Anti-SIRP alpha antibody

Abstract

Anti-SIRPα antibodies are provided 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. The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα, which comprises a combination of a heavy chain sequence containing a specific amino acid sequence and a light chain sequence containing another specific amino acid sequence.

Description

[Technical Field]

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

[0002] FIELD OF THE INVENTION The present invention relates to anti-SIRPα antibodies and the use of these antibodies in the treatment of disease. [Background technology]

[0003] Background of the Invention Signal-regulatory protein alpha (SIRPα) is a membrane glycoprotein of the SIRP family. SIRP family members share certain common structural motifs: a transmembrane segment and an N-terminal extracellular domain containing three Ig-like loops connected by three pairs of disulfide bonds. However, the C-terminal intracellular domain differs among SIRP family members. SIRPα has an extended intracellular domain containing four tyrosine residues that form two immunoreceptor tyrosine-dependent inhibitory motifs (ITIMs), whereas SIRPβ1 contains a lysine residue within the transmembrane domain followed by a short intracellular tail lacking ITIMs, which act as a receptor for DAP12. Eight SIRPα single-nucleotide polymorphisms have been identified, with SIRPαV1 and SIRPαV2 being the most common (Takenaka et al., Nat. Immunol. 2007, 8:1313-23).

[0004] "Eat me" signals (i.e., modified self) are extracellular players specifically produced and displayed on the surface of apoptotic cells, but not in healthy cells, that activate phagocyte receptors and then signal transduction cascades, making them important for the initiation of phagocytosis. Eat me signals require extracellular trafficking to be displayed on apoptotic cells. A specific category of eat me signals is provided by membrane-bound proteins such as phosphatidylserine (PtdSer) and calreticulin (CRT). Externalized PtdSer binds to receptors on phagocytes to facilitate 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-related protein 1 (LRP1) on phagocytes, thereby mediating phagocytosis.

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

[0006] Disruption of CD47-SIRPα signaling (e.g., by antagonistic monoclonal antibodies that bind to either CD47 or SIRPα) reportedly 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. Summary of the Invention

[0007] Summary of the Invention In a first aspect, the present invention provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising the structural and functional features specified below.

[0008] In various embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα, comprising one, two, or all three of (i), (ii), and (iii): (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that differs from SEQ ID NO:1 by one, two, three, or more conservative substitutions; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that differs from SEQ ID NO:2 by one, two, three, or more conservative substitutions; and / or (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that differs 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 thereof that binds to human SIRPα, comprising one, two, or all three of (i), (ii), and (iii): (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:69, or an amino acid sequence that differs from SEQ ID NO:1 by one, two, three, or more conservative substitutions; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:70, or an amino acid sequence that differs from SEQ ID NO:2 by one, two, three, or more conservative substitutions; and / or (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that differs from SEQ ID NO:3 by one, two, three, or more conservative substitutions.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof SEQ ID NO:75, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:78, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:80, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:82, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:84, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:86, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:88, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO: 102, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:7, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 12, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 16, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO:30, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; The heavy chain variable region comprises an amino acid sequence selected from the group consisting of:

[0011] In various embodiments, the present invention also provides an antibody or antigen-binding fragment thereof that binds to human SIRPα, comprising one, two, or all three of (i), (ii), and (iii), namely: (i) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that differs from SEQ ID NO:4 by one, two, three, or more conservative substitutions; (ii) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that differs from SEQ ID NO:5 by one, two, three, or more conservative substitutions; and / or (iii) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that differs 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 thereof that binds to human SIRPα, comprising one, two, or all three of (i), (ii), and (iii), namely: (i) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that differs from SEQ ID NO:4 by one, two, three, or more conservative substitutions; (ii) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:73, or an amino acid sequence that differs from SEQ ID NO:5 by one, two, three, or more conservative substitutions; and / or (iii) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:74, or an amino acid sequence that differs from SEQ ID NO:6 by one, two, three, or more conservative substitutions.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof SEQ ID NO:76, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:90, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:92, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:94, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:96, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:98, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 100, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 104, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:8, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:20, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:22, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:24, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:26, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:28, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO:32, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; The antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of:

[0014] In various embodiments, the present invention provides a method for treating a cancer cell comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence that differs from SEQ ID NO:1 by one, two, three or more conservative substitutions; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence that differs from SEQ ID NO:2 by one, two, three or more conservative substitutions; and / or (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence that differs from SEQ ID NO:3 by one, two, three or more conservative substitutions; and (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence that differs from SEQ ID NO:4 by one, two, three or more conservative substitutions; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence that differs from SEQ ID NO:5 by one, two, three or more conservative substitutions; and / or (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by one, two, three or more conservative substitutions. The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα, comprising the following:

[0015] In various other embodiments, the present invention provides a method for treating a cancer cell comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:69, or an amino acid sequence that differs from SEQ ID NO:1 by one, two, three or more conservative substitutions; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:70, or an amino acid sequence that differs from SEQ ID NO:2 by one, two, three or more conservative substitutions; and / or (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that differs from SEQ ID NO:3 by one, two, three or more conservative substitutions; and (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:72 or an amino acid sequence that differs from SEQ ID NO:4 by one, two, three or more conservative substitutions; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that differs from SEQ ID NO:5 by one, two, three or more conservative substitutions; and / or (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:74 by one, two, three or more conservative substitutions. The present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα, comprising the following:

[0016] In yet another embodiment, the present invention provides SEQ ID NO:7, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 12, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 16, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO:30, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:8, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:20, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:22, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:24, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:26, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:28, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO:32, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; a light chain variable region comprising 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, comprising:

[0017] In yet another embodiment, the present invention provides SEQ ID NO:75, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:78, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:80, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:82, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:84, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:86, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:88, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO: 102, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:76, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:90, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:92, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:94, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:96, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:98, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:100, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO: 104, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; a light chain variable region comprising 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, comprising:

[0018] In the context of this specification, "sequence similarity" is based on the degree of identity combined with the degree of conservative changes. The percentage of "sequence similarity" is the percentage of amino acids or nucleotides that are either identical or conservatively changed, i.e., "sequence similarity" = % sequence identity + % conservative changes. Therefore, for the purposes of the present invention, "conservative changes" and "identity" are considered to be species of the broader term "similarity." Thus, whenever the term sequence "similarity" is used, it encompasses sequence "identity" and "conservative changes." According to certain embodiments, conservative changes are disregarded, and the % sequence similarity refers to the % sequence identity. In certain embodiments, the changes in a sequence that are allowed by the referenced % sequence identity are all or almost all conservative changes; i.e., if the sequences are 90% identical, the remaining 10% are all or almost all conservative changes. In the context of this specification, the term "substantially all" refers to at least 75%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the allowed sequence variations being conservative variations. In certain embodiments of antibody heavy and / or light chains, the allowed sequence variations are within the framework regions and not within the CDRs.

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

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

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

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

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

[0024] The antibodies or antigen-binding fragments of the present invention may be derived from various species. For example, the antibodies of the present invention may comprise immunoglobulin sequences that are rabbit, mouse, rat, guinea pig, chicken, goat, sheep, donkey, human, llama, or camel sequences, or a combination of these sequences (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 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments connected by a disulfide bridge at the hinge region; and (iii) a V H and C H (iv) an Fd fragment consisting of the I domain; (iv) a V fragment of one arm of an antibody; L and V H (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Single-chain antibodies are also encompassed by reference to the term "antibody." Preferred therapeutic antibodies are intact IgG antibodies. As used herein, the term "intact IgG" refers to a polypeptide belonging to the class of antibodies substantially encoded by recognized immunoglobulin gamma genes. 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, VH ... H , Cγ1, Cγ2, Cγ3, V L , and C L is.

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

[0027] In any of the above embodiments, the antibody or antigen-binding fragment thereof of the present invention may comprise any of the above-described light chain variable regions, a human kappa or lambda light chain constant domain, and an IgG1, IgG2, or IgG4 heavy chain constant domain. Exemplary light (kappa) and heavy (IgG2 and IgG4) chain constant region sequences that can be used in accordance with the present invention are listed in SEQ ID NOs:63, 65, 67 (nucleotide sequences, respectively), 64, 66, and 68 (polypeptide sequences, respectively).

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

[0029] In some preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises SEQ ID NO:10 and each light chain comprises SEQ ID NO:20, or in each case at least 90%, 95%, 97%, 98% or 99% similar or identical to the respective SEQ ID NO, and most preferably each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises 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, wherein each heavy chain comprises SEQ ID NO: 16 and each light chain comprises SEQ ID NO: 28, or in each case at least 90%, 95%, 97%, 98% or 99% similar or identical to the respective SEQ ID NO, and most preferably each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises a human IgG1, IgG2 or IgG4 constant region.

[0031] In yet other preferred embodiments, the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises SEQ ID NO: 18 and each light chain comprises SEQ ID NO: 20, or in each case at least 90%, 95%, 97%, 98% or 99% similar or identical to the respective SEQ ID NO, and most preferably each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises 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, wherein each heavy chain comprises SEQ ID NO:80 and each light chain comprises SEQ ID NO:90, or in each case at least 90%, 95%, 97%, 98% or 99% similar or identical to the respective SEQ ID NO, and most preferably each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises 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, wherein each heavy chain comprises SEQ ID NO:80 and each light chain comprises SEQ ID NO:92, or in each case at least 90%, 95%, 97%, 98% or 99% similar or identical to the respective SEQ ID NO, and most preferably each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises 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, wherein each heavy chain comprises SEQ ID NO:80 and each light chain comprises SEQ ID NO:96, or in each case at least 90%, 95%, 97%, 98% or 99% similar or identical to the respective SEQ ID NO, and most preferably each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises a human IgG1, IgG2 or IgG4 constant region.

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

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

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

[0038] In certain embodiments, an antibody or antigen-binding fragment of the invention has one, two, three, four or more, and preferably each, of the following functional characteristics: EC 50 Binds to human SIRPαV1 protein having the sequence of SEQ ID NO:34 with <1 nM; EC2 at least 100-fold higher than SIRPαV1(P74A) having the sequence of SEQ ID NO:62 50 and optionally also exhibiting at least 100-fold higher EC 50(In each case, a decreased EC 50 is the EC for human SIRPαV1 protein having the sequence of SEQ ID NO:34 50 and in each instance preferably as measured by cellular ELISA (CELISA) as described hereinafter); <10 nM, preferably <5 nM, more preferably <1.5 nM, even more preferably <1.0 nM, even more preferably <0.5 nM, and most preferably an EC 50 binds to cells expressing human SIRPαV1 protein; <10 nM, preferably <5 nM, more preferably <1.5 nM, even more preferably <1.0 nM, even more preferably <0.5 nM, and most preferably an EC 50 binds to cells expressing human SIRPαV2 protein; At an antibody concentration of 50 nM, preferably 67 nM, more preferably 100 nM, or an antibody EC 50 does not appreciably bind to SIRPβ1 protein at concentrations 10-fold greater, preferably 50-fold greater, more preferably 100-fold greater, and even more preferably 200-fold greater than <10.0 nM, more preferably <5.0 nM, even more preferably <2.5 nM, and most preferably about 1.0 nM or less 50 inhibits the binding of human SIRPα to CD47; and exhibiting a T20 "humanness" score of at least 79, more preferably 85.

