Anti-SIRPα monoclonal antibody and its use

Abstract

To provide anti-SIRP α monoclonal antibodies and uses thereof.SOLUTION: Provided are antibodies or fragments thereof having binding specificity to both variant 1 and variant 2 of the signal regulatory protein alpha (SIRPα) protein. The antibodies and fragments can dose-dependently and efficiently block the interaction between SIRPα and CD47, and effectively induce macrophage-mediated phagocytosis of tumor cells expressing CD47. By contrast, known anti-SRPLa antibodies can only recognize variant 1.SELECTED DRAWING: None

Description

Background Art

[0001] Background Signal regulatory protein alpha (SIRPα) is a member of the signal regulatory protein (SIRP) family and also belongs to the immunoglobulin superfamily. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signal transduction processes. SIRPα can be phosphorylated by tyrosine kinases. This phosphotyrosine residue of the PTP has been shown to recruit tyrosine phosphatases (PTPs) containing SH2 domains and to act as a substrate for the PTP. SIRPα has been found to be involved in signal transduction mediated by various growth factor receptors. CD47 has been demonstrated to be a ligand. SIRPα shares very high similarity with some other members of the SIRP family. SIRPα is mainly expressed by myeloid cells and also by stem cells or neurons.

[0002] SIRPα functions as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47 (also called the "don't eat me" signal). This interaction negatively regulates the effector functions of innate immune cells (such as host cell phagocytosis). SIRPα diffuses laterally on the macrophage membrane, accumulates at the phagocytic synapse, binds to CD47 and emits a "self" signal, which inhibits the cytoskeletal aggregation process of phagocytosis by macrophages.

[0003] Upon CD47 ligation, SIRPα is phosphorylated and recruits phosphatases such as SHP1 and SHP2. The extracellular region contains three immunoglobulin superfamily domains, namely a single V-set and two C1-set IgSF domains. SIRPβ and γ have similar extracellular structures but different cytoplasmic regions that give rise to contrasting types of signals.

[0004] SIRPα recognizes CD47 (an anti-phagocytic signal that discriminates live cells from cells that are dying). The extracellular domain of SIRPα binds to CD47 and transmits an intracellular signal through its cytoplasmic domain. CD47 binding is mediated by the NH2-terminal V-like domain of SIRPα. The cytoplasmic region contains four ITIMs that are phosphorylated after ligand binding. This phosphorylation mediates the activation of the tyrosine phosphatase SHP2. SIRPα also binds the phosphatase SHP1, the adapter protein SCAP2, and the FYN-binding protein. Recruitment of the SHP phosphatase to the membrane causes inhibition of myosin accumulation at the cell surface, resulting in inhibition of phagocytosis.

[0005] Cancer cells highly express CD47, which activates SIRPα and inhibits macrophage-mediated destruction. A high-affinity variant of SIRPα that antagonizes CD47 on cancer cells has been shown to increase phagocytosis of cancer cells. Anti-SIRPα antibodies have also been shown to help macrophages reduce cancer growth and metastasis, both alone and in synergy with other cancer treatments. Summary of the Invention Means for Solving the Problems

[0006] Summary Anti-SIRPα antibodies with high affinity for both variants v1 and v2 have been found herein that can dose-dependently and efficiently block the interaction between SIRPα and CD47 and can efficiently induce macrophage-mediated phagocytosis of cells expressing CD47. In contrast, known anti-SIRPα antibodies can only recognize variant 1.

[0007] According to one embodiment of the present disclosure, an antibody or a fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein is provided, and the antibody or the fragment thereof includes a heavy-chain variable region including heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain including complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein (a) the CDRH1 includes the amino acid sequence of SEQ ID NO: 15, the CDRH2 includes the amino acid sequence of SEQ ID NO: 16, 21, or 22, the CDRH3 includes the amino acid sequence of SEQ ID NO: 17, the CDRL1 includes the amino acid sequence of SEQ ID NO: 18, the CDRL2 includes the amino acid sequence of SEQ ID NO: 19, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 20; (b) the CDRH1 includes the amino acid sequence of SEQ ID NO: 30, the CDRH2 includes the amino acid sequence of SEQ ID NO: 31, 36, 37, or 38, the CDRH3 includes the amino acid sequence of SEQ ID NO: 32, the CDRL1 includes the amino acid sequence of SEQ ID NO: 33, the CDRL2 includes the amino acid sequence of SEQ ID NO: 34, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 35; (c) the CDRH1 includes the amino acid sequence of SEQ ID NO: 47, the CDRH2 includes the amino acid sequence of SEQ ID NO: 48, 53, or 54, the CDRH3 includes the amino acid sequence of SEQ ID NO: 49, the CDRL1 includes the amino acid sequence of SEQ ID NO: 50, the CDRL2 includes the amino acid sequence of SEQ ID NO: 51, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 52; (d) the CDRH1 includes the amino acid sequence of SEQ ID NO: 63, the CDRH2 includes the amino acid sequence of SEQ ID NO: 64, the CDRH3 includes the amino acid sequence of SEQ ID NO: 65, the CDRL1 includes the amino acid sequence of SEQ ID NO: 66, the CDRL2 includes the amino acid sequence of SEQ ID NO: 67, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 68;(e) The CDRH1 includes the amino acid sequence of SEQ ID NO: 77, the CDRH2 includes the amino acid sequence of SEQ ID NO: 78, the CDRH3 includes the amino acid sequence of SEQ ID NO: 79, the CDRL1 includes the amino acid sequence of SEQ ID NO: 80, the CDRL2 includes the amino acid sequence of SEQ ID NO: 81, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 82; (f) The CDRH1 includes the amino acid sequence of SEQ ID NO: 91, the CDRH2 includes the amino acid sequence of SEQ ID NO: 92, the CDRH3 includes the amino acid sequence of SEQ ID NO: 93, the CDRL1 includes the amino acid sequence of SEQ ID NO: 94, the CDRL2 includes the amino acid sequence of SEQ ID NO: 95, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 96; or (g) The CDRH1 includes the amino acid sequence of SEQ ID NO: 103, the CDRH2 includes the amino acid sequence of SEQ ID NO: 104, the CDRH3 includes the amino acid sequence of SEQ ID NO: 105, the CDRL1 includes the amino acid sequence of SEQ ID NO: 106, the CDRL2 includes the amino acid sequence of SEQ ID NO: 107, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 108.;

[0008] In one embodiment, the present disclosure provides an antibody or a fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof includes a heavy chain variable region including heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region including complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 includes the amino acid sequence of SEQ ID NO: 15, the CDRH2 includes the amino acid sequence of SEQ ID NO: 16, 21, or 22, the CDRH3 includes the amino acid sequence of SEQ ID NO: 17, the CDRL1 includes the amino acid sequence of SEQ ID NO: 18, the CDRL2 includes the amino acid sequence of SEQ ID NO: 19, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 20.

[0009] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOs: 23-27, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOs: 23-27.

[0010] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NOs: 28-29, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NOs: 28-29.

[0011] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29.

[0012] In another embodiment, an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, the antibody or fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, 36, 37, or 38, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35.

