Multispecific binding agents and uses thereof

Abstract

The present disclosure provides multispecific binding agents (e.g., antibodies, such as bispecific antibodies) having a first binding domain that binds CD47, including human CD47, and one or more additional binding domains that bind one or more targets that are not CD47, e.g., PD-L1, and uses thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 425,960, filed November 16, 2022, and U.S. Provisional Patent Application No. 63 / 308,485, filed February 9, 2022, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a computer-readable Sequence Listing that has been submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence XML file submitted with this application is titled "14529-129-228_SEQ_LISTING.xml", was created on February 8, 2023, and is 96,794 bytes in size.

[0003] Field The present disclosure relates generally to multispecific binding agents, e.g., 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 their use. [Background technology]

[0004] Tumor cells have been shown to utilize both innate and adaptive checkpoints to evade antitumor immune responses. CD47 and PD-L1 are two targets widely expressed on the cell surface of tumor cells, which can coordinately suppress innate and adaptive sensing, respectively, to evade immune control.

[0005] 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 interacts with SIRPα on the surface of macrophages and dendritic cells, inducing a "don't eat me" signal, thereby inhibiting phagocytosis.

[0006] CD47 expression allows tumor cells to evade phagocytosis and escape innate immune surveillance. Therefore, CD47 has been identified as a potential therapeutic target. However, CD47 is widely expressed on normal cells, such as hematopoietic cells, red blood cells (RBCs), and platelets. Widespread expression of CD47 by healthy cells raises safety and efficacy concerns because targeting CD47 with neutralizing antibodies may affect healthy cells and potentially lead to toxic effects. Furthermore, widespread CD47 expression may lead to rapid clearance of CD47-binding agents, resulting in poor pharmacokinetics and reduced efficacy.

[0007] Furthermore, activation or loss of CD47 can result in enhanced proliferation in a cell type-dependent manner. For example, increased proliferation of astrocytoma cells, but not normal astrocytes, has been shown after activation of CD47 and TSP-1. It has also been proposed that CD47 can promote cancer cell proliferation through the PI3K / Akt pathway.

[0008] Many of the reported anti-CD47 antibodies are known to cause RBC aggregation upon inhibition of CD47 binding to SIRPα, thereby significantly reducing the therapeutic efficacy of such antibodies.

[0009] Programmed death-ligand 1 (PD-L1) is a cell surface glycoprotein ligand that specifically binds to the key immune checkpoint receptor, programmed death receptor 1 (PD-1). PD-1 is upregulated on activated T cells, B cells, and monocytes and mediates immunosuppression. The other PD-1 ligand, PD-L2, is primarily expressed on activated antigen-presenting cells (APCs), whereas PD-L1 is broadly expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, and other hematopoietic lineage cells, as well as peripheral tissues such as the heart, skeletal muscle, placenta, lung, kidney, and liver.

[0010] Binding of PD-L1 to PD-1 is a negative checkpoint that can activate downstream signaling of the PD-1 receptor in T cells, thus inhibiting T cell proliferation, cytokine production and release, and cytotoxicity. This inhibition of T cell activation and effector cytokine secretion can prevent autoimmunity 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 its overexpression is often associated with poor prognosis. In cancer, PD-1 / PD-L1 interaction stimulates downstream signals to suppress T cell activation and result in tumor cell survival.

[0012] Blocking PD-1 interaction with its ligand has been proposed as an immunotherapeutic method to enhance T cell immune responses against tumor cells. Current strategies of PD-1 / PD-L1-based immunotherapy have shown efficacy in treating some advanced cancers, but have limited effectiveness against many solid tumors and certain specific PD-L1 functions. Therefore, there remains an urgent need in the art for agents that can inhibit or prevent PD-1 / PD-L1 interaction.

[0013] Thus, there remains a need for agents that overcome these concerns and target CD47 and other targets, e.g., PD-L1, to treat, prevent, or ameliorate immune cell dysfunction diseases, disorders, or conditions, including those involving tumor cells that express CD47 and / or PD-L1. The multispecific binding agents, compositions, and methods provided herein fulfill this need and offer related advantages. Summary of the Invention

[0014] The present disclosure provides multispecific binding agents (e.g., antibodies such as bispecific antibodies) that have 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 that are not CD47 (e.g., PD-L1). Such agents include multispecific antibodies (e.g., antibodies such as bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1), such as multispecific antibodies that have 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 that are not CD47 (e.g., PD-L1). In some embodiments, such agents include multispecific antibodies (e.g., bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1), e.g., multispecific 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 that are not CD47 (e.g., PD-L1), where the first binding domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences set forth in Table 1, and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences set forth in Table 1, or the multispecific antibody competes for binding to human CD47 with an antibody having a heavy chain variable region and a light chain variable region described herein (e.g., Table 1).

[0015] The present disclosure also provides nucleic acids encoding the multispecific binding agents provided herein (e.g., antibodies or fragments thereof), vectors containing one or more of such nucleic acids, and cells expressing them.

[0016] The present disclosure also provides compositions comprising the multispecific binding agents described herein. In some embodiments, such compositions include multispecific antibodies (e.g., antibodies such as bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1), e.g., multispecific 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 that are not CD47 (e.g., PD-L1). In some embodiments, such compositions include multispecific antibodies (e.g., antibodies such as bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1), e.g., multispecific 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 that are not CD47 (e.g., PD-L1), where the first binding domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences set forth in Table 1, and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences set forth in Table 1, or the multispecific antibody competes for binding to human CD47 with an antibody having a heavy chain variable region and a light chain variable region described herein (e.g., Table 1).

[0017] 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 an immune cell dysfunction disease, disorder, or condition, using a multispecific binding agent, including a bispecific antibody or a composition comprising a bispecific antibody, or a composition comprising a multispecific binding agent, 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 that are not 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 that are not CD47 (e.g., PD-L1). In some embodiments, such compositions include multispecific antibodies (e.g., antibodies such as bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1), e.g., multispecific antibodies that have 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 that are not CD47 (e.g., PD-L1), and that compete for binding to human CD47 with antibodies having heavy chain and light chain variable regions described herein (e.g., in Table 1). [Brief explanation of the drawings]

[0018] [Figure 1A] 1 shows exemplary results from a cell binding assay, further described in Example 2. Binding of bsAb1 to PD-L1-expressing MDA-MB-231 cells (referred to herein as PDL1-MDA-MB-231 cells) is shown. [Figure 1B] 1 shows exemplary results from a cell binding assay, further described in Example 2. Binding of bsAb1 to NCI-H292 is shown. [Figure 1C]1 shows exemplary results from a cell binding assay, further described in Example 2. Binding of bsAb1 to HT-1080 is shown. [Figure 1D] 1 shows exemplary results from a cell binding assay, further described in Example 2. Illustrates the lack of binding of bsAb1 to RBCs. [Figure 1E] 1 shows exemplary results from a cell binding assay, further described in Example 2. Binding of bsAb1 to NCI-H292 with or without RBCs is shown.

[0019] [Figure 2] Two exemplary results from a CD47 / SIRPα inhibition assay, further described in Example 3, are shown.

[0020] [Figure 3] Two exemplary results from a PD-L1 / PD-1 inhibition assay, further described in Example 3, are shown.

[0021] [Figure 4A] 1 shows exemplary results from a phagocytosis assay, further described in Example 4. [Figure 4B] 1 shows exemplary results from a phagocytosis assay, further described in Example 4. [Figure 4C] 1 shows exemplary results from a phagocytosis assay, further described in Example 4. [Figure 4D] 1 shows exemplary results from a phagocytosis assay, further described in Example 4.

[0022] [Figure 5A] 1 shows exemplary results from a hemagglutination assay, further described in Example 5. [Figure 5B] 1 shows exemplary results from a hemagglutination assay, further described in Example 5. [Figure 5C] 1 shows exemplary results from a hemagglutination assay, further described in Example 5. [Figure 5D] 1 shows exemplary results from a hemagglutination assay, further described in Example 5.

[0023] [Figure 6] 1 shows exemplary results from SEC chromatography, as further described in Example 6.

[0024] [Figure 7] 1 shows exemplary results from HIC chromatography, as further described in Example 6.

[0025] [Figure 8] 1 shows exemplary results from SMAC chromatography, as further described in Example 6.

[0026] [Figure 9A] 1 shows exemplary results from an in vivo efficacy study, further described in Example 7. [Figure 9B] 1 shows exemplary results from an in vivo efficacy study, further described in Example 7.

[0027] [Figure 10A] Illustrative results are shown from testing in non-human primates (NHPs), as further described in Example 8. [Figure 10B] Illustrated are exemplary results tested in non-human primates (NHPs), as further described in Example 8. [Figure 10C] Illustrated are exemplary results tested in non-human primates (NHPs), as further described in Example 8.

[0028] [Figure 11] Figure 1 shows an exemplary Biacore sensorgram of bsAb1 binding to human PD-L1, as further described in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure provides multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1). Such multispecific binding agents include antibodies (e.g., antibodies, e.g., bispecific antibodies) that bind to CD47, including antibodies that bind to human CD47 and one or more additional targets that are not CD47 (e.g., PD-L1). Such multispecific binding agents are useful in compositions and methods that treat, prevent, or alleviate immune cell dysfunction diseases, disorders, or conditions (e.g., phagocyte dysfunction diseases, disorders, or conditions or T cell dysfunction diseases, disorders, or conditions), including one or more symptoms of the disease, disorder, or condition. Phagocytic cell dysfunction diseases, disorders, and conditions include immuno-oncology and related cancers, including, but not limited to, any cancer in which tumor cells express or overexpress CD47. T cell dysfunction diseases, disorders, and conditions include immuno-oncology and related cancers, including, but not limited to, any cancer in which tumor cells express or overexpress PD-L1. Such CD47- and / or PD-L1-expressing tumor cells may aid the tumor cells in evading immune surveillance and clearance (e.g., tumor immunity). Furthermore, the multispecific binding agents described herein, e.g., multispecific antibodies (e.g., 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, enhancing phagocyte function and / or immune surveillance, and enhancing tumor cell elimination. The multispecific binding agents (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 phagocyte function and T cell function, including upregulation of cell-mediated immune responses.

[0030] Provided herein, inter alia, are binding agents comprising (i) a first means for inhibiting the interaction of PD-L1 with PD-1, and (ii) a second means for inhibiting the interaction of CD47 with SIRPα. In some embodiments, the first means has high affinity for PD-L1 (e.g., human PD-L1 or cynoPD-L1), and the second means has detuned and / or intermediate affinity for CD47 (e.g., human CD47 or cynoCD47). In some embodiments, the second means enables tight binding to tumor cells while minimizing binding to red blood cells (RBCs). In some embodiments, the first means has a KD of less than 1 nM or less than 0.1 nM, e.g., about 0.05 nM, for interaction with PD-L1, and the second means has a KD of (i) greater than 0.1 nM, greater than 1 nM, greater than 5 nM, greater than 10 nM, greater than 15 nM, or greater than 20 nM, and (ii) less than 1 μM, less than 500 nM, less than 100 nM, less than 50 nM, or less than 30 nM, for interaction with CD47. In some embodiments, the second means has a KD of about 0.1 nM to about 1 μM (including each number and range therebetween), e.g., about 1 nM to about 100 nM, or about 10 nM to about 50 nM, or about 20 to about 30 nM, for interaction with CD47. In some embodiments, the binding agent is a bispecific antibody comprising a binding domain that binds PD-L1 and a binding domain that binds CD47. Also provided herein are nucleic acids encoding the binding agents, pharmaceutical compositions comprising the binding agents or nucleic acids, and uses of the binding agents and pharmaceutical compositions.

[0031] The techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed by those skilled in the art using conventional methodologies, such as the widely used methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For purposes of interpreting this specification, the following terminology applies, and whenever appropriate, terms used in the singular also include the plural and vice versa. In the event that any of the explanations of terms provided conflict with any document incorporated herein by reference, the explanations of terms provided below shall control.

[0032] The terms "CD47," "cluster of differentiation 47," or "CD47 polypeptide," and similar terms, unless otherwise specified, refer to a polypeptide ("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 (cynos)), dogs, and rodents (e.g., mice and rats). CD47, also known in the art as integrin-associated protein (IAP), has an extracellular N-terminal IgV domain, five transmembrane domains, 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 the four known alternatively spliced ​​isoforms of CD47 that differ in the length of their intracellular tails. 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, which leads to cell signaling, including, inter alia, inhibition of phagocytosis by macrophages. The full-length amino acid sequence of human CD47 is provided below (exemplary extracellular domain = underlined letters): [ka]

[0033] Other related CD47 polypeptides, also encompassed by the term CD47, include fragments, derivatives (e.g., substitution, deletion, truncation, and insertion variants), fusion polypeptides, and interspecies homologs sufficient to retain CD47 activity and / or generate an anti-CD47 immune response. As those skilled in the art will appreciate, the multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) described herein can bind to CD47 polypeptides, CD47 polypeptide fragments, CD47 antigens, and / or CD47 epitopes. An epitope can be part of a larger CD47 antigen, which in turn can be part of a larger CD47 polypeptide fragment, which can be part of a larger CD47 polypeptide. CD47 can exist in its native or denatured form. The CD47 polypeptides described herein can be isolated from various sources, e.g., from human tissue types or other sources, or prepared by recombinant or synthetic methods. CD47 polypeptides can include polypeptides having the same amino acid sequence as the corresponding CD47 polypeptide derived from nature. Orthologs to CD47 polypeptides are also well known in the art.

[0034] The terms "SIRPα," "signal-regulatory protein alpha," or "signal-regulatory protein α," and similar terms, unless otherwise specified, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native SIRPα from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynos)), dogs, and rodents (e.g., mice and rats). SIRPα has an extracellular region containing three immunoglobulin superfamily domains—a single V-set and two C1-set IgSF domains, a transmembrane domain, and a cytoplasmic region containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The term SIRPα also encompasses naturally occurring variants of SIRPα, such as SNP variants, splice variants, and allelic variants. It is known in the art that SIRPα interacts with CD47, leading to phosphorylation of the ITIMs, which in turn mediates its association with the phosphatase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2). The full-length amino acid sequence of human SIRPα is provided below: [ka]

[0035] The terms "programmed death-ligand-1 (PD-L1)," "programmed death-ligand-1," "PD-1 ligand 1," or similar terms, unless otherwise indicated, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native PD-L1 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 cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein in humans that is encoded by the CD274 gene. PD-L1 is one of two naturally occurring cell surface glycoprotein ligands for PD-1 (the other is 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. PD-L1 is known in the art to down-regulate T cell activation and cytokine secretion upon binding to PD-1. The term PD-L1 encompasses "full-length" PD-L1, as well as any form of PD-L1 resulting from processing in cells, or any fragment thereof. 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 text): [ka]

[0036] Other related PD-L1 polypeptides that are also encompassed by the term PD-L1 include fragments, derivatives (e.g., substitution, deletion, truncation, and insertion variants), fusion polypeptides, and interspecies homologs that retain PD-L1 activity and / or are sufficient to generate an anti-PD-L1 immune response. As one of skill in the art will appreciate, the PD-L1 binding agents (e.g., antibodies) described herein may bind to a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 antigen, and / or a PD-L1 epitope. An epitope may be part of a larger PD-L1 antigen that may be part of a larger PD-L1 polypeptide fragment, which in turn may be part of a larger PD-L1 polypeptide. PD-L1 may exist in its native form or in a denatured form. The PD-L1 polypeptides described herein may be isolated from a variety of sources, for example, from human tissue types or another source, or prepared by recombinant or synthetic methods. PD-L1 polypeptides may include polypeptides having the same amino acid sequence as the corresponding PD-L1 polypeptide derived from nature. Orthologues to PD-L1 polypeptides are also well known in the art.

[0037] The terms "programmed cell death-1 (PD-1)," "programmed death-1," "PD-1 receptor," or similar terms, unless otherwise indicated, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native PD-1 from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys (cynos)), dogs, and rodents (e.g., mice and rats). PD-1, also known as CD279 (cluster of differentiation 279), is an immunoinhibitory receptor belonging to the CD28 family. PD-1 is predominantly expressed in vivo on preactivated 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: an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-dependent switch motif (ITSM). The term PD-1 encompasses "full-length" PD-1 as well as any form of PD-1 resulting from processing in cells or any fragment thereof. The term PD-1 also encompasses naturally occurring variants of PD-1, such as SNP variants, splice variants, and allelic variants. It is 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]

[0038] As used herein, the term "binding agent" or grammatical equivalents refers to a molecule (e.g., an antibody, e.g., a bispecific antibody) having one or more antigen-binding sites that bind to an antigen. In some embodiments, the multispecific binding agents described herein are antibodies, antibody fragments, or other peptide-based molecules that bind to CD47 and / or PD-L1, e.g., human CD47 and / or PD-L1.

[0039] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and in the broadest sense to specifically cover, 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 with multi-epitope specificity or single-epitope specificity, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies. The present disclosure also includes antibody fragments (and / or polypeptides comprising antibody fragments) that retain the characteristic of binding to CD47 and / or PD-L1. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, dual-variable region antibodies, single-variable region antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, di-diabodies, disulfide-linked Fvs (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments consisting of non-covalently coupled heavy and light chain variable regions). Generally speaking, the variable (V) region domains can be immunoglobulin heavy chain (VH) and / or light chain (VL) variable domains in any suitable arrangement. For example, the present disclosure also includes tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region domain may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers that bind to CD47 or PD-L1. If desired, the VH and VL chains may be covalently coupled, either directly or through a linker, to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein under the category of "antibody fragments." Another form of antibody fragment is a peptide comprising one or more complementarity-determining regions (CDRs) of an antibody.CDR (also referred to as "minimal recognition unit" or "hypervariable region") can be obtained by constructing a polynucleotide encoding the desired CDR. Such a polynucleotide can be 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," 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," 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, intrabodies, diabodies, triabodies, tetrabodies, variable domains of neo-antigen receptors (v-NARs), and bis-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). Binding agents, in some embodiments, contain one or more constant regions, including light and / or heavy chain constant regions, e.g., one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies can comprise any of the epitope-binding fragments described above.The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibodies can be agonist or antagonist antibodies.

[0040] The term "monospecific," as used herein, when used in reference to a binding agent (e.g., an antibody), refers to a binding agent that has one or more binding sites that each bind to the same epitope of the same antigen.

[0041] The term "multispecific," when used in reference to a binding agent (e.g., an antibody), means that the binding agent has binding specificities for at least two different antigens or at least two different epitopes on the same antigen (e.g., a bispecific antibody to CD47 having a first binding site for a first epitope of CD47 and a second binding site for a second epitope of CD47).