[0039] Preferably, the anti-SIRPα antibodies or antigen-binding fragments of the invention bind to cells expressing human SIRPαV1 protein with an EC50 <10 nM, while exhibiting an antibody concentration of 100 nM or an antibody EC50 <10 nM for SIRPαV1 or SIRPαV2. 50Most preferably, each light chain comprises a human kappa light chain or a human lambda light chain constant domain, and each heavy chain comprises a human IgG1, IgG2, or IgG4 constant region.

[0040] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof of the present invention can be conjugated to at least one therapeutic agent, which in one embodiment 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, enzyme, radiolabel, hormone, antisense oligonucleotide, or cytotoxic agent, or a combination thereof.

[0041] The present invention also provides isolated polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: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 a fragment of any of said sequences, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

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

[0043] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: SEQ ID NO:75, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:78, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:80, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:82, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:84, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:86, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:88, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO: 102, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 12, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 16, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO:30, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; The present invention provides an isolated nucleic acid encoding an amino acid sequence selected from the group consisting of:

[0044] In certain embodiments, the amino acid sequence of SEQ ID NO:10, or an amino acid sequence 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0057] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: SEQ ID NO:76, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:90, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:92, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:94, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:96, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:98, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 100, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO: 104, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:8, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:20, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:22, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:24, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:26, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; SEQ ID NO:28, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto, and SEQ ID NO:32, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% similar or identical thereto; The present invention provides an isolated nucleic acid encoding an amino acid sequence selected from the group consisting of:

[0058] In certain embodiments, the amino acid sequence of SEQ ID NO:20, or an amino acid sequence 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0071] In certain embodiments, the isolated nucleic acids of the invention can optionally include a leader sequence.

[0072] Such nucleic acids may include one or more of the following nucleic acid sequences: the nucleic acid sequence of SEQ ID NO:77, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:79, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:81, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:83, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:85, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 101, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:89, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:91, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:93, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:95, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:97, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:99, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 101, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 103, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:9, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:29, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 19, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:21, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:23, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:25, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:27, or a nucleic acid sequence 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 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 an IgG. In a preferred embodiment, the antibody is an IgG1, IgG2, or IgG4, preferably a 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 comprise the following combination of heavy and light chain variable region nucleic acid sequences: SEQ ID NO:9 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H1L1) SEQ ID NO:9 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H1L2) SEQ ID NO:9 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H1L3) SEQ ID NO:9 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H1L4) SEQ ID NO:9 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H1L5) SEQ ID NO:11 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H2L1) SEQ ID NO:11 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H2L2) SEQ ID NO:11 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H2L3) SEQ ID NO:11 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H2L4) SEQ ID NO:11 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H2L5) SEQ ID NO:13 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H3L1) SEQ ID NO:13 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H3L2) SEQ ID NO:13 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H3L3) SEQ ID NO:13 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H3L4) SEQ ID NO:13 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H3L5) SEQ ID NO:15 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H4L1) SEQ ID NO:15 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H4L2) SEQ ID NO:15 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H4L3) SEQ ID NO:15 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H4L4) SEQ ID NO:15 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H4L5) SEQ ID NO:17 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H5L1) SEQ ID NO:17 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H5L2) SEQ ID NO:17 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H5L3) SEQ ID NO:17 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H5L4) SEQ ID NO:17 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H5L5) SEQ ID NO:77 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H1L1) SEQ ID NO:77 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H1L2) SEQ ID NO:77 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H1L3) SEQ ID NO:77 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H1L4) SEQ ID NO:77 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H1L5) SEQ ID NO:77 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H1L6) SEQ ID NO:79 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H2L1) SEQ ID NO:79 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H2L2) SEQ ID NO:79 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H2L3) SEQ ID NO:79 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H2L4) SEQ ID NO:79 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H2L5) SEQ ID NO:79 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H2L6) SEQ ID NO:81 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H3L1) SEQ ID NO:81 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H3L2) SEQ ID NO:81 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H3L3) SEQ ID NO:81 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H3L4) SEQ ID NO:81 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H3L5) SEQ ID NO:81 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H3L6) SEQ ID NO:83 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H4L1) SEQ ID NO:83 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H4L2) SEQ ID NO:83 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H4L3) SEQ ID NO:83 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H4L4) SEQ ID NO:83 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H4L5) SEQ ID NO:83 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H4L6) SEQ ID NO:85 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H5L1) SEQ ID NO:85 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H5L2) SEQ ID NO:85 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H5L3) SEQ ID NO:85 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H5L4) SEQ ID NO:85 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H5L5) SEQ ID NO:85 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H5L6) SEQ ID NO:87 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H6L1) SEQ ID NO:87 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H6L2) SEQ ID NO:87 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H6L3) SEQ ID NO:87 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H6L4) SEQ ID NO:87 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H6L5) SEQ ID NO:87 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H6L6) Or, in each instance, at least 90%, 95%, 97%, 98% or 99% identical to the respective SEQ ID NO.

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

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

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

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

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

[0080] In some preferred embodiments, the nucleic acid comprises SEQ ID NO:79 and SEQ ID NO:95, or in each instance 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 required for the transcription of a target nucleic acid in a host cell. Typically, the target nucleic acid is placed 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 linked to" a regulatory element when the regulatory element controls the expression of a gene.

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

[0083] Such expression vectors may include one or more of the following nucleic acid sequences operably linked to regulatory elements: the nucleic acid sequence of SEQ ID NO:77, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:79, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:81, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:83, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:85, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 101, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:89, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:91, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:93, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:95, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:97, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:99, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 103, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:29, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO: 19, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:21, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:23, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:25, or a nucleic acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; the nucleic acid sequence of SEQ ID NO:27, or a nucleic acid sequence 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 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 and light chain sequences of an anti-SIRPα antibody of the invention. Preferably, such an expression vector comprises the following combination of heavy and light chain variable region nucleic acid sequences: SEQ ID NO:9 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H1L1) SEQ ID NO:9 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H1L2) SEQ ID NO:9 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H1L3) SEQ ID NO:9 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H1L4) SEQ ID NO:9 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H1L5) SEQ ID NO:11 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H2L1) SEQ ID NO:11 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H2L2) SEQ ID NO:11 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H2L3) SEQ ID NO:11 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H2L4) SEQ ID NO:11 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H2L5) SEQ ID NO:13 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H3L1) SEQ ID NO:13 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H3L2) SEQ ID NO:13 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H3L3) SEQ ID NO:13 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H3L4) SEQ ID NO:13 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H3L5) SEQ ID NO:15 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H4L1) SEQ ID NO:15 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H4L2) SEQ ID NO:15 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H4L3) SEQ ID NO:15 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H4L4) SEQ ID NO:15 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H4L5) SEQ ID NO:17 / SEQ ID NO:19 (referred to herein as hSIRPα.50A.H5L1) SEQ ID NO:17 / SEQ ID NO:21 (referred to herein as hSIRPα.50A.H5L2) SEQ ID NO:17 / SEQ ID NO:23 (referred to herein as hSIRPα.50A.H5L3) SEQ ID NO:17 / SEQ ID NO:25 (referred to herein as hSIRPα.50A.H5L4) SEQ ID NO:17 / SEQ ID NO:27 (referred to herein as hSIRPα.50A.H5L5) SEQ ID NO:77 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H1L1) SEQ ID NO:77 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H1L2) SEQ ID NO:77 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H1L3) SEQ ID NO:77 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H1L4) SEQ ID NO:77 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H1L5) SEQ ID NO:77 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H1L6) SEQ ID NO:79 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H2L1) SEQ ID NO:79 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H2L2) SEQ ID NO:79 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H2L3) SEQ ID NO:79 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H2L4) SEQ ID NO:79 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H2L5) SEQ ID NO:79 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H2L6) SEQ ID NO:81 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H3L1) SEQ ID NO:81 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H3L2) SEQ ID NO:81 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H3L3) SEQ ID NO:81 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H3L4) SEQ ID NO:81 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H3L5) SEQ ID NO:81 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H3L6) SEQ ID NO:83 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H4L1) SEQ ID NO:83 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H4L2) SEQ ID NO:83 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H4L3) SEQ ID NO:83 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H4L4) SEQ ID NO:83 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H4L5) SEQ ID NO:83 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H4L6) SEQ ID NO:85 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H5L1) SEQ ID NO:85 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H5L2) SEQ ID NO:85 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H5L3) SEQ ID NO:85 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H5L4) SEQ ID NO:85 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H5L5) SEQ ID NO:85 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H5L6) SEQ ID NO:87 / SEQ ID NO:89 (referred to herein as hSIRPα.40A.H6L1) SEQ ID NO:87 / SEQ ID NO:91 (referred to herein as hSIRPα.40A.H6L2) SEQ ID NO:87 / SEQ ID NO:93 (referred to herein as hSIRPα.40A.H6L3) SEQ ID NO:87 / SEQ ID NO:95 (referred to herein as hSIRPα.40A.H6L4) SEQ ID NO:87 / SEQ ID NO:97 (referred to herein as hSIRPα.40A.H6L5) SEQ ID NO:87 / SEQ ID NO:99 (referred to herein as hSIRPα.40A.H6L6) Or, in each instance, at least 90%, 95%, 97%, 98% or 99% identical to the respective SEQ ID NO.

[0085] In any of the above embodiments, the expression vector can encode a human or humanized antibody for expression and includes both heavy and light chain nucleic acid sequences. In one embodiment, the antibody is an IgG. In a preferred embodiment, the antibody is an IgG1, IgG2, or IgG4, preferably a human IgG1, IgG2, or IgG4. In a specific 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 for expression a human or humanized antibody, wherein the heavy chain nucleic acid sequence comprises SEQ ID NO:9 and the light chain nucleic acid sequence comprises SEQ ID NO:19, or in each case at least 90%, 95%, 97%, 98% or 99% identical to the respective SEQ ID NO, and is most preferably of the IgG1, IgG2 or IgG4 isotype.

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

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

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

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

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

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

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

[0094] In one embodiment, the composition comprises one or more additional therapeutic agents, such as an anti-CD27 antibody or antigen-binding fragment thereof, an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-APRIL antibody or antigen-binding fragment thereof, an anti-TIGIT antibody or antigen-binding fragment thereof, an anti-VISTA antibody or antigen-binding fragment thereof, an anti-BTLA antibody or antigen-binding fragment thereof, an anti-TIM3 antibody or antigen-binding fragment thereof, an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-HVEM antibody or antigen-binding fragment thereof, an anti-CD70 antibody or antigen-binding fragment thereof, an anti-CD137 antibody or antigen-binding fragment thereof, or an anti-OX40 antibody. or antigen-binding fragment thereof; anti-CD28 antibody or antigen-binding fragment thereof; anti-PD1 antibody or antigen-binding fragment thereof; anti-PDL1 antibody or antigen-binding fragment thereof; anti-PDL2 antibody or antigen-binding fragment thereof; anti-GITR antibody or antigen-binding fragment thereof; anti-ICOS antibody or antigen-binding fragment thereof; anti-ILT2 antibody or antigen-binding fragment thereof; anti-ILT3 antibody or antigen-binding fragment thereof; anti-ILT4 antibody or antigen-binding fragment thereof; anti-ILT5 antibody or antigen-binding fragment thereof; anti-4-1BB antibody or antigen-binding fragment thereof; anti-NKG2A antibody or antigen-binding fragment thereof; anti NKG2C antibodies or antigen-binding fragments thereof; anti-NKG2E antibodies or antigen-binding fragments thereof; anti-TSLP antibodies or antigen-binding fragments thereof; anti-IL-10 antibodies or antigen-binding fragments thereof; IL-10 or PEGylated IL-10; agonists of TNF receptor proteins (e.g., agonistic antibodies or antigen-binding fragments thereof, or soluble fusions); immunoglobulin-like proteins; cytokine receptors; integrins; signaling lymphocyte activation molecules (SLAM proteins); activating 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;CD8alpha;CD8beta;IL2Rbeta;IL2Rgamma;IL7Ralpha;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 ); CD69; SLAMF6 (NTB-A; Lyl08); SLAM (SLAMF1, CD150, IPO-3); SLAM7; BLAME (SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; PAG / Cbp; CD19a; a ligand that specifically binds to CD83; an inhibitor of CD47, PD-1, PD-L1; PD-L2; CTLA4; TIM3; LAG3; CEACAM (e.g., CEACAM-1, -3, and / or -5); VISTA; BTLA; TIGIT; LAIRl; IDO; TDO; CD160; TGFR beta; and a cyclic dinucleotide or other STING pathway agonist.

