecific binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes a first binding domain comprising VL CDR3 bound to CD47 and having one of the amino acid sequences of SEQ ID NOs. 6, 17, 23, 32, 43, 49, 58, 69, and 75. In some embodiments, the multispecific binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes a first binding domain comprising VL CDR1 and / or VL CDR2 and / or VL CDR3 bound to CD47 and independently selected from VL CDR1, VL CDR2, and VL CDR3, which are represented by one of the amino acid sequences shown in Tables 1-3.

[0139] In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain that binds to PD-L1 and contains VH having one or more (e.g., one, two, or three) VH CDRs listed in Tables 4-6. In other embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain that binds to PD-L1 and contains VL having one or more (e.g., one, two, or three) VL CDRs listed in Tables 4-6. In yet another embodiment, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain that binds to PD-L1 and contains one or more (e.g., one, two, or three) VH CDRs listed in Tables 4-6, and one or more VL CDRs listed in Tables 4-6. Accordingly, in some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain comprising VH CDR1 that binds to PD-L1 and has one of the amino acid sequences of SEQ ID NOs. 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, and 148. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain comprising VH CDR2 that binds to PD-L1 and has one of the amino acid sequences of SEQ ID NOs. 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 44, 149, and 154. In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain comprising a VH CDR3 that binds to PD-L1 and has one of the amino acid sequences of SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, and 150.In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain comprising VH CDR1 and / or VH CDR2 and / or VH CDR3, which bind to PD-L1 and are independently selected from VH CDR1, VH CDR2, and VH CDR3, which are represented by any one of the amino acid sequences shown in Tables 4-6. In some embodiments, the multispecific binding agonists described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain comprising VL CDR1, which binds to PD-L1 and has any one of the amino acid sequences of SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, and 151. In another embodiment, the multispecific binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes a second binding domain comprising VL CDR2 that binds to PD-L1 and has one of the amino acid sequences of SEQ ID NOs. 83, 89, 100, 109, 115, 126, 135, 141, and 152. In some embodiments, the multispecific binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes a second binding domain comprising VL CDR3 that binds to PD-L1 and has one of the amino acid sequences of SEQ ID NOs. 84, 95, 101, 110, 121, 127, 136, 147, and 153. In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) include a second binding domain that binds to PD-L1 and comprises VL CDR1 and / or VL CDR2 and / or VL CDR3, independently selected from VL CDR1, VL CDR2, and VL CDR3, which are represented by any one of the amino acid sequences shown in Tables 4-6.

[0140] In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to CD47 and form VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1, 27, or 53, (ii) SEQ ID NOs: 7, 33, or 59, (iii) SEQ ID NOs: 12, 38, or 64, (iv) SEQ ID NOs: 13, 39, or 65, and (v) SEQ ID NOs: 18, 44, or 70; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 2, 28, or 54, (ii) SEQ ID NOs: 8, 34, or 60, (iii) SEQ ID NOs: 14, 40, or 66, (iv) SEQ ID NOs: 19, 45, or 71, and (v) SEQ ID NOs: 24, 50, or 76 CDR2; and a heavy chain variable (VH) region including VH CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 3, 29, or 55, (ii) SEQ ID NOs: 9, 35, or 61, (iii) SEQ ID NOs: 15, 41, or 67, and (iv) SEQ ID NOs: 20, 46, or 72, and / or VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 4, 30, or 56, (ii) SEQ ID NOs: 10, 36, or 62, (iii) SEQ ID NOs: 16, 42, or 68, and (iv) SEQ ID NOs: 21, 47, or 73; (2) VL having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 5, 31, or 57, (ii) SEQ ID NOs: 11, 37, or 63, and (iii) SEQ ID NOs: 22, 48, or 74 The first binding domain comprises CDR2; and a light chain variable (VL) region including a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 6, 32, or 58, (ii) SEQ ID NOs: 17, 43, or 69, and (iii) SEQ ID NOs: 23, 49, or 75.

[0141] In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to CD47 and form VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1, 27, or 53, (ii) SEQ ID NOs: 7, 33, or 59, (iii) SEQ ID NOs: 12, 38, or 64, (iv) SEQ ID NOs: 13, 39, or 65, and (v) SEQ ID NOs: 18, 44, or 70; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 2, 28, or 54, (ii) SEQ ID NOs: 8, 34, or 60, (iii) SEQ ID NOs: 14, 40, or 66, (iv) SEQ ID NOs: 19, 45, or 71, and (v) SEQ ID NOs: 24, 50, or 76. CDR2; and a first binding domain comprising a heavy chain variable (VH) region including a VH CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 3, 29, or 55, (ii) SEQ ID NOs: 9, 35, or 61, (iii) SEQ ID NOs: 15, 41, or 67, and (iv) SEQ ID NOs: 20, 46, or 72.

[0142] In some embodiments, the multispecific binding activator described herein (e.g., an antibody such as a bispecific antibody) includes a first binding domain comprising a light chain variable (VL) region that binds to CD47 and includes: (1) VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 4, 30, or 56, (ii) SEQ ID NO: 10, 36, or 62, (iii) SEQ ID NO: 16, 42, or 68, and (iv) SEQ ID NO: 21, 47, or 73; (2) VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 5, 31, or 57, (ii) SEQ ID NO: 11, 37, or 63, and (iii) SEQ ID NO: 22, 48, or 74; and (3) VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 6, 32, or 58, (ii) SEQ ID NO: 17, 43, or 69, and (iii) SEQ ID NO: 23, 49, or 75.

[0143] In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to PD-L1 and form VH CDR1 having an amino acid sequence selected from the group consisting of (a) (1) (i) SEQ ID NO: 79, 105, or 131, (ii) SEQ ID NO: 85, 111, or 137, (iii) SEQ ID NO: 90, 116, or 142, (iv) SEQ ID NO: 91, 117, or 143, (v) SEQ ID NO: 96, 122, or 148; (2) (i) SEQ ID NO: 80, 106, or 132, (ii) SEQ ID NO: 86, 112, or 138, (iii) SEQ ID NO: 92, 118, or 144, (iv) SEQ ID NO: 97, 123, or 149, (v) SEQ ID NO: 102, 128, or 159 CDR2; and a heavy chain variable (VH) region including VH CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 81, 107, or 133, (ii) SEQ ID NOs: 87, 113, or 139, (iii) SEQ ID NOs: 93, 119, or 145, (iv) SEQ ID NOs: 98, 124, or 150, and / or (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 82, 108, or 134, (ii) SEQ ID NOs: 88, 114, or 140, (iii) SEQ ID NOs: 94, 120, or 146, (iv) SEQ ID NOs: 99, 125, or 151; (2) VL having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 83, 109, or 135, (ii) SEQ ID NOs: 89, 115, or 141, (iii) SEQ ID NOs: 100, 126, or 152 CDR2; (3) comprising a second binding domain including a light chain variable (VL) region containing a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 84, 110, or 136, (ii) SEQ ID NOs: 95, 121, or 147, and (iii) SEQ ID NOs: 101, 127, or 153.

[0144] In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to PD-L1 and form VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 79, 105, or 131, (ii) SEQ ID NOs: 85, 11, 1, or 137, (iii) SEQ ID NOs: 90, 116, or 142, (iv) SEQ ID NOs: 91, 117, or 143, (v) SEQ ID NOs: 96, 122, or 148; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 80, 106, or 132, (ii) SEQ ID NOs: 86, 112, or 138, (iii) SEQ ID NOs: 92, 118, or 144, (iv) SEQ ID NOs: 97, 123, or 149, (v) SEQ ID NOs: 102, 128, or 159 CDR2; and a second binding domain comprising a heavy chain variable (VH) region containing a VH CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 81, 107, or 133, (ii) SEQ ID NOs: 87, 113, or 139, (iii) SEQ ID NOs: 93, 119, or 145, and (iv) SEQ ID NOs: 98, 124, or 150.

[0145] In some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to PD-L1 and form VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 82, 108, or 134, (ii) SEQ ID NOs: 88, 114, or 140, (iii) SEQ ID NOs: 94, 120, or 146, (iv) SEQ ID NOs: 99, 125, or 151; (2) VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 83, 109, or 135, (ii) SEQ ID NOs: 89, 115, or 141, (iii) SEQ ID NOs: 100, 126, or 152; (3) VL having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 84, 110, or 136, (ii) SEQ ID NOs: 95, 121, or 147, (iii) SEQ ID NOs: 101, 127, or 153 It includes a second binding domain, which includes a light chain variable (VL) region containing CDR3.

[0146] Multispecific binding agonists (e.g., antibodies such as bispecific antibodies) comprising a first binding domain that binds to CD47 and includes one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-3 are also described herein. In particular, multispecific antibodies (e.g., bispecific antibodies) including the following are described herein: VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73);VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74);VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75);VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR3(SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, ; 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, and 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, and 75);Or any combination of VH CDRs (sequences 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, 70, 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, 76, 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72) and VL CDRs (sequences 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, 73, 5, 11, 22, 31, 37, 48, 57, 63, 74, 6, 17, 23, 32, 43, 49, 58, 69, and 75) listed in Tables 1-3.

[0147] Multispecific binding agonists (e.g., antibodies) comprising a second binding domain that binds to PD-L1 and includes one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 4-6 are also described herein. In particular, multispecific binding agonists (e.g., antibodies) including the following are described herein: VH CDR1 (SEQ ID NOs. 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148) and VL CDR1 (SEQ ID NOs. 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151); VH CDR1 (SEQ ID NOs. 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148) and VL CDR2 (SEQ ID NOs. 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR1 (sequences 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148) and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154) and VL CDR1 (sequences 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151); VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154) and VL CDR2 (sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154) and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153);VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151); VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR3 (sequences 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (sequences 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154) and VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR1 (sequences 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153);VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR1 (sequences 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VL CDR1 (sequences 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151), and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153);VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR2 (sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152) and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR3 (sequences 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (sequences 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152);VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR2(sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152);VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR1 (Sequence IDs 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH C; DR2 (Sequence codes 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR1 (Sequence codes 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR3 (Sequence codes 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (Sequence codes 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR3 (sequences 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (sequences 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151), and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153);VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR2 (sequence keys 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (sequence keys 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR2 (sequence keys 83, 89, 100, 109, 115, 126, 135, 141, or 152) and VL CDR3 (sequence keys 84, 95, 101, 110, 121, 127, 136, 147, or 153);VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151) and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (sequences 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR2 (sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152), and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153);VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR2 (SEQ ID NOs: 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151), VL CDR2 (SEQ ID NOs: 83, 89, 100, 109, 115, 126, 135, 141, or 152), and VL CDR3 (SEQ ID NOs: 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR1 (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, or 148), VH CDR3 (SEQ ID NOs: 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151), VL CDR2 (sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152), and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153); VH CDR2 (sequences 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, or 154), VH CDR3 (sequences 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150), VL CDR1 (sequences 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, or 151), VL CDR2 (sequences 83, 89, 100, 109, 115, 126, 135, 141, or 152), and VL CDR3 (sequences 84, 95, 101, 110, 121, 127, 136, 147, or 153);Or any combination thereof of the VH CDRs (SEQ ID NOs: 79, 85, 90, 91, 96, 105, 11, 116, 117, 122, 131, 137, 142, 143, 148, 80, 86, 92, 97, 102, 106, 112, 118, 123, 128, 132, 138, 144, 149, 154, 81, 87, 93, 98, 107, 113, 119, 124, 133, 139, 145, or 150) and VL CDRs (SEQ ID NOs: 82, 88, 94, 99, 108, 114, 120, 125, 134, 140, 146, 151, 83, 89, 100, 109, 115, 126, 135, 141, 152, 84, 95, 101, 110, 121, 127, 136, 147, or 153) listed in Tables 4-6.;

