Anti-SIRPα monoclonal antibodies and uses thereof

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 technology]

[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-linked signal transduction processes. SIRPα can be phosphorylated by tyrosine kinases. Phosphotyrosine residues of this PTP have been shown to recruit SH2 domain-containing tyrosine phosphatases (PTPs) and serve as substrates for the PTPs. 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 a great deal of similarity with several other members of the SIRP family. SIRPα is expressed primarily by myeloid cells, but also by stem cells or neurons.

[0002] SIRPα acts as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47 (also known as 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 and accumulates at the phagocytic synapse, where it binds and emits a "self" signal to CD47, which inhibits the cytoskeleton-intensive process of phagocytosis by macrophages.

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

[0004] SIRPα recognizes CD47, an anti-phagocytic signal that distinguishes live from dying cells. The extracellular domain of SIRPα binds to CD47 and transmits intracellular signals 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 kinase SHP2. SIRPα also binds the phosphatase SHP1, the adaptor protein SCAP2, and FYN-binding protein. Recruitment of 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. High-affinity variants of SIRPα that antagonize CD47 on cancer cells have 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 problem]

[0006] overview Anti-SIRPα antibodies with high affinity for both variants v1 and v2 are found herein, which can block the interaction between SIRPα and CD47 in a dose-dependent and efficient manner, 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, there is provided an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, said 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 comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein (a) 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; (b) 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, 37, or 38, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 20. (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 CDRH1 comprises the amino acid sequence of SEQ ID NO: 50, the CDRH2 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, and 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, and the CDRH3 comprises the amino acid sequence of SEQ ID NO:93, and the CDRL or (g) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 94, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 95, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 96; or (g) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 103, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 104, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 105, said CDRL1 comprises the amino acid sequence of SEQ ID NO: 106, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 107, and said CDRL3 comprises the amino acid sequence of SEQ ID NO: 108;

[0008] In one embodiment, the disclosure provides an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or fragment thereof comprises 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 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.

[0009] 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.

[0010] 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.

[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 fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, said 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 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, 37, 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.

[0013] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39 to 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 to 44.

[0014] In some embodiments, 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.

[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 fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or fragment thereof comprises 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 CDRH1 comprises the amino acid sequence of SEQ ID NO: 47, CDRH2 comprises the amino acid sequence of SEQ ID NO: 48, 53, or 54, CDRH3 comprises the amino acid sequence of SEQ ID NO: 49, CDRL1 comprises the amino acid sequence of SEQ ID NO: 50, CDRL2 comprises the amino acid sequence of SEQ ID NO: 51, and 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 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.

[0018] In some embodiments, 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.

[0019] In some embodiments, the antibodies or fragments disclosed herein are capable of binding 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 are also provided that include the antibody or fragment thereof and a pharma- ceutically acceptable carrier. In some embodiments, the composition further includes 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 is provided comprising administering to the patient an antibody or fragment thereof of the present disclosure. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows cross-binding of antibodies to SIRPα v1 and v2.

[0023] [Diagram 2] FIG. 2 shows the binding affinity of antibodies to SIRPa v1.

[0024] [Diagram 3] FIG. 3 shows competition of SIRPα interaction with CD47 by antibodies.

[0025] [Figure 4] FIG. 4 shows induction of macrophage-mediated phagocytosis by antibodies.

[0026] [Diagram 5]FIG. 5 shows increased macrophage-mediated phagocytosis of tumor cells by antibody treatment.

[0027] [Figure 6] FIG. 6 shows animal scanning images and charts demonstrating that 03-hz51 synergized with rituximab in completely inhibiting tumor growth in the Raji lymphoma tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description definition The terms "a" or "an" entity refer to one or more of that entity. For example, "an antibody" refers to one or more of that entity. It should be understood to refer to more than one antibody. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0029] As used herein, the term "polypeptide" refers to the singular "poly The term "polypeptide" is intended to encompass "peptide", as well as "polypeptides", and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or 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 chain or chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of 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 or produced by recombinant technology, but not necessarily translated from a specified nucleic acid sequence. Polypeptides may be produced in any manner, including chemical synthesis.

[0030] "Homology", or "identity", or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence (which may be aligned for purposes of comparison). If a position in the compared sequences is 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. An "unrelated" or "non-homologous" sequence shares less than 40% identity (although 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, meaning that, when aligned, that percentage of bases (or amino acids) are identical in comparing the two sequences.