[0013] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NOs: 39-44, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NOs: 39-44.

[0014] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 45-46, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 45-46.

[0015] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 45.

[0016] Yet another embodiment provides an antibody or a fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 47, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 48, 53, or 54, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 49, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 50, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 51, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 52.

[0017] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 55-60, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 55-60.

[0018] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 61-62, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 61-62.

[0019] In some embodiments, the antibodies or fragments disclosed herein can bind to SIRPα variant 1 and variant 2. In some embodiments, the antibodies or fragments are humanized. In some embodiments, the antibodies or fragments disclosed herein further have binding specificity for a second target protein.

[0020] In some embodiments, compositions comprising the antibody or fragment thereof and a pharmaceutically acceptable carrier are also provided. In some embodiments, the composition further comprises a secondary antibody having specificity for a tumor antigen. In some embodiments, the secondary antibody is a tumor opsonizing antibody.

[0021] Methods and uses for the treatment of diseases and conditions are also provided. In one embodiment, a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an antibody or fragment thereof of the present disclosure is provided. BRIEF DESCRIPTION OF THE DRAWINGS

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[0028] **Detailed Description** **Definitions** The term "a" or "an" entity refers to one or more than one of that entity. For example, "an antibody" should be understood to represent one or more than one antibody. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein. As used herein, the term "polypeptide" refers to the singular "poly

[0029] peptide" "Peptide", and is intended to include a plurality of "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a single or multiple chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of any of these terms or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources, may be produced by recombinant techniques, but do not necessarily have to be translated from a specified nucleic acid sequence. Polypeptides may be produced by any method, including chemical synthesis.

[0030] "Homology", or "identity", or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence (which may be aligned for purposes of comparison). If the positions in the sequences being compared are occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching, or homologous, positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity (preferably less than 25% identity) with one of the sequences of the present disclosure.

[0031] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, which means that when aligned, that percentage of bases (or amino acids) is identical in the comparison of the two sequences.

[0032] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity to the nucleotide sequence of a nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology to its complement or with the complement thereof (with or with the complement thereof). In one embodiment, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide having a certain degree of homology or sequence identity to the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, an equivalent polypeptide or polynucleotide has 1, 2, 3, 4, or 5 additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some embodiments, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0033] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody, as well as any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide that includes a molecule that contains at least a portion of an immunoglobulin molecule having biological activity to bind an antigen. Examples of such include, but are not limited to, complementarity determining regions (CDRs) of the heavy or light chains, or ligand-binding portions thereof, variable regions of the heavy or light chains, constant regions of the heavy or light chains, framework (FR) regions, or any portion thereof, or at least a portion of a binding protein.

[0034] The terms "antibody fragment" or "antigen-binding fragment", as used herein, are portions of an antibody, such as, for example, F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0035] "Single-chain variable region fragment" or "scFv" refers to a fusion protein of the variable region of the heavy chain (V H ) and the variable region of the light chain (V L ). In some embodiments, this region is linked by a short linker peptide of 10 to about 25 amino acids. This linker can be rich in glycine for flexibility and serine or threonine for solubility, and the N-terminus of V H to V LIt can be either linked to the C-terminus of [protein name] or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0036] The term antibody encompasses a variety of broad classes of polypeptides that can be biologically distinguished. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), each with several subclasses (e.g., γ1-γ4). The "class" of an antibody is determined by the nature of this chain, designated as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and are known to confer functional specialization. Modified forms of each of these classes and isotypes are readily distinguishable to those skilled in the art in view of the present disclosure and are thus within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion is generally directed to immunoglobulin molecules of the IgG class. For IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 - 70,000. These four chains are typically linked by disulfide bonds and are in a "Y" configuration, with the light chains flanking the heavy chains starting at the mouth of the "Y" and continuing through the variable regions.

[0037] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab’ and F(ab’)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-bonded Fv (sdFv), fragments containing either the VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0038] Light chains are classified into either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by either a covalent disulfide bond or a non-covalent bond when the immunoglobulin is produced by any of a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain.

[0039] Both the light chain and the heavy chain are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, the variable domain (VK) of the light chain portion and the variable domain (VH) of the heavy chain portion both determine antigen recognition and specificity. Conversely, the constant domain of the light chain (CK) and the constant domains of the heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of the constant region domains increases as one moves away from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxy termini of the heavy and light chains, respectively.

[0040] As shown above, the variable region enables the antibody to selectively recognize and specifically bind to the epitope of an antigen. That is, the VK domain and VH domain of the antibody, or a subset of the complementarity-determining regions (CDRs), combine to form the variable region that defines the three-dimensional antigen-binding site. The quaternary structure of this antibody forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of each of the VH and VK chains. In some examples, for instance, in certain immunoglobulin molecules derived from Camelidae species or engineered based on Camelidae immunoglobulins, the complete immunoglobulin molecule can consist of only the heavy chain without a light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0041] In a naturally occurring antibody, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous amino acid sequences that occupy specific positions so as to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids of the antigen-binding domain (referred to as the "framework" region) exhibit less inter-molecular variability. The framework region predominantly assumes a β-sheet structure, and the CDRs form loops that connect the β-sheet structures and, in some cases, form part of the β-sheet structure. Thus, the framework region serves to form a scaffold that provides the correct orientation of the CDRs through inter-chain non-covalent interactions. The antigen-binding domain formed by the arranged CDRs determines the surface complementarity to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework region are precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)), and can thus be readily determined by one of ordinary skill in the art for any given heavy or light chain variable region.

[0042] In cases where there are two or more definitions of a term used and / or accepted in the art, the definition of that term used herein is intended to encompass all of those meanings unless otherwise clearly indicated. A specific example is the use of the term "complementary determining region" ("CDR") to describe the discontinuous antigen-binding sites found within the variable regions of both heavy chain and light chain polypeptides. This particular region was described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins" Described by Kabat et al., "Sequence of Proteins of Immunological Interest" (1983), and Chothia et al., J. Mol. Biol. 196:901 - 917 (1987) (which are hereby incorporated by reference in their entirety). The definitions of CDRs by Kabat and Chothia, when compared to each other, include an overlap or subset of amino acid residues. Nevertheless, the application of either definition for referring to the CDRs of an antibody or its variant is intended to be within the scope of the terms defined and used herein. The appropriate amino acid residues encompassing the CDRs defined in each of the references cited above are listed in the table below for comparison. The exact number of residues encompassing a particular CDR varies depending on the sequence and size of that CDR. One of ordinary skill in the art can routinely determine which residues are included in a particular CDR given the amino acid sequence of the variable region of an antibody. [Table 11]

[0043] Kabat et al. also defined a numbering system for variable domain sequences that can be applied to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0044] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), contains approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, contains approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; contains 3-25 amino acids; and ends at the sequence W-G-X-G (where X is any amino acid). CDR-L1 begins at approximately residue 24 (i.e., after the cysteine residue); contains approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins at approximately the 33rd residue (i.e., after the cysteine residue) after the end of CDR-L2; contains approximately 7-11 residues; and ends at the sequence F or W-G-X-G (where X is any amino acid).