[0148] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises (a)(1)(i) an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 27, or 53, (ii) SEQ ID NO: 7, 33, or 59, (iii) SEQ ID NO: 12, 38, or 64, (iv) SEQ ID NO: 13, 39, or 65, and (v) SEQ ID NO: 18, 44, or 70 for V H CDR1; (2)(i) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 28, or 54, (ii) SEQ ID NO: 8, 34, or 60, (iii) SEQ ID NO: 14, 40, or 66, (iv) SEQ ID NO: 19, 45, or 71, and (v) SEQ ID NO: 24, 50, or 76 for V H CDR2; and (3)(i) an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 29, or 55, (ii) SEQ ID NO: 9, 35, or 61, (iii) SEQ ID NO: 15, 41, or 67, and (iv) SEQ ID NO: 20, 46, or 72 for V H CDR3-containing heavy chain variable (V H ) region, and (b)(1)(i) an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 30, or 56, (ii) SEQ ID NO: 10, 36, or 62, (iii) SEQ ID NO: 16, 42, or 68, and (iv) SEQ ID NO: 21, 47, or 73 for V LCDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 5, 31, or 57, (ii) SEQ ID NOs: 11, 37, or 63, and (iii) SEQ ID NOs: 22, 48, or 74 L CDR2; and V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 6, 32, or 58, (ii) SEQ ID NOs: 17, 43, or 69, and (iii) SEQ ID NOs: 23, 49, or 75. L Light chain variable (V) including CDR3 L A multispecific antibody or fragment thereof, comprising a region, is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or fragment is a bispecific antibody. In some embodiments, the second binding domain has an amino acid sequence selected from the group consisting of (a)(1)(i)SEQ ID NO: 79, 105, or 131, (ii)SEQ ID NO: 85, 111, or 137, (iii)SEQ ID NO: 90, 116, or 142, (iv)SEQ ID NO: 91, 117, or 143, and (v)SEQ ID NO: 96, 122, or 148. H CDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 80, 106, or 132, (ii) SEQ ID NOs: 86, 112, or 138, (iii) SEQ ID NOs: 92, 118, or 144, (iv) SEQ ID NOs: 97, 123, or 149, and (v) SEQ ID NOs: 102, 128, or 159 H CDR2; and (3)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 81, 107, or 133, (ii) SEQ ID NOs: 87, 113, or 139, (iii) SEQ ID NOs: 93, 119, or 145, (iv) SEQ ID NOs: 98, 124, or 150 H Heavy chain variable (V) including CDR3 H (b)(1)(i)Sequence ID 82, 108, or 134, (ii)Sequence ID 88, 114, or 140, (iii)Sequence ID 94, 120, or 146, and (iv)Sequence ID 99, 125, or 151, having an amino acid sequence selected from the group. LCDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 83, 109, or 135, (ii) SEQ ID NOs: 89, 115, or 141, and (iii) SEQ ID NOs: 100, 126, or 152 L CDR2; and (3)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 84, 110, or 136, (ii) SEQ ID NOs: 95, 121, or 147, and (iii) SEQ ID NOs: 101, 127, or 153. L Light chain variable (V) including CDR3 L ) region and. In some embodiments, a multispecific antibody or fragment having a first binding domain that binds to CD47, wherein the first binding domain has an amino acid sequence selected from the group consisting of (1)(i) SEQ ID NOs: 1, 27, or 53, (ii) SEQ ID NOs: 7, 33, or 59, (iii) SEQ ID NOs: 12, 38, or 64, (iv) SEQ ID NOs: 13, 39, or 65, and (v) SEQ ID NOs: 18, 44, or 70. H CDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 2, 28, or 54, (ii) SEQ ID NOs: 8, 34, or 60, (iii) SEQ ID NOs: 14, 40, or 66, (iv) SEQ ID NOs: 19, 45, or 71, and (v) SEQ ID NOs: 24, 50, or 76 H CDR2; and (3)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 3, 29, or 55, (ii) SEQ ID NOs: 9, 35, or 61, (iii) SEQ ID NOs: 15, 41, or 67, and (iv) SEQ ID NOs: 20, 46, or 72 H Heavy chain variable (V) including CDR3 HMultispecific antibodies or fragments thereof, including the ) region, are described herein. In some embodiments, the multispecific antibody includes a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or fragment thereof is a bispecific antibody. In some embodiments, the second binding domain has an amino acid sequence selected from the group consisting of (a)(1)(i)SEQ ID NO: 79, 105, or 131, (ii)SEQ ID NO: 85, 111, or 137, (iii)SEQ ID NO: 90, 116, or 142, (iv)SEQ ID NO: 91, 117, or 143, and (v)SEQ ID NO: 96, 122, or 148. H CDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 80, 106, or 132, (ii) SEQ ID NOs: 86, 112, or 138, (iii) SEQ ID NOs: 92, 118, or 144, (iv) SEQ ID NOs: 97, 123, or 149, and (v) SEQ ID NOs: 102, 128, or 159 H CDR2; and (3)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 81, 107, or 133, (ii) SEQ ID NOs: 87, 113, or 139, (iii) SEQ ID NOs: 93, 119, or 145, and (iv) SEQ ID NOs: 98, 124, or 150. H Heavy chain variable (V) including CDR3 H ) Includes the region.

[0149] In some embodiments, a multispecific antibody or fragment having a first binding domain that binds to CD47, wherein the first binding domain has an amino acid sequence selected from the group consisting of (1)(i) SEQ ID NOs: 4, 30, or 56, (ii) SEQ ID NOs: 10, 36, or 62, (iii) SEQ ID NOs: 16, 42, or 68, and (iv) SEQ ID NOs: 21, 47, or 73. L CDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 5, 31, or 57, (ii) SEQ ID NOs: 11, 37, or 63, and (iii) SEQ ID NOs: 22, 48, or 74 LCDR2; (3) V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 6, 32, or 58, (ii) SEQ ID NOs: 17, 43, or 69, and (iii) SEQ ID NOs: 23, 49, or 75 L Light chain variable (V) including CDR3 L Multispecific antibodies or fragments thereof, including the ) region, are described herein. In some embodiments, the multispecific antibody includes a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or fragment thereof is a bispecific antibody. In some embodiments, the second binding domain has an amino acid sequence selected from the group consisting of (a)(1)(i)SEQ ID NOs: 82, 108, or 134, (ii)SEQ ID NOs: 88, 114, or 140, (iii)SEQ ID NOs: 94, 120, or 146, and (iv)SEQ ID NOs: 99, 125, or 151. L CDR1;(2)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 83, 109, or 135, (ii) SEQ ID NOs: 89, 115, or 141, and (iii) SEQ ID NOs: 100, 126, or 152 L CDR2; and (3)V having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 84, 110, or 136, (ii) SEQ ID NOs: 95, 121, or 147, and (iii) SEQ ID NOs: 101, 127, or 153. L Light chain variable (V) including CDR3 L ) Includes the region.

[0150] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47 is described herein, wherein the first binding domain comprises all three heavy chain complementarity-determining regions (CDRs) or all three light chain CDRs from an antibody referred to as C40, comprising a VH sequence of SEQ ID NO: 25 and a VL sequence of SEQ ID NO: 26; a VH sequence of SEQ ID NO: 51 and a VL sequence of SEQ ID NO: 52; or a VH sequence of SEQ ID NO: 77 and a VL sequence of SEQ ID NO: 78 from an antibody referred to as C59. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the second binding domain includes all three heavy chain complementarity determining regions (CDRs) or all three light chain CDRs from an antibody referred to as P22, including the VH sequence of SEQ ID NO: 103 and the VL sequence of SEQ ID NO: 104; from an antibody referred to as P24, including the VH sequence of SEQ ID NO: 129 and the VL sequence of SEQ ID NO: 130; or from an antibody referred to as P31.2, including the VH sequence of SEQ ID NO: 155 and the VL sequence of SEQ ID NO: 156.

[0151] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated as C40, is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or fragment thereof is a bispecific antibody. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated as P22. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated as P24. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated as P31.2.

[0152] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises all three heavy chain CDRs or all three light chain CDRs from an antibody designated as C56, is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or fragment thereof is a bispecific antibody. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated as P22. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated as P24. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated as P31.2.

[0153] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises all three heavy chain CDRs or all three light chain CDRs from an antibody designated C59, is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or fragment thereof is a bispecific antibody. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated P22. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated P24. In some embodiments, the second binding domain comprises all three heavy chain CDRs and all three light chain CDRs from an antibody designated P31.2.

[0154] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain is (a) V shown in Tables 1-3H CDR1, V H CDR2, and V H Heavy chain variable (V) containing CDR3 amino acid sequence H ) region, or (b) V shown in Tables 1-3 L CDR1, V L CDR2, and V L Light chain variable (V) containing CDR3 amino acid sequence L Multispecific antibodies or fragments thereof, including the ) region, are described herein. In some embodiments, the first binding domain is (a) V shown in Tables 1-3 H CDR1, V H CDR2, and V H Heavy chain variable (V) containing CDR3 amino acid sequence H ) region, and (b) V shown in Tables 1-3 L CDR1, V L CDR2, and V L Light chain variable (V) containing CDR3 amino acid sequence L ) Includes the region. In some embodiments, the multispecific antibody includes a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or a fragment thereof is a bispecific antibody. In some embodiments, the second binding domain is (a) V shown in Tables 4-6 H CDR1, V H CDR2, and V H Heavy chain variable (V) containing CDR3 amino acid sequence H ) region, and / or (b) V shown in Tables 4-6 L CDR1, V L CDR2, and V L Light chain variable (V) containing CDR3 amino acid sequence L ) Includes the region. In some embodiments, the first binding domain is V shown in Tables 1-3. H CDR1, V H CDR2, and V H Heavy chain variable (V) containing CDR3 amino acid sequence H) Includes the region. In some embodiments, the multispecific antibody includes a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or a fragment thereof is a bispecific antibody. In some embodiments, the second binding domain is V shown in Tables 4-6. H CDR1, V H CDR2, and V H Heavy chain variable (V) containing CDR3 amino acid sequence H ) Includes the region. In some embodiments, the first binding domain is V shown in Tables 1-3. L CDR1, V L CDR2, and V L Light chain variable (V) containing CDR3 amino acid sequence L ) Includes the region. In some embodiments, the multispecific antibody includes a second binding domain that binds to PD-L1. In some embodiments, the multispecific antibody or a fragment thereof is a bispecific antibody. In some embodiments, the second binding domain is V shown in Tables 4-6. L CDR1, V L CDR2, and V L Light chain variable (V) containing CDR3 amino acid sequence L ) Includes the region.