[0032] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. A homolog of a double-stranded nucleic acid is a nucleic acid that is identical to or similar to its complement. It is intended to include nucleic acids having nucleotide sequences with a degree of homology (e.g., with the complement thereof). In one embodiment, a nucleic acid homolog is capable of hybridizing to a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a 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 one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, compared to a reference polypeptide or polynucleotide. In some embodiments, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) 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 to an antigen. An antibody can be a whole antibody, and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide, including molecules that contain at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, at least a portion of a heavy or light chain complementarity determining region (CDR), or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or a binding protein.

[0034] The term "antibody fragment" or "antigen-binding fragment" as used herein is a portion 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 with the same antigen 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] A "single chain variable fragment" or "scFv" is a fragment of the variable region of an immunoglobulin heavy chain (V H ) and the light chain variable region (V L In some embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, as well as V H N-terminus of V LThe ScFv molecule may be linked to the C-terminus of either the ScFv or the C-terminus of the ScFv polypeptide, or vice versa. The 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 wide variety of biologically distinguishable polypeptide classes. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses therein (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to provide functional specialization. Modified forms of each of these classes and isotypes are readily discernible to one of skill in the art in light of the present disclosure, and thus are within the scope of the present disclosure. All immunoglobulin classes are expressly 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 polypeptide chains with a molecular weight of approximately 23,000 daltons, and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0037] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, 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-linked Fv (sdFv), fragments comprising either the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin 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 as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequences run from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0039] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, the variable domain of the light chain portion (VK) and the variable domain of the heavy chain portion (VH) both determine antigen recognition and specificity. Conversely, the constant domain of the light chain (CK) and the constant domain 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 they move away from the antigen-binding site or 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 comprise the carboxy termini of the heavy and light chains, respectively.

[0040] As shown above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope of an antigen. That is, the VK domain and the VH domain of an antibody, or a subset of complementarity determining regions (CDRs), combine to form the variable region that defines a three-dimensional antigen-binding site. The quaternary structure of the antibody forms the antigen-binding site present at the end 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 cases, for example, certain immunoglobulin molecules derived from Camelidae species or engineered based on Camelidae immunoglobulins, the complete immunoglobulin molecule may be composed of only heavy chains without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0041] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous amino acid sequences that occupy specific positions such that the antibody forms an antigen-binding domain when it adopts a three-dimensional arrangement in an aqueous environment. The remaining amino acids of the antigen-binding domain (called the "framework" regions) show less inter-molecular variability. The framework regions adopt a predominantly β-sheet structure, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions serve to form a scaffold that provides for the positioning of the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs determines a surface complementarity to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids which comprise the CDRs and framework regions, respectively, have been precisely defined (see Sequences of Proteins of Immunological Interest,'Kabat, E. et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)), and can therefore be readily determined for any given heavy or light chain variable region by one of ordinary skill in the art.

[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 as used herein is intended to include all of those meanings unless expressly indicated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found in the variable regions of both the heavy and light chain polypeptides. This particular region is described in Kabat et al., US Pept. of Health and Human Services, "Sequences of Proteins, of Immunological Interest'' (1983), and Chothia et al., J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDRs by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, application of either definition to refer to the CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined in each of the above cited references are set forth in the table below as a comparison. The exact number of residues that encompass a particular CDR will vary depending on the sequence and size of that CDR. One skilled in the art can routinely determine which residues encompass a particular CDR given the variable region amino acid sequence of an antibody. [Table 11]

[0043] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., US 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 about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence WGXG (where X is any amino acid). CDR-L1 begins at about residue 24 (i.e., after the cysteine ​​residue); includes about 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at about the 16th residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins at approximately the 33rd residue after the end of CDR-L2 (i.e., after the cysteine ​​residue); contains approximately 7 to 11 residues; and ends at the sequence F or WGXG (where X is any amino acid).

[0045] The antibodies disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.In another embodiment, the variable region can be of condricthoid origin (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 that includes 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 that includes a CH1 domain; a polypeptide chain that includes a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain that includes a CH1 domain and a CH3 domain; a polypeptide chain that includes a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain that includes a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure includes a polypeptide chain that includes a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, it will be appreciated by those skilled in the art that the heavy chain constant region may be modified so that it differs in amino acid sequence from that of a naturally occurring immunoglobulin molecule.

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

[0049] A "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0050] As indicated above, the subunit structures and three-dimensional configurations 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 to the hinge region of an immunoglobulin heavy chain molecule.

