Anti-SIRP alpha antibody

Abstract

To provide novel anti-SIRPα antibodies that result in enhanced phagocytosis of both solid and hematopoietic tumor cells, including increased phagocytosis of glioblastoma cells in vitro and significant anti-tumor activity in vivo.SOLUTION: Provided is an antibody or an antigen-binding fragment thereof that binds to human SIRPα, comprising: a heavy chain variable region comprising CDR1 to CDR3 of a specific amino acid sequence; and a light chain variable region comprising CDR1 to CDR3 of another specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the interests of Dutch Patent Application No. 2018708 filed on 13 April 2017 and Dutch Patent Application No. 2019166 filed on 3 July 2017, each of which, including all tables, figures and claims, is incorporated herein by reference in its entirety.

[0002] Field of Invention This invention relates to anti-SIRPα antibodies and the use of these antibodies in the treatment of diseases. [Background technology]

[0003] Background of the Invention Signal-regulating protein alpha (SIRPα) is a membrane glycoprotein belonging to the SIRP family. Members of the SIRP family share a specific common structural motif. These include a transmembrane segment and an N-terminal extracellular domain containing three Ig-like loops linked by three pairs of disulfide bonds. However, the C-terminal intracellular domain differs among SIRP family membranes. SIRPα has an extended intracellular domain containing four tyrosine residues that form two immunoreceptor tyrosine-dependent repressive motifs (ITIMs), while SIRPβ1 has a short intracellular tail lacking a lysine residue within the transmembrane domain, followed by an ITIM that acts as a receptor for DAP12. Eight SIRPα single-nucleotide polymorphisms have been identified, the most common being SIRPαV1 and SIRPαV2 (Takenaka et al., Nat. Immunol. 2007, 8:1313-23).

[0004] The "eat-me" signal (i.e., modified self) is an extracellular player specifically produced and presented on the surface of apoptotic cells, but not so produced and presented in healthy cells. It is crucial for the initiation of phagocytosis by activating phagocytic receptors and subsequently the signaling cascade. The eat-me signal requires extracellular trafficking to be presented on apoptotic cells. Specific categories of eat-me signals are provided by membrane-bound proteins such as phosphatidylserine (PtdSer) and calreticulin (CRT). Externalized PtdSer binds to receptors on phagocytic cells, facilitating the clearance of apoptotic cells (a process also known as efferocytosis). Similarly, CRT is upregulated on the surface of apoptotic cells and binds to LDL receptor-associated protein 1 (LRP1) on phagocytic cells, thereby mediating phagocytosis.

[0005] SIRPα is widely expressed on phagocytic cells (e.g., macrophages, granulocytes, and dendritic cells) and acts as an inhibitory receptor through interaction with the transmembrane protein CD47. This interaction mediates a response known as the "don't eat me" signaling pathway. This interaction negatively modulates the effector functions of innate immune cells, such as phagocytosis of host cells. Since CD47 is often present on tumor cells, this "don't eat me" signaling pathway is thought to contribute to tumor resistance to phagocytic clearance. Despite the similarity of the extracellular domains of SIRPα and SIRPβ1, functional differences exist among members of the SIRP family. For example, SIRPβ1 does not bind to CD47 at detectable levels and therefore does not mediate the "don't eat me" signaling pathway. Instead, SIRPβ1 is involved in the activation of myeloid cells.

[0006] Reportedly, disruption of CD47-SIRPα signaling (e.g., by antagonistic monoclonal antibodies that bind to either CD47 or SIRPα) results in enhanced phagocytosis of both solid and hematopoietic tumor cells, including increased phagocytosis of glioblastoma cells in vitro and significant antitumor activity in vivo. [Overview of the Initiative]

[0007] Summary of the Invention In a first embodiment, the present invention provides an anti-SIRPα antibody and its antigen-binding fragment comprising the structural and functional features specified below.

[0008] In various embodiments, the present invention provides an antibody or antigen-binding fragment that binds to human SIRPα comprising one, two, or all three of the following: (i), (ii), and (iii), namely (i) heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence different from SEQ ID NO:1 by one, two, three or more conservative substitutions; (ii) heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence different from SEQ ID NO:2 by one, two, three or more conservative substitutions; and / or (iii) heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence different from SEQ ID NO:3 by one, two, three or more conservative substitutions.

[0009] In various other embodiments, the present invention provides an antibody or antigen-binding fragment that binds to human SIRPα comprising one, two, or all three of the following: (i), (ii), and (iii), namely (i) heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence different from SEQ ID NO: 1 by one, two, three or more conservative substitutions; (ii) heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence different from SEQ ID NO: 2 by one, two, three or more conservative substitutions; and / or (iii) heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence different from SEQ ID NO: 3 by one, two, three or more conservative substitutions.

[0010] In a particular embodiment, the antibody or its antigen-binding fragment is SEQ ID NO: 75, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 78, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 80, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:82, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:84, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 86, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 88, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:102, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:7, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto. SEQ ID NO:10, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:12, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:14, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:16, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:18, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:30, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. It includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of the following.

[0011] In various embodiments, the present invention also provides an antibody or antigen-binding fragment that binds to human SIRPα comprising one, two, or all three of the following: (i), (ii), and (iii), namely (i) light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence different from SEQ ID NO:4 by one, two, three or more conservative substitutions; (ii) light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence different from SEQ ID NO:5 by one, two, three or more conservative substitutions; and / or (iii) light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence different from SEQ ID NO:6 by one, two, three or more conservative substitutions.

[0012] In various other embodiments, the present invention also provides an antibody or antigen-binding fragment that binds to human SIRPα comprising one, two or all three of the following: (i), (ii), and (iii), namely (i) light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence different from SEQ ID NO: 4 by one, two, three or more conservative substitutions; (ii) light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence different from SEQ ID NO: 5 by one, two, three or more conservative substitutions; and / or (iii) light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence different from SEQ ID NO: 6 by one, two, three or more conservative substitutions.

[0013] In a particular embodiment, the antibody or its antigen-binding fragment is SEQ ID NO:76, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 90, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:92, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:94, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:96, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 100, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:104, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to it. SEQ ID NO:8, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:20, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:22, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:24, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:26, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:28, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:32, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. It includes a light chain variable region containing an amino acid sequence selected from the group consisting of the following.

[0014] In various embodiments, the present invention (i) Heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO:1, or an amino acid sequence different from SEQ ID NO:1 due to 1, 2, 3 or more conservative substitutions; (ii) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO:2, or an amino acid sequence different from SEQ ID NO:2 due to 1, 2, 3 or more conservative substitutions; and / or (iii) Heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO:3, or an amino acid sequence different from SEQ ID NO:3 due to 1, 2, 3 or more conservative substitutions; and (iv) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO:4, or an amino acid sequence different from SEQ ID NO:4 due to 1, 2, 3 or more conservative substitutions; (v) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO:5, or an amino acid sequence different from SEQ ID NO:5 due to 1, 2, 3 or more conservative substitutions; and / or (vi) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO:6, or an amino acid sequence different from SEQ ID NO:6 due to 1, 2, 3 or more conservative substitutions. The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα containing [specific component].

[0015] In various other embodiments, the present invention is (i) Heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO:69, or an amino acid sequence different from SEQ ID NO:1 due to 1, 2, 3 or more conservative substitutions; (ii) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO:70, or an amino acid sequence different from SEQ ID NO:2 due to 1, 2, 3 or more conservative substitutions; and / or (iii) Heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO:71, or an amino acid sequence different from SEQ ID NO:3 due to 1, 2, 3 or more conservative substitutions; and (iv) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO:72, or an amino acid sequence different from SEQ ID NO:4 by 1, 2, 3 or more conservative substitutions; (v) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO:73, or an amino acid sequence different from SEQ ID NO:5 by 1, 2, 3 or more conservative substitutions; and / or (vi) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO:6, or an amino acid sequence different from SEQ ID NO:74 by 1, 2, 3 or more conservative substitutions. The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα containing [specific component].

[0016] In yet another embodiment, the present invention is SEQ ID NO:7, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto. SEQ ID NO:10, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:12, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:14, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:16, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:18, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:30, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. A heavy chain variable region containing an amino acid sequence selected from the group consisting of, SEQ ID NO:8, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:20, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:22, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:24, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:26, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:28, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:32, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. A light chain variable region containing an amino acid sequence selected from the group consisting of, The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα.

[0017] In yet another embodiment, the present invention is SEQ ID NO: 75, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 78, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 80, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:82, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:84, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 86, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:88, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:102, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. A heavy chain variable region containing an amino acid sequence selected from the group consisting of, SEQ ID NO:76, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 90, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:92, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:94, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:96, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:100, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:104, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to it. A light chain variable region containing an amino acid sequence selected from the group consisting of, The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα.

[0018] In the context of this specification, “sequence similarity” is based on the degree of identity tied to the degree of conservative transformations. The percentage of “sequence similarity” is the percentage of amino acids or nucleotides that are either identical or conservative transformations, i.e., “sequence similarity” = sequence identity% + conservative transformation%. Therefore, for the purposes of this invention, “conservative transformation” and “identity” are considered to be a broader category of the term “similarity.” Thus, whenever the term sequence “similarity” is used, it always encompasses sequence “identity” and “conservative transformation.” According to certain embodiments, conservative transformations are disregarded, and sequence similarity% refers to sequence identity%. In certain embodiments, all or nearly all transformations in a sequence permitted by the referenced sequence identity% are conservative transformations; i.e., if the sequences are 90% identical, the remaining 10% are all or nearly all conservative transformations. In the context of this specification, the term “nearly all” means that at least 75%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the permitted sequence transformations are conservative transformations. In certain embodiments of antibody heavy and / or light chains, the permitted sequence transformations are within the framework region and not within the CDR.

[0019] Preferably, the antibody has a heavy chain according to SEQ ID NO: 7. More preferably, the antibody has a light chain according to SEQ ID NO: 8. More preferably, the heavy chain is selected from SEQ ID NO: 10, 12, 14, 16, 18, or 30. More preferably, the light chain is selected from SEQ ID NO: 20, 22, 24, 26, 28, or 32.

[0020] Alternatively, the antibody has a heavy chain according to SEQ ID NO: 75. More preferably, the antibody has a light chain according to SEQ ID NO: 76. More preferably, the heavy chain is selected from SEQ ID NO: 78, 80, 82, 84, 86, 88, or 102. More preferably, the light chain is selected from SEQ ID NO: 90, 92, 94, 96, 98, 100, or 104.

[0021] In any of the above embodiments, the antibody or its antigen-binding fragment may be isolated as the term is defined herein.

[0022] In any of the embodiments described above, the antibody or its antigen-binding fragment is a recombinant antibody, as the term is defined herein.

[0023] In any of the embodiments described above, the antibody or its antigen-binding fragment is a full-length antibody, as the term is defined herein.

[0024] The antibodies or antigen-binding fragments of the present invention may be obtained from a variety of species. For example, the antibodies of the present invention may contain immunoglobulin sequences that are sequences from rabbits, mice, rats, guinea pigs, chickens, goats, sheep, donkeys, humans, llamas, or camels, or combinations thereof (so-called chimeric antibodies). Most preferably, the antibodies or antigen-binding fragments are human or humanized antibodies or antigen-binding fragments.

[0025] The term antibody refers to the antigen-binding portion, i.e., (i)VL 、V H 、C L and C H a Fab fragment, which is a monovalent fragment consisting of a V H and C H l domain; (ii) an F(ab’)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of a V L and C H l domain; (iv) an Fv fragment consisting of the V L and V H domains of one arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an "antigen-binding site" that retains the ability to bind to an antigen, including, for example, a fragment, a subsequence, a complementarity-determining region (CDR), etc. A single-chain antibody is also included by reference to the term "antibody". Preferred therapeutic antibodies are intact IgG antibodies. As used herein, the term "intact IgG" means a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3. Known Ig domains in the IgG class of antibodies are V H , Cγ1, Cγ2, Cγ3, V L and C L .

[0026] In any of the above embodiments, the antibody or its antigen-binding fragment is a human or humanized antibody comprising two heavy chains and two light chains. In one embodiment, the antibody is IgG. In a preferred embodiment, the antibody is IgG1, IgG2, or IgG4, preferably human IgG1, IgG2, or IgG4.

[0027] In any of the embodiments described above, the antibody or antigen-binding fragment of the present invention may comprise the light chain variable region, the human kappa or lambda light chain constant domain, and the IgG1, IgG2, or IgG4 heavy chain constant domain. Exemplary light chain (kappa) and heavy chain (IgG2 and IgG4) constant region sequences that can be used in the present invention are listed in SEQ ID NO: 63, 65, 67 (nucleotide sequences, respectively), 64, 66, and 68 (polypeptide sequences, respectively).

[0028] In various embodiments, such antibodies or their antigen-binding fragments may be the following combinations of heavy chain sequences / light chain variable region sequences: SEQ ID NO:10 / SEQ ID NO:20 (referred to as hSIRPα.50A.H1L1 in this specification) SEQ ID NO:10 / SEQ ID NO:22 (referred to as hSIRPα.50A.H1L2 in this specification) SEQ ID NO:10 / SEQ ID NO:24 (referred to as hSIRPα.50A.H1L3 in this specification) SEQ ID NO:10 / SEQ ID NO:26 (referred to as hSIRPα.50A.H1L4 in this specification) SEQ ID NO:10 / SEQ ID NO:28 (referred to as hSIRPα.50A.H1L5 in this specification) SEQ ID NO:12 / SEQ ID NO:20 (referred to as hSIRPα.50A.H2L1 in this specification) SEQ ID NO:12 / SEQ ID NO:22 (referred to as hSIRPα.50A.H2L2 in this specification) SEQ ID NO:12 / SEQ ID NO:24 (referred to as hSIRPα.50A.H2L3 in this specification) SEQ ID NO:12 / SEQ ID NO:26 (referred to as hSIRPα.50A.H2L4 in this specification) SEQ ID NO:12 / SEQ ID NO:28 (referred to as hSIRPα.50A.H2L5 in this specification) SEQ ID NO:14 / SEQ ID NO:20 (referred to as hSIRPα.50A.H3L1 in this specification) SEQ ID NO:14 / SEQ ID NO:22 (referred to as hSIRPα.50A.H3L2 in this specification) SEQ ID NO:14 / SEQ ID NO:24 (referred to as hSIRPα.50A.H3L3 in this specification) SEQ ID NO:14 / SEQ ID NO:26 (referred to as hSIRPα.50A.H3L4 in this specification) SEQ ID NO:14 / SEQ ID NO:28 (referred to as hSIRPα.50A.H3L5 in this specification) SEQ ID NO:16 / SEQ ID NO:20 (referred to as hSIRPα.50A.H4L1 in this specification) SEQ ID NO:16 / SEQ ID NO:22 (referred to as hSIRPα.50A.H4L2 in this specification) SEQ ID NO:16 / SEQ ID NO:24 (referred to as hSIRPα.50A.H4L3 in this specification) SEQ ID NO:16 / SEQ ID NO:26 (referred to as hSIRPα.50A.H4L4 in this specification) SEQ ID NO:16 / SEQ ID NO:28 (referred to as hSIRPα.50A.H4L5 in this specification) SEQ ID NO:18 / SEQ ID NO:20 (referred to as hSIRPα.50A.H5L1 in this specification) SEQ ID NO:18 / SEQ ID NO:22 (referred to as hSIRPα.50A.H5L2 in this specification) SEQ ID NO:18 / SEQ ID NO:24 (referred to as hSIRPα.50A.H5L3 in this specification) SEQ ID NO:18 / SEQ ID NO:26 (referred to as hSIRPα.50A.H5L4 in this specification) SEQ ID NO:18 / SEQ ID NO:28 (referred to as hSIRPα.50A.H5L5 in this specification) SEQ ID NO:78 / SEQ ID NO:90 (referred to as hSIRPα.40A.H1L1 in this specification) SEQ ID NO:78 / SEQ ID NO:92 (referred to as hSIRPα.40A.H1L2 in this specification) SEQ ID NO:78 / SEQ ID NO:94 (referred to as hSIRPα.40A.H1L3 in this specification) SEQ ID NO:78 / SEQ ID NO:96 (referred to as hSIRPα.40A.H1L4 in this specification) SEQ ID NO:78 / SEQ ID NO:98 (referred to as hSIRPα.40A.H1L5 in this specification) SEQ ID NO:78 / SEQ ID NO:100 (referred to as hSIRPα.40A.H1L6 in this specification) SEQ ID NO:80 / SEQ ID NO:90 (referred to as hSIRPα.40A.H2L1 in this specification) SEQ ID NO:80 / SEQ ID NO:92 (referred to as hSIRPα.40A.H2L2 in this specification) SEQ ID NO:80 / SEQ ID NO:94 (referred to as hSIRPα.40A.H2L3 in this specification) SEQ ID NO:80 / SEQ ID NO:96 (referred to as hSIRPα.40A.H2L4 in this specification) SEQ ID NO:80 / SEQ ID NO:98 (referred to as hSIRPα.40A.H2L5 in this specification) SEQ ID NO:80 / SEQ ID NO:100 (referred to as hSIRPα.40A.H2L6 in this specification) SEQ ID NO:82 / SEQ ID NO:90 (referred to as hSIRPα.40A.H3L1 in this specification) SEQ ID NO:82 / SEQ ID NO:92 (referred to as hSIRPα.40A.H3L2 in this specification) SEQ ID NO:82 / SEQ ID NO:94 (referred to as hSIRPα.40A.H3L3 in this specification) SEQ ID NO:82 / SEQ ID NO:96 (referred to as hSIRPα.40A.H3L4 in this specification) SEQ ID NO:82 / SEQ ID NO:98 (referred to as hSIRPα.40A.H3L5 in this specification) SEQ ID NO:82 / SEQ ID NO:100 (referred to as hSIRPα.40A.H3L6 in this specification) SEQ ID NO:84 / SEQ ID NO:90 (referred to as hSIRPα.40A.H4L1 in this specification) SEQ ID NO:84 / SEQ ID NO:92 (referred to as hSIRPα.40A.H4L2 in this specification) SEQ ID NO:84 / SEQ ID NO:94 (referred to as hSIRPα.40A.H4L3 in this specification) SEQ ID NO:84 / SEQ ID NO:96 (referred to as hSIRPα.40A.H4L4 in this specification) SEQ ID NO:84 / SEQ ID NO:98 (referred to as hSIRPα.40A.H4L5 in this specification) SEQ ID NO:84 / SEQ ID NO:100 (referred to as hSIRPα.40A.H4L6 in this specification) SEQ ID NO:86 / SEQ ID NO:90 (referred to as hSIRPα.40A.H5L1 in this specification) SEQ ID NO:86 / SEQ ID NO:92 (referred to as hSIRPα.40A.H5L2 in this specification) SEQ ID NO:86 / SEQ ID NO:94 (referred to as hSIRPα.40A.H5L3 in this specification) SEQ ID NO:86 / SEQ ID NO:96 (referred to as hSIRPα.40A.H5L4 in this specification) SEQ ID NO:86 / SEQ ID NO:98 (referred to as hSIRPα.40A.H5L5 in this specification) SEQ ID NO:86 / SEQ ID NO:100 (referred to as hSIRPα.40A.H5L6 in this specification) SEQ ID NO:88 / SEQ ID NO:90 (referred to as hSIRPα.40A.H6L1 in this specification) SEQ ID NO:88 / SEQ ID NO:92 (referred to as hSIRPα.40A.H6L2 in this specification) SEQ ID NO:88 / SEQ ID NO:94 (referred to as hSIRPα.40A.H6L3 in this specification) SEQ ID NO:88 / SEQ ID NO:96 (referred to as hSIRPα.40A.H6L4 in this specification) SEQ ID NO:88 / SEQ ID NO:98 (referred to as hSIRPα.40A.H6L5 in this specification) SEQ ID NO:88 / SEQ ID NO:100 (referred to as hSIRPα.40A.H6L6 in this specification) Or, in each example, each SEQ ID NO includes at least one of the following: 90%, 95%, 97%, 98%, or 99% similar or identical.

[0029] In some preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, each heavy chain comprising SEQ ID NO:10 and each light chain comprising SEQ ID NO:20, or in each example comprising at least 90%, 95%, 97%, 98%, or 99% similar to or identical to each SEQ ID NO, most preferably each light chain comprising a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprising a human IgG1, IgG2, or IgG4 constant region.

[0030] In other preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, each heavy chain comprising SEQ ID NO:16 and each light chain comprising SEQ ID NO:28, or in each example comprising at least 90%, 95%, 97%, 98%, or 99% similar to or identical to each SEQ ID NO, most preferably each light chain comprising a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprising a human IgG1, IgG2, or IgG4 constant region.

[0031] In yet another preferred embodiment, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, each heavy chain comprising SEQ ID NO:18 and each light chain comprising SEQ ID NO:20, or in each example comprising at least 90%, 95%, 97%, 98%, or 99% similar to or identical to each SEQ ID NO, most preferably each light chain comprising a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprising a human IgG1, IgG2, or IgG4 constant region.

[0032] In some preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 80 and each light chain comprising SEQ ID NO: 90, or in each example comprising at least 90%, 95%, 97%, 98%, or 99% similar to or identical to each SEQ ID NO, most preferably each light chain comprising a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprising a human IgG1, IgG2, or IgG4 constant region.

[0033] In some preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 80 and each light chain comprising SEQ ID NO: 92, or in each example comprising at least 90%, 95%, 97%, 98%, or 99% similar to or identical to each SEQ ID NO, most preferably each light chain comprising a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprising a human IgG1, IgG2, or IgG4 constant region.

[0034] In some preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 80 and each light chain comprising SEQ ID NO: 96, or in each example comprising at least 90%, 95%, 97%, 98%, or 99% similar to or identical to each SEQ ID NO, most preferably each light chain comprising a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprising a human IgG1, IgG2, or IgG4 constant region.

[0035] In one embodiment, the anti-SIRPα antibody of the present invention comprises a full-length antibody structure having two light chains and two heavy chains as previously listed, each light chain comprising a human kappa light chain or human lambda light chain constant domain, and each heavy chain comprising a human IgG1 constant region.

[0036] In one embodiment, the anti-SIRPα antibody of the present invention comprises a full-length antibody structure having two light chains and two heavy chains as previously listed, each light chain comprising a human kappa light chain or human lambda light chain constant domain, and each heavy chain comprising a human IgG2 constant region.

[0037] In one embodiment, the anti-SIRPα antibody of the present invention comprises a full-length antibody structure having two light chains and two heavy chains as previously listed, each light chain comprising a human kappa light chain or human lambda light chain constant domain, and each heavy chain comprising a human IgG4 constant region.

[0038] In certain embodiments, the antibody or antigen-binding fragment of the present invention has one, two, three, four or more of the following functional features, preferably each of the following: EC 50 <1nM human SIRPαV1 protein with sequence SEQ ID NO:34; at least 100 times higher EC than SIRPαV1 (P74A) with sequence SEQ ID NO:62. 50 This indicates that, in some cases, human SIRPβ1 protein with sequence SEQ ID NO:38 also has at least 100 times higher EC. 50This shows (in each example, the EC decreased) 50 This refers to the EC of human SIRPαV1 protein having sequence SEQ ID NO:34. 50 This is the case for which the measurement was preferably performed by cell ELISA (CELISA) as described later in this specification; <10nM, preferably <5nM, more preferably <1.5nM, even more preferably <1.0nM, even more preferably <0.5nM, most preferably about 0.3nM or less EC 50 It binds to cells expressing the human SIRPαV1 protein; <10nM, preferably <5nM, more preferably <1.5nM, even more preferably <1.0nM, even more preferably <0.5nM, most preferably about 0.3nM or less EC 50 It binds to cells expressing the human SIRPαV2 protein; Antibody concentration of 50 nM, preferably 67 nM, more preferably 100 nM, or antibody EC of SIRPαV1 or SIRPαV2 50 It does not bind to the SIRPβ1 protein in a sense at concentrations 10 times greater than, preferably 50 times greater, more preferably 100 times greater, and even more preferably 200 times greater than; ICs with a molecular weight of <10.0nM, more preferably <5.0nM, even more preferably <2.5nM, and most preferably about 1.0nM or less. 50 And it inhibits the binding of human SIRPα to CD47; and It exhibits a T20 "humanity" score of at least 79, more preferably 85.

[0039] Preferably, the anti-SIRPα antibody or antigen-binding fragment of the present invention binds to cells expressing human SIRPαV1 protein at an EC50 < 10 nM, while at an antibody concentration of 100 nM, or to SIRPαV1 or SIRPαV2 antibody EC50 < 10 nM. 50At antibody concentrations 200 times greater than that, the antibodies do not bind enough to sense one or both of the SIRPαV1(P74A) and SIRPβ1 proteins. Most preferably, each light chain contains a human kappa light chain or a human lambda light chain constant domain, and each heavy chain contains a human IgG1, IgG2, or IgG4 constant region.

[0040] In certain embodiments, the anti-SIRPα antibody or its antigen-binding fragment of the present invention can be conjugated to at least one therapeutic agent. In one embodiment, the therapeutic agent is a second antibody or fragment thereof, an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a radionuclide, or a second antibody conjugated to at least one immunomodulator, an enzyme, a radiolabeled agent, a hormone, an antisense oligonucleotide, or a cytotoxic agent, or a combination thereof.

[0041] The present invention also provides isolated polypeptides comprising any one amino acid sequence from SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 76, 90, 92, 94, 96, 98, 100, 102, 104, 7, 10, 12, 14, 16, 18, 30, 8, 20, 22, 24, 26, 28, and 32, or any one of any fragments of said sequence, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to them.

[0042] The present invention also provides isolated nucleic acids encoding any one of the anti-SIRPα antibodies or antigen-binding fragments of the present invention.

[0043] In one embodiment, the present invention is SEQ ID NO: 75, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 78, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 80, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:82, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:84, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 86, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:88, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:102, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:10, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:12, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:14, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:16, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:18, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:30, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. This provides isolated nucleic acids encoding amino acid sequences selected from the group consisting of [specific amino acids].

[0044] In certain embodiments, the amino acid sequence of SEQ ID NO:10, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:9, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0045] In certain embodiments, the amino acid sequence of SEQ ID NO:12, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0046] In certain embodiments, the amino acid sequence of SEQ ID NO:14, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:13, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0047] In certain embodiments, the amino acid sequence of SEQ ID NO:16, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:15, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0048] In certain embodiments, the amino acid sequence of SEQ ID NO:18, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:17, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0049] In certain embodiments, the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:29, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0050] In certain embodiments, the amino acid sequence of SEQ ID NO:78, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:77, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0051] In certain embodiments, the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:79, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0052] In certain embodiments, the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:81, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0053] In certain embodiments, the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:83, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0054] In certain embodiments, the amino acid sequence of SEQ ID NO:86, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:85, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0055] In certain embodiments, the amino acid sequence of SEQ ID NO:88, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0056] In certain embodiments, the amino acid sequence of SEQ ID NO:102, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:101, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0057] In one embodiment, the present invention is SEQ ID NO:76, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 90, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:92, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:94, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:96, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO: 100, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:104, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:8, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:20, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:22, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:24, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:26, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. SEQ ID NO:28, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it, and SEQ ID NO:32, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical to it. This provides isolated nucleic acids encoding amino acid sequences selected from the group consisting of [specific amino acids].

[0058] In certain embodiments, the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:19, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0059] In certain embodiments, the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:21, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0060] In certain embodiments, the amino acid sequence of SEQ ID NO:24, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:23, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0061] In certain embodiments, the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:25, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0062] In certain embodiments, the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:27, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0063] In certain embodiments, the amino acid sequence of SEQ ID NO:32, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:31, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0064] In certain embodiments, the amino acid sequence of SEQ ID NO:90, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:89, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0065] In certain embodiments, the amino acid sequence of SEQ ID NO:92, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:91, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0066] In certain embodiments, the amino acid sequence of SEQ ID NO:94, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:93, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0067] In certain embodiments, the amino acid sequence of SEQ ID NO:96, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:95, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0068] In certain embodiments, the amino acid sequence of SEQ ID NO:98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:97, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0069] In certain embodiments, the amino acid sequence of SEQ ID NO:100, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:99, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0070] In certain embodiments, the amino acid sequence of SEQ ID NO:104, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, is encoded by the nucleic acid sequence of SEQ ID NO:103, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0071] In certain embodiments, the isolated nucleic acid of the present invention may optionally include a leader sequence.