[0155] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain has an amino acid sequence selected from the group consisting of (a)(1)SEQ ID NOs: 1, 7, 12, 13, and 18. H (2) V having an amino acid sequence selected from the group consisting of CDR1, (2) SEQ ID NOs: 2, 8, 14, 19, and 24 H CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20. H Heavy chain variable (V) including CDR3 H (b)(1)V having an amino acid sequence selected from the group consisting of sequence numbers 4, 10, 16, and 21 L (2) V having an amino acid sequence selected from the group consisting of CDR1, (2) SEQ ID NOs: 5, 11, and 22 LCDR2 and a V having an amino acid sequence selected from the group consisting of (3) SEQ ID NO: 6, 17, and 23 L light chain variable (V L ) region containing CDR3, a multispecific antibody or a fragment thereof is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the first binding domain is (a) a V having the amino acid sequence of (1) SEQ ID NO: 1 H CDR1, (2) a V having the amino acid sequence of SEQ ID NO: 2 H CDR2, and (3) a V having the amino acid sequence of SEQ ID NO: 3 H heavy chain variable (V H ) region containing CDR3, and (b) a V having the amino acid sequence of (1) SEQ ID NO: 4 L CDR1, (2) a V having the amino acid sequence of SEQ ID NO: 5 L CDR2, and (3) a V having the amino acid sequence of SEQ ID NO: 6 L light chain variable (V L ) region containing CDR3. In some embodiments, the second binding domain is (a) a V having the amino acid sequence of (1) SEQ ID NO: 79 H CDR1, (2) a V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) a V having the amino acid sequence of SEQ ID NO: 81 H heavy chain variable (V H ) region containing CDR3, and (b) a V having the amino acid sequence of (1) SEQ ID NO: 82 L CDR1, (2) a V having the amino acid sequence of SEQ ID NO: 83 L CDR2, and (3) a V having the amino acid sequence of SEQ ID NO: 84 L light chain variable (V L ) region containing CDR3. In some embodiments, the second binding domain is (a) a V having the amino acid sequence of (1) SEQ ID NO: 105 H CDR1, (2) a V having the amino acid sequence of SEQ ID NO: 106 H CDR2, and (3) a V having the amino acid sequence of SEQ ID NO: 107 H heavy chain variable (V H(b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 7 H CDR1, (2) V having the amino acid sequence of sequence number 8 H CDR2 and (3) V having the amino acid sequence of SEQ ID NO: 9 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 10 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 85 H CDR1, (2) V having the amino acid sequence of sequence number 86 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 87 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 88 L CDR1, (2) V having the amino acid sequence of sequence number 89 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 111 H CDR1, (2) V having the amino acid sequence of sequence number 112 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 113 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 114 L CDR1, (2) V having the amino acid sequence of sequence number 115 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 137 H CDR1, (2) V having the amino acid sequence of sequence number 138 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 139 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 140 L CDR1, (2) V having the amino acid sequence of sequence number 141 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 12. H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of SEQ ID NO: 4 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 90 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 116 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 142. H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 13. H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 14 H CDR2 and (3) V having the amino acid sequence of SEQ ID NO: 15 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 16 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 17 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 91. H CDR1, (2) V having the amino acid sequence of sequence number 92 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 93 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 94 L CDR1, (2) V having the amino acid sequence of sequence number 89 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 95 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 117 H CDR1, (2) V having the amino acid sequence of sequence number 118 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 119 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 120 L CDR1, (2) V having the amino acid sequence of sequence number 115 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 128 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 143. H CDR1, (2) V having the amino acid sequence of sequence number 144 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 145 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 146 L CDR1, (2) V having the amino acid sequence of sequence number 141 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 147 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 18 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 19 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 20 H Heavy-chain CDR3 is available. V H (b)(1)V having the amino acid sequence of Sequence ID No. 21 L CDR1, (2) V having the amino acid sequence of sequence number 22 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 23 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 96. H CDR1, (2) V having the amino acid sequence of sequence number 97 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 98 H Heavy chain variable (V) including CDR3H (b)(1)V having the amino acid sequence of sequence number 99 L CDR1, (2) V having the amino acid sequence of sequence number 100 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 101 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 122. H CDR1, (2) V having the amino acid sequence of sequence number 123 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 124 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 125 L CDR1, (2) V having the amino acid sequence of sequence number 126 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 127 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 148 H CDR1, (2) V having the amino acid sequence of sequence number 149 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 150 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 151 L CDR1, (2) V having the amino acid sequence of sequence number 152 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 153 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 1. H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 24 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of SEQ ID NO: 4 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of sequence number 102 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 128 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 154 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the above multispecific antibody or fragment is a bispecific antibody.

[0156] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain has an amino acid sequence selected from the group consisting of (a)(1)SEQ ID NOs: 27, 33, 38, 39, and 44. H (2) V having an amino acid sequence selected from the group consisting of CDR1, (2) SEQ ID NOs: 28, 34, 40, 45, and 50 H CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 35, 41, and 46. H Heavy chain variable (V) including CDR3 H (b)(1)V having an amino acid sequence selected from the group consisting of sequence numbers 30, 36, 42, and 47 L (2) V having an amino acid sequence selected from the group consisting of CDR1, (2) SEQ ID NOs: 31, 37, and 48 L (3) V having an amino acid sequence selected from the group consisting of CDR2 and (4) SEQ ID NOs: 32, 43, and 49 L Light chain variable (V) including CDR3 L A multispecific antibody or fragment thereof, comprising the ) region, is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the first binding domain comprises (a)(1)V having the amino acid sequence of SEQ ID NO: 27 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of SEQ ID NO: 30 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 33 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 34 H CDR2 and (3) V having the amino acid sequence of SEQ ID NO: 35 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of SEQ ID NO: 36 L CDR1, (2) V having the amino acid sequence of sequence number 37 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 38 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of SEQ ID NO: 30 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 39 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 40 H CDR2 and (3) V having the amino acid sequence of SEQ ID NO: 41 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 42 L CDR1, (2) V having the amino acid sequence of sequence number 37 L CDR2 and (3) V having the amino acid sequence of SEQ ID NO: 43 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 44 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 45 H CDR2 and (3) the amino acid sequence of SEQ ID NO: 46 V with columns H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 47 L CDR1, (2) V having the amino acid sequence of sequence number 48 L CDR2 and (3) V having the amino acid sequence of SEQ ID NO: 49 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 27 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 50 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of SEQ ID NO: 30 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the above multispecific antibody or fragment is a bispecific antibody.

[0157] In some embodiments, a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain has an amino acid sequence selected from the group consisting of (a)(1)SEQ ID NOs: 53, 59, 64, 65, and 70. H (2) V having an amino acid sequence selected from the group consisting of CDR1, (2) SEQ ID NOs: 54, 60, 66, 71, and 76 H (3) V having an amino acid sequence selected from the group consisting of CDR2 and (3) SEQ ID NOs: 55, 61, 67, and 72 H Heavy chain variable (V) including CDR3 H (b)(1)V having an amino acid sequence selected from the group consisting of sequence numbers 56, 62, 68, and 73 L (2) V having an amino acid sequence selected from the group consisting of CDR1, (2) SEQ ID NOs: 57, 63, and 74 L (3) V having an amino acid sequence selected from the group consisting of CDR2 and (3) SEQ ID NOs: 58, 69, and 75 L Light chain variable (V) including CDR3 L A multispecific antibody or fragment thereof, comprising the ) region, is described herein. In some embodiments, the multispecific antibody comprises a second binding domain that binds to PD-L1. In some embodiments, the first binding domain comprises (a)(1)V having the amino acid sequence of SEQ ID NO: 53 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 55 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of SEQ ID NO: 56 L CDR1, (2) V having the amino acid sequence of sequence number 57 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 HCDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 59 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 60 HCDR2, and (3) V having the amino acid sequence of SEQ ID NO: 61 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 62 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 HCDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 64 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 55 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of SEQ ID NO: 56 L CDR1, (2) V having the amino acid sequence of sequence number 57 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 HCDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 65 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 66 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 67 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 68 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 69 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 HCDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 70 H CDR1, (2) V having the amino acid sequence of sequence number 71 HCDR2 and (3) the amino acid sequence of SEQ ID NO: 72 V with columns H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 73 L CDR1, (2) V having the amino acid sequence of sequence number 74 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 75 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 HCDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, including light chain variable (V L ) including a region, and in some embodiments, the first binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 53 H CDR1, (2) V having the amino acid sequence of sequence number 76 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 55 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of SEQ ID NO: 56 L CDR1, (2) V having the amino acid sequence of sequence number 57 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of Sequence ID No. 79 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 82 L CDR1, (2) V having the amino acid sequence of sequence number 83 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 105 H CDR1, (2) V having the amino acid sequence of sequence number 106 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 H Heavy chain variable (V) including CDR3 H(b)(1)V having the amino acid sequence of sequence number 108 L CDR1, (2) V having the amino acid sequence of sequence number 109 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 L Light chain variable (V) including CDR3 L ) includes the region. In some embodiments, the second binding domain is (a)(1)V having the amino acid sequence of SEQ ID NO: 131 H CDR1, (2) V having the amino acid sequence of sequence number 132 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 H Heavy chain variable (V) including CDR3 H (b)(1)V having the amino acid sequence of sequence number 134 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 L Light chain variable (V) including CDR3 L ) region and. In some embodiments, the above multispecific antibody or fragment is a bispecific antibody.

[0158] In some embodiments, multispecific antibodies comprising the VH region and / or VL region described herein, further comprising human framework sequences, are described herein. In some embodiments, the VH region and / or VL region further comprises framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

[0159] In some embodiments, the multispecific antibodies described herein are monoclonal antibodies. In some embodiments, the monoclonal antibodies are humanized antibodies, human antibodies, or chimeric antibodies. In some embodiments, the multispecific antibodies described herein are multispecific antibodies formed from Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate region antibodies, single variable region antibodies, linear antibodies, V regions, or antibody fragments. In some embodiments, the multispecific antibodies described herein are recombinant antibodies, which are, if necessary, humanized antibodies, human antibodies, or chimeric antibodies.

[0160] In some embodiments, the CDRs disclosed herein for a first binding domain that binds to CD47 include consensus sequences derived from a group of relevant antibodies (see, for example, Tables 1-3). As described herein, “consensus sequence” refers to an amino acid sequence having conserved amino acids common among several sequences and variable amino acids that vary within a given amino acid sequence. The provided CDR consensus sequences include CDRs corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. Consensus sequences of CDRs for multispecific binding agonists (e.g., antibodies such as bispecific antibodies) for a first binding domain that binds to CD47 are shown in Figures 16A and 16B. Therefore, in some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to CD47 and include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence GFTFX1X2YYIH (SEQ ID NO: 194) (wherein X1 and X2 are independently naturally occurring amino acids); (2) VH CDR2 having the amino acid sequence X1IDX2X3X4X5X6TX7YADSVKG (SEQ ID NO: 195) (wherein X1, X2, X3X4, X5, X6, and X7 are independently naturally occurring amino acids); and (3) VH CDR3 having the amino acid sequence GGX1X2AX3DY (SEQ ID NO: 196) (wherein X1, X2, and X3 are independently naturally occurring amino acids), and / or (b) (1) VL having the amino acid sequence RASQSVSSAVA (SEQ ID NO: 197). The first binding domain includes a light chain variable (VL) region comprising CDR1; (2) VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO: 198); and (3) VL CDR3 having the amino acid sequence QQX1X2X3X4LX5T (SEQ ID NO: 199) (wherein X1, X2, X3, X4, and X5 are each independently naturally occurring amino acids). In some embodiments, the VH CDR1 of the multispecific binding agent described herein has the amino acid sequence GFTFX1X2YYIH (SEQ ID NO: 216) (wherein X1 is S or T and X2 is Y or S).In some embodiments, the VH CDR2 of the multispecific binding activator described herein has the amino acid sequence X1IDX2X3X4X5X6TX7YADSVKG (SEQ ID NO: 206) (wherein X1 is W, F, or Y; X2 is P or S; X3 is Y or K; X4 is G, S, or H; X5 is H or G; X6 is S or T; and X7 is T, E, or Q). In some embodiments, the VH CDR3 of the multispecific binding activator described herein has the amino acid sequence GGX1X2AX3DY (SEQ ID NO: 207) (wherein X1 is R or L; X2 is G, Y, or S; and X3 is M or L). In some embodiments, the VL CDR1 of the multispecific binding activator described herein has the amino acid sequence RASQSVSSAVA (SEQ ID NO: 197). In some embodiments, the VL CDR2 of the multispecific binding activator described herein has the amino acid sequence SASSLYS (SEQ ID NO: 198). In some embodiments, the VL CDR3 of the multispecific binding activator described herein has the amino acid sequence QQX1X2X3X4LX5T (SEQ ID NO: 208) (wherein X1 is R or G, X2 is Y, R or T, X3 is S or T, X4 is S or D, and X5 is L or R).