[0042] The term "bispecific," when used with respect to a binding agent (e.g., an antibody), means that the binding agent is capable of specifically binding to two distinct antigenic determinants (e.g., epitopes), e.g., two binding sites formed by pairing an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), each binding to different antigens or different epitopes on the same antigen. Such bispecific binding agents may have a 1+1 format. Other bispecific binding agent (e.g., antibody) formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., an antibody) comprises two antigen-binding sites, each may bind to a different antigenic determinant. Such bispecific binding agents (eg, antibodies) can bind to two different epitopes on the same antigen (eg, epitopes on CD47).

[0043] The term "identical" or percent "identity," in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align 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 variations thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that when compared and aligned for maximum 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%, or 99% nucleotide or amino acid residue identity, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the 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 therebetween in length. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding regions of target proteins or antibodies. In some embodiments, the identity exists over a region of nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60-80 bases, e.g., over a region of at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as nucleotide sequences encoding proteins of interest.

[0044] " Conservative amino acid substitution " refers to the substitution of one amino acid residue with another amino acid residue that has a side chain with similar chemical characteristics.The family of amino acid residues with similar side chains is generally defined in the art, and includes 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), non-polar 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 with phenylalanine is a conservative substitution. In general, conservative substitutions within the sequence of a polypeptide, soluble protein, and / or antibody of the disclosure do not abrogate binding of the polypeptide, soluble protein, or antibody containing that amino acid sequence to its target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0045] 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, and may contain non-amino acids (e.g., may be interrupted by non-amino acids). The term also encompasses amino acid polymers that are modified, either naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e.g., linked or conjugated (directly or indirectly) to a moiety such as a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as other modifications known in the art, are also included within this definition. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in some embodiments, the polypeptide may exist as a single chain.

[0046] As used herein, an "antigen" is a moiety or molecule that contains 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 a binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein binds is CD47 (e.g., human CD47) and / or PD-L1 (e.g., human PD-L1), or their respective fragments.

[0047] As used herein, "epitope" (used interchangeably with "antigenic determinant") is a term used in the art and refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a structural, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more non-contiguous regions of a polypeptide, e.g., human CD47 and / or PD-L1 (a "structural," "non-linear," or "discontinuous" epitope). In general, those of skill in the art will understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether they are folded into their native three-dimensional protein structure. In other embodiments, the amino acid residues in an epitope must assume a particular conformation (e.g., a bend, twist, turn, or fold) in order for the antibody to recognize and bind to the epitope.

[0048] An antibody binds to an "epitope" or binds to "essentially the same epitope" or "the same epitope" as a reference antibody, when the two antibodies recognize the same, overlapping, or adjacent epitopes in three-dimensional space. The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competitive assay, which can be configured into several different formats, for example, by using either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radiolabel, a fluorescent label, or an enzyme label.

[0049] "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 using a combination of competitive assays and computational processes to cluster antibodies based on their epitope recognition characteristics and identify antibodies with distinct binding specificities.

[0050] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," "selectively bind," "immunospecifically recognize," and "immunospecific" are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope), and such binding is understood by those of skill 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, with a longer duration, with higher affinity, or some combination of the above, than alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may generally bind other peptides or polypeptides with lower affinity, as determined, for example, by immunoassay, 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 or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise, and may be more than 10 times the background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In some embodiments, the extent 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 specific target antigen, as determined, e.g., by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen binds to that antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs, or more greater than the Ka of the molecule when bound to another antigen. In some embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins.In another specific embodiment, a molecule that specifically binds to an antigen does not cross-react with other non-CD47 and / or non-PD-L1 proteins. In some embodiments, "specifically binds" 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 about 1 μM. In some embodiments, "specifically binds" 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 the polypeptide sequences of different proteins, specific binding may include polypeptides or molecules that recognize proteins or targets in more than one species. It is understood that in some embodiments, a polypeptide or molecule that specifically binds to a first target may or may not also specifically bind to a second target. Thus, "specific binding" does not necessarily require (but can include) exclusive binding, e.g., binding to a single target. Thus, a polypeptide or molecule may, in some embodiments, specifically bind to more than one target. In some embodiments, multiple targets may be bound by the same antigen-binding site of a polypeptide or molecule. For example, an antibody may, in certain cases, contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody may be bispecific, containing at least two antigen-binding sites with different specificities. Generally, but not necessarily, reference to "binding" means "specific binding."

[0051] "Binding affinity" generally refers to the strength of the sum total of 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 a 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 determined by the dissociation constant (K D ) Affinity can be generally indicated by the affinity of the antibody to the antigen. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate quickly, whereas high affinity antibodies generally tend to bind antigen faster and remain bound longer. Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure. In one embodiment, "K" is used to measure affinity of the antibody to the antigen. D " or "K D The "K value" reported herein can be measured by assays known in the art, for example, by binding assays. D Values ​​were determined by biolayer interferometry (BLI) using, for example, an OctetQK384 system (ForteBio, Menlo Park, CA). D 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 using a surface plasmon resonance (SPR) assay by Biacore, for example, using a BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). "On-rate" or "rate of association" or "association rate" or "k on " and "off-rate" or "rate of dissociation" or "dissociation rate" or "k off" can also be determined using the same SPR or BLI techniques described above, for example, using the OctetQK384 system (ForteBio, Menlo Park, CA) or the BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ), respectively.

[0052] The term "compete," when used in the context of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), means binding agents that compete for the same epitope or binding site on a target, including competition between such binding agents as determined by an assay in which the binding agent under study prevents or inhibits specific binding of a reference molecule (e.g., a reference ligand, or reference antigen-binding protein, e.g., a reference antibody) to a common antigen (e.g., CD47 or PD-L1). Many types of competitive binding assays can be used to determine whether a test binding agent competes with a reference molecule for binding to CD47 (e.g., human CD47) or PD-L1 (e.g., human PD-L1). Examples of assays that can be used include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahl 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 and Cheung, et al., (1990) Virology 176:546-552), 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 RIAs using I-125 labels (see, e.g., Morel et al., (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); al., (1988) Molec. Immunol. 25:7-15); and direct labeling 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 cells bearing either an unlabeled test antigen-binding protein (e.g., a test CD47 antibody) or a labeled reference antigen-binding protein (e.g., a reference CD47 antibody).Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cell in the presence of a test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and / or antibodies that bind to adjacent epitopes (e.g., similar or overlapping epitopes) that are close enough to the epitope bound by the reference antibody to cause steric hindrance. Typically, when a competing antibody is present in excess, it inhibits the specific binding of the reference antibody to a 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.

[0053] As used herein, the term "constant region" or "constant domain" is a well-known antibody terminology and refers to portions of an antibody, e.g., the carboxyl-terminal portions of the light and / or heavy chains, that are not directly involved in binding the antibody 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 than immunoglobulin variable domains.

[0054] Antibody "effector function" refers to the biological activity attributable to the Fc region of an antibody (e.g., a native sequence Fc region or an amino acid sequence variant Fc region) 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 cytotoxicity (ADCC); phagocytosis (such as antibody-dependent cellular phagocytosis (ADCP)); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0055] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined as the stretch from the amino acid residue at position Cys226 (according to the EU numbering system), or from Pro230 (according to the EU numbering system), to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are provided below (CH2 domain = bold; CH3 domain = underlined letters): [ka]

[0056] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (e.g., ADCP); down-regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain), and can be assessed using a variety of assays disclosed.

[0057] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature, and has not been manipulated, modified, and / or altered (e.g., isolated, purified, selected, included in, or combined with other sequences such as variable region sequences) by human manipulation, modification, and / or alteration. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A allotypes and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0058] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region described herein can possess at least about 80% homology with the native-sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology therewith, e.g., at least about 95% homology therewith. The variant Fc region described herein can also lack effector function (e.g., a silent Fc). Exemplary variant Fc region ("silent Fc") sequences are provided below (CH2 domain = bold, amino acid changes underlined; CH3 domain = underlined letters): [ka] Exemplary variant CH2 domain (e.g., silent CH2) sequences useful for the multispecific (e.g., bispecific) binding agents described herein are provided below (amino acid changes are underlined): [ka]

[0059] As used herein, the term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of about 50 to 70 kDa, in which the amino-terminal portion contains a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion contains one or more constant regions. Based on the amino acid sequence of the constant domain, "heavy chain" can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to antibodies of the IgA, IgD, IgE, IgG (including subclasses, e.g., IgG1, IgG2, IgG3, and IgG4), and IgM classes, respectively.

[0060] As used herein, the term "light chain," when used in reference to an antibody, can refer to a polypeptide chain of approximately 25 kDa, in which the amino-terminal portion contains a variable region of about 100 to about 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.

[0061] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that contains amino acid residues (e.g., CDRs) that interact with an antigen and confer specificity and affinity to the binding fragment, domain, or region for the antigen. "Antigen-binding fragment," as used herein, includes "antibody fragments," which include a portion of an antibody that contains one or more CDRs, such as the antigen-binding region or variable region of the antibody.

[0062] Antibodies as described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including, e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0063] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions 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).

[0064] An antibody can 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 antibodies described herein are IgG antibodies (e.g., human IgG), or classes thereof (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclasses thereof.

[0065] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In some embodiments, the H chain and L chain comprise 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 comprises a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., an IgG1, IgG2, IgG3, and / or IgG4 constant region).

[0066] An antibody or fragment thereof may preferentially bind to CD47, e.g., human CD47, meaning that the antibody or fragment thereof binds to CD47 with greater affinity than it binds to an unrelated control protein and / or binds to human CD47 with greater affinity than 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 thereof binds to CD47 with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, an antibody or fragment thereof may bind substantially exclusively to CD47 (e.g., CD47 can be distinguished from other known polypeptides, e.g., by a measurable difference in binding affinity). In some embodiments, a multispecific binding agent (eg, an antibody, eg, a bispecific antibody) may react with a CD47 sequence other than the human CD47 sequence (eg, a cynomolgus monkey CD47 sequence).

[0067] Additionally or alternatively, the antibody or fragment thereof may preferentially bind to PD-L1, e.g., human PD-L1, meaning that the antibody or fragment thereof binds to PD-L1 with greater affinity than it binds to an unrelated control protein and / or binds to human PD-L1 with greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind to PD-L1 or a portion thereof. "Specific binding" means that the antibody or fragment thereof binds to PD-L1 with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof may bind substantially exclusively to PD-L1 (e.g., PD-L1 can be distinguished from other known polypeptides, e.g., by a measurable difference in binding affinity). In some embodiments, the multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) may react with a PD-L1 sequence other than the human PD-L1 sequence (e.g., the cynomolgus monkey PD-L1 sequence).

[0068] The term "variable region" or "variable domain" generally refers to the portion of an antibody light or heavy chain located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, that is used for binding and specificity of each particular antibody to its specific antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that the sequence of certain segments of the variable region varies extensively among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for 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 stretches of approximately 15-30 amino acids, termed framework regions (FRs), that are less variable (e.g., relatively invariant), separated by shorter regions of higher variability (e.g., extreme variability), termed "hypervariable regions," or "complementarity-determining regions." The heavy and light chain variable regions each contain four FRs (FR1, FR2, FR3, and FR4), which are primarily in a β-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions within each chain are held together in close proximity by the FRs, and hypervariable regions from other chains contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC), ADCP, and complement-dependent cytotoxicity (CDC). The sequences of the variable regions vary widely among different antibodies. Sequence variability is concentrated in the CDRs, while the less variable portions of the variable regions are called framework regions (FRs).The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In certain embodiments, the variable regions are human variable regions.

[0069] The terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR," as used herein, refer to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Antibodies generally contain six hypervariable regions: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). Several hypervariable region delineations are in use and are encompassed by 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, instead, refers to the location of the 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 length of the loop (this is due to insertions 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 regions are a compromise between the Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable regions are based on analysis of available complex crystal structures. Residues from each of these hypervariable regions or CDRs are listed below.

[0070] A universal numbering system, the ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)), has been developed and widely adopted. IMGT is an integrated information system specializing in immunoglobulins (IGs), T-cell receptors (TRs), and major histocompatibility complexes (MHCs) from humans 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 readily identified by using a numbering system that aligns variable domain sequences according to structural features, CDRs, and framework residues, represented by structures called loops. This information can be used for grafting and substituting CDR residues from one species of immunoglobulin into an acceptor framework, typically derived from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-670 (2001). For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra), and is also exemplified below. The exemplary system shown herein combines Kabat and Chothia. [Table 13]

[0071] The hypervariable region may comprise "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-determining region," or "CDR" are used interchangeably.

[0072] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. Cells producing the binding molecules of the present disclosure may include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is called the transcription direction, and the region of the DNA strand 5' to the 5' end of the RNA transcript that has the same sequence as the RNA transcript is called the "upstream sequence"; the region of the DNA strand 3' to the 3' end of the RNA transcript that has the same sequence as the RNA transcript is called the "downstream sequence."

[0073] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, those intended for introducing a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers operable for stable integration into a host cell chromosome. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included include, for example, those that confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply essential nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are to be co-expressed (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 from a single vector, the encoding nucleic acids can be operationally linked to a common expression control sequence, or can be linked to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods for testing the expression of introduced nucleic acid sequences or their corresponding gene products. It will be understood by those skilled in the art that the nucleic acid molecules will be expressed in amounts sufficient to produce the desired product (e.g., a multispecific binding agent described herein), and further that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0074] The term "treat" or grammatical variations thereof refers to reducing and / or ameliorating the severity and / or duration of a particular disease, disorder, or condition, and / or symptoms associated therewith, such as (i) reducing, delaying, or ameliorating the progression or worsening of a given disease, disorder, or condition; (ii) reducing, delaying, or ameliorating the recurrence, occurrence, or onset of a given disease, disorder, or condition; (iii) improving or enhancing the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of a multispecific binding agent described herein).

[0075] "Immune cell dysfunction disease" and "immune cell dysfunction disorder" and "immune cell dysfunction state" are used interchangeably and refer to any disease, disorder, or condition that is caused in whole or in part by or is the result of inappropriate signaling to immune cells, and / or any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of the interaction of an immune cell receptor (e.g., SIRPα or PD-1) with its ligand (e.g., CD47 or PD-L1). Immune cell dysfunction diseases include phagocyte dysfunction diseases and T cell dysfunction diseases.

[0076] The terms "phagocyte dysfunction disease," "phagocyte dysfunction disorder," and "phagocyte dysfunction state" are used interchangeably and refer to any disease, disorder, or condition that is caused in whole or in part by or is a result of CD47 (e.g., aberrant expression of CD47) or the interaction of CD47 with SIRPα, and / or alternatively, any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of the interaction of CD47 with SIRPα. Phagocyte dysfunction diseases include diseases, disorders, or conditions characterized by or associated with decreased activity of phagocytic immune cells (e.g., neutrophils, macrophages, dendritic cells, B lymphocytes). In some embodiments, a phagocyte dysfunction disease is a disease, disorder, or condition specifically associated with inappropriately increased signaling through SIRPα. In some embodiments, a phagocyte dysfunction disease is a decreased ability of phagocytes (e.g., macrophages) to ingest or engulf other cells (e.g., tumor cells) or particles. In some embodiments, the reduced ability to ingest or engulf other cells or particles results in ineffective control of pathogens or tumors, including but not limited to, tumors that express CD47. Examples of phagocyte dysfunction disorders characterized by phagocyte dysfunction include unresolved acute infections, chronic infections, and tumor immunity (e.g., any cancer, including but not limited to, cancers that express or overexpress CD47).

[0077] "T cell dysfunction disease," "T cell dysfunction disorder," and "T cell dysfunction state" are used interchangeably and refer to any disease, disorder, or condition of T cells characterized by reduced responsiveness to antigenic stimulation. T cell dysfunction diseases include diseases, disorders, or conditions that are caused in whole or in part by or are the result of PD-L1 (e.g., aberrant expression of PD-L1) or the interaction of PD-L1 with PD-1, and / or alternatively, any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of PD-L1 interaction with PD-1. In some embodiments, a T cell dysfunction disease is a disease, disorder, or condition specifically associated with inappropriate increased signaling through PD-1. In some embodiments, a T cell dysfunction disease is a disease in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or carry out cytolytic activity. In some embodiments, the reduced responsiveness results in ineffective control of pathogens or tumors, including, but not limited to, tumors that express PD-L1. Examples of T cell dysfunction diseases characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity (e.g., from any cancer, including but not limited to, cancers that express or overexpress PD-L1).

[0078] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is weakened and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0079] "Enhancing T cell function" means inducing T cells to have a sustained or increased biological function, causing T cells to have a sustained or increased biological function, or stimulating T cells to have a sustained or increased biological function, or regenerating or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include: increasing CD8 + 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 alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Manners for measuring this enhancement are known to those skilled in the art.

[0080] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms resulting from or associated with a disease, disorder, or condition, eliminate the symptoms and / or their underlying causes, prevent the appearance of the symptoms and / or their underlying causes, and / or ameliorate or correct damage. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0081] The term "therapeutically effective amount," as used herein, refers to an amount of an agent (e.g., an antibody described herein or any other agent described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition, and / or symptoms associated therewith. A therapeutically effective amount of an agent, including a therapeutic agent, may be the amount necessary to (i) reduce or ameliorate the progression or worsening of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, occurrence, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of an antibody described herein). A "therapeutically effective amount" of a substance / molecule / agent (e.g., a binding agent such as a monoclonal antibody, monospecific antibody, or bispecific antibody) of the present disclosure may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (e.g., or drug) effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0082] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, e.g., prevents or delays the onset (or reoccurrence) of a disease, disorder, or condition, or reduces the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition, or associated symptom(s). A complete therapeutic or prophylactic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

[0083] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency for use in animals, and more particularly in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0084] As used herein, the term "carrier" includes carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to the carrier at the dosages and concentrations used. In many cases, the carrier is an aqueous pH buffer solution. Examples of carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium, and / or non-ionic surfactants, such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered. Such carriers can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a typical carrier when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, and the like.Oral compositions containing formulations may contain standard carriers, such as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions containing pharmaceutical compounds may contain a prophylactically or therapeutically effective amount of a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody), e.g., in isolated or purified form, together with a suitable amount of carrier so as to provide the form for proper administration to a subject (e.g., a patient). The formulation should suit the mode of administration.

[0085] The terms "about" and "approximately" refer to a variation of within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0086] As used herein, comparative terms such as "reduce," "decrease," "increase," or any grammatical variation thereof can refer to a certain change from the baseline. In some embodiments, such a change can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1-fold, or about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 10-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 100-fold, or more of the baseline. In some embodiments, such a change may refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the baseline.

[0087] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0088] In some embodiments, the terms "first," "second," "third," "fourth," and the like in component names are used to distinguish and identify multiple components that share a particular identity in their names. For example, "first antibody" and "second antibody" are used to distinguish between two antibodies.