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

[0052] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be divided into three separate domains: 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 a disulfide bond, 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" refers to any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, a portion, 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 it by the total number of amino acids and multiplying by 100.

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

[0057] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow down (alleviate) an undesired physiological change or disorder (e.g., the progression of cancer). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliative of the disease state, and remission (whether partial or complete remission). "Treatment" can also mean prolonging survival compared to the expected survival if not treated. Subjects in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to 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, sport, or pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.).

[0059] As used herein, phrases such as "to a patient in need of treatment" or "subject in need of treatment" include subjects (e.g., mammalian subjects) who would benefit from the administration of an antibody or composition of the disclosure, e.g., used for detection, for diagnostic procedures, and / or for treatment. Anti-SIRPα antibody

[0060] The present disclosure provides anti-SIRPα antibodies and fragments with high affinity for both variants v1 and v2. Variant 1 (hSIRPα V1) is the predominant variant among Europeans, Africans, mixed Americans, and South Asians. Variant 2 (hSIRPα V2) is the predominant variant among East Asians. hSIRPα V1 and hSIRPα V2 differ in 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 allows them to be effective among the broadest patient population.

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

[0062] According to one embodiment of the present disclosure, therefore, there is provided an antibody and antigen-binding fragment thereof capable of binding to both variants 1 and 2 of SIRPα. Exemplary antibodies include the murine antibodies shown in Table 1, and the humanized antibodies 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 epitopes 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 is provided an antibody or fragment thereof, and biological equivalents thereof, comprising a heavy chain variable domain and a light chain variable domain having the CDR regions disclosed herein.

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

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

[0066] One embodiment provides an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, wherein the antibody or fragment thereof comprises 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 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 fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, said 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 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: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.

[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 CDRs are those of 300A6A6, as illustrated in Tables 3B and 3D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, 36, 37, or 38 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRH3 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRL1 comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRL2 comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0072] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, said 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 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, 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.

[0073] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, said 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 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: 36, 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.

[0074] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, said 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 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: 37, 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.

[0075] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, said 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 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: 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.

[0076] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39 to 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 to 44.

[0077] In some embodiments, 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.

[0078] 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.

[0079] In one embodiment, the CDRs are those of 102A10F2, as illustrated in Tables 4B and 4D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO:47 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRH2 comprises the amino acid sequence of SEQ ID NO:48, 53, or 54 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRH3 comprises the amino acid sequence of SEQ ID NO:49 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRL1 comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; CDRL2 comprises the amino acid sequence of SEQ ID NO:51 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof; and CDRL3 comprises the amino acid sequence of SEQ ID NO:52 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

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

[0081] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, said 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 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: 53, 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.

[0082] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein is provided, wherein the antibody or fragment thereof comprises 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, and wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 47, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 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.

[0083] In some embodiments, 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.

[0084] In some embodiments, 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.

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

[0086] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 69-72, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 69-72.

[0087] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 73-76, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 73-76.

[0088] In one embodiment, the CDRs are those of 211F8E11, as illustrated in Tables 6B and 6D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 77 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO: 78 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO: 79 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 80 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO: 81 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 82 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0089] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 9 and 83-86.

[0090] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 10 and 87-90.

[0091] In one embodiment, the CDRs are those of 217D11E5, as illustrated in Tables 7B and 7D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO:91 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO:92 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO:93 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO:94 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO:95 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO:96 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0092] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 12 and 101-102.

[0094] In one embodiment, the CDRs are those of 234B7D5, as illustrated in Tables 8B and 8D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 103 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO: 104 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO: 105 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 106 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO: 107 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 108 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0095] In some embodiments, the heavy chain variable region comprises 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 comprises 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 strong 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 for carrying post-translational modifications (PTMs). Such modified CDRs can be referred to as affinity matured or de-risked CDRs.

[0098] Non-limiting examples of risk-reducing CDRs are shown in Tables 2B, 3B, and 4B. Modified CDRs can include those with 1, 2, or 3 amino acid additions, deletions, and / or substitutions. In some embodiments, the 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 have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). 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, the string of amino acids may be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0100] Non-limiting examples of conservative amino acid substitutions are shown in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids. Table A. Amino acid similarity matrix [Table A] Table B. Conservative Amino Acid Substitutions [Table B]

[0101] Those skilled in the art will also understand that the antibodies disclosed herein may be modified to differ in amino acid sequence from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., be 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 one or more moieties that are not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibody of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0103] The antibodies, variants, or derivatives of the present disclosure include derivatives that are modified, i.e., modified by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to its epitope. For example, but not by way of limitation, the antibody may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody may contain one or more non-classical amino acids.