[0072] Such nucleic acids may contain one or more of the following nucleic acid sequences: The nucleic acid sequence of SEQ ID NO:77, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:79, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:81, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:83, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:85, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:101, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:89, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:91, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:93, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:95, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:97, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. Nucleic acid sequence of SEQ ID NO:99, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:101, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:103, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:9, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:13, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:15, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:17, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:29, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:19, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:21, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:23, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:25, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:27, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto, and / or The nucleic acid sequence of SEQ ID NO:31, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0073] In certain embodiments, the nucleic acid can encode a human or humanized antibody and includes both heavy and light chain nucleic acid sequences. In one embodiment, the antibody is IgG. In preferred embodiments, the antibody is IgG1, IgG2, or IgG4, preferably human IgG1, IgG2, or IgG4. In certain embodiments, the light chain sequence includes a human kappa light chain or human lambda light chain constant domain sequence, and each heavy chain sequence includes a human IgG1, IgG2, or IgG4 constant region sequence.

[0074] Preferably, such nucleic acids are combinations of the following heavy chain and light chain variable region nucleic acid sequences: SEQ ID NO:9 / SEQ ID NO:19 (referred to as hSIRPα.50A.H1L1 in this specification) SEQ ID NO:9 / SEQ ID NO:21 (referred to as hSIRPα.50A.H1L2 in this specification) SEQ ID NO:9 / SEQ ID NO:23 (referred to as hSIRPα.50A.H1L3 in this specification) SEQ ID NO:9 / SEQ ID NO:25 (referred to as hSIRPα.50A.H1L4 in this specification) SEQ ID NO:9 / SEQ ID NO:27 (referred to as hSIRPα.50A.H1L5 in this specification) SEQ ID NO:11 / SEQ ID NO:19 (referred to as hSIRPα.50A.H2L1 in this specification) SEQ ID NO:11 / SEQ ID NO:21 (referred to as hSIRPα.50A.H2L2 in this specification) SEQ ID NO:11 / SEQ ID NO:23 (referred to as hSIRPα.50A.H2L3 in this specification) SEQ ID NO:11 / SEQ ID NO:25 (referred to as hSIRPα.50A.H2L4 in this specification) SEQ ID NO:11 / SEQ ID NO:27 (referred to as hSIRPα.50A.H2L5 in this specification) SEQ ID NO:13 / SEQ ID NO:19 (referred to as hSIRPα.50A.H3L1 in this specification) SEQ ID NO:13 / SEQ ID NO:21 (referred to as hSIRPα.50A.H3L2 in this specification) SEQ ID NO:13 / SEQ ID NO:23 (referred to as hSIRPα.50A.H3L3 in this specification) SEQ ID NO:13 / SEQ ID NO:25 (referred to as hSIRPα.50A.H3L4 in this specification) SEQ ID NO:13 / SEQ ID NO:27 (referred to as hSIRPα.50A.H3L5 in this specification) SEQ ID NO:15 / SEQ ID NO:19 (referred to as hSIRPα.50A.H4L1 in this specification) SEQ ID NO:15 / SEQ ID NO:21 (referred to as hSIRPα.50A.H4L2 in this specification) SEQ ID NO:15 / SEQ ID NO:23 (referred to as hSIRPα.50A.H4L3 in this specification) SEQ ID NO:15 / SEQ ID NO:25 (referred to as hSIRPα.50A.H4L4 in this specification) SEQ ID NO:15 / SEQ ID NO:27 (referred to as hSIRPα.50A.H4L5 in this specification) SEQ ID NO:17 / SEQ ID NO:19 (referred to as hSIRPα.50A.H5L1 in this specification) SEQ ID NO:17 / SEQ ID NO:21 (referred to as hSIRPα.50A.H5L2 in this specification) SEQ ID NO:17 / SEQ ID NO:23 (referred to as hSIRPα.50A.H5L3 in this specification) SEQ ID NO:17 / SEQ ID NO:25 (referred to as hSIRPα.50A.H5L4 in this specification) SEQ ID NO:17 / SEQ ID NO:27 (referred to as hSIRPα.50A.H5L5 in this specification) SEQ ID NO:77 / SEQ ID NO:89 (referred to as hSIRPα.40A.H1L1 in this specification) SEQ ID NO:77 / SEQ ID NO:91 (referred to as hSIRPα.40A.H1L2 in this specification) SEQ ID NO:77 / SEQ ID NO:93 (referred to as hSIRPα.40A.H1L3 in this specification) SEQ ID NO:77 / SEQ ID NO:95 (referred to as hSIRPα.40A.H1L4 in this specification) SEQ ID NO:77 / SEQ ID NO:97 (referred to as hSIRPα.40A.H1L5 in this specification) SEQ ID NO:77 / SEQ ID NO:99 (referred to as hSIRPα.40A.H1L6 in this specification) SEQ ID NO:79 / SEQ ID NO:89 (referred to as hSIRPα.40A.H2L1 in this specification) SEQ ID NO:79 / SEQ ID NO:91 (referred to as hSIRPα.40A.H2L2 in this specification) SEQ ID NO:79 / SEQ ID NO:93 (referred to as hSIRPα.40A.H2L3 in this specification) SEQ ID NO:79 / SEQ ID NO:95 (referred to as hSIRPα.40A.H2L4 in this specification) SEQ ID NO:79 / SEQ ID NO:97 (referred to as hSIRPα.40A.H2L5 in this specification) SEQ ID NO:79 / SEQ ID NO:99 (referred to as hSIRPα.40A.H2L6 in this specification) SEQ ID NO:81 / SEQ ID NO:89 (referred to as hSIRPα.40A.H3L1 in this specification) SEQ ID NO:81 / SEQ ID NO:91 (referred to as hSIRPα.40A.H3L2 in this specification) SEQ ID NO:81 / SEQ ID NO:93 (referred to as hSIRPα.40A.H3L3 in this specification) SEQ ID NO:81 / SEQ ID NO:95 (referred to as hSIRPα.40A.H3L4 in this specification) SEQ ID NO:81 / SEQ ID NO:97 (referred to as hSIRPα.40A.H3L5 in this specification) SEQ ID NO:81 / SEQ ID NO:99 (referred to as hSIRPα.40A.H3L6 in this specification) SEQ ID NO:83 / SEQ ID NO:89 (referred to as hSIRPα.40A.H4L1 in this specification) SEQ ID NO:83 / SEQ ID NO:91 (referred to as hSIRPα.40A.H4L2 in this specification) SEQ ID NO:83 / SEQ ID NO:93 (referred to as hSIRPα.40A.H4L3 in this specification) SEQ ID NO:83 / SEQ ID NO:95 (referred to as hSIRPα.40A.H4L4 in this specification) SEQ ID NO:83 / SEQ ID NO:97 (referred to as hSIRPα.40A.H4L5 in this specification) SEQ ID NO:83 / SEQ ID NO:99 (referred to as hSIRPα.40A.H4L6 in this specification) SEQ ID NO:85 / SEQ ID NO:89 (referred to as hSIRPα.40A.H5L1 in this specification) SEQ ID NO:85 / SEQ ID NO:91 (referred to as hSIRPα.40A.H5L2 in this specification) SEQ ID NO:85 / SEQ ID NO:93 (referred to as hSIRPα.40A.H5L3 in this specification) SEQ ID NO:85 / SEQ ID NO:95 (referred to as hSIRPα.40A.H5L4 in this specification) SEQ ID NO:85 / SEQ ID NO:97 (referred to as hSIRPα.40A.H5L5 in this specification) SEQ ID NO:85 / SEQ ID NO:99 (referred to as hSIRPα.40A.H5L6 in this specification) SEQ ID NO:87 / SEQ ID NO:89 (referred to as hSIRPα.40A.H6L1 in this specification) SEQ ID NO:87 / SEQ ID NO:91 (referred to as hSIRPα.40A.H6L2 in this specification) SEQ ID NO:87 / SEQ ID NO:93 (referred to as hSIRPα.40A.H6L3 in this specification) SEQ ID NO:87 / SEQ ID NO:95 (referred to as hSIRPα.40A.H6L4 in this specification) SEQ ID NO:87 / SEQ ID NO:97 (referred to as hSIRPα.40A.H6L5 in this specification) SEQ ID NO:87 / SEQ ID NO:99 (referred to as hSIRPα.40A.H6L6 in this specification) Alternatively, each example may include at least 90%, 95%, 97%, 98%, or 99% identical SEQ ID NO.

[0075] In some preferred embodiments, the nucleic acid includes SEQ ID NO:9 and SEQ ID NO:19, or in each example includes at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO.

[0076] In some preferred embodiments, the nucleic acid includes SEQ ID NO:15 and SEQ ID NO:27, or, in each example, includes at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO.

[0077] In some preferred embodiments, the nucleic acid includes SEQ ID NO:17 and SEQ ID NO:19, or, in each example, includes one that is at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO.

[0078] In some preferred embodiments, the nucleic acid includes SEQ ID NO:79 and SEQ ID NO:89, or, in each example, includes one that is at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO.

[0079] In some preferred embodiments, the nucleic acid includes SEQ ID NO:79 and SEQ ID NO:91, or in each example includes at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO.

[0080] In some preferred embodiments, the nucleic acid includes SEQ ID NO:79 and SEQ ID NO:95, or in each example includes at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO.

[0081] The present invention also provides an expression vector comprising one or more nucleic acids of the present invention. An expression vector is a DNA molecule containing regulatory elements necessary for the transcription of a target nucleic acid within a host cell. Typically, the target nucleic acid is positioned under the control of specific regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancer elements. Such a target nucleic acid is said to be "operably ligated to" the regulatory elements if the regulatory elements control gene expression.

[0082] These isolated nucleic acids and expression vectors containing them may be used to express the antibodies or antigen-binding fragments of the present invention in recombinant host cells. Thus, the present invention also provides host cells containing the expression vectors of the present invention.

[0083] Such an expression vector may contain one or more of the following nucleic acid sequences operably ligated to a regulatory element: The nucleic acid sequence of SEQ ID NO:77, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:79, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:81, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:83, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:85, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:101, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:89, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:91, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:93, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:95, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:97, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. Nucleic acid sequence of SEQ ID NO:99, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:103, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:13, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:15, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:17, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:29, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:19, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:21, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:23, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:25, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. The nucleic acid sequence of SEQ ID NO:27, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto, and / or The nucleic acid sequence of SEQ ID NO:31, or a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0084] In certain embodiments, the expression vector comprises nucleic acid sequences encoding both the heavy chain and light chain sequences of the anti-SIRPα antibody of the present invention. Preferably, such an expression vector is a combination of the following heavy chain and light chain variable region nucleic acid sequences: SEQ ID NO:9 / SEQ ID NO:19 (referred to as hSIRPα.50A.H1L1 in this specification) SEQ ID NO:9 / SEQ ID NO:21 (referred to as hSIRPα.50A.H1L2 in this specification) SEQ ID NO:9 / SEQ ID NO:23 (referred to as hSIRPα.50A.H1L3 in this specification) SEQ ID NO:9 / SEQ ID NO:25 (referred to as hSIRPα.50A.H1L4 in this specification) SEQ ID NO:9 / SEQ ID NO:27 (referred to as hSIRPα.50A.H1L5 in this specification) SEQ ID NO:11 / SEQ ID NO:19 (referred to as hSIRPα.50A.H2L1 in this specification) SEQ ID NO:11 / SEQ ID NO:21 (referred to as hSIRPα.50A.H2L2 in this specification) SEQ ID NO:11 / SEQ ID NO:23 (referred to as hSIRPα.50A.H2L3 in this specification) SEQ ID NO:11 / SEQ ID NO:25 (referred to as hSIRPα.50A.H2L4 in this specification) SEQ ID NO:11 / SEQ ID NO:27 (referred to as hSIRPα.50A.H2L5 in this specification) SEQ ID NO:13 / SEQ ID NO:19 (referred to as hSIRPα.50A.H3L1 in this specification) SEQ ID NO:13 / SEQ ID NO:21 (referred to as hSIRPα.50A.H3L2 in this specification) SEQ ID NO:13 / SEQ ID NO:23 (referred to as hSIRPα.50A.H3L3 in this specification) SEQ ID NO:13 / SEQ ID NO:25 (referred to as hSIRPα.50A.H3L4 in this specification) SEQ ID NO:13 / SEQ ID NO:27 (referred to as hSIRPα.50A.H3L5 in this specification) SEQ ID NO:15 / SEQ ID NO:19 (referred to as hSIRPα.50A.H4L1 in this specification) SEQ ID NO:15 / SEQ ID NO:21 (referred to as hSIRPα.50A.H4L2 in this specification) SEQ ID NO:15 / SEQ ID NO:23 (referred to as hSIRPα.50A.H4L3 in this specification) SEQ ID NO:15 / SEQ ID NO:25 (referred to as hSIRPα.50A.H4L4 in this specification) SEQ ID NO:15 / SEQ ID NO:27 (referred to as hSIRPα.50A.H4L5 in this specification) SEQ ID NO:17 / SEQ ID NO:19 (referred to as hSIRPα.50A.H5L1 in this specification) SEQ ID NO:17 / SEQ ID NO:21 (referred to as hSIRPα.50A.H5L2 in this specification) SEQ ID NO:17 / SEQ ID NO:23 (referred to as hSIRPα.50A.H5L3 in this specification) SEQ ID NO:17 / SEQ ID NO:25 (referred to as hSIRPα.50A.H5L4 in this specification) SEQ ID NO:17 / SEQ ID NO:27 (referred to as hSIRPα.50A.H5L5 in this specification) SEQ ID NO:77 / SEQ ID NO:89 (referred to as hSIRPα.40A.H1L1 in this specification) SEQ ID NO:77 / SEQ ID NO:91 (referred to as hSIRPα.40A.H1L2 in this specification) SEQ ID NO:77 / SEQ ID NO:93 (referred to as hSIRPα.40A.H1L3 in this specification) SEQ ID NO:77 / SEQ ID NO:95 (referred to as hSIRPα.40A.H1L4 in this specification) SEQ ID NO:77 / SEQ ID NO:97 (referred to as hSIRPα.40A.H1L5 in this specification) SEQ ID NO:77 / SEQ ID NO:99 (referred to as hSIRPα.40A.H1L6 in this specification) SEQ ID NO:79 / SEQ ID NO:89 (referred to as hSIRPα.40A.H2L1 in this specification) SEQ ID NO:79 / SEQ ID NO:91 (referred to as hSIRPα.40A.H2L2 in this specification) SEQ ID NO:79 / SEQ ID NO:93 (referred to as hSIRPα.40A.H2L3 in this specification) SEQ ID NO:79 / SEQ ID NO:95 (referred to as hSIRPα.40A.H2L4 in this specification) SEQ ID NO:79 / SEQ ID NO:97 (referred to as hSIRPα.40A.H2L5 in this specification) SEQ ID NO:79 / SEQ ID NO:99 (referred to as hSIRPα.40A.H2L6 in this specification) SEQ ID NO:81 / SEQ ID NO:89 (referred to as hSIRPα.40A.H3L1 in this specification) SEQ ID NO:81 / SEQ ID NO:91 (referred to as hSIRPα.40A.H3L2 in this specification) SEQ ID NO:81 / SEQ ID NO:93 (referred to as hSIRPα.40A.H3L3 in this specification) SEQ ID NO:81 / SEQ ID NO:95 (referred to as hSIRPα.40A.H3L4 in this specification) SEQ ID NO:81 / SEQ ID NO:97 (referred to as hSIRPα.40A.H3L5 in this specification) SEQ ID NO:81 / SEQ ID NO:99 (referred to as hSIRPα.40A.H3L6 in this specification) SEQ ID NO:83 / SEQ ID NO:89 (referred to as hSIRPα.40A.H4L1 in this specification) SEQ ID NO:83 / SEQ ID NO:91 (referred to as hSIRPα.40A.H4L2 in this specification) SEQ ID NO:83 / SEQ ID NO:93 (referred to as hSIRPα.40A.H4L3 in this specification) SEQ ID NO:83 / SEQ ID NO:95 (referred to as hSIRPα.40A.H4L4 in this specification) SEQ ID NO:83 / SEQ ID NO:97 (referred to as hSIRPα.40A.H4L5 in this specification) SEQ ID NO:83 / SEQ ID NO:99 (referred to as hSIRPα.40A.H4L6 in this specification) SEQ ID NO:85 / SEQ ID NO:89 (referred to as hSIRPα.40A.H5L1 in this specification) SEQ ID NO:85 / SEQ ID NO:91 (referred to as hSIRPα.40A.H5L2 in this specification) SEQ ID NO:85 / SEQ ID NO:93 (referred to as hSIRPα.40A.H5L3 in this specification) SEQ ID NO:85 / SEQ ID NO:95 (referred to as hSIRPα.40A.H5L4 in this specification) SEQ ID NO:85 / SEQ ID NO:97 (referred to as hSIRPα.40A.H5L5 in this specification) SEQ ID NO:85 / SEQ ID NO:99 (referred to as hSIRPα.40A.H5L6 in this specification) SEQ ID NO:87 / SEQ ID NO:89 (referred to as hSIRPα.40A.H6L1 in this specification) SEQ ID NO:87 / SEQ ID NO:91 (referred to as hSIRPα.40A.H6L2 in this specification) SEQ ID NO:87 / SEQ ID NO:93 (referred to as hSIRPα.40A.H6L3 in this specification) SEQ ID NO:87 / SEQ ID NO:95 (referred to as hSIRPα.40A.H6L4 in this specification) SEQ ID NO:87 / SEQ ID NO:97 (referred to as hSIRPα.40A.H6L5 in this specification) SEQ ID NO:87 / SEQ ID NO:99 (referred to as hSIRPα.40A.H6L6 in this specification) Alternatively, each example may include at least 90%, 95%, 97%, 98%, or 99% identical SEQ ID NO.

[0085] In any of the embodiments described above, the expression vector may encode a human or humanized antibody for expression and may include both heavy and light chain nucleic acid sequences. In one embodiment, the antibody is IgG. In a preferred embodiment, the antibody is IgG1, IgG2, or IgG4, preferably human IgG1, IgG2, or IgG4. In a particular embodiment, the light chain sequence includes a human kappa light chain or human lambda light chain constant domain sequence, and each heavy chain sequence includes a human IgG4 constant region sequence.

[0086] In some preferred embodiments, the expression vector encodes a human or humanized antibody for expression, the heavy chain nucleic acid sequence comprising SEQ ID NO:9, and the light chain nucleic acid sequence comprising SEQ ID NO:19, or, in each example, comprising at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO, most preferably an IgG1, IgG2, or IgG4 isotype.

[0087] In some preferred embodiments, the expression vector encodes a human or humanized antibody for expression, the heavy chain nucleic acid sequence comprising SEQ ID NO: 15, and the light chain nucleic acid sequence comprising SEQ ID NO: 27, or, in each example, comprising at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO, most preferably an IgG1, IgG2, or IgG4 isotype.

[0088] In some preferred embodiments, the expression vector encodes a human or humanized antibody for expression, the heavy chain nucleic acid sequence comprising SEQ ID NO:17, and the light chain nucleic acid sequence comprising SEQ ID NO:19, or, in each example, comprising at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO, most preferably an IgG1, IgG2, or IgG4 isotype.

[0089] In some preferred embodiments, the expression vector encodes a human or humanized antibody for expression, the heavy chain nucleic acid sequence comprising SEQ ID NO:79, and the light chain nucleic acid sequence comprising SEQ ID NO:89, or, in each example, comprising at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO, most preferably an IgG1, IgG2, or IgG4 isotype.

[0090] In some preferred embodiments, the expression vector encodes a human or humanized antibody for expression, the heavy chain nucleic acid sequence comprising SEQ ID NO: 79, and the light chain nucleic acid sequence comprising SEQ ID NO: 91, or, in each example, comprising at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO, most preferably an IgG1, IgG2, or IgG4 isotype.

[0091] In some preferred embodiments, the expression vector encodes a human or humanized antibody for expression, the heavy chain nucleic acid sequence comprising SEQ ID NO: 79, and the light chain nucleic acid sequence comprising SEQ ID NO: 95, or, in each example, comprising at least 90%, 95%, 97%, 98%, or 99% identical to each SEQ ID NO, most preferably an IgG1, IgG2, or IgG4 isotype.

[0092] In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In another embodiment, the host cell is a mammalian cell (e.g., a human cell such as HEK293 cells, a hamster cell such as CHO cells, etc.), a bacterial cell (e.g., an E. coli cell), a yeast cell (e.g., a Pichia pastris cell, etc.), a plant cell (e.g., a Nicotiana benthamiana cell, etc.). Mammalian cells are preferred due to their most suitable glycosylation pattern.

[0093] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier or diluent.

[0094] In one embodiment, the composition comprises one or more further therapeutic agents. In one embodiment, the further therapeutic agents include an anti-CD27 antibody or its antigen-binding fragment; an anti-LAG3 antibody or its antigen-binding fragment; an anti-APRIL antibody or its antigen-binding fragment; an anti-TIGIT antibody or its antigen-binding fragment; an anti-VISTA antibody or its antigen-binding fragment; an anti-BTLA antibody or its antigen-binding fragment; an anti-TIM3 antibody or its antigen-binding fragment; an anti-CTLA4 antibody or its antigen-binding fragment; an anti-HVEM antibody or its antigen-binding fragment; an anti-CD70 antibody or its antigen-binding fragment; an anti-CD137 antibody or its antigen-binding fragment; and an anti-OX40 antibody. or its antigen-binding fragment; anti-CD28 antibody or its antigen-binding fragment; anti-PD1 antibody or its antigen-binding fragment; anti-PDL1 antibody or its antigen-binding fragment; anti-PDL2 antibody or its antigen-binding fragment; anti-GITR antibody or its antigen-binding fragment; anti-ICOS antibody or its antigen-binding fragment; anti-ILT2 antibody or its antigen-binding fragment; anti-ILT3 antibody or its antigen-binding fragment; anti-ILT4 antibody or its antigen-binding fragment; anti-ILT5 antibody or its antigen-binding fragment; anti-4-1BB antibody or its antigen-binding fragment; anti-NKG2A antibody or its antigen-binding fragment; anti NKG2C antibody or its antigen-binding fragment; anti-NKG2E antibody or its antigen-binding fragment; anti-TSLP antibody or its antigen-binding fragment; anti-IL-10 antibody or its antigen-binding fragment; IL-10 or PEGylated IL-10; TNF receptor protein agonists (e.g., agonistic antibodies or their antigen-binding fragments, or soluble fusions); immunoglobulin-like proteins; cytokine receptors; integrins; signaling lymphocyte-activating molecules (SLAM proteins); activated NK cell receptors; Toll-like receptors; OX40; CD2; CD7; CD2 7;CD28;CD30;CD40;ICAM-1;LFA-1(CD11a / CD18);4-1BB(CD137);B7-H3;ICOS(CD278);GITR;BAFFR;LIGHT;HVEM(LIGHTR);KIRDS2;SLAMF7;NKp80(KLRF1);NKp44;NKp30;NKp46;CD19;CD4;CD8 Alpha;CD8 Beta;IL2R Beta;IL2R Gamma;IL7R Alpha;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB2;CD18;ITGB7;NKG2D;NKG2C;TNFR2;TRANCE / RANKL;DNAM1(CD226);SLAMF4(CD244;2B4);CD84;CD96(Tactile);CEACAMl;CRTAM;Ly9(CD229);CD160(BY55);PSGL1;CD100(SEMA4D Selected from the group consisting of );CD69;SLAMF6(NTB-A;Lyl08);SLAM(SLAMF1, CD150, IPO-3);SLAM7;BLAME(SLAMF8);SELPLG(CD162);LTBR;LAT;GADS;PAG / Cbp;CD19a;Ligamids that specifically bind to CD83;CD47, PD-1, PD-L1 inhibitors;PD-L2;CTLA4;TIM3;LAG3;CEACAM(e.g., CEACAM-1, -3 and / or -5);VISTA;BTLA;TIGIT;LAIRl;IDO;TDO;CD160;TGFR beta; and cyclic dinucleotides or other STING pathway agonists.

[0095] The present invention also includes a combination comprising the antibody or antigen-binding fragment of the present invention and a second antibody that induces ADCC, wherein the antibody or antigen-binding fragment of the present invention enhances antibody-mediated destruction of cells by the second antibody. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells to which membrane surface antigens have been bound by specific antibodies. ADCC is often thought to be mediated by natural killer (NK) cells, but dendritic cells, macrophages, monocytes, and granulocytes can also mediate ADCC.

[0096] The present invention also comprises a combination of the antibody or antigen-binding fragment of the present invention and a second antibody that induces ADCP, wherein the antibody or antigen-binding fragment of the present invention enhances antibody-mediated phagocytosis of cells by the second antibody. Antibody-dependent cell-mediated phagocytosis (ADCP) is a cell-mediated immune defense mechanism that kills target cells via granulocyte, monocyte, dendritic cell, or macrophage-mediated phagocytosis.

[0097] Natural killer (NK) cells play a major role in cancer immunotherapy, including tumor antigen targeting with monoclonal antibodies (mAbs). In cell targeting situations, NK cells can be "specifically activated" through specific Fc receptors expressed on their cell surface. NK cells may express FcγRIIIA and / or FcγRIIC, which can bind to the Fc portion of immunoglobulins and transmit activation signals within the NK cell. Once activated through the Fc receptor by an antibody bound to a target cell, NK cells can lyse the target cell without priming and secrete cytokines such as interferon-gamma to mobilize adaptive immune cells. Similarly, tumor-associated macrophages (TAMs) express surface receptors that bind to the Fc fragment of an antibody, engaging the antibody in antibody-dependent cell-mediated cytotoxicity / phagocytosis (ADCC / ADCP). Since SIRPα / CD47 signaling induces a "don't eat me" response that reduces ADCC / ADCP, blocking this signaling with the anti-SIRPα antibody or antigen-binding fragment of the present invention may enhance ADCC in tumor cells containing antigenic determinants to which therapeutic antibodies are directed.

[0098] As a mechanism of action, this ADCC / ADCP may be used to treat various cancers and infectious diseases. Exemplary lists of ADCC / ADCP-inducible antibodies and antibody conjugates that can be combined with the antibody or antigen-binding fragment of the present invention include: rituximab, ubrituximab, margetuximab, IMGN-529, SCT400, vertuzumab, obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, dinutuximab, trastuzumab, cetuximab, rituximab-RLI, c. 60C3-RLI, Hul4.18-IL2, KM2812, AFM13, (CD20)2xCD16, Erlotinib (Tarceva), Daratumumab, Alemtuzumab, Pertuzumab, Brentuximab, Elotuzumab, Ibritumomab, Ifabotuzumab, Farletuzumab, Otlertuzumab, Carotuximab, Epratuzumab, Inevilizumab, Lumuletuzumab, 4G 7SDIE, AFM21, AFM22, LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20, Mogamulizumab, ChiLob-7 / 4, Leukotuximab, Isatuximab, DS-8895, FPA144, GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213, P Examples include, but are not limited to, ankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202, MOR-208, oportuzumab, encituximab, vedotin (Adcetris), ibritumomab / tiuxetan, ABBV-838, HuMax-AXL-ADC, and adtrastuzumab / emtansine (Kadcyla).Exemplary target antigens for such ADCC / ADCP-inducing antibodies include, but are not limited to, AMHR2, AXL, BCMA, CA IX, CD4, CD16, CD19, CD20, CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII, endoglin, EPHA2, EphA3, FGFR2b, folate receptor alpha, fucosyl-GM1, HER2, HER3, IL1RAP, kappa myeloma antigen, MS4A1, prolactin receptor, TA-MUC1, and PSMA.

[0099] In certain embodiments, the second antibody or its antigen-binding fragment induces ADCP. Such antibodies may be selected from the group consisting of, but are not limited to, rituximab, ubrituximab, margetuximab, IMGN-529, SCT400, bertuzumab, obinutuzumab, trastuzumab, cetuximab, alemtuzumab, ibritumomab, farletuzumab, inebilizumab, lumuretuzumab, 4G7SDIE, BMS-986012, BVX-20, mogamulizumab, ChiLob-7 / 4, GM102, GSK-2857916, PankoMab-GEX, chKM-4927, MDX-1097, MOR202, and MOR-208.