[0089] Where embodiments are described herein with the term "comprising," it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided. It is also understood that where embodiments are described herein with the phrase "consisting essentially of," it is also provided that analogous embodiments described with the term "consisting of."

[0090] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0091] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0092] The term "optionally" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the description includes cases where the circumstance occurs and cases where the circumstance does not occur.

[0093] Binding Agent-Binding Domain In some embodiments, the present disclosure provides multispecific binding agents that can be used herein as therapeutic agents. Such agents include multispecific antibodies (e.g., antibodies, e.g., bispecific antibodies) that comprise a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more additional targets that are not CD47 (e.g., PD-L1). Exemplary antibodies include humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugate antibodies, as well as variants thereof with increased or decreased affinity or other properties.

[0094] In some embodiments, described herein are multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) that comprise a first binding domain that binds to CD47, including a CD47 polypeptide, a CD47 polypeptide fragment, a CD47 peptide, or a CD47 epitope. In some embodiments, the multispecific binding agent is a human or humanized antibody (e.g., comprising a human constant region) that comprises a first binding domain that binds to CD47, including a CD47 polypeptide, a CD47 polypeptide fragment, a CD47 peptide, or a CD47 epitope. In some embodiments, the multispecific binding agent (e.g., antibody, e.g., bispecific antibody) can bind to CD47 expressed on the surface of mammalian (e.g., human) cells, including CD47-expressing tumor cells. In some embodiments, the multispecific binding agent (e.g., antibody, e.g., bispecific antibody) binds to a CD47 extracellular epitope (e.g., a CD47 epitope) exposed on a cell, such as a tumor cell. In some embodiments, described herein are multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) that bind to CD47, e.g., human CD47, or a portion thereof. In some embodiments, the CD47 is human CD47. In some embodiments, provided herein are multispecific binding agents that bind to CD47 (e.g., antibodies that bind to human CD47). An exemplary amino acid sequence of human CD47 is described herein.

[0095] Multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1) described herein, can be monospecific, bispecific, trispecific, or of greater multispecificity. Such agents can include antibodies. Multispecific antibodies, e.g., bispecific antibodies, are monoclonal antibodies that have binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody directed against 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 can be constructed based on the sequences of the antibodies described herein, e.g., the CDR sequences listed in Table 1. In some embodiments, the second binding domain of a multispecific (e.g., bispecific) antibody described herein may be constructed based on the sequence of an antibody described herein, e.g., the CDR sequences listed in Table 2. In some embodiments, a multispecific antibody described herein is a bispecific antibody. In some embodiments, the bispecific antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, one of the binding specificities of the multispecific antibody is for CD47 and the other is for any other target (e.g., an antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may comprise more than one target binding domain, wherein different domains are specific for 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), such as an immune checkpoint regulator (e.g., a negative checkpoint regulator)). In some embodiments, a multispecific (e.g., bispecific) antibody may bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen).In some embodiments, one of the binding specificities is for CD47 and the other is for any other target (e.g., antigen). In some embodiments, one of the binding specificities is for CD47 and the others are 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-activation 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 These antibodies are directed against one or more of the following: T cell immunoreceptor with ITIM domain (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig suppressor of T cell activation (VISTA), glucocorticoid-inducible tumor necrosis factor receptor-related (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137.CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0096] In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) are described herein that comprise 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 agent is a humanized antibody (e.g., comprising a human constant region) that comprises 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 agent (e.g., antibody, e.g., bispecific antibody) can 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 agent (e.g., antibody, e.g., bispecific antibody) binds to a PD-L1 extracellular epitope (e.g., a PD-L1 epitope) exposed on cells, such as tumor cells. In some embodiments, described herein are multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) that include a second binding domain that binds to PD-L1, e.g., human PD-L1, or a portion thereof. In some embodiments, the PD-L1 is human PD-L1. In some embodiments, provided herein are multispecific binding agents that bind to human PD-L1 (e.g., antibodies that bind to human PD-L1). Exemplary amino acid sequences of human PD-L1 are described herein.

[0097] In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) provided herein have a cytotoxicity of ≦1 μM, ≦500 nM, ≦100 nM, ≦50 nM, ≦30 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 Less than M, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K DAdditionally or alternatively, the multispecific binding agents (e.g., bispecific antibodies) provided herein bind to CD47 (e.g., human CD47) with a dissociation constant (K) of ≥ 0.1 nM, ≥ 1 nM, ≥ 5 nM, ≥ 10 nM, ≥ 15 nM, or ≥ 20 nM. D In further embodiments, the multispecific binding agents (e.g., bispecific antibodies) provided herein bind to CD47 (e.g., human CD47) with a dissociation constant (K) of about 0.1 nM to about 1 μM (including every number and range therebetween, e.g., about 1 nM to about 100 nM, or about 10 nM to about 50 nM, or about 20 nM to about 30 nM, etc.). D Additionally or alternatively, the multispecific binding agents (e.g., bispecific antibodies) provided herein bind to CD47 (e.g., human CD47) with a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D ) and binds to PD-L1 (e.g., human PD-L1).

[0098] Various methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, such as, for example, an RIA performed using a Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays with OCTET®, for example, using the OCTET® Red 96 system, or by BIACORE®, for example, using a BIACORE® TM-2000 or BIACORE® TM-3000. The "on-rate" or "rate of association" or "association rate" or "k" can also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, for example, using OCTET® Red96, a BIACORE® TM-2000, a BIACORE® TM-3000 system, a BIACORE® TM-8K, or a BIACORE® TM-8K+ system.

[0099] In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein competes for binding to CD47, e.g., human CD47, with a binding agent (e.g., an antibody, e.g., a bispecific antibody) that comprises the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, e.g., the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Table 1. Thus, in some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein competes for binding to CD47, e.g., human CD47, with a binding agent (e.g., an antibody, e.g., a bispecific antibody) that comprises one, two, and / or three VH CDRs and / or one, two, and / or three VL CDRs from the antibody designated mAb-C shown in Table 1. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein competes for binding to CD47, e.g., human CD47, with a binding agent (e.g., an antibody, e.g., a bispecific antibody) that comprises one, two, and / or three VH CDRs and one, two, and / or three VL CDRs from the antibody designated mAb-C shown in Table 1. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein competes for binding to CD47, e.g., human CD47, with a binding agent (e.g., an antibody, e.g., a bispecific antibody) comprising a VH region and a VL region from the antibody designated mAb-C, shown in Table 1. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein competes for binding to CD47, e.g., human CD47, with a binding agent (e.g., an antibody, e.g., a bispecific antibody) comprising a VH region comprising the amino acid sequence of SEQ ID NO: 25 and a VL region comprising the amino acid sequence of SEQ ID NO: 26.

[0100] In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, e.g., the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Tables 1-2. Thus, in some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from the antibody designated mAb-C, shown in Table 1. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from the antibody designated mAb-P shown in Table 2. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from the antibody designated mAb-C shown in Table 1, 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 the antibody designated mAb-P shown in Table 2. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises a first binding domain comprising one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from the antibody designated mAb-C shown in Table 1, and a second binding domain comprising one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from the antibody designated mAb-P shown in Table 2.

[0101] In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) comprises a first binding domain that binds CD47 and comprises a VH region comprising the VH CDR1, VH CDR2, and / or VH CDR3 and a VL region comprising the VL CDR1, VL CDR2, and / or VL CDR3 of any one of the binding agents listed in Table 1, and a second binding domain that binds PD-L1 and comprises a VH region comprising the VH CDR1, VH CDR2, and / or VH CDR3 and a VL region comprising the VL CDR1, VL CDR2, and / or VL CDR3 of any one of the binding agents listed in Table 2. Thus, in some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises a first binding domain that comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from Table 1. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein comprises a second binding domain that comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from Table 2. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) described herein is bispecific and comprises a first binding domain that comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from Table 1, and a second binding domain that comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from a binding agent that binds to a second target antigen that is not CD47. In some embodiments, the multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) described herein are bispecific and comprise a first binding domain comprising one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from Table 1, and a second binding domain comprising one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from Table 2.

[0102] The antibody designated mAb-C comprises a VH sequence that is SEQ ID NO:25 and a VL sequence that is SEQ ID NO:26.

[0103] The antibody designated mAb-P comprises a VH sequence that is SEQ ID NO:46 and a VL sequence that is SEQ ID NO:47.

[0104] In some embodiments, a multispecific binding agent (e.g., an antibody) that binds to CD47 comprises (i) a VH domain comprising the VH sequence that is SEQ ID NO: 25, and (ii) a VL domain comprising the VL sequence that is SEQ ID NO: 26. In some embodiments, such VH and VL domains are used to construct bispecific binding agents (e.g., antibodies) that each have a first binding domain that binds to CD47, including where the first binding domain comprises the VH sequence that is SEQ ID NO: 26 and the VL sequence that is SEQ ID NO: 26.

[0105] In some embodiments, a multispecific binding agent (e.g., an antibody) that binds to PD-L1 comprises (i) a VH domain comprising the VH sequence that is SEQ ID NO: 46, and (ii) a VL domain comprising the VL sequence that is SEQ ID NO: 47. In some embodiments, such VH and VL domains are used to construct bispecific binding agents (e.g., antibodies) that each have a first binding domain that binds to PD-L1, including where the first binding domain comprises the VH sequence that is SEQ ID NO: 46 and the VL sequence that is SEQ ID NO: 47.

[0106] In some embodiments, the multispecific binding agent (e.g., a bispecific antibody) that binds to CD47 and PD-L1 comprises: (a) a first binding domain comprising (i) a VH domain comprising the VH sequence that is SEQ ID NO: 25, and (ii) a VL domain comprising the VL sequence that is SEQ ID NO: 26; and (b) a second binding domain comprising (i) a VH domain comprising the VH sequence that is SEQ ID NO: 46, and (ii) a VL domain comprising the VL sequence that is SEQ ID NO: 47.

[0107] In some embodiments, such VH and VL domains are used to construct a bispecific antibody, the bispecific antibody comprising a first polypeptide chain comprising a VL domain (e.g., the VL sequence of SEQ ID NO: 26) and a second polypeptide chain comprising a VH domain (e.g., the VH sequence of SEQ ID NO: 25), where the VL and VH domains form a first binding domain that binds CD47; and the bispecific antibody further comprising a third polypeptide chain comprising a VL domain (e.g., the VL sequence of SEQ ID NO: 47), and a fourth polypeptide chain comprising a VH domain (e.g., the VH sequence of SEQ ID NO: 46), where the VL and VH domains form a second binding domain that binds PD-L1.

[0108] In an exemplary embodiment of a multispecific binding agent (e.g., a bispecific antibody) comprising four polypeptide chains, the first polypeptide chain has the amino acid sequence of SEQ ID NO: 48, the second polypeptide chain has the amino acid sequence of SEQ ID NO: 49, the third polypeptide chain has the amino acid sequence of SEQ ID NO: 50, and the fourth polypeptide chain has the amino acid sequence of SEQ ID NO: 51. In another exemplary embodiment of a multispecific binding agent (e.g., a bispecific antibody) comprising four polypeptide chains, the first polypeptide chain has the amino acid sequence of SEQ ID NO: 52, the second polypeptide chain has the amino acid sequence of SEQ ID NO: 49, the third polypeptide chain has the amino acid sequence of SEQ ID NO: 53, and the fourth polypeptide chain has the amino acid sequence of SEQ ID NO: 51. [Table 1] [Table 2]

[0109] In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), and the first binding domain and / or second binding domain comprise a VH region or domain. In other embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), and the first binding domain and / or second binding domain comprise a VL region or domain. In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets that are not 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 region, and / or (ii) a VL domain or region.

[0110] In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47, including human CD47, and a second binding domain that binds to one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), wherein the first binding domain comprises one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, identified in Table 1.

[0111] In some embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies), including multispecific binding agents described herein that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), comprise a first binding domain that comprises one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, as listed in Table 1. In other embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies), including multispecific binding agents described herein that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), comprise a first binding domain that comprises one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3, as listed in Table 1. In still other embodiments, multispecific binding agents (e.g., antibodies, such as bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), described herein, comprise a first binding domain that comprises one or more CDRs, including three VH CDRs, e.g., a VH CDR1, a VH CDR2, a VH CDR3, listed in Table 1, and one or more CDRs, including three VL CDRs, e.g., a VL CDR1, a VL CDR2, and / or a VL CDR3, listed in Table 1.

[0112] In some embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies), including multispecific binding agents that bind CD47, including human CD47, and PD-L1, including human PD-L1, described herein, comprise a second binding domain that comprises one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, listed in Table 2. In other embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies), including multispecific binding agents that bind CD47, including human CD47, and PD-L1, including human PD-L1, described herein, comprise a second binding domain that comprises one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3, listed in Table 2. In still other embodiments, multispecific binding agents (e.g., antibodies, such as bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and PD-L1, including human PD-L1, described herein, comprise a second binding domain that comprises one or more CDRs, including three VH CDRs, e.g., a VH CDR1, a VH CDR2, a VH CDR3, listed in Table 2, and one or more CDRs, including three VL CDRs, e.g., a VL CDR1, a VL CDR2, and / or a VL CDR3, listed in Table 2.

[0113] In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47 and comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24. In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47 and comprises two or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24. In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47 and comprises three or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24. In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-45. In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises two or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-45. In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises three or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-45.

[0114] In some embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47 and comprises a VH with one or more (e.g., one, two, or three) VH CDRs listed in Table 1. In other embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47 and comprises a VL with one or more (e.g., one, two, or three) VL CDRs listed in Table 1. In yet other embodiments, multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a first binding domain that binds to CD47 and comprises one or more (e.g., one, two, or three) VH CDRs listed in Table 1, and one or more VL CDRs listed in Table 1. Thus, in some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VH CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1, 7, 12, 13, and 18. In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VH CDR2 having the amino acid sequence of any one of SEQ ID NOs: 2, 8, 14, 19, and 24. In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VH CDR3 having the amino acid sequence of any one of SEQ ID NOs: 3, 9, 15, and 20. In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VH CDR1 and / or a VH CDR2 and / or a VH CDR3 independently selected from the VH CDR1, VH CDR2, VH CDR3 depicted in any one of the amino acid sequences depicted in Table 1.In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VL CDR1 having the amino acid sequence of any one of SEQ ID NOs: 4, 10, 16, and 21. In another embodiment, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VL CDR2 having the amino acid sequence of any one of SEQ ID NOs: 5, 11, and 22. In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VL CDR3 having the amino acid sequence of any one of SEQ ID NOs: 6, 17, and 23. In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a first binding domain that binds to CD47 and comprises a VL CDR1 and / or a VL CDR2 and / or a VL CDR3 independently selected from a VL CDR1, a VL CDR2, a VL CDR3 depicted in any one of the amino acid sequences depicted in Table 1.

[0115] In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VH with one or more (e.g., one, two, or three) VH CDRs listed in Table 2. In other embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VL with one or more (e.g., one, two, or three) VL CDRs listed in Table 2. In yet other embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises one or more (e.g., one, two, or three) VH CDRs listed in Table 2, and one or more VL CDRs listed in Table 2. Thus, in some embodiments, a multispecific binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a second binding domain that binds to PD-L1 and comprises a VH CDR1 having the amino acid sequence of any one of SEQ ID NOs: 27, 32, 35, 36, and 40. In some embodiments, a multispecific binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a second binding domain that binds to PD-L1 and comprises a VH CDR2 having the amino acid sequence of any one of SEQ ID NOs: 28, 33, 37, 41, and 45. In some embodiments, a multispecific binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a second binding domain that binds to PD-L1 and comprises a VH CDR3 having the amino acid sequence of any one of SEQ ID NOs: 29, 34, 38, and 42. In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VH CDR1 and / or a VH CDR2 and / or a VH CDR3 independently selected from the VH CDR1, VH CDR2, VH CDR3 depicted in any one of the amino acid sequences depicted in Table 2.In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VL CDR1 having the amino acid sequence of any one of SEQ ID NOs: 4, 10, 16, and 21. In other embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VL CDR2 having the amino acid sequence of any one of SEQ ID NOs: 11, 30, and 43. In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VL CDR3 having the amino acid sequence of any one of SEQ ID NOs: 31, 39, and 44. In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a second binding domain that binds to PD-L1 and comprises a VL CDR1 and / or a VL CDR2 and / or a VL CDR3 independently selected from the VL CDR1, VL CDR2, VL CDR3 depicted in any one of the amino acid sequences depicted in Table 2.

[0116] In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein binds to CD47 and comprises a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:1, (ii) SEQ ID NO:7, (iii) SEQ ID NO:12, (iv) SEQ ID NO:13, and (v) SEQ ID NO:18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:2, (ii) SEQ ID NO:8, (iii) SEQ ID NO:14, (iv) SEQ ID NO:19, and (v) SEQ ID NO:24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:3, (ii) SEQ ID NO:9, (iii) SEQ ID NO:15, and (iv) SEQ ID NO:20; and / or (1) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, and (iv) SEQ ID NO:21. (2) a VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:5, (ii) SEQ ID NO:11, and (iii) SEQ ID NO:22; and (3) a light chain variable (VL) region comprising a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:6, (ii) SEQ ID NO:17, and (iii) SEQ ID NO:23.

[0117] In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein binds to CD47 and comprises a first binding domain comprising a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:1, (ii) SEQ ID NO:7, (iii) SEQ ID NO:12, (iv) SEQ ID NO:13, and (v) SEQ ID NO:18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:2, (ii) SEQ ID NO:8, (iii) SEQ ID NO:14, (iv) SEQ ID NO:19, and (v) SEQ ID NO:24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:3, (ii) SEQ ID NO:9, (iii) SEQ ID NO:15, and (iv) SEQ ID NO:20.

[0118] In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein binds to CD47 and comprises a first binding domain comprising a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, and (iv) SEQ ID NO:21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:5, (ii) SEQ ID NO:11, and (iii) SEQ ID NO:22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:6, (ii) SEQ ID NO:17, and (iii) SEQ ID NO:23.

[0119] In some embodiments, the multispecific binding agents (e.g., antibodies, such as bispecific antibodies) described herein bind to PD-L1 and comprise: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:27, (ii) SEQ ID NO:32, (iii) SEQ ID NO:35, (iv) SEQ ID NO:36, (v) SEQ ID NO:40; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:28, (ii) SEQ ID NO:33, (iii) SEQ ID NO:37, (iv) SEQ ID NO:41, (v) SEQ ID NO:45; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:29, (ii) SEQ ID NO:34, (iii) SEQ ID NO:38, (iv) SEQ ID NO:42; and / or (b) a VL CDR3 having an amino acid sequence selected from the group consisting of: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, (iv) SEQ ID NO:21. (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 30, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 43; and (3) a light chain variable (VL) region comprising a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 31, (ii) SEQ ID NO: 39, and (iii) SEQ ID NO: 44.