[0095] The present invention also includes combinations comprising an antibody or antigen-binding fragment of the present invention and a second antibody that induces ADCC, where 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 whose 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 includes a combination comprising an antibody or antigen-binding fragment of the present invention and a second antibody that induces ADCP, where 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 key role in cancer immunotherapy, including tumor antigen targeting with monoclonal antibodies (mAbs). In the context of cell targeting, NK cells can be "specifically activated" through specific Fc receptors expressed on the cell surface. NK cells can 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 Fc receptors by antibodies bound to target cells, NK cells can lyse target cells without priming and secrete cytokines such as interferon gamma to recruit adaptive immune cells. Similarly, tumor-associated macrophages (TAMs) express surface receptors that bind the Fc fragment of antibodies, engaging the antibodies in Ab-dependent cellular cytotoxicity / phagocytosis (ADCC / ADCP). Because SIRPα / CD47 signaling induces a "don't eat me" response that reduces ADCC / ADCP, blocking this signaling with the anti-SIRPα antibodies or antigen-binding fragments of the invention may enhance ADCC of tumor cells that contain the antigenic determinant against which the therapeutic antibody is directed.

[0098] This ADCC / ADCP as a mechanism of action may be used to treat various cancers and infectious diseases. An exemplary list of ADCC / ADCP-inducing antibodies and antibody conjugates that may be combined with the antibodies or antigen-binding fragments of the invention includes rituximab, ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab, 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, ifavotuzumab, farletuzumab, otlertuzumab, carotuximab, epratuzumab, inebilizumab, lumletuzumab, 4G 7SDIE, AFM21, AFM22, LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20, mogamulizumab, ChiLob-7 / 4, leukoximab, isatuximab, DS-8895, FPA144, GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213, P These include, but are not limited to, ankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202, MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomab tiuxetan, ABBV-838, HuMax-AXL-ADC, and adotrastuzumab emtansine (Kadcyla).An exemplary list of target antigens for such ADCC / ADCP-inducing antibodies includes, but is 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 antigen-binding fragment thereof induces ADCP. By way of example only, such an antibody may be selected from the group consisting of rituximab, ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab, obinutuzumab, trastuzumab, cetuximab, alemtuzumab, ibritumomab, farletuzumab, inebilizumab, lumletuzumab, 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 antibodies or antigen-binding fragments of the invention are combined with one or more ADCC / ADCP-inducing antibodies and antibody conjugates, such combinations may be used, optionally in conjunction with additional therapeutic agents or procedures. In one embodiment, the additional therapeutic agents are an anti-LAG3 antibody or antigen-binding fragment thereof; an anti-APRIL antibody or antigen-binding fragment thereof; an anti-TIGIT antibody or antigen-binding fragment thereof; an anti-VISTA antibody or antigen-binding fragment thereof; an anti-BTLA antibody or antigen-binding fragment thereof; an anti-TIM3 antibody or antigen-binding fragment thereof; an anti-CTLA4 antibody or antigen-binding fragment thereof; an anti-HVEM antibody or antigen-binding fragment thereof; an anti-CD70 antibody or antigen-binding fragment thereof; an anti-CD137 antibody or antigen-binding fragment thereof; an anti-OX40 antibody or antigen-binding fragment thereof; an anti-CD28 antibody or antigen-binding fragment thereof; an anti-PD1 antibody or antigen-binding fragment thereof; or an anti-PDL1 antibody or antigen-binding fragment thereof. 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 invention also provides a container or injection device comprising any one of the anti-SIRPα antibodies or antigen-binding fragments of the 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 a host cell containing a polynucleotide encoding the heavy chain and / or light chain of an antibody (or antigen-binding fragment thereof) of the present invention under conditions suitable for expression of the polynucleotide; and optionally recovering the antibody or antigen-binding fragment from the host cell and / or culture medium. In one embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in a single 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 of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an anti-SIRPα antibody or antigen-binding fragment of the present invention, optionally in association with an additional therapeutic agent or procedure.

[0104] In one embodiment, the subject to be treated is a human subject. In one embodiment, the additional therapeutic agent is an anti-LAG3 antibody or antigen-binding fragment thereof; an anti-APRIL antibody or antigen-binding fragment thereof; an anti-TIGIT antibody or antigen-binding fragment thereof; an anti-VISTA antibody or antigen-binding fragment thereof; an anti-BTLA antibody or antigen-binding fragment thereof; an anti-TIM3 antibody or antigen-binding fragment thereof; an anti-CTLA4 antibody or antigen-binding fragment thereof; an anti-HVEM antibody or antigen-binding fragment thereof; an anti-CD70 antibody or antigen-binding fragment thereof; an anti-CD137 antibody or antigen-binding fragment thereof; an anti-OX40 antibody or antigen-binding fragment thereof; an anti-CD28 antibody or antigen-binding fragment thereof; an anti-PD1 antibody or antigen-binding fragment thereof; or an anti-PDL1 antibody or antigen-binding fragment thereof. 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 of 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, optionally in association with an additional therapeutic agent or procedure. In one embodiment, the subject being treated is a human subject.

[0106] In one embodiment, the additional therapeutic agent is an anti-LAG3 antibody or antigen-binding fragment thereof; an anti-APRIL antibody or antigen-binding fragment thereof; an anti-TIGIT antibody or antigen-binding fragment thereof; an anti-VISTA antibody or antigen-binding fragment thereof; an anti-BTLA antibody or antigen-binding fragment thereof; an anti-TIM3 antibody or antigen-binding fragment thereof; an anti-CTLA4 antibody or antigen-binding fragment thereof; an anti-HVEM antibody or antigen-binding fragment thereof; an anti-CD70 antibody or antigen-binding fragment thereof; an anti-CD137 antibody or antigen-binding fragment thereof; an anti-OX40 antibody or antigen-binding fragment thereof; an anti-CD28 antibody or antigen-binding fragment thereof; an anti-PD1 antibody or antigen-binding fragment thereof; or an anti-PDL1 antibody or antigen-binding 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 between the antibody or fragment and the peptide, wherein detection of the complex indicates the presence of the SIRPα peptide. [Brief explanation of the drawings]

[0108] [Figure 1] 1 depicts the cross-reactivity of commercially available anti-hSIRPα antibodies with hSIRPβ1 and allele-specific binding to hSIRPαV1 and hSIRPαV2. [Figure 2] 1 shows the reactivity of KWAR23 antibody with hSIRPαV1, hSIRPαV2, hSIRPβ1, and hSIRPγ. [Figure 3] 1 shows the reactivity of antibody clone hSIRPα.50A with various hSIRPα alleles. [Figure 4] 1 depicts the ability of hSIRPα.50A antibody to block recombinant hCD47 / Fc protein binding to cell surface expressed hSIRPα. [Figure 5A] Binding of hSIRPα.50A antibody to primary human CD14+ enriched monocytes is shown. [Figure 5B] Binding of hSIRPα.50A antibody to primary human CD14+ enriched monocytes is shown. [Figure 5C] 1 depicts the ability of hSIRPα.50A antibodies to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 5D] 1 depicts the ability of hSIRPα.50A antibodies to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 6A] Binding of hSIRPα.50A antibody to primary human granulocytes. [Figure 6B] Phagocytosis of tumor cells by primary human granulocytes in the presence of rituximab + or - hSIRPα.50A antibody. [Figure 6C] Figure 1 depicts the phagocytosis of tumor cells by primary human granulocytes in the presence of daratumumab + or - hSIRPα.50A antibody. [Figure 6D] Phagocytosis of tumor cells by primary human granulocytes in the presence of alemtuzumab + or - hSIRPα.50A antibody. [Figure 6E] Phagocytosis of tumor cells by primary human granulocytes in the presence of cetuximab + or - hSIRPα.50A antibody. [Figure 7] Phagocytosis of tumor cells by human macrophages in the presence of the indicated antibody (rituximab or daratumumab) + or - hSIRPα.50A antibody. [Figure 8] 1 depicts blockade of the hSIRPα / hCD47 interaction by mouse hSIRPα.50A and humanized hSIRPα.50A antibodies to hSIRPα. [Figure 9]Figure 1 depicts hSIRPα.50A antibody binding to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPα-VβC1αC2α, hSIRPα-VαC1βC2α, and hSIRPα-VαC1αC2β. [Figure 10A] 1 shows an alignment of hSIRPα and hSIRPβ1 IgV domain amino acid sequences. [Figure 10B] Figure 1 shows loss of hSIRPα.50A antibody binding to hSIRPαV1(P74A). [Figure 11] Binding of hSIRPα.40A and hSIRPα.50A antibodies to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPβL, and hSIRPγ is shown. [Figure 12] Binding of hSIRPα.40A and hSIRPα.50A antibodies to hSIRPαV1, hSIRPαV2, hSIRPαV3, hSIRPαV4, hSIRPαV5, hSIRPαV6, hSIRPαV8, and hSIRPαV9 is shown. [Figure 13] 1 depicts the ability of hSIRPα.40A and hSIRPα.50A antibodies to block recombinant hCD47 / Fc protein binding to cell surface expressed hSIRPα. [Figure 14A] Binding of hSIRPα.40A antibody to primary human CD14+ enriched monocytes is shown. [Figure 14B] Binding of hSIRPα.40A antibody to primary human CD14+ enriched monocytes is shown. [Figure 14C] 1 depicts the ability of hSIRPα.40A antibodies to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 14D] 1 depicts the ability of hSIRPα.40A antibodies to block the binding of hCD47 to primary human CD14+ enriched monocytes. [Figure 15A] Binding of hSIRPα.40A and hSIRPα.50A antibodies to primary human granulocytes. [Figure 15B] 1 depicts phagocytosis of Ramos cells by primary human granulocytes in the presence of rituximab + or - hSIRPα.40A and hSIRPα.50A antibodies. [Figure 16] 1 shows enhancement of rituximab-induced Raji cell phagocytosis by hSIRPα.40A and hSIRPα.50A antibodies. [Figure 17] Binding of murine hSIRPα.40A and humanized hSIRPα.40A antibodies to hSIRPα is shown. [Figure 18] 1 shows blockade of hCD47 binding to hSIRPα in the presence of the humanized hSIRPα.40A antibody variant. [Figure 19] Binding of hSIRPα.40A and hSIRPα.50A antibodies to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRP-VγC1βC2β, hSIRP-VβC1γC2β, and hSIRP-VβC1βC2γ is shown. [Figure 20] Figure 1 shows loss of hSIRPα.40A and hSIRPα.50A antibody binding to hSIRPαV1(P74A). [Figure 21] 1 depicts the ability of chimeric hSIRPα.40A antibody variants to affect rituximab-mediated phagocytosis. [Figure 22] 1 depicts the ability of humanized hSIRPα.40A antibody variants to affect rituximab-mediated phagocytosis. [Figure 23A] 1 depicts the ability of murine hSIRPα.50A and chimeric hSIRPα.50A hIgG2 and hIgG4 antibody variants to affect rituximab-mediated phagocytosis. [Figure 23B] 1 depicts the ability of chimeric hSIRPα.50A hIgG2 and hIgG4 antibody variants to affect rituximab-mediated phagocytosis. [Figure 23C] 1 depicts the ability of chimeric hSIRPα.50A hIgG2 and hIgG4 antibody variants to affect daratumumab-mediated phagocytosis. [Figure 23D] 1 depicts the ability of murine hSIRPα.50A and chimeric hSIRPα.50A hIgG2 antibody variants to affect rituximab-mediated phagocytosis in granulocytes. [Figure 24A]1 depicts the ability of murine 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] Figure 1 depicts the ability of murine 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] 1 depicts 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. DETAILED DESCRIPTION OF THE INVENTION