[0045] The antibodies disclosed herein can be of any animal origin, including birds and mammals. Preferably, the antibody is a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody. In another embodiment, the variable region can be of chondrichthoid (e.g., shark) origin.

[0046] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may include a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a part of the hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a part of the hinge domain, and a CH3 domain, or a polypeptide chain containing a CH1 domain, at least a part of the hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. Further, an antibody for use in the present disclosure may lack at least a part (e.g., all or a part of the CH2 domain). As described above, it will be understood by those skilled in the art that the heavy chain constant region may be modified such that its amino acid sequence is different from that of a naturally occurring immunoglobulin molecule.

[0047] The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion may include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0048] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0049] "Light chain-heavy chain pair" refers to a collection of a light chain and a heavy chain that can form a dimer by a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0050] As shown above, the subunit structures and three-dimensional arrangements of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminus of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal with respect to the hinge region of the immunoglobulin heavy chain molecule.

[0051] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule that extends from approximately residue 244 to residue 360 (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, ‘‘Sequences of Proteins of Immunological Interest’’ (1983)) of an antibody using a conventional numbering scheme. The CH2 domain is unique in that it does not pair closely with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. It has also been well demonstrated that the CH3 domain extends from the CH2 domain to the C terminus of the IgG molecule and contains approximately 108 residues.

[0052] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains, namely the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0053] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 (positions 226 or 229, EU numbering system) using the Kabat numbering system.

[0054] As used herein, the term "chimeric antibody" means any antibody in which the immunoreactive region or site is obtained from or derived from a first species and the constant region (which may be intact, partial, or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is human.

[0055] As used herein, "percent humanization" is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain, subtracting this number from the total number of amino acids, then dividing by the total number of amino acids and multiplying by 100.

[0056] "Specifically binds" or "is specific for" generally means that an antibody binds to an epitope by its antigen-binding domain and that the binding requires some complementarity between the antigen-binding domain and the epitope. Consistent with this definition, an antibody is said to "specifically bind" to an epitope if it binds more readily to that epitope compared to the case where it would bind to a random and unrelated epitope by its antigen-binding domain. The term "specificity" is used herein to qualify the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B", or it may be said that antibody "A" binds to epitope "C" with higher specificity than the specificity it has for a related epitope "D".

[0057] As used herein, the terms "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down (mitigate) an undesired physiological change or disorder (e.g., cancer progression). Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, diminishment of the degree of the disease, stabilization (i.e., not getting worse) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging the survival period as compared to the expected survival period if the treatment were not received. Subjects in need of treatment include subjects already having a condition or disorder, as well as subjects having a tendency to have a condition or disorder, or subjects in which a condition or disorder should be prevented.

[0058] "Subject", or "individual", or "animal", or "patient", or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals, sports animals, or pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.).

[0059] As used herein, expressions such as "for a patient in need of treatment" or "a subject in need of treatment" include subjects (e.g., mammalian subjects) that would benefit from administration of the antibodies or compositions of the present disclosure, which are used, for example, for detection, for diagnostic procedures, and / or for treatment. Anti-SIRPα antibody

[0060] The present disclosure provides anti-SIRPα antibodies and fragments that have high affinity for both variants v1 and v2. Variant 1 (hSIRPα V1) is the predominant variant among Europeans, Africans, admixed Americans, and South Asians. Variant 2 (hSIRPα V2) is the predominant variant among East Asians. hSIRPα V1 and hSIRPα V2 differ in the sequences within the extracellular Ig-like V-like (IgV) domain. The ability of the antibodies and fragments of the present disclosure to recognize both variants enables them to be effective among the broadest patient populations.

[0061] Furthermore, the antibodies and fragments of the present disclosure exhibit excellent binding affinity, strong induction of macrophage-mediated phagocytosis, and excellent chemistry, manufacturing, and control (CMC) developability.

[0062] According to one embodiment of the present disclosure, there are provided antibodies and antigen-binding fragments thereof that are capable of binding to both variants 1 and 2 of SIRPα. Exemplary antibodies include the mouse antibodies shown in Table 1 and the humanized antibodies shown in Tables 2-8. Also included are antibodies that contain the same CDRs as those described herein. In some embodiments, the antibodies and fragments of the present disclosure include antibodies and fragments that bind to the same epitope as those exemplified herein, as well as antibodies and fragments that compete with those disclosed herein for binding to SIRPα.

[0063] According to one embodiment of the present disclosure, there are provided antibodies or fragments thereof that contain a heavy-chain variable domain and a light-chain variable domain having the CDR regions disclosed herein, as well as their biological equivalents.

[0064] In one embodiment, the CDRs are those of 248G3F6, as exemplified in Tables 2B and 2D. In one embodiment, CDRH1 includes the amino acid sequence of SEQ ID NO: 15, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 includes the amino acid sequence of SEQ ID NO: 16, 21, or 22, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 includes the amino acid sequence of SEQ ID NO: 17, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 includes the amino acid sequence of SEQ ID NO: 18, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 includes the amino acid sequence of SEQ ID NO: 19, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 includes the amino acid sequence of SEQ ID NO: 20, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0065] One embodiment provides an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy-chain variable region comprising heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 16, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 20.

[0066] One embodiment provides an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy-chain variable region comprising heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 21, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 20.

[0067] One embodiment provides an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy-chain variable region comprising heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 22, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 20.

[0068] In some embodiments, the heavy-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-27, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-27.

[0069] In some embodiments, the light-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-29, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-29.

[0070] In some embodiments, the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 29.

[0071] In one embodiment, the CDR is 300A6A6, as exemplified in Tables 3B and 3D. In one embodiment, CDRH1 includes the amino acid sequence of SEQ ID NO: 30, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 includes the amino acid sequence of SEQ ID NO: 31, 36, 37, or 38, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 includes the amino acid sequence of SEQ ID NO: 32, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 includes the amino acid sequence of SEQ ID NO: 33, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 includes the amino acid sequence of SEQ ID NO: 34, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 includes the amino acid sequence of SEQ ID NO: 35, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0072] In one embodiment, there is provided an antibody or a fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein, the antibody or the fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35.

[0073] In one embodiment, an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, the antibody or the fragment thereof comprising a heavy-chain variable region comprising heavy-chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35.

[0074] In one embodiment, an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, the antibody or the fragment thereof comprising a heavy-chain variable region comprising heavy-chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 37, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35.

[0075] In one embodiment, an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, and the antibody or the fragment thereof includes a heavy-chain variable region including heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain including complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 includes the amino acid sequence of SEQ ID NO: 30, the CDRH2 includes the amino acid sequence of SEQ ID NO: 38, the CDRH3 includes the amino acid sequence of SEQ ID NO: 32, the CDRL1 includes the amino acid sequence of SEQ ID NO: 33, the CDRL2 includes the amino acid sequence of SEQ ID NO: 34, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 35.

[0076] In some embodiments, the heavy-chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39-44, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39-44.