[0100] In embodiments in which the antibody or antigen-binding fragment of the present invention is combined with one or more ADCC / ADCP-inducible antibodies and antibody conjugates, such combinations may be used in conjunction with further therapeutic agents or therapeutic procedures, as may be. In one embodiment, the further therapeutic agent is an anti-LAG3 antibody or its antigen-binding fragment; an anti-APRIL antibody or its antigen-binding fragment; an anti-TIGIT antibody or its antigen-binding fragment; an anti-VISTA antibody or its antigen-binding fragment; an anti-BTLA antibody or its antigen-binding fragment; an anti-TIM3 antibody or its antigen-binding fragment; an anti-CTLA4 antibody or its antigen-binding fragment; an anti-HVEM antibody or its antigen-binding fragment; an anti-CD70 antibody or its antigen-binding fragment; an anti-CD137 antibody or its antigen-binding fragment; an anti-OX40 antibody or its antigen-binding fragment; an anti-CD28 antibody or its antigen-binding fragment; an anti-PD1 antibody or its antigen-binding fragment; an anti-PDL1 antibody or its antigen-binding fragment A fragment is selected from the group consisting of: anti-PDL2 antibody or its antigen-binding fragment; anti-GITR antibody or its antigen-binding fragment; anti-ICOS antibody or its antigen-binding fragment; anti-ILT2 antibody or its antigen-binding fragment; anti-ILT3 antibody or its antigen-binding fragment; anti-ILT4 antibody or its antigen-binding fragment; anti-ILT5 antibody or its antigen-binding fragment; anti-4-1BB antibody or its antigen-binding fragment; anti-NKG2A antibody or its antigen-binding fragment; anti-NKG2C antibody or its antigen-binding fragment; anti-NKG2E antibody or its antigen-binding fragment; anti-TSLP antibody or its antigen-binding fragment; anti-IL-10 antibody or its antigen-binding fragment; and IL-10 or PEGylated IL-10.

[0101] The present invention also provides a container or injection device comprising any one of the anti-SIRPα antibody or antigen-binding fragments of the present invention.

[0102] The present invention also provides a method for producing an anti-SIRPα antibody or antigen-binding fragment of the present invention, comprising culturing host cells containing polynucleotides encoding the heavy chain and / or light chain of the antibody (or antigen-binding fragment thereof) under conditions suitable for the expression of the polynucleotides; and optionally recovering the antibody or antigen-binding fragment from the host cells and / or culture medium. In one embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in one vector. In another embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in different vectors.

[0103] The present invention also provides a method for treating cancer in a subject requiring treatment, comprising administering to the subject an effective amount of the anti-SIRPα antibody or antigen-binding fragment of the present invention, which may be related to further therapeutic agents or treatment procedures.

[0104] In one embodiment, the subject to be treated is a human subject. In one embodiment, the further therapeutic agent is an anti-LAG3 antibody or its antigen-binding fragment; an anti-APRIL antibody or its antigen-binding fragment; an anti-TIGIT antibody or its antigen-binding fragment; an anti-VISTA antibody or its antigen-binding fragment; an anti-BTLA antibody or its antigen-binding fragment; an anti-TIM3 antibody or its antigen-binding fragment; an anti-CTLA4 antibody or its antigen-binding fragment; an anti-HVEM antibody or its antigen-binding fragment; an anti-CD70 antibody or its antigen-binding fragment; an anti-CD137 antibody or its antigen-binding fragment; an anti-OX40 antibody or its antigen-binding fragment; an anti-CD28 antibody or its antigen-binding fragment; an anti-PD1 antibody or its antigen-binding fragment; an anti-PDL1 antibody or its antigen-binding fragment A fragment is selected from the group consisting of: anti-PDL2 antibody or its antigen-binding fragment; anti-GITR antibody or its antigen-binding fragment; anti-ICOS antibody or its antigen-binding fragment; anti-ILT2 antibody or its antigen-binding fragment; anti-ILT3 antibody or its antigen-binding fragment; anti-ILT4 antibody or its antigen-binding fragment; anti-ILT5 antibody or its antigen-binding fragment; anti-4-1BB antibody or its antigen-binding fragment; anti-NKG2A antibody or its antigen-binding fragment; anti-NKG2C antibody or its antigen-binding fragment; anti-NKG2E antibody or its antigen-binding fragment; anti-TSLP antibody or its antigen-binding fragment; anti-IL-10 antibody or its antigen-binding fragment; and IL-10 or PEGylated IL-10.

[0105] The present invention also provides a method for treating an infection or infectious disease in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of the present invention, which may be related to a further therapeutic agent or treatment procedure. In one embodiment, the subject to be treated is a human subject.

[0106] In one embodiment, the further therapeutic agent is an anti-LAG3 antibody or its antigen-binding fragment; an anti-APRIL antibody or its antigen-binding fragment; an anti-TIGIT antibody or its antigen-binding fragment; an anti-VISTA antibody or its antigen-binding fragment; an anti-BTLA antibody or its antigen-binding fragment; an anti-TIM3 antibody or its antigen-binding fragment; an anti-CTLA4 antibody or its antigen-binding fragment; an anti-HVEM antibody or its antigen-binding fragment; an anti-CD70 antibody or its antigen-binding fragment; an anti-CD137 antibody or its antigen-binding fragment; an anti-OX40 antibody or its antigen-binding fragment; an anti-CD28 antibody or its antigen-binding fragment; an anti-PD1 antibody or its antigen-binding fragment; an anti-PDL1 antibody or its antigen-binding fragment A fragment is selected from the group consisting of: anti-PDL2 antibody or its antigen-binding fragment; anti-GITR antibody or its antigen-binding fragment; anti-ICOS antibody or its antigen-binding fragment; anti-ILT2 antibody or its antigen-binding fragment; anti-ILT3 antibody or its antigen-binding fragment; anti-ILT4 antibody or its antigen-binding fragment; anti-ILT5 antibody or its antigen-binding fragment; anti-4-1BB antibody or its antigen-binding fragment; anti-NKG2A antibody or its antigen-binding fragment; anti-NKG2C antibody or its antigen-binding fragment; anti-NKG2E antibody or its antigen-binding fragment; anti-TSLP antibody or its antigen-binding fragment; anti-IL-10 antibody or its antigen-binding fragment; and IL-10 or PEGylated IL-10.

[0107] The present invention also provides a method for detecting the presence of a SIRPα peptide or a fragment thereof in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof of the present invention, and detecting the presence of a complex of the antibody or fragment with the peptide, wherein the detection of the complex indicates the presence of the SIRPα peptide. [Brief explanation of the drawing]

[0108] [Figure 1] This shows the cross-reactivity of a commercially available anti-hSIRPα antibody with hSIRPβ1, as well as allele-specific binding to hSIRPαV1 and hSIRPαV2. [Figure 2] This shows the reactivity of the KWAR23 antibody with hSIRPαV1, hSIRPαV2, hSIRPβ1, and hSIRPγ. [Figure 3] Represents the reactivity of the antibody clone hSIRPα.50A with various hSIRPα alleles. [Figure 4] Represents the ability of the hSIRPα.50A antibody to block the binding of recombinant hCD47 / Fc protein to hSIRPα expressed on the cell surface. [Figure 5A] Represents the binding of the hSIRPα.50A antibody to primary human CD14+ enriched monocytes. [Figure 5B] Represents the binding of the hSIRPα.50A antibody to primary human CD14+ enriched monocytes. [Figure 5C] Represents the ability of the hSIRPα.50A antibody to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 5D] Represents the ability of the hSIRPα.50A antibody to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 6A] Represents the binding of the hSIRPα.50A antibody to primary human granulocytes. [Figure 6B] Represents phagocytosis of tumor cells by primary human granulocytes in the presence of rituximab + or - hSIRPα.50A antibody. [Figure 6C] Represents phagocytosis of tumor cells by primary human granulocytes in the presence of daratumumab + or - hSIRPα.50A antibody. [Figure 6D] Represents phagocytosis of tumor cells by primary human granulocytes in the presence of alemtuzumab + or - hSIRPα.50A antibody. [Figure 6E] Represents phagocytosis of tumor cells by primary human granulocytes in the presence of cetuximab + or - hSIRPα.50A antibody. [Figure 7] Represents phagocytosis of tumor cells by human macrophages in the presence of the indicated antibody (rituximab or daratumumab) + or - hSIRPα.50A antibody. [Figure 8] Represents the blockade of hSIRPα / hCD47 interaction by mouse hSIRPα.50A and humanized hSIRPα.50A antibodies to hSIRPα. [Figure 9]This represents the binding of the hSIRPα.50A antibody to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPα-VβC1αC2α, hSIRPα-VαC1βC2α, and hSIRPα-VαC1αC2β. [Figure 10A] This shows the alignment of the amino acid sequences of the hSIRPα and hSIRPβ1 IgV domains. [Figure 10B] This represents the loss of binding of the hSIRPα.50A antibody to hSIRPαV1(P74A). [Figure 11] This shows the binding of hSIRPα.40A and hSIRPα.50A antibodies to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPβL, and hSIRPγ. [Figure 12] This shows the binding of hSIRPα.40A and hSIRPα.50A antibodies to hSIRPαV1, hSIRPαV2, hSIRPαV3, hSIRPαV4, hSIRPαV5, hSIRPαV6, hSIRPαV8, and hSIRPαV9. [Figure 13] This demonstrates the ability of hSIRPα.40A and hSIRPα.50A antibodies to block recombinant hCD47 / Fc protein binding to hSIRPα expressed on the cell surface. [Figure 14A] This shows the binding of the hSIRPα.40A antibody to primary human CD14+ enriched monocytes. [Figure 14B] This shows the binding of the hSIRPα.40A antibody to primary human CD14+ enriched monocytes. [Figure 14C] This demonstrates the ability of the hSIRPα.40A antibody to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 14D] This demonstrates the ability of the hSIRPα.40A antibody to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 15A] This shows the binding of hSIRPα.40A and hSIRPα.50A antibodies to primary human granulocytes. [Figure 15B] This shows the phagocytosis of Ramos cells by primary human granulocytes in the presence of rituximab-+ or -hSIRPα.40A and hSIRPα.50A antibodies. [Figure 16] Represents the enhancement of rituximab-induced large cell phagocytosis by hSIRPα.40A and hSIRPα.50A antibodies. [Figure 17] Represents the binding of mouse hSIRPα.40A and humanized hSIRPα.40A antibodies to hSIRPα. [Figure 18] Represents the blockade of hCD47 binding to hSIRPα in the presence of humanized hSIRPα.40A antibody variants. [Figure 19] Represents the binding of hSIRPα.40A and hSIRPα.50A antibodies to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRP-VγC1βC2β, hSIRP-VβC1γC2β, and hSIRP-VβC1βC2γ. [Figure 20] Represents the loss of hSIRPα.40A and hSIRPα.50A antibody binding to hSIRPαV1(P74A). [Figure 21] Represents the ability of chimeric hSIRPα.40A antibody variants to affect rituximab-mediated phagocytosis. [Figure 22] Represents the ability of humanized hSIRPα.40A antibody variants to affect rituximab-mediated phagocytosis. [Figure 23A] Represents the ability of mouse hSIRPα.50A and chimeric hSIRPα.50A hIgG2 and hIgG4 antibody variants to affect rituximab-mediated phagocytosis. [Figure 23B] Represents the ability of chimeric hSIRPα.50A hIgG2 and hIgG4 antibody variants to affect rituximab-mediated phagocytosis. [Figure 23C] Represents the ability of chimeric hSIRPα.50A hIgG2 and hIgG4 antibody variants to affect daratumumab-mediated phagocytosis. [Figure 23D] Represents the ability of mouse hSIRPα.50A and chimeric hSIRPα.50A hIgG2 antibody variants to affect rituximab-mediated phagocytosis in granulocytes. [Figure 24A]This demonstrates the ability of mouse hSIRPα.50A and chimeric hSIRPα.50A.hIgG1.N297Q, hSIRPα.50A.hIgG4.N297Q, or hSIRPα.50A.hIgG2 antibody variants to affect rituximab-mediated phagocytosis. [Figure 24B] This demonstrates the ability of mouse hSIRPα.50A and chimeric hSIRPα.50A.hIgG1.N297Q, hSIRPα.50A.hIgG4.N297Q, or hSIRPα.50A.hIgG2 antibody variants to affect daratumumab-mediated phagocytosis. [Figure 25] This describes the ability of chimeric hSIRPα.50A.hIgG1.N297Q, hSIRPα.50A hIgG1.L234A.L235A.P329G, and hSIRPα.50A hIgG2 or hIgG4 antibody variants to affect rituximab-mediated phagocytosis. [Modes for carrying out the invention]

[0109] Detailed description Abbreviation Throughout the detailed description and examples of this invention, the following abbreviations are used: ADCC antibody-dependent cytotoxicity ADCP antibody-dependent cellular phagocytosis CDC complement-dependent cell injury Complementarity-determining regions within immunoglobulin variable regions defined using the CDR Kabat numbering system. CHO Chinese hamster ovaries Concentration at which 50% of the EC50 fully bound signal was observed. ELISA (Enzyme-mediated Immunoassay) FR Antibody Framework Region: Immunoglobulin Variable Region excluding the CDR Region HRP (Horseradiol Peroxidase) IFN (Interferon) Concentration that produces 50% inhibition of IC50 IgG (Immunoglobulin G) Immunoglobulin alignment and numbering system developed by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) mAb or Mab or MAb monoclonal antibody SEB Staphylococcus enterotoxin B TT Tetanus Toxoid The V region is a segment of the Ig chain whose sequence is variable between different antibodies. It extends from Kabat residue 109 in the light chain to 113 in the heavy chain. VH (Variable Region of Immunoglobulin Heavy Chain) VK Immunoglobulin Kappa Light Chain Variable Region VL (Variable Region of Immunoglobulin Light Chain)

[0110] definition To enable a more immediate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise specifically defined elsewhere in this document, all other technical and scientific terms used herein have meanings that are commonly understood by those skilled in the art to which the present invention pertains.

[0111] As used herein, including in the attached claims, singular forms of words such as "a," "an," and "the" encompass multiple corresponding subjects unless otherwise clearly indicated in the context.

[0112] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, “administration” and “treatment” refer to the contact of exogenous pharmaceuticals, therapeutic agents, diagnostic agents, or compositions with said animals, humans, subjects, cells, tissues, organs, or biological fluids. Treatment of cells includes contact of reagents with cells and contact of reagents with fluids that have come into contact with cells. “Administration” and “treatment” also mean, for example, in vitro and ex vivo treatment of cells with reagents, diagnostic agents, conjugates, or other cells.

[0113] "To treat" or "to treat" means to administer, either internally or externally, a therapeutic agent, such as a composition containing either the antibody or antigen-binding fragment of the present invention, to a subject or patient having one or more disease symptoms for which the therapeutic agent has therapeutic activity, or to a subject or patient suspected of having the disease. Typically, the agent is administered in an amount effective to improve one or more disease symptoms in the subject or population being treated, whether by inducing recovery of or inhibiting the progression of such symptoms(s) to any clinically measurable degree. The amount of therapeutic agent effective to improve any particular disease symptom may vary depending on factors such as the disease status, the patient's age and weight, and the drug's ability to induce the desired response in the subject. Whether or not the disease symptoms have improved can typically be assessed by any clinical scale used by a physician or other skilled healthcare provider assessing the severity or progression of the symptoms.

[0114] "Recombinant expression" of a protein means the transcription and translation of an exogenous gene within a host organism to produce a protein referred to herein as a "recombinant protein."

[0115] SIRPα and related proteins SIRPα belongs to a class of membrane proteins known as "paired receptors," which include multiple genes encoding proteins (e.g., SIRPα, SIRPβ1, and SIRPγ) that have similar extracellular domains and different transmembrane and / or cytoplasmic domains with opposite signaling capabilities (activation or inhibition). Similar to SIRPα, several paired receptors, including the SIRP and CD200 receptor families, are found on NK cells, and some are found on bone marrow cells (Hatherley et al., Mol Cell. 2008; 31:266-277).

[0116] SIRPα contains an extracellular domain that can be subdivided into three distinct domains: Ig-like (immunoglobulin-like) type V (IgV), Ig-like type C1 (IgC1), and Ig-like type C2 (IgC2) domains. The IgV domain is also known as the ligand-binding N-terminal domain of SIRPα. Similar to SIRPα, the related proteins SIRPβ1 and SIRPγ also contain extracellular domains that can be subdivided into IgV, IgC1, and IgC2 domains. However, SIRPα, SIRPβ1, and SIRPγ have different cytoplasmic domains. SIRPβ1 has a very short cytoplasmic domain with only six amino acids and lacks a signaling motif for association with phosphatases. Instead, this protein can associate with DNAX-activating protein 12 (DAP12), a dimeric adapter protein that binds amino acids to basic side chains within the transmembrane domain of SIRPβ1, and transmit activation signals through the immunoreceptor tyrosine activation motif (ITAM). SIRPγ also has a short cytoplasmic region of four amino acids, but it lacks a charged amino acid side chain within its transmembrane domain and therefore does not associate with DAP12. For this reason, SIRPγ is noted as a non-signaling protein (Barclay, AN and Brown, MH, Nat Rev Immunol. 2006; 6: 457-464).

[0117] The primary ligand for SIRPα is CD47, which consists of one extracellular IgV domain, a 5x transmembrane domain, and a short cytoplasmic tail. CD47 functions as a cellular ligand that binds to SIRPα via its NH2-terminal IgV domain. Evidence that CD47 contributes to self-recognition is that spleen macrophages from CD47-expressing mice are linked to CD47. - / - This was obtained by observing the removal of blood cells infused from mice (Oldenborg et al., Science. 2000; 288: 2051-2054).

[0118] In addition to CD47, two other SIRPα ligands, also known as surfactant proteins A and D (Sp-A and Sp-D), have been reported, both belonging to the collectin family. Sp-D has been reported to bind to the membrane-proximal IgC2 domain of SIRPα in a calcium-dependent and glucose-dependent manner. Sp-A and Sp-D are thought to maintain an anti-inflammatory environment in the lungs by stimulating SIRPα on alveolar macrophages (Gardai et al., Cell. 2003; 115: 13-23).

[0119] The amino acid sequences of eight human SIRPα variants are listed under SEQ ID NO:34, 36, 44, 46, 48, 50, 52, and 54; and the exemplary nucleic acid sequences encoding these variants are listed under SEQ ID NO:33, 35, 43, 45, 47, 49, 51, and 53, respectively.

[0120] For comparison, the amino acid sequences of human SIRPβ1 and SIRPγ are listed under SEQ ID NO:38 and 40, respectively, and exemplary nucleic acid sequences are listed under SEQ ID NO:37 and 39, respectively.

[0121] The amino acid sequence of human CD47 is listed under SEQ ID NO:42, and the exemplary nucleic acid sequence is listed under SEQ ID NO:41.

[0122] The modified SIRPα polypeptides hSIRPα-VβC1αC2α, hSIRPα-VαC1βC2α, hSIRPα-VαC1αC2β, and hSIRPαV1(P74A), which will be discussed later in this specification, are listed under SEQ ID NO: 56, 58, 60, and 62; exemplary nucleic acid sequences encoding these variants are listed under SEQ ID NO: 55, 57, 59, and 61, respectively.

[0123] Anti-SIRPα antibody and its antigen-binding fragment The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα, and the use of such an antibody or fragment. In some embodiments, the anti-SIRPα antibody is isolated.

[0124] Whether an antibody specifically binds to a polypeptide sequence (e.g., human SIRPα, hSIRPβ1, etc.) can be determined using any assay known in the art. Examples of assays known in the art for measuring binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET).

[0125] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. The term antibody refers to the antigen-binding portion, i.e., (i)V L , V H , C L and C H (ii) A monovalent fragment consisting of an l domain, the Fab fragment; (ii) A bivalent fragment containing two Fab fragments linked by disulfide bridges in a hinge region, the F(ab')2 fragment; (iii) V H and C H (iv) V of one arm of the antibody L and V HThis includes "antigen-binding sites" (e.g., fragments, subsequences, complementarity-determining regions (CDRs)) that possess the ability to bind to an antigen, including (v) a VH domain-based Fv fragment; (v) a VH domain-based dAb fragment (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR). Single-chain antibodies are also included by reference to the term "antibody." Preferred therapeutic antibodies are intact IgG antibodies. As used herein, the term "intact IgG" means polypeptides belonging to a class of antibodies substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3. Known Ig domains in the IgG class of antibodies are V H , Cγ1, Cγ2, Cγ3, V L , and C L That is the case.

[0126] This invention encompasses anti-SIRPα antigen-binding fragments and methods of using them.

[0127] As used herein, "full-length antibody" refers to a bivalent molecule containing two heavy chains and two light chains, in the example of IgG. Each heavy chain is V H Domain and the subsequent constant domain (C H1 ), hinge region, and two more steady-state (C H2 and C H3 ) Contains a domain; each light chain contains one V L Domain and one stationary (C L ) contains a domain. In the case of IgM, a full-length antibody is a decavalent or dodecavalent molecule containing 5 or 6 linked immunoglobulins, in which each monomer has two antigen-binding sites that form a heavy chain and a light chain.

[0128] As used herein, unless otherwise specified, “antibody fragment” or “antigen-binding fragment” refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, for example, a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies; nanobodies formed from antibody fragments and multispecific antibodies.

[0129] This invention encompasses anti-SIRPα Fab fragments and methods of using them. A "Fab fragment" consists of one light chain and one heavy chain of C H It consists of a variable region and a Fab molecule. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. The "Fab fragment" may be a product of papain cleavage of an antibody.

[0130] The present invention encompasses an anti-SIRPα antibody containing an Fc region, its antigen-binding fragment, and a method of using the same. The "Fc" region is the C region of the antibody. H 3 and C H It contains two heavy chain fragments, each containing two domains. These two heavy chain fragments are connected by two or more disulfide bonds, and C H It is held together by hydrophobic interactions between three domains.

[0131] This invention encompasses anti-SIRPα Fab' fragments and methods of using the same. The "Fab' fragment" consists of one light chain and V H Domain and C H A portion or fragment of a heavy chain containing one domain, and C H 1 and C H Because it includes a region between the two domains, an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0132] This invention encompasses the anti-SIRPα F(ab')2 fragment and methods of using the same. The "F(ab')2 fragment" consists of two light chains and C H1Domain and C H2 Because it contains two heavy chains, each containing part of the constant region, an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "F(ab')2 fragment" can be a product of pepsin cleavage of an antibody.

[0133] This invention encompasses anti-SIRPα Fv fragments and methods of using the same. The "Fv region" includes variable regions from both the heavy and light chains, but lacks a constant region.

[0134] This invention encompasses anti-SIRPα scFv fragments and methods of using the same. The term "single-stranded Fv" or "scFv" antibody refers to the V of the antibody. H and V L This refers to antibody fragments containing domains, which are located within a single polypeptide chain. Generally, Fv polypeptides cause scFv to form structures desirable for antigen binding. H Domain and V L Further includes polypeptide linkers between domains. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269–315. Also see International Patent Application Publication WO88 / 01649, and U.S. Patents 4,946,778 and 5,260,203.

[0135] This invention encompasses anti-SIRPα domain antibodies and methods of using the same. A "domain antibody" is an immunoglobulin fragment of immunological function that contains only the variable region of the heavy chain or the variable region of the light chain. In some examples, two or more V H The regions are covalently joined by a peptide linker to produce a bivalent domain antibody. The two Vs of the bivalent domain antibody HThe region may target the same or different antigens.

[0136] This invention encompasses a bivalent anti-SIRPα antibody and methods of using the same. A "bivalent antibody" contains two antigen-binding sites. In some examples, these two binding sites have the same antigen specificity. However, a bivalent antibody may be bispecific (see below).

[0137] This invention encompasses the anti-SIRPα diabody and methods of using the same polypeptide chain (V). The term "diabody" as used herein refers to the same polypeptide chain (V H -V L or V L -V H ) Light chain variable domain (V L ) linked to a heavy chain variable domain (V H This refers to a small antibody fragment having two antigen-binding sites, including a diabody. By using an excessively short linker to allow pairing between two domains on the same chain, the domains are facilitated to pair with a complementary domain on another chain and create two antigen-binding sites. Diabodies are described more fully, for example, in EP404,097;WO93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. Duobodies are described in Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13):5145-5150. For a review of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0138] Typically, the antibody or antigen-binding fragments of the present invention, modified in several ways, retain at least 10% of the binding activity (compared to the parent antibody) when the binding activity is expressed on a molar basis. Preferably, the antibody or antigen-binding fragments of the present invention retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100%, or more, of the SIRPα binding affinity as the parent antibody. The antibody or antigen-binding fragments of the present invention are also intended to include conserved or non-conserved amino acid substitutions ("conserved variants" or "functionally conserved variants" of the antibody) that do not substantially alter the biological activity.

[0139] The present invention encompasses isolated anti-SIRPα antibodies and their antigen-binding fragments, as well as methods of use thereof. In this specification, the term “isolated” does not refer to the complete absence of such biomolecules, or the absence of water, buffers, or salts, or to components of a pharmaceutical formulation containing the antibody or fragment. “Isolated” antibodies, antigen-binding fragments, nucleic acids, etc., are identified and isolated and / or recovered from one or more components of the natural environment. In preferred embodiments, the antibodies, antigen-binding fragments, nucleic acids, etc., are purified to 75% by weight or more, more preferably 90% by weight or more, even more preferably 95% by weight or more, and even more preferably 98% by weight or more. Thus, the “isolated” biomolecules are at least partially free from other biomolecules from the resulting cells or cell cultures. Such biomolecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell fragments and growth media. The isolated antibodies or antigen-binding fragments may further be at least partially free from components of the expression system, such as biomolecules from host cells or in their growth media.

[0140] The present invention encompasses anti-SIRPα chimeric antibodies (e.g., human constant domain / mouse variable domain) and methods of use thereof. As used herein, a “chimeric antibody” is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies belong to different species (U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). Typically, the variable domain is obtained from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant domain sequence is obtained from a human antibody; therefore, the resulting chimeric antibody is unlikely to induce an adverse immune response in human subjects other than the parent (e.g., mouse) antibody.

[0141] The present invention encompasses anti-SIRPα humanized antibodies and their antigen-binding fragments (e.g., humanized rat or mouse antibodies), as well as methods of using them. As used herein, the term "humanized antibody" refers to a form of antibody comprising sequences from both human and non-human (e.g., mouse or rat) antibodies. Generally, such humanized antibodies consist of substantially at least one, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of the framework (FR) region being a human immunoglobulin sequence. The humanized antibody may optionally include at least a portion of the human immunoglobulin constant region (Fc). For further details on humanized antibodies, see, for example, Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000).

[0142] Generally, the structural unit of basic antibodies consists of a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminus of the heavy chain may define a constant region, primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. For general information, please refer to Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0143] The variable region of each light / heavy chain pair forms an antibody binding site. Therefore, intact antibodies generally have two binding sites. Except for bifunctional or bispecific antibodies, these two binding sites are generally identical.

[0144] Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within a relatively conserved framework region (FR). These CDRs are usually aligned by the framework region to enable binding to specific epitopes. Generally, from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain are generally described in *Sequences of Proteins of Immunological Interest*, Kabat, et al.; National Institutes of Health, Bethesda, MD; 5 thed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917, or as defined by Chothia, et al., (1989) Nature 342:878-883.

[0145] As used herein, the term "hypervariable region" refers to amino acid residues of an antibody or its antigen-binding fragment responsible for antigen binding. The hypervariable region includes amino acid residues from the "complementarity-determining region" or "CDR" (i.e., CDRL1, CDRL2, and CDRL3 of the light chain variable domain, and CDRH1, CDRH2, and CDRH3 of the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Defining the CDR region of antibodies by sequence); also see Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (Defining the CDR region of antibodies by structure). As used herein, the term "framework" or "FR" residues refer to variable domains other than the hypervariable region residues defined herein as CDR residues.