[0109] Detailed Description Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: ADCC antibody-dependent cytotoxicity ADCP antibody-dependent cellular phagocytosis CDC Complement-dependent cytotoxicity CDR Complementarity-determining regions within immunoglobulin variable regions defined using the Kabat numbering system CHO Chinese hamster ovary EC50: The concentration at which 50% of the total binding signal is observed ELISA enzyme-linked immunosorbent assay FR antibody framework region: immunoglobulin variable region excluding CDR regions HRP horseradish peroxidase IFN Interferon IC50: Concentration that produces 50% inhibition IgG immunoglobulin G The 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 Staphylococcal enterotoxin B TT tetanus toxoid V region The segment of an Ig chain that is variable in sequence among different antibodies, spanning Kabat residues 109 in the light chain and 113 in the heavy chain. VH immunoglobulin heavy chain variable region VK immunoglobulin kappa light chain variable region VL immunoglobulin light chain variable region

[0110] definition In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0111] As used in this specification, including the appended claims, the singular forms of language such as "a," "an," and "the" include the corresponding plural referents unless the context clearly dictates otherwise.

[0112] "Administration" and "treatment," as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell and contact of a reagent with a fluid in contact with the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatments of a cell, for example, with a reagent, diagnostic agent, binding compound, or another cell.

[0113] "Treat" or "treating" refers to the administration, either internally or externally, of a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments 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 a disease. Typically, the agent is administered in an amount effective to improve one or more disease symptoms in the treated subject or population, whether by any clinically measurable degree, whether inducing recovery or inhibiting progression of such symptom(s). The amount of therapeutic agent effective to improve any particular disease symptom may vary depending on factors such as the state of the disease, the age and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been improved can be assessed by any clinical scale typically used by a physician or other skilled health care provider to assess the severity or progression of the symptom.

[0114] "Recombinant expression" of a protein refers to the transcription and translation of a foreign 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 includes multiple genes encoding proteins (e.g., SIRPα, SIRPβ1, and SIRPγ) with similar extracellular domains and distinct transmembrane and / or cytoplasmic domains with opposing signaling capacities (activation or inhibition). Similar to SIRPα, several paired receptors are present on NK cells, including the SIRP and CD200 receptor families, and some on myeloid cells (Hatherley et al., Mol Cell. 2008; 31:266-277).

[0116] SIRPα contains an extracellular region that can be subdivided into three distinct domains: the Ig-like (immunoglobulin-like) V (IgV), Ig-like C1 (IgC1), and Ig-like 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 regions that can be subdivided into IgV, IgC1, and IgC2 domains. However, SIRPα, SIRPβ1, and SIRPγ have distinct cytoplasmic regions. SIRPβ1 has a very short cytoplasmic region of only six amino acids and lacks a signaling motif for phosphatase association. Instead, this protein associates with DNAX-activation protein 12 (DAP12), a dimeric adaptor protein that binds amino acids with basic side chains within the transmembrane region of SIRPβ1, allowing it to transmit activation signals through immunoreceptor tyrosine-based activation motifs (ITAMs). SIRPγ also has a short cytoplasmic domain of four amino acids, but lacks charged amino acid side chains within the transmembrane domain and therefore does not associate with DAP12, leading to its annotated status 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 a single extracellular IgV domain, a fivefold transmembrane domain, and a short cytoplasmic tail. CD47 functions as a cellular ligand, binding via the NH2-terminal IgV domain of SIRPα. Evidence that CD47 contributes to self-recognition is provided by the fact that splenic macrophages from CD47-expressing mice express CD47. - / - This comes from the observation that mice clear transfused blood cells (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 of which belong to the collectin family. Sp-D has been reported to bind to the membrane-proximal IgC2 domain of SIRPα in a calcium- and glucose-dependent manner. Sp-A and Sp-D are thought to maintain an anti-inflammatory environment in the lung 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 in SEQ ID NOs: 34, 36, 44, 46, 48, 50, 52, and 54; exemplary nucleic acid sequences encoding these variants are listed in SEQ ID NOs: 33, 35, 43, 45, 47, 49, 51, and 53, respectively.

[0120] For comparison, the amino acid sequences of human SIRPβ1 and SIRPγ are listed in SEQ ID NOs:38 and 40, respectively, and exemplary nucleic acid sequences are listed in SEQ ID NOs:37 and 39, respectively.

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

[0122] The modified SIRPα polypeptides hSIRPα-VβC1αC2α, hSIRPα-VαC1βC2α, hSIRPα-VαC1αC2β, and hSIRPαV1(P74A) discussed hereinafter are listed in SEQ ID NOs:56, 58, 60, and 62; exemplary nucleic acid sequences encoding these variants are listed in SEQ ID NOs:55, 57, 59, and 61, respectively.

[0123] Anti-SIRPα antibodies and antigen-binding fragments thereof The present invention provides antibodies or antigen-binding fragments thereof that bind to human SIRPα, and uses of such antibodies or fragments. In some embodiments, the anti-SIRPα antibodies are 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 technologies (e.g., KinExa or OCTET).

[0125] The term "antibody" as used herein 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 F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge at the hinge region; and (iii) a V H and C H (iv) an Fd fragment consisting of the I domain; (iv) a V fragment of one arm of an antibody; L and V H(v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Single-chain antibodies are also encompassed by reference to the term "antibody." Preferred therapeutic antibodies are intact IgG antibodies. As used herein, the term "intact IgG" refers to a polypeptide belonging to the class of antibodies substantially encoded by recognized immunoglobulin gamma genes. 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, VH ... H , Cγ1, Cγ2, Cγ3, V L , and C L is.

[0126] The present invention encompasses anti-SIRPα antigen-binding fragments and methods of use thereof.

[0127] As used herein, a "full antibody" is a bivalent molecule comprising, for example, IgG, two heavy chains and two light chains. Each heavy chain has a V H domain followed by a constant domain (C H1 ), the hinge region, and two more constant (C H2 and C H3 ) domains; each light chain contains one V L domain and one constant (C L ) domains. In the case of IgM, a full-length antibody is a decavalent or dodecavalent molecule containing five or six linked immunoglobulin monomers, each with two antigen-binding sites forming a heavy and light chain.

[0128] As used herein, unless otherwise indicated, "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 the antigen bound by the full-length antibody, e.g., 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] The present invention encompasses anti-SIRPα Fab fragments and methods of using same. A "Fab fragment" is a fragment consisting of one light chain and one heavy chain. H 1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fab fragment" can be the product of papain cleavage of an antibody.

[0130] The present invention encompasses anti-SIRPα antibodies and antigen-binding fragments thereof that contain an Fc region, and methods of using the same. H 3 and C H The two heavy chain fragments contain two domains, which are connected by two or more disulfide bonds and H The three domains are held together by hydrophobic interactions.

[0131] The present invention encompasses anti-SIRPα Fab′ fragments and methods of using same. A “Fab′ fragment” is a fragment that contains one light chain and one V H Domain and C H a portion or fragment of one heavy chain containing one domain and a C H 1 and C H and the region between the two domains, so that interchain disulfide bonds can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0132] The present invention encompasses anti-SIRPα F(ab')2 fragments and methods of using same. "F(ab')2 fragment" refers to a fragment consisting of two light chains and a C H1Domain and C H2 Since the F(ab')2 fragment contains two heavy chains with a portion of the constant region between the domains, an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains. The "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody.

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

[0134] The present invention encompasses anti-SIRPα scFv fragments and methods of use thereof. The term "single chain Fv" or "scFv" antibody refers to the V of an antibody. H and V L Fv refers to an antibody fragment containing V domains, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide contains V domains that allow the scFv to form the desired structure for antigen binding. H Domains and V L The scFv domains further comprise a polypeptide linker between them. For a review of scFvs, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88 / 01649, and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0135] The present invention encompasses anti-SIRPα domain antibodies and methods of use thereof. A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The two V domains are covalently joined by a peptide linker to create a bivalent domain antibody. HThe regions may target the same or different antigens.

[0136] The present invention encompasses anti-SIRPα bivalent antibodies and methods of use thereof. A "bivalent antibody" comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificity. However, a bivalent antibody may also be bispecific (see below).

[0137] The present invention encompasses anti-SIRPα diabodies and methods of use thereof. As used herein, the term "diabody" refers to a diabody comprising a plurality of polypeptide chains (V H -V L or V L -V H ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H (Antibody) refers to a small antibody fragment having two antigen-binding sites, each containing a nucleotide sequence identical to that of the nucleotide sequence of the antibody fragment. The use of a linker that is too short to allow pairing between the two domains on the same chain encourages pairing of the domains with complementary domains on another chain and the creation of two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 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 generally Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0138] Typically, antibodies or antigen-binding fragments of the invention modified in some way retain at least 10% of their binding activity (compared to the parent antibody) when expressed on a molar basis. Preferably, antibodies or antigen-binding fragments of the invention retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100%, or more, of the SIRPα-binding affinity of the parent antibody. It is also intended that antibodies or antigen-binding fragments of the invention may include conservative or non-conservative amino acid substitutions ("conservative variants" or "function-conservative variants" of antibodies) that do not substantially alter biological activity.

[0139] The present invention encompasses isolated anti-SIRPα antibodies and antigen-binding fragments thereof, as well as methods for their use. As used herein, the term "isolated" does not refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of pharmaceutical formulations containing the antibody or fragment. An "isolated" antibody, antigen-binding fragment, nucleic acid, etc., has been identified and separated and / or recovered from one or more components of its natural environment. In preferred embodiments, the antibody, antigen-binding fragment, nucleic acid, etc., is purified to at least 75% by weight, more preferably at least 90% by weight, even more preferably at least 95% by weight, and even more preferably at least 98% by weight. Thus, an "isolated" biological molecule is at least partially free from other biological molecules from the cell or cell culture from which it is produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free from components of the expression system, such as biological molecules from the host cell or in its growth medium.

[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 are of 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 (the "parent antibody"), and the constant domain sequence is obtained from a human antibody, so the resulting chimeric antibody will be less likely to induce an adverse immune response in a human subject than the parent (e.g., mouse) antibody.