[0077] In some embodiments, the light-chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 45-46, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 45-46.

[0078] In some embodiments, the heavy-chain variable region includes the amino acid sequence of SEQ ID NO: 43, and the light-chain variable region includes the amino acid sequence of SEQ ID NO: 45.

[0079] In one embodiment, the CDR is of 102A10F2 as exemplified in Tables 4B and 4D. In one embodiment, CDRH1 includes the amino acid sequence of SEQ ID NO: 47, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 includes the amino acid sequence of SEQ ID NO: 48, 53, or 54, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 includes the amino acid sequence of SEQ ID NO: 49, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 includes the amino acid sequence of SEQ ID NO: 50, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 includes the amino acid sequence of SEQ ID NO: 51, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 includes the amino acid sequence of SEQ ID NO: 52, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0080] In one embodiment, there is provided an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, the antibody or the fragment thereof including a heavy chain variable region including heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain including complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 includes the amino acid sequence of SEQ ID NO: 47, the CDRH2 includes the amino acid sequence of SEQ ID NO: 48, the CDRH3 includes the amino acid sequence of SEQ ID NO: 49, the CDRL1 includes the amino acid sequence of SEQ ID NO: 50, the CDRL2 includes the amino acid sequence of SEQ ID NO: 51, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 52.

[0081] In one embodiment, an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, and the antibody or the fragment thereof includes a heavy chain variable region including heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain including complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 includes the amino acid sequence of SEQ ID NO: 47, the CDRH2 includes the amino acid sequence of SEQ ID NO: 53, the CDRH3 includes the amino acid sequence of SEQ ID NO: 49, the CDRL1 includes the amino acid sequence of SEQ ID NO: 50, the CDRL2 includes the amino acid sequence of SEQ ID NO: 51, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 52.

[0082] In one embodiment, an antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, and the antibody or the fragment thereof includes a heavy chain variable region including heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain including complementarity determining regions CDRL1, CDRL2, and CDRL3. Also, wherein the CDRH1 includes the amino acid sequence of SEQ ID NO: 47, the CDRH2 includes the amino acid sequence of SEQ ID NO: 54, the CDRH3 includes the amino acid sequence of SEQ ID NO: 49, the CDRL1 includes the amino acid sequence of SEQ ID NO: 50, the CDRL2 includes the amino acid sequence of SEQ ID NO: 51, and the CDRL3 includes the amino acid sequence of SEQ ID NO: 52.

[0083] In some embodiments, the heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55 - 60, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55 - 60.

[0084] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NOs: 61-62, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NOs: 61-62.

[0085] In one embodiment, the CDRs are those of 62D2H6 as exemplified in Tables 5B and 5D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 63, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 comprises the amino acid sequence of SEQ ID NO: 64, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 comprises the amino acid sequence of SEQ ID NO: 65, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 comprises the amino acid sequence of SEQ ID NO: 66, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 comprises the amino acid sequence of SEQ ID NO: 67, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 68, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0086] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NOs: 69-72, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NOs: 69-72.

[0087] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NOs: 73-76, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NOs: 73-76.

[0088] In one embodiment, the CDR is that of 211F8E11 as exemplified in Tables 6B and 6D. In one embodiment, CDRH1 includes the amino acid sequence of SEQ ID NO: 77, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 includes the amino acid sequence of SEQ ID NO: 78, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 includes the amino acid sequence of SEQ ID NO: 79, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 includes the amino acid sequence of SEQ ID NO: 80, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 includes the amino acid sequence of SEQ ID NO: 81, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 includes the amino acid sequence of SEQ ID NO: 82, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0089] In some embodiments, the heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and 83 - 86, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and 83 - 86.

[0090] In some embodiments, the light chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 87 - 90, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 87 - 90.

[0091] In one embodiment, the CDR is of 217D11E5 as exemplified in Tables 7B and 7D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 91, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 comprises the amino acid sequence of SEQ ID NO: 92, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 comprises the amino acid sequence of SEQ ID NO: 93, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 comprises the amino acid sequence of SEQ ID NO: 94, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 comprises the amino acid sequence of SEQ ID NO: 95, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 96, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0092] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and 97 - 100, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and 97 - 100.

[0093] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 101 - 102, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 101 - 102.

[0094] In one embodiment, the CDR is of 234B7D5 as exemplified in Tables 8B and 8D. In one embodiment, CDRH1 includes the amino acid sequence of SEQ ID NO: 103, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH2 includes the amino acid sequence of SEQ ID NO: 104, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRH3 includes the amino acid sequence of SEQ ID NO: 105, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL1 includes the amino acid sequence of SEQ ID NO: 106, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; CDRL2 includes the amino acid sequence of SEQ ID NO: 107, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and CDRL3 includes the amino acid sequence of SEQ ID NO: 108, or variants thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0095] In some embodiments, the heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 109 - 112, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 109 - 112.

[0096] In some embodiments, the light chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 113 - 118, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 113 - 118.

[0097] Antibodies containing these CDR regions (whether murine, humanized, or chimeric) had potent SIRPα binding and inhibitory activity. As shown in Example 5, certain residues within the CDRs can be modified to maintain or improve their properties or to reduce their potential to have post-translational modifications (PTMs). Such modified CDRs can be referred to as affinity matured or risk de-reduced CDRs.

[0098] Non-limiting examples of risk de-reduced CDRs are shown in Tables 2B, 3B, and 4B. Modified CDRs can include those having 1, 2, or 3 amino acid additions, deletions, and / or substitutions. In some embodiments, such substitutions can be conservative substitutions.

[0099] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string having a different order and / or composition of side chain family members.

[0100] Non-limiting examples of conservative amino acid substitutions are shown in the following table, where a similarity score of 0 or higher indicates that the substitution between the two amino acids is a conservative substitution. Table A. Amino Acid Similarity Matrix

Table A

Table B

[0101] Moreover, those skilled in the art will also understand that the antibodies disclosed herein can be modified such that their amino acid sequences are different from those of the binding polypeptides of natural origin from which they are derived. For example, the polypeptides or amino acid sequences derived from a specified protein can be similar, for example, having a certain percent identity to the starting sequence, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0102] In certain embodiments, the antibody comprises an amino acid sequence or portion that is not normally associated with an antibody, or one or more than one portion. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure can include a flexible linker sequence or can be modified to add a functional moiety (e.g., PEG, drug, toxin, or label).

[0103] The antibodies, variants, or derivatives of the present disclosure include derivatives that are modified, i.e., derivatives in which any type of molecule is covalently attached to the antibody such that the covalent attachment does not interfere with the binding of the antibody to its epitope. For example, but not by way of limitation, the antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to a cell ligand or other protein, etc. Any of a number of chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, the antibody can include one or more non-classical amino acids.

[0104] In some embodiments, the antibody can be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical, or PEG.

[0105] The antibody can be conjugated or fused to a therapeutic agent (which may include a detectable label such as a radiolabel), an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which can be a drug or a toxin), an ultrasonic enhancer, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0106] The antibody can be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescently tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, and oxalate esters.