[0146] "Isolated nucleic acid molecule" or "isolated polynucleotide" means genomic DNA or RNA, mRNA, cDNA, or a combination thereof of synthetic origin, that is not associated with all or part of a polynucleotide, and that is isolated polynucleotide is linked to a naturally occurring or naturally unlinked polynucleotide. For the purposes of this disclosure, it should be understood that "nucleic acid molecule containing" a particular nucleotide sequence does not include intact chromosomes. An isolated nucleic acid molecule "containing" a specified nucleic acid sequence may, in addition to the specified sequence, contain up to 10 or up to 20 or more coding sequences of other proteins, or parts or fragments thereof, or may contain operablely linked regulatory sequences that control the expression of the coding region of the listed nucleic acid sequence, and / or may contain a vector sequence.

[0147] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a coding sequence that is functionally linked within a specific host organism. For example, regulatory sequences suitable for prokaryotes include promoters, sometimes operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0148] Nucleic acids or polynucleotides are “operably ligated” when considering their functional relevance to another nucleic acid sequence. For example, DNA for a precursor sequence or secretory leader is operably ligated to the DNA for a polypeptide if it is expressed as a precursor protein participating in the secretion of the polypeptide; a promoter or enhancer is operably ligated to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably ligated to a coding sequence if it is positioned to facilitate translation. Generally, though not always, “operably ligated” means that the ligated DNA sequences are adjacent and, in the case of a secretory leader, adjacent and in the reading phase. Enhancers, however, do not need to be adjacent. Ligation is completed by ligation at a convenient control site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0149] The terms "cells," "cell lines," and "cell cultures" used herein are interchangeable, and all such terms include offspring. Therefore, the terms "transformant" and "transformed cells" encompass primary target cells and cultures derived therefrom, regardless of the number of introductions. Similarly, it should be understood that not all offspring will have exactly the same amount of DNA due to planned or unintended mutations. This includes mutant offspring that possess the same function and biological activity as those screened in the originally transformed cells. Where different terminology is intended, it will be evident from the context.

[0150] As used herein, “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences can be obtained, for example, from the JOINSOLVER germline database on the website of the National Institute of Arthritis, Skeletal Musculoskeletal Diseases, National Institutes of Health. Mouse germline sequences can be obtained, for example, as described in Giudicelli et al. (2005) Nucleic Acids Res. 33: D256-D261.

[0151] Physical and functional properties of exemplary anti-SIRPα antibodies The present invention provides an anti-SIRPα antibody having specified structural and functional characteristics, its antigen-binding fragment, and a method for using the antibody or its antigen-binding fragment in the treatment or prevention of a disease (e.g., cancer or infectious disease).

[0152] As previously stated, antibodies and fragments that bind to the same epitope as either the anti-SIRPα antibody or its antigen-binding fragment of the present invention also form part of the present invention. In one embodiment, the present invention provides an antibody or its antigen-binding fragment that binds to the same human SIRPα epitope as an antibody containing one of the following heavy chain / light chain sequence combinations (or, in the case of an amino acid sequence, at least 90%, 95%, 97%, 98%, or 99% identical thereto): SEQ ID NO:10 / SEQ ID NO:20 (referred to as hSIRPα.50A.H1L1 in this specification) SEQ ID NO:10 / SEQ ID NO:22 (referred to as hSIRPα.50A.H1L2 in this specification) SEQ ID NO:10 / SEQ ID NO:24 (referred to as hSIRPα.50A.H1L3 in this specification) SEQ ID NO:10 / SEQ ID NO:26 (referred to as hSIRPα.50A.H1L4 in this specification) SEQ ID NO:10 / SEQ ID NO:28 (referred to as hSIRPα.50A.H1L5 in this specification) SEQ ID NO:12 / SEQ ID NO:20 (referred to as hSIRPα.50A.H2L1 in this specification) SEQ ID NO:12 / SEQ ID NO:22 (referred to as hSIRPα.50A.H2L2 in this specification) SEQ ID NO:12 / SEQ ID NO:24 (referred to as hSIRPα.50A.H2L3 in this specification) SEQ ID NO:12 / SEQ ID NO:26 (referred to as hSIRPα.50A.H2L4 in this specification) SEQ ID NO:12 / SEQ ID NO:28 (referred to as hSIRPα.50A.H2L5 in this specification) SEQ ID NO:14 / SEQ ID NO:20 (referred to as hSIRPα.50A.H3L1 in this specification) SEQ ID NO:14 / SEQ ID NO:22 (referred to as hSIRPα.50A.H3L2 in this specification) SEQ ID NO:14 / SEQ ID NO:24 (referred to as hSIRPα.50A.H3L3 in this specification) SEQ ID NO:14 / SEQ ID NO:26 (referred to as hSIRPα.50A.H3L4 in this specification) SEQ ID NO:14 / SEQ ID NO:28 (referred to as hSIRPα.50A.H3L5 in this specification) SEQ ID NO:16 / SEQ ID NO:20 (referred to as hSIRPα.50A.H4L1 in this specification) SEQ ID NO:16 / SEQ ID NO:22 (referred to as hSIRPα.50A.H4L2 in this specification) SEQ ID NO:16 / SEQ ID NO:24 (referred to as hSIRPα.50A.H4L3 in this specification) SEQ ID NO:16 / SEQ ID NO:26 (referred to as hSIRPα.50A.H4L4 in this specification) SEQ ID NO:16 / SEQ ID NO:28 (referred to as hSIRPα.50A.H4L5 in this specification) SEQ ID NO:18 / SEQ ID NO:20 (referred to as hSIRPα.50A.H5L1 in this specification) SEQ ID NO:18 / SEQ ID NO:22 (referred to as hSIRPα.50A.H5L2 in this specification) SEQ ID NO:18 / SEQ ID NO:24 (referred to as hSIRPα.50A.H5L3 in this specification) SEQ ID NO:18 / SEQ ID NO:26 (referred to as hSIRPα.50A.H5L4 in this specification) SEQ ID NO:18 / SEQ ID NO:28 (referred to as hSIRPα.50A.H5L5 in this specification) SEQ ID NO:78 / SEQ ID NO:90 (referred to as hSIRPα.40A.H1L1 in this specification) SEQ ID NO:78 / SEQ ID NO:92 (referred to as hSIRPα.40A.H1L2 in this specification) SEQ ID NO:78 / SEQ ID NO:94 (referred to as hSIRPα.40A.H1L3 in this specification) SEQ ID NO:78 / SEQ ID NO:96 (referred to as hSIRPα.40A.H1L4 in this specification) SEQ ID NO:78 / SEQ ID NO:98 (referred to as hSIRPα.40A.H1L5 in this specification) SEQ ID NO:78 / SEQ ID NO:100 (referred to as hSIRPα.40A.H1L6 in this specification) SEQ ID NO:80 / SEQ ID NO:90 (referred to as hSIRPα.40A.H2L1 in this specification) SEQ ID NO:80 / SEQ ID NO:92 (referred to as hSIRPα.40A.H2L2 in this specification) SEQ ID NO:80 / SEQ ID NO:94 (referred to as hSIRPα.40A.H2L3 in this specification) SEQ ID NO:80 / SEQ ID NO:96 (referred to as hSIRPα.40A.H2L4 in this specification) SEQ ID NO:80 / SEQ ID NO:98 (referred to as hSIRPα.40A.H2L5 in this specification) SEQ ID NO:80 / SEQ ID NO:100 (referred to as hSIRPα.40A.H2L6 in this specification) SEQ ID NO:82 / SEQ ID NO:90 (referred to as hSIRPα.40A.H3L1 in this specification) SEQ ID NO:82 / SEQ ID NO:92 (referred to as hSIRPα.40A.H3L2 in this specification) SEQ ID NO:82 / SEQ ID NO:94 (referred to as hSIRPα.40A.H3L3 in this specification) SEQ ID NO:82 / SEQ ID NO:96 (referred to as hSIRPα.40A.H3L4 in this specification) SEQ ID NO:82 / SEQ ID NO:98 (referred to as hSIRPα.40A.H3L5 in this specification) SEQ ID NO:82 / SEQ ID NO:100 (referred to as hSIRPα.40A.H3L6 in this specification) SEQ ID NO:84 / SEQ ID NO:90 (referred to as hSIRPα.40A.H4L1 in this specification) SEQ ID NO:84 / SEQ ID NO:92 (referred to as hSIRPα.40A.H4L2 in this specification) SEQ ID NO:84 / SEQ ID NO:94 (referred to as hSIRPα.40A.H4L3 in this specification) SEQ ID NO:84 / SEQ ID NO:96 (referred to as hSIRPα.40A.H4L4 in this specification) SEQ ID NO:84 / SEQ ID NO:98 (referred to as hSIRPα.40A.H4L5 in this specification) SEQ ID NO:84 / SEQ ID NO:100 (referred to as hSIRPα.40A.H4L6 in this specification) SEQ ID NO:86 / SEQ ID NO:90 (referred to as hSIRPα.40A.H5L1 in this specification) SEQ ID NO:86 / SEQ ID NO:92 (referred to as hSIRPα.40A.H5L2 in this specification) SEQ ID NO:86 / SEQ ID NO:94 (referred to as hSIRPα.40A.H5L3 in this specification) SEQ ID NO:86 / SEQ ID NO:96 (referred to as hSIRPα.40A.H5L4 in this specification) SEQ ID NO:86 / SEQ ID NO:98 (referred to as hSIRPα.40A.H5L5 in this specification) SEQ ID NO:86 / SEQ ID NO:100 (referred to as hSIRPα.40A.H5L6 in this specification) SEQ ID NO:88 / SEQ ID NO:90 (referred to as hSIRPα.40A.H6L1 in this specification) SEQ ID NO:88 / SEQ ID NO:92 (referred to as hSIRPα.40A.H6L2 in this specification) SEQ ID NO:88 / SEQ ID NO:94 (referred to as hSIRPα.40A.H6L3 in this specification) SEQ ID NO:88 / SEQ ID NO:96 (referred to as hSIRPα.40A.H6L4 in this specification) SEQ ID NO:88 / SEQ ID NO:98 (referred to as hSIRPα.40A.H6L5 in this specification) SEQ ID NO:88 / SEQ ID NO:100 (referred to as hSIRPα.40A.H6L6 in this specification).

[0153] Several methods exist for mapping antigenic epitopes on target antigens, including H / D-Ex mass spectrometry, crosslinked and linked mass spectrometry, X-ray crystallography, Pepscan analysis, and site-directed mutagenesis. For example, HDX (hydrogen-deuterium exchange) linked to proteolysis and mass spectrometry can be used to determine the epitopes of antibodies on specific antigen Y. HDX-MS relies on the accurate measurement and comparison of the degree of deuterium uptake by the antigen when incubated in D2O, both alone and in the presence of the antibody, at various time intervals. Deuterium is exchanged for hydrogen on the amide backbone of the protein in the exposed area, but the region of the antigen bound to the antibody will be protected and will show little to no exchange after analysis of the proteolytic fragment by LC-MS / MS. Crosslinked and linked mass spectrometry is initiated by conjugating the antibody and antigen with a mass-labeled chemical crosslinking agent. The presence of the complex is then confirmed using high-mass MALDI detection. Following the chemical reaction of crosslinking, the Ab / Ag complex is extremely stable, allowing for the application of numerous different enzymes and digestion conditions to the complex and yield a wide variety of overlapping peptides. Identification of these peptides is performed using high-resolution mass spectrometry and MS / MS techniques. Crosslinked peptides are identified using mass tags attached to the crosslinking reagents. After MS / MS fragmentation and data analysis, both epitopes and paratopes are determined in the same experiment.

[0154] The scope of the present invention also includes isolated anti-SIRPα antibodies and their antigen-binding fragments (e.g., humanized antibodies) comprising variants of the immunoglobulin chains shown herein, wherein the variants exhibit one or more of the following characteristics: EC 50 <1nM human SIRPαV1 protein with sequence SEQ ID NO:34; at least 100 times higher EC than SIRPαV1 (P74A) with sequence SEQ ID NO:62. 50This indicates that, in some cases, human SIRPβ1 protein with sequence SEQ ID NO:38 also has at least 100 times higher EC. 50 This shows (in each example, the EC decreased) 50 This refers to the EC of human SIRPαV1 protein having sequence SEQ ID NO:34. 50 This is the case for which the measurement was preferably performed by cell ELISA (CELISA) as described later in this specification; <10nM, preferably <5nM, more preferably <1.5nM, even more preferably <1.0nM, even more preferably <0.5nM, most preferably about 0.3nM or less EC 50 It binds to cells expressing the human SIRPαV1 protein; <10nM, preferably <5nM, more preferably <1.5nM, even more preferably <1.0nM, even more preferably <0.5nM, most preferably about 0.3nM or less EC 50 It binds to cells expressing the human SIRPαV2 protein; Antibody concentration of 50 nM, preferably 67 nM, more preferably 100 nM, or antibody EC of SIRPαV1 or SIRPαV2 50 It does not bind to the SIRPβ1 protein in a sense at concentrations 10 times greater than, preferably 50 times greater, more preferably 100 times greater, and even more preferably 200 times greater than; ICs with a molecular weight of <10.0nM, more preferably <5.0nM, even more preferably <2.5nM, and most preferably about 1.0nM or less. 50 And it inhibits the binding of human SIRPα to CD47; and It exhibits at least 79%, more preferably 85%, of T20 "humanity."

[0155] In another embodiment, the present invention relates to a V that binds to human SIRPα (e.g., a humanized antibody) and has at least 90% sequence identity with SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. H Domain and V L The present invention provides an antibody or its antigen-binding fragment having a domain. In another embodiment, the present invention provides a V that binds to human SIRPα (e.g., a humanized antibody) and has at least 95% sequence identity with SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. H Domain and V L The present invention provides an antibody or its antigen-binding fragment having a domain. In another embodiment, the present invention provides a V that binds to human SIRPα (e.g., a humanized antibody) and has at least 97% sequence identity with SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. H Domain and V L The present invention provides an antibody or its antigen-binding fragment having a domain. In another embodiment, the present invention provides a V that binds to human SIRPα (e.g., a humanized antibody) and has at least 98% sequence identity with SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. H Domain and V LThe present invention provides an antibody or its antigen-binding fragment having a domain. In another embodiment, the present invention provides a V that binds to human SIRPα (e.g., a humanized antibody) and has at least 99% sequence identity with SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. H Domain and V L The present invention provides an antibody or its antigen-binding fragment having a domain. Preferably, in each example, the sequence differences between the variant and SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32 consist of conserved substitutions, most preferably limited to substitutions within framework residues.

[0156] The following references pertain to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Camacho, C. et al. (2009): BMC Bioinformatics 10:421; Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, SF, et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, TL, et al., (1996) Meth. Enzymol. 266:131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, JC, et al., (1993) Comput. Chem. 17:149-163; Hancock, JM et al., (1994) Comput. Appl. Biosci. 10:67-70;ALIGNMENT SCORING SYSTEMS: Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), pp. 345-352, Dayhoff, MO, et al., “A model of evolutionary change in proteins.” in Natl. Biomed. Res. Found., Washington, DC; Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Schwartz, RM, et al., Washington, DC., “Matrices for detecting distant relationships.”; Altschul, SF, (1991) J. Mol. Biol. 219:555-565; States, DJ, et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, SF, et al., (1993) J. Mol. Evol. 36:290-300;ALIGNMENT STATISTICS:Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Altschul, SF, in Plenum, New York, “Evaluating the statistical significance of multiple distinct local alignments.”. In this application, % identity comparison is preferably performed by the BLAST algorithm, and the algorithm parameters are selected to give the maximum match between each sequence over the entire length of each reference sequence (e.g., prediction threshold: 10; language size: 6; maximum match within query range: 0; BLOSUM 62 matrix; gap cost: extension 11, extension 1; conditional compositional score matrix adjustment).

[0157] A “conservatively modified variant” or “conservative substitution” refers to the substitution of an amino acid within a protein by another amino acid with similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, backbone conformation and stiffness), thereby often being carried out without altering the protein’s biological activity. Those skilled in the art generally recognize that single amino acid substitutions within non-essential regions of polypeptides do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are shown in Table 1 below. [Table 1]

[0158] Functionally conserved variants of the antibody of the present invention are also intended by the present invention. As used herein, “functionally conserved variant” refers to an antibody or fragment in which one or more amino acid residues are modified without altering desired properties such as antigen affinity and / or specificity. Such variants include, but are not limited to, exchanges of an amino acid with an amino acid having similar properties to a given amino acid, such as the conservative amino acid substitutions in Table 1. Also provided are V variants of the anti-SIRPα antibody of the present invention having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions. L Isolated polypeptides containing domains (e.g., SEQ ID NO: 76, 90, 92, 94, 96, 98, 100, 8, 20, 22, 24, 26, 28, and 32), and the V of the anti-SIRPα antibody of the present invention. H These are isolated polypeptides containing domains (e.g., SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 7, 10, 12, 14, 16, 18, and 30).

[0159] The present invention further comprises polynucleotides encoding either a polypeptide or an immunoglobulin chain of the anti-SIRPα antibody of the present invention and its antigen-binding fragment. For example, the present invention comprises polynucleotides encoding amino acids listed in SEQ ID NO: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and any one of SEQ ID NO: 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32.

[0160] In one embodiment, an isolated polynucleotide, such as DNA, encoding the polypeptide chain of an isolated antibody or antigen-binding fragment as described herein is provided. In one embodiment, the isolated polynucleotide encodes an antibody or antigen-binding fragment comprising at least one mature immunoglobulin light chain variable (VL) domain and / or at least one mature immunoglobulin heavy chain variable (VH) domain according to the present invention. In some embodiments, the isolated polypeptide encodes both the light and heavy chains on a single polynucleotide molecule, while in other embodiments, the light and heavy chains are encoded on separate polynucleotide molecules. In yet another embodiment, the polynucleotide further encodes a signal sequence.

[0161] The present invention also provides vectors, such as expression vectors, including plasmids containing isolated polynucleotides of the present invention, which are operably linked to a control sequence recognized by the host cell when the host cell is transfected with the vector. Also provided are host cells containing the vector of the present invention, and methods for producing an antibody or antigen-binding fragment or polypeptide disclosed herein, comprising culturing a host cell harboring an expression vector or nucleic acid encoding the immunoglobulin chain of the antibody or antigen-binding fragment in a culture medium, and isolating the antigen or antigen-binding fragment from the host cell or culture medium.

[0162] binding affinity By way of example and not limitation, the antibodies and antigen-binding fragments disclosed herein can bind bivalently to human SIRPα with a K -9 value of 10×10 D M or less in measurements by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or biolayer interferometry (OCTET)). In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind to human SIRPα or bivalently with a K -9 value of about 5-10×10 D M in measurements by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET). Affinity is calculated as K D =K off / k on (where k off is the dissociation rate constant and K on is the association rate constant and K D is the equilibrium constant). Affinity can be measured at equilibrium by measuring the fraction (r) of bound labeled ligand at various concentrations (c). The data is graphed using the Scatchard equation: r / c = K(n - r), where r = number of moles of bound ligand / moles of receptor at equilibrium; c = free ligand concentration at equilibrium; K = equilibrium binding constant; n = number of ligand binding sites per receptor molecule. Analysis of the graph plots r / c on the Y-axis against r on the X-axis, thereby creating a Scatchard plot. Measurement of antibody affinity by Scatchard analysis is well known in the art. See, for example, van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

[0163] Human nature For the purposes of this paper, "humanity" is measured using a T20 score analyzer and quantifies the humanity of the variable region of a monoclonal antibody as described in Gao SH, Huang K, Tu H, Adler AS. Monoclonal antibody humanness score and its applications. BMC Biotechnology. 2013: 13:55. doi:10.1186 / 1472-6750-13-55).

[0164] A web-based tool is provided to calculate the T20 score of an antibody sequence using the T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com. When calculating the T20 score, the input VH, VK, or VL variable region protein sequence is first assigned a Kabat number, and CDR residues are identified. The full-length sequence or a framework-only sequence (with CDR residues removed) is compared to the corresponding sequence in each antibody database using the blastp protein-protein BLAST algorithm. Sequence identity between each pairwise comparison is isolated, and after each sequence in the database is analyzed, it is classified from high to low based on its sequence identity to the input sequence. The T20 score is obtained by averaging the % identity of the top 20 matching sequences.

[0165] Each antibody sequence was scored in each database using a T20 score analyzer for the chain type (VH, VK, VL) and sequence length (full length or framework only) within the "All Human Databases." After excluding the input sequence itself, T20 scores were obtained for the top 20 matched sequences (since sequence 1 was always the input antibody itself, the % identity of sequences 2-21 was averaged). The T20 scores for each group were classified from high to low. The decrease in score was generally linear for most sequences, but the T20 scores of the bottom 15% of antibodies began to drop sharply. Therefore, the bottom 15% of sequences were removed to form T20 Cutoff Human Databases with the remaining sequences (where the T20 score cutoff represents the lowest T20 score of the sequence in the new database).

[0166] The “human” antibodies used herein have a T20 human sex score of at least 79%, more preferably at least 85%.

[0167] The ability of anti-hSIRPα antibodies to block binding to CD47 In some embodiments, the anti-SIRPα antibody or its antigen-binding fragment of the present invention can block the binding of human SIRPα to human CD47. The ability to block the binding of human SIRPα to human CD47 can be measured using any method known in the art. In one embodiment, the ability of the antibody to block the binding of human SIRPα to human CD47 is measured using an ELISA assay.

[0168] Method for producing antibodies and their antigen-binding fragments Accordingly, the present invention encompasses a method for producing the anti-SIRPα antibody or its antigen-binding fragment, comprising culturing hybridoma cells expressing the antibody or fragment under conditions suitable for such expression, and optionally isolating the antibody or fragment from the hybridoma and / or growth medium (e.g., cell medium).

[0169] The anti-SIRPα antibodies disclosed herein may also be generated by recombinant means (e.g., in an E. coli / T7 expression system, a mammalian cell expression system, or a lower eukaryote expression system). In this embodiment, the nucleic acid encoding the antibody immunoglobulin molecule of the present invention (e.g., V H or V L The T7 RNA polymerase may be inserted into a pET-based plasmid and expressed in an E. coli / T7 system. For example, the present invention encompasses a method for expressing an antibody or its antigen-binding fragment or its immunoglobulin chain in a host cell (e.g., a bacterial host cell such as E. coli, such as BL21 or BL21DE3), which includes expressing T7 RNA polymerase in a cell that also contains a polynucleotide encoding a T7 RNA polymerase gene operably linked to a lac promoter, and the expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

[0170] Several methods exist for producing recombinant antibodies known in the art. One example of a method for recombinant antibody production is disclosed in U.S. Patent No. 4,816,567.

[0171] Transformation may be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, bioristic injection of DNA into the nucleus, and direct microinjection. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patents 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

[0172] Accordingly, the present invention encompasses a recombinant method for producing the anti-SIRPα antibody or its antigen-binding fragment or its immunoglobulin chain, comprising: introducing a polynucleotide encoding one or more immunoglobulin chains (e.g., heavy chain and / or light chain immunoglobulin chain) of the antibody or fragment; culturing host cells (e.g., CHO or Pichia or Pichia pastrius) under conditions suitable for such expression; and optionally isolating the antibody or fragment or chain from the host cells and / or the medium in which the host cells were grown.

[0173] Anti-SIRPα antibodies can also be synthesized by any of the methods described in U.S. Patent No. 6,331,415.

[0174] The eukaryotic and prokaryotic host cells, including mammalian cells, used as hosts for the expression of antibodies, fragments, or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, in particular, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Examples of fungal cells include Pitia pastris, Pitia finlandica, Pitia trehalophila, Pitia cocrame, Pitia membranefaciens, Pitia minuta (Ogataea minuta, Pitia lindoneri), Pitia opuntia, Pitia thermotolerance, Pitia salicaria, Pitia guelcum, Pitia pieperi, Pitia stipityis, Pitia metanorica, Pitia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hanzenula polymorpha, Cluiveromyces sp., Cluiveromyces lactis, Candida albicans, Aspergillus nijurans, Aspergillus Examples include yeast and filamentous fungal cells such as Russ niger, Aspergillus oryzae, Trichoderma resei, Chrysosporium lucnowens, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Fiscomitrella patens and Neurospora crassae, Pichia sp., any Saccharomyces sp., Hanzenula polymorpha, any Cliveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma resei, Chrysosporium lucnowens, any Fusarium sp., Yarowia liporitica, and Neurospora crassae.When a recombinant expression vector encoding a heavy chain or its antigen-binding portion or fragment, and / or a light chain or its antigen-binding fragment, is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow expression of the antibody, fragment, or chain in the host cell, or secretion into the culture medium in which the host cell is grown.

[0175] Antibodies, their antigen-binding fragments, and immunoglobulin chains can be recovered from culture media using standard protein purification methods. Furthermore, the expression of the antibodies, their antigen-binding fragments, and immunoglobulin chains (or other parts thereof) of the present invention from the generating cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under specific conditions. The GS system has been discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, 0323997, and 0338841. Thus, in one embodiment of the present invention, the mammalian host cell (e.g., CHO) lacks the glutamine synthetase gene and is grown in culture media in the absence of glutamine, but the polynucleotide encoding the immunoglobulin chain contains the glutamine synthetase gene, compensating for the gene deficiency in the host cell.

[0176] The present invention comprises a method for purifying the anti-SIRPα antibody or its antigen-binding fragment, comprising: introducing a sample containing the antibody or fragment into a purification medium (e.g., cation exchange medium, anion exchange medium, hydrophobic exchange medium, affinity purification medium (e.g., Protein-A, Protein-G, Protein-A / G, Protein-L)); and recovering the purified antibody or fragment from a flow-through fraction of the sample that does not bind to the medium; or discarding the flow-through fraction to elute the bound antibody or fragment from the medium and recovering the eluent. In one embodiment of the present invention, the medium is present in a column to which the sample is applied. In one embodiment of the present invention, the purification method is carried out following recombinant expression of the antibody or fragment in a host cell, for example, the host cell is first lysed, and optionally the lysate is purified from insoluble material before purification in the medium.

[0177] Generally, glycoproteins produced in individual cell lines or transgenic animals have glycosylation patterns characteristic of the glycoproteins produced in those cell lines or transgenic animals. Therefore, the specific glycosylation pattern of an antibody will depend on the individual cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by nucleic acid molecules provided herein, or antibodies containing amino acid sequences provided herein, are included in the present invention, independently of the glycosylation pattern they may have. Similarly, in special embodiments, antibodies with glycosylation patterns consisting only of non-fucosylated N-glycans may be advantageous because they have typically been shown to exhibit potent efficacy over fucosylated equivalents both in vitro and in vivo (see, for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patents 6,946,292 and 7,214,775). These antibodies, which contain non-fucosylated N-glycans, are unlikely to be immunogenic because their carbohydrate structures are normal components of the population present in human serum IgG.

[0178] The present invention encompasses bispecific and bifunctional antibodies and antigen-binding fragments having binding specificity to SIRPα and other antigens, such as CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, c-Met, CEA, EGFR, EpCAM, HER2, HER3, PSMA, PTHR2, mesothelin, PD-1, PD-L1, and TIM3, as well as methods for their use. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linking. For example, see Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321 and Kostelny, et al., (1992) J Immunol. 148:1547-1553. In addition, bispecific antibodies may be formed as "diabodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52). This includes "Duobody," a bispecific antibody with a normal IgG structure (Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150).

[0179] The present invention further encompasses anti-SIRPα antigen-binding fragments of anti-SIRPα antibodies disclosed herein. These antibody fragments include F(ab)2 fragments, which may be produced by enzymatic degradation of IgG, for example, with pepsin. Fab fragments may be produced by reduction of F(ab)2 with dithiothreitol or mercaptoethylamine, for example.

[0180] Immunoglobulins can be assigned to different classes according to the amino acid sequence of the constant domain of the heavy chain. In some embodiments, the different constant domains may be humanized V derived from the CDRs provided herein L and V H regions may be added. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of these can be further subclassified (isotypes), such as IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The present invention includes antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.

[0181] In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant region, such as a human constant region, such as a γ1, γ2, γ3 or γ4 human heavy chain constant region or variants thereof. In another embodiment, the antibody or antigen-binding fragment includes a light chain constant region, such as a lambda or kappa human light chain region or variants thereof, such as a human light chain constant region. By way of example and not limitation, the human heavy chain constant region can be γ4 and the human light chain constant region can be kappa. In an alternative embodiment, the Fc region of the antibody is γ4 having a Ser228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).