[0141] The present invention encompasses anti-SIRPα humanized antibodies and antigen-binding fragments thereof (e.g., humanized rat or mouse antibodies), as well as methods for their use. As used herein, the term "humanized antibody" refers to a form of antibody that contains sequences from both human and non-human (e.g., mouse or rat) antibodies. Generally, such humanized antibodies are substantially composed of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions being those of a human immunoglobulin sequence. The humanized antibody may optionally also contain at least a portion of a human immunoglobulin constant region (Fc). For further details on humanized antibodies, see, e.g., 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] The structural unit of a basic antibody typically comprises 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-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Human heavy chains are further classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype 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. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0143] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are generally identical.

[0144] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions to enable binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is generally based on the information 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 the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen binding. The hypervariable region comprises 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 antibody CDR regions by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining antibody CDR regions by structure). As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0146] An "isolated nucleic acid molecule" or "isolated polynucleotide" means genomic DNA or RNA, mRNA, cDNA, or synthetic origin or some combination thereof, unassociated with all or a portion of a polynucleotide, whether the isolated polynucleotide is found in nature or linked to a polynucleotide to which it is not naturally linked. For purposes of this disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass an intact chromosome. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or up to 20 or more other proteins, or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0147] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0148] A nucleic acid or polynucleotide is "operably linked" by virtue of its functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, but not necessarily, "operably linked" means that the linked DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient control sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0149] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is also understood that not all progeny will have the exact same amount of DNA, due to deliberate or inadvertent mutations. Mutant progeny that have the same function and biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, it will be clear from the context.

[0150] As used herein, "germline sequence" refers to a sequence of unrearranged immunoglobulin DNA sequences. 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 and Musculoskeletal and Skin Diseases of the 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 anti-SIRPα antibodies and antigen-binding fragments thereof having specified structural and functional features, and methods of using the antibodies or antigen-binding fragments thereof in the treatment or prevention of disease (e.g., cancer or infectious disease).

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

[0153] Several methods are available for mapping antigenic epitopes on target antigens, including H / D-Ex mass spectrometry, cross-linking-coupled mass spectrometry, X-ray crystallography, Pepscan analysis, and site-directed mutagenesis. For example, HDX (hydrogen-deuterium exchange) coupled with proteolysis and mass spectrometry can be used to determine the epitope of an antibody on a specific antigen Y. HDX-MS relies on the precise measurement and comparison of the degree of deuterium incorporation by the antigen when incubated in DO alone and in the presence of the antibody for various time intervals. While deuterium exchanges with hydrogen on the amide backbone of the protein in exposed areas, regions of the antigen bound to the antibody are protected and will show little or no exchange after LC-MS / MS analysis of proteolytic fragments. Cross-linking-coupled mass spectrometry begins by conjugating the antibody and antigen with a mass-labeled chemical cross-linker. The presence of the complex is then confirmed using high-mass MALDI detection. Because the Ab / Ag complex is extremely stable after cross-linking chemistry, many different enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high-resolution mass spectrometry and MS / MS techniques. The identity of the cross-linked peptide is determined using a mass tag attached to the cross-linking reagent. After MS / MS fragmentation and data analysis, both the epitope and paratope are determined in the same experiment.

[0154] The scope of the present invention also includes isolated anti-SIRPα antibodies and antigen-binding fragments thereof (e.g., humanized antibodies) comprising variants of the immunoglobulin chains set forth herein, which variants exhibit one or more of the following properties: EC 50 Binds to human SIRPαV1 protein having the sequence of SEQ ID NO:34 with <1 nM; EC2 at least 100-fold higher than SIRPαV1(P74A) having the sequence of SEQ ID NO:62 50and optionally also exhibiting at least 100-fold higher EC 50 (In each case, a decreased EC 50 is the EC for human SIRPαV1 protein having the sequence of SEQ ID NO:34 50 and in each instance preferably as measured by cellular ELISA (CELISA) as described hereinafter); <10 nM, preferably <5 nM, more preferably <1.5 nM, even more preferably <1.0 nM, even more preferably <0.5 nM, and most preferably an EC 50 binds to cells expressing human SIRPαV1 protein; <10 nM, preferably <5 nM, more preferably <1.5 nM, even more preferably <1.0 nM, even more preferably <0.5 nM, and most preferably an EC 50 binds to cells expressing human SIRPαV2 protein; At an antibody concentration of 50 nM, preferably 67 nM, more preferably 100 nM, or an antibody EC 50 does not appreciably bind to SIRPβ1 protein at concentrations 10-fold greater, preferably 50-fold greater, more preferably 100-fold greater, and even more preferably 200-fold greater than <10.0 nM, more preferably <5.0 nM, even more preferably <2.5 nM, and most preferably about 1.0 nM or less 50 inhibits the binding of human SIRPα to CD47; and Exhibits a T20 "humanity" of at least 79, more preferably 85%.

[0155] In other embodiments, the invention provides antibodies that bind to human SIRPα (e.g., humanized antibodies) and have at least 90% sequence identity to SEQ ID NOs: 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 Domains and V L In other embodiments, the present invention provides antibodies or antigen-binding fragments thereof that bind to human SIRPα (e.g., humanized antibodies) and have a V domain that has at least 95% sequence identity to SEQ ID NOs: 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 Domains and V L In another embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα (e.g., a humanized antibody) and has a V domain that has at least 97% sequence identity to SEQ ID NOs: 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 Domains and V L In other embodiments, the present invention provides antibodies or antigen-binding fragments thereof that bind to human SIRPα (e.g., humanized antibodies) and have a V domain that has at least 98% sequence identity to SEQ ID NOs: 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 Domains and V LIn another embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to human SIRPα (e.g., a humanized antibody) and has a V domain that has at least 99% sequence identity to SEQ ID NOs: 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 Domains and V L Preferably, in each instance, sequence differences between the variant and SEQ ID NOs: 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 conservative substitutions, most preferably limited to substitutions within framework residues.

[0156] The following references relate to BLAST algorithms that are 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. in 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 Altschul, S. F. "Evaluating the statistical significance of multiple distinct local alignments," in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York. In the present application, percent identity comparisons are preferably performed using the BLAST algorithm, with algorithm parameters selected to maximize matches between each sequence over the entire length of each reference sequence (e.g., expectation threshold: 10; word size: 6; maximum matches within query: 0; BLOSUM 62 matrix; gap cost: extension 11, extension 1; conditional compositional score matrix adjustment).

[0157] "Conservatively modified variants" or "conservative substitutions" refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), whereby such substitutions can often be made without altering the biological activity of the protein. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally 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 unlikely to destroy biological activity. Exemplary conservative substitutions are shown in Table 1 below. [Table 1]

[0158] Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. As used herein, "function-conservative variant" refers to an antibody or fragment in which one or more amino acid residues have been altered without altering desired properties, such as antigen affinity and / or specificity. Such variants include, but are not limited to, replacing an amino acid with an amino acid having similar properties, such as the conservative amino acid substitutions in Table 1. Also provided are V variants of the anti-SIRPα antibodies of the invention with up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, preferably conservative substitutions. L and isolated polypeptides comprising the V domain (e.g., SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100, 8, 20, 22, 24, 26, 28, and 32) of the anti-SIRPα antibodies of the present invention. H and isolated polypeptides comprising the domains (e.g., SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 7, 10, 12, 14, 16, 18, and 30).

[0159] The present invention further includes polynucleotides encoding any of the polypeptides or immunoglobulin chains of the anti-SIRPα antibodies and antigen-binding fragments thereof of the present invention. For example, the present invention encompasses polynucleotides that encode the amino acids set forth in any one of SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32.

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

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

[0162] binding affinity By way of example and not limitation, the antibodies and antigen-binding fragments disclosed herein exhibit a 10×10 affinity to human SIRPα as measured by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or Biolayer Interferometry (OCTET)). -9 K below M D In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind bivalently at a value of about 5-10 x 10 as measured by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET). -9 K of M D The affinity can be expressed as K D =K off / k on (k off is the dissociation rate constant, K on is the binding rate constant, K D The affinity is calculated as (r / c = K(nr)), where r = moles of bound ligand / moles of receptor at equilibrium; c = concentration of free ligand at equilibrium; K = equilibrium binding constant; n = number of ligand binding sites per receptor molecule. Graph analysis involves plotting r / c on the Y-axis against r on the X-axis, thereby creating a Scatchard plot. Antibody affinity measurement 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] humanity For purposes of this document, "humanity" is measured using the T20 score analyzer to quantify the humanness of the variable regions 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 protein sequence of the input VH, VK, or VL variable region is first assigned Kabat numbering, and the CDR residues are identified. The full-length sequence or the framework-only sequence (with the CDR residues removed) is compared with each sequence in each antibody database using the blastp protein-protein BLAST algorithm. The sequence identity between each pairwise comparison is isolated, and after each sequence in the database is analyzed, the sequence is classified from high to low based on its sequence identity to the input sequence. The percent identity of the top 20 matching sequences is averaged to obtain the T20 score.

[0165] For each chain type (VH, VK, VL) and sequence length (full-length or framework only) in the "All Human Database," each antibody sequence was scored in each database using the T20 score analyzer. 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 percent identity of sequences 2–21 was averaged). The T20 scores for each group were categorized from high to low. The score decline was roughly linear for most of the sequences, but the T20 scores for approximately the bottom 15% of antibodies began to decline sharply. Therefore, the bottom 15% of sequences were removed, and the remaining sequences formed the T20 Cutoff Human Databases (where the T20 score cutoff indicates the minimum T20 score for sequences in the new database).

[0166] As used herein, a "human" antibody is one that has a T20 humanity score of at least 79%, more preferably at least 85%.

[0167] Ability of anti-hSIRPα antibodies to block binding to CD47 In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof 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] Methods for Producing Antibodies and Antigen-Binding Fragments Thereof Thus, the present invention encompasses methods for producing an anti-SIRPα antibody or antigen-binding fragment thereof of the present invention, comprising culturing a hybridoma cell 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 culture medium).

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

[0170] There are several methods of making recombinant antibodies known in the art. One example of a method for the recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567.

[0171] Transformation can be by any known method for introducing polynucleotide into host cell.Methods for introducing heterologous polynucleotide into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposome, biolistic injection of DNA into nucleus and direct microinjection.In addition, nucleic acid molecules can be introduced into mammalian cells by viral vector.Methods for transforming cells are well known in the art.See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

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

[0173] Anti-SIRPα antibodies can also be synthesized by any of the methods set forth in US Pat. No. 6,331,415.

[0174] Eukaryotic and prokaryotic host cells, including mammalian cells, for expression of the antibodies or fragments or immunoglobulin chains disclosed herein are well known in the art and include the many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, 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, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia cochlamy, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercoum, Pichia pieri, Pichia stipitis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus Yeast and filamentous fungal cells, including Rhus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucnowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa, Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucnowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa.When a recombinant expression vector encoding a heavy chain or antigen-binding portion or fragment thereof, and / or a light chain or antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow for expression of the antibody or fragment or chain in the host cell or secretion into the medium in which the host cell is grown.

[0175] Antibodies, antigen-binding fragments thereof, and immunoglobulin chains can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibodies, antigen-binding fragments thereof, and immunoglobulin chains of the present invention (or other portions thereof) from production cell lines 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 certain conditions. The GS system is 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 a glutamine synthetase gene and is grown in the absence of glutamine in the culture medium, while the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene that complements the absence of the gene in the host cell.