[0107] The antibody can also be 152It can be detectably labeled using a fluorescent-emitting metal such as Eu or another metal of the lanthanide series. These metals can be conjugated to an antibody using a metal chelate group such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known (e.g., Arnon et al., ''Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy'', in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985)); Hellstrom et al., ''Antibodies For Drug Delivery'', in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, ''Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review'', in Monoclonal Antibodies ’84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); ''Analysis, Results, And Future Prospective “Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy’’, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., ’’The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates’’, Immunol. Rev. (52: 119-58 (1982)). Bifunctional Molecules and Combinatorial Therapy

[0108] An important component of the innate immune system is the macrophage. Macrophages inhibit tumor growth by phagocytosis of tumor cells. Tumor cells can evade macrophage surveillance by upregulating CD47, a “don't eat me” signal on their cell surface. CD47 is ubiquitously expressed but is upregulated in various tumor types. By interacting with various ligands, CD47 has a role in the regulation of cell motility, adhesion, migration, and platelet activation. SIRPα is another member of the immunoglobulin superfamily and is mainly expressed on the surface of neurons and myeloid cells (including macrophages, granulocytes, monocytes, and dendritic cells). The CD47 / SIRPα system is a known major pathway for immune evasion by tumor cells.

[0109] Agents targeting CD47 can cause antibody opsonization and activate antibody-dependent cell cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP), which promotes tumor cell destruction. However, the ubiquitous expression of CD47 can lead to RBC toxicity and other hematological adverse effects due to the presence of this antigen on blood cells. Molecules targeting either SIRPα or a bispecific agent targeting both SIRPα and CD47 can be designed to induce ADCC and ADCP effects by processing a functional Fc domain.

[0110] Since SIRPα has a more restricted expression compared to CD47, these molecules should not have as many hematological events as molecules targeting CD47 alone. Therefore, anti-SIRPα antibodies may be safer than anti-CD47 antibodies. Another option is the development of bispecific agents that target both the CD47 / SIRPα system and another tumor antigen. These agents can be any of bispecific antibodies, fusion proteins, and combination therapies.

[0111] Examples of tumor antigens, particularly those that can induce tumor opsonization, include, but are not limited to, CD19, CD20, EGFR, HER2, CD3, CD16, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, VEGFR, TIGIT, Claudin 18.2, CD24, GPC3, Il13RA2, 4-1BB, CCR8, and CMET.

[0112] Different formats of bispecific antibodies are also provided. In some embodiments, each of the anti-SIRPα fragment and the second fragment is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment. Polynucleotides encoding antibodies, and methods for preparing antibodies

[0113] The present disclosure also provides an isolated polynucleotide or nucleic acid molecule encoding an antibody, variant or derivative thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant or derivative thereof, either on the same polynucleotide molecule or on separate polynucleotide molecules. Further, the polynucleotides of the present disclosure may encode portions of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant or derivative thereof, either on the same polynucleotide molecule or on separate polynucleotide molecules.

[0114] Methods of making antibodies are well known in the art and are also described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen exposure, but whose endogenous locus has been inactivated. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584; 6,458,592; 6,420,140 (which are incorporated by reference in their entirety). Methods of treatment

[0115] As described herein, the antibodies, variants, or derivatives of the present disclosure can be used in certain methods of treatment and diagnosis.

[0116] The present disclosure is also directed to antibody-based therapies that include administering to a patient, such as an animal, mammal, and human, an antibody of the present disclosure to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including their variants and derivatives described herein), and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives described herein).

[0117] The antibodies of the present disclosure can also be used to treat or inhibit cancer. As noted above, SIRPα can be overexpressed in tumor cells, particularly gastric tumors, pancreatic tumors, esophageal tumors, ovarian tumors, and lung tumors. Inhibition of SIRPα has been shown to be useful for treating said tumors.

[0118] Accordingly, in some embodiments, a method for treating cancer in a patient in need thereof is provided. This method, in one embodiment, involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, in the patient, at least one cancer cell (e.g., stromal cell) overexpresses SIRPα.

[0119] Cell therapies (e.g., chimeric antigen receptor (CAR) T cell therapy) are also provided in the present disclosure. Suitable cells contacted with (or alternatively engineered to express) an anti-SIRPα antibody of the present disclosure can be used. After such contacting or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient can have any type of cancer disclosed herein. The cells (e.g., T cells) can be, but are not limited to, for example, tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0120] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided by a donor or from a cell bank. When the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0121] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0122] Additional disorders or conditions that can be treated, prevented, diagnosed, and / or prognosed by an antibody, variant, or derivative thereof of the present disclosure related to an increase in the lifespan of cells include, but are not limited to, leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)), and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors (including, but not limited to, sarcomas and cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma, etc.)), progression and / or metastasis of malignant tumors and related disorders are mentioned.

[0123] The specific dosing and treatment regimen for any particular patient depends on a variety of factors, including the specific antibody, variant, or derivative used, the patient's age, weight, general health, gender, and diet, as well as the time of administration, rate of excretion, drug combination, and severity of the particular disease being treated. The determination of such factors by a healthcare provider is within the scope of those skilled in the art. The amount also depends on the individual patient being treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0124] Examples of methods for administering the antibodies and variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition can be administered by any conventional route, such as by infusion or bolus injection, by absorption through the epithelium or mucosal skin layer (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can also be administered together with other biologically active agents. Thus, the pharmaceutical composition comprising the antigen-binding polypeptide of the present disclosure can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (by powder, ointment, drops, or transdermal patch), bucally, or as an oral or nasal spray.

[0125] As used herein, the term "parenteral" refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0126] Administration can be systemic or local. Further, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intracerebroventricular and intrathecal injections; intracerebroventricular injection can be facilitated by an intracerebroventricular catheter (e.g., attached to a reservoir such as an Ommaya reservoir). Lung administration can also be employed (e.g., by using an inhaler or nebulizer and a formulation using an aerosolizing agent).

[0127] It may sometimes be desirable to administer the antigen-binding polypeptides or compositions of the present disclosure locally to the extent that treatment is needed; this can be achieved, for example (and without limitation), by local injection during surgery, by topical application (e.g., combined) using a postoperative wound dressing, by injection, by catheter, by suppository, or by an implant (wherein the implant is a porous, non-porous, or gelatinous material (such as a membrane like a sialastic membrane, or containing fibers)). Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken in the use of materials that do not absorb the protein.

[0128] The amount of the antibody of the present disclosure that is effective in the treatment, inhibition, and prevention of inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. Additionally, in vitro assays can be employed as needed to assist in the confirmation of the optimal dosage range. The exact dosage to be employed in the formulation also depends on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the judgment of the physician and the circumstances of each patient. The effective dosage can be estimated from a dose-response curve derived from in vitro or animal model test systems.