[0120] In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein bind to PD-L1 and comprise a second binding domain comprising a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:27, (ii) SEQ ID NO:32, (iii) SEQ ID NO:35, (iv) SEQ ID NO:36, (v) SEQ ID NO:40; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:28, (ii) SEQ ID NO:33, (iii) SEQ ID NO:37, (iv) SEQ ID NO:41, (v) SEQ ID NO:45; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:29, (ii) SEQ ID NO:34, (iii) SEQ ID NO:38, (iv) SEQ ID NO:42.

[0121] In some embodiments, the multispecific binding agents (e.g., antibodies such as bispecific antibodies) described herein bind to PD-L1 and comprise a second binding domain comprising a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, (iv) SEQ ID NO:21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:30, (ii) SEQ ID NO:11, (iii) SEQ ID NO:43; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:31, (ii) SEQ ID NO:39, (iii) SEQ ID NO:44.

[0122] Also described herein are multispecific binding agents (e.g., antibodies, such as bispecific antibodies) that bind to CD47 and comprise a first binding domain that comprises 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 Table 1. In particular, described herein are multispecific binding agents (e.g., antibodies, such as bispecific antibodies) that include: VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20) and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20) and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20) and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), and VL CDR3 (SEQ ID NOs: 6, 17, and 23);VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23);VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 5, 11, and 22); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, and 18), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, and 24), VH CDR3 (SEQ ID NOs: 3, 9, 15, and 20), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), VL CDR2 (SEQ ID NOs: 5, 11, and 22), and VL CDR3 (SEQ ID NOs: 6, 17, and 23); or the VH CDRs (SEQ ID NOs: 1, 2, 3, 7, 8, 9, 12, 13, 14, 15, 18, 19, 20, and 24) and VL CDRs listed in Table 1. Any combination thereof of CDRs (SEQ ID NOs: 4, 5, 6, 10, 11, 16, 17, 21, 22, and 23);

[0123] Also described herein are multispecific binding agents (e.g., antibodies) that bind to PD-L1 and comprise a second binding domain that comprises 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 Table 2. In particular, described herein are multispecific binding agents (e.g., antibodies) that include: VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40) and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40) and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40) and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45) and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45) and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45) and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42) and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42) and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42) and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45) and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), and VL CDR3 (SEQ ID NOs: 31, 39, and 44);VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 31, 39, and 44);VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), and VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 11, 30, and 43);VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21) and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR2 (SEQ ID NOs: 11, 30, and 43) and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21) and VL CDR2 (SEQ ID NOs: 11, 30, and 43); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21) and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR2 (SEQ ID NOs: 11, 30, and 43) and VL CDR3 (SEQ ID NOs: 31, 39, and 44);VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR1 (SEQ ID NOs: 27, 32, 35, 36, and 40), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); VH CDR2 (SEQ ID NOs: 28, 33, 37, 41, and 45), VH CDR3 (SEQ ID NOs: 29, 34, 38, and 42), VL CDR1 (SEQ ID NOs: 4, 10, 16, and 21), VL CDR2 (SEQ ID NOs: 11, 30, and 43), and VL CDR3 (SEQ ID NOs: 31, 39, and 44); or any combination thereof of VH CDRs (SEQ ID NOs: 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, and 45) and VL CDRs (SEQ ID NOs: 4, 30, 31, 10, 11, 16, 39, 21, 43, and 44) ​​listed in Table 2.

[0124] 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) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:1, (ii) SEQ ID NO:7, (iii) SEQ ID NO:12, (iv) SEQ ID NO:13, and (v) SEQ ID NO:18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:2, (ii) SEQ ID NO:8, (iii) SEQ ID NO:14, (iv) SEQ ID NO:19, and (v) SEQ ID NO:24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:3, (ii) SEQ ID NO:9, (iii) SEQ ID NO:15, and (iv) SEQ ID NO:20; and (b) a VL CDR3 having an amino acid sequence selected from the group consisting of: (1) (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, and (iv) SEQ ID NO:21. and (3) a light chain variable (VL) region comprising a CDR1; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:5, (ii) SEQ ID NO:11, and (iii) SEQ ID NO:22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:6, (ii) SEQ ID NO:17, and (iii) SEQ ID NO:23. 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 comprises: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:27, (ii) SEQ ID NO:32, (iii) SEQ ID NO:35, (iv) SEQ ID NO:36, and (v) SEQ ID NO:40; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:28, (ii) SEQ ID NO:33, (iii) SEQ ID NO:37, (iv) SEQ ID NO:41, and (v) SEQ ID NO:45; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:29, (ii) SEQ ID NO:34, (iii) SEQ ID NO:38, and (iv) SEQ ID NO:42; and (b) a VL CDR3 having an amino acid sequence selected from the group consisting of: (1) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, and (iv) SEQ ID NO:21. (2) a VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 30, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 43; and (3) a light chain variable (VL) region comprising a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 31, (ii) SEQ ID NO: 39, and (iii) SEQ ID NO: 44.

[0023] In some embodiments, described herein are multispecific antibodies or fragments thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:1, (ii) SEQ ID NO:7, (iii) SEQ ID NO:12, (iv) SEQ ID NO:13, and (v) SEQ ID NO:18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:2, (ii) SEQ ID NO:8, (iii) SEQ ID NO:14, (iv) SEQ ID NO:19, and (v) SEQ ID NO:24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:3, (ii) SEQ ID NO:9, (iii) SEQ ID NO:15, and (iv) SEQ ID NO:20. 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 a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:27, (ii) SEQ ID NO:32, (iii) SEQ ID NO:35, (iv) SEQ ID NO:36, and (v) SEQ ID NO:40; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:28, (ii) SEQ ID NO:33, (iii) SEQ ID NO:37, (iv) SEQ ID NO:41, and (v) SEQ ID NO:45; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:29, (ii) SEQ ID NO:34, (iii) SEQ ID NO:38, and (iv) SEQ ID NO:42.

[0125]

[0023] In some embodiments, described herein is a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, and (iv) SEQ ID NO:21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:5, (ii) SEQ ID NO:11, and (iii) SEQ ID NO:22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:6, (ii) SEQ ID NO:17, and (iii) SEQ ID NO:23. 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 a light chain variable (VL) region comprising: (a) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:4, (ii) SEQ ID NO:10, (iii) SEQ ID NO:16, and (iv) SEQ ID NO:21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:30, (ii) SEQ ID NO:11, and (iii) SEQ ID NO:43; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:31, (ii) SEQ ID NO:39, and (iii) SEQ ID NO:44.

[0126] In some embodiments, described herein is 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 complementarity determining regions (CDRs) or all three light chain CDRs from an antibody designated mAb-C, which comprises a VH sequence of SEQ ID NO: 25 and a VL sequence of SEQ ID NO: 26. 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 comprises all three heavy chain complementarity determining regions (CDRs) or all three light chain CDRs from an antibody designated mAb-P, which comprises a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 47.

[0127] In some embodiments, described herein is 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 mAb-C. 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 mAb-P.

[0128] In some embodiments, described herein are multispecific antibodies or fragments thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences set forth in Table 1, or (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences set forth in Table 1. In some embodiments, the first binding domain comprises (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences set forth in Table 1, and (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences set forth in Table 1. 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 (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences depicted in Table 2, and / or (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences depicted in Table 2. In some embodiments, the first binding domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences depicted in Table 1. 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 a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences depicted in Table 2. In some embodiments, the first binding domain comprises a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences depicted in Table 1. 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 a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences shown in Table 2.

[0129] In some embodiments, described herein is a multispecific antibody or fragment thereof having a first binding domain that binds to CD47, wherein the first binding domain comprises: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23. 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) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 27, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 31.In some embodiments, the first binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:7, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:8, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:9; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6. In some embodiments, the second binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 32, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 33, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 34; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 31. In some embodiments, the first binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 12, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 35, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 31.In some embodiments, the first binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 17. In some embodiments, the second binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 36, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 37, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 38; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 39. In some embodiments, the first binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 18, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 19, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 20; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the second binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 40, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 41, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 42; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 43, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 44.In some embodiments, the first binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 24, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second binding domain comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 27, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 45, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 31. In some embodiments, the above-described multispecific antibody or fragment thereof is a bispecific antibody.

[0130] In some embodiments, described herein are binding agents that bind to essentially the same epitope as any one of the antibodies described herein, or fragments thereof. In some embodiments, described herein are binding agents that compete with any one of the antibodies described herein, or fragments thereof, for binding to PD-L1 (e.g., human PD-L1). Additionally or alternatively, described herein are binding agents that compete with any one of the antibodies described herein, or fragments thereof, for binding to CD47 (such as human CD47). In some embodiments, the binding agent is an antibody or fragment thereof.

[0131] In certain embodiments, the CDRs of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not 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, USDapartment of Health and Human Services, NIH Publication No. 91-3242).

[0132] In certain embodiments, the CDRs of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), may be determined according to the Chothia system and are referred to herein as "Chothia CDRs" (see, 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. 215(1):175-82; and U.S. Patent No. 7,709,226).

[0133] In certain embodiments, the CDRs of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), can be determined according to the ImMunoGeneTics (IMGT) system described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDRs").

[0134] In certain embodiments, the CDRs of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), can be determined according to the AbM system described, e.g., in MacCallum et al., 1996, J. Mol. Biol., 262:732-745, and are referred to herein as "AbM CDRs." See also, e.g., 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).

[0135] In certain embodiments, the CDRs of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), can be determined according to the Contact system and are referred to herein as "Contact CDRs" (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5:732-745). Contact CDRs are based on analysis of available complex crystal structures.

[0136] In some embodiments, the position 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 agent (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, may be varied by one, two, three, four, five, or six amino acid positions, so 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%, or at least 95%). For example, in some embodiments, the positions defining the CDRs in Table 1 may be varied by shifting the N-terminal and / or C-terminal boundaries of the CDRs by 1, 2, 3, 4, 5, or 6 amino acids compared to the current CDR 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%, or at least 95%). Additionally or alternatively, 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 agent (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, can be varied by one, two, three, four, five, or more amino acids (e.g., shorter or longer), so 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%, or 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, or more amino acids shorter than one or more of the CDRs described by SEQ ID NOs: 1-24, 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%, or 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, or more amino acids longer than one or more of the CDRs described by SEQ ID NOs: 1-24, so 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%, or at least 95%). In some embodiments, the amino terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein may be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, so 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%, or at least 95%). Additionally or alternatively, the carboxy terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein may be extended or shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, so 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 terminus 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, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, so 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%, or at least 95%). Additionally or alternatively, the carboxy terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the first binding domain described herein can be extended or shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, so 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, such as the binding assays and conditions described in the "Examples" section herein, can be used to determine whether binding to CD47 (e.g., human CD47) is maintained.

[0137] In some embodiments, the position 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 agent (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agent that binds to CD47, including human CD47, and PD-L1, including human PD-L1, described herein, may be varied by one, two, three, four, five, or six amino acid positions, so 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%, or at least 95%). For example, in some embodiments, the positions defining the CDRs in Table 2 may be varied by shifting the N-terminal and / or C-terminal boundaries of the CDRs by 1, 2, 3, 4, 5, or 6 amino acids compared to the current CDR positions, so 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%, or at least 95%). Additionally or alternatively, 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 agent (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agent that binds to CD47, including human CD47, and PD-L1, including human PD-L1, described herein, can be varied (e.g., shorter or longer) by 1, 2, 3, 4, 5, or more amino acids, so 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%, or 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, or more amino acids shorter than one or more of the CDRs set forth by SEQ ID NOs: 27-45, 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%, or 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, or more amino acids longer than one or more of the CDRs described by SEQ ID NOs: 27-45, so 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%, or at least 95%). In some embodiments, the amino terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domains described herein may be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 27-45, so 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%, or at least 95%). Additionally or alternatively, the carboxy terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domains described herein may be extended or shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 27-45, so 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 terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domains described herein may be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 27-45, so 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%, or at least 95%). Additionally or alternatively, the carboxy terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 of the second binding domains described herein may be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 27-45, so 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 may be used to determine whether binding to PD-L1 (e.g., human PD-L1) is maintained, such as the binding assays and conditions described in the "Examples" section herein.

[0138] In some embodiments, multispecific antibodies are described herein that include the VH and / or VL regions described herein that further comprise human framework sequences. In some embodiments, the VH and / or VL regions further comprise framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

[0139] In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), provided herein, comprise one or more conservative sequence modifications. With respect to polypeptides that are multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), e.g., multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not 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 with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, a predicted non-essential amino acid residue in a binding agent described herein is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., 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, conservative sequence modifications described herein alter 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% of the amino acid sequence of a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1). In some embodiments, the nucleotide and amino acid sequence modifications refer to the substitution of at most 1, 2, 3, 4, 5, or 6 amino acids relative to the CDRs set forth in Table 1 or Table 2. Thus, for example, each such CDR may contain up to five conservative amino acid substitutions, e.g., up to four (or less than four) conservative amino acid substitutions, e.g., up to three (or less than three) conservative amino acid substitutions, e.g., up to two (or less than two) conservative amino acid substitutions, or no more than one conservative amino acid substitution. In some embodiments, the binding agents described herein comprise conservative amino acid substitutions outside of any CDR. In further embodiments, the binding agents described herein comprise conservative amino acid substitutions in the FRs. Additionally or alternatively, the binding agents described herein comprise conservative amino acid substitutions in constant regions or constant domains, such as Cl, CH1, CH2, or CH3.

[0140] The present disclosure provides humanized antibodies that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1). A humanized antibody of the present disclosure may comprise a first binding domain that binds to CD47 and comprises one or more CDRs set forth in Table 1. A humanized antibody of the present disclosure may comprise a second binding domain that binds to PD-L1 and comprises one or more CDRs set forth in Table 2. 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 introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanized antibodies that bind to CD47 can be made 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. Patent Nos. 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).

[0141] In some cases, humanized antibodies are constructed by CDR grafting, which involves grafting the amino acid sequences of six complementarity-determining regions (CDRs) of parent non-human antibodies (e.g., rodents) onto human antibody frameworks.For example, Padlan et al. (FASEB J.9:133-139,1995) have determined that only about one-third of the residues in CDRs actually contact antigen, and these are referred to as "specificity-determining residues" or SDRs.In the technique of SDR grafting, only SDR residues are grafted onto human antibody frameworks (see, for example, Kashmiri et al., Methods 36:25-34,2005).

[0142] The selection of human variable domains, both light and heavy, used to create a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another method uses a specific framework derived from the consensus sequence of all human antibodies whose light or heavy chains belong 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 selected from the most abundant human subclass, V L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0143] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. This method involves comparing non-human sequences with functional human germline gene repertoires. Genes encoding the same or closely related canonical structures as the mouse sequences are then selected. Next, among the genes that share canonical structures with non-human antibodies, those with the highest homology within the CDRs are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., J. Immunol. 169:1119-1125, 2002).

[0144] Furthermore, it is generally desirable for antibodies to be humanized while retaining their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display likely 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). Inspection of these displays permits the analysis of residues likely to play a role in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues from the recipient and import sequences can be selected and combined so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0145] Another method for antibody humanization is based on a metric for antibody humanity called human string content (HSC). This method compares the mouse sequence with the human germline gene repertoire and scores differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using a global identity metric to generate a large number of diverse humanized variants (Lazar et al., Mol. Immunol. 44:1986-1998, 2007).

[0146] In addition to the above methods, 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 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).

[0147] In the FR library approach, a collection of residue variants is introduced into a specific position of the FR, and then library selection is performed to select the FR that best supports the grafted CDR. The substituted residues can include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, for example, Foote and Winter J. Mol. Biol. 224: 487-499, 1992), or from a more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0148] 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 humanizing the VL and then the 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, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0149] The "humaneering" method is based on experimental identification of essential minimal specificity determinants (MSDs), followed by sequentially replacing non-human fragments with a library of human FRs and assessing binding. Starting with the CDR3 regions of the non-human VH and VL chains, other regions, including CDR1 and CDR2 of both the VH and VL of the non-human antibody, are gradually replaced with human FRs. This methodology typically results in the identification of antibodies from multiple subclasses that retain epitopes and have distinct human V-segment CDRs. Humaneering makes it possible to isolate antibodies that are 91-96% homologous to human germline antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0150] "Human engineering" methods involve modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by making specific changes to the antibody's amino acid sequence to generate modified antibodies that retain the desired binding characteristics of the original non-human antibody while reducing immunogenicity in humans. Generally, this technique involves classifying amino acid residues in non-human (e.g., murine) antibodies into "low risk," "moderate risk," or "high risk" residues. Classification is performed using a comprehensive risk / reward calculation that assesses the predicted benefit of making a particular substitution (e.g., in terms of immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody and / or be substituted with a human residue. Specific human amino acid residues (e.g., low or moderate risk) to be substituted at a given position in a non-human (e.g., murine) antibody sequence can be selected by aligning amino acid sequences from the variable regions of the non-human antibody with the corresponding regions of specific or consensus human antibody sequences. Amino acid residues at low or moderate risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence according to the alignment. Techniques for producing engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication No. WO 93 / 11794.

[0151] A "humanized antibody" is an antibody in which the CDRs of the heavy and light variable chains of a non-human immunoglobulin have been transferred into human variable domains. The constant regions need not be present, but, if present, optionally are substantially identical to those of a human immunoglobulin, e.g., at least about 85-90%, about 95%, 96%, 97%, 98%, 99% or more identical in some embodiments. Thus, in some cases, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of native human immunoglobulin sequences. For example, humanized antibodies are human immunoglobulins (e.g., host antibodies) in which hypervariable region residues of the host antibody are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity.

[0152] Multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), can be genetically engineered. For example, multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), comprise variable region domains generated, for example, by recombinant DNA engineering techniques. In this regard, including as described above, the variable regions are modified as needed by insertions, deletions, or changes in the amino acid sequence of the antibody to generate the antibody of interest. Polynucleotides encoding the desired complementarity-determining regions (CDRs) are prepared, for example, by using mRNA from antibody-producing cells as a template to synthesize the variable regions using the polymerase chain reaction (see, for example, 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); 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); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106-110, 1991). Current antibody manipulation techniques allow for the construction of engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remaining variable region domain from a second antibody.Such techniques are used, for example, to humanize antibodies or to improve the affinity of antibodies for their binding targets.

[0153] In certain embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein comprise an amino acid sequence having a particular percent identity (e.g., at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) to any antibody or fragment thereof provided herein, e.g., a CDR, VH, or VL in Tables 1-2. In some embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein comprise the CDRs of any antibody or fragment thereof provided herein, e.g., shown in Tables 1-2. In further embodiments, the bispecific antibodies provided herein comprise an amino acid sequence having a particular percent identity (e.g., at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or more) to any antibody or fragment thereof provided herein, e.g., a VH or VL of Tables 1-2.