[0176] The present invention encompasses methods for purifying an anti-SIRPα antibody or antigen-binding fragment thereof of the present invention, comprising introducing a sample containing the antibody or fragment into a purification medium (e.g., a cation exchange medium, an anion exchange medium, a hydrophobic exchange medium, or an affinity purification medium (e.g., Protein-A, Protein-G, Protein-A / G, Protein-L)) and either recovering the purified antibody or fragment from the flow-through fraction of the sample that does not bind to the medium, or discarding the flow-through fraction and eluting the bound antibody or fragment from the medium and recovering the eluate. 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 performed following recombinant expression of the antibody or fragment in host cells, e.g., the host cells are first lysed, and optionally the lysate is purified from insoluble material prior to purification in the medium.

[0177] Generally, glycoproteins produced in a particular cell line or transgenic animal have a glycosylation pattern characteristic of the glycoprotein produced in that cell line or transgenic animal. Therefore, the specific glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are encompassed by the present invention, regardless of the glycosylation pattern the antibody may have. Similarly, in particular embodiments, antibodies with a glycosylation pattern containing only nonfucosylated N-glycans may be advantageous, as these antibodies have been shown to typically exhibit stronger efficacy than fucosylated counterparts both in vitro and in vivo (see, e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These antibodies with nonfucosylated 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 that have binding specificity for SIRPα and another antigen, 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. Bispecific or bifunctional antibodies are artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321; Kostelny, et al., (1992) J Immunol. 148:1547- 1553. Additionally, bispecific antibodies may be formed as "diabodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52). These include "Duobodies," which are bispecific antibodies with 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 the anti-SIRPα antibodies disclosed herein. Such antibody fragments include F(ab)2 fragments that can be generated by enzymatic digestion of IgG, for example, with pepsin. Fab fragments can also be generated by reduction of F(ab)2 with, for example, dithiothreitol or mercaptoethylamine.

[0180] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. In some embodiments, the different constant domains are humanized V sequences derived from the CDRs provided herein. L and V H The antibody may be attached to a region. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., 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 comprises a heavy chain constant region, e.g., a human constant region, such as a γ1, γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a human light chain constant region, such as a lambda or kappa human light chain region or a variant thereof. 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 with a Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38: 1-8, 2001).

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

[0183] Antibody manipulation Also included are embodiments in which the anti-SIRPα antibodies and antigen-binding fragments thereof are engineered antibodies containing modifications to the framework region within the variable domain of the antibody, e.g., to improve the properties of the antibody or fragment. 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 present in the immune repertoire of the species to which the antibody is to be used, e.g., human residues in the case of a human therapeutic. Such antibodies or fragments are referred to as "humanized" antibodies or fragments. In some instances, it is desirable to increase the affinity or alter the specificity of an engineered (e.g., humanized) antibody. One approach is to mutate one or more framework residues to the corresponding germline sequence. More specifically, antibodies or fragments that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the framework sequence of the antibody or fragment to the germline sequence from which the antibody or fragment was derived. Another approach is to restore the original parental (e.g., rodent) residue at one or more positions in an engineered (e.g., humanized) antibody, e.g., to restore binding affinity that may be lost in the process of exchanging framework residues (see, e.g., U.S. Patent Nos. 5,693,762, 5,585,089, and 5,530,101).

[0184] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof are engineered (e.g., humanized) to include modifications within the framework and / or CDRs that improve properties. Such engineered changes can be based on molecular modeling. Molecular models for the variable regions of the parent (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. Traditional 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 coworkers carefully examined the conformations of loops in antibody crystal structures and proposed hypervariable loops. 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 "CDRs" and "hypervariable loops." In a later study (Raghunathan et al., (2012) J. Mol Recog. 25, 3, 103-113), multiple antibody-antigen crystal complexes were analyzed and it was observed that the antigen-binding region within an antibody does not necessarily strictly follow the "CDR" residues or "hypervariable" loops. A molecular model of the variable region of a non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In fact, potential antigen-binding regions based on the model differ from traditional "CDR" or "hypervariable" loops. Commercial scientific software such as Discovery Studio (BIOVIA, Dassault Systems) can be used for molecular modeling. A human framework can be selected based on the best match with the non-human sequence in both the framework and CDR. For FR4 (framework 4) in VH, the VJ region of the human germline sequence is compared with the corresponding non-human region. For FR4 (framework 4) in VL, 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 CDRs are grafted onto the selected human framework. In some cases, certain residues within the VL-VH interface can be retained as in the non-human (parent) sequence. Molecular models can also be used to identify residues that can potentially alter the CDR conformation and thereby bind to the antigen. In some cases, these residues are retained as in the non-human (parent) sequence.Molecular modeling can also be used to identify solvent-exposed amino acids that may lead to unwanted effects such as glycosylation, deamidation, and oxidation. Developability filters can be introduced early in the design phase to eliminate / minimize these potential issues.

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

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

[0187] For example, asparagine (Asn) residues may be converted to Gln or Ala, particularly within CDRs, to reduce the possibility of isoaspartate formation in any Asn-Gly sequence. Similar problems may also occur in Asp-Gly sequences. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation may weaken or completely prevent antibody binding to target antigens. 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 acid adjacent to the asparagine (Asn) or glutamine (Gln) residue to reduce the possibility of deamidation, which occurs 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 also contribute to molecular heterogeneity in the final antibody preparation, any methionine residues (typically solvent-exposed Met) in the CDRs may be converted to Lys, Leu, Ala, or Phe, or other amino acids (ibid.). In addition, to prevent or minimize potential cleavage of the Asn-Pro peptide bond, it may be desirable to modify any Asn-Pro combinations found in the CDRs to Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are then screened to ensure that the substitutions do not reduce the affinity or specificity of the antibody for SIRPα or other desired biological activity to an unacceptable level. [Table 2]

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

[0189] Antibody engineering of the Fc region The antibodies (e.g., humanized antibodies) and antigen-binding fragments thereof disclosed herein can also be engineered to contain modifications within the Fc region, typically to alter 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 cellular cytotoxicity). Additionally, the antibodies and antigen-binding fragments thereof disclosed herein can be chemically modified (e.g., capable of attaching one or more chemical moieties to the antibody) or modified to alter its glycosylation, again to alter one or more properties of the antibody or fragment. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0190] The antibodies and antigen-binding fragments thereof disclosed herein also include antibodies and fragments with modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent No. 5,624,821; WO 2003 / 086310; WO 2005 / 120571; WO 2006 / 0057702. Such modifications can be used to enhance or suppress various immune system responses, with potential beneficial effects in diagnosis and therapy. Modifications to the Fc region include amino acid alterations (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc regions. Fc modifications can also alter the half-life of therapeutic antibodies, allowing for less frequent administration and thereby improved convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731, p. 734-35.

[0191] In one embodiment, the antibody or antigen-binding fragment of the invention is an IgG4 isotype antibody or fragment comprising a serine to proline mutation at the position corresponding to position 228 in the hinge region of the heavy chain constant region (S228P; EU index; SEQ ID NO:66). This mutation has been reported to eliminate heterogeneity of inter-heavy chain disulfide bridges in 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 in the hinge region. This approach is described in further detail in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease 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 decrease the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface 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, as described in U.S. Patent No. 6,277,375, one or more of the following mutations can be introduced: T252L, T254S, T256F. Alternatively, as described in U.S. Patent Nos. 5,869,046 and 6,121,022, to increase biological half-life, the antibody can be modified within the CH1 or CL region to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the IgG Fc region.

[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 modify 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 can be replaced with a different amino acid residue so that the antibody has modified affinity for the effector ligand and retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 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 can be replaced with another amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551.

[0197] In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication WO 94 / 29351.

[0198] The proteins of the present invention, which are preferably antibodies, most preferably IgG antibodies or fragments thereof, may have altered (e.g., compared to unmodified antibodies) FcγR binding properties (e.g., binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (k off and k on ), binding affinity and / or avidity), certain modifications may be more or less desirable. D ) is k off / k on is defined as K a K D It is known in the art that .times. ...

[0199] The affinity and binding properties of an Fc region for a ligand may be measured by a variety of in vitro assays (biochemical or immunologically based assays) known in the art for measuring Fc-FcγR interactions, i.e., specific binding of an Fc region to an FcγR, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE®, Octet®, or KinExa® analysis), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being tested and / or may utilize a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels.

[0200] In specific embodiments, proteins of the invention bind to one or more human FcγR selected from the group consisting of FcγRI, FcγRIIB, FcγRIIC, FcγRIIIA-F158, and FcγRIIIA-V158 with an affinity that is at least 10-fold, preferably at least 30-fold, and more preferably at least 100-fold less than a comparable protein 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.

[0201] In various embodiments, the proteins of the present invention comprise an immunoglobulin Fc region comprising 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 certain preferred embodiments, the proteins comprise two immunoglobulin Fc regions, each comprising an immunoglobulin C2 region, an immunoglobulin C3 region, and an immunoglobulin hinge region, with the hinge region of one of the immunoglobulin Fc regions being joined to the hinge region of the other immunoglobulin Fc region to form a dimeric Fc structure. Most preferably, such proteins are human or humanized IgG proteins.

[0202] In a specific embodiment, the protein of the invention comprises a mutant IgG4 Fc region, preferably an IgG comprising two mutant IgG4 Fc regions forming a dimeric Fc structure. By way of example, the mutant IgG4 Fc region may comprise one of the mutations or a combination of mutations listed in Table 3. The numbering system for constant regions referred to in this table is that of the EU index set forth in 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. For those entries containing more than one mutation combination, each mutation in the combination is separated by a " / ". [Table 3]

[0203] In a specific embodiment, the protein of the invention comprises a mutant IgG1 Fc region, preferably an IgG comprising two mutant IgG1 Fc regions to form a dimeric Fc structure. By way of example, the mutant IgG1 Fc region may comprise one of the mutations listed in Table 4. The numbering system for constant regions referred to in this table is that of the EU index set forth in 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] TIFF0007745527000005.tif230159TIFF0007745527000006.tif62160

[0204] In certain embodiments, the mutant IgG1 Fc region may comprise one of the mutation combinations listed in Table 5. The numbering system for constant regions referred to in this table is that of the EU index set forth in 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 more than one mutation combinations, each mutation in the combination is separated by a " / " and deletions are indicated by a "△". [Table 5] TIFF0007745527000008.tif25160

[0205] In a specific embodiment, 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 forming a dimeric Fc structure. The mutant IgG2 Fc region may comprise one of the mutations or a combination of mutations listed in Table 6. The numbering system for constant regions referred to in this table is that of the EU index set forth in 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. For those entries containing more than one mutation combination, each mutation in the combination is separated by a " / ". [Table 6]

[0206] Generation of antibodies with modified glycosylation In yet another embodiment, the antibody or antigen-binding fragment of the present invention comprises a particular glycosylation pattern. For example, an afucosylated or aglycosylated antibody or fragment can be generated (i.e., the antibody lacks fucose or glycosylation, respectively). The glycosylation pattern of the antibody or fragment can be altered to, for example, enhance the affinity or avidity of the antibody or fragment for a SIRPα antigen. Such modifications can be accomplished, for example, by altering 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 that site. Such deglycosylation can enhance the affinity or avidity of the antibody or fragment for an antigen. See, e.g., U.S. Patent Nos. 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 with mammalian- or human-like glycosylation patterns (see, e.g., 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)). A particular advantage of these genetically modified host cells over currently used mammalian cell lines is their ability to control the glycosylation profile of glycoproteins produced in the cells, thereby generating glycoprotein compositions in which specific N-glycan structures predominate (see, e.g., U.S. Patent Nos. 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies that predominantly have specific N-glycan structures (see, e.g., Li et al., (2006) Nat. Biotechnol. 24:210-215).