[0182] In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant region of the IgG1 subtype. In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant region of the IgG2 subtype. In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant region of the IgG4 subtype.

[0183] Engineering of antibodies Further embodiments include those in which the anti-SIRPα antibody and its antigen-binding fragment are engineered antibodies, which include modifications to the framework region within the antibody's variable domain to improve the properties of the antibody or fragment, for example. Typically, such framework modifications are made to reduce the immunogenicity of the antibody or fragment. This is usually accomplished by replacing non-CDR residues within the variable domain (i.e., framework residues) of the parent (e.g., rodent) antibody or fragment with similar residues in the immune repertoire of the species in which the antibody is to be used, for example, human residues in the case of human therapeutics. Such antibodies or fragments are referred to as “humanized” antibodies or fragments. In some cases, it is desirable to increase the affinity or modify the specificity of the engineered (e.g., humanized) antibody. One approach is to mutate one or more framework residues into the corresponding germline sequence. More specifically, an antibody or fragment that has undergone somatic mutation may contain framework residues different from the germline sequence from which the antibody was obtained. Such residues can be identified by comparing the framework sequence of the antibody or fragment with the germline sequence from which the antibody or fragment was obtained. Another approach is to revert one or more positions of the manipulated (e.g., humanized) antibody back to the original parent (e.g., rodent) residues, for example, to restore binding affinity that may be lost in the process of exchanging framework residues (see, for example, U.S. Patents 5,693,762, 5,585,089 and 5,530,101).

[0184] In certain embodiments, the anti-SIRPα antibody and its antigen-binding fragment are manipulated (e.g., humanized) to include modifications within the framework and / or CDR that improve their properties. Such manipulated modifications can be obtained based on molecular modeling. Molecular models for the variable region of the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and can be used to identify potential regions on the antibody that may interact with the antigen. Conventional CDRs are based on the alignment of immunoglobulin sequences and the identification of variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, MD; 5 thed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and collaborators proposed hypervariable loops by carefully examining the conformation of loops in the antibody crystal structure. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There is variation between the regions classified as "CDR" and "hypervariable loops". Subsequent studies (Raghunathan et al, (2012) J. Mol Recog. 25, 3, 103-113) analyzed multiple antibody-antigen crystal complexes and observed that the antigen-binding region within the antibody does not necessarily strictly follow the "CDR" residue or "hypervariable" loop. Molecular models for the variable region of non-human antibodies can be used to guide the selection of regions that can potentially bind to the antigen. In fact, potential antigen-binding regions based on this model differ from conventional "CDR" or "hypervariable" loops. Commercial scientific software such as Discovery Studio (BIOVIA, Dassault Systems) can be used for molecular modeling. The human framework can be selected based on the best match with non-human sequences in both the framework and the CDR. For FR4 in VH (framework 4), the VJ region of the human germline is compared with the corresponding non-human region. For FR4 in VL (framework 4), the J-kappa and J-lambda regions of the human germline sequence are compared with the corresponding non-human region. Once a suitable human framework is identified, the CDR is transplanted into the selected human framework. In some cases, certain residues within the VL-VH boundary may be retained similarly to the non-human (parent) sequence. Molecular models can also be used to identify residues that can potentially modify the CDR conformation and thereby enable antigen binding. In some cases, these residues are retained similarly to the non-human (parent) sequence.Molecular models can also be used to identify amino acids exposed to solvents that may have undesirable effects such as glycosylation, deamidation, and oxidation. These potential problems can be eliminated or minimized by introducing developmental filters early in the design phase.

[0185] Another type of framework modification involves mutating one or more residues within a framework region or within one or more CDR regions to remove a T cell epitope, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in more detail in U.S. Patent No. 7,125,689.

[0186] In certain embodiments, to avoid deamidation or isomerization, it may be desirable to replace certain amino acids containing exposed side chains with other amino acid residues to provide greater chemical stability of the final antibody. Deamidation of asparagine can occur on NG, DG, NG, NS, NA, NT, QG, or QS sequences, potentially leading to the formation of isoaspartic acid residues, which introduce kinks into the polypeptide chain and reduce its stability (isoaspartic acid effect). Isomerization may occur on DG, DS, DA, or DT sequences. In certain embodiments, the antibodies of this disclosure do not contain deamidated or asparagine isomerized sites.

[0187] For example, particularly within a CDR, asparagine (Asn) residues may be converted to Gln or Ala to reduce the likelihood of isoaspartate formation in any Asn-Gly sequence. Similar problems can occur in Asp-Gly sequences. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation may attenuate or completely block antibody binding to the target antigen. See Presta (2005) J. Allergy Clin. Immunol. 116:731, p. 734. In one embodiment, the asparagine is converted to glutamine (Gln). When small amino acids are present adjacent to asparagine or glutamine, it may be desirable to modify the amino acids adjacent to the asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation occurring at a higher rate. See Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, to reduce the possibility of methionine sulfur oxidation, which can reduce antigen-binding affinity and contribute to molecular heterogeneity in the final antibody preparation, any methionine residue in the CDR (typically Met in solvent exposure) may be converted to Lys, Leu, Ala, or Phe or other amino acids (ibid.). In addition, to prevent or minimize potential cleavage of Asn-Pro peptide bonds, it may be desirable to modify any Asn-Pro combination found in the CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Next, antibodies with such substitutions are screened to confirm that the substitutions do not reduce the antibody's affinity or specificity or other desired biological activity against SIRPα to an unacceptable level. [Table 2]

[0188] Another type of framework modification involves mutating one or more residues within a framework region to prevent aggregation. The risk of antibody aggregation can be assessed using spatial aggregation tendency. See Chennamsetty, N et al (2010) J. Phys. Chem. 114, 6614-6624. This method requires calculating the solvent exposure area (SAA) of each atom. The molecular aggregation score is then calculated as the sum of all atomic scores. For a given radius and size of the molecule, this is an approximate indicator of the overall aggregation tendency. Residues with high aggregation scores are replaced by residues with lower scores (e.g., more hydrophilic amino acids).

[0189] Antibody manipulation of the Fc region Antibodies (e.g., humanized antibodies) and their antigen-binding fragments disclosed herein can also be manipulated to modify one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or effector function (e.g., antigen-dependent cell-mediated cytotoxicity), typically including modifications within the Fc region. Furthermore, antibodies and their antigen-binding fragments disclosed herein can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody), or modified to modify their glycosylation in order to again modify one or more properties of the antibody or fragment. Each of these embodiments is described in further detail below. The numbering of residues within the Fc region is according to the Kabat EU index.

[0190] The antibodies and antigen-binding fragments disclosed herein also include antibodies and fragments having modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702. Using such modifications, various responses of the immune system can be enhanced or suppressed with possible beneficial effects in diagnosis and therapy. Modifications of the Fc region include amino acid conversion (substitution, deletion, and insertion), glycosylation or deglycosylation, and the addition of multiple Fc regions. Conversion to Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent administration and thus improving convenience and reducing material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731, pp. 734-35.

[0191] In one embodiment, the antibody or antigen-binding fragment of the present invention is an IgG4 isotype antibody or fragment containing a serine-to-proline mutation (S228P; EU index; SEQ ID NO: 66) at the position corresponding to position 228 in the hinge region of the heavy chain constant region. This mutation has been reported to eliminate heterogeneity of inter-heavy chain disulfide crosslinks within the hinge region (Angal et al (1993). Mol. Immunol. 30:105-108; position 241 is based on the Kabat numbering system).

[0192] In one embodiment of the present invention, the hinge region of CH1 is modified to increase or decrease the number of cysteine ​​residues within the hinge region. This approach is described in further detail in U.S. Patent No. 5,677,425. The number of cysteine ​​residues within the hinge region of CH1 is modified, for example, to facilitate the assembly of light and heavy chains, or to enhance or reduce the stability of the antibody.

[0193] In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the present invention is mutated to reduce the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced within the CH2-CH3 domain boundary region of the Fc-hinge fragment so that the antibody or fragment has reduced Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745.

[0194] In another embodiment, the antibody or antigen-binding fragment of the present invention is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced, as described in U.S. Patent No. 6,277,375: T252L, T254S, T256F. Alternatively, as described in U.S. Patents No. 5,869,046 and No. 6,121,022, the antibody may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope taken from two loops of the CH2 domain in the Fc region of IgG in order to increase its biological half-life.

[0195] In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be replaced with a different amino acid residue so that the antibody has a modified affinity for an effector ligand and retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260.

[0196] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced with other amino acid residues so that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cell-mediated cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551.

[0197] In another example, one or more amino acid residues at positions 231 and 239 are modified to alter the complement-fixing ability of an antibody. This approach is further described in PCT Publication WO94 / 29351.

[0198] The protein of the present invention, preferably an antibody, most preferably an IgG antibody or a fragment thereof, may have modified (for example, compared to an unmodified antibody) FcγR binding properties (examples of binding properties include binding specificity and equilibrium dissociation constant (K). D ), dissociation and binding rates (k each) off and k on Certain modifications are desirable, to varying degrees, including but not limited to binding affinity and / or avidity. Equilibrium dissociation constant (K D ) is k off / k on Defined as, K a ga K D It is known in the relevant technical field that it is the reciprocal of .

[0199] The affinity and binding properties of the Fc region for a ligand can be measured by various in vitro assays (biochemical or immunological assays) known in the art to measure the Fc-FcγR interaction, i.e., specific binding of the Fc region to FcγR, including non-limiting equilibrium assays (e.g., enzyme immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic assays (e.g., BIACORE®, Octet®, or KinExa® analysis), as well as other methods such as indirect binding assays, antagonistic inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling of one or more of the components being examined and / or may utilize various detection methods, including non-limiting colorimetric, fluorescence, luminescence, or isotopic labeling.

[0200] In certain embodiments, the protein of the present invention binds to one or more human FcγR selected from the group consisting of FcγRI, FcγRIIB, FcγRIIC, FcγRIIIA-F158, and FcγRIIIA-V158 with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times lower than an equivalent protein having a wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119) Fc domain or a wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66) Fc domain.

[0201] In various embodiments, the protein of the present invention comprises an immunoglobulin Fc region including an immunoglobulin C2 region, an immunoglobulin C3 region, and an immunoglobulin hinge region. For example, the immunoglobulin Fc region may be an IgG Fc region, an IgE Fc region, or an IgA Fc region. In a particular preferred embodiment, the protein comprises two immunoglobulin Fc regions, each immunoglobulin Fc region comprising an immunoglobulin C2 region, an immunoglobulin C3 region, and an immunoglobulin hinge region, wherein one hinge region of the immunoglobulin Fc region is bound to the hinge region of the other immunoglobulin Fc region to form a dimeric Fc structure. Most preferably, such a protein is a human or humanized IgG protein.

[0202] In certain embodiments, the protein of the present invention comprises a mutant IgG4 Fc region, preferably an IgG comprising two mutant IgG4 Fc regions to form a dimeric Fc structure. For example, the mutant IgG4 Fc region may contain one or a combination of such mutations listed in Table 3. The constant region numbering system referenced in this table is that of the EU index shown by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In this table, the first letter and number represent the unmodified amino acid and its position, and the second letter represents the substituted amino acid at that position. In cases where entries contain more than one combination of mutations, each mutation in the combination is separated by a " / ". [Table 3]

[0203] In certain embodiments, the protein of the present invention comprises a mutant IgG1 Fc region, preferably an IgG comprising two mutant IgG1 Fc regions to form a dimeric Fc structure. For example, the mutant IgG1 Fc region may contain one of the mutations listed in Table 4. The constant region numbering system referenced in this table is the EU index shown by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In this table, the first letter and number represent the unmodified amino acid and its position, and the second letter represents the substituted amino acid at that position. [Table 4] TIFF0007871364000005.tif230159TIFF0007871364000006.tif62160

[0204] In certain embodiments, the mutant IgG1 Fc region may contain one of the mutation combinations listed in Table 5. The constant region numbering system referenced in this table is that of the EU index presented by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In this table, the first letter and number represent the unmodified amino acid and its position, and the second letter represents the substituted amino acid at that position. In each of the mutation combinations of one or more, each mutation in the combination is separated by " / ", and deletions are indicated by "△". [Table 5] TIFF0007871364000008.tif25160

[0205] In certain embodiments, the protein of the present invention comprises a wild-type or mutant IgG2 Fc region, preferably an IgG comprising two wild-type or mutant IgG2 Fc regions to form a dimeric Fc structure. The mutant IgG2 Fc region may contain one or a combination of the mutations listed in Table 6. The numbering system for constant regions referenced in this table is that of the EU index shown by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In this table, the first letter and number represent the unmodified amino acid and its position, and the second letter represents the substituted amino acid at that position. In cases where entries contain more than one combination of mutations, each mutation in the combination is separated by a " / ". [Table 6]

[0206] Production of antibodies with modified glycosylation In yet another embodiment, the antibody or antigen-binding fragment of the present invention comprises a special glycosylation pattern. For example, afucosylated or aglycosylated antibody or fragment can be produced (i.e., the antibody lacks fucose or glycosylation, respectively). The glycosylation pattern of the antibody or fragment can be modified to enhance, for example, the affinity or avidity of the antibody or fragment against a SIRPα antigen. Such modifications can be carried out, for example, by modifying one or more glycosylation sites within the antibody or fragment sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such deglycosylation can enhance the affinity or avidity of the antibody or fragment against an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0207] The antibodies and antigen-binding fragments disclosed herein further include those produced in lower eukaryotic host cells, particularly fungal host cells, such as yeast and filamentous fungi, that have been genetically engineered to produce glycoproteins having mammalian-like or human-like glycosylation patterns (see, for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100:5022-5027; Hamilton et al, (2003) Science 301:1244-1246; Hamilton et al, (2006) Science 313:1441-1443; Nett et al, Yeast 28(3):237-52 (2011); Hamilton et al, Curr Opin Biotechnol. 18(5):387-92 (2007)). The special advantage of these genetically modified host cells over currently used mammalian cell lines is their ability to control the glycosylation profile of glycoproteins produced within the cell, thereby enabling the creation of glycoprotein compositions predominantly composed of specific N-glycan structures (see, e.g., U.S. Patents 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies primarily composed of specific N-glycan structures (see, e.g., Li et al., (2006) Nat. Biotechnol. 24:210-215).

[0208] In certain embodiments, the antibodies and their antigen-binding fragments disclosed herein further include those produced in inferior eukaryotic host cells, and non-limited to GlcNAc (1-4) Man3GlcNAc2;Gal (1-4) GlcNAc (1-4) Man3GlcNAc2;NANA (1-4) Gal (1-4) GlcNAc (1-4) This includes fucosylated and non-fucosylated hybrid and composite N-glycans, including bifidant and multiantennary species such as N-glycans Man3GlcNAc2.

[0209] In a particular embodiment, the antibody and its antigen-binding fragment provided herein may contain an antibody or fragment having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In a particular embodiment, the hybrid N-glycan is the dominant N-glycan species in the composition.

[0210] In a special embodiment, the antibody and its antigen-binding fragment provided herein comprises an antibody and fragment having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2;GalGlcNAcMan3GlcNAc2;NANAGalGlcNAcMan3GlcNAc2;GlcNAc2Man3GlcNAc2;GalGlcNAc2Man3GlcNAc2;Gal2GlcNAc2Man3GlcNAc2;NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In a special embodiment, the complex N-glycan is the dominant N-glycan species in the composition. In a further embodiment, the complex N-glycan is a specific N-glycan species that constitutes about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycan in the composition. In one embodiment, the antibody and its antigen-binding fragment provided herein comprises a complex N-glycan in which at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% comprises the structure NANA2Gal2GlcNAc2Man3GlcNAc2, and such a structure is afucosylated. Such a structure may be produced, for example, in engineered Pichia pastris host cells.

[0211] In a particular embodiment, the N-glycan is fucosylated. Generally, the fucose is located in an α1,3-bond to GlcNAc at the reducing end of the N-glycan, an α1,6-bond to GlcNAc at the reducing end of the N-glycan, an α1,2-bond to Gal at the non-reducing end of the N-glycan, an α1,3-bond to GlcNAc at the non-reducing end of the N-glycan, or an α1,4-bond to GlcNAc at the non-reducing end of the N-glycan.

[0212] Therefore, in a special embodiment of the above glycoprotein composition, the glycoform is selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc) in the α1,3-linkage or α1,6-linkage that produces a glycoform selected from the group consisting of GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc 1-2 )Man3GlcNAc2,GalGlcNAc2(Fuc 1-2 )Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc 1-2 )Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc 1-2 )A glycoform is generated from the group consisting of Man3GlcNAc2, in the form of an α1,3-linked or α1,4-linked structure; or Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2, NANAGal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc 1-2It is located in the α1,2-linked fucose that generates glycoforms selected from the group consisting of GlcNAc2Man3GlcNAc2.

[0213] In a further embodiment, the antibody (e.g., a humanized antibody) or its antigen-binding fragment may include, but not limited to, high-mannose N-glycans, including N-glycans consisting of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or Man3GlcNAc2 N-glycan structures.

[0214] In a further embodiment described above, the composite N-glycan further comprises fucosylated and unfucosylated bifid and polyantenna species.

[0215] As used herein, the terms “N-glycan” and “glycoform” are interchangeable and refer to N-linked oligosaccharides, such as N-linked oligosaccharides that are linked to an asparagine residue in a polypeptide via an asparagine-N-acetylglucosamine linkage. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein. The main sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (e.g., N-acetylneuraminic acid (NANA)). Processing of sugar groups occurs in the lumen of the ER simultaneously with translation, and then in the Golgi apparatus post-translation for N-linked glycoproteins.

[0216] N-glycans share a common pentasaccharide core, Man3GlcNAc2 (where "Man" refers to mannose, "Glc" to glucose, "NAc" to N-acetyl, and GlcNAc to N-acetylglucosamine). Typically, N-glycan structures are represented with the non-reducing end on the left and the reducing end on the right. The reducing end of an N-glycan is the end attached to an Asn residue containing the glycosylation site on the protein. N-glycans differ in the number of branches (antennas) containing peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) attached to the Man3GlcNAc2 ("Man3") core structure, also called the "trimannose core," "pentasaccharide core," or "pouch mannose core." N-glycans are classified according to the components of their branches (e.g., high-mannose, complex, or hybrid). "High-mannose" N-glycans have five or more mannose residues. "Complex" N-glycans typically have at least one GlcNAc bonded to the 1,3-mannose arm of a "trimannose" core and at least one GlcNAc bonded to the 1,6-mannose arm. Complex N-glycans may also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues optionally modified with sialic acid or a derivative (e.g., "NANA" or "NeuAc," where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N-glycans may also have intrachain substitutions, including "bibranched" GlcNAc and core fucose ("Fuc"). Complex N-glycans may also have multiple antennae on the "trimannose core," which are often referred to as "multi-antennae glycans." "Hybrid" N-glycans have at least one GlcNAc at the terminal end of the 1,3-mannose arm of the trimannose core and zero or more mannoses on the 1,6-mannose arm of the trimannose core. These various N-glycans are also referred to as "glycoforms."

[0217] With respect to complex N-glycans, the terms "G-2", "G-1", "G0", "G1", "G2", "A1", and "A2" mean the following: "G-2" refers to an N-glycan structure that can be characterized as Man3GlcNAc2; "G-1" refers to an N-glycan structure that can be characterized as GlcNAcMan3GlcNAc2; "G0" refers to an N-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2; "G1" refers to an N-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2; "G2" refers to an N-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2; "A1" refers to an N-glycan structure that can be characterized as NANAGal2GlcNAc2Man3GlcNAc2; and "A2" refers to an N-glycan structure that can be characterized as NANA2Gal2GlcNAc2Man3GlcNAc2. Unless otherwise indicated, the terms “G-2”, “G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan species lacking fucose bound to the GlcNAc residue at the reducing end of the N-glycan. Where the term includes “F”, “F” indicates that the N-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all indicate that the N-glycan further contains fucose bound to the GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes such as yeast and filamentous fungi do not typically produce N-glycans that generate fucose.

[0218] Regarding multi-antenna N-glycans, the term "multi-antenna N-glycan" refers to an N-glycan that further contains GlcNAc residues of mannose residues containing the non-reducing ends of the 1,6-arm or 1,3-arm of the N-glycan, or each of the GlcNAc residues of mannose residues containing the non-reducing ends of the 1,6-arm and 1,3-arm of the N-glycan. Therefore, a multi-antenna N-glycan is defined by the formula GlcNAc (2-4) Man3GlcNAc2, Gal (1-4) GlcNAc (2-4)Man3GlcNAc2, or NANA (1-4) Gal (1-4) GlcNAc (2-4) It may be characterized by Man3GlcNAc2. The term "1-4" refers to 1, 2, 3, or 4 residues.

[0219] Regarding branched N-glycans, the term "branched N-glycan" refers to an N-glycan in which a GlcNAc residue is bound to a mannose residue at the reducing end of the N-glycan. A branched N-glycan may be characterized by the formula GlcNAc3Man3GlcNAc2, where each mannose residue is bound to the non-reducing end of a GlcNAc residue. In contrast, when a multi-antenna N-glycan is characterized by GlcNAc3Man3GlcNAc2, the formula indicates that two GlcNAc residues are bound to the mannose residues at the non-reducing end of one of the two arms of the N-glycan, and one GlcNAc residue is bound to the mannose residue at the non-reducing end of the other arm of the N-glycan.

[0220] In certain embodiments, the protein of the present invention includes an aglycosylated Fc region. For example, the IgG1 Fc region may be aglycosylated by deleting or substituting residue N297.

[0221] Physical properties of antibodies The antibodies and antigen-binding fragments disclosed herein may further contain one or more glycosylation sites within the immunoglobulin variable region of either the light chain or the heavy chain. Such glycosylation sites may result in enhanced immunogenicity of the antibody or fragment, or a change in the pK of the antibody due to modification of antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing the NXS / T sequence.

[0222] Each antibody or antigen-binding fragment will have a specific isoelectric point (pI), which is generally within a pH range of 6 to 9.5. The pI of IgG1 antibodies is typically within a pH range of 7 to 9.5, while the pI of IgG4 antibodies is typically within a pH range of 6 to 8.

[0223] Each antibody or antigen-binding fragment has a characteristic melting temperature, and higher melting temperatures indicate greater overall in vivo stability (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). Generally, T M1 The initial unfolding temperature can be higher than 60°C, higher than 65°C, or higher than 70°C. The melting point of the antibody or fragment can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al (2002) J. Chromatogr Sci 40:343-9).

[0224] In further embodiments, antibodies and their antigen-binding fragments that do not degrade rapidly are selected. The degradation of the antibody or fragment can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0225] In further embodiments, antibodies and their antigen-binding fragments are selected that exhibit minimal agglutination, which may lead to the induction of an undesirable immune response and / or altered or unsuitable pharmacokinetic properties. Generally, antibodies and fragments exhibiting agglutination of ≤25%, ≤20%, ≤15%, ≤10%, or ≤5% are acceptable. Agglutination can be measured by several techniques, including size exclusion column (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0226] Antibody conjugate The anti-SIRPα antibodies and their antigen-binding fragments disclosed herein may be conjugated with a chemical moiety. The chemical moiety may, among other things, be a polymer, a radionuclide, or a cytotoxic factor. In a particular embodiment, the chemical moiety is a polymer that increases the half-life of the antibody or fragment in the body of the subject. Suitable polymers include, but are not limited to, hydrophilic polymers such as polyethylene glycol (PEG) (e.g., PEG having molecular weights of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, and monomethoxypolyethylene glycol (mPEG). Lee et al (1999) (Bioconj.Chem.10:973-981) disclose PEG-conjugated single-chain antibodies. Wen et al (2001) (Bioconj.Chem.12:545-553) disclose the conjugation of an antibody against PEG conjugated to a radioactive metal chelating agent (diethylenetriaminepentaacetic acid (DTPA)).

[0227] The antibodies and their antigen-binding fragments disclosed herein are also, for example, 99 Tc, 90 Y, 111 In, 32 P, 14 C, 125 I, 3 H, 131 I, 11 C, 15 O, 13 N, 18 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 EU, 67 CU, 217 Carbon, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th and 40 K, 157 Gd, 55 Mn, 52 Tr and 56 It may also be conjugated with markers such as Fe.

[0228] The antibodies and antigen-binding fragments disclosed herein may also be PEGylated, for example, to increase their biological (e.g., serum) half-life. To PEGylate an antibody or fragment, the antibody or fragment is typically reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups bind to the antibody or antibody fragment. In specific embodiments, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term “polyethylene glycol” as used herein encompasses any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In specific embodiments, the antibody or fragment to be PEGylated is an aglycosylated antibody or fragment. Methods for PEGylation of proteins are known in the art and may be applied to the antibodies of the present invention. See, for example, EP0154316 and EP0401384.

[0229] The antibodies and antigen-binding fragments disclosed herein also include rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescein, 152 They may be conjugated with fluorescent or chemiluminescent labels, including fluorophores such as Eu, dansyl, umbelliferone, luciferin, luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridimium salt labels, oxalate ester labels, aequorin labels, 2,3-dihydrophthalazinedione, biotin / avidin, spin labels, and stable free radicals.

[0230] The antibodies of the present invention and their antigen-binding fragments may also be conjugated to cytotoxic factors such as diphtheria toxin, Pseudomonas erginosa exotoxin A chain, lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein and compounds (e.g., fatty acids), dianthin protein, Phytoiacca americana protein PAPI, PAPII and PAP-S, Momordica caranchia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogenin, restrictosin, phenomycin and enomycin.

[0231] Any method known in the art for conjugating the antibody and its antigen-binding fragment of the present invention to various parts may be used, including the methods described in Hunter et al., (1962) Nature 144:945; David et al., (1974) Biochemistry 13:1014; Pain et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J. (1982) Histochem. and Cytochem. 30:407. The methods for conjugating the antibody and fragment are conventional and well known in the art.

[0232] Therapeutic use of anti-SIRPα antibodies Also provided is a method for treating subjects such as human subjects that require treatment with isolated antibodies or antigen-binding fragments disclosed herein. In one embodiment of the present invention, such subjects are infected or suffering from an infectious disease.

[0233] In another embodiment of the present invention, such subjects are suffering from cancer. In one embodiment, the cancer is, for example, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilms' cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, kidney These include rhabdoid tumors of the viscera, Ewing's sarcoma, chondrosarcoma, brain tumors, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumors, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, or liver cancer, breast cancer, or gastric cancer. In one embodiment of the present invention, the cancer is, for example, a metastatic cancer of one of the various cancers described above.

[0234] Cancers that can be treated by the antibody or antigen-binding fragment, composition and method of the present invention include: heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma and teratoma; lung: bronchogenic carcinoma (squamous cell carcinoma, anaplastic small cell carcinoma, anaplastic large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal tract: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (cancer, lymphoma, leiomyoma), pancreas (tubular adenocarcinoma, insulinoma, glucagonoma, gastrinocarcinoma) Malignant tumors (carcinoid tumors, bipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colorectal tract; urogenital system: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testes (seminoma, teratoma, fetal carcinoma, malignant teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma); liver: hepatoma (liver Cellular carcinoma, intrahepatic cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticuloma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondrosis), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; nervous system: skull (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), meninges (meningioma, meningiosarcoma, glioma), brain (astrocytoma, medulloblastoma, glioma, ependymocyte tumor, germ cell tumor [pineal gland tumor], pleomorphoma) Gliablastoma, oligodendroglioma, Schwann cell tumor, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma; Gynecology: Uterus (endometrial cancer), cervix (cervical cancer, preneoplastic cervical dysplasia), ovaries (ovarian cancer [serous adenocarcinoma, mucinous adenocarcinoma, unclassified cancer], granulosa-meningiocyte tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, staphyloid sarcoma (fetal rhabdomyoma), fallopian tube (cancer), breast;Hematology: Blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin lymphoma [malignant lymphoma]; Skin: Malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and Adrenal gland: Neuroblastoma, but not limited to these. Therefore, the term “cancer cell” as provided herein encompasses cells affected by any one of the previously identified conditions.