[0129] As a general proposal, the dosage of the antigen-binding polypeptide of the present disclosure administered to a patient is typically 0.1 mg / kg to 100 mg / kg (patient body weight), 0.1 mg / kg to 20 mg / kg (patient body weight), or 1 mg / kg to 10 mg / kg (patient body weight). Generally, human antibodies have a longer half-life in the human body than antibodies derived from other species due to the immune response to foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administrations are often possible. Additionally, the dosage and frequency of administration of the antibody of the present disclosure can be reduced by enhancing the uptake and tissue penetration of the antibody (e.g., into the brain) by modification (such as lipidation, etc.).

[0130] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0131] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens (e.g., radiation therapy, etc.). Diagnostic methods

[0132] Overexpression of SIRPα has been observed in certain tumor samples, and patients with SIRPα-overexpressing cells may be responsive to treatment with the anti-SIRPα antibodies of the present disclosure. Accordingly, the antibodies of the present disclosure can also be used for diagnostic and prognostic purposes.

[0133] Preferably, the sample containing cells can be obtained from a patient (which can be a cancer patient or a patient desiring a diagnosis). The cells can be tumor tissue or mass, blood sample, urine sample, or cells from any sample from the patient. After pre-treating the sample if necessary, the sample can be incubated with the antibody of the present disclosure under conditions that allow interaction between the potentially present SIRPα protein in the sample and the antibody. To detect the presence of SIRPα protein in the sample, methods such as ELISA can be used using the anti-SIRPα antibody.

[0134] The presence (optionally with amount or concentration) of SIRPα protein in the sample can be used for cancer diagnosis as an indicator that the patient is suitable for treatment with the antibody or as an indicator that the patient has responded (or not responded) to cancer treatment. For prognostic methods, the detection can be performed once, twice, or more than once at certain stages to indicate the progress of the treatment after starting cancer treatment. Compositions

[0135] The present disclosure also provides pharmaceutical compositions. Such compositions include an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further includes a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0136] In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and, more particularly, in humans. Further, a "pharmaceutically acceptable carrier" generally is a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0137] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, corn, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. The composition can also, if necessary, include small amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic pressure such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository using conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E.W. Martin (incorporated herein by reference). Such compositions contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide a form for appropriate administration to a patient. The formulation should be suitable for the mode of administration.A parenteral preparation can be enclosed in an ampoule, disposable syringe, or multi-dose vial made of glass or plastic.

[0138] In certain embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, a composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also contain solubilizing agents and local anesthetics such as lignocaine to relieve pain at the injection site. Generally, the components may be supplied individually or together in unit dosage forms (e.g., as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachette displaying the amount of the active agent). If the composition is to be administered by infusion, it can be dosed in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components can be mixed prior to administration.

[0139] The compounds of the present disclosure can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Example

[0140] Example 1: Generation of a Mouse Monoclonal Antibody Against Human SIRPα Human SIRPα protein was used to immunize mice of different strains, thereby generating hybridomas. Eight fusions were made to generate a sufficient number of hybridoma clones. SIRPα v1 / v2 positive binders (SIRPa v1 / v2 positive binders) were selected and subcloned. Subsequently, in vitro binding and functional screening were performed using approximately 30 purified antibodies to identify lead antibodies with the highest binding affinity and the strongest functional ability. The lead antibodies were humanized.

[0141] The VH / VL sequences of the lead mouse antibodies are shown in the following table. Table 1. VH / VL sequences of lead mouse antibodies [Table 1] Example 2. Cross-binding of SIRPα v1 (SIRPa v1) and v2

[0142] In this example, the dose-response of ELISA binding of mouse anti-SIRPα mAbs to recombinant human SIRPα variant 1 and variant 2 proteins (0.5 μg / ml in 100 μl) was measured. Recombinant human SIRPα v1 or v2 protein (Biointron) was coated on microtiter plates at 0.5 μg / ml (in PBS) for 2 hours at room temperature. After coating the antigen, the wells were blocked with PBS / 0.05% Tween (PBST) and 1% BSA for 1 hour at room temperature.

[0143] After washing the wells with PBST, different concentrations of anti-SIRPα antibodies were added to the wells and incubated at room temperature for 1 hour. To detect the bound antibodies, an HRP-conjugated secondary antibody against mouse Fc (Jackson Immuno Research) was added, followed by a chemiluminescent substrate (Roche). During all incubation steps, the wells of the plate were washed 3 times with PBST. Fluorescence was measured using a TECAN Spectrafluor plate reader.

[0144] The results are shown in Figure 1. Both antibodies 248G3F6 and 300A6A6 tested showed nanogram-level affinity for both variants 1 and 2.

[0145] The binding kinetics assay for antibody variant 1 was performed using a Biacore 8K system by a human antibody capture approach. Anti-mouse Fc IgG was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The test antibody was injected and captured by the immobilized anti-human Fc IgG. A series of concentrations of antigen were injected individually, and the binding profiles were recorded for each concentration of antigen analyte.

[0146] The assay system was regenerated by injecting 10 mM glycine-HCl pH 1.5 for 30 seconds. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20). The assay temperature was 25 °C, and the binding time and dissociation time were 180 seconds and 600 seconds, respectively. Biacore data were fitted according to a 1:1 binding model using Biacore K8 evaluation software 1.0 to calculate the association rate constant (ka), dissociation rate constant (kd), and equilibrium constant (KD).

[0147] The results are shown in Figure 2 and summarized in the following table. Both of the tested antibodies showed excellent binding affinity.

Table 12

[0148] In this example, the ability of the anti-SIRPα antibody to compete with CD47 in binding to SIRPα was tested.

[0149] The recombinant CD47-Fc fusion protein (Acrobiosystems) was coated on microtiter plates at 1 μg / ml (in PBS) at 4°C for 16 hours. After blocking with 1% BSA (in PBST) at room temperature for 1 hour, 1 μg / ml of SIRPα-His protein was added at room temperature for 1 hour in the presence or absence of different concentrations of anti-SIRPα antibody. The plates were then washed three times and incubated with an HRP-conjugated anti-His secondary antibody at room temperature for 1 hour. After washing, TMB solution was added to each well for 30 minutes, the reaction was stopped with 2 M H2SO4, and the OD was measured at 490 nm.

[0150] As shown in Figure 3, both 248G3F6 and 300A6A6 potently and dose-dependently inhibited the binding of CD47 to SIRPα. Example 4. Induction of macrophage-mediated phagocytosis

[0151] In this example, the ability of anti-SIRPα antibody to induce macrophage-mediated phagocytosis was tested.

[0152] PBMCs were isolated from human blood, and monocytes were differentiated into macrophages over 6 days. Monocyte-derived macrophages (MDMs) were scraped off and reseeded into 24-well dishes and allowed to adhere for 24 hours. The human tumor cell line Raji, which endogenously expresses CD47, was transfected with human PD-L1 to overexpress human PD-L1 on the surface. These PD-L1-overexpressing Raji cells were selected as target cells, labeled with 1 μM CFSE for 10 minutes, and then added to MDMs at a tumor cell to phagocyte ratio of 5:1.

[0153] Anti-SIRPα antibody and anti-PD-L1 antibody were added to the culture system. After 3 hours of incubation, unphagocytosed target cells were washed away with PBS, the remaining phagocytes were scraped off, stained with the macrophage marker C76 antibody, and analyzed by flow cytometry. Phagocytosis was measured by + gating on C76 +The percentage of cells was evaluated.