[0208] In certain embodiments, the antibodies and antigen-binding fragments thereof disclosed herein further include those produced in lower eukaryotic host cells, including, but not limited to, GlcNAc (1-4) Man3GlcNAc2;Gal (1-4) GlcNAc (1-4) Man3GlcNAc2;NANA (1-4) Gal (1-4) GlcNAc (1-4) These include fucosylated and nonfucosylated hybrid and complex N-glycans, including biantennary and multiantennary species, including N-glycans such as Man3GlcNAc2.

[0209] In particular embodiments, the antibodies and antigen-binding fragments thereof provided herein may include antibodies or fragments having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In particular embodiments, the hybrid N-glycan is the predominant N-glycan species in the composition.

[0210] In particular embodiments, the antibodies and antigen-binding fragments thereof provided herein include antibodies and fragments having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In particular embodiments, the complex N-glycan is the predominant N-glycan species in the composition. In further embodiments, the complex N-glycan is a particular N-glycan species that constitutes about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans in the composition. In one embodiment, the antibodies and antigen-binding fragments thereof provided herein comprise complex N-glycans, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of which comprise the structure NANA2Gal2GlcNAc2Man3GlcNAc2, and which are afucosylated. Such structures can be produced, for example, in engineered Pichia pastoris host cells.

[0211] In particular embodiments, the N-glycan is fucosylated. Typically, the fucose is in an α1,3-linkage with GlcNAc at the reducing end of the N-glycan, an α1,6-linkage with GlcNAc at the reducing end of the N-glycan, an α1,2-linkage with Gal at the non-reducing end of the N-glycan, an α1,3-linkage with GlcNAc at the non-reducing end of the N-glycan, or an α1,4-linkage with GlcNAc at the non-reducing end of the N-glycan.

[0212] Thus, in particular embodiments of the above glycoprotein compositions, the glycoforms are in α1,3- or α1,6-linkages generating glycoforms 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); 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 )Man3GlcNAc2 in α1,3- or α1,4-linkages to produce glycoforms selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2, NANAGal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc 1-2)GlcNAc2Man3GlcNAc2.

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

[0214] In a further embodiment of the above, the complex N-glycans further comprise fucosylated and non-fucosylated biantennary and multiantennary species.

[0215] As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to N-linked oligosaccharides, e.g., N-linked oligosaccharides attached to asparagine residues of polypeptides via asparagine-N-acetylglucosamine linkages. N-linked glycoproteins contain N-acetylglucosamine residues attached to the amide nitrogen of asparagine residues in the protein. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). Processing of sugar groups occurs cotranslationally in the lumen of the ER and subsequently posttranslationally in the Golgi apparatus for N-linked glycoproteins.

[0216] N-glycans share a common pentasaccharide core of Man3GlcNAc2 ("Man" refers to mannose, "Glc" refers to glucose, "NAc" refers to N-acetyl, and GlcNAc refers to N-acetylglucosamine). N-glycan structures are typically 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 that contains the glycosylation site on the protein. N-glycans differ in the number of branches (antennae) containing peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) attached to the Man3GlcNAc2 ("Man3") core structure, also referred to as the "trimannose core," "pentasaccharide core," or "paucimannose 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 attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a "trimannose" core. Complex N-glycans may also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues optionally modified with sialic acid or derivatives (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 "biantennary" GlcNAc and core fucose ("Fuc"). Complex N-glycans may also have multiple antennae on the "trimannose core," and are often referred to as "multiantennary glycans." "Hybrid" N-glycans have at least one GlcNAc at the terminal 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. The 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" refer to the following: The term "G-2" refers to an N-glycan structure that can be characterized as Man3GlcNAc2; the term "G-1" refers to an N-glycan structure that can be characterized as GlcNAcMan3GlcNAc2; the term "G0" refers to an N-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2; the term "G1" refers to an N-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2; the term "G2" refers to an N-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2; the term "A1" refers to an N-glycan structure that can be characterized as NANAGal2GlcNAc2Man3GlcNAc2; and the term "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 that lack fucose attached to the GlcNAc residue at the reducing end of the N-glycan. When 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 attached to the GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes, such as yeast and filamentous fungi, do not usually produce N-glycans that produce fucose.

[0218] With respect to multiantennary N-glycans, the term "multiantennary N-glycan" refers to an N-glycan that further comprises a GlcNAc residue on the mannose residue comprising the non-reducing end of the 1,6 arm or the 1,3 arm of the N-glycan, or a GlcNAc residue on each of the mannose residues comprising the non-reducing end of the 1,6 arm and the 1,3 arm of the N-glycan. Thus, a multiantennary N-glycan can be of the formula GlcNAc (2-4) Man3GlcNAc2, Gal (1-4) GlcNAc (2-4)Man3GlcNAc2, or NANA (1-4) Gal (1-4) GlcNAc (2-4) The term "1-4" refers to 1, 2, 3, or 4 residues.

[0219] With respect to biantennary N-glycans, the term "biantennary N-glycan" refers to an N-glycan in which a GlcNAc residue is linked to a mannose residue at the reducing end of the N-glycan. A biantennary N-glycan can be characterized by the formula GlcNAc3Man3GlcNAc2, in which each mannose residue is linked to the non-reducing end of a GlcNAc residue. In contrast, when a multiantennary N-glycan is characterized by GlcNAc3Man3GlcNAc2, the formula indicates that two GlcNAc residues are linked to the mannose residue at the non-reducing end of one of the two arms of the N-glycan, and one GlcNAc residue is linked to the mannose residue at the non-reducing end of the other arm of the N-glycan.

[0220] In certain embodiments, the proteins of the invention comprise an aglycosylated Fc region. By way of example, an IgG1 Fc region may be aglycosylated by deleting or substituting residue N297.

[0221] Antibody physical properties The antibodies and antigen-binding fragments thereof disclosed herein may further comprise one or more glycosylation sites within the immunoglobulin variable region of either the light or heavy chain. Such glycosylation sites may enhance the immunogenicity of the antibody or fragment or alter the pK of the antibody by altering 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 at motifs containing NXS / T sequences.

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

[0223] Each antibody or antigen-binding fragment has a characteristic melting temperature, with a higher melting temperature indicating greater overall in vivo stability (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). M1 (the temperature of initial unfolding) can be greater than 60° C., greater than 65° C., or greater 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 antigen-binding fragments thereof are selected that do not rapidly degrade. 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 antigen-binding fragments thereof are selected that exhibit minimal aggregation, which may lead to the elicitation of unwanted immune responses and / or altered or unfavorable pharmacokinetic properties. Generally, antibodies and fragments that exhibit less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% aggregation are acceptable. Aggregation can be measured by several techniques, including size exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0226] antibody conjugates The anti-SIRPα antibodies and antigen-binding fragments thereof disclosed herein may be conjugated to a chemical moiety. The chemical moiety may be, among others, a polymer, a radionuclide, or a cytotoxic factor. In a specific embodiment, the chemical moiety is a polymer that increases the half-life of the antibody or fragment in the subject's body. Suitable polymers include, but are not limited to, hydrophilic polymers, including polyethylene glycol (PEG) (e.g., PEG with a molecular weight 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) discloses PEG-conjugated single-chain antibodies. Wen et al (2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies to PEG linked to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA).

[0227] The antibodies and antigen-binding fragments thereof disclosed herein can also be used in, 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 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th and 40 K. 157 Gd, 55 Mn, 52 Tr and 56 It may be conjugated to a label 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 result in one or more PEG groups being attached to the antibody or antibody fragment. In particular embodiments, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the PEGylated antibody or fragment is an aglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. See, e.g., EP 0154316 and EP 0401384.

[0229] The antibodies and antigen-binding fragments disclosed herein also include compounds that bind to rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 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 and antigen-binding fragments thereof of the present invention may also be conjugated to cytotoxic agents such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordi proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogenin, restrictocin, phenomycin, and enomycin.

[0231] Any method known in the art for conjugating the antibodies and antigen-binding fragments of the present invention to various moieties may be utilized, including those 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. Methods for conjugating antibodies and fragments are conventional and very well known in the art.

[0232] Therapeutic Uses of Anti-SIRPα Antibodies Further provided is a method for treating a subject, such as a human subject, in need of treatment with the isolated antibody or antigen-binding fragment thereof disclosed herein. In one embodiment of the present invention, such a subject is suffering from an infection or infectious disease.

[0233] In another embodiment of the present invention, such a subject is suffering from cancer. In one embodiment, the cancer is, for example, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilms' carcinoma, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, colon cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, renal cell carcinoma, nephr ... The cancer is selected from the group consisting of rhabdoid tumor of the liver, Ewing's sarcoma, chondrosarcoma, brain tumor, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, 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, and gastric cancer. In one embodiment of the present invention, the cancer is metastatic cancer, such as those described above.

[0234] Cancers that may be treated with the antibodies or antigen-binding fragments, compositions, and methods of the invention include: cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyoma), pancreas (tubular adenocarcinoma, insulinoma, glucagonoma, gastrinocarcinoma, esophageal sarcoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colorectum; genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, malignant teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoroid tumor, lipoma); liver: hepatoma (hepatic cell carcinoma), intrahepatic cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondroma), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumor; nervous system: skull (osteoma, hemangioma, granuloma, xanthomatosis, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], pleomorph Glioblastoma, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibroma, meningioma, glioma, sarcoma; Gynecology: uterus (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovary (ovarian carcinoma [serous adenocarcinoma, mucinous adenocarcinoma, unclassified carcinoma], granulosa-meningioma, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyoma), fallopian tube (carcinoma), breast;Hematology: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma. Thus, the term "cancer cell" as used herein encompasses cells afflicted by any one of the above-identified conditions.

[0235] In one embodiment, cancers that may be treated by the antibodies or antigen-binding fragments thereof disclosed herein, 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 myelocytic leukemia, non-Hodgkin's lymphoma, B-cell non-Hodgkin's 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, multiple myeloma.

[0236] In one embodiment, the antibodies or antigen-binding fragments thereof disclosed herein may be used to treat infections and infectious diseases. As used herein, the term "infection" refers to any condition in which at least one cell of an organism (e.g., a subject) is infected with an infectious agent (e.g., the subject has an intracellular pathogenic infection, e.g., a chronic intracellular pathogenic infection). As used herein, the term "infectious agent" refers to a foreign organism (i.e., a pathogen) that induces CD47 expression (e.g., increased CD47 expression) in at least one cell of the infected organism. For example, 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 induce unrecognized syndromes or diseases under certain conditions, and have the potential to induce syndromes or diseases under changing conditions. The methods of the present invention can be used to treat chronic pathogenic infections, including, but not limited to, viral infections, such as retroviruses, lentiviruses, hepadnaviruses, herpesviruses, poxviruses, and human papillomaviruses; intracellular bacterial infections, such as Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp., and Hercobacter pylori; and intracellular protozoan pathogens, such as Plasmodium sp., Trypanosoma sp., Giardia sp., Toxoplasma sp., and Leishmania sp.