[0161] In some embodiments, the consensus CDR sequence of a multispecific binding agonist (e.g., an antibody such as a bispecific antibody) for a second binding domain that binds to PD-L1 is shown in Figures 17A and 17B. Therefore, in some embodiments, the multispecific binding activators described herein (e.g., antibodies such as bispecific antibodies) bind to PD-L1 and include: (a) (1) VH CDR1 having the amino acid sequence GFTFX1X2YYIH (SEQ ID NO: 200) (wherein X1 and X2 are independently naturally occurring amino acids); (2) VH CDR2 having the amino acid sequence X1IX2X3X4GX5X6TX7YADSVKG (SEQ ID NO: 201) (wherein X1, X2, X3, X4, X5, X6, and X7 are independently naturally occurring amino acids); and (3) VH having the amino acid sequence X1X2X3X4X5X6X7X8LDY (SEQ ID NO: 202) (wherein X1, X2, X3, X4, X5, X6, X7, and X8 are independently naturally occurring amino acids). The second binding domain comprises a heavy chain variable (VH) region containing CDR3 and / or (b) a light chain variable (VL) region containing (1) VL CDR1 having the amino acid sequence RASQSVSSAVA (SEQ ID NO: 197); (2) VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO: 198); and (3) VL CDR3 having the amino acid sequence QQX1X2X3X4PX5T (SEQ ID NO: 203) (wherein X1, X2, X3, X4, and X5 are each independently naturally occurring amino acids). In some embodiments, the VH CDR1 of the multispecific binding agent described herein has the amino acid sequence GFTFX1X2YYIH (SEQ ID NO: 217) (wherein X1 is D or S and X2 is Q or S). In some embodiments, the multispecific binding activator VH CDR2 described herein has the amino acid sequence X1IX2X3X4GX5X6TX7YADSVKG (SEQ ID NO: 209) (wherein X1 is E, W, or T; X2 is Y, T, or S; X3 is P or S; X4 is A, H, or G; X5 is S, Y, or G; X6 is Y, S, or F; and X7 is Y or K).In some embodiments, the VH CDR3 of the multispecific binding activator described herein has the amino acid sequence X1X2X3X4X5X6X7X8LDY (SEQ ID NO: 210) (wherein X1 is G or D, X2 is P, S, or Y, X3 is Y, V, or T, X4 is S, I, or L, X5 is V, Y, or T, X6 is R, G, or P, X7 is Y or V (or absent), and X8 is A (or absent)). In some embodiments, the VL CDR1 of the multispecific binding activator described herein has the amino acid sequence RASQSVSSAVA (SEQ ID NO: 197). In some embodiments, the VL CDR2 of the multispecific binding activator described herein has the amino acid sequence SASSLYS (SEQ ID NO: 198). In some embodiments, the VL CDR3 of the multispecific binding activator described herein has the amino acid sequence QQX1X2X3X4PX5T (SEQ ID NO: 211) (wherein X1 is V, Y, or F; X2 is S, Y, or G; X3 is Y, T, or A; X4 is S or E; and X5 is Y or I).

[0162] In some embodiments, a binding agent is described herein that binds to essentially the same epitope as one of the antibodies or fragments described herein. In some embodiments, a binding agent is described herein that competes with one of the antibodies or fragments described herein for binding to human PD-L1. In some embodiments, the binding agent is an antibody or fragment thereof.

[0163] In certain embodiments, the CDR of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) can be determined according to the Kabat system (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0164] In certain embodiments, the CDR of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) may be determined according to the Chothia system, which is referred to herein as the "Chothia CDR" (e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol.). See 215(1):175-82; and U.S. Patent No. 7,709,226.

[0165] In certain embodiments, the CDR of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) may be determined according to the ImMunoGeneTics (IMGT) system described, for example, Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDR").

[0166] In certain embodiments, the CDR of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) may be determined according to the AbM system described, for example, MacCallum et al., 1996, J. Mol. Biol., 262:732-745, which is referred to herein as the “AbM CDR”. See also, for example, Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0167] In certain embodiments, the CDR of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) may be determined according to the Contact system, which is referred to herein as "Contact CDR" (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5: 732-745). The Contact CDR is based on the analysis of available complex crystal structures.

[0168] In some embodiments, the positions of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the first binding domain of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1), can vary by one, two, three, four, five, or six amino acid positions, as long as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the position defining any of the CDRs in Tables 1, 2, or 3 may be varied by shifting the N-terminal and / or C-terminal boundary of the CDR by 1, 2, 3, 4, 5, or 6 amino acids compared to the current CDR position, as long as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the first binding domain of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1), can vary by 1, 2, 3, 4, 5 amino acids, or more (e.g., shorter or longer), as long as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).For example, in some embodiments, the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be 1, 2, 3, 4, 5 amino acids or more shorter than one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, as long as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be 1, 2, 3, 4, 5 amino acids or more longer than one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, as long as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino-terminuses of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein may be elongated by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, insofar as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the carboxyl termini of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein may be elongated by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, insofar as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino-terminuses of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein may be shortened by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, insofar as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl termini of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein may be shortened by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, as long as binding to CD47 (e.g., human CD47) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art, e.g., the binding assays and conditions described in the “Examples” section described herein, may be used to determine whether binding to CD47 (e.g., human CD47) is maintained.

[0169] In some embodiments, the positions of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the second binding domain of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1 as described herein may vary by one, two, three, four, five, or six amino acid positions, as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the position defining any of the CDRs in Tables 4, 5, or 6 may be varied by shifting the N-terminal and / or C-terminal boundary of the CDR by 1, 2, 3, 4, 5, or 6 amino acids compared to the current CDR position, as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the second binding domain of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) including a multispecific binding agonist that binds to CD47 including human CD47 and PD-L1 including human PD-L1 as described herein may vary by 1, 2, 3, 4, 5 amino acids, or more (e.g., shorter or longer), as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).For example, in some embodiments, the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be 1, 2, 3, 4, 5 amino acids or more shorter than one or more of the CDRs described by SEQ ID NOs. 79-102, 105-128, or 131-154, as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be 1, 2, 3, 4, 5 amino acids or more longer than one or more of the CDRs described by SEQ ID NOs. 79-102, 105-128, or 131-154, as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino-terminuses of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domain described herein may be elongated by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs. 79-102, 105-128, or 131-154, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the carboxyl termini of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domain described herein may be elongated by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs. 79-102, 105-128, or 131-154, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino-terminuses of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domain described herein may be shortened by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs. 79-102, 105-128, or 131-154, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl termini of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domain described herein may be shortened by 1, 2, 3, 4, 5 amino acids or more compared to one or more of the CDRs described by SEQ ID NOs. 79-102, 105-128, or 131-154, as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art, e.g., the binding assays and conditions described in the “Examples” section described herein, may be used to determine whether binding to PD-L1 (e.g., human PD-L1) is maintained.

[0170] In other embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) present herein, including multispecific binding agonists that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), include conservative sequence modifications. With respect to polypeptides that are multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), e.g., multispecific binding agonists that bind to one or more targets other than CD47, including human CD47 (e.g., PD-L1, including human PD-L1), conservative sequence modifications include conservative amino acid substitutions, including substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in some embodiments, predicted non-essential amino acid residues in CD47 or PD-L1 are replaced with other amino acid residues from the same side chain family. Methods for identifying conserved nucleotide and amino acid substitutions without excluding antigen binding are well-known in the field (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).In some embodiments, the conservative sequence modifications described herein involve modifying the amino acid sequence of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agonist that binds to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, nucleotide and amino acid sequence modifications refer to substitutions of at most one, two, three, four, five, or six amino acids in the CDRs listed in Tables 1, 2, 3, 4, 5, or 6. Therefore, for example, each such CDR may contain up to five conservative amino acid substitutions, for example, up to four (or less) conservative amino acid substitutions, for example, up to three (or less) conservative amino acid substitutions, for example, up to two (or less) conservative amino acid substitutions, or one or fewer conservative amino acid substitutions.

[0171] This disclosure provides multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) including multispecific binding agonists that bind to one or more targets other than human CD47, including human CD47 and PD-L1, including human PD-L1, having a masking portion and / or a cleavable portion, wherein one or more of the CD47 and / or other target binding domains of the multispecific binding agonist (e.g., antibody) are masked (e.g., via the masking portion) and / or activatable (e.g., via the cleavable portion). Techniques for masking multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) are well known in the art, including SAFE body masking techniques (see, for example, U.S. Patent Application Publication 2019 / 0241886) and Probody masking techniques (see, for example, U.S. Patent Application Publication 2015 / 0079088). Such techniques may be used to generate masked and / or activatable multispecific conjugates (e.g., antibodies, e.g., bispecific antibodies). Such masked and / or activatable multispecific conjugates (e.g., antibodies, e.g., bispecific antibodies), including multispecific conjugates that conjugate to CD47 including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), are useful for the preparation of conjugates including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable antibody-drug conjugates (AADCs), which include any one of the multispecific conjugates (e.g., antibodies, e.g., bispecific antibodies) that conjugate to CD47 including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), and include another activator that is directly or indirectly linked, such as a drug.For example, multispecific binding agonists of this disclosure that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), such as multispecific binding agonists including human CD47 binding agonists (e.g., antibodies, e.g., bispecific antibodies), can be covalently bound to one or more agonists, such as drugs, by a synthetic linker.

[0172] If desired, a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) may be linked or conjugated (directly or indirectly) with an effector function, e.g., a moiety having cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or a moiety having immunorecruitment activity. The moiety linked or conjugated (directly or indirectly) may include, in the form of a masked conjugate, a cytotoxic drug (e.g., a toxin, e.g., aurostatin) or a non-cytotoxic drug (e.g., a signaling modulator, e.g., a kinase) or a masking moiety that masks one or more binding domains of a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody), or a cleavable moiety that activates the multispecific binding agonist by cleaving a cleavable moiety to unmask one or more binding domains of the multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) in the tumor microenvironment. The portion that promotes immune recruitment may include other antigen-binding agonists, such as viral proteins that selectively bind to cells of the innate immune system. Alternatively, a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) may be linked or conjugated (directly or indirectly) with a portion that facilitates isolation from the mixture (e.g., a tag) or a portion having reporter activity (e.g., a detection label or reporter protein), as needed. It is understood that the features of the multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) described herein can also be extended to polypeptides containing binding agonist fragments.

[0173] In some embodiments, a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agonist that binds to one or more targets other than human CD47 (e.g., PD-L1, including human PD-L1), may be linked or conjugated (directly or indirectly) with a polypeptide, which may result in the production of an activatable antibody. In some embodiments, the multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) is linked or conjugated (directly or indirectly) with the agonist. In some embodiments, the agonist is a drug that produces an ADC or AADC, where the antibody of the ADC contains a masking moiety and a cleavable moiety.

[0174] In some embodiments, multispecific conjugate agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific conjugate agonists that conjugate to one or more targets other than human CD47 (e.g., PD-L1, including human PD-L1) as described herein, are conjugated (directly or indirectly) or linked by recombination with therapeutic agents (e.g., cytotoxic agents) or diagnostic or detection agents. Conjugated or recombination-linked antibodies, including masked or activatable conjugates, may be useful, for example, to treat or prevent diseases or disorders, e.g., immunodeficiency disorders, disorders, or conditions. Conjugated or recombinantly linked multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agonists that bind to one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), including masked or activatable conjugates, may be useful, for example, for monitoring or prognosticating the onset, development, progression, and / or severity of immunodeficiency disorders.