[0154] The percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be determined using a mathematical algorithm.A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc.Natl.Acad.Sci.USA 87:2264 2268 (1990) and the revised version of Karlin and Altschul, Proc.Natl.Acad.Sci.USA 90:5873 5877 (1993).Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J.Mol.Biol.215:403 (1990).BLAST nucleotide searches can be performed using NBLAST nucleotide program parameters set, for example, score=100, word length=12, to obtain nucleotide sequences that are homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameters set, for example, to score=50 and word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percent identity between two sequences is calculated by dividing the number of residues that vary between the two sequences in the alignment (excluding or including conservative amino acid substitution(s) or degenerate nucleotide substitution(s)) by the number of residues in one of the following: (i) the total length of the shorter sequence; (ii) the total length of the longer sequence; (iii) the average length of the two sequences; (iv) the total length of the ungapped portion of the alignment; (v) the alignment length excluding overhangs; or (vi) the alignment length including overhangs. As used herein, an overhang with respect to a sequence alignment refers to one or both ends of the alignment where residues of one sequence are considered not aligned relative to residues in the other sequence (e.g., a gap).Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web (ncbi.nlm.nih.gov)). Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, typically, only exact matches are counted.

[0155] In some embodiments, the multispecific binding agents provided herein comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:25, and / or to the amino acid sequence of SEQ ID NO:26. and a VL domain having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and binding of the multispecific binding agent 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%, or at least 95%).

[0156] In some embodiments, the multispecific binding agents provided herein comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 46, and / or at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 47. and a VL domain having 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and binding of the multispecific binding agent to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0157] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning, to identify amino acids in the CD47 (or PD-L1) protein required for interaction with a multispecific binding agent, its CD47 (or PD-L1) binding domain, and / or an anti-CD47 (or anti-PD-L1) antibody provided herein. In some embodiments, conformational and crystal structures of a multispecific binding agent, its CD47 (or PD-L1) binding domain, and / or an anti-CD47 (or anti-PD-L1) antibody bound to CD47 (or PD-L1) can be used to identify epitopes. In some embodiments, the present disclosure provides multispecific antibodies comprising a CD47 (or PD-L1) binding domain that specifically binds to the same epitope as any of the multispecific binding agents disclosed herein and a CD47 (or PD-L1) binding domain thereof, and / or an anti-CD47 (or anti-PD-L1) antibody or fragment thereof provided herein.

[0158] Binding Agents - Scaffolds and Generation In some embodiments, a multispecific binding agent (e.g., an antibody such as a bispecific antibody) described herein has 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 VH domain or VH region described herein in combination with one or more heavy chain constant domains or regions (e.g., CH1, hinge, CH2, and CH3), and (ii) a light chain having a VL domain or VL region described herein in combination with a light chain constant domain or region (CL). An exemplary IgG heavy chain comprises any of the VH domains described herein, and the following CH1, hinge, CH2, and CH3 amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65). Another exemplary IgG heavy chain comprises any VH domain described herein, as well as the following CH1, hinge, CH2, and CH3 amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66). An exemplary light chain (e.g., for pairing with an IgG heavy chain) comprises any of the VL domains described herein and the following CL amino acid sequence: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67).

[0159] In some embodiments, mutations are introduced into one or more of the heavy chain constant regions (such as CH1, CH2, and / or CH3) and / or one or more light chain constant regions (such as CL) to accomplish one or more of the following: (i) destabilize homodimers formed by the polypeptides of the multispecific antibodies; (ii) stabilize the multispecific antibodies described herein (also referred to herein as heterodimers); (iii) promote proper assembly of the multispecific antibodies described herein; (iv) favor heterodimerization over homodimerization of the constituent polypeptide chains; (v) improve the yield of the multispecific antibodies described herein; and (vi) improve the purity of the multispecific antibodies described herein. Various mutations have been developed that promote preferential heterodimerization, such as knob-in-hole (KIH or KiH) mutations (see, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; and 8,216,805), disulfide-stabilized KIH mutations (see, e.g., U.S. Pat. Nos. 7,951,917; 8,642,745; and 9,409,989), and others (see, e.g., WO2022 / 125986). Each patent and / or patent publication cited herein is incorporated herein by reference in its entirety.

[0160] In certain embodiments, the multispecific antibodies provided herein comprise four polypeptides: a first polypeptide comprising, from N- to C-terminus, a first VL, a first CH3, an optional hinge, a first CH2, and a second CH3; a second polypeptide comprising, from N- to C-terminus, a first VH and a third CH3; a third polypeptide comprising, from N- to C-terminus, a second VL, a CL (optionally a kappa CL, also referred to herein as CK), an optional hinge, a second CH2, and a fourth CH3; and a fourth polypeptide comprising, from N- to C-terminus, a second VH and a CH1. These four polypeptides form two binding domains. In some embodiments, the first and second polypeptides (e.g., their first VL and first VH) form a binding domain that binds to PD-L1, and the third and fourth polypeptides (e.g., their second VL and second VH, etc.) form a binding domain that binds to CD47. In other embodiments, the first and second polypeptides (e.g., their first VL and first VH) form a binding domain that binds to CD47, and the third and fourth polypeptides (e.g., their second VL and second VH, etc.) form a binding domain that binds to PD-L1. In some embodiments, the amino acid sequences of the first CH3, the second CH3, the third CH3, and the fourth CH3, or any subgroup thereof, are identical to one another. In some embodiments, the amino acid sequences of the first CH3, the second CH3, the third CH3, and the fourth CH3, or any subgroup thereof, are different from one another. In some embodiments, the second CH3 and the fourth CH3 provide a knob-in-hole assembly. Additionally or alternatively, the amino acid sequences of the first CH2 and the second CH2 are identical to each other. In other embodiments, the amino acid sequences of the first CH2 and the second CH2 are different from each other.

[0161] In some embodiments, any one or more of the CH3 sequences comprises GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70; aa 116 to aa 222 of SEQ ID NO: 62).

[0162] In some embodiments, the isoallotypic mutations D356E and L358M are made in the CH3 sequence as disclosed herein. In some embodiments, one or more isoallotypic mutations (e.g., either or both of D356E and L358M) are present in one or more of the CH3 sequences immediately adjacent to the C-terminus of the CH2 sequence. Additionally or alternatively, any one or more of the CH3 sequences are engineered to reduce the risk of antibody immunogenicity by replacing specific amino acids of one allotype with amino acids of another allotype, which is referred to herein as isoallotypic mutation and is described in detail by Stickler et al. (Genes Immun. 2011 Apr;12(3):213-221).

[0163] Additionally or alternatively, the CH3 sequence is engineered to contain knob-into-hole mutations. In some embodiments, in adjacent CH3 / CH3 pairs C-terminal to the CH2 sequence (e.g., one CH3 in one polypeptide dimerizes with another CH3 in a different polypeptide when forming a binding agent containing the polypeptide), one CH3 contains a knob mutation (e.g., T366W) and the other CH3 contains a hole mutation (e.g., any one, any two, or all three of T366S, L368A, and Y407V). In other embodiments, in a VH / VL pair (e.g., where the VH and VL form a binding domain in a binding agent comprising the VH and VL), one CH3 immediately adjacent to the C-terminus of the VH or VL comprises a knob mutation (e.g., T366W), and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) comprises a hole mutation (e.g., any one or any two or all three of T366S, L368A, and Y407V).

[0164] As will be understood by one of skill in the art, a domain pair, e.g., a CH3 / CH3 pair, a VH / VL pair, a VL / VH pair, a CH2 / CH2 pair, a CH1 / CL pair, or a CL / CH1 pair, refers to one antibody domain (e.g., VH, VL, CH1, CH2, CH3, or CL) in one polypeptide chain that is dimerized to another antibody domain (such as VL, VH, CL, CH2, CH3, or CH, respectively) in a different polypeptide chain when forming a binding agent that includes the polypeptide.

[0165] In some embodiments, in a CH3 / CH3 pair (e.g., one CH3 in one polypeptide dimerizes with another CH3 in a different polypeptide when forming a binding agent comprising the polypeptides), one CH3 comprises a Y349C mutation and the other CH3 comprises a S354C mutation. In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair comprises a S354C mutation, and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) comprises a Y349C mutation. Additionally or alternatively, the CH3 sequence is engineered to allow for disulfide bond formation in the antibody, e.g., to stabilize the knob-into-hole mutations described above.

[0166] Additionally or alternatively, any one or more of the CH3 sequences are engineered to include other mutation(s), provided that the mutation(s) do not significantly reduce the affinity and / or stability of the antibody or significantly increase the risk of immunogenicity of the antibody. In some embodiments, any one or more of the CH3 sequences are engineered to include the mutations described in WO2022 / 125986.

[0167] In some embodiments, in a CH3 / CH3 pair, one CH3 comprises a S354C mutation and the other CH3 comprises a Y349C mutation. Additionally or alternatively, in a CH3 / CH3 pair, E357 of one CH3 is substituted with a hydrophobic amino acid or an aromatic amino acid. In various embodiments, the hydrophobic amino acid residue is selected from the group consisting of isoleucine (I), leucine (L), methionine (M), proline (P), and valine (V). In various embodiments, the aromatic amino acid is selected from the group consisting of histidine (H), tryptophan (W), phenylalanine (F), and tyrosine (Y). In some embodiments, E357 of the CH3 is substituted with W. Additionally or alternatively, in some embodiments, a CH (e.g., CH3) comprises a K370R mutation that is dimerized to the E357 mutation-containing CH3 in the binding agent. In some embodiments, one CH3 of a CH3 / CH3 pair comprises a K370R mutation. Additionally or alternatively, the other CH3 of the same CH3 / CH3 pair comprises an E357W mutation. In some embodiments, one CH3 of a CH3 / CH3 pair comprises a K370R mutation, and the other CH3 of the same CH3 / CH3 pair comprises an E357W mutation. In some embodiments, one CH3 of a CH3 / CH3 pair comprises S354C and E357W, and the other CH3 comprises Y349C and K370R. In some embodiments, each CH3 of a CH3 / CH3 pair is immediately adjacent to the C-terminus of the CH2 sequence. In other embodiments, one CH3 of a CH3 / CH3 pair is immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair, and the other CH3 of a CH3 / CH3 pair is immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair. In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair contains E357W, and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) contains K370R. In other embodiments, in a CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence, one CH3 contains both S354C and E357W, and the other CH3 contains both Y349C and K370R.

[0168] Additionally or alternatively, one or more amino acid residues in CH3 are swapped with one or more corresponding amino acid residues in CH1. In some embodiments, as used herein, a first amino acid residue in a first peptide corresponding to a second amino acid residue in a second peptide refers to the first amino acid residue aligned with the second amino acid residue in a sequence alignment between the first and second peptides. Alignment methods, such as BLAST and / or Clustal Omega, as disclosed herein, are available to those skilled in the art. In some embodiments, CH3 is immediately adjacent to the C-terminus of VH or VL. Additionally or alternatively, one or more amino acid residues are in an N-terminal fragment of CH1, e.g., selected from the first to tenth amino acids (e.g., first to fifth, first to third, first or third, etc., including each range or integer therebetween) of CH1. In some embodiments, CH3 comprises a first amino acid residue swapped with a first amino acid residue in CH1, which is also referred to herein as the N-terminal amino acid residue swapped with CH1. In some embodiments, the first amino acid residue of CH3 that is G is substituted with the first amino acid residue of CH1 that is A. Such a substitution is also referred to herein as G341A for ease of reference. In some embodiments, the CH3 immediately adjacent to the C-terminus of the VH or VL comprises G341A. Without wishing to be bound by theory, a CH3 immediately adjacent to the C-terminus of the VH or VL and engineered to comprise a CH1 N-terminal fragment can improve the assembly and / or purity of the binding agents disclosed herein. In some embodiments, in a CH3 / CH3 pair, each CH3 is immediately adjacent to the C-terminus of the VH or VL, and one CH3 comprises S354C and E357W mutations, and the other CH3 comprises Y349C and K370R mutations. In some embodiments, in a CH3 / CH3 pair, each CH3 is immediately adjacent to the C-terminus of a VH or VL, and one CH3 includes G341A, S354C, and E357W mutations, and the other CH3 includes Y349C and K370R mutations.In some embodiments, in a CH3 / CH3 pair, each CH3 is immediately adjacent to the C-terminus of a VH or VL, and one CH3 comprises G341A, S354C, and E357W mutations, and the other CH3 comprises G341A, Y349C, and K370R mutations. In some embodiments, in a CH3 / CH3 pair, each CH3 is immediately adjacent to the C-terminus of a VH or VL, and one CH3 comprises S354C and E357W mutations, and the other CH3 comprises G341A, Y349C, and K370R mutations.

[0169] In some embodiments, the multispecific binding agents described herein comprise one or more CH3 mutations as disclosed in WO2022 / 125986, which is incorporated herein by reference in its entirety. In some embodiments, the multispecific binding agents described herein comprise one or more CH3 domains disclosed in WO2022 / 125986.

[0170] Thus, any one or more of the CH3 sequences includes any one of the following: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70; aa 116 to aa 222 of SEQ ID NO: 62); GQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71; Y349C and K370R); AQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72; N-terminal amino acid residues swapped with CH1, Y349C and K370R); GQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73; S354C and E357W); AQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74; N-terminal amino acid residues swapped with CH1, S354C and E357W); GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 75; D356E, L358M, T366S, L368A and Y407V); or GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 76; T366W).

[0171] In some embodiments, a binding agent disclosed herein comprises a CH3 sequence disclosed herein (e.g., any one of SEQ ID NOs: 70-74), but the CH3 lacks the C-terminal lysine (K) amino acid residue. In further embodiments, the CH3 is at the C-terminus of any one or more polypeptides of the binding agent. In some embodiments, the C-terminal lysine has been truncated in one or more polypeptides of the binding agent, e.g., any one, or any two, or any three of a first polypeptide of a binding agent disclosed herein, a second polypeptide of a binding agent disclosed herein, and a third polypeptide of a binding agent disclosed herein.

[0172] In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair comprises GQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71; Y349C and K370R); and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) comprises AQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74; N-terminal amino acid residues swapped with CH1, S354C, E357W).

[0173] In some embodiments, in the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence, one CH3 comprises GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 75; D356E, L358M, T366S, L368A and Y407V), and the other CH3 comprises GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 76; T366W).

[0174] As will be understood by those skilled in the art, and unless otherwise specified, EU numbering (also referred to herein as EU indexing) is referred to herein when describing mutations of antibodies or fragments thereof, such as Fc or CH3. For further details, see www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs, which is incorporated herein by reference in its entirety, and identifies residues according to their position in the endogenous constant region sequence, regardless of the residue's physical location within the chains of the antibody constructs described herein. For example, in a CH3 consisting of aa116 to aa222 of SEQ ID NO: 62, the first aa of CH3 (e.g., the 116th aa of SEQ ID NO: 62) is numbered as 341 and is referred to herein as G341; the 9th aa of CH3 is referred to herein as Y349; the 14th aa of CH3 is referred to herein as S354; the 16th aa of CH3 is referred to herein as D356; the 17th aa of CH3 is referred to herein as E357; the 18th aa of CH3 is referred to herein as L358; the 26th aa of CH3 is referred to herein as T366; the 28th aa of CH3 is referred to herein as L368; the 30th aa of CH3 is referred to herein as K370; and the 67th aa of CH3 is referred to herein as Y407. Therefore, the mutated aa residues can be added after the EU numbering to identify the mutation, such as S354C, E357W, Y349C, K370R, D356E, L358M, T366W, T366S, L368A, and Y407V.

[0175] In some embodiments, any one or more of the CH2 sequences comprises APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAK (SEQ ID NO: 77; aa 6 to aa 115, L234A, L235A and P329K of SEQ ID NO: 63); or APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 78; aa 6 to aa 115, L234, L235 and P329 of SEQ ID NO: 62). In some embodiments, any one or more of the CH2 sequences lack a mutation that reduces (including significantly reduces and eliminates) the effector function of the multispecific binding agent. In further embodiments, any one or more of the CH2 sequences lack any one, any two, or all three of the following mutations: L234A, L235A, and P329K (according to the EU numbering system). In some embodiments, any one or more of the CH2 sequences include any one, any two, or all three of the following: L234, L235, and P329 (according to the EU numbering system). In some embodiments, any one or more of the CH2 sequences lack a mutation that reduces the effector function of the multispecific binding agent. In some embodiments, the multispecific binding agents described herein comprise one or more CH2 domains disclosed in WO2022 / 125986.

[0176] In some embodiments, one or more light chain constant regions (CL) are RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 79) or RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67). In some embodiments, the multispecific antibodies described herein comprise one or more CL mutations disclosed in WO2022 / 125986. In some embodiments, the multispecific binding agents described herein comprise one or more CL domains disclosed in WO2022 / 125986.

[0177] In some embodiments, one or more CH1 sequences are ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 80; aa1 to aa103 of SEQ ID NO: 65), ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 81), or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDRKVEPKSC (SEQ ID NO: 82). In some embodiments, the multispecific binding agents described herein comprise one or more CH1 mutations disclosed in WO 2022 / 125986. In some embodiments, the multispecific binding agents described herein comprise one or more CH1 domains disclosed in WO 2022 / 125986.

[0178] In some embodiments, the hinge domain is immediately adjacent to the N-terminus of the CH2 domain, e.g., between CH3 and CH2, or between the light chain matching domain (CL) and CH2. Additionally or alternatively, the hinge domain is immediately adjacent to the C-terminus of the CL and the N-terminus of the light chain variable domain (VL). In further embodiments, the hinge domain comprises DKTHTCPPCP (SEQ ID NO: 83). In some embodiments, the multispecific binding agents described herein comprise one or more domain junctions disclosed in WO2022 / 125986.

[0179] In some embodiments, the multispecific antibodies described herein comprise the antibody constructs disclosed in WO2022 / 125986.

[0180] In some aspects, provided herein are binding agents comprising four polypeptide chains: a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. In some embodiments, the first polypeptide comprises (i) the amino acid sequence of SEQ ID NO: 48, or (ii) the amino acid sequence of SEQ ID NO: 48 lacking the C-terminal lysine (K). Additionally or alternatively, the second polypeptide comprises (i) the amino acid sequence of SEQ ID NO: 49, or (ii) the amino acid sequence of SEQ ID NO: 49 lacking the C-terminal lysine (K). Additionally or alternatively, the third polypeptide chain comprises (i) the amino acid sequence of SEQ ID NO: 50, or (ii) the amino acid sequence of SEQ ID NO: 50 lacking the C-terminal lysine (K). Additionally or alternatively, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51.