[0154] The phagocytosis results of PD-L1-expressing tumor cells by the combined treatment of anti-SIRPα antibody and anti-PD-L1 antibody are shown in Figure 4. The combination of anti-PD-L1 antibody and any anti-SIRPα antibody showed the highest phagocytosis (the two rightmost columns). Example 5. Humanization of mouse mAbs

[0155] Mouse antibody variable region genes were utilized to generate humanized mAbs. In the first step of this process, the amino acid sequences of VH and VL of the mAb were compared with the available database of human Ig gene sequences to find the human germline Ig gene sequences that best matched overall.

[0156] The amino acid sequence of the humanized antibody is shown below. Humanized sequence A.248G3F6 Table 2 Humanization of A.248G3F6 - VH [Table 2A] Table 2B. CDR sequences [Table 2B] Table 2C. Humanization of 248G3F6 - VL [Table 2C-1] [Table 2C-2] Table 2D. CDR sequences [Table 2D] Table 2E. Humanized antibody [Table 2E] B.300A6A6 Table 3 Humanization of 300A6A6 - VH

Table 3A

Table 3B

Table 3C

Table 3D

Table 3E - 1

Table 4A - 1

Table 4A - 2

Table 4B

Table 4C

Table 4D

Table 3E - 2

Table 5A

[0157] In this example, several humanized antibodies were tested for their ability to block the interaction between SIRPα and CD47.

[0158] The recombinant CD47-Fc fusion protein (Acrobiosystems) was coated on microtiter plates at 1 μg / ml (in PBS) at 4°C for 16 hours. After blocking with 1% BSA (in PBST) at room temperature for 1 hour, 1 μg / ml of SIRPα-His protein was added at room temperature for 1 hour in the presence or absence of different concentrations of anti-SIRPα antibodies. The plates were then washed three times and incubated with an HRP-conjugated anti-His secondary antibody at room temperature for 1 hour. After washing, TMB solution was added to each well for 30 minutes, the reaction was stopped with 2M H2SO4, and the OD was measured at 490 nm.

[0159] All antibodies listed in Table 2E (248G3F6), 3E (300A6A6), and 4E (102A10F2) were tested and showed high IC50 (Table 9). Table 9. Activity of humanized antibodies to block the interaction between SIRPα and CD47

Table 9-1

Table 9-2

[0160] In this example, several humanized antibodies were tested for their ability to increase macrophage-mediated phagocytosis of tumor cells.

[0161] PBMCs were isolated from human blood, and monocytes were differentiated into macrophages using a standard protocol. Monocyte-derived macrophages (MDMs) were scraped and reseeded into 24-well dishes and allowed to adhere for 24 hours. The human tumor cell line Raji, which endogenously expresses CD47, was selected as the target cell, labeled with 1 μM CFSE for 10 minutes, and then added to the MDMs at a tumor cell to phagocyte ratio of 5:1, and different concentrations of anti-SIRPα antibody were added at the indicated concentrations. After 3 hours of incubation, non-phagocytosed target cells were washed away with PBS, the remaining phagocytes were scraped, stained with the C76 antibody, and analyzed by flow cytometry. Phagocytosis was measured by gating on C76 + cells, and then the percentage of CFSE + cells was evaluated.

[0162] The results are shown in Figure 5. Among the antibodies tested, 02-hz52 (248G3F6) and 03-hz51 (300A6A6) showed the highest activity, and all the others also showed good activity as well. Example 8. Binding affinity for SIRPα v1 and v2

[0163] In this example, the humanized antibodies 02-hz52 (248G3F6) and 03-hz51 (300A6A6) were tested for their binding affinity for SIRPα v1 and v2.

[0164] The binding kinetics assay of the antibody to the antigen was performed using a Biacore 8K system by a human antibody capture approach. Anti-mouse Fc IgG was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The test antibody was injected and captured by the immobilized anti-human Fc IgG. Then, a series of concentrations of human SIRPα v1 or SIRPα v2 protein were individually injected, and the binding profiles were recorded for each concentration of the antigen analyte. The assay system was regenerated by injecting 10 mM glycine-HCl pH 1.5 for 30 seconds. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20). The assay temperature was 25 °C, and the binding time and dissociation time were 180 seconds and 600 seconds, respectively. The Biacore data was fitted according to a 1:1 binding model using Biacore K8 evaluation software 1.0, and the association rate constant (ka), dissociation rate constant (kd), and equilibrium constant (KD) were calculated.

[0165] The test results are shown in Tables 10A - B. Table 10A. Binding affinity for SIRPα v1

Table 10A-1

Table 10B

[0166] Antibodies 02-hz52 (248G3F6) and 03-hz51 (300A6A6) (fused to the human IgG4 heavy chain constant region and kappa light chain constant region) were tested for development. Neither antibody contains free cysteine. The test results showed that both antibodies could be purified until they reached clinical-grade purity and had appropriate Tm, acid / base peak ratio, hydrophobicity, and pI. The test results are shown in Table 11.

[0167] Table 10A. Binding affinity for SIRPα v1

Table 10A-2

[0168] In this example, the efficacy of the humanized antibody 03-hz51 (300A6A6) alone or in combination with rituximab in mice was tested.

[0169] Raji-Luc cells resuspended in PBS were inoculated into the tail vein of B-NDG-hSIRPα humanized mice at a concentration of 1×10 5 cells / 0.2 mL and a volume of 0.2 mL / mouse. Using a small animal imaging device, on the 3rd day after inoculation, the tumor imaging signal value was measured. When the average imaging signal intensity reached 1.35×10 6 p / s, the animals were grouped according to the tumor imaging signal value and the body weight of the animals and evenly assigned to 4 experiments. There were 6 mice in each experimental group.

[0170] Rituximab showed a significant inhibitory effect on Raji-Luc lymphoma at a dose level of 10 mg / kg without clinically harmful conditions. The combination of 03-hz51 and rituximab (10 mg / kg + 0.1 mg / kg) showed a significant inhibitory effect on Raji-Luc lymphoma. This data indicates that 03-hz51 synergized with rituximab in the complete suppression of tumor growth in the Raji lymphoma tumor model.

[0171] The present disclosure is not limited, in scope, to the specific embodiments described (which are intended to be one illustration of the individual aspects of the present disclosure), and any compositions and methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the disclosure. Accordingly, the present disclosure is intended to embrace such modifications and variations, provided they are within the scope of the appended claims or the equivalents thereof.