[0175] Such diagnosis and detection can be achieved, for example, by coupling a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) to a detectable substance, for example, including: enzymes including but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups including but not limited to streptavidin / biotin or avidin / biotin; fluorescent materials including but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein chloride, or phycoerythrin; luminescent materials including but not limited to luminol; bioluminescent materials including but not limited to luciferase, luciferin, or aequorin; and acridin Chemiluminescent materials including but not limited to ium-based compounds or HALOTAGs; iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine ( Radioactive materials including, but not limited to, 18F, 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn; positron-emitting metals using various positron emission tomography techniques; and non-radioactive paramagnetic metal ions.

[0176] The use of a heterologous protein, polypeptide, or peptide is also described herein, and a polyspecific binding agonist (e.g., antibody, e.g., bispecific antibody) linked or conjugated by recombination (covalent or noncovalent conjugation, direct or indirect) with a polyspecific binding agonist (e.g., antibody, e.g., bispecific antibody) to generate a fusion protein. In particular, the use of a heterologous protein, polypeptide, or peptide is also described herein, and a polyspecific binding agonist (e.g., antibody, e.g., bispecific antibody) linked or conjugated by recombination with a polyspecific binding agonist (e.g., antibody, e.g., bispecific antibody) that binds to one or more targets other than human CD47 (e.g., PD-L1, including human CD47) is also described herein. In some embodiments, heterogeneous proteins, polypeptides, or peptides to which a multispecific binding agonist (e.g., an antibody, e.g., a bispecific antibody) is ligated are useful for targeting the multispecific binding agonist to specific cells (e.g., CD47-expressing cells and / or PD-L1-expressing cells, including tumor cells).

[0177] Furthermore, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agonists that bind to one or more targets other than human CD47 and PD-L1 (e.g., human PD-L1), as described herein, may be (directly or indirectly) linked to a marker or "tag" sequence, e.g., a peptide, to facilitate purification. In some embodiments, the amino acid sequence of the marker or tag is, among other things, a hexa-histidine peptide, e.g., a tag provided in pQE vectors (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexa-histidine provides convenient purification of fusion proteins. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag (Wilson et al., 1984, Cell 37:767-78), which corresponds to an epitope derived from the influenza hemagglutinin protein, and the "FLAG" tag.

[0178] Methods for linking or conjugating a portion (including polypeptides) with an antibody (directly or indirectly) are well known in the art, and any one of these may be used to produce the antibody-drug conjugates or fusion proteins described herein.

[0179] In some embodiments, the multispecific binding agonists (e.g., antibodies) described herein are fusion proteins. The term “fusion protein,” as used herein, refers to a polypeptide comprising the amino acid sequence of a binding agonist (e.g., an antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not typically part of that antibody (e.g., a non-CD47 binding antibody or a non-PD-L1 binding antibody)). In certain embodiments, the fusion protein retains the biological activity of the multispecific binding agonist. In certain embodiments, the fusion protein comprises a first binding domain including the VH region, VL region, VH CDR (one, two, or three VH CDRs), and / or VL CDR (one, two, or three VL CDRs) of the CD47 antibody, and the fusion protein binds to the CD47 epitope, CD47 fragment, and / or CD47 polypeptide. In a particular embodiment, the fusion protein comprises a second binding domain containing the VH region, VL region, VH CDR (one, two, or three VH CDRs), and / or VL CDR (one, two, or three VL CDRs) of the PD-L1 antibody, and the fusion protein binds to a PD-L1 epitope, a PD-L1 fragment, and / or a PD-L1 polypeptide.

[0180] Fusion proteins can be generated, for example, through techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1, including human PD-L1), including multispecific binding agonists having higher affinity and lower dissociation rates (e.g., U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patentten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. See 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). In some embodiments, multispecific binding agonists, including those that bind to CD47 including human CD47 and one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), may be modified before recombination by subjecting them to error-prone PCR, random nucleotide insertion, or random mutagenesis by other means. The polynucleotides encoding the multispecific binding agonists described herein may be recombined with one or more components, motifs, regions, parts, domains, fragments, etc., of one or more heterologous molecules.

[0181] Multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) as described herein may also be conjugated to solid supports useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0182] Multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) as described herein may also be linked or conjugated (directly or indirectly) with a second antibody to form an antibody heteroconjugate.

[0183] A linker may be a “cleavable portion” that facilitates the intracellular release of a linked or conjugated activator, but non-cleavable linkers are also intended herein. Linkers for use in the conjugates of this disclosure (e.g., antibody-drug conjugates) include, but are not limited to, acid-unstable linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, e.g., valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photosensitive linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid resistance mediated by multidrug transporters.

[0184] Antibody-agonist conjugates, including cases where the agonist is a drug for preparing ADCs or AADCs, can be prepared using various bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoic acid). This disclosure further intends that antibody-agonist conjugates, including cases where the agonist is a drug for preparing ADCs or AADCs, can be prepared using any suitable method disclosed in the Art (see, for example, Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0185] Antibody-agonist conjugation strategies, including those where the agonist is a drug for preparing ADCs or AADCs, have traditionally relied on random conjugation chemistry resulting in heterogeneous conjugates involving ε-amino groups on Lys residues or thiol groups on Cys residues. Recently developed techniques enable site-specific conjugation with antibodies, thereby resulting in a uniform load and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics. These techniques involve "thiomab" manipulations, which include cysteine ​​substitutions at positions on the heavy and light chains that yield reactive thiol groups without disrupting immunoglobulin folding and assembly or altering antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26: 925-32). Alternatively, selenocysteine ​​can be inserted into the antibody sequence in a manner that allows for co-translation, by recoding the stop codon UGA from termination to selenocysteine ​​insertion, thereby enabling site-directed covalent conjugation of selenocysteine ​​at the nucleophilic selenool group in the presence of other native amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105: 12451-56; and Hofer et al., 2009, Biochemistry 48(50): 12047-57).

[0186] In some embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1), are conjugated with cytotoxic agents. In some embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1), as disclosed herein, may be conjugated as necessary with one or more cytotoxic agents disclosed herein or known in the art to produce ADCs or AADCs. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, including, but not limited to, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other inserts. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin, including, but not limited to, diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain, lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecenes. In some embodiments, the cytotoxic agent is a radioactive conjugate or a radioisotope for making a radioactively conjugated agent. While not limited to these, various radionuclides, including 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re, and 212Bi, can be used to create radioconjugated agents.Conjugates of polypeptides or molecules with one or more small molecule toxins, such as calitiamycin, maytansinoids, trichothene, and CC1065, as well as derivatives of these toxins having toxic activity, can also be used. Conjugates of polypeptides or molecules with cytotoxic agents are created using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol))propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disaxinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0187] In some embodiments, multispecific conjugate agonists (e.g., antibodies, e.g., bispecific antibodies) including multispecific conjugate agonists that conjugate to one or more targets other than human CD47 and PD-L1 (e.g., PD-L1 including human PD-L1) as described herein are conjugated with drugs such as signaling modulators, apoptosis promoters, mitotic inhibitors, antitumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agonists, chemoprotective agents, hormones, antihormone agents, corticosteroids, phototherapeutic agents, oligonucleotides, radionuclide agonists, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors. In some embodiments, the mitotic inhibitors are drastatin, auristatin, mytansinoids, and plant alkaloids. In some embodiments, the drugs are drastatin, auristatin, mytansinoids, and plant alkaloids. Examples of auristatins are monomethylaurisatin F (MMAF) or monomethylaurisatin E (MMAE). Examples of mytansinoids include, but are not limited to, DM1, DM2, DM3, and DM4. In some embodiments, the antitumor antibiotic is selected from the group consisting of actinomycin, anthracyclines, calicheamicin, and duocalmycin. In some embodiments, actinomycin is pyrrolobenzodiazepine (PBD).

[0188] Multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agonists that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), may be monospecific, bispecific, tripspecific, or have greater multispecificity. Such agonists may include antibodies. A multispecific antibody, e.g., a bispecific antibody, is a monoclonal antibody having binding specificity to at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody for CD47 having a first binding domain for a first epitope of CD47 and a second binding domain for a second epitope of CD47). In some embodiments, the first binding domain of a multispecific (e.g., bispecific) antibody described herein may be constructed based on the sequence of the antibody described herein, e.g., the CDR sequences listed in Tables 1, 2, and 3. In some embodiments, the second binding domain of the multispecific (e.g., bispecific) antibody described herein may be constructed based on the sequence of the antibody described herein, e.g., the CDR sequences listed in Tables 4, 5, and 6. In some embodiments, the multispecific antibody described herein is a bispecific antibody. In some embodiments, the bispecific antibody is a mouse antibody, a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, one of the binding specificities of the multispecific antibody is to CD47, and the other is to any other target (e.g., an antigen). In some embodiments, the multispecific (e.g., bispecific) antibody may contain more than one target-binding domain, where the different domains are specific to different targets (e.g., a first binding domain that binds to CD47 and a second binding domain that binds to another target (e.g., an antigen), e.g., an immune checkpoint regulator (e.g., a negative checkpoint regulator)). In some embodiments, the multispecific (e.g., bispecific) antibody may bind to more than one (e.g., two or more) epitopes on the same target (e.g., an antigen).In some embodiments, one of the binding specificities is for CD47, and the other is for any other target (e.g., an antigen). In some embodiments, one of the binding specificities is for CD47, and the other is for cytotoxic T lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), lymphocyte activator gene-3 (LAG-3; also known as CD223), galectin-3, B and T lymphocyte attenuator (BTLA), T cell membrane protein 3 (TIM3), galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, Ig and This targets one or more of the following: T cell immune receptors with an ITIM domain (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig inhibitor of T cell activation (VISTA), glucocorticoid-induced tumor necrosis factor receptor-associated (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0189] Methods for producing multispecific antibodies are known in the art, for example, by simultaneously expressing two immunoglobulin heavy-light chain pairs in which the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305: 537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0190] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10: 1-18; Brinkman et al., 2017, MABS, 9:2, 182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0191] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. As a non-limiting example, BsIgG formats may include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-hole assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0192] In some embodiments, BslgG includes a heavy chain manipulated for heterodimerization. For example, the heavy chain can be manipulated for heterodimerization by using a "knobs-into-holes" strategy, a SEED platform, a common heavy chain (e.g., one in the κλ-body), and the use of a heterodimer Fc region. Strategies to avoid homodimer heavy chain pairing in BsIgG are known in the art, including knob-into-holes, duobody, azymetric, charge pairing, HA-TF, SEEDbody, and differential protein A affinity.

[0193] Another bispecific antibody format is IgG with an additional antigen-binding moiety. For example, monospecific IgG can be manipulated to be bispecific by adding an additional antigen-binding unit to monospecific IgG, for example, to the N-terminus or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or variable light chains), manipulated protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). Non-limiting examples of additional IgG formats include bivariable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67 (2015): 95-106. In some embodiments, exemplary antibody formats are the B-Body format for monospecific or multispecific (e.g., bispecific antibodies) described, for example, in International Patent Application Publication WO2018 / 075692 and U.S. Patent Application Publication 2018 / 0118811.