[0181] In some embodiments, a binding agent, referred to herein as bsAb1, is provided. That is, the binding agent comprises a first polypeptide chain represented by SEQ ID NO:48, a second polypeptide chain represented by SEQ ID NO:49, a third polypeptide chain represented by SEQ ID NO:50, and a fourth polypeptide chain represented by SEQ ID NO:51. In further embodiments, the C-terminal lysine of the first polypeptide chain of bsAb1 is removed (e.g., cleaved) from the binding agent. Additionally or alternatively, the C-terminal lysine of the second polypeptide chain of bsAb1 is removed (e.g., cleaved) from the binding agent. Additionally or alternatively, the C-terminal lysine of the third polypeptide chain of bsAb1 is removed (e.g., cleaved) from the binding agent. Additionally or alternatively, the C-terminal lysine of the first polypeptide chain and the C-terminal lysine of the second polypeptide chain of bsAb1 are removed (e.g., cleaved) from the binding agent. Additionally or alternatively, the C-terminal lysine of the first polypeptide chain and the C-terminal lysine of the third polypeptide chain of bsAb1 are removed (e.g., cleaved) from the binding agent. Additionally or alternatively, the C-terminal lysine of the second polypeptide chain and the C-terminal lysine of the third polypeptide chain of bsAb1 are removed (e.g., cleaved) from the binding agent. Additionally or alternatively, the C-terminal lysine of each of the first, second, and third polypeptide chains are removed (e.g., cleaved) from the binding agent. As used herein, a bsAb1 from which any one, any two, or all three of its C-terminal lysine amino acid residues have been removed is referred to as a C-terminal variant of bsAb1. Accordingly, compositions comprising bsAb1 and one or more C-terminal variants thereof, or compositions comprising one or more C-terminal variants of bsAb1, are also provided herein. As will be appreciated by one of skill in the art, the compositions, methods, uses or any other embodiments relating to bsAb1 disclosed herein also extend to compositions, methods, uses or embodiments of (i) C-terminal variants of bsAb1 or (ii) compositions comprising any one or more of bsAb1 and / or its C-terminal variants.

[0182] In some embodiments, the multispecific antibodies described herein are monoclonal antibodies. In some embodiments, the monoclonal antibodies are humanized, human, 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, dual variable 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 optionally humanized, human, or chimeric antibodies.

[0183] In some embodiments, the multispecific binding agents described herein comprise a non-antibody protein scaffold. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, anticalins, adnectins, affibodies, DARPins, finomers, affitins, affilins, avimers, cysteine-rich knottin peptides, or engineered Kunitz-type inhibitors. Methods for producing such non-antibody protein scaffolds are well known in the art, any one of which can be used to produce a multispecific binding agent comprising a non-antibody protein scaffold (see, e.g., 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)).

[0184] Methods for producing multispecific antibodies are known in the art, such as by co-expressing two immunoglobulin heavy chain-light chain pairs, where 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, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0185] 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.

[0186] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with additional antigen-binding moieties appended, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. As non-limiting examples, BsIgG formats can 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, orthogonal Fab.

[0187] In some embodiments, the BsIgG comprises a heavy chain engineered for heterodimer formation. For example, the heavy chain can be engineered for heterodimer formation by utilizing a "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., in κλ-body), and the use of heterodimeric Fc regions. Strategies for avoiding homodimeric heavy chain pairing in BsIgG are known in the art, including knobs-into-holes, duobodies, azymetric, charge-pair, HA-TF, SEEDbodies, and differential Protein A affinity.

[0188] Another bispecific antibody format is an IgG with an additional antigen-binding moiety added. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the 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 chains or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). Non-limiting examples of adjunct IgG formats include dual variable 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, an exemplary antibody format is the B-Body format for monospecific or multispecific (e.g., bispecific) antibodies, as described, for example, in International Patent Application Publication Nos. WO2018 / 075692, WO2022 / 125986, and U.S. Patent Application Publication No. 2018 / 0118811.

[0189] Bispecific antibody fragments are formats of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some bispecific antibody fragments lack the Fc region. In some embodiments, bispecific antibody fragments comprise heavy and light chain regions connected by a peptide linker, allowing for efficient expression of the bispecific antibody fragment in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody-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 intrabody.

[0190] Bispecific fusion proteins include 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 high-valency bispecific antibody molecules. For example, fusing bispecific antibodies to albumin-binding proteins or human serum albumin can extend the serum half-life of antibody fragments. In some embodiments, chemical conjugation, e.g., of antibodies and / or antibody fragments, can be used to create bispecific antibody fragment molecules. Exemplary bispecific antibody conjugates include the CovX-body format, which site-specifically conjugates a low-molecular-weight drug to a single reactive lysine within each Fab arm or antibody or fragment thereof. In some embodiments, conjugation improves serum half-life.

[0191] 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 the component antibodies in different host cells and then purifying / assembling them, or by expressing the component 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.

[0192] In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind CD47, including human CD47, as disclosed herein, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), may be provided in any antibody format disclosed herein or known in the art. By way of non-limiting example, in some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), may be provided in any antibody format disclosed herein or known in the art. Tandem single-chain Fv fragments; TandAb, trispecific Ab (Affimed); Darts dual affinity retargeting (Macrogenics); bispecific Xmab (Xencor); bispecific T cell engager (Bites; Amgen; 55 kDa); triplebody; tribody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (CreativeBiolabs); duobody platform (Genmab); dock and lock and lock platform; knobs-into-holes (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-Ig = dual variable domain immunoglobulin (Abbott); kappa-lambda body; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0193] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise 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 comprise a CD47-binding domain comprising a VH and / or VL amino acid sequence of Table 1.

[0194] In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that bind to CD47 and comprise binding domains comprising the VH and VL CDRs depicted in Table 1.

[0195] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise a CD47-binding domain and a PD-L1-binding domain. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise a CD47-binding domain comprising a VH and / or VL amino acid sequence in Table 1, and a PD-L1-binding domain comprising a VH and / or VL amino acid sequence in Table 2.

[0196] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise a CD47-binding domain comprising a VH and VL amino acid sequence in Table 1, and a PD-L1-binding domain comprising a VH and VL amino acid sequence in Table 2.

[0197] In some embodiments, the antibody is a human antibody, including, but not limited to, antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences, e.g., as described in Kabat et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242. If the antibody contains a constant region, it is preferred that the constant region also be derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., to enhance the activity of the antibody, but do not contain CDRs derived from other species (e.g., murine CDRs positioned within human variable framework regions).

[0198] Antibodies that bind CD47 and / or PD-L1 can be obtained by any suitable method, including (but not limited to) immunization with whole tumor cells containing CD47 and / or PD-L1 and 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 a variety of known techniques (see, e.g., 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 producing monoclonal antibodies involves immunizing an animal with human CD47 antigen and producing hybridomas from spleen cells obtained from the animal. The hybridomas can produce monoclonal antibodies or antibody fragments that bind to CD47. One exemplary technique for producing monoclonal antibodies involves immunizing an animal with human PD-L1 antigen and producing hybridomas from spleen cells obtained from the animal. The hybridomas can produce monoclonal antibodies or antibody fragments that bind to PD-L1.

[0199] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques such as those described in "Antibody Phage Display: Methods and Protocols," P.M.O. Brien and R. Aitken, eds., Humana Press, Totawa, NJ, 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones can then be eluted from the antigen and further enriched by additional antigen adsorption / elution cycles.

[0200] For example, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994), variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which VH and VL are covalently linked by a short, flexible peptide, or as Fab fragments in which VH and VL are each fused to a constant domain and interact non-covalently.

[0201] As described in Winter et al., supra, VH gene repertoires and VL gene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding clones. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self antigens and self antigens, without any immunization. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described, for example, in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0202] Screening of the library can be accomplished by various techniques known in the art. For example, CD47 (e.g., a CD47 polypeptide, fragment, or epitope) or PD-L1 (e.g., a PD-L1 polypeptide, fragment, or epitope) can be used to coat the wells of an adsorption plate, expressed on host cells immobilized on 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. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by the use of extended washes and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690, and by the use of low coating densities of antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0203] Multispecific binding agents can be obtained by designing an appropriate antigen screening procedure to select for phage clones 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 full-length multispecific binding agent (e.g., antibody) clones using VH and / or VL sequences (e.g., Fv sequences) or various CDR sequences from the VH and VL sequences from the phage clones of interest and appropriate constant region (e.g., Fc) sequences.

[0204] Similarly, human antibodies that bind CD47 and / or PD-L1 can be produced by any of a number of 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 splenocytes 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 the present disclosure. Methods for obtaining human antibodies from transgenic animals are further 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 U.S. Patent No. 5,877,397.

[0205] 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 antigen challenge. For example, International Patent Publication No. WO 98 / 24893 discloses transgenic animals with human Ig loci that do not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain loci. Also described are transgenic non-primate mammalian hosts capable of mounting an immune response to immunogens, in which the antibodies have primate constant and / or variable regions and in which the endogenous immunoglobulin-encoding loci have been replaced or inactivated. International Patent Publication No. WO 96 / 30498 discloses the use of the Cre / Lox system to modify immunoglobulin loci in mammals, e.g., to replace all or part of the constant or variable regions to form modified antibody molecules. International Patent Publication No. WO 94 / 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 transgenic mice lacking endogenous heavy chains and expressing exogenous immunoglobulin loci containing one or more heterologous constant regions. Transgenic animals, such as those described herein, can be used to generate an immune response to a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to generate hybridomas secreting human-derived monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art and have been used, for example, to immunize transgenic mice, as described in International Patent Publication No. WO 96 / 33735. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.

[0206] Fusion Proteins and Conjugates The present disclosure provides multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), with masking moieties and / or cleavable moieties, wherein one or more of the CD47 and / or other target binding domains of the multispecific binding agent (e.g., antibody) are masked (e.g., via a masking moiety) and / or activatable (e.g., via a cleavable moiety). Techniques for masking multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) are well known in the art, including SAFE body masking technology (see, e.g., U.S. Patent Application Publication No. 2019 / 0241886) and Probody masking technology (see, e.g., U.S. Patent Application Publication No. 2015 / 0079088). Such techniques can be used to generate masked and / or activatable multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies). In some embodiments, such masked and / or activatable multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), comprise any one of the multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) of the present disclosure that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), e.g., human CD47 binding agents, and are useful for the preparation of conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable antibody-drug conjugates (AADCs).In some embodiments, for example, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) of the present disclosure that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), including human CD47 binding agents, is directly or indirectly linked to another agent, such as a drug. In further embodiments, the multispecific binding agents described herein may be covalently linked to one or more agents, such as drugs, by a synthetic linker.

[0207] Optionally, the multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) is linked or conjugated (directly or indirectly) to an effector function, e.g., a moiety having cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or a moiety having immunomobilizing activity (e.g., a cytokine). Moieties that are linked or conjugated (directly or indirectly) include drugs that are cytotoxic (e.g., a toxin, e.g., an aurostatin) or non-cytotoxic (e.g., a signal transduction modulator, e.g., a kinase, or a masking moiety that masks one or more binding domains of the multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody), or a cleavable moiety that activates the multispecific binding agent by cleavage of the cleavable moiety to unmask one or more binding domains of the multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) in the tumor microenvironment). Moieties that promote immune recruitment can include other antigen-binding agents, for example, viral proteins that selectively bind to cells of the innate immune system. Alternatively, or in addition, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) are optionally linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from a mixture (e.g., a tag) or a moiety that has reporter activity (e.g., a detectable label or reporter protein). It is understood that the features of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) described herein also extend to polypeptides, including binding agent fragments.

[0208] In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, may be linked or conjugated (directly or indirectly) to a polypeptide, which may result in the generation of an activatable antibody. In some embodiments, a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) is linked or conjugated (directly or indirectly) to an agent. In some embodiments, the agent is a drug that generates an ADC or AADC when the antibody of the ADC comprises a masking moiety and a cleavable moiety.

[0209] In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including those multispecific binding agents that bind CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detection agent. Conjugated or recombinantly linked antibodies, including masked or activatable conjugates, may be useful, for example, for treating or preventing a disease or disorder, e.g., an immune cell dysfunction disease, disorder, or condition. Conjugated or recombinantly linked multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), including masked or activatable conjugates, may be useful, for example, for monitoring or prognosing the onset, development, progression, and / or severity of immune cell dysfunction disorders.

[0210] Such diagnosis and detection can be accomplished, for example, by coupling the multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody) to a detectable substance, including, for example: an enzyme, including but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; a prosthetic group, including but not limited to, streptavidin / biotin or avidin / biotin; a fluorescent material, including but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent material, including but not limited to, luminol; a bioluminescent material, including but not limited to, luciferase, luciferin, or aequorin; a chemiluminescent material, including but not limited to, acridinium-based compounds or HALOTAG; iodine ( 131 I, 125 I, 123 I, and 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, and 111 In), technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga and 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, or 117 Radioactive materials include, but are not limited to, Sn; various positron-emitting metals using positron emission tomography; and non-radioactive paramagnetic metal ions.

[0211] Also described herein are multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) recombinantly linked or conjugated (covalently or non-covalently, directly or indirectly) to a heterologous protein or polypeptide or fragment thereof, e.g., to a polypeptide (e.g., of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate a fusion protein, and uses thereof. In particular, described herein are fusion proteins comprising an antigen-binding fragment (e.g., comprising CDR1, CDR2, and / or CDR3 of the VH and / or VL) of a multispecific binding agent (e.g., an antibody, e.g., a bispecific antibody), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, and a heterologous protein, polypeptide, or peptide. In some embodiments, heterologous proteins, polypeptides, or peptides to which multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies) are linked are useful for targeting the multispecific binding agents to particular cells (e.g., CD47- and / or PD-L1-expressing cells, including tumor cells).

[0212] Additionally, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including those that bind CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, can be linked (directly or indirectly) 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, inter alia, a hexa-histidine peptide, such as the tag provided in a pQE vector (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 for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the "FLAG" tag.

[0213] Methods for linking or conjugating (directly or indirectly) moieties (including polypeptides) to antibodies are well known in the art, any one of which can be used to generate the antibody-drug conjugates or fusion proteins described herein.

[0214] In some embodiments, the multispecific binding agents (e.g., antibodies) described herein are fusion proteins. The term "fusion protein," as used herein, refers to a polypeptide that comprises the amino acid sequence of a binding agent (e.g., an antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not normally 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 agent. In certain embodiments, the fusion protein comprises a first binding domain comprising a VH region, a VL region, a VH CDR (one, two, or three VH CDRs), and / or a VL CDR (one, two, or three VL CDRs) of a CD47 antibody, and the fusion protein binds to a CD47 epitope, CD47 fragment, and / or CD47 polypeptide. In certain embodiments, the fusion protein comprises a second binding domain comprising a VH region, a VL region, a VH CDR (one, two, or three VH CDRs), and / or a VL CDR (one, two, or three VL CDRs) of a PD-L1 antibody, and the fusion protein binds to a PD-L1 epitope, PD-L1 fragment, and / or PD-L1 polypeptide.

[0215] Fusion proteins can be generated, for example, through gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling techniques (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), as described herein, including multispecific binding agents with higher affinity and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1998 ... (See, e.g., Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). In some embodiments, multispecific binding agents, including those that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding the multispecific binding agents described herein may be recombined with one or more components, motifs, regions, portions, domains, fragments, etc., of one or more heterologous molecules.

[0216] Multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including those that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, can also be attached to solid supports useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0217] Multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including those multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0218] The linker may be a "cleavable moiety" that facilitates intracellular release of the linked or conjugated agent, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates (e.g., antibody-drug conjugates) of the present disclosure include, without limitation, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance.

[0219] Conjugates of antibodies and agents, including those in which the agent is a drug for preparing an ADC or AADC, can be made using a variety of 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)benzoate). The present disclosure further contemplates that conjugates of antibodies and agents, including those in which the agent is a drug for preparing an ADC or AADC, can be prepared using any suitable method disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0220] Conjugation strategies for antibodies and agents, including those where the agent is a drug for preparing an ADC or AADC, have traditionally been based on random conjugation chemistry involving the ε-amino group of a Lys residue or the thiol group of a Cys residue, resulting in heterogeneous conjugates. Recently developed techniques allow for site-specific conjugation with antibodies, thereby resulting in uniform loading and avoiding conjugate subpopulations with altered antigen binding or pharmacokinetics. These techniques include engineering "thiomabs," which contain cysteine ​​substitutions at positions on the heavy and light chains that result in reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter binding to antigens (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332:41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another method, selenocysteine ​​is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from termination to selenocysteine ​​insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural 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).

[0221] In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1) described herein, are conjugated to a cytotoxic agent. In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1) disclosed herein, can be optionally conjugated to one or more cytotoxic agent(s) disclosed herein or known in the art to generate 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 intercalating agents. 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, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene. In some embodiments, the cytotoxic agent is a radioactive isotope for making a radioconjugate or a radioconjugated agent. 90 Y, 125 I, 131 I, 123 I, 111 In, 131 In, 105 Rh, 153 Sm, 67Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re, and 212 A variety of radionuclides, including Bi, are available for making radioconjugated agents. Conjugates of a polypeptide or molecule with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothenes, and CC1065, and the toxic derivatives of these toxins, can also be used. Conjugates of polypeptides or molecules and cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido 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).

[0222] In some embodiments, multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), as described herein, are conjugated to drugs such as signal transduction modulators, pro-apoptotic agents, antimitotic agents, antitumor antibiotics, immunomodulatory agents, nucleic acids for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormone agents, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors. In some embodiments, the antimitotic agent is a dolastatin, auristatin, maytansinoid, or plant alkaloid. In some embodiments, the drug is a dolastatin, auristatin, maytansinoid, or plant alkaloid. Examples of auristatins are monomethylaurisatin F (MMAF) or monomethyauristatin E (MMAE). Examples of maytansinoids 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, anthracycline, calicheamicin, and duocarmycin. In some embodiments, the actinomycin is a pyrrolobenzodiazepine (PBD).

[0223] Polynucleotides, vectors, and production Materials for producing multispecific binding agents and fragments thereof are further provided. For example, isolated cells (e.g., hybridomas) can produce multispecific binding agents (e.g., antibodies or antibody fragments). In this regard, cells (e.g., isolated cells) can produce antibodies or fragments thereof that include a first binding domain comprising the VH and VL shown in Table 1 for mAb-C. Alternatively, or in addition, cells (e.g., isolated cells) can produce antibodies or fragments thereof that include a second binding domain comprising the VH and VL shown in Table 2 for mAb-P.