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

[0105] Antibodies may be conjugated or fused to therapeutic agents (which may include a detectable label, such as a radioactive label), immunomodulatory agents, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, 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 chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0107] The antibody may also be 152Fluorescence-emitting metals such as Eu, or other metals of the lanthanum series may be used to detectably label the antibody. These metals may be attached to the antibody using metal chelating groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known (see, 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 See, "Of The Therapeutic Use Of Radiolabeled Antibodies 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 combination therapies

[0108] An important component of the innate immune system is the macrophage. Macrophages inhibit tumor growth by phagocytosis of tumor cells. Tumor cells can escape surveillance by macrophages 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 regulating cell motility, adhesion, migration, and platelet activation. SIRPα is another member of the immunoglobulin superfamily and is expressed primarily 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 trigger antibody opsonization and activate antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), which promotes tumor cell destruction. However, the ubiquitous expression of CD47 can result in RBC toxicity and other hematological adverse effects due to the presence of this antigen on blood cells. Molecules targeting either SIRPα, or bispecific agents targeting both SIRPα and CD47, can be designed to induce ADCC and ADCP effects by processing functional Fc domains.

[0110] Because SIRPα has a more restricted expression compared to CD47, these molecules should not have as many hematological events as molecules that target only CD47. 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 either bispecific antibodies, fusion proteins, and combination therapies.

[0111] Examples of tumor antigens, particularly those capable of inducing 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 isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0114] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be produced 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 disabled. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent Nos. 6,150,584; 6,458,592; and 6,420,140, ​​which are incorporated by reference in their entirety. Treatment

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

[0116] The present disclosure is also directed to antibody-based therapies, including administering the antibodies of the present disclosure to patients, such as animals, mammals, and humans, 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 as described herein), and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives as described herein).

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

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

[0119] Cell therapy (e.g., chimeric antigen receptor (CAR) T cell therapy) is also provided in the present disclosure. Appropriate cells that have been contacted with the anti-SIRPα antibody of the present disclosure (or alternatively, engineered to express the anti-SIRPα antibody of the present disclosure) can be used. Once such contacted or engineered, 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, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination 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. If the cells are isolated from the cancer patient, unwanted immune responses may 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 associated with increased cell survival that may be treated, prevented, diagnosed, and / or prognosed by the disclosed antibodies or variants or derivatives thereof include, but are not limited to, leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)), and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (including, but not limited to, sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and sarcoma). , chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, 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 and / or metastasis of malignant tumors and related disorders, including, but not limited to, tumors, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0123] The specific administration and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the particular disease being treated. The judgment of such factors by the medical caregiver is within the skill of the art. The amount will also depend 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] Methods of administration of antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions may be administered by any conventional route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Thus, pharmaceutical compositions comprising antigen-binding polypeptides of the present disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powders, ointments, drops, or transdermal patches), bucally, or as an oral or nasal spray.

[0125] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

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

[0127] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area requiring treatment; this may be achieved, for example (and not by way of limitation), by localized infusion during surgery, by topical application after surgery (e.g., in combination with) a wound dressing, by injection, by catheter, by suppository, or by implant (said implants being porous, non-porous, or gelatinous materials, including membranes such as sialastic membranes, or fibers). Preferably, when administering proteins (including antibodies) of the disclosure, care must be taken to use materials to which the proteins do not absorb.

[0128] The amount of the antibody of the present disclosure that is effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays can be employed as necessary to help determine optimal dosage ranges. The exact dose 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 each patient's circumstances. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0129] As a general proposition, the dosage of an antigen-binding polypeptide of the present disclosure administered to a patient is typically 0.1 mg / kg to 100 mg / kg (patient's body weight), 0.1 mg / kg to 20 mg / kg (patient's body weight), or 1 mg / kg to 10 mg / kg (patient's body weight). In general, human antibodies have a longer half-life in the human body than antibodies derived from other species due to the immune response to the foreign polypeptide. This often allows for lower dosages of human antibodies and less frequent administration. Additionally, the dosage and frequency of administration of an antibody of the present disclosure can be reduced by enhancing uptake (e.g., into the brain) and tissue passage of the antibody through modifications (e.g., lipidation, etc.).