[0194] A bispecific antibody fragment (BsAb) is a format of bispecific antibody molecule that lacks some or all of the antibody constant domain. For example, some BsAbs lack the Fc region. In several embodiments, the bispecific antibody fragment comprises a heavy chain region and a light chain region linked by a peptide linker, thereby enabling efficient expression of the BsAb in a single host cell. Non-exclusive examples of bispecific antibody fragments include, but are not limited to, nanobodies, nanobodies-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intracellular antibodies.

[0195] Bispecific fusion proteins contain antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or functionality. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with high binding titers. For example, the serum half-life of an antibody fragment can be extended by fusing a bispecific antibody with an albumin-binding protein or human serum albumin. In several embodiments, BsAb molecules can be created using chemical conjugation, such as chemical conjugation of an antibody and / or antibody fragment. An exemplary bispecific antibody conjugate is the CovX-body format, in which a low molecular weight drug is site-specifically conjugated to a single reactive lysine within each Fab arm or antibody or fragment. In several embodiments, conjugation improves the serum half-life.

[0196] Methods for producing multispecific antibodies, including bispecific antibodies, are known in the art. For example, multispecific antibodies, including bispecific antibodies, can be produced by separately expressing constituent antibodies in different host cells and then purifying / assembling them, or by expressing constituent antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be carried out by various methods known in the art, including affinity chromatography.

[0197] In some embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) as disclosed herein may be provided in any antibody form disclosed herein or known in the art. In non-limiting examples, in some embodiments, multispecific binding agonists (e.g., antibodies, e.g., bispecific antibodies) including multispecific binding agonists that bind to one or more targets other than human CD47 (e.g., PD-L1 including human PD-L1) may include Fab-in-tandem-Ig (FIT-Ig); DVD-Ig; hybrid hybridoma (quadroma or tetradoma); anticarin platform (Pieris); diabody; single-chain Diabody; Tandem single-chain Fv fragment; TandAb, Triple-specific Ab (Affimed); Darts biaffinity retargeting (Macrogenics); Bi-specific XmAb (Xencor); Bi-specific T cell engager (Bites; Amgen; 55kDa); Triplebody; Tribody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (Creative Biolabs); Duobody platform (Genmab); Dock and lock The following may be selected: and lock platform; knob-into-holes (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-Ig = bivariable domain immunoglobulin (Abbott); kappa-lambda body; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0198] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain and one or more additional binding domains that bind to one or more targets other than CD47. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequences of Table 1. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequences of Table 2. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequences of Table 3.

[0199] In some embodiments, multispecific (e.g., bispecific) antibodies comprising binding domains that bind to CD47, including VH and VL CDRs, as listed in Table 1, are described herein. In some embodiments, multispecific (e.g., bispecific) antibodies comprising binding domains that bind to CD47, including VH and VL CDRs, as listed in Table 2, are described herein. In some embodiments, multispecific (e.g., bispecific) antibodies comprising binding domains that bind to CD47, including VH and VL CDRs, as listed in Table 3, are described herein.

[0200] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain and a PD-L1-binding domain. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 1 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 1 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 5. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 1 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 6. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 2 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 2 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 5. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 2 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 6. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequence of Table 3 and a PD-L1-binding domain containing the VH and / or VL amino acid sequence of Table 4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain comprising the VH and / or VL amino acid sequences of Table 3 and a PD-L1-binding domain comprising the VH and / or VL amino acid sequences of Table 5.In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and / or VL amino acid sequences of Table 3 and a PD-L1-binding domain containing the VH and / or VL amino acid sequences of Table 6.

[0201] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 1 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 1 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 5. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 1 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 6. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 2 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 2 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 5. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 2 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 6. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 3 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 3 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 5. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a CD47-binding domain containing the VH and VL amino acid sequences of Table 3 and a PD-L1-binding domain containing the VH and VL amino acid sequences of Table 6.

[0202] Antibodies that bind to CD47 and / or PD-L1 can be obtained by any suitable method, for example (but not limited to), immunization with whole tumor cells containing CD47 and / or PD-L1, as well as by antibody collection, recombinant techniques, or screening of libraries of antibodies or antibody fragments using CD47 extracellular domain epitopes or PD-L1 extracellular domain epitopes. Monoclonal antibodies can be produced using various known techniques (see, for example, Coligan et al. (eds.), Current Protocols in Immunology, 1: 2.5.12.6.7 (John Wiley & Sons 1991); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). One exemplary technique for generating monoclonal antibodies involves immunizing an animal with human CD47 antigen and generating a hybridoma from spleen cells obtained from that animal. The hybridoma can be made to produce a monoclonal antibody or antibody fragment that binds to CD47. Another exemplary technique for generating monoclonal antibodies involves immunizing an animal with human PD-L1 antigen and generating a hybridoma from spleen cells taken from the animal.Hybridomas can produce monoclonal antibodies or antibody fragments that bind to PD-L1.

[0203] In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques described, for example, in *Antibody Phage Display: Methods and Protocols*, PM O'Brien and R. Aitken, eds, Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening a phage library containing phages displaying various fragments of the antibody variable region (Fv) fused to a phage coat protein. Such phage libraries are then screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen adsorb to the antigen and are therefore separated from non-binding clones in the library. The binding clones can then be eluted from the antigen and further enriched by additional cycles of antigen adsorption / elution.

[0204] For example, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994), the variable domain can be functionally displayed on the phage either as a single-chain Fv(scFv) fragment in which VH and VL are covalently linked by a short, flexible peptide, or as a Fab fragment in which VH and VL are fused to a constant domain and interact noncovalently.

[0205] As described by Winter et al., the VH gene repertoire and the VL gene repertoire can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then searched for antigen-binding clones. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), cloning a naive repertoire can provide a single source of human antibodies against a wide range of non-self antigens and similarly against self antigens without any immunization. Finally, as described, for example, in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), naive libraries can also be synthetically created by cloning unreconstituted V gene segments derived from stem cells and using PCR primers containing random sequences, in order to encode a highly variable CDR3 region and to enable reconstitution in vitro.

[0206] Library screening can be achieved by various techniques known in the art. For example, CD47 (e.g., CD47 polypeptide, fragment, or epitope) or PD-L1 (e.g., PD-L1 polypeptide, fragment, or epitope) can be used to coat wells of an adsorption plate, expressed on host cells attached to an adsorption plate, used in cell sorting, conjugated with biotin for capture using streptavidin-coated beads, or used in any other method for panning a display library. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by the use of prolonged washing and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and WO92 / 09690, as well as by the use of low antigen coating densities as described in Marks et al., Biotechnol., 10: 779-783 (1992).

[0207] Multiple specificity-binding agonists can be obtained by designing an appropriate antigen screening procedure for selecting a phage clone of interest, as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3, and then constructing a full-length multiple specificity-binding agonist (e.g., antibody) clone using VH and / or VL sequences (e.g., Fv sequences) from the phage clone of interest, or various CDR sequences from VH and VL sequences, along with an appropriate constant region (e.g., Fc) sequence.

[0208] Similarly, human antibodies that bind to CD47 and / or PD-L1 can be generated by any of several techniques, including, but not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells derived from immunized transgenic mice into which human immunoglobulin genes have been inserted, isolation from a human immunoglobulin V region phage library, or other procedures known in the art and based on this disclosure. Methods for obtaining human antibodies from transgenic animals are described, for example, in Bruggemann et al., Curr. Opin. Biotechnol., 8: 455 58, 1997; Jakobovits et al., Ann. NY Acad. Sci., 764: 525 35, 1995; Green et al., Nature Genet., 7: 13-21, 1994; Lonberg et al., Nature, 368: 856-859, 1994; Taylor et al., Int. Immun. 6: 579-591, 1994; and further in U.S. Patent No. 5,877,397.

[0209] For example, human antibodies that bind to CD47 and / or PD-L1 can be obtained from transgenic animals engineered to produce specific human antibodies in response to antigenic attack. For example, International Patent Publication WO98 / 24893 discloses a transgenic animal having a human Ig locus that does not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain loci. Transgenic non-primate mammalian hosts capable of initiating an immune response to an immunogen are also described, in which the antibody has primate constant and / or variable regions, and the endogenous immunoglobulin encoding the locus is substituted or inactivated. International Patent Publication WO96 / 30498 discloses the use of a Cre / Lox system to modify an immunoglobulin locus in a mammal, for example, by replacing all or part of the constant or variable region to form a modified antibody molecule. International Patent Publication WO94 / 02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. U.S. Patent No. 5,939,598 discloses a method for producing a transgenic mouse that lacks an endogenous heavy chain and expresses an exogenous immunoglobulin locus expressing one or more heterologous constant regions. Transgenic animals, such as the transgenic animals described herein, can be used to induce an immune response to selected antigenic molecules, and antibody-producing cells can be isolated from the animals and used to produce hybridomas that secrete human-derived monoclonal antibodies. Immunization protocols, adjuvants, etc., are known in the art and have been used, for example, in the immunization of transgenic mice as described in International Patent Publication WO96 / 33735. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effects of the corresponding protein.

[0210] This disclosure provides humanized antibodies that bind to CD47, including human CD47, and one or more non-CD47 targets (e.g., PD-L1, including human PD-L1). The humanized antibodies of this disclosure may include a first binding domain that binds to CD47 and include one or more CDRs shown in Tables 1-3. The humanized antibodies of this disclosure may include a second binding domain that binds to PD-L1 and include one or more CDRs shown in Tables 4-6. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues from a non-human source introduced into it. These non-human amino acid residues are often referred to as “implant” residues and are typically obtained from “implant” variable domains. Humanized antibodies that bind to CD47 can be prepared using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Patents 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567, all of which are expressly incorporated herein by reference).

[0211] In some cases, humanized antibodies are constructed by CDR grafting, which involves grafting the amino acid sequences of six complementarity-determining regions (CDRs) of a parent non-human antibody (e.g., rodents) onto a human antibody framework. For example, Padlan et al. (FASEB J. 9: 133-139, 1995) determined that only about one-third of the residues within the CDR actually come into contact with the antigen; these are called "specificity-determining residues" or SDRs. In SDR grafting techniques, only the SDR residues are grafted onto the human antibody framework (see, for example, Kashmiri et al., Methods 36: 25-34, 2005).

[0212] The selection of human variable domains in both the light and heavy chains used to create humanized antibodies can be crucial for reducing antigenicity. For example, the sequences of variable domains of non-human (e.g., rodent) antibodies are screened against an entire library of known human variable domain sequences according to the so-called "best fit" method. Human sequences that are closest to rodent sequences can be selected as the human framework for humanized antibodies (Sims et al. (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mol. Biol. 196: 901). Alternatively, a specific framework derived entirely from the consensus sequence of human antibodies is used, where either the light or heavy chain belongs to a particular subgroup. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623). In some cases, the framework is used for the most abundant human subclass, V L 6 Subgroup I(V L 6I) and V H Subgroup III (V H It is derived from the consensus sequence of (III). Alternatively, human germline genes are used as a source for the framework region.

[0213] In an alternative paradigm based on CDR comparison, known as superhumanization, FR homology is irrelevant. This method involves comparing non-human sequences with a functional human germline gene repertoire. Next, genes encoding canonical structures identical to or closely related to the mouse sequence are selected. Then, among the genes sharing canonical structures with non-human antibodies, those with the highest homology within the CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol. 169: 1119-1125, 2002).

[0214] Furthermore, it is generally desirable that antibodies be humanized while retaining their affinity for their antigens and other favorable biological properties. To achieve this objective, according to one method, humanized antibodies are prepared by a process in which the parental sequence and various conceptual humanized products are analyzed using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display highly accurate three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Examination of these indications allows for the analysis of residues that are likely to play a role in the function of candidate immunoglobulin sequences, for example, residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and transfer sequences, resulting in the realization of desired antibody characteristics, such as increased affinity for the target antigen(s). Generally, hypervariable region residues are most directly and substantially involved in influencing antigen binding.