[0224] In some embodiments, provided herein are polynucleotides encoding the multispecific binding agents disclosed herein, polypeptide chains thereof, or fusion polypeptides disclosed herein, or polynucleotides complementary thereto. In some embodiments, the polynucleotides described herein may comprise one or more nucleic acid sequences encoding a multispecific binding agent (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotides are isolated and / or recombinant polynucleotides. In various aspects, the isolated polynucleotides comprise nucleotide sequences encoding an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL), wherein the VH and VL comprise complementarity-determining regions (CDRs) identical to the CDRs set forth in Table 1. In various aspects, the isolated polynucleotides comprise nucleotide sequences encoding an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL), wherein the VH and VL comprise complementarity-determining regions (CDRs) identical to the CDRs set forth in Table 2.

[0225] As used herein, the term "complementary" refers to the specific binding between polynucleotides based on the sequence of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, for example, if they produce a given or detectable level of signal in a hybridization assay. Portions of polynucleotides are complementary to each other when they follow conventional base pairing rules (e.g., A pairs with T (or U), G pairs with C), but there may be small regions of mismatch, insertion, or deletion sequence (e.g., less than about 3 bases). The term "stringent assay conditions" refers to conditions that are suitable for producing binding pairs of nucleic acids, such as probes and target mRNAs, that have sufficient complementarity to provide the desired level of specificity in the assay, but are generally not suitable for the formation of binding pairs between binding members that are not sufficiently complementary to provide the desired specificity. The term "stringent assay conditions" generally refers to the combination of hybridization conditions and washing conditions.

[0226] In some embodiments, one or more vectors (e.g., expression vectors) may comprise one or more polynucleotides disclosed herein, e.g., for expressing one or more polynucleotides in a suitable host cell and / or for producing one or more polynucleotides disclosed herein. Such vectors are useful, e.g., for amplifying the polynucleotides in a host cell to generate useful quantities thereof, and for expressing binding agents such as antibodies and / or antibody fragments using recombinant techniques.

[0227] In some embodiments, the one or more vectors are expression vectors, in which one or more polynucleotides are operatively linked to one or more polynucleotides comprising expression control sequences. Autonomously replicating recombinant expression constructs, such as plasmid and viral DNA vectors, incorporating one or more polynucleotides encoding antibody sequences that bind to CD47 are specifically contemplated. Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct will be utilized. 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 contain sequences encoding one or more selectable markers that allow identification of host cells carrying the construct. The expression construct may also contain sequences that facilitate, and preferably promote, homologous recombination in the host cell. In some embodiments, the expression constructs disclosed herein may also contain sequences necessary for replication in the host cell.

[0228] Exemplary expression control 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); the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther. 4:151, 1993); the Tie promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995); the simian virus 40 promoter; DRA (downregulated in adenomas; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293:G923-934, 2007); MCT1 (monocarboxylate transporter 1; Cuff et al., J. Physiol. Gastrointest. Liver Physiol., 293:G923-934, 2007); al., Am. J. Physiol. Gastrointet. Liver Physiol., G977-G979, 2005); and Math1 (mouse atonal homolog 1; Shroyer et al., Gastroenterology, 132:2477-2478, 2007), where the promoter is operably linked upstream (e.g., 5') of the polypeptide coding sequence. In another variant, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include an appropriate polyadenylation sequence (e.g., SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0229] If desired, the one or more polynucleotides may also optionally contain a nucleotide sequence encoding a secretory signal peptide fused in-frame with the polypeptide sequence. The secretory signal peptide directs secretion of the antibody polypeptide by cells expressing the one or more polynucleotides and is cleaved from the secreted polypeptide by the cells. The one or more polynucleotides may also optionally further contain a sequence intended to function solely to facilitate large-scale production of the vector. Production and administration of polynucleotides for gene therapy can be carried out using procedures described in various transgene literature. See, for example, Isner et al., Circulation, 91:2687-2692, 1995; and Isner et al., Human Gene Therapy, 7:989-1011, 1996.

[0230] In some embodiments, the polynucleotide may further comprise additional sequences to facilitate uptake and expression of the antibody or fragment thereof (and / or any other peptide) by the host cell. In some embodiments, a "naked" transgene (e.g., a transgene without a virus, liposome, or other vector to facilitate transfection) encoding an antibody or fragment thereof described herein is used.

[0231] Any suitable vector can be used to introduce one or more polynucleotides encoding the antibody or fragment thereof into the host. Exemplary vectors that have been described include, but are not limited to, 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); parvoviral vectors, such as adeno-associated viral (AAV) vectors (U.S. Pat. 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 ... al., J. Invest. Med., 45: 87-98, 1997); adenovirus (AV) vectors (U.S. 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. al., Cell, 68:143-155, 1992); adenovirus-adeno-associated virus chimeras (U.S. Pat. No. 5,856,152) or vaccinia virus or herpes virus vectors (U.S. Pat. Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688); lipofectin-mediated gene transfer (BRL); liposomal vectors (U.S. Pat. No. 5,631,237); and combinations thereof.All of these expression vectors can be prepared using standard recombinant DNA techniques, as described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd 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 desired, the viral vector is made replication-deficient, for example, by deleting or disrupting a selection gene required for viral replication.

[0232] Other contemplated non-viral 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), and gene bombardment using high-velocity particle bombardment (Yang et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987). 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).

[0233] The expression vector (or antibody or fragment thereof discussed herein) can be encapsulated in a liposome. 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" technology are also contemplated. In some embodiments, liposomes are complexed with Sendai virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989). In some embodiments, the liposomes are complexed with or used in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., J. Biol. Chem., 266:3361-3364, 1991). In some embodiments, the liposomes are complexed with or used in conjunction with both HVJ and HMG-1. Such expression constructs have been successfully used for the transfer and expression of nucleic acids in vitro and in vivo. In some embodiments, multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), are included in the liposomes to target the liposomes to cells (e.g., tumor cells) that express CD47 and / or PD-L1 on their surface.

[0234] Further provided is a cell comprising any one or more of the binding agents disclosed herein, the nucleic acids disclosed herein, or the vectors disclosed herein. In some embodiments, the cell expresses the binding agents provided herein. In some embodiments, the cell replicates the nucleic acid or vector.

[0235] The cells can comprise one or more polynucleotides or one or more vectors, for example, the cells are transformed or transfected with one or more polynucleotides encoding multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1). In some embodiments, the cells express a multispecific binding agent, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), that has at least 75% identity with the CDRs of mAb-C (see, e.g., Table 1). In some embodiments, the cells express a multispecific binding agent, including a multispecific binding agent that binds to CD47, including human CD47, and PD-L1, including human PD-L1, that has at least 75% identity with the CDRs of mAb-P (see, e.g., Table 2). The cells can be prokaryotic cells, such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178:497-515, 1989), or eukaryotic cells, such as animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or hybridoma cells), yeast (e.g., Saccharomyces cerevisiae), or plant cells (e.g., tobacco, corn, soybean, or rice cells). The use of mammalian host cells can result in translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on the recombinant expression product.Similarly, polypeptides (e.g., multispecific binding agents (e.g., antibodies, e.g., bispecific antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1)) can be glycosylated or non-glycosylated and / or covalently modified to include the attachment of one or more water soluble polymers, e.g., polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0236] Methods for introducing DNA or RNA into host cells are well known, including transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are useful for amplifying polynucleotides and for expressing polypeptides encoded by polynucleotides. In this regard, the process for producing multispecific binding agents (e.g., antibodies) can include culturing host cells and isolating the multispecific binding agents. The transfer of naked DNA expression constructs into cells can be achieved using particle bombardment, which relies on the ability to accelerate DNA-coated microprojectiles to high velocities, allowing them to penetrate cell membranes and enter the cells without killing them (Klein et al., Nature, 327:70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high-voltage discharge to generate an electric current, which in turn provides the motive force (Yang et al., Proc. Natl. Acad Sci USA, 87:9568-9572, 1990). The particle bombardment used consisted of biologically inert materials such as tungsten or gold beads. Host cells can be isolated and / or purified. Host cells can also be cells transformed in vivo to cause transient or permanent expression of a polypeptide in vivo. Host cells can also be isolated cells transformed ex vivo, e.g., introduced after transformation to produce a polypeptide in vivo for therapeutic purposes. Transgenic humans are specifically excluded from the definition of host cells.

[0237] Various methods for producing antibodies from polynucleotides are generally well known.For example, basic molecular biology procedures are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (also see Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001).In addition, many publications describe techniques suitable for manipulating DNA, creating expression vectors, and transforming and culturing suitable cells to prepare antibodies (see, for example, Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andover, UK, 1992; and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0238] Multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), are produced using any suitable method, including those described above, e.g., isolated from immunized animals, recombinantly or synthetically produced, or genetically engineered. Antibody fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody. For example, papain or pepsin digestion of a whole antibody produces a 5S fragment, termed F(ab')2, or two monovalent Fab fragments, and an Fc fragment, respectively. F(ab)2 can be further cleaved using a thiol reducing agent to produce a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described, for example, in Edelman et al., Methods in Enzymology, 1:422 Academic Press (1967); Nisonoff et al., Arch. Biochem. Biophys., 89:230-244, 1960; Porter, Biochem. J., 73:119-127, 1959; U.S. Patent No. 4,331,647; and by Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5.

[0239] Uses and Methods In another aspect, provided herein are methods for using binding agents, such as the multispecific binding agents or compositions provided herein.

[0240] In some embodiments, provided herein are methods of inhibiting the interaction between CD47 (e.g., expressed on and / or in a first cell) and SIRPα (e.g., expressed on and / or in a second cell), the method comprising contacting CD47 (e.g., a first cell expressing CD47) with a multispecific binding agent provided herein (e.g., a multispecific antibody or fragment thereof). In some embodiments, provided herein are uses of multispecific binding agents provided herein to inhibit the interaction between CD47 (e.g., expressed on and / or in a first cell) and SIRPα (e.g., expressed on and / or in a second cell), wherein the use comprises contacting CD47 (e.g., a first cell expressing CD47) with a multispecific binding agent provided herein (e.g., an antibody or fragment thereof).

[0241] In some embodiments of any method or any use disclosed herein, the multispecific binding agent does not substantially inhibit the interaction of CD47 expressed on erythrocytes with SIRPα expressed on immune cells (e.g., macrophages, neutrophils, dendritic cells, or B lymphocytes). In further embodiments, the multispecific binding agent does not inhibit the interaction of CD47 expressed on erythrocytes with SIRPα expressed on immune cells (e.g., macrophages, neutrophils, dendritic cells, or B lymphocytes). In other embodiments, the multispecific binding agent inhibits the interaction between CD47 expressed on erythrocytes and SIRPα expressed on immune cells (e.g., macrophages, neutrophils, dendritic cells, or B lymphocytes), but the inhibition is significantly less (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% less) than inhibition with a benchmark anti-CD47 antibody.

[0242] As used herein, the term "inhibit" means to decrease or reduce. This can refer to any decrease or reduction, such as a significant decrease or reduction (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%), as well as 100% elimination. For example, in some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) with SIRPα by 10% to 99%. In other embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) with SIRPα by 100% (e.g., completely eliminate the interaction as measured by an assay). In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) with SIRPα by at least 10%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 20%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 30%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 40%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 50%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 60%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 70%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 80%.In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by at least 90%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by about 10% to 90%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by about 20% to 80%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by about 30% to 70%. In some embodiments, the binding agents provided herein inhibit the interaction of CD47 (or its extracellular domain) and SIRPα by about 40% to 60%.

[0243] In some embodiments, CD47 and SIRPα are expressed on different cells. In some embodiments, CD47 is expressed on a first cell, such as a tumor cell. In further embodiments, the first cell is not an erythrocyte. In even further embodiments, the first cell is not an erythrocyte precursor cell. Additionally or alternatively, SIRPα is expressed on a second cell, such as an immune cell. In further embodiments, the immune cell is a macrophage, neutrophil, dendritic cell, or B lymphocyte.

[0244] In some embodiments, provided herein are methods of inhibiting the interaction between PD-1 (e.g., expressed on and / or in a first cell) and PD-L1 (e.g., expressed on and / or in a second cell), the method comprising contacting PD-L1 (and / or the second cell expressing PD-L1) with a binding agent (e.g., a multispecific binding agent, a multispecific antibody, or fragment thereof) provided herein. In some embodiments, provided herein is a use of a binding agent (e.g., a multispecific binding agent, a multispecific antibody, or a fragment thereof) provided herein to inhibit the interaction between PD-1 (e.g., expressed on and / or in a first cell) and PD-L1 (e.g., expressed on and / or in a second cell), the use comprising contacting PD-L1 (and / or a second cell expressing PD-L1) with a binding agent (e.g., a multispecific binding agent, a multispecific antibody, or a fragment thereof) provided herein.

[0245] For example, in some embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by 10% to 99%. In other embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by 100% (e.g., completely abolish the interaction as measured by an assay). In some embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by at least 10%. In some embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by at least 20%. In some embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by at least 30%. In some embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by at least 40%. In some embodiments, the binding agents provided herein inhibit the interaction between PD-L1 and PD-1 by at least 50%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by at least 60%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by at least 70%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by at least 80%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by at least 90%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by about 10% to 90%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by about 20% to 80%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by about 30% to 70%. In some embodiments, the binding agents provided herein inhibit the interaction of PD-L1 and PD-1 by about 40% to 60%.

[0246] In some embodiments, PD-1 and PD-L1 are expressed on different cells. In some embodiments, PD-1 is expressed on a first cell, such as an immune cell. In further embodiments, the immune cell is a T cell. In still further embodiments, the T cell is a cytotoxic T cell, such as a CD8+ T cell. In some embodiments, the immune cell is an NK cell. Additionally or alternatively, PD-L1 is expressed on a second cell, such as a tumor cell.

[0247] Immune cells are cells of the immune system and can be lymphoid cells. Non-limiting examples of lymphoid cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and lymphocytes (B cells and T cells). T cells are a type of lymphocyte and can be characterized by expressing a T cell receptor (TCR). T cells play a central role in adaptive immune responses. T cell subtypes have various important functions in controlling and shaping immune responses. For example, cytotoxic T cells (also called cytotoxic T lymphocytes and killer T cells) are T lymphocytes that kill specific cells, such as tumor cells, cells infected with intracellular pathogens (such as viruses or bacteria), or cells that have been damaged in other ways. Most cytotoxic T cells express a T cell receptor (TCR) that can recognize a specific antigen. CD8+ T cells are a subpopulation of T cells restricted by MHC class I and are important mediators of adaptive immunity for killing cancer cells or virus-infected cells. NK cells are a type of cytotoxic lymphocyte that belongs to the innate lymphoid cell (ILC) family and are important in the innate immune system. In some embodiments, NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3-). NK cells have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune response.

[0248] In some embodiments, provided herein are methods of inhibiting the interaction of CD47 (or its extracellular domain) (e.g., expressed on and / or in a first cell) and SIRPα (e.g., expressed on and / or in a second cell) and inhibiting the interaction of PD-1 (e.g., expressed on and / or in a third cell) and PD-L1 (e.g., expressed on and / or in a fourth cell), the method comprising contacting CD47 (or the extracellular domain of each thereof, or a first cell expressing CD47) and PD-L1 (and / or a fourth cell expressing PD-L1) with a binding agent provided herein (e.g., a multispecific binding agent, a multispecific antibody, or fragment thereof). In some embodiments, provided herein is a use of a binding agent (e.g., a multispecific binding agent, a multispecific antibody, or fragment thereof) provided herein to inhibit the interaction of CD47 (or its extracellular domain) (e.g., expressed on and / or in a first cell) and SIRPα (e.g., expressed on and / or in a second cell) and inhibit the interaction of PD-1 (e.g., expressed on and / or in a third cell) and PD-L1 (e.g., expressed on and / or in a fourth cell), the use comprising contacting CD47 (or each of its extracellular domains, or a first cell expressing CD47) and PD-L1 (and / or a fourth cell expressing PD-L1) with a binding agent (e.g., a multispecific binding agent, a multispecific antibody, or fragment thereof) provided herein. In some embodiments, CD47 (or its extracellular domain) and PD-L1 are expressed on and / or in the same cell, such as a cancer or tumor cell (e.g., the first cell and the fourth cell are the same cell). Additionally or alternatively, the cell expressing CD47 (or its extracellular domain) is not an erythrocyte. In yet further embodiments, the cell expressing CD47 (or its extracellular domain) is not an erythrocyte progenitor cell.Additionally or alternatively, SIRPα and PD-1 are expressed on and / or in the same cell, such as an immune cell (e.g., the second cell and the third cell are the same cell). In some embodiments, SIRPα and PD-1 are expressed on and / or in different cells, e.g., different immune cells. In one embodiment, SIRPα is expressed on macrophages, and PD-L1 is expressed on T cells (such as cytotoxic T cells) or NK cells.

[0249] In further embodiments, the method does not inhibit the interaction of CD47 (or its extracellular domain) substantially expressed in or on red blood cells (RBCs) with SIRPα (e.g., expressed on and / or in a second cell). In other embodiments, the method inhibits the interaction of CD47 (or its extracellular domain) substantially expressed in or on red blood cells (RBCs) with SIRPα (e.g., expressed on and / or in a second cell), but the inhibition is significantly (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%) less than a benchmark anti-CD47 antibody. In one embodiment, SIRPα is expressed on immune cells such as macrophages. In some embodiments, the RBCs do not substantially express PD-L1.

[0250] In some embodiments, provided herein are methods of preventing (e.g., alleviating or decreasing (including but not limited to, eliminating)) or inhibiting immune cell suppression, such as tumor / cancer-associated immune suppression. The method comprises (i) inhibiting the interaction between CD47 (or its extracellular domain) (e.g., expressed on and / or in a first cell) and SIRPα (e.g., expressed on and / or in a second cell), and (ii) inhibiting the interaction between PD-1 (e.g., expressed on and / or in a third cell) and PD-L1 (e.g., expressed on and / or in a fourth cell). In some embodiments, the method comprises contacting CD47 (or each of its extracellular domains, or a first cell expressing CD47) and PD-L1 (and / or a fourth cell expressing PD-L1) with a binding agent provided herein (e.g., a multispecific binding agent, a multispecific antibody, or fragment thereof). In some embodiments, CD47 (or its extracellular domain) and PD-L1 are expressed on and / or in the same cell, such as a cancer or tumor cell (e.g., the first cell and the fourth cell are the same cell). Additionally or alternatively, SIRPα and PD-1 are expressed on and / or in the same cell, such as an immune cell (e.g., the second cell and the third cell are the same cell). In some embodiments, SIRPα and PD-1 are expressed on and / or in different cells, e.g., different immune cells. In one embodiment, SIRPα is expressed on macrophages and PD-L1 is expressed on T cells (such as cytotoxic T cells) or NK cells.