[0130] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines that may be administered with the compositions of the present disclosure, including, but 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, compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as radiation therapy. Diagnostic methods

[0132] Overexpression of SIRPα is 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. Thus, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

[0133] A sample, preferably including cells, can be obtained from a patient, which can be a cancer patient or a patient seeking diagnosis. The cell can be a cell from a tumor tissue or tumor mass, a blood sample, a urine sample, or any sample from a patient. After sample pretreatment as required, the sample can be incubated with an antibody of the present disclosure under conditions that allow the antibody to interact with SIRPα protein potentially present in the sample. To detect the presence of SIRPα protein in a sample, a method such as ELISA can be used utilizing an anti-SIRPα antibody.

[0134] The presence of SIRPα protein in the sample (along with the amount or concentration, if necessary) can be used to diagnose cancer, as an indication that the patient is suitable for treatment with the antibody, or as an indication that the patient has responded (or has not responded) to cancer treatment. For prognostic methods, detection can be performed once, twice, or more times at a certain stage, once cancer treatment has been started, to indicate the progress of treatment. composition

[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 "pharmaceutical acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopeia or a generally recognized pharmacopoeia for use in animals, and more specifically, in humans. Moreover, a "pharmaceutical acceptable carrier" is generally 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, and the like). 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, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if necessary, can also contain minor 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 regulating tonicity such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions may be formulated as suppositories, using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW 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 the form for proper administration to the patient. The formulation should be suitable for the mode of administration.The parental preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0138] In some embodiments, the composition is formulated according to the usual procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. In general, the ingredients may be supplied separately or mixed together in unit dosage form (e.g., as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachette, indicating the amount of active agent). If the composition is to be administered by injection, it may be dispensed 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 ingredients can be mixed prior to administration.

[0139] The compounds of the present disclosure can be formulated in neutral or salt form. 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. EXAMPLES

[0140] Example 1: Generation of mouse monoclonal antibodies against human SIRPα Human SIRPα protein was used to immunize different strains of mice, thereby generating hybridomas. Eight fusions were made to generate a sufficient number of hybridoma clones. SIRPα SIRPa v1 / v2 positive binders were selected and subcloned. Approximately 30 purified antibodies were then used for in vitro binding and functional screening to identify lead antibodies with the highest binding affinity and strongest functional capacity. The lead antibodies were humanized.

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

[0142] In this example, the dose response of ELISA binding of mouse anti-SIRPα mAb 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 proteins (Biointron) were coated at 0.5 μg / ml (in PBS) onto microtiter plates for 2 hours at room temperature. After coating the antigen, wells were blocked with PBS / 0.05% Tween (PBST) with 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. To detect bound antibodies, an HRP-conjugated secondary antibody against mouse Fc (Jackson Immuno Research) was added, followed by a fluorogenic substrate (Roche). Between all incubation steps, the wells of the plate were washed three times with PBST. Fluorescence was measured on a TECAN Spectrafluor plate reader.

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

[0145] Binding kinetics assay for antibody variant 1 was carried out using a Biacore 8K system with a human antibody capture approach. Anti-mouse Fc IgG was immobilized on a CM5 sensor chip according to the manufacturer's instructions. Test antibodies were injected and captured by immobilized anti-human Fc IgG. A series of antigen concentrations were injected separately, and 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 association and dissociation times were 180 and 600 seconds, respectively. The Biacore data were fitted according to a 1:1 binding model using Biacore K8 evaluation software 1.0 to calculate the association and dissociation rate constants (ka) and (kd), as well as the equilibrium constant (KD).

[0147] The results are shown in Figure 2 and summarized in the table below. Both antibodies tested showed excellent binding affinity. [Table 12] Example 3. Competition with CD47

[0148] In this example, the ability of anti-SIRPα antibodies to compete with CD47 for binding to SIRPα was examined.

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

[0150] As shown in FIG. 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 antibodies to induce macrophage-mediated phagocytosis was examined.

[0152] PBMCs were isolated from human blood, and monocytes were differentiated into macrophages for 6 days. Monocyte-derived macrophages (MDMs) were scraped, replated in 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 its surface. The PD-L1-overexpressing Raji cells were selected as target cells, labeled with 1 μM CFSE for 10 min, and then added to the MDMs at a tumor cell to phagocyte ratio of 5:1.

[0153] Anti-SIRPα and anti-PD-L1 antibodies were added to the culture system. After 3 hours of incubation, non-phagocytosed target cells were washed away with PBS, and the remaining phagocytic cells were scraped off and stained with the macrophage marker C76 antibody and analyzed by flow cytometry. Phagocytosis was determined by C76 antibody. + CFSE +The percentage of cells was assessed.