[0215] Another method for antibody humanization is based on a metric for antibody humanization called Human String Content (HSC). This method compares mouse sequences with a repertoire of human germline genes and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using an overall identity metric to generate a large number of diverse humanized variants (Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

[0216] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as by bacterial colony screening (see, for example, Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60, 2004).

[0217] In the FR library method, a collection of residue variants is introduced at specific positions in the FR, and then library selection is performed to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the "Vernier" residues from a more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997) or those identified as potentially contributing to the CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992).

[0218] In FR shuffling, instead of creating a combinatorial library of selected residue variants, the entire FR is combined with a non-human CDR (see, e.g., Dall'Acqua et al., Methods 36: 43-60, 2005). The library can be screened for binding in a two-step selection process, first for humanization of the VL, and then for VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and increased thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

[0219] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and involves sequentially replacing non-human fragments with human FR libraries and assessing binding. Starting with the CDR3 region of non-human VH and VL chains, other regions, including CDR1 and CDR2 of both VH and VL of the non-human antibody, are progressively replaced with human FR. This methodology typically leads to the identification of antibodies from multiple subclasses possessing epitope retention and distinct human V-segment CDRs. Humaneering makes it possible to isolate antibodies that are 91–96% homologous to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0220] Human engineering methods involve modifying non-human antibodies or antibody fragments, such as mouse or chimeric antibodies or antibody fragments, by making specific changes to the amino acid sequence of the antibody, thereby creating modified antibodies that retain the desired binding properties of the original non-human antibody, even though their immunogenicity in humans is reduced. Generally, this technique involves classifying amino acid residues in non-human (e.g., mouse) antibodies into "low-risk," "medium-risk," or "high-risk" residues. The classification is performed using a comprehensive risk / reward calculation that evaluates the expected benefit of making a particular substitution (e.g., in terms of immunogenicity in humans) against the impact of the substitution on the folding of the resulting antibody and / or the risk of substitution with human residues. Specific human amino acid residues (e.g., low or medium risk) to be substituted at a given position in the non-human (e.g., mouse) antibody sequence can be selected by aligning the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or consensus human antibody sequence. Amino acid residues at low or medium-risk positions in the non-human sequence can be substituted with corresponding residues in the human antibody sequence according to the alignment. Techniques for creating human-engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994), U.S. Patents No. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Publication No. WO93 / 11794.

[0221] In some embodiments, the multispecific binding agonists described herein include non-antibody protein scaffolds. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, antikalin, adonectin, afibody, DARPin, finomer, afitin, afirin, avimer, cysteine-rich Nottin peptide, or engineered Knitz-type inhibitors. Methods for generating such non-antibody protein scaffolds are well known in the art, and any one of them may be used to generate multispecific binding agonists containing non-antibody protein scaffolds (see, for example, Simeon and Chen, Protein Cell, 9(1):3-14 (2018); Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1):293-320 (2017)).

[0222] Further materials are provided for generating multispecific binding agonists and fragments thereof. For example, isolated cells (e.g., hybridomas) may produce multispecific binding agonists (e.g., antibodies or antibody fragments). In this regard, cells (e.g., isolated cells) may produce antibodies or fragments thereof comprising a first binding domain containing VH and VL as shown in Tables 1, 2, or 3 for C40, C56, or C59, respectively. Alternatively, cells (e.g., isolated cells) may further produce antibodies or fragments thereof comprising a second binding domain containing VH and VL as shown in Tables 4, 5, or 6 for P22, P24, or P31.2, respectively. In some embodiments, the polynucleotides described herein may comprise one or more nucleic acid sequences encoding a multispecific binding agonist (e.g., antibodies or antibody fragments). In some embodiments, the polynucleotides are isolated and / or recombinant polynucleotides. In various embodiments, the isolated polynucleotides comprise an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VH). L It contains a nucleotide sequence that codes for V H and V LThis includes a complementarity-determining region (CDR) identical to the CDR shown in Table 1, the CDR shown in Table 2, or the CDR shown in Table 3. In various embodiments, the isolated polynucleotide contains the antibody heavy chain variable region (VH) and / or the antibody light chain variable region (VH). L It contains a nucleotide sequence that codes for V H and V L This includes a complementarity determination region (CDR) identical to the CDR shown in Table 4, the CDR shown in Table 5, or the CDR shown in Table 6.

[0223] In some embodiments, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides for expressing one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying polynucleotides in a host cell to produce a useful quantity thereof, and for expressing binding agents such as antibodies or antibody fragments using recombinant techniques.

[0224] In some embodiments, one or more vectors are expression vectors, where one or more polynucleotides are operatively ligated to one or more polynucleotides containing expression regulatory sequences. Autonomous recombinant expression constructs, such as plasmids and viral DNA vectors, incorporating one or more polynucleotides encoding antibody sequences that bind to CD47 are particularly intended. Expression regulatory DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct is used. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, while operator sequences are generally selected for their ability to regulate gene expression. The expression construct may also include sequences encoding one or more selection markers that enable the identification of the host cell carrying the construct. The expression construct may also include sequences that facilitate, preferably promote, homologous recombination in the host cell. In some embodiments, the expression construct may also include sequences necessary for replication in the host cell.

[0225] Examples of regulatory sequences include promoter / enhancer sequences for expression in mammalian cells, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41: 521-530, 1985); Roussarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); Simian virus 40 promoter; DRA (downregulated in adenoma; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293: G923-G934). Examples include MCT1 (monocarboxylic acid transporter 1; Cuff et al., Am. J. Physiol. Gastrointet. Liver Physiol., G977-G979. 2005); and Math1 (mouse atnal homolog 1; Shroyer et al., Gastroenterology, 132: 2477-2478, 2007), where the promoter is operatively linked upstream (e.g., 5') of the polypeptide coding sequence. In other variants, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. Polynucleotides may also optionally include a suitable polyadenylated sequence (e.g., SV40 or the human growth hormone gene polyadenylated sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0226] If desired, one or more polynucleotides may also optionally include nucleotide sequences encoding a secretion signal peptide fused in-frame with the polypeptide sequence. The secretion signal peptide directs the secretion of an antibody polypeptide by cells expressing one or more polynucleotides, which are then cleaved from the secreted polypeptide by the cells. One or more polynucleotides may also optionally include further sequences intended solely to facilitate the large-scale production of the vector. The production and administration of polynucleotides for gene therapy can be carried out using procedures described in the literature on various transgenes. See, for example, Isner et al., Circulation, 91: 2687-2692, 1995; and Isner et al., Human Gene Therapy, 7: 989-1011, 1996.

[0227] In some embodiments, the polynucleotide may further include additional sequences to facilitate the uptake and expression of the antibody or its fragment (and / or any other peptide) by the host cell. In some embodiments, a “naked” transgene encoding the antibody or its fragment described herein (e.g., a transgene without a virus, liposome, or other vector to facilitate transfection) is used.

[0228] Using any suitable vector, one or more polynucleotides encoding an antibody or a fragment thereof can be introduced into a host. The exemplary vectors listed are not limited to those listed above, but include replication-deficient retroviral vectors, including lentiviral vectors (Kim et al., J. Virol., 72(1): 811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46); parvovirus vectors, such as adeno-associated virus (AAV) vectors (US Patent Nos. 5,474,935; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko et al., J. Invest.). Med., 45: 87-98, 1997); Adenovirus (AV) vector (US Patent Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, 1992; Stratford Perricaudet et al., J. Clin. Invest., 90: 626-630, 1992; and Rosenfeld et al., Cell, 68: 143-155, Examples include adenovirus adeno-associated virus chimeras (U.S. Patent No. 5,856,152) or vaccinia virus or herpesvirus vectors (U.S. Patents No. 5,879,934; No. 5,849,571; No. 5,830,727; No. 5,661,033; No. 5,328,688); lipofectin-mediated gene transfer (BRL); liposomal vectors (U.S. Patent No. 5,631,237); and combinations thereof.These expression vectors can all be prepared using standard recombinant DNA techniques described, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). If necessary, replication deficiency can be conferred to the viral vector, for example, by deleting or disrupting select genes necessary for viral replication.

[0229] Other intended nonviral delivery mechanisms include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10: 689-695, 1990), DEAE-dextran (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81: 7161-7165, 1984), and direct microinjection (Harland and Weintraub, J. Cell Biol., 101: 1094-1099, 1985, DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979; Felgner, Sci Am., 276(6): 102-6, 1997; Felgner, Hum Gene Ther., 7(15): 1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 8463-8467, 1987), gene guns using high-speed microparticle guns (Yang et al., Examples include Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990, and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167, 1993).

[0230] Expression vectors (or antibodies or fragments thereof as discussed herein) can be encapsulated in liposomes. See, for example, Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275 (5301): 810-814, 1997). Various commercial methods involving “lipofection” techniques are also intended. In some embodiments, liposomes are complexed with Sendai virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote the entry of liposome-encapsulated DNA into cells (Kaneda et al., Science, 243: 375-378, 1989). In some embodiments, liposomes are complexed with or used in combination with nuclear nonhistone chromosome protein (HMG-1) (Kato et al., J. Biol. Chem., 266: 3361-3364, 1991). In some embodiments, liposomes are complexed with or used in combination with both HVJ and HMG-1. Such expression constructs have been successfully used for nucleic acid transfer and expression in vitro and in vivo. In some embodiments, multispecific binding agonists (e.g., antibodies), including multispecific binding agonists that bind to CD47, including human CD47, and one or more non-CD47 targets (e.g., PD-L1, including human PD-L1), are included in the liposomes to target cells expressing CD47 and / or PD-L1 on their surface (e.g., tumor cells).

[0231] Cells may contain one or more polynucleotides or one or more vectors. For example, cells may be transformed or transfected with one or more polynucleotides encoding a multispecific binding agonist (e.g., an antibody) that includes a multispecific binding agonist that binds to one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), including a multispecific binding agonist, including a multispecific binding agonist that binds to one or more targets other than CD47 (e.g., PD-L1 including human PD-L1), including a multispecific binding agonist having at least 75% identity with the C40 CDR (see, for example, Table 1). In some embodiments, cells express a multispecific binding agonist that includes a multispecific binding agonist that binds to CD47, including human CD47, and one or more non-CD47 targets (e.g., PD-L1, including human PD-L1), having at least 75% identity with the C56 CDR (see, for example, Table 2). In some embodiments, cells express a multispecific binding agonist that includes a multispecific binding agonist that binds to CD47, including human CD47, and one or more non-CD47 targets (e.g., PD-L1, including human PD-L1), having at least 75% identity with the C59 CDR (see, for example, Table 3). In some embodiments, cells express a multispecific binding agonist that includes a multispecific binding agonist that binds to CD47, including human CD47, and PD-L1, including human PD-L1, having at least 75% identity with the P22 CDR (see, for example, Table 4). In some embodiments, the cells express multispecific binding agonists, including multispecific binding agonists that bind to CD47, including human CD47, and PD-L1, including human PD-L1, having at least 75% identity with the P24 CDR (see, for example, Table 5).In some embodiments, cells express multispecific binding...