[0251] In other embodiments, provided herein are methods of preventing or inhibiting immune cell suppression, e.g., suppression mediated by the interaction between SIRPα expressed on an immune cell and CD47 (or its extracellular domain) (e.g., expressed on a cancer or tumor cell) and / or suppression mediated by the interaction between PD-1 (e.g., expressed on an immune cell) and PD-L1 (e.g., expressed on a cancer or tumor cell). In still other embodiments, provided herein are methods of activating an immune cell-mediated response, e.g., an anti-tumor response. In some embodiments, the immune cell suppression is tumor / cancer-associated immune cell suppression, such as in the tumor microenvironment. Additionally or alternatively, the immune cell suppression is suppression of ADCP and / or ADCC. In some embodiments, the method comprises contacting an immune cell with a binding agent (e.g., a multispecific antibody or fragment thereof) provided herein. Additionally or alternatively, the method comprises contacting a tumor cell with a binding agent (e.g., a multispecific antibody or fragment thereof) provided herein. In some embodiments, provided herein is a use of a binding agent provided herein to prevent suppression of immune cells or to activate responses mediated by immune cells. In some embodiments, the immune cells are macrophages, neutrophils, dendritic cells, or B lymphocytes. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CD8 + The immune cells are cytotoxic T cells, such as T cells. In some embodiments, the immune cells express SIRPα. Additionally or alternatively, the immune cells express PD-1. In some embodiments, the immune cells mediate an anti-cancer / tumor response. Additionally or alternatively, the immune cells mediate ADCC and / or ADCP. In further embodiments, the cancer or tumor cells express PD-L1. Additionally or alternatively, the cancer or tumor cells express CD47.

[0252] In some embodiments, the immunosuppression (also referred to herein as suppression of immune cells) is suppression of antibody-dependent cellular cytotoxicity (ADCC). In further embodiments, ADCC is mediated by PD-L1 / PD-1 signaling. In some embodiments, ADCC is mediated by T cells, such as T cells that express PD-1. Additionally or alternatively, the immunosuppression is suppression of antibody-dependent cellular phagocytosis (ADCP). In further embodiments, ADCP is mediated by CD47 / SIRPα signaling. In some embodiments, ADCP is mediated by macrophages, such as macrophages that express SIRPα.

[0253] In one aspect, provided herein is a method of inducing ADCC of tumor cells. In further embodiments, the tumor cells express PD-L1. In some embodiments, the method comprises contacting the tumor cells with a multispecific binding agent disclosed herein. In some embodiments, the method comprises administering a multispecific binding agent or composition disclosed herein to a subject having the tumor cells. Also provided herein is a use of a multispecific binding agent provided herein for inducing ADCC of tumor cells. In some embodiments, the use comprises contacting the tumor cells with a multispecific binding agent disclosed herein. In some embodiments, the use comprises administering a multispecific binding agent or composition disclosed herein to a subject having the tumor cells. In further embodiments, the tumor cells express PD-L1.

[0254] In another aspect, provided herein is a method of inducing ADCP of tumor cells. In further embodiments, the tumor cells express CD47. In some embodiments, the method comprises contacting the tumor cells with a multispecific binding agent as disclosed herein. In some embodiments, the method comprises administering a multispecific binding agent or composition disclosed herein to a subject having the tumor cells. Also provided herein is a use of a multispecific binding agent provided herein to induce ADCP of tumor cells. In some embodiments, the use comprises contacting the tumor cells with a multispecific binding agent disclosed herein. In some embodiments, the use comprises administering a multispecific binding agent or composition disclosed herein to a subject having the tumor cells. In further embodiments, the tumor cells express CD47.

[0255] In yet another aspect, provided herein are methods for inducing ADCC and ADCP in tumor cells and / or methods for reducing the suppression of ADCC and ADCP in tumor cells. In some embodiments, the methods comprise contacting tumor cells with a multispecific binding agent disclosed herein. In some embodiments, the methods comprise administering a multispecific binding agent or composition disclosed herein to a subject having tumor cells. Also provided herein are uses of multispecific binding agents provided herein for inducing ADCC and ADCP in tumor cells and / or methods for reducing the suppression of ADCC and ADCP in tumor cells. In some embodiments, the use comprises contacting tumor cells with a multispecific binding agent disclosed herein. In some embodiments, the use comprises administering a multispecific binding agent or composition disclosed herein to a subject having tumor cells.

[0256] In further embodiments, each of the tumor cells expresses PD-L1 and CD47. In other embodiments, a first population of tumor cells (e.g., at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%) express PD-L1, and a second population of tumor cells (e.g., at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%) express CD47. In further embodiments, a substantial proportion of the tumor cells (e.g., at least 80%, or at least 90%, or at least 95%, 100%, etc.) express PD-L1, CD47, or both.

[0257] In some embodiments, the methods disclosed herein are in vitro or ex vivo methods. In other embodiments, the methods disclosed herein are in vivo methods. In some embodiments, the uses disclosed herein are in vitro or ex vivo uses. In other embodiments, the uses disclosed herein are in vivo uses.

[0258] Further provided herein are methods of treating a disease, disorder, or condition (such as those disclosed herein) in a subject in need thereof. In some embodiments, the methods comprise administering to the subject effective amounts of (i) a first means for inhibiting the interaction between PD-L1 and PD-1, and (ii) a second means for inhibiting the interaction between CD47 and SIRPα. Also provided herein are uses of multispecific binding agents provided herein for treating a disease, disorder, or condition (such as those disclosed herein) in a subject in need thereof. In some embodiments, the use comprises administering to the subject effective amounts of (i) a first means for inhibiting the interaction between PD-L1 and PD-1, and (ii) a second means for inhibiting the interaction between CD47 and SIRPα.

[0259] In some embodiments, the first means has high affinity for PD-L1 (such as human PD-L1 or cyno PD-L1). In further embodiments, the first means binds to PD-L1 (such as human PD-L1 or cyno PD-L1) competitively with a multispecific binding agent disclosed herein, such as a bsAb1. Additionally or alternatively, the first means has a K for interaction with PD-L1 of less than 1 nM, or less than 0.1 nM, for example, about 0.05 nM. D It has.

[0260] Additionally or alternatively, the second means has a deregulated and / or intermediate affinity for CD47 (such as human CD47 or cyno CD47). In some embodiments, the second means binds to CD47 (e.g., human CD47 or cyno CD47) competitively with a multispecific binding agent disclosed herein, such as bsAb1. Additionally or alternatively, the second means has a K for interaction with CD47 of greater than 0.1 nM, greater than 1 nM, greater than 5 nM, greater than 10 nM, greater than 15 nM, or greater than 20 nM. DIn further embodiments, the second means has a K of less than 1 μM, less than 500 nM, less than 100 nM, less than 50 nM, or less than 30 nM for interaction with CD47. D In some embodiments, the second means has a K of about 0.1 nM to about 1 μM (including every number and range therebetween, e.g., about 1 nM to about 100 nM, or about 10 nM to about 50 nM, or about 20 to about 30 nM) for interaction with CD47. D In some embodiments, the method or use comprises administering a binding agent disclosed herein or a composition disclosed herein. D is measured by the methods disclosed herein, such as Biacore.

[0261] In some embodiments, multispecific binding agents (e.g., antibodies), including those multispecific binding agents that bind CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1) described herein, are useful in compositions and methods for treating, preventing, or ameliorating immune cell dysfunction diseases, disorders, or conditions (e.g., phagocyte dysfunction diseases, disorders, or conditions or T cell dysfunction diseases, disorders, or conditions), including one or more symptoms of the disease, disorder, or condition. Phagocytic cell dysfunction diseases, disorders, and conditions include tumor immunity and related cancers, including, but not limited to, any cancer in which tumor cells express or overexpress CD47. T cell dysfunction diseases, disorders, and conditions include tumor immunity and related cancers, including, but not limited to, any cancer in which tumor cells express or overexpress PD-L1. Such CD47- and / or PD-L1-expressing tumor cells may aid tumor cells in evading immune surveillance and clearance (e.g., tumor immunity). Additionally, the multispecific binding agents described herein, e.g., multispecific antibodies (e.g., antibodies, e.g., bispecific antibodies) that bind to CD47 and one or more additional targets that are not CD47 (e.g., PD-L1), are useful for inhibiting SIRPα signaling and / or PD-1 signaling, for enhancing phagocyte function and / or immune surveillance, and for enhancing tumor cell elimination.

[0262] Described herein, in some embodiments, are methods for treating tumor immunity in a subject, the methods comprising administering to the subject a multispecific binding agent (e.g., an antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, or a fragment thereof, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0263] Described herein, in some embodiments, are methods for treating cancer or tumor in a subject, the methods comprising administering to the subject a multispecific binding agent (e.g., an antibody), or fragment thereof, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0264] In some embodiments, described herein are methods for alleviating one or more symptoms associated with cancer or a tumor in a subject, the method comprising administering to the subject a multispecific binding agent (e.g., an antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, or a fragment thereof, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0265] Described herein, in some embodiments, are methods for reducing tumor size in a subject having a tumor, the method comprising administering to the subject a multispecific binding agent (e.g., an antibody), or fragment thereof, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0266] Described herein, in some embodiments, are methods for enhancing tumor cell elimination in a subject having a tumor, the method comprising administering to the subject a multispecific binding agent (e.g., an antibody), or fragment thereof, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0267] In some embodiments, described herein are methods for treating a phagocyte dysfunction disease, disorder, or condition in a subject, the method comprising administering to the subject a multispecific binding agent (e.g., an antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), as described herein, or a fragment thereof, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0268] In some embodiments, described herein are methods for increasing phagocytosis by immune cells in a subject, the method comprising administering to the subject a multispecific binding agent (e.g., an antibody) or fragment thereof, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein. In some embodiments, the immune cell is a macrophage, neutrophil, dendritic cell, or B lymphocyte. In some embodiments, the subject has been diagnosed with cancer or a tumor.

[0269] In some embodiments, described herein are methods for treating a T cell dysfunction disease, disorder, or condition in a subject, the methods comprising administering to the subject a multispecific binding agent (e.g., an antibody), including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1), as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein. In some embodiments, the T cell dysfunction disease, disorder, or condition is tumor immunity.

[0270] In some embodiments, described herein are methods for enhancing T cell function in a subject, the methods comprising administering to the subject a multispecific binding agent (e.g., an antibody) or fragment thereof, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein. In some embodiments, the T cell function is cytokine secretion. In some embodiments, the T cell function is tumor cell elimination. In some embodiments, the subject has been diagnosed with cancer or a tumor.

[0271] The subject of the above methods may be administered one or more therapeutic agents described herein in combination with a multispecific binding agent (e.g., an antibody) or fragment thereof, including a multispecific binding agent that binds to CD47, including human CD47, and one or more targets that are not CD47 (e.g., PD-L1, including human PD-L1) described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0272] In some embodiments, multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1), increase phagocytosis and / or enhance phagocytic activity of cells in a cell culture. For example, such cell cultures may include tumor cells that express or overexpress CD47. In some embodiments, multispecific binding agents (e.g., antibodies), including multispecific binding agents that bind to CD47, including human CD47, and PD-L1, including human PD-L1, increase T cell function and / or enhance cytolytic activity of cells in a cell culture. Such cell cultures may include tumor cells that express or overexpress PD-L1. In some embodiments, multispecific binding agents, including multispecific binding agents (e.g., antibodies), that bind to CD47, including human CD47, and PD-L1, including human PD-L1, increase phagocytosis, enhance phagocytic activity, increase T cell function, and / or enhance cytolytic activity of cells in cell culture. Such cell cultures can contain tumor cells that express or overexpress CD47 and PD-L1. Tumor cells include, but are not limited to, breast cancer cells, bladder cancer cells, melanoma cells, prostate cancer cells, mesothelioma cells, lung cancer cells, testicular cancer cells, thyroid cancer cells, squamous cell carcinoma cells, glioblastoma cells, neuroblastoma cells, uterine cancer cells, colorectal cancer cells, and pancreatic cancer cells.

[0273] In some embodiments, methods of inhibiting tumor cell growth in a subject are described herein. For example, the method includes administering an amount of a multispecific binding agent (e.g., an antibody), e.g., a multispecific binding agent that binds to CD47, including human CD47, and one or more targets other than CD47 (e.g., PD-L1, including human PD-L1) described herein, effective to inhibit tumor cell growth. In some embodiments, the method includes administering a multispecific binding agent (e.g., an antibody) that competes with antibody mAb-C (see, e.g., the CDRs and VH / VL in Table 1) for binding to human CD47 and / or binds to a region of CD47 recognized by antibody mAb-C (see, e.g., the CDRs and VH / VL in Table 1), resulting in enhanced elimination of tumor cells. In some embodiments, the method comprises administering a multispecific binding agent (e.g., an antibody) that competes with antibody mAb-P (see, e.g., the CDRs and VH / VL in Table 2) for binding to human PD-L1 and / or binds to a region of PD-L1 recognized by antibody mAb-P (see, e.g., the CDRs and VH / VL in Table 2), resulting in enhanced elimination of tumor cells. In some embodiments, the one or more polynucleotides, vectors, and / or cells may be used in methods of enhancing elimination of tumor cells in vivo (e.g., in methods of treating cancer in a subject).

[0274] Also provided are methods of modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject. For example, the method includes administering to the subject a composition comprising a multispecific binding agent (e.g., an antibody) in an amount effective to modulate tumor growth in the subject.

[0275] As used herein, "tumor" refers to any neoplastic cell growth or proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, breast cancer, colon cancer, renal cancer, lung cancer, squamous cell myeloid leukemia, hemangioma, melanoma, astrocytoma, and glioblastoma, as well as other cell proliferative disease conditions, including, but not limited to: Heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung: bronchogenic carcinoma (squamous cell, small undifferentiated cell, anaplastic lung cancer, pulmonary fibrosis ... Differentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, inesothelioma; gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarc...

Claims

1. A multispecific antibody or fragment thereof comprising a first binding domain that binds to CD47 and a second binding domain that binds to PD-L1, wherein the first binding domain is (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences represented in the VH comprising the amino acid sequence of SEQ ID NO: 25; and (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences represented in the VL comprising the amino acid sequence of SEQ ID NO: 26; The multispecific antibody or fragment thereof comprising:

2. the first binding domain comprises: (a) (1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and (3) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20. and a VH region comprising: (b) (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 22 or the amino acid sequence of SAS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23. and a VL region comprising:

2. The multispecific antibody or fragment thereof of claim 1, comprising:

3. The first binding domain comprises (i) to (vi): (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SAS, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SAS, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and (vi) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, a VH CDR2 having the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO:

6.

2. The multispecific antibody or fragment thereof of claim 1, comprising any one or more of:

4. 2. The multispecific antibody or fragment thereof of claim 1, wherein the first binding domain further comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence, optionally as depicted in SEQ ID NO: 25 or 26.

5. A multispecific antibody or fragment thereof described in claim 1, wherein the first binding domain further comprises a human framework sequence.

6. The multispecific antibody or fragment thereof of claim 1, wherein the first binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO:

26.

7. The method of claim 1, wherein the second binding domain comprises: (a) a VH region comprising a VH CDR1, a VH CDR2, and a VH CDR3 represented in the VH comprising the amino acid sequence of SEQ ID NO: 46; and (b) a VL region comprising a VL CDR1, a VL CDR2, and a VL CDR3 represented in the VL comprising the amino acid sequence of SEQ ID NO: 47; The multispecific antibody or fragment thereof according to any one of claims 1 to 6, comprising:

8. The method of claim 7, wherein the second binding domain comprises: (a) (1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 32, 35, 36, and 40; (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 33, 37, 41, and 45; and (3) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 34, 38, and 42. and a VH region comprising: (b) (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 43 or the amino acid sequence of SAS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 39, and 44. and a VL region comprising 8. The multispecific antibody or fragment thereof of claim 7, comprising:

9. The second binding domain comprising: (i) to (vi): (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 34; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SAS, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SAS, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 39; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 44; and (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

31.

8. The multispecific antibody or fragment thereof of claim 7, comprising any one or more of:

10. The multispecific antibody or fragment thereof of claim 7, wherein the second binding domain further comprises framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences, optionally as set forth in SEQ ID NO: 46 or 47.

11. A multispecific antibody or fragment thereof described in claim 7, wherein the second binding domain further comprises a human framework sequence.

12. The multispecific antibody or fragment thereof of claim 7, wherein the second binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO:

47.

13. A multispecific antibody or fragment thereof comprising a first binding domain that binds to CD47 and a second binding domain that binds to PD-L1, wherein the second binding domain is: (a) a VH region comprising a VH CDR1, a VH CDR2, and a VH CDR3 represented in the VH comprising the amino acid sequence of SEQ ID NO: 46; and (b) a VL region comprising a VL CDR1, a VL CDR2, and a VL CDR3 represented in the VL comprising the amino acid sequence of SEQ ID NO: 47; A multispecific antibody or fragment thereof comprising:

14. The second binding domain comprising: (a) (1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 32, 35, 36, and 40; (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 33, 37, 41, and 45; and (3) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 34, 38, and 42; and a VH region comprising: (b) (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 43 or the amino acid sequence of SAS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 39, and 44. and a VL region comprising 14. The multispecific antibody or fragment thereof of claim 13, comprising:

15. The second binding domain comprising: (i) to (vi): (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 34; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SAS, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SAS, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 39; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 44; and (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

31.

14. The multispecific antibody or fragment thereof of claim 13, comprising any one or more of:

16. The multispecific antibody or fragment thereof of claim 13, wherein the second binding domain further comprises framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences, optionally as set forth in SEQ ID NO: 46 or 47.

17. The multispecific antibody or fragment thereof of claim 13, wherein the second binding domain further comprises a human framework sequence.

18. The multispecific antibody or fragment thereof of claim 13, wherein the second binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO:

47.

19. A multispecific antibody or a fragment thereof described in any one of claims 1 to 6 and 13 to 18, which is a bispecific antibody.

20. One or more polynucleotides encoding a multispecific antibody or fragment thereof described in any one of claims 1 to 6 and 13 to 18.

21. 21. One or more vectors comprising one or more polynucleotides of claim 20.

22. A cell comprising one or more polynucleotides described in claim 20.

23. A pharmaceutical composition comprising the multispecific antibody or fragment thereof according to any one of claims 1 to 6 and 13 to 18, and a pharmaceutically acceptable carrier.

24. 20. A composition comprising the multispecific antibody or fragment thereof of any one of claims 1 to 6 and 13 to 18 for treating cancer or a tumor in a subject, for alleviating one or more symptoms associated with cancer or a tumor in a subject, for reducing tumor size in a tumor-bearing subject, for enhancing tumor cell removal in a tumor-bearing subject, for treating a phagocyte dysfunction disease, disorder or condition in a subject, for increasing phagocytosis by immune cells in a subject, for treating a T-cell dysfunction disease, disorder or condition in a subject, or for enhancing T-cell function in a subject, wherein optionally the subject is administered one or more therapeutic agents in combination with the composition.

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