[0172] All publications and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) An antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy-chain variable region comprising heavy-chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein (a) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 16, 21, or 22, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 20; (b) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, 36, 37, or 38, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35; (c) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 47, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 48, 53, or 54, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 49, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 50, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 51, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 52; (d) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 63, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 64, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 65, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 66, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 67, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 68; (e) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 77, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 78, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 79, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 80, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 81, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 82; (f) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 91, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 92, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 93, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 94, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 95, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 96; or (g) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 103, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 104, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 105, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 106, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 107, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 108, an antibody or a fragment thereof. (Item 2) An antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region light chain comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 16, 21, or 22, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 20, Antibody or a fragment thereof. (Item 3) The antibody or the fragment thereof according to item 2, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23 to 27, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23 to 27. (Item 4) The antibody or the fragment thereof according to item 2 or 3, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28 to 29, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28 to 29. (Item 5) The antibody or the fragment thereof according to item 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29. (Item 6) An antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy-chain variable region comprising heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, 36, 37, or 38, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35. Antibody or a fragment thereof. (Item 7) The antibody or fragment thereof according to item 6, wherein the heavy-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39-44, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39-44. (Item 8) The antibody or fragment thereof according to item 6 or 7, wherein the light-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 45-46, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 45-46. (Item 9) The antibody or fragment thereof according to item 6, wherein the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 43, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 45. (Item 10) An antibody or a fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or the fragment thereof comprises a heavy-chain variable region comprising heavy-chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light-chain variable region light-chain comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 47, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 48, 53, or 54, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 49, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 50, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 51, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 52, Antibody or fragment thereof. (Item 11) The antibody or fragment thereof according to item 10, wherein the heavy-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55 to 60, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55 to 60. (Item 12) The antibody or fragment thereof according to item 10 or 11, wherein the light-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 61 to 62, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 61 to 62. (Item 13) The antibody or fragment thereof according to any one of items 1 to 12, which is capable of binding to SIRPα variant 1 and variant 2. (Item 14) The antibody or fragment thereof according to any one of items 1 to 13, which is humanized. (Item 15) The antibody or fragment thereof according to any one of items 1 to 24, which further has binding specificity for a second target protein. (Item 16) A composition comprising an antibody or a fragment thereof according to any one of items 1 to 15, and a pharmaceutically acceptable carrier. (Item 17) The composition according to item 16, further comprising a secondary antibody having specificity for a tumor antigen. (Item 18) The composition according to item 17, wherein the secondary antibody is a tumor opsonizing antibody. (Item 19) A method for treating cancer in a patient in need of treating cancer, comprising administering to the patient an antibody or a fragment thereof according to any one of items 1 to 15. (Item 20) The method according to item 19, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. (Item 21) The method according to item 19, further comprising administering to the patient a secondary antibody targeting a protein that induces tumor opsonization. (Item 22) The method according to item 21, wherein the protein is selected from the group consisting of CD19, CD20, EGFR, HER2, CD3, CD16, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, VEGFR, TIGIT, Claudin 18.2, CD24, GPC3, Il13RA2, 4-1BB, CCR8, and CMET.

Claims

1. 1. An antibody or antigen-binding fragment thereof having binding specificity for a wild-type human signal-regulatory protein alpha (SIRPα) protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, (a) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 16, 21, or 22, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 20; (b) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, 36, or 38, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 35; (c) the CDRH1 comprises the amino acid sequence of SEQ ID NO:47, the CDRH2 comprises the amino acid sequence of SEQ ID NO:48, 53, or 54, the CDRH3 comprises the amino acid sequence of SEQ ID NO:49, the CDRL1 comprises the amino acid sequence of SEQ ID NO:50, the CDRL2 comprises the amino acid sequence of SEQ ID NO:51, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:52; (d) the CDRH1 comprises the amino acid sequence of SEQ ID NO:63, the CDRH2 comprises the amino acid sequence of SEQ ID NO:64, the CDRH3 comprises the amino acid sequence of SEQ ID NO:65, the CDRL1 comprises the amino acid sequence of SEQ ID NO:66, the CDRL2 comprises the amino acid sequence of SEQ ID NO:67, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:68; (e) the CDRH1 comprises the amino acid sequence of SEQ ID NO:77, the CDRH2 comprises the amino acid sequence of SEQ ID NO:78, the CDRH3 comprises the amino acid sequence of SEQ ID NO:79, the CDRL1 comprises the amino acid sequence of SEQ ID NO:80, the CDRL2 comprises the amino acid sequence of SEQ ID NO:81, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:82; (f) the CDRH1 comprises the amino acid sequence of SEQ ID NO:91, the CDRH2 comprises the amino acid sequence of SEQ ID NO:92, the CDRH3 comprises the amino acid sequence of SEQ ID NO:93, the CDRL1 comprises the amino acid sequence of SEQ ID NO:94, the CDRL2 comprises the amino acid sequence of SEQ ID NO:95, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:96; or (g) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 103, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 104, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 105, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 106, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 107, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:

108. An antibody or an antigen-binding fragment thereof.

2. 1. An antibody or antigen-binding fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein said CDRH1 comprises the amino acid sequence of SEQ ID NO: 15, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 16, 21, or 22, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 17, said CDRL1 comprises the amino acid sequence of SEQ ID NO: 18, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:

20. An antibody or an antigen-binding fragment thereof.

3. 3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-27, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-27.

4. 4. The antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-29, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-29.

5. The antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:27 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

29.

6. An antibody or antigen-binding fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein said CDRH1 comprises the amino acid sequence of SEQ ID NO: 30, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, 36, or 38, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 32, said CDRL1 comprises the amino acid sequence of SEQ ID NO: 33, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 34, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:

35. An antibody or an antigen-binding fragment thereof.

7. 7. The antibody or antigen-binding fragment thereof of claim 6, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, and 39-42, and 44, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, and 39-42, and 44.

8. The antibody or antigen-binding fragment thereof of claim 6 or 7, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 46, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 46.

9. 1. An antibody or antigen-binding fragment thereof having binding specificity for a wild-type human signal regulatory protein alpha (SIRPα) protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein said CDRH1 comprises the amino acid sequence of SEQ ID NO: 47, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 48, 53, or 54, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 49, said CDRL1 comprises the amino acid sequence of SEQ ID NO: 50, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 51, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:

52. An antibody or an antigen-binding fragment thereof.

10. 10. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55-60, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55-60.

11. The antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 61-62, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 61-62.

12. An antibody or fragment thereof according to any one of claims 1 to 11, capable of binding to SIRPα variant 1 and variant 2.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is humanized.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, further having binding specificity for a second target protein.

15. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

16. The composition of claim 15, further comprising a second antibody having specificity for a tumor antigen.

17. The composition of claim 16 , wherein the secondary antibody is a tumor-opsonizing antibody.

18. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14 for treating cancer in a patient in need thereof.

19. 19. The composition of claim 18, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

20. 20. The composition of claim 18, further characterized in that the patient is administered a secondary antibody that targets a protein that induces tumor opsonization.

21. 21. The composition of claim 20, wherein the protein is selected from the group consisting of CD19, CD20, EGFR, HER2, CD3, CD16, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, VEGFR, TIGIT, claudin 18.2, CD24, GPC3, Il13RA2, 4-1BB, CCR8, and CMET.

22. 15. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 for the manufacture of a medicament for treating cancer in a patient in need thereof.

23. 23. The use of claim 22, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

24. 24. The use according to claim 23, characterized in that the patient is administered a secondary antibody that targets a protein that induces tumor opsonization.

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