[0235] In one embodiment, cancers that can be treated by the antibodies or antigen-binding fragments disclosed herein, the compositions and methods of the present invention include breast cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, osteosarcoma, neuroblastoma, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, lung cancer, squamous cell carcinoma, melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, kidney cancer, renal cell carcinoma, thyroid cancer, glioblastoma multiforme, fallopian tube cancer, peritoneal cancer, and angiosarcoma. Hepatocellular carcinoma, choriocarcinoma, soft tissue sarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia, T-cell lymphoma, natural killer cell lymphoma, extranodal marginal zone B-cell lymphoma, acute lymphocytic leukemia, and multiple myeloma are examples, but are not limited to these.

[0236] In one embodiment, antibodies or antigen-binding fragments disclosed herein may be used for the treatment of infections and infectious diseases. As used herein, “infection” refers to any state in at least one cell of an organism (e.g., a subject) infected with an infectious agent (e.g., the subject has an intracellular pathogenic infection, e.g., a chronic intracellular pathogenic infection). As used herein, “infectious agent” refers to an alien organism (i.e., a pathogen) that induces CD47 expression (e.g., increased CD47 expression) in at least one cell of an infected organism. Examples of infectious agents include, but are not limited to, bacteria, viruses, protozoa, and fungi.

[0237] Intracellular pathogens are particularly relevant. Infectious diseases are disorders induced by infectious agents. Some infectious agents can induce unrecognizable syndromes or diseases under certain conditions and can induce syndromes or diseases with changes in conditions. The methods of the present invention can be used, non-limitingly, to the treatment of chronic pathogenic infections, including, but not limited to, viral infections such as retroviruses, lentiviruses, hepadnaviruses, herpesviruses, poxviruses, and human papillomavirus; intracellular bacterial infections such as Mycobacterium, Chlamydophila, Erlitia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp., Helicobacter pylori, and intracellular protist pathogens such as Plasmodium sp., Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmania sp., and others.

[0238] In one embodiment, the present invention provides a method for treating a subject with the anti-SIRPα antibody or an antigen-binding fragment thereof, wherein the subject is suffering from a viral infection. In one embodiment, the viral infection is caused by a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis viruses (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus.

[0239] In one embodiment, the present invention provides a method for treating a subject with the anti-SIRPα antibody or an antigen-binding fragment thereof, wherein the subject is suffering from a bacterial infection. In one embodiment, the bacterial infection is caused by bacteria selected from the group consisting of Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio corere, Clostridium tetan, Clostridium botulinum, Bacillus anthrissis, Yersinia pestis, Mycobacterium leple, Mycobacterium lepromatosis and Boriera.

[0240] In one embodiment, the present invention provides a method for treating a subject with an anti-SIRPα antibody or an antigen-binding fragment thereof, wherein the subject is suffering from a fungal infection. In one embodiment, the fungal infection is caused by a fungus selected from the group consisting of Candida (e.g., Candida albicans, Candida glabrata, Candida tropicalis), Cryptococcus neoformans, Aspergillus (e.g., Aspergillus fumigatus, Aspergillus niger), Mucorales (e.g., Mucor, Aspergillus abscisia, Aspergillus rhizopsus), Sporosricus schenkyi, Blastomyces dermatichidis, Paracoccidioides brassiliensis, Coccidioides imitis, and Histoplasma capsulatum.

[0241] In one embodiment, the present invention provides a method for treating a subject with an anti-SIRPα antibody or an antigen-binding fragment thereof, wherein the subject is suffering from a parasitic infection. In one embodiment, the parasitic infection is caused by a parasite selected from the group consisting of Entermoeva historia, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microttii, Trypanosoma bursei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nipostronjirus brassiliensis.

[0242] The "subject" may be a mammal such as a human, dog, cat, horse, cattle, mouse, rat, monkey (e.g., cynomolgus macaque, e.g., Macaca fasciculis), or rabbit. In a preferred embodiment of the present invention, the subject is a human subject.

[0243] The term "in conjunction with" indicates that the components administered in the method of the present invention (e.g., an anti-SIRPα antibody (e.g., a humanized antibody) or its antigen-binding fragment accompanied by an anticancer agent) may be formulated in a single composition for simultaneous delivery, or they may be formulated separately in two or more compositions (e.g., a kit). Each component may be administered to the subject at a different time than the other components are administered, for example, each administration may be carried out asynchronously (e.g., separately or sequentially) at multiple intervals over a given period. Furthermore, the other components may be administered to the subject via the same or different routes.

[0244] In particular embodiments, the antibodies or antigen-binding fragments disclosed herein may be used alone or in conjunction with other further therapeutic agents and / or therapeutic procedures to treat or prevent any disease, such as cancer, as discussed herein, in subjects requiring such treatment or prevention. Compositions comprising such antibodies and fragments in relation to further therapeutic agents, such as pharmaceutical compositions comprising a pharmaceutically acceptable carrier, are also part of the present invention.

[0245] Therefore, the present invention provides a method for treating cancer in a human subject, comprising administering an effective amount of the antibody or antigen-binding fragment disclosed herein to the subject, optionally in conjunction with further therapeutic agents or treatment procedures. The present invention also provides a method for treating infection or infectious disease in a human subject, comprising administering an effective amount of the antibody or antigen-binding fragment disclosed herein to the subject, optionally in conjunction with further therapeutic agents or treatment procedures. The present invention also provides a method for increasing the activity of immune cells, comprising administering an effective amount of the antibody or antigen-binding fragment disclosed herein to a subject requiring such action. In one embodiment, the method is used for the treatment of cancer, infection or infectious disease, or as a vaccine adjuvant.

[0246] In specific embodiments, the antibodies or antigen-binding fragments disclosed herein may be used alone or in conjunction with oncology vaccines. Examples of oncology vaccines include: vaccines for human papillomavirus (HPV) infection such as Gardasil®, Gardasil9®, and Cervarix®; vaccines for preventing hepatitis B virus-induced liver cancer such as Engerix-B® and Recombivax HB®; oncolytic virus therapies that induce an immune response such as Imlygic®; DNA vaccines such as Synchotrope MA2M plasmid DNA vaccine and ZYC101; mammoglobin-a DNA vaccine (see Clinical Cancer Res. 2014 20(23):5964-75); vector-based vaccines such as PSA-TRICOM (prostvac) and PANVAC-VF; and vaccines based on Listeria monocytogenes (e.g., Therapeutic Advances in Vaccines, 2014). See 2(5)137-148), Listeria-based vaccines (Listeria expressing one or more cancer vaccines such as Listeria mesothelin (e.g., CRS-207), ADXS-HPV, AXALIMODINE Phyllorisvac, Listeria-HER2 / Neu, Listeria-EGFRvIII); Adeno-CEA; GVAX, BLP-25 (anti-ankaramin 1), Bellagen Pumazucel-L, TG4010, CIMAvax epidermal growth factor vaccine, NY-E Examples of allogeneic vaccines include SO and GM.CD40L-CCL21; autologous vaccines such as Adeno-CD40L, BCG, and INGN-225; dendritic cell vaccines such as Provenge® (Cipuroisel-T) and rF-CEA-MUC1-TRICOM (Panvac-DC); and antigenic vaccines such as MUC-1 (Stimuvax), NY-ESO-1, GP-100, MAGE-A3 (gene A3 encoding melanoma antigen), and INGN-225 (see Pharmacology & Therapeutics 153 (2015) 1-9). However, these are not limited to these.

[0247] The Eat Me signal can be increased, non-limitingly, by cytotoxic therapies such as radiotherapy and chemotherapeutic agents, including anthracyclines (doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone), oxaliplatin, bortezomib, cyclophosphamide, bleomycin, vorinostat, paclitaxel, 5-fluorouracil, cytarabine, prednisolone, docetaxel, mitomycin C, topotecan / camptothecin, etoposide, zoledronic acid, methotrexate, ibrutinib, aflibercept, bevacizumab, toremifene, vinblastine, vincristine, idelalisib, mercaptopurine, satidomid, and sorafenib. Therefore, in certain embodiments, the antibodies or antigen-binding fragments disclosed herein may be used in conjunction with chemotherapeutic agents, in conjunction with radiotherapy, etc. In a particular embodiment, the antibodies or antigen-binding fragments disclosed herein may be used alone or in conjunction with targeted therapy.Examples of targeted therapies include hormone therapy, signaling inhibitors (e.g., EGFR inhibitors such as cetuximab (Erbitux) and erlotinib (Tarceva)); CD20 inhibitors (e.g., rituximab (Rituxan) and afatumumab (Arzerra)); and CD38 inhibitors (e.g., daratumumab (DARZALEX)). CD52 inhibitors (e.g., alemtuzumab (Campath)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (e.g., imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (e.g., crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (e.g., vemurafenib (Zelboraf) and dabrafenib (Tafinlar)); gene expression regulators (e.g., decitabine (Dacogen)) Examples include vorinostat (Zolinza), apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)), immunomodulatory imides (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast), and monoclonal antibodies conjugated to toxins (e.g., brentuximab vedotin (Adcetris) and adtrastuzumab emtansine (Kadcyla)).

[0248] The antibodies or antigen-binding fragments disclosed herein may preferably find applications related to targeted therapies mediated by ADCC / ADCP using the antibodies. Functional bioassays can be used to analyze the mechanism of action of antibody drugs and to distinguish ADCP from ADCC as a mechanism of action. As an example, antibody-dependent cell-mediated cytotoxicity (ADCC) assays typically utilize normal human peripheral blood mononuclear cells (PBMCs) or effector cells isolated therefrom. The variability of the assay can be reduced by using a selective donor pool that includes defined copy number variations (CNVs) of the Fcγ receptor IIa (FcγRIIa / CD32a), IIIa (FcγRIIIa / CD16a), or IIIb (FcγRIIIb / CD16b) gene, or genotypes such as FcγRIIIa-158 V / V for V / F or F / F, FcγRIIIa-131 H / H for H / R or R / R, and FcγRIIIb-NA1 and -NA2 polymorphic variants. Alternatively, effector cells such as PBMCs, PBMC-derived natural killer (NK) cells, granulocytes, monocytes, monocyte-derived macrophages, or dendritic cells (DCs) can be replaced with FcγRIIIa-expressing cell lines (e.g., engineered NK92). Killing of target cells is 51 Chromium (Cr 51 This can be assessed by measuring the release of a specific probe from pre-labeled target cells using a fluorescent dye, such as calcein-acetoxymethyl (calcein-AM), carboxyfluorescein succinimidyl ester (CFSE), 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), europium (Eu), or propidium iodide (PI), or by measuring the release of cytosolic enzymes such as lactate dehydrogenase (LDH) or nucleoside triphosphate (ATP).

[0249] In contrast, antibody-dependent phagocytosis (ADCP) may be assessed by measuring the destruction of target cells via granulocyte, monocyte, dendritic cell, or macrophage-mediated phagocytosis. ADCP assays utilize PBMC-derived cells or myeloid cell lines such as HL-60, THP-1, and U937 cells that differentiate into macrophages or granulocytes. Common stimuli used to induce macrophage differentiation in monocyte cell lines include phorbol-12-myristo-13-acetate (PMA), 1,25-dihydroxyvitamin D3 (VD3), and retinoic acid (RA). RA is also known to induce terminal granulocyte differentiation in HL-60 cells, for example. Target cell phagocytosis can be assessed by monitoring effector cells for the internalization of specific probes from target cells pre-labeled with fluorescent dyes such as the cell proliferation dye eFluor450 and CFSE, as well as pH-sensitive dyes including pHrodo and CypHer5E. Phagocytosis is measured by the increase in fluorescently labeled effector cells using flow cytometry or fluorescence microscopy. A "reporter gene" assay is also available to assess ADCP. To measure ADCP function in a reporter gene assay, target cells are initially incubated with a corresponding antibody at a constant titer. Once the antibody has bound to a congeneral target on the surface of the target cells, engineered Jurkat effector cells are added. If activation of the ADCP pathway occurs as a result, the Jurkat cells produce a luciferase product by expressing the reporter gene NFAT-RE-luc2. Subsequently, luciferase activity is measured after the addition of the luciferase assay reagent, following an induction period of 4–24 hours. Dose-dependent responses in microtiter plate-based assays can be used to quantify the relative bioactivity of therapeutic antibodies by comparing them to a dose-dependent curve of a suitable reference substance.

[0250] In a particular embodiment, the anti-SIRPα antibody or its antigen-binding fragment of the present invention may be used in combination with an immunomodulatory agent such as an anticancer drug or an immunomodulatory receptor inhibitor, for example, an antibody or its antigen-binding fragment that specifically binds to a receptor.

[0251] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen binding is TNF receptor protein agonists (e.g., agonistic antibodies or their antigen-binding fragments, or soluble fusions), immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, Toll-like receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D , ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD1 8, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACA Ligands that specifically bind to Ml, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), SLAM7, BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, PAG / Cbp, CD19a, and CD83; or Inhibitors of CD47, PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, CEACAM (e.g., CEACAM-1, -3 and / or -5), VISTA, BTLA, TIGIT, LAIR1, IDO, TDO, CD160 and / or TGFR beta. It will be linked with one or more of the following.

[0252] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with one or more cyclic dinucleotides (dinculeotides) or other STING pathway agonists. STING (an interferon gene stimulant also known as TMEM173, MITA, ERIS, and MPYS) is an ER-localized transmembrane protein that undergoes conformational changes in response to direct binding of cyclic dinucleotides (CDNs), resulting in a downstream signaling cascade including TBK1 activation, IRF-3 phosphorylation, and production of IFN-β and other cytokines. In tumor-resident host antigen-presenting c3ellss, the STING pathway is involved in inducing a spontaneous CD8+ T cell response to tumor-derived antigens. Activation of this pathway and subsequent IFN-β production also reportedly contribute to the antitumor effect of radioactivity. STING agonists and their use are, for example, US20060040887, US20080286296, US20120041057, US20140205653, WO2014179335, WO2014179760, US20150056224, WO2015185565, WO2016096174, WO2016145102, WO2017011444, WO2017027645, Listed in WO2017027646, WO2017123657, WO2017123669, WO2017175147, WO2017175156, WO2018045204, WO2018009648, WO2018006652, WO2018013887, WO2018013908, US20180002369, US20180092937, and US20180093964.

[0253] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is an anti-CD47 antibody, an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PDL1 antibody, an anti-TIGIT antibody, an anti-APRIL antibody, an anti-CTLA4 antibody, an anti-CS1 antibody (e.g., elotuzumab), an anti-KIR2DL1 / 2 / 3 antibody (e.g., lirilumab), an anti-CD137 antibody (e.g., urelumab), an anti-GITR antibody (e.g., TRX518), an anti-PD-L1 antibody (e.g., BMS-936559, MSB0010718C or MPDL3280A), an anti-PD-L2 antibody, an anti-ILT1 antibody, an anti-ILT2 antibody, an anti-ILT3 antibody, an anti-ILT It can be linked with one or more of the following small organic molecule inhibitors of such targets: 4 antibodies, anti-ILT5 antibodies, anti-ILT6 antibodies, anti-ILT7 antibodies, anti-ILT8 antibodies, anti-CD40 antibodies, anti-OX40 antibodies, anti-ICOS, anti-KIR2DL1 antibodies, anti-KIR2DL2 / 3 antibodies, anti-KIR2DL4 antibodies, anti-KIR2DL5A antibodies, anti-KIR2DL5B antibodies, anti-KIR3DL1 antibodies, anti-KIR3DL2 antibodies, anti-KIR3DL3 antibodies, anti-NKG2A antibodies, anti-NKG2C antibodies, anti-NKG2E antibodies, anti-4-1BB antibodies (e.g., PF-05082566), anti-TSLP antibodies, anti-IL-10 antibodies, IL-10 or PEGylated IL-10, or any other small organic molecule inhibitors of such targets.

[0254] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is linked with an anti-CD20 antibody (e.g., rituximab, ofatumumab, ocrelizumab, obinutuzumab, okalatuzumab, ubrituximab, vertuzumab, ibritumomab / tiuxetan, tositumomab, BVX-20, SCT-400, or PRO131921).

[0255] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD38 antibody (e.g., daratumumab, isatuximab, or MOR202).

[0256] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-EGFR antibody (e.g., cetuximab, CetuGEX, panitumumab, nimotuzumab, depatuxizumab, or AFM-21).

[0257] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-HER2 antibody (e.g., trastuzumab, TrasGEX, pertuzumab, margetuximab, or ADCT-502).

[0258] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-HER3 antibody (e.g., lumuletuzumab, patrizumab, or LJM716).

[0259] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD19 antibody (e.g., ricebilizumab, blinatumomab, DI-B4, MDX-1342, MEDI-551, MOR208, or 4-G7SDIE).

[0260] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD52 antibody (e.g., alemutuzumab).

[0261] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-EpCAM antibody (e.g., adecatumumab, catumaxomab, edrecolomab, or ING-1).

[0262] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-SLAMF7 antibody (e.g., elotuzumab or ABBV-838).

[0263] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab).

[0264] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-PD-L1 antibody (e.g., BMS-936559, MSB0010718C, or MPDL3280A).

[0265] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CTLA4 antibody (e.g., ipilimumab or tremelimumab).

[0266] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD137 antibody (e.g., urelumab).

[0267] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-GITR antibody (e.g., TRX518 or FPA154).

[0268] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-OX40 antibody (e.g., MEDI6469, MOXR0916, or INCAGN1949).

[0269] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD40 antibody (e.g., lucatumumab, dacetuzumab, APX005M, ChiLob7 / 4, CP-870, 893, or JNJ-64457107). In another embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CS1 antibody.

[0270] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR2DL1 / 2 / 3 antibody.

[0271] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD137 antibody (e.g., urelumab).

[0272] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-GITR (e.g., TRX518) antibody.

[0273] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-PD-L2 antibody.

[0274] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL1 antibody.

[0275] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL2 antibody.

[0276] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL3 antibody.

[0277] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL4 antibody.

[0278] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL5 antibody.

[0279] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL6 antibody.

[0280] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL7 antibody.

[0281] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ITL8 antibody.

[0282] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-CD40 antibody.

[0283] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-OX40 antibody.

[0284] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR2DL1 antibody.

[0285] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR2DL2 / 3 antibody.

[0286] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR2DL4 antibody.

[0287] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR2DL5A antibody.

[0288] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with the anti-KIR2DL5B antibody.

[0289] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR3DL1 antibody.

[0290] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR3DL2 antibody.

[0291] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-KIR3DL3 antibody.

[0292] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-NKG2A antibody.

[0293] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-NKG2C antibody.

[0294] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-ICOS antibody.

[0295] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-4-1BB antibody.

[0296] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-IL-10 antibody.

[0297] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-TSLP antibody.

[0298] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is coupled with an anti-IL-10 or PEGylated IL-10 antibody.

[0299] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is linked with one or more inhibitors (e.g., small organic molecules or antibodies or their antigen-binding fragments) such as MTOR (mammalian target of rapamycin) inhibitors, cytotoxic agents, platinum agents, EGFR inhibitors, VEGF inhibitors, microtubule stabilizers, taxanes, CD20 inhibitors, CD52 inhibitors, CD30 inhibitors, RANK (receptor activator for nuclear factor kappa-B) inhibitors, RANKL (receptor activator for nuclear factor kappa-B ligand) inhibitors, ERK inhibitors, MAP kinase inhibitors, AKT inhibitors, MEK inhibitors, PI3K inhibitors, HER1 inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, Bcl2 inhibitors, CD22 inhibitors, CD79b inhibitors, ErbB2 inhibitors, or farnesyl protein transferase inhibitors.

[0300] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is 13-cis-retinoic acid, 3-[5-(methylsulfonylpiperazine methyl)-indolyl]-quinolone, 4-hydroxytamoxifen, 5-deoxyuridine, 5'-deoxy-5-fluorouridine, 5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654, abiraterone acetate, abraxane, ABT-578, acorbifen, ADS-100380, ALT-110, A Lutretamin, Amifostin, Aminoglutethimide, Amrubicin, Amsacrin, Anagrelide, Anastrozole, Angiostatin, AP-23573, ARQ-197, Alzoxifen, AS-252424, AS-605240, Asparaginase, AT-9263, Atrasentan, Axitinib, AZD1152, Calmette-Guérin (BCG) vaccine, Batablin, BC-210, Besodutox, Bevacizumab, Bicalutamide, Bio111, BIO140, Bleomycin, BMS-214662, BM S-247550, BMS-275291, BMS-310705, Bortezomib, Buserelin, Busulfan, Calcitriol, Camptothecin, Canertinib, Capecitabine, Carboplatin, Carmustine, CC8490, Cedilanib, CG-1521, CG-781, Chlamydosine, Chlorambucil, Chlorotoxin, Silenditide, Cimitidine, Cisplatin, Cladribine, Clodronate, COL-3, CP-724714, Cyclophosphamide, Cyproterone, Cyproterone Acetate, Cytarabine, Cytosine Alkaline Vinoside, dacarbazine, dacinostat, dactinomycin, darotuzumab, danucertib, dasatanib, daunorubicin, decatanib, deguerin, denileukin, deoxycoformycin, depsipeptide, diallylpropionitrile, diethylstilbestrol, difutitox, docetaxel, dovitinib, doxorubicin, droroxifene, edotecarin, yttrium-90 labeled edtreotide, edtreotide, EKB-569, EMD121974, endostatin, enzalutamide,Enzastaurin, Epirubicin, Epithilone B, ERA-923, Erbitux, Erlotinib, Estradiol, Estramustine, Etoposide, Everolimus, Exemestane, Ficlatuzumab, Finasteride, Flavopyridol, Fulocuridine, Fludarabine, Fludrocortisone, Fluoxymesterone, Flutamide, FOLFOX regimen, Fulvestrant, Galeterone, Gefitinib, Gemcitabine, Jaimatecan, Goserelin, Goserelin acetate, Gossypol, GSK461364, GSK690693, HMR-3339, Hydroxycaproate Cyprogesterone, Hydroxyurea, IC87114, Idarubicin, Idoxifene, Ifosfamide, IM862, Imatinib, IMC-1C11, INCB24360, INO1001, Interferon, Interleukin-12, Ipilimumab, Irinotecan, JNJ-16241199, Ketoconazole, KRX-0402, Thalidomide, Lenalidomide, Pomalidomide, Apremilast, Lapatinib, Lasofoxifene, Letrozole, Leucovorin, Leuprolide, Leuprolide Acetate, Levamysol, Liposa Paclitaxel, Lomustine, Ronafarnib, Lucanton, LY292223, LY292696, LY293646, LY293684, LY294002, LY317615, Marimast, Mechloretamine, Medroxyprogesterone acetate, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitramycin, Mitomycin, Mitotan, Mitoxantrone, Tozacertib, MLN8054, Neovastat, Neratinib, Neurabrab, Nilotinib, Nil Nilutimide, Noratexed, NVP-BEZ235, Oblimersen, Octreotide, Ofatumumab, Olegobomab, Orteronel, Oxaliplatin, Paclitaxel, Palbociclib, Pamidronate, Panitumumab, Pazopanib, PD0325901, PD184352, PEG-Interferon, Pemetrexed, Pentostatin, Perifosine, Phenylalanine Mustard, PI-103, Pictilisib, PIK-75, Pipendoxifene, PKI-166, Plicamycin, Porfimer,Prednisone, procarbazine, progestins, PX-866, R-763, raloxifene, larcitrexed, razoxin, ridafololimus, rituximab, romidepsin, RTA744, rubitecan, scriptaid, Sdx102, celicyclib, selumetinib, semacusanib, SF1126, sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668, streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib, synthetic estrogen, talampanel, tarimodine / laharpalepbec, tamoxifen, temozolomide, temsirolimus, teniposide, tesmirifen, testosterone, tetrandrin, T GX-221, thalidomide, thioguanine, thiotepa, tremelimumab, tipifarnib, tivozanib, TKI-258, TLK286, topotecan, toremifene citrate, trabectedin, trastuzumab, tretinoin, trichostatin A, trisilibine phosphate monohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproic acid, valul It is used in conjunction with one or more of the following: Bicin, vandetanib, batalanib, VEGF trap, vinblastine, vincristine, vindesine, vinorelbine, vitaxin, vitespan, vorinostat, VX-745, wartmannin, Xr311, zanorimumab, ZK186619, ZK-304709, ZM336372, and ZSTK474.

[0301] Suitable anticancer agents to be used in combination with the anti-SIRPα antibody or its antigen-binding fragment of the present invention include, but are not limited to, cell division inhibitors, immunomodulatory imides, cytotoxic agents, and targeted therapies (small molecules, biologics, siRNA and microRNA) for cancer and tumor diseases. 1) Antimetabolites (methotrexate, 5-fluorouracil, gemcitabine, fludarabine, capecitabine, etc.) 2) Alkylating agents, such as temozolomide, cyclophosphamide, 3) DNA interaction and DNA damaging agents, e.g., cisplatin, oxaliplatin, doxorubicin, 4) Ionizing radiation, for example, radiation therapy, 5) Topoisomerase II inhibitors, e.g., etoposide, doxorubicin, 6) Topoisomerase I inhibitors, e.g., irinotecan, topotecan, 7) Tubulin interaction agents, such as paclitaxel, docetaxel, abraxane, epotilon, 8) Kinesin spindle protein inhibitors, 9) Spindle checkpoint inhibitors, 10) Poly(ADP-ribose) polymerase (PARP) inhibitors, e.g., olaparib, MK-4827 and veliparib, 11) Matrix metalloproteinase (MMP) inhibitors, 12) Protease inhibitors, such as cathepsin D and cathepsin K inhibitors, 13) Proteosome or ubiquitination inhibitors, e.g., bortezomib, 14) Activators of mutant p53 that restore wild-type p53 activity, 15) Adenobiral-p53, 16) Bcl-2 inhibitors, e.g., ABT-263, 17) Heat shock protein (HSP) modifiers, e.g., geldanamycin and 17-AAG, 18) Histone deacetylase (HDAC) inhibitors, such as vorinostat (SAHA), 19) Sex hormone regulators, a. Anti-estrogens, e.g., tamoxifen, fulvestrant, b. Selective estrogen receptor modulators (SERMs), e.g., raloxifene c. Antiandrogens, e.g., bicalutamide, flutamide, d. LHRH agonists, for example, leuprolide, e. 5α-reductase inhibitors, such as finasteride, f. Cytochrome P450 C17 lyase (also known as CYP450c17 or 17αC), g. Aromatase inhibitors, e.g. letrozole, anastrozole, exemestane, 20) EGFR kinase inhibitors, e.g., geftinib, erlotinib, laptinib, 21) Dual erbB1 and erbB2 inhibitors, e.g., lapatinib, 22) Multi-target kinase (serine / threonine and / or tyrosine kinase) inhibitors, a. ABL kinase inhibitors, imatinib and nilotinib, dasatinib, b. VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK, and ERK inhibitors, e.g., sunitinib, sorafenib, vandetanib, pazopanib, PLX-4032, axitinib, PTK787, GSK-1120212, c. Polo-like kinase inhibitors, d. Aurora kinase inhibitors, e.JAK inhibitors, fc-MET kinase inhibitors, g. Cyclin-dependent kinase inhibitors, such as the CDK1 and CDK2 inhibitor dinaciclib SCH727965 (see Parry et al, Molecular Cancer Therapeutics 9(8):2344-53(2010)) and CDK4 / 6 inhibitors, such as ribociclib, palbociclib, abemaciclib and trilaciclib. h.PI3K and mTOR inhibitors, e.g., GDC-0941, BEZ-235, BKM-120 and AZD-8055, i. Rapamycin and its analogues, e.g., temsirolimus, everolimus and deforolimus, 23) and other anticancer drugs (also known as antitumor drugs), for example, non-limitedly, Ala-C, Adriamycin, Cytoxan, Carboplatin, Uracil mustard, Chlormetine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, Furoxuridine, Cytarabine, 6-Me Lucaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, vinblastine, vincristine, vindesine, vinorelbine, navelbine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, teniposide, cytarabine, pemetrexed, idarubicin, mitramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide, ethinylestradiol Diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianicene, hydroxyprogesterone, aminoglutethimide, estramustine, flutamide, medroxyprogesterone acetate, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisol, drolloxafine, hexamethylmelamine, Bexxar, Zevalin, Trisenox, Profimer, thiotepa, altoretamine, Doxil, Ontak, Depocyt, Aranesp, Neupogen, Neulasta, Kepivance, 24) Farnesyl protein transferase inhibitors, e.g., SARASAR™ (4-(2-(4-((11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine-11-yl-]-1-piperidinyl]-2-oxoethyl]-piperidinecarboxamide), tipifarnib, 25) Interferons, e.g., Intron A, Peg-Intron, 26) Anti-erbB1 antibodies, e.g., cetuximab, panitumumab, 27) Anti-erbB2 antibodies, e.g., trastuzumab, 28) Anti-CD52 antibodies, e.g., alemtuzumab, 29) Anti-CD20 antibodies, e.g., rituximab, 30) Anti-CD33 antibodies, e.g., gemtuzumab ozogamicin, 31) Anti-VEGF antibodies, e.g., Avastin, 32) TRIAL ligands, e.g., lexatumumab, mapatumumab, and AMG-655, 33) Anti-CTLA-4 antibodies, e.g., ipilimumab, 34) Antibodies against CTA1, CEA, CD5, CD19, CD22, CD30, CD44, CD44V6, CD55, CD56, EpCAM, FAP, MHCII, HGF, IL-6, MUC1, PSMA, TAL6, TAG-72, TRAILR, VEGFR, IGF-2, and FGF. 35) Anti-IGF-1R antibodies, e.g., dalotuzumab (MK-0646) and lobatumumab (SCH717454), These are some examples.