Claims

1. A multispecific antibody or its antigen-binding fragment having a first binding domain that binds to CD47 and a second binding domain that binds to PD-L1, The first binding domain is From an antibody containing the VH sequence, which is sequence number 25, and the VL sequence, which is sequence number 26; From an antibody containing the VH sequence, which is sequence number 51, and the VL sequence, which is sequence number 52; or From an antibody containing the VH sequence (SEQ ID NO: 77) and the VL sequence (SEQ ID NO: 78) It includes all three heavy chain complementarity-determining regions (CDRs) and all three light chain CDRs. The second binding domain described above is From an antibody containing the VH sequence, which is sequence number 155, and the VL sequence, which is sequence number 156; From an antibody containing the VH sequence, which is sequence number 103, and the VL sequence, which is sequence number 104; or The antibody contains all three heavy chain CDRs and all three light chain CDRs from the antibody containing the VH sequence (SEQ ID NO: 129) and the VL sequence (SEQ ID NO: 130), A multispecific antibody or its antigen-binding fragment.

2. The first binding domain is (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18 Ｈ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24 Ｈ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21 Ｌ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22 Ｌ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 1, comprising:

3. The first binding domain is (i) (a) V having the amino acid sequence of SEQ ID NO: 1 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 4 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 Light chain variable (V) Ｌ )region, (ii) (a) (1) V having the amino acid sequence of SEQ ID NO: 7 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 8 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 9 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 10 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iii) (a) (1) V having the amino acid sequence of SEQ ID NO: 12 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 4 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iv) (a) (1) V having the amino acid sequence of SEQ ID NO: 13 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 14 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 15 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 16 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 17 Ｌ CDR3 Light chain variable (V) Ｌ )region, (v) (a) (1) V having the amino acid sequence of SEQ ID NO: 18 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 19 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 20 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 21 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 22 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 23 Ｌ CDR3 Light chain variable (V) Ｌ ) area, or (vi) (a) (1) V having the amino acid sequence of SEQ ID NO: 1 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 24 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 4 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 Light chain variable (V) Ｌ )region, A multispecific antibody or its antigen-binding fragment according to claim 2, comprising:

4. The first binding domain is (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 33, 38, 39, and 44 Ｈ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 34, 40, 45, and 50 Ｈ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 35, 41, and 46 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, 42, and 47 Ｌ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 37, and 48 Ｌ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 43, and 49 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 1, comprising:

5. The first binding domain is (i) (a) (1) V having the amino acid sequence of SEQ ID NO: 27 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 30 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 Light chain variable (V) Ｌ )region, (ii) (a) (1) V having the amino acid sequence of SEQ ID NO: 33 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 34 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 35 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 36 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 37 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iii) (a) (1) V having the amino acid sequence of SEQ ID NO: 38 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 30 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iv) (a) (1) V having the amino acid sequence of SEQ ID NO: 39 Ｈ CDR1, (2)V having the amino acid sequence of SEQ ID NO: 40 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 41 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 42 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 37 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 43 Ｌ CDR3 Light chain variable (V) Ｌ )region, (v) (a) (1) V having the amino acid sequence of SEQ ID NO: 44 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 45 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 46 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 47 Ｌ CDR1, (2) V having the amino acid sequence of Sequence ID No. 48 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 49 Ｌ CDR3 Light chain variable (V) Ｌ ) area, or (vi) (a) (1) V having the amino acid sequence of SEQ ID NO: 27 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 50 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 30 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 4, comprising:

6. The first binding domain is (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 59, 64, 65, and 70 Ｈ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 60, 66, 71, and 76 Ｈ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 61, 67, and 72 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 62, 68, and 73 Ｌ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 63, and 74 Ｌ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 69, and 75 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 1, comprising:

7. The first binding domain is (i) (a) (1) V having the amino acid sequence of SEQ ID NO: 53 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 55 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 56 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 Light chain variable (V) Ｌ )region, (ii) (a) (1) V having the amino acid sequence of SEQ ID NO: 59 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 60 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 61 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 62 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iii) (a) (1) V having the amino acid sequence of SEQ ID NO: 64 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 55 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 56 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iv) (a) (1) V having the amino acid sequence of SEQ ID NO: 65 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 66 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 67 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 68 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 69 Ｌ CDR3 Light chain variable (V) Ｌ )region, (v) (a) (1) V having the amino acid sequence of SEQ ID NO: 70 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 71 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 72 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 73 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 74 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 75 Ｌ CDR3 Light chain variable (V) Ｌ ) area, or (vi) (a) (1) V having the amino acid sequence of SEQ ID NO: 53 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 76 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 55 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 56 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 6, comprising:

8. The second binding domain described above is (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 131, 137, 142, 143, and 148 Ｈ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 132, 138, 144, 149, and 154 Ｈ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 133, 139, 145, and 150 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 140, 146, and 151 Ｌ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 135, 141, and 152 Ｌ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 136, 147, and 153 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 7, comprising:

9. The second binding domain described above is (i) (a) (1) V having the amino acid sequence of SEQ ID NO: 131 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 132 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 134 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 Ｌ CDR3 Light chain variable (V) Ｌ )region, (ii) (a) (1) V having the amino acid sequence of SEQ ID NO: 137 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 138 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 139 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 140 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 141 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iii) (a) (1) V having the amino acid sequence of SEQ ID NO: 142 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 132 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 134 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iv) (a) (1) V having the amino acid sequence of SEQ ID NO: 143 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 144 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 145 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 146 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 141 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 147 Ｌ CDR3 Light chain variable (V) Ｌ )region, (v) (a) (1) V having the amino acid sequence of SEQ ID NO: 148 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 149 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 150 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 151 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 152 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 153 Ｌ CDR3 Light chain variable (V) Ｌ ) area, or (vi) (a) (1) V having the amino acid sequence of SEQ ID NO: 131 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 154 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 133 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 134 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 135 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 136 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 8, comprising:

10. The second binding domain described above is (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 85, 90, 91 and 96 Ｈ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 86, 92, 97 and 102 Ｈ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 87, 93, and 98 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 88, 94, and 99 Ｌ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 83, 89 and 100. Ｌ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 95 and 101 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 7, comprising:

11. The second binding domain described above is (i) (a) (1) V having the amino acid sequence of SEQ ID NO: 79 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 82 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 83 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 Ｌ CDR3 Light chain variable (V) Ｌ )region, (ii) (a) (1) V having the amino acid sequence of SEQ ID NO: 85 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 86 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 87 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 88 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 89 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iii) (a) (1) V having the amino acid sequence of SEQ ID NO: 90 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 80 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 82 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 83 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iv) (a) (1) V having the amino acid sequence of SEQ ID NO: 91 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 92 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 93 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 94 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 89 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 95 Ｌ CDR3 Light chain variable (V) Ｌ )region, (v) (a) (1) V having the amino acid sequence of SEQ ID NO: 96 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 97 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 98 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 99 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 100 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 101 Ｌ CDR3 Light chain variable (V) Ｌ ) area, or (vi) (a) (1) V having the amino acid sequence of SEQ ID NO: 79 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 102 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 81 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 82 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 83 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 84 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 10, comprising:

12. The second binding domain described above is (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 105, 111, 116, 117, and 122 Ｈ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 112, 118, 123 and 128 Ｈ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 113, 119, and 124 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 108, 114, 120, and 125 Ｌ CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 115 and 126 Ｌ CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 121 and 127 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 7, comprising:

13. The second binding domain described above is (i) (a) (1) V having the amino acid sequence of SEQ ID NO: 105 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 106 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 108 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 109 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 Ｌ CDR3 Light chain variable (V) Ｌ )region, (ii) (a) (1) V having the amino acid sequence of SEQ ID NO: 111 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 112 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 113 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 114 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 115 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iii) (a) (1) V having the amino acid sequence of SEQ ID NO: 116 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 106 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 108 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 109 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 Ｌ CDR3 Light chain variable (V) Ｌ )region, (iv) (a) (1) V having the amino acid sequence of SEQ ID NO: 117 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 118 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 119 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 120 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 115 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 121 Ｌ CDR3 Light chain variable (V) Ｌ )region, (v) (a) (1) V having the amino acid sequence of SEQ ID NO: 122 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 123 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 124 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 125 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 126 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 127 Ｌ CDR3 Light chain variable (V) Ｌ ) area, or (vi) (a) (1) V having the amino acid sequence of SEQ ID NO: 105 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 128 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 107 Ｈ CDR3 Heavy chain variable (V) Ｈ ) domain, and (b) (1) V having the amino acid sequence of SEQ ID NO: 108 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 109 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 110 Ｌ CDR3 Light chain variable (V) Ｌ )region A multispecific antibody or its antigen-binding fragment according to claim 12, comprising:

14. The first binding domain V Ｈ Area and / or V Ｌ A multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the region further comprises (i) a human framework sequence and / or (ii) framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

15. The multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 14, wherein the first binding domain comprises VH containing the amino acid sequence of SEQ ID NO: 25 and VL containing the amino acid sequence of SEQ ID NO:

26.

16. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the first binding domain comprises VH containing the amino acid sequence of SEQ ID NO: 51 and VL containing the amino acid sequence of SEQ ID NO:

52.

17. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the first binding domain comprises VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO:

78.

18. The V of the second bonding domain Ｈ Area and / or V Ｌ A multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the region further comprises (i) a human framework sequence and / or (ii) framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

19. The multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 18, wherein the second binding domain comprises VH containing the amino acid sequence of SEQ ID NO: 155 and VL containing the amino acid sequence of SEQ ID NO:

156.

20. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the second binding domain comprises VH containing the amino acid sequence of SEQ ID NO: 103 and VL containing the amino acid sequence of SEQ ID NO:

104.

21. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the second binding domain comprises VH containing the amino acid sequence of SEQ ID NO: 129 and VL containing the amino acid sequence of SEQ ID NO:

130.

22. The multispecific antibody or antigen-binding fragment according to any one of claims 1 to 21, wherein the multispecific antibody is a recombinant antibody.

23. The multispecific antibody or antigen-binding fragment according to any one of claims 1 to 22, wherein the multispecific antibody is a bispecific antibody.

24. The multispecific antibody or antigen-binding fragment according to any one of claims 1 to 23, wherein the multispecific antibody or antigen-binding fragment thereof is conjugated with a diagnostic agent, detection agent or therapeutic agent, or is fused with it by recombination.

25. The multispecific antibody or antigen-binding fragment according to claim 24, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxic agent, or a drug.

26. One or more polynucleotides encoding a multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 25.

27. One or more vectors comprising one or more polynucleotides encoding a multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 25.

28. A cell comprising one or more polynucleotides according to claim 26, or one or more vectors according to claim 27.

29. A pharmaceutical composition comprising a multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 25, and a pharmaceutically acceptable carrier.

30. A composition or pharmaceutical composition according to claim 29, for use in a method for treating cancer or a tumor in a subject, alleviating one or more symptoms associated with cancer or a tumor in a subject, reducing tumor size in a subject having a tumor, enhancing tumor cell removal in a subject having a tumor, treating a phagocytosis disorder, impairment or condition in a subject, increasing phagocytosis by immune cells in a subject, treating a T cell dysfunction disorder, impairment or condition in a subject, or enhancing T cell function in a subject, wherein the method comprises the step of administering the composition or pharmaceutical composition to the subject.

31. The composition for use according to claim 30, wherein the target is administered in combination with one or more therapeutic agents, the composition or the pharmaceutical composition.

32. Use of a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 25 or a pharmaceutical composition according to claim 29 in the manufacture of a pharmaceutical for the purpose of treating cancer or tumors in a subject, alleviating one or more symptoms associated with cancer or tumors in a subject, reducing tumor size in a subject having a tumor, enhancing tumor cell removal in a subject having a tumor, treating phagocytosis, disorder or condition in a subject, increasing phagocytosis by immune cells in a subject, treating T cell dysfunction, disorder or condition in a subject, or enhancing T cell function in a subject, wherein the pharmaceutical is administered to the subject.

33. The use according to claim 32, wherein the subject is administered in combination with one or more therapeutic agents.

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