[0154] The phagocytosis results of PD-L1-expressing tumor cells by combined treatment with anti-SIRPα antibody and anti-PD-L1 antibody are shown in Figure 4. The combination of anti-PD-L1 antibody and either anti-SIRPα antibody showed the highest phagocytosis (the two columns on the right). Example 5. Humanization of Mouse mAb

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

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

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

[0159] All antibodies listed in Tables 2E (248G3F6), 3E (300A6A6), and 4E (102A10F2) were tested and demonstrated high IC50s (Table 9). Table 9. Activity of humanized antibodies to block the interaction of SIRPα with CD47 [Table 9-1] [Table 9-2] Example 7. Increased macrophage-mediated phagocytosis of tumor cells

[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 standard protocols. Monocyte-derived macrophages (MDMs) were scraped, replated in 24-well dishes and allowed to adhere for 24 hours. Human tumor cell line Raji, which endogenously expresses CD47, was selected as target cells and labeled with 1uM CFSE for 10 minutes, then added to 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, unphagocytosed target cells were washed away with PBS, and remaining phagocytic cells were scraped, stained with C76 antibody and analyzed by flow cytometry. Phagocytosis was assessed using C76 antibody. + CFSE + The percentage of cells was assessed.

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

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

[0164] The binding kinetics assay of antibodies to antigens 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. Test antibodies were injected and captured by the immobilized anti-human Fc IgG. A series of concentrations of human SIRPα v1 or SIRPα v2 protein were then injected separately and binding profiles were recorded for each concentration of antigen analyte, respectively. 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 association and dissociation times were 180 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 and dissociation rate constants (ka) and (kd), as well as the equilibrium constant (KD).

[0165] The test results are shown in Tables 10A-B. Table 10A. Binding affinity to SIRPα v1 [Table 10A-1] Table 10B. Binding affinity to SIRPα v2 [Table 10B] Example 9. Development Study

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

[0167] Table 10A. Binding affinity to SIRPα v1 [Table 10A-2] Example 10. In vivo testing

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

[0169] Raji-Luc cells resuspended in PBS were diluted to 1 × 10 5 The cells were inoculated into the tail vein of B-NDG-hSIRPα humanized mice at a concentration of 0.2 mL per cell and a volume of 0.2 mL per mouse. The tumor imaging signal value was measured on the third day after inoculation using a small animal imaging device. The average imaging signal intensity was 1.35 × 10 6 Once p / sec was reached, the animals were grouped according to tumor imaging signal values ​​and animal weights and equally allocated into four experiments, with six mice per experimental group.

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

[0171] The present disclosure is not limited in scope by the specific embodiments described (which are intended to be an illustration of one 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 is clear 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 present disclosure. Thus, the present disclosure is intended to cover modifications and variations of the present disclosure, provided that such modifications and variations are within the scope of the appended claims or their equivalents.

[0172] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent 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) 1. An antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, said 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 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, 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 fragment thereof. (Item 2) An antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, said 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 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 fragment thereof. (Item 3) 3. The antibody or 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) 4. The antibody or 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) 3. The antibody or 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 fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, said 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 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, 37, 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 fragment thereof. (Item 7) 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 to 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 to 44. (Item 8) 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 to 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 to 46. (Item 9) 7. 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 fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPα) protein, said 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 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 fragment thereof. (Item 11) 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) 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) 13. The antibody or fragment thereof according to any one of items 1 to 12, capable of binding to SIRPα variant 1 and variant 2. (Item 14) 14. The antibody or fragment thereof according to any one of items 1 to 13, which is humanized. (Item 15) 25. The antibody or fragment thereof according to any one of items 1 to 24, further having binding specificity for a second target protein. (Item 16) 16. A composition comprising the antibody or fragment thereof according to any one of items 1 to 15, and a pharma- ceutically acceptable carrier. (Item 17) 17. The composition of claim 16, further comprising a secondary antibody having specificity for a tumor antigen. (Item 18) 18. The composition of claim 17, wherein the secondary antibody is a tumor-opsonizing antibody. (Item 19) 16. A method for treating cancer in a patient in need thereof, comprising administering to the patient the antibody or fragment thereof according to any one of items 1 to 15. (Item 20) 20. The method of claim 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, renal cancer, melanoma, prostate cancer, and thyroid cancer. (Item 21) 20. The method of claim 19, further comprising administering to the patient a secondary antibody that targets a protein that induces tumor opsonization. (Item 22) 22. The method of claim 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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