[0302] "Estrogen receptor modulators" refer to compounds that interact with or inhibit estrogen binding to receptors, regardless of the mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifen, idoxifen, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4'-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

[0303] "Androgen receptor modulators" refer to compounds that interact with or inhibit androgen binding to receptors, regardless of the mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, rialozol, and abiraterone acetate.

[0304] "Retinoid receptor modifiers" refer to compounds that interact with or inhibit retinoid binding to receptors, regardless of the mechanism. Examples of such retinoid state modifiers include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, α-difluoromethylornithine, ILX23-7553, trans-N-(4'-hydroxyphenyl)retinamide, and N-4-carboxyphenylretinamide.

[0305] "Cytotoxicity / cell division inhibitors" refer to compounds that induce cell death or inhibit cell proliferation, primarily by directly interfering with cell function or inhibiting or interfering with cell mitosis. These include alkylating agents, tumor necrosis factors, intercalators, hypoxia-activating compounds, microtubule inhibitors / microtubule stabilizers, mitotic kinesin inhibitors, histone deacetylase inhibitors, kinase inhibitors involved in the progression of mitosis, kinase inhibitors involved in growth factor and cytokine signaling pathways, antimetabolites, bioresponse modifiers, hormone / antihormone drugs, hematopoietic growth factors, monoclonal antibody-targeted therapies, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

[0306] Examples of cytotoxic / cell division inhibitors include platinum-coordinate compounds, certenef, kaketin, ifosfamide, tasonelmin, ronidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosylate, trophosphamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satoraplatin, prophylromycin, cisplatin, ilofluben, dexyphosphamide, cis-amine dichloro(2-methylpyridine)platinum, benzylguanine, glucosphamide, GPX100, (trans,trans,trans)bis-mu-(hexane-)tetrachloride Examples include, but are not limited to, 1,6-diamine)-mu-[diamineplatinum(II)]bis[diamine(chloro)platinum(II)], diariridinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, solubicin, idarubicin, daunorubicin, bisanthren, mitoxantrone, pirarubicin, pinafide, barurubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, erinafide, MEN10755, and 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulfonyl-daunorubicin (see WO00 / 50032).

[0307] An example of a hypoxia-activating compound is tirapazamine.

[0308] Examples of proteosome inhibitors include, but are not limited to, lactacystin and MLN-341 (Velcade).

[0309] Examples of microtubule inhibitors / microtubule stabilizers generally include taxanes. Specific compounds include paclitaxel (Taxol®), vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-norvincaloicoblastine, docetaxol (Taxotere®), rhizoxin, drastatin, isethionate mybobrin, auristatin, semadin, RPR109881, BMS184476, vinflunin, cryptophycin, 2,3,4 Examples include 5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, anhydrous vinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, TDX258, epotilone (see, for example, U.S. Patent Nos. 6,284,781 and 6,288,237) and BMS188797.

[0310] Some examples of topoisomerase inhibitors include topotecan, hicaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-O-exobenzylidene-cartholeucine, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridin-2-(6H)propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':b,7]-indolidino[1,2b]quinoline-10, 13(9H,15H)dione, lulutotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'-dimethylamino-2'-deoxyetoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, aslacrin, (5a,5aB,8aa,9b)- 9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofloflo(3',4':6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenantridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione, 5-(3-aminopro These are pyramino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridine-6-one, N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthene-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinoline-7-one, and dimesna.

[0311] Examples of inhibitors of mitotic kinesin, specifically human mitotic kinesin KSP, are listed in Announcements WO03 / 039460, WO03 / 050064, WO03 / 050122, WO03 / 049527, WO03 / 049679, WO03 / 049678, WO04 / 039774, WO03 / 079973, WO03 / 099211, WO As described in 03 / 105855, WO03 / 106417, WO04 / 037171, WO04 / 058148, WO04 / 058700, WO04 / 126699, WO05 / 018638, WO05 / 019206, WO05 / 019205, WO05 / 018547, WO05 / 017190, and US2005 / 0176776. In one embodiment, mitotic kinesin inhibitors include, but are not limited to, KSP inhibitors, MKLP1 inhibitors, CENP-E inhibitors, MCAK inhibitors, and Rab6-KIFL inhibitors.

[0312] Examples of "histone deacetylase inhibitors" include, but are not limited to, SAHA, TSA, oxamfratin, PXD101, MG98, and scriptides. Further references to other histone deacetylase inhibitors can be found in the following manuscript: Miller, TA et al. J. Med. Chem. 46(24):5097-5116 (2003).

[0313] Examples of "inhibitors of kinases involved in the progression of mitosis" include, but are not limited to, inhibitors of aurora kinase, Polo-like kinase (PLK), and more specifically, inhibitors of PLK-1, bub-1, and bub-R1. An example of an "aurora kinase inhibitor" is VX-680.

[0314] Examples of "antiproliferative agents" include antisense RNA and DNA oligonucleotides, such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, as well as antimetabolites, such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimethrexate, fludarabine, capecitabine, gallocitabine, cytarabine ocphosphonate, fosteabin sodium hydrate, and larcitre. Xed, Palitrexide, Emitefur, Thiazofulin, Decitabine, Nolatrexed, Pemetrexed, Nerzarabine, 2'-Deoxy-2'-Methylidencytidine, 2'-Fluoromethylene-2'-Deoxycytidine, N-[5-(2,3-Dihydrobenzofuryl)sulfonyl]-N'-(3,4-Dichlorophenyl)urea, N6-[4-Deoxy-4-[N2-[2(E),4(E)-Tetradecadienoyl]glycylamino] -L-glycero-BL-mannoheptopyranosyl]adenine, apridin, ectinacidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]-thiadin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-fluorouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formi Examples include ru-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetate, swinesonin, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-1-BD-arabinofuranosilcytosine, 3-aminopyridine-2-carboxyaldehyde thiosemicarbazone, and trastuzumab.

[0315] Examples of therapeutic agents that target monoclonal antibodies include cytotoxic agents or radioisotopes of monoclonal antibodies that are specific to cancer cells or target cells. Bexxar is one example.

[0316] A "prenyl protein transferase inhibitor" refers to a compound that inhibits any one or any combination of prenyl protein transferase enzymes, including farnesyl protein transferase (FPTase), geranylgeranyl protein transferase type I (GGPTase-I), and geranylgeranyl protein transferase type II (GGPTase-II; also known as Rab GGPTase).

[0317] Examples of prenyl protein transferase inhibitors can be found in the following publications and patents: WO96 / 30343, WO97 / 18813, WO97 / 21701, WO97 / 23478, WO97 / 38665, WO98 / 28980, WO98 / 29119, WO95 / 32987, U.S. Patents Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510, 5,589,485, and 5,6 Patent No. 02,098, European Patent Publication No. 0618221, No. 0675112, No. 0604181, No. 0696593, WO94 / 19357, WO95 / 08542, WO95 / 11917, WO95 / 12612, WO95 / 12572, WO95 / 10514, U.S. Patent No. 5,661,152, WO95 / 10515, WO95 / 10516, WO95 / 24612, WO95 / 34535, WO95 / 25086, WO96 / 05529, WO96 / 06138, WO96 / 06193, WO96 / 16443, WO96 / 21701, WO96 / 21456, WO96 / 22278, WO96 / 24611, WO96 / 24612, WO96 / 05168, WO96 / 05169, WO96 / 00736, US Patent No. 5,571,792, WO96 / 17861, WO96 / 33159, WO96 / 34850, WO96 / 34851, WO96 / 30017, WO96 / 30018, W O96 / 30362, WO96 / 30363, WO96 / 31111, WO96 / 31477, WO96 / 31478, WO96 / 31501, WO97 / 00252, WO97 / 03047, WO97 / 03050, WO97 / 04785, WO97 / 02920, WO97 / 17070, WO97 / 23478, WO97 / 26246, WO97 / 30053, WO97 / 44350, WO98 / 02436, and U.S. Patent No. 5,532,359. For an example of the role of prenyl protein transferase inhibitors in angiogenesis, see European J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).

[0318] "Angiogenesis inhibitors" refer to compounds that inhibit the formation of new blood vessels, regardless of the mechanism. Examples of angiogenesis inhibitors include tyrosine kinase inhibitors, such as inhibitors of tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2); inhibitors of epithelial, fibroblast, or platelet-derived growth factors; MMP (matrix metalloproteinase) inhibitors; integrin blockers; nonsteroidal anti-inflammatory drugs (NSAIDs) such as interferon-α, interleukin-12, pentosan polysulfate, aspirin, and ibuprofen; and cyclooxygenase inhibitors, including selective cyclooxygenase-2 inhibitors such as celecoxib and rofecoxib. (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68) (1994);FEBS Letters, Vol. 372, p. 83 (1995);Clin, Orthop. Vol. 313, p. 76 (1995);J. Mol. Endocrinol., Vol. 16, p. 107 (1996);Jpn. J. Pharmacol., Vol. 75, p. 105 (1997);Cancer Res., Vol. 57, p. 1625 (1997);Cell, Vol. 93, p. 705 (1998);Intl. J. Mol. Med., Vol. 2, p. 715 (1998);J. Biol. Chem., Vol. 274, p. 9116 (1999)) Steroidal anti-inflammatory drugs (corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone, etc.), carboxamide triazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagirol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (Fernandez et al., J. Lab. Clin. Med.)Examples include, but are not limited to, antibodies against VEGF (see 105: 141-145 (1985)) and antibodies against VEGF (see Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO00 / 44777; and WO00 / 61186).

[0319] Other examples of angiogenesis inhibitors include endostatin, Ukrain, lampirase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxyranyl]-1-oxaspiro[2,5]octo-6-yl(chloroacetyl)carbamate, acetyldinanaline, and 5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxate Examples include, but are not limited to, Mido, CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrole-5-yl)methylene]-2-indolinone (SU5416).

[0320] Other therapeutic agents that modulate or inhibit angiogenesis and may be used in combination with the compounds of the present invention include agents that modulate or inhibit the coagulation and fibrinolytic systems (see the review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolytic pathways include, but are not limited to, heparin (Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparin, and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin-activated fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). TAFIa inhibitors are described in U.S. Patent Applications 60 / 310,927 (filed August 8, 2001) and 60 / 349,925 (filed January 18, 2002).

[0321] "Cell cycle checkpoint disrupting agents" refer to compounds that inhibit protein kinases that transmit cell cycle checkpoint signals, thereby causing cancer cells to sense DNA damaging agents. Such agents include inhibitors of ATR, ATM, CHK11 and CHK12 kinases, as well as cdk and cdc kinase inhibitors, specifically exemplified by 7-hydroxystaurosporine, flavopyridol, CYC202 (Cyclacel), and BMS-387032.

[0322] "Receptor tyrosine kinase (RTK) inhibitors" refer to compounds that inhibit RTKs, thereby inhibiting mechanisms involved in oncogenesis and tumor progression. Examples of such drugs include inhibitors of c-Kit, Eph, PDGF, Flt3, and c-Met. Further examples include RTK inhibitors described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

[0323] "Inhibitors of cell proliferation and survival signaling pathways" refer to drugs that inhibit the signaling cascade downstream of cell surface receptors. Such drugs include serine / threonine kinase (non-limited) inhibitors of Akt, e.g., WO02 / 083064, WO02 / 083139, WO02 / 083140, US2004-0116432, WO02 / 083138, US 2004-0102360, WO03 / 086404, WO03 / 086279, WO03 / 086394, WO03 / 084473, WO03 / 086403, WO2004 / 041162, WO2004 / 096131 , WO2004 / 096129, WO2004 / 096135, WO2004 / 096130, WO2005 / 100356, WO2005 / 100344, US2005 / 029941, US2005 / 44294, US2 Examples include inhibitors of Raf kinase (e.g., PLX-4032), inhibitors of MEK (e.g., Arry-162, RO-4987655, and GSK-1120212), inhibitors of mTOR (e.g., AZD-8055, BEZ-235, and everolimus), and inhibitors of PI3K (e.g., GDC-0941, BKM-120).

[0324] The previously used "integrin blockers" were α v Compounds that selectively antagonize, inhibit, or interfere with the binding of physiological ligands to β3 integrin, compounds that selectively antagonize, inhibit, or interfere with the binding of physiological ligands to αvβ5 integrin, α v β3 integrin and α v This term refers to compounds that selectively antagonize, inhibit, or interfere with the binding of physiological ligands to both β5 integrins, as well as compounds that selectively antagonize, inhibit, or interfere with the activity of specific integrins expressed on capillary endothelial cells. The term also refers to α v β6, α v This refers to antagonists of β8, α1β1, α2β1, α5β1, α6β1, and α6β4 integrins. The term is also α v β3, α v β5, αv This refers to antagonists of any combination of β8, α1β1, α2β1, α5β1, α6β1, and α6β4 integrins.

[0325] Some specific examples of tyrosine kinase inhibitors include N-(trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide, 3-[(2,4-dimethylpyrrole-5-yl)methylidenyl]indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolineamine, BIBX1382, and 2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl Examples include ru-9,12-epoxy-1H-diindro[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazosin-1-one, SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazineamine, and EMD121974.

[0326] The combination of antibodies or antigen-binding fragments claimed herein with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists may be useful in the treatment of certain malignancies. PPAR-γ and PPAR-δ are nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis have been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31: 909-913; J. Biol. Chem. 1999; 274: 9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41: 2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit retinal neovascularization in mice (Arch. Ophthamol. 2001; 119: 709-717). Examples of PPAR-γ agonists and PPAR-γ / α agonists include Lynparza®, Rucaparib®, Talazoparib®, niraparib, Veliparib®, thiazolidinediones (DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone, etc.), fenofibrate, genfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, and MCC-55. Examples include, but are not limited to, 5, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzoisoxazole-6-yl)oxy]-2-methylpropionic acid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchroman-2-carboxylic acid.

[0327] The antibody or antigen-binding fragment of the present invention may also be useful in combination with an aromatase inhibitor to treat or prevent breast cancer. Examples of aromatase inhibitors include, but are not limited to, anastrozole, letrozole, and exemestane.

[0328] The antibodies or antigen-binding fragments of the present invention may also be useful in treating cancer in combination with the following chemotherapy agents: Avalerix (Plenaxis depot®); Aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumab (Campath®); Alitretinoin (Panretin®); Allopurinol (Zyloprim®); Altretamine (Hexalen®); Amifostin (Ethyol®); Anastrozole (Arimidex®); Arsenic trioxide (Trisenox®); Asparaginase (Elspar®); Azacitidine (Vidaza®); Bendamustine hydrochloride (Treanda®); Bevacizumab (Avastin®); Bexarotene Capsules (Targretin®); Bexarotene Gel (Targretin®); Bleomycin (Blenoxane®); Bortezomib (Velcade®); Breferzin A; Busulfan for Injection (Busulfex®); Busulfan for Oral Use (Myleran®); Carsterone (Methosarb®); Capecitabine (Xeloda®); Carboplatin (Paraplatin®); Carmustine (BCNU®, BiCNU®); Carmustine (Gliadel®); Implantable Polyfeprosan 20 Carmustine (Gliadel Wafer (registered trademark); Celecoxib (Celebrex (registered trademark)); Cetuximab (Erbitux (registered trademark)); Chlorambucil (Leukeran (registered trademark)); Cisplatin (Platinol (registered trademark)); Cladribine (Leustatin (registered trademark), 2-CdA (registered trademark)); Clofarabine (Clolar (registered trademark)); Cyclophosphamide (Cytoxan (registered trademark), Neosar (registered trademark)); Cyclophosphamide (Cytoxan Injection (registered trademark)); Cyclophosphamide (Cytoxan Tablet (registered trademark)); Cytarabine (Cytosar-U (registered trademark));Liposomal cytarabine (DepoCyt®); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection (Fragmin®); daratumumab (DARZALEX®); darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); liposomal daunorubicin (DanuoXome®); daunorubi Syn, Daunorubicin (registered trademark); Daunorubicin, Daunorubicin (Cerbidine (registered trademark)); Degarelix (Firmagon (registered trademark)); Denileukin-Defitox (Ontak (registered trademark)); Dexrazoxane (Zinecard (registered trademark)); Dexrazoxane hydrochloride (Totect (registered trademark)); Didemnin B; 17-DMAG; Docetaxel (Taxotere (registered trademark)); Doxorubicin (Adriamycin) PFS (registered trademark); Doxorubicin (Adriamycin (registered trademark), Rubex (registered trademark)); Doxorubicin (Adriamycin PFS Injection (registered trademark)); Liposomal doxorubicin (Doxil (registered trademark)); Dromostanolone propionate (registered trademark); Dromostanolone propionate (Masterone Injection (registered trademark)); Eculizumab injection (Soliris (registered trademark)); Elliott's B solution Solution (registered trademark); Eltrombopag (Promacta (registered trademark)); Epirubicin (Ellence (registered trademark)); Epoetin alfa (epogen (registered trademark)); Erlotinib (Tarceva (registered trademark)); Estramustine (Emcyt (registered trademark)); Ethinylestradiol; Etoposide phosphate (Etopophos (registered trademark)); Etoposide, VP-16 (Vepesid (registered trademark)); Everolimus tablets (Afinitor (registered trademark)); Exemestane (Aromasin (registered trademark)); Fermoxyitol (Feraheme Injection (registered trademark));Filgrastim (Neupogen®); floxuridine (intra-arterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin implant(registered trademark); Hydroxyurea(registered trademark); Ibritumomab / Tiuxetan(Zevalin(registered trademark); Idamycin(registered trademark); Ifosfamide(IFEX(registered trademark)); Imatinib mesylate(Gleevec(registered trademark)); Interferon alpha-2a(Roferon A(registered trademark)); Interferon alpha-2b(Intron A(registered trademark)); Iobenguan I 123 Injection (AdreView®); Irinotecan (Camptosar®); Ixabepyrone (Ixempra®); Lapatinib Tablets (Tykerb®); Lenalidomide (Revlimid®); Letrozole (Femara®); Leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®); Levamisol (Ergamisol®); Lomustine, CCNU (CeeBU®); Mechloretamine, Nitrogen Mustard (Mustargen®); Megestrol Acetate (Megace®); Melphalan, L-PAM (Alkeran®); Mercaptopurine, 6-MP (Purinethol®); Mesna (Mesnex®); Mesna (Mesnex tabs(registered trademark); Methotrexate(registered trademark); Methoxsalen(registered trademark)); 8-Methoxypsoralen; Mitomycin(registered trademark));Mitotan (Lysodren®); Mitoxantrone (Novantrone®); Mitramycin; Nandrolone fenpropionate (Durabolin-50®); Neralabine (Arranon®); Nilotinib (Tasigna®); Nofetumomab (Verluma®); Ofatumumab (Arzerra®); Oprelbequine (Neumega®); Oxaliplatin (Eloxatin®); Paclitaxel (Paxene®) (Trademark)); Paclitaxel (Taxol (Registered Trademark)); Paclitaxel Protein-Binding Particles (Abraxane (Registered Trademark)); Palifermin (Kepivance (Registered Trademark)); Pamidronate (Aredia (Registered Trademark)); Panitumumab (Vectibix (Registered Trademark)); Pazopanib Tablets (Votrienttm (Registered Trademark)); Pegademase (Adagen (Bovine Pegademase) (Registered Trademark)); Pegasparagase (Oncaspar (Registered Trademark)); Pegfilgrastim (Neulasta (Registered Trademark)); Pemet Lexed disodium (Alimta®); Pentostatin (Nipent®); Pipobroman (Vercyte®); Prelixafor (Mozobil®); Plicamycin, Mitramycin (Mithracin®); Porfimer sodium (Photofrin®); Pralatrexate injection (Folotyn®); Procarbazine (Matulane®); Quinacrine (Atabrine®); Rapamycin; Rasburicase (E litek(registered trademark); raloxifene hydrochloride (Evista(registered trademark)); rituximab (Rituxan(registered trademark)); romidepsin (Istodax(registered trademark)); romiprostim (Nplate(registered trademark)); salglamostin (Leukine(registered trademark)); salglamostin (Prokine(registered trademark)); sorafenib (Nexavar(registered trademark)); streptozosin (Zanosar(registered trademark)); sunitinib maleate (Sutent(registered trademark)); talc (Sclerosol(registered trademark));Tamoxifen (Nolvadex®); Temozolomide (Temodar®); Temsirolimus (Torisel®); Teniposide, VM-26 (Vumon®); Testolactone (Teslac®); Thioguanine, 6-TG (Thioguanine®); Thiopurine; Thiotepa (Thioplex®); Topotecan (Hycamtin®); Toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab / I-131; Tositumomab (Bexxar®); Trans-retinoic acid; Tratuzumab (Herceptin®); Tretinoin, ATRA (Vesanoid®); Triethylenemelamine; Uracil Mustard Capsules®); Valrubicin (Valstar®); Vinblastine (Velban®); Vincristine (Oncovin®); Vinorelbine (Navelbine®); Vorinostat (Zolinza®); Waltmannin; and Zoledronate (Zometa®).

[0329] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is, without limitation, casopitant (GlaxoSmithKline), netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (marketed as Aloxi by MGI Pharma), aprepitant (marketed as Emend by Merck and Co.; Rahway, NJ), diphenhydramine (marketed as Benadryl® by Pfizer; New York, NY), hydroxyzine (marketed as Atarax® by Pfizer; New York, NY), metoclopramide (marketed as Reglan® by AH Robins Co.; Richmond, VA), lorazepam (marketed as Ativan® by Wyeth; Madison, NJ), and alprazolam (marketed as Xanax® by Pfizer; New (Sold by York, NY), Haloperidol (Haldol®, sold by Ortho-McNeil; Raritan, NJ), Droperidol (Inapsine®), Dronabinol (Marinol®, sold by Solvay Pharmaceuticals, Inc.; Marietta, GA), Dexamethasone (Decadron®, sold by Merck and Co.; Rahway, NJ), Methylprednisolone (Medrol®, sold by Pfizer; New York, NY), Prochlorperazine (Compazine®, sold by Glaxosmithkline; Research Triangle Park, NC), Granisetron (Kytril®, sold by Hoffmann-La Roche Inc.)It is used in conjunction with one or more antiemetics, including (marketed by Nutley, NJ), ondansetron (marketed as Zofran® by Glaxosmithkline; Research Triangle Park, NC), drasetron (marketed as Anzemet® by Sanofi-Aventis; New York, NY), and tropisetron (marketed as Navoban® by Novartis; East Hanover, NJ).

[0330] Other side effects of cancer treatment include deficiencies in red and white blood cells. Therefore, in one embodiment of the present invention, an anti-SIRPα antibody or its antigen-binding fragment is coupled with a drug that treats or prevents such deficiencies, such as filgrastim, pegfilgrastim, erythropoietin, epoetin alfa, or darbepoetin alfa.

[0331] In one embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment is administered in conjunction with anti-cancer radiotherapy. For example, in one embodiment of the present invention, the radiotherapy is an external beam radiation therapy (EBT), a method for delivering high-energy X-ray beams to the site of a tumor. The beams are generated outside the patient's body (e.g., by a linear accelerator) and target the tumor site. These X-rays can destroy cancer cells, and with careful treatment planning, damage to surrounding healthy tissue can be avoided. The radiation source is not placed inside the patient's body. In one embodiment of the present invention, the radiotherapy is a type of conformal irradiation called proton beam therapy, which uses protons instead of X-rays to target diseased tissue. In one embodiment of the present invention, the radiotherapy is a procedure that utilizes advanced technology to tailor radiotherapy to individual body structures, called conformal external beam radiation therapy. In one embodiment of the present invention, the radiotherapy is a temporary placement of radioactive material inside the body, which is typically used to deliver an excess dose or large amount of radiation to a particular area.

[0332] In one embodiment of the present invention, a surgical procedure, which is surgical tumor removal, is performed in conjunction with an anti-SIRPα antibody or its antigen-binding fragment.

[0333] Experimental and diagnostic use The anti-SIRPα antibodies and their antigen-binding fragments disclosed herein may be used as affinity purifiers. In this step, the anti-SIRPα antibodies and their antigen-binding fragments are immobilized on a solid phase such as Sephadex, glass, or agarose resin or filter paper using methods well known in the art. The immobilized antibody or fragment is brought into contact with a sample containing the SIRPα protein to be purified, and the support is then washed with a suitable solvent to remove substantially all of the material in the sample other than the SIRPα protein bound to the immobilized antibody or fragment. Finally, the support is washed with a solvent that elutes the bound SIRPα (e.g., protein A). Such immobilized antibodies and fragments form part of the present invention.

[0334] Also provided are antigens for producing secondary antibodies useful, for example, for performing Western blotting and other immunoassays discussed herein.

[0335] Anti-SIRPα antibodies (e.g., humanized antibodies) and their antigen-binding fragments can also be useful in diagnostic assays for the SIRPα protein, such as detecting its expression in specific cells, tissues, or serum, including bone marrow cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, and dendritic cells. Such diagnostic methods can be useful in diagnosing a variety of diseases.

[0336] The present invention encompasses an ELISA assay (enzyme-mediated immunosorbent assay) that incorporates the use of an anti-SIRPα antibody or its antigen-binding fragment disclosed herein.

[0337] For example, such a method includes the following steps: (a) The step of coating a substrate (e.g., the wells of a microtiter plate, e.g., the surface of a plastic plate) with an anti-SIRPα antibody or its antigen-binding fragment; (b) The step of coating the substrate with a sample to be tested for the presence of SIRPα; (c) Wash the plate to remove any unbound material from the sample; (d) The step of applying a detectable labeled antibody (e.g., an enzyme-binding antibody) that is also specific to the SIRPα antigen; (e) Washing the substrate to remove the unbound labeled antibody; (f) Once the labeled antibody is bound to the enzyme, a chemical is applied to it to convert it into a fluorescent signal by the enzyme; and (g) A step of detecting the presence of the labeled antibody.

[0338] Detection of a label associated with the substrate indicates the presence of the SIRPα protein.

[0339] In further embodiments, the labeled antibody or its antigen-binding fragment is labeled with a peroxidase and reacted with ABTS (e.g., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or 3,3',5,5'-tetramethylbenzidine to produce a detectable color change. Alternatively, the labeled antibody or fragment is reacted with a detectable radioisotope (e.g., 3 When labeled with H), it can be detected by a scintillation counter in the presence of scintillant.

[0340] The anti-SIRPα antibody of the present invention or its antigen-binding fragment may be used in Western blotting or immunoprotein blotting procedures. Such procedures form part of the present invention, for example, (1) If applicable, a protein from a sample to be tested for the presence of SIRPα (e.g., from PAGE or SDS-PAGE electrophoresis separation of proteins in the sample) is transferred to a membrane or other solid support using a method known in the art (e.g., semi-dry blotting or tank blotting); the membrane or other solid substrate to be tested for the presence of bound SIRPα or a fragment thereof is brought into contact with the anti-SIRPα antibody or antigen-binding fragment of the present invention. (2) Wash the membrane once or multiple times to remove unbound anti-SIRPα antibody or fragments and other unbound substances, (3) To detect the conjugated anti-SIRPα antibody or fragment, Includes.

[0341] Such membranes may be in the form of nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membranes on which proteins to be tested for the presence of SIRPα in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., after electrophoretic separation in the gel). The membrane may be blocked before contact with the anti-SIRPα antibody or fragment, for example, by skim milk powder that binds to nonspecific protein binding sites on the membrane.

[0342] The detection of the bound antibody or fragment indicates the presence of the SIRPα protein on the membrane or substrate and in the sample. The detection of the bound antibody or fragment may also be performed by binding the antibody or fragment to a detectably labeled secondary antibody (anti-immunoglobulin antibody) and then detecting the presence of the secondary antibody.

[0343] The anti-SIRPα antibodies and their antigen-binding fragments disclosed herein may be used in immunohistochemical measurements. Such methods form part of the present invention, for example. (1) Contacting cells to be tested for the presence of SIRPα protein (e.g., a sample containing bone marrow cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, and dendritic cells) with the anti-SIRPα antibody of the present invention or its antigen-binding fragment, and (2) To detect the antibody or fragment on or within the cell, Includes.

[0344] If the antibody or fragment is detectably labeled, it can be detected directly. Alternatively, the antibody or fragment may be conjugated with a detectably labeled secondary antibody that is to be detected.

[0345] Certain anti-SIRPα antibodies and their antigen-binding fragments disclosed herein may be used in vivo oncography. Such a method may include injecting a radiolabeled anti-SIRPα antibody or its antigen-binding fragment into the body of a patient to be tested for the presence of a tumor associated with SIRPα expression (e.g., expressing SIRPα, e.g., on the surface of tumor cells), followed by nuclear imaging of the patient's body to detect the presence of the labeled antibody or fragment at a site containing a high concentration of the antibody or fragment, e.g., the tumor-binding antibody or fragment. The detection of that site is SIRPα + This indicates the presence of tumors and tumor cells.

[0346] Contrast enhancement techniques include SPECT contrast (single-photon emission tomography) or PET contrast (positron emission tomography). Labeling methods include, for example, iodine-123 in combination with SPECT contrast. 123 I) and technetium-99m( 99m Tc), or, for example, combined with PET contrast or indium-111. 11 C, 13 N, 15 O or 18 F is one example (see, for example, Gordon et al., (2005) International Rev. Neurobiol. 67:385-440).

[0347] Pharmaceutical composition and administration To prepare a pharmaceutical or sterile composition of the anti-SIRPα antibody and antigen-binding fragment of the present invention, the antibody or antigen-binding fragment is miscible with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0348] Therapeutic and diagnostic formulations may be prepared by mixing acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions (e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; see Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0349] The toxicity and therapeutic efficacy of the antibodies of the present invention, administered alone or in combination with other therapeutic agents, are, for example, LD 50 (The dose at which 50% of the population becomes lethal) and ED 50 The dose at which 50% of the population exhibits therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose-to-toxicity ratio is the therapeutic index (LD50). 50 / ED 50 ) The data obtained from these cell culture assays and animal studies may be used to formulate a range of dosages for use in humans. The dosage of such compounds is preferably ED 50 It is included within the circulating concentration range and has little to no toxicity. The dose may vary within this range depending on the dosage form and route of administration used.

[0350] In further embodiments, additional therapeutic agents are administered to the subject in conjunction with the anti-SIRPα antibody or its antigen-binding fragment according to the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

[0351] The mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, nasal, intraocular, inhalation, inhalation, topical, cutaneous, percutaneous, or intra-arterial.

[0352] In a particular embodiment, the anti-SIRPα antibody or its antigen-binding fragment of the present invention may be administered by an invasive route such as injection. In a further embodiment of the present invention, the anti-SIRPα antibody or its antigen-binding fragment, or a pharmaceutical composition thereof, may be administered intravenously, subcutaneously, intramuscularly, intra-arterially, intratumorally, or by inhalation or aerosol delivery. Administration by a non-invasive route (e.g., orally, in pills, capsules, or tablets) is also within the scope of the present invention.

[0353] The present invention provides a container (e.g., a plastic or glass vial, e.g., with a cap, or a chromatography column, a hollow needle, or a cylindrical syringe) containing any of the antibodies or antigen-binding fragments of the present invention, or any of the pharmaceutical compositions thereof. The present invention also provides an injection device containing any of the antibodies or antigen-binding fragments of the present invention, or any of the pharmaceutical compositions thereof. An injection device is a device for introducing a substance into a patient's body via a parenteral route, e.g., intramuscular, subcutaneous, or intravenous route. For example, an injection device may be a syringe (e.g., an autoinjector pre-filled with the pharmaceutical composition) comprising, for example, a syringe or barrel for holding the fluid to be injected (e.g., an antibody or antigen-binding fragment, or any of the pharmaceutical compositions thereof), a needle for puncturing the skin and / or blood vessels for injecting the fluid, and a plunger for pushing the fluid out of the syringe through the needle hole. In one embodiment of the present invention, the injection device containing the antibodies or antigen-binding fragments of the present invention, or any of the pharmaceutical compositions thereof, is an intravenous (IV) injection device. Such a device may contain the antibody or fragment, or their pharmaceutical composition, in a cannula or sheath needle / needle that can be attached to a tube, and the tube may be attached to a bag or reservoir for holding a fluid (e.g., saline solution, or lactated Ringer's solution containing NaCl, sodium lactate, KCl, CaCl2, and optionally glucose) to be introduced into the patient's body through the cannula or sheath needle / needle. In one embodiment of the present invention, the antibody or fragment, or their pharmaceutical composition, may be introduced into the device once the sheath needle and cannula have been inserted into a target vein, and the sheath needle may be removed from the inserted cannula. The IV device may be inserted, for example, into a peripheral vein (e.g., in the hand or arm); into the superior or inferior vena cava or the right atrium (e.g., into a central vein); or into the subclavian, internal jugular, or femoral vein and advanced toward the heart until it reaches the superior vena cava or the right atrium (e.g., a central venous line). In one embodiment of the present invention, the injection device is an auto-injector, a jet injector, or an extracorporeal infusion pump.A jet injector utilizes a high-pressure, narrow jet of liquid to introduce the antibody or fragment, or their pharmaceutical composition, into the patient's body through the epidermis. An extracorporeal infusion pump is a medical device that delivers the antibody or fragment, or their pharmaceutical composition, to the patient's body in a controlled amount. Extracorporeal infusion pumps may be electrically or mechanically operated. Different pumps operate in different ways; for example, a syringe pump holds fluid in a syringe reservoir, and a movable piston controls fluid delivery; an elastomer pump holds fluid in an expandable balloon reservoir, and pressure from the elastic wall of the balloon drives fluid delivery; in a peristaltic pump, a set of rollers squeeze along the length of a flexible tube structure to push the fluid forward; in a multi-channel pump, fluid can be delivered from multiple reservoirs at multiple speeds.

[0354] The pharmaceutical compositions disclosed herein may also be administered by needle-free subcutaneous injection devices such as those disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Such needle-free devices containing the pharmaceutical compositions are also part of the present invention. The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules for administering the pharmaceutical composition include those disclosed in U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; and those disclosed in Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment. Many other such implants, delivery systems, and modules are well-known to those skilled in the art and include the pharmaceutical composition of the present invention within the scope of the present invention.

[0355] Alternatively, the anti-SIRPα antibody or antigen-binding fragment of the present invention may be administered locally rather than systemically, for example, by directly injecting the antibody or fragment into the tumor. Furthermore, the antibody or fragment may be administered in a targeted drug delivery system, for example, in liposomes coated with a tissue-specific antibody and targeting a tumor. The liposomes would selectively target and take up the affected tissue. Such methods and liposomes are part of the present invention.

[0356] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody or antigen-binding fragment, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target tissue in the biological matrix. Preferably, the administration regimen delivers enough therapeutic antibody or fragment to improve the target disease condition while minimizing undesirable side effects. Therefore, the amount of biological agent delivered depends in part on the individual therapeutic antibody and the severity of the disease being treated. Guidelines for selecting an appropriate dose of therapeutic antibody or fragment are available (e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

[0357] The determination of the appropriate dose is made by a physician, for example, by utilizing parameters or factors known or thought to affect the treatment in the art. Generally, the dose is started at a slightly lower amount than the optimal dose and then increased in small increments until the desired or optimal effect is achieved compared to any negative side effects. Important diagnostic measures include, for example, the degree of inflammation, the measure of symptoms, or the level of inflammatory cytokines produced. Generally, it is desirable that the biological agent used is derived from the same species as the animal being targeted by the treatment, thereby minimizing the immune response to the reagent. In the case of human subjects, humanized antibodies and fully human antibodies may be desirable.

[0358] The antibodies or antigen-binding fragments disclosed herein may be provided by continuous infusion or by the administered dose, for example, once daily, 1 to 7 times per week, once per week, twice per week, once per month, twice per month, four times per year, every six months, once per year, etc. The dose may be provided, for example, intravenously, subcutaneously, topically, orally, intranasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, more generally at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, or more (e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67: 451-456; Portielji, et al. (2003) Cancer Immunol. Immunother. (See 52: 151-144). Doses may be provided to achieve a predetermined target concentration of anti-SIRPα antibody in the serum of the subject, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / mL or more. In other embodiments, the anti-SIRPα antibody of the present invention is administered, for example, subcutaneously or intravenously, at doses of 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg / subject, on a basis of once a week, twice a week, "every four weeks", once a month, twice a month, or four times a year.

[0359] As used herein, the term “effective dose” refers to the amount of the anti-SIRPα antibody or its antigen-binding fragment of the present invention that, when administered alone or in combination with additional therapeutic agents to a cell, tissue, or subject, is effective in inducing a measurable improvement in one or more symptoms of a disease, such as cancer or cancer progression. The effective dose also refers to the amount of antibody or fragment sufficient to result in at least partial improvement of symptoms, such as tumor regression or elimination, absence of tumor growth, or increased survival time. When applied to individual active ingredients administered alone, the effective dose refers to that ingredient alone. When applied in combination, the effective dose refers to the amount of the combination of active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective dose of a therapeutic agent will result in an improvement of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in a diagnostic measure or parameter. The effective dose may also result in an improvement in a subjective measure when disease severity is assessed using a subjective scale.

[0360] kit Further provided are kits comprising one or more components, including an anti-SIRPα antibody or antigen-binding fragment, as discussed herein, in conjunction with one or more additional components, including a pharmaceutically acceptable carrier and / or therapeutic agent, as discussed herein without limitation. The antibody or fragment, and / or the therapeutic agent, may be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

[0361] In one embodiment, the kit comprises the anti-SIRPα antibody of the present invention or its antigen-binding fragment, or a pharmaceutical composition, in one container (e.g., a sterile glass or plastic vial), and / or a therapeutic agent and its pharmaceutical composition in another container (e.g., a sterile glass or plastic vial).

[0362] In another embodiment, the kit comprises a combination of the present invention, which optionally includes the anti-SIRPα antibody of the present invention or its antigen-binding fragment, together with a pharmaceutically acceptable carrier, in combination optionally with one or more therapeutic agents formulated together in a pharmaceutical composition, which may optionally be contained in a single common container.

[0363] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for carrying out such administration. For example, the kit may include one or more subcutaneous needleless or other injection devices as previously discussed.

[0364] The kit may include a package insert containing information about the pharmaceutical composition and dosage form. Generally, such information assists patients and physicians in the effective and safe use of the enclosed pharmaceutical composition and dosage form. For example, the following information regarding the combination of the present invention may be provided in the package insert: pharmacokinetics, pharmacodynamic properties, clinical trials, efficacy parameters, indications and uses, contraindications, warnings, precautions for use, adverse reactions, overdose, appropriate dosage and administration, method of delivery, appropriate storage conditions, references, manufacturer / distributor information and patent information.

[0365] The kit also contains a second therapeutic agent, such as anti-CD47 antibody, anti-APRIL antibody, anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), anti-PDL1 antibody, anti-TIGIT antibody, anti-CTLA4 antibody, anti-CS1 antibody (e.g., elotuzumab), anti-KIR2DL1 / 2 / 3 antibody (e.g., lirirumab), anti-CD137 antibody (e.g., urelumab), anti-GITR antibody (e.g., TRX518), anti-PD-L1 antibody (e.g., BMS-936559, MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILT1 antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody, anti-ILT5 antibody, anti-ILT6 antibody, and anti-ILT7 antibody. The antibody may include one or more of the following: anti-ILT8 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS, anti-KIR2DL1 antibody, anti-KIR2DL2 / 3 antibody, anti-KIR2DL4 antibody, anti-KIR2DL5A antibody, anti-KIR2DL5B antibody, anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E antibody, anti-4-1BB antibody (e.g., PF-05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 or PEGylated IL-10, or any small organic molecule inhibitor of such a target; the antibody or its antigen-binding fragment may be AMHR2, AXL, BCMA, CA IX, CD4, CD16, CD19, CD20, CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b, Folate receptor alpha, Fucosyl-GM1, HER2, HER3, IL1RAP, Kappa myeloma antigen, MS4A1, Prolactin receptor, TA-MU Antigens selected from the group consisting of C1 and PSMA; rituximab, ubrituximab, margetuximab, IMGN-529, SCT400, bertuzumab, obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, dinitruximab, trastuzumab, cetuximab, rituximab-RLI, c.60C3-RLI, Hul4.18-IL2, KM2812, AFM13, and (CD20) 2xCD16, Erlotinib (Tarceva), Daratumumab, Aretuzumab, Pertuzumab, Brentuximab, Elotuzumab, Ibritumumab, Ifabotuzumab, Falletuzumab, Falletuzumab, Otreltuzumab, Carotuximab, Epratuzumab, Inevilizumab, Lumuletuzumab, 4G7SDIE, AFM21, AFM22, LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986 012, BVX-20, mogamulizumab, ChiLob-7 / 4, leukotuximab, isatuximab, DS-8895, FPA144, GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213, PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202, MOR-208, opotuzumab, enshituki Simab, vedotin (Adcetris), ibritumomab / tiuxetan, ABBV-838, HuMax-AXL-ADC, and adtrastuzumab / emtansine (Kadcyla); non-limited anthracyclines (doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone), oxaliplatin, bortezomib, cyclophosphamide, bleomycin, vorinostat, paclitaxel, 5-fluoro Radiotherapy or chemotherapy drugs, including racil, cytarabine, prednisolone, docetaxel, mitomycin C, topotecan / camptothecin, etoposide, zoledronic acid, methotrexate, ibrutinib, aflibercept, bevacizumab, toremifene, vinblastine, vincristine, idelalisib, mercaptopurine, thalidomide, and sorafenib; bind to cyclic dinucleotides or other STING pathway agonists.

[0366] Detection kits and treatment kits For convenience, the anti-SIRPα antibody or its antigen-binding fragment of the present invention may be provided in a kit, i.e., a packaged combination of predetermined amounts of reagents, along with instructions for use in performing a diagnostic or detection assay. If the antibody or fragment is enzyme-labeled, the kit will include substrates and cofactors required by the enzyme (e.g., substrate precursors that provide detectable chromophore or fluorophore). In addition, other additives such as stabilizers and buffers (e.g., blocking buffers or solubilizing buffers) may be included. The relative amounts of the various reagents may vary widely to provide solution concentrations of reagents that substantially optimize the sensitivity of the assay. In particular, the reagents may be provided as dry powders, usually lyophilized, including excipients that, when dissolved, provide a reagent solution of appropriate concentration.

[0367] Also provided are diagnostic or detection reagents for use in various detection assays, such as immunoassays (sandwich or antagonistic), and kits containing one or more such reagents. The components of the kit may be pre-attached to a solid carrier or coated onto the surface of the solid carrier when the kit is used. In some embodiments of the present invention, the signal generating means may be pre-associated with the antibody or fragment of the present invention before use, or may require a combination of one or more components, such as a buffer, an antibody-enzyme conjugate, or an enzyme substrate. The kit may also contain additional reagents, such as blocking reagents to reduce nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, etc. The solid phase surface may be in the form of tubes, beads, microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In a particular embodiment, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product, or the reduction of a chemiluminescent or chromogenic substrate, is a component of the signal generating means. Such enzymes are well known in the art. The kit may contain any of the capture agents and detection reagents described herein. Depending on the circumstances, the kit may also include instructions for use in carrying out the method of the present invention.

[0368] Also provided is a kit comprising an anti-SIRPα antibody (e.g., a humanized antibody) or its antigen-binding fragment packaged in a container such as a vial or bottle, and further comprising a label attached to or packaged on the container, the label describing the contents of the container and providing instructions and / or instructions for use regarding the use of the contents of the container to treat one or more disease conditions described herein.

[0369] In one embodiment, the kit is for treating cancer and comprises an anti-SIRPα antibody (e.g., a humanized antibody) or its antigen-binding fragment and a further therapeutic agent or vaccine. The kit may optionally further comprise a syringe for parenteral administration, for example, intravenous administration. In another embodiment, the kit comprises an anti-SIRPα antibody (e.g., a humanized antibody) or its antigen-binding fragment and a label attached to or packaged with the container describing the use of the antibody or fragment with the vaccine or further therapeutic agent. In yet another embodiment, the kit comprises a vaccine or further therapeutic agent and a label attached to or packaged with the container describing the use of the vaccine or further therapeutic agent with the anti-SIRPα antibody or fragment. In certain embodiments, the anti-SIRPα antibody and the vaccine or further therapeutic agent are present in separate vials or mixed together in the same pharmaceutical composition.

[0370] As discussed earlier in the section on combination therapy, when administering two therapeutic drugs simultaneously, it is not necessary to administer the drugs at the same time or via the same route, as long as there is an overlap in the periods during which the drugs exert their therapeutic effects. Simultaneous or consecutive administration is intended, as is administration on different days or weeks.

[0371] A therapeutic and detection kit disclosed herein may also be prepared, comprising at least one of the antibodies, peptides, antigen-binding fragments, or polynucleotides disclosed herein, and instructions for use for using the composition as a detection reagent or therapeutic agent. The container for use in such a kit typically comprises at least one vial, test tube, flask, bottle, syringe or other suitable container, in which one or more of the detection and / or therapeutic compositions are placed, preferably dispensed as appropriate. If a second therapeutic agent is also provided, the kit may also comprise a second separate container in which the second detection and / or therapeutic composition may be placed. Alternatively, multiple compounds may be prepared in a single pharmaceutical composition or packaged in a single-container means such as a vial, flask, syringe, bottle or other suitable single container. The kits disclosed herein also typically comprise means for containing vials(or vials) sealed for commercial use, such as injection- or blow-molded plastic containers in which the desired vials(or vials) are held. If the kit includes a radioactive label, colorimetric, fluorescent, or other type of detectable label or detection means, the label may be provided in the same container as the single detection or therapeutic composition, or it may be provided in a second, separate container means in which the second composition is placed and which can be appropriately dispensed. Alternatively, the detection reagent and label may be prepared in a single container means, and in most examples, the kit will also include means containing sealed vials, typically for commercial use and / or for convenient packaging and delivery.

[0372] Devices or apparatus for performing the detection or monitoring methods described herein are also provided. Such apparatus may include a chamber or tube into which a sample can be introduced, a fluid handling system optionally including a valve or pump for directing the flow of the sample through the device, a filter for optionally separating plasma or serum from blood, a mixing chamber for adding a capture agent or detection reagent, and optionally a detection device for detecting the amount of a detectable label bound to an immunocomplex of the capture agent. The flow of the sample may be passive (e.g., by capillary, hydrostatic pressure, or other force, which does not require further operation of the device once the sample is applied), active (e.g., by the application of force generated through a mechanical pump, electroosmotic pump, centrifugal force, or high pneumatic pressure), or a combination of active and passive forces.

[0373] In a further embodiment, also provided are a processor, computer-readable memory, and machine operations stored in the computer-readable memory and adapted to be executed by the processor, for carrying out any of the methods described herein. Examples of suitable computer systems, environments, and / or forms include personal computers, server computers, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable home appliances, network PCs, minicomputers, mainframe computers, distributed computing environments including any of the above systems or devices, or any other systems known in the art.

[0374] Preferred Embodiment Embodiment 1. a. The amino acid sequence of SEQ ID NO:69, or the heavy chain variable region CDR1 containing an amino acid sequence different from SEQ ID NO:1 due to one, two, or three conservative substitutions, b. The amino acid sequence of SEQ ID NO:70, or the heavy chain variable region CDR2 containing an amino acid sequence different from SEQ ID NO:2 due to one, two, or three conservative substitutions, c. The amino acid sequence of SEQ ID NO:71, or the heavy chain variable region CDR3 containing an amino acid sequence different from SEQ ID NO:3 due to one, two, or three conservative substitutions. d. Light chain variable region CDR1 containing an amino acid sequence different from that of SEQ ID NO:4 by the amino acid sequence of SEQ ID NO:72, or by 1, 2, or 3 conservative substitutions. e. Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO:73, or an amino acid sequence different from SEQ ID NO:5 due to one, two, or three conservative substitutions, and f. Light chain variable region CDR3 containing an amino acid sequence different from that of SEQ ID NO:6 by one, two, or three conservative substitutions, the amino acid sequence of SEQ ID NO:74. One or more of these, and depending on the case, including each of them, g. The amino acid sequence of SEQ ID NO:1, or the heavy chain variable region CDR1 containing an amino acid sequence different from SEQ ID NO:1 due to one, two, or three conservative substitutions. h. The amino acid sequence of SEQ ID NO:2, or the heavy chain variable region CDR2 containing an amino acid sequence different from SEQ ID NO:2 due to one, two, or three conservative substitutions, i. The amino acid sequence of SEQ ID NO:3, or the heavy chain variable region CDR3 containing an amino acid sequence different from SEQ ID NO:3 due to one, two, or three conservative substitutions, j. The amino acid sequence of SEQ ID NO:4, or the light chain variable region CDR1 containing an amino acid sequence different from SEQ ID NO:4 due to one, two, or three conservative substitutions, The light chain variable region CDR2 contains the amino acid sequence of k.SEQ ID NO:5, or an amino acid sequence different from SEQ ID NO:5 due to one, two, or three conservative substitutions, and l. The amino acid sequence of SEQ ID NO:6, or the light chain variable region CDR3 containing an amino acid sequence different from SEQ ID NO:6 due to one, two, or three conservative substitutions, One or more of these, and in some cases including each of them, An antibody or its antigen-binding fragment that binds to human SIRPα.

[0375] Embodiment 2. The amino acid sequence of SEQ ID NO:69, or an amino acid sequence different from SEQ ID NO:69 due to one, two, or three conservative substitutions; the amino acid sequence of SEQ ID NO:70, or an amino acid sequence different from SEQ ID NO:70 due to one, two, or three conservative substitutions; the amino acid sequence of SEQ ID NO:71, or an amino acid sequence different from SEQ ID NO:71 due to one, two, or three conservative substitutions; Each of the heavy chain sequences containing; and / or The amino acid sequence of SEQ ID NO:72, or an amino acid sequence different from SEQ ID NO:72 due to one, two, or three conservative substitutions; the amino acid sequence of SEQ ID NO:73, or an amino acid sequence different from SEQ ID NO:73 due to one, two, or three conservative substitutions; the amino acid sequence of SEQ ID NO:74, or an amino acid sequence different from SEQ ID NO:74 due to one, two, or three conservative substitutions; Each of the light chain sequences containing, or The amino acid sequence of SEQ ID NO:1, or an amino acid sequence different from SEQ ID NO:1 due to 1, 2, or 3 conservative substitutions; the amino acid sequence of SEQ ID NO:2, or an amino acid sequence different from SEQ ID NO:2 due to 1, 2, or 3 conservative substitutions; the amino acid sequence of SEQ ID NO:3, or an amino acid sequence different from SEQ ID NO:3 due to 1, 2, or 3 conservative substitutions; Each of the heavy chain sequences containing, and / or The amino acid sequence of SEQ ID NO:4, or an amino acid sequence different from SEQ ID NO:4 due to 1, 2, or 3 conservative substitutions; the amino acid sequence of SEQ ID NO:5, or an amino acid sequence different from SEQ ID NO:5 due to 1, 2, or 3 conservative substitutions; the amino acid sequence of SEQ ID NO:6, or an amino acid sequence different from SEQ ID NO:6 due to 1, 2, or 3 conservative substitutions; An antibody or antigen-binding fragment of Embodiment 1, comprising each of the light chain sequences including the following.

[0376] Embodiment 3. SEQ ID NO:75, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:78, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO: 80, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:82, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:84, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:86, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:88, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto, and SEQ ID NO:102, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. A heavy chain variable region containing an amino acid sequence selected from the group consisting of, SEQ ID NO:76, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:90, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:92, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:94, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:96, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. SEQ ID NO:100, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto, and SEQ ID NO:104, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical thereto. A light chain variable region containing an amino acid sequence selected from the group consisting of, It includes one or both of the above, or SEQ ID NO:7, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical there...

Claims

1. A first antigen-binding region comprising heavy chain CDR1, 2, and 3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1, 2, and 3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6 respectively; and Second antigen-binding region; A bispecific antibody containing, The first antigen-binding region and the second antigen-binding region bind to different antigens. The first antigen-binding region binds to the human SIRPαV1 protein. Bispecific antibodies.

2. The bispecific antibody according to claim 1, wherein the first antigen-binding region is a Fab antigen-binding region.

3. The bispecific antibody according to claim 1, wherein the first antigen-binding region is an scFv antigen-binding region.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the second antigen-binding region binds to human CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, c-Met, CEA, EGFR, EpCAM, HER2, HER3, PSMA, PTHR2, mesothelin, PD-1, PD-L1, or TIM3.

5. The bispecific antibody according to claim 4, wherein the second antigen-binding region binds to human PD-L1.

6. The bispecific antibody according to claim 5, wherein the second antigen-binding region is the antigen-binding region of BMS-936559, MSB0010718C (avelumab) or MPDL3280A (atezolizumab).

7. The bispecific antibody according to claim 6, wherein the second antigen-binding region is the antigen-binding region of MPDL3280A (atezolizumab).

8. The bispecific antibody according to any one of claims 1 to 7, wherein the bispecific antibody is IgG.

9. The bispecific antibody according to any one of claims 1 to 7, wherein the bispecific antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

10. The bispecific antibody according to any one of claims 1 to 9, wherein the bispecific antibody comprises one or more Fc region mutations that modify Fc receptor binding, thereby reducing effector function and / or complement-dependent cell-mediated cytotoxicity (CDC) compared to the corresponding bispecific antibody having a wild-type Fc region.

11. The bispecific antibody according to claim 10, wherein the bispecific antibody binds to one or more human FcγR selected from the group consisting of FcγRI, FcγRIIB, FcγRIIC, FcγRIIIIA-F158, and FcγRIIIIA-V158 with an affinity at least 10 times lower than an equivalent bispecific antibody having a wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119) Fc region or a wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66) Fc region.

12. The bispecific antibody according to claim 11, wherein the bispecific antibody comprises a heavy chain constant domain having the L234A / L235A (LALA) (SEQ ID NO: 123) or L234A / L235A / P329G (LALAPG) (SEQ ID NO: 125) mutation.

13. The bispecific antibody according to claim 11 or 12, wherein the bispecific antibody comprises a heavy chain constant domain having the N297Q (SEQ ID NO: 126) mutation.

14. The bispecific antibody according to claim 12, wherein the bispecific antibody comprises an IgG1 heavy chain constant domain having the L234A / L235A (LALA) (SEQ ID NO: 123) or L234A / L235A / P329G (LALAPG) (SEQ ID NO: 125) mutation.

15. The bispecific antibody according to claim 14, wherein the IgG1 heavy chain constant domain has the N297Q (SEQ ID NO: 126) mutation.

16. A composition comprising a bispecific antibody according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier or diluent.

17. A composition comprising the bispecific antibody described in claim 5 and a pharmaceutically acceptable carrier or diluent.

18. A composition comprising the bispecific antibody described in claim 6 and a pharmaceutically acceptable carrier or diluent.

19. A composition comprising the bispecific antibody described in claim 7 and a pharmaceutically acceptable carrier or diluent.

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