[0238] In one embodiment, the present invention provides a method for treating a subject with an anti-SIRPα antibody or antigen-binding fragment thereof of the present invention, wherein the subject is suffering from a viral infection. In one embodiment, the viral infection is an infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, 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 invention provides a method for treating a subject with an anti-SIRPα antibody or antigen-binding fragment thereof of the invention, wherein the subject is suffering from a bacterial infection. In one embodiment, the bacterial infection is an infection caused by a bacterium selected from the group consisting of chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, corynebacterium diphtheriae, salmonella, bacillus, vibrio cholerae, clostridium tetanus, clostridium botulinum, bacillus anthracis, yersinia pestis, mycobacterium leprae, mycobacterium lepromatosis, and borrela.

[0240] In one embodiment, the invention provides a method for treating a subject with an anti-SIRPα antibody or antigen-binding fragment thereof of the invention, wherein the subject is suffering from a fungal infection. In one embodiment, the fungal infection is an infection caused by a fungus selected from the group consisting of Candida (e.g., Candida albicans, Krusei, Glabrata, Tropicalis), Cryptococcus neoformans, Aspergillus (e.g., Fumigatus, Niger), Mucorales (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0241] In one embodiment, the invention provides a method for treating a subject with an anti-SIRPα antibody or antigen-binding fragment thereof of the invention, wherein the subject is suffering from a parasitic infection. In one embodiment, the parasitic infection is an infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microtti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongilus brasiliensis.

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

[0243] The term "in conjunction with" indicates that the components administered in the methods of the present invention (e.g., an anti-SIRPα antibody (e.g., a humanized antibody) or antigen-binding fragment thereof together with an anti-cancer agent) can be formulated into a single composition for simultaneous delivery, or can be formulated separately into two or more compositions (e.g., a kit). Each component can be administered to a subject at a time different from the time at which the other components are administered, for example, each administration can be non-simultaneous (e.g., separate or sequential) at multiple intervals over a given period of time. Furthermore, the other components can be administered to a subject by the same route or by different routes.

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

[0245] Thus, the present invention provides a method for treating cancer in a human subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment disclosed herein, optionally in conjunction with an additional therapeutic agent or procedure. The present invention also provides a method for treating infection or infectious disease in a human subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment disclosed herein, optionally in conjunction with an additional therapeutic agent or procedure. The present invention also provides a method for increasing immune cell activity, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment disclosed herein. In one embodiment, the method is used in the treatment of cancer, the treatment of an infection or infectious disease, or as a vaccine adjuvant.

[0246] In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used alone or in conjunction with tumor vaccines. Examples of tumor vaccines include vaccines for human papillomavirus (HPV) infection, such as Gardasil®, Gardisil9®, and Cervarix®; vaccines for preventing hepatitis B virus-induced liver cancer, such as Engerix-B® and Recombivax HB®; oncolytic virotherapy that elicits an immune response, such as Imlygic®; DNA vaccines, such as Synchotrope MA2M plasmid DNA vaccine and ZYC101; mammaglobin-a DNA vaccines (see Clinical Cancer Res. 2014 20(23):5964-75); vector-based vaccines, such as PSA-TRICOM (prostvac) and PANVAC-VF; and Listeria monocytogenes-based vaccines (see, e.g., Therapeutic Advances in Vaccines, 2014, 2(5)137-148), Listeria-based vaccines (Listeria expressing one or more cancer vaccines such as Listeria mesothelin (e.g., CRS-207), ADXS-HPV, Axalimogene phyllosvac, Listeria-HER2 / Neu, Listeria-EGFRvIII); adeno-CEA; GVAX, BLP-25 (anti-ancaramucin 1), Veragenpmatucel-L, TG4010, CIMAvax epidermal growth factor vaccine, NY-E These vaccines include, but are not limited to, allogeneic vaccines such as SO and GM.CD40L-CCL21; autologous vaccines such as Adeno-CD40L, BCG, and INGN-225; dendritic cell vaccines such as Provenge® (sipuleucel-T) and rF-CEA-MUC1-TRICOM (Panvac-DC); and antigen vaccines such as MUC-1 (Stimuvax), NY-ESO-1, GP-100, MAGE-A3 (melanoma antigen-encoding gene A3), and INGN-225 (see Pharmacology & Therapeutics 153 (2015) 1-9).

[0247] The eat-me signal can be increased by cytotoxic therapy such as radiation therapy and chemotherapeutic agents, including, but not limited to, 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, satidomide, sorafenib. Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used in conjunction with chemotherapeutic agents, in conjunction with radiation therapy, etc. In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used alone or in conjunction with targeted therapies.Examples of targeted therapies include hormone therapy, signal transduction inhibitors (e.g., EGFR inhibitors such as cetuximab (Erbitux) and erlotinib (Tarceva)); CD20 inhibitors (e.g., rituximab (Rituxan) and afatumumab (Arzerra)); CD38 inhibitors (e.g., daratumumab (DARZALEX)); CD52 inhibitors (e.g., alemtuzumab (Campath)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (such as imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators (e.g., decitabine (Dacogen)) and vorinostat (Zolinza)), apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)), immunomodulatory agents (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast), and monoclonal antibodies conjugated to toxins (e.g., brentuximab vedotin (Adcetris) and adotrastuzumab emtansine (Kadcyla)).

[0248] The antibodies or antigen-binding fragments thereof disclosed herein may find use in connection with targeted therapies, preferably using antibodies to mediate ADCC / ADCP. 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. By way of example, antibody-dependent cell-mediated cytotoxicity (ADCC) assays typically utilize normal human peripheral blood mononuclear cells (PBMCs) or effector cells isolated therefrom. Assay variability can be reduced by utilizing selected donor pools containing defined Fcγ receptor IIa (FcγRIIa / CD32a), IIIa (FcγRIIIa / CD16a), or IIIb (FcγRIIIb / CD16b) gene copy number variants (CNVs), 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). Target cell killing can be achieved by: 51 Chromium (Cr 51 ) or fluorescent dyes, 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), by measuring the release of specific probes from pre-labeled target cells, or by measuring the release of cytosolic enzymes such as lactate dehydrogenase (LDH) or nucleoside triphosphate (ATP).

[0249] In contrast, antibody-dependent cellular 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 bone marrow cell lines, such as HL-60, THP-1, and U937 cells, which are differentiated into macrophages or granulocytes. Commonly used stimuli to induce macrophage differentiation in monocytic cell lines include phorbol-12-myristate-13-acetate (PMA), 1,25-dihydroxyvitamin D3 (VD3), and retinoic acid (RA). RA is also known to induce terminal granulocytic differentiation of, for example, HL-60 cells. Target cell phagocytosis can be assessed by monitoring effector cells for internalization of specific probes from target cells prelabeled with fluorescent dyes, such as the cell proliferation dye eFluor 450, CFSE, and pH-sensitive dyes, including pHrodo and CypHer5E. Phagocytosis is measured by the expansion of fluorescently labeled effector cells using flow cytometry or fluorescence microscopy. A "reporter gene" assay can also be used to assess ADCP. To measure ADCP function in a reporter gene assay, target cells are first incubated with a fixed titer of the appropriate antibody. Once the antibody binds to its cognate target on the target cell surface, engineered Jurkat effector cells are added. Activation of the ADCP pathway results in Jurkat cells producing luciferase product through expression of the reporter gene NFAT-RE-luc2. Luciferase activity is then measured after the addition of luciferase assay reagents following a 4- to 24-hour induction period. The dose-dependent response in the microtiter plate-based assay can be used to quantify the relative bioactivity of therapeutic antibodies by comparing them to a dose-response curve of an appropriate reference substance.

[0250] In particular embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof of the present invention may be used in combination with anti-cancer therapeutic agents or immunomodulatory agents such as immunomodulatory receptor inhibitors, e.g., antibodies or antigen-binding fragments thereof that specifically bind to a receptor.

[0251] In one embodiment of the invention, the anti-SIRPα antibody or antigen binding thereof of the invention is Agonists of TNF receptor proteins (e.g., agonistic antibodies or antigen-binding fragments thereof, or soluble fusions), immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating 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 a ligand that specifically binds to Ml, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), 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, LAIRl, IDO, TDO, CD160 and / or TGFR beta; It is linked to one or more of the following:

[0252] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof is combined with one or more cyclic dinucleotides or other STING pathway agonists. STING (also known as TMEM173, MITA, ERIS, and MPYS) is an ER-localized transmembrane protein that undergoes a conformational change in response to direct binding of cyclic dinucleotides (CDNs), resulting in downstream signaling cascades including TBK1 activation, IRF-3 phosphorylation, and the production of IFN-β and other cytokines. The STING pathway in tumor-resident host antigen-presenting cells is involved in the induction of natural CD8+ T cell responses against tumor-derived antigens. Activation of this pathway and subsequent production of IFN-β also reportedly contributes to the antitumor effects of radiation. STING agonists and uses thereof are described in, for example, US20060040887, US20080286296, US20120041057, US20140205653, WO2014179335, WO2014179760, US20150056224, WO2015185565, WO2016096174, WO2016145102, WO2017011444, WO2017027645, and US20180093964.

[0253] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of an anti-CD47 antibody, an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, anti-PDL1 antibody, anti-TIGIT antibody, anti-APRIL antibody, anti-CTLA4 antibody, 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-ILT4 antibody, an anti-ILT5 antibody, an anti-ILT6 antibody, an anti-ILT7 antibody, an anti-ILT8 antibody, an anti-ILT9 antibody, an anti-ILT10 antibody, an anti-ILT11 antibody, an anti-ILT22 antibody, an anti-ILT3 antibody, an anti-ILT4 antibody, an anti-ILT5 antibody, an anti-ILT6 antibody, an anti-ILT7 antibody, an anti-ILT8 antibody, an anti-ILT9 antibody, an anti-ILT12 antibody, an anti-ILT13 antibody, an anti-ILT14 antibody, an anti-ILT15 antibody, an anti-ILT16 antibody, an anti-ILT17 antibody, an anti-ILT18 antibody, an anti-ILT19 antibody, an anti-ILT21 antibody, an anti-ILT22 antibody, an anti-ILT23 antibody, an anti-ILT24 antibody, an anti-ILT25 antibody, an anti-ILT26 antibody, an anti- 4 antibody, anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody, 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 targets.

[0254] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is linked to an anti-CD20 antibody (e.g., rituximab, ofatumumab, ocrelizumab, obinutuzumab, ocaratuzumab, ublituximab, veltuzumab, ibritumomab tiuxetan, tositumomab, BVX-20, SCT-400, or PRO131921).

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

[0256] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is conjugated 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 antigen-binding fragment thereof of the present invention is combined with an anti-HER2 antibody (eg, trastuzumab, TrasGEX, pertuzumab, margetuximab, or ADCT-502).

[0258] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is combined with an anti-HER3 antibody (e.g., lumletuzumab, patritumab, or LJM716).

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

[0260] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is conjugated with an anti-CD52 antibody (eg, alemutuzumab).

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

[0262] In one embodiment of the present invention, an anti-SIRPα antibody or antigen-binding fragment thereof of the invention 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 antigen-binding fragment thereof of the present invention is linked to an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab).

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

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

[0266] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is conjugated with an anti-CD137 antibody (eg, urelumab).

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

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

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

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

[0271] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the invention is conjugated with an anti-CD137 (eg, urelumab) antibody.

[0272] In one embodiment of the present invention, an anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is conjugated with an anti-GITR (eg, TRX518) antibody.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0300] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is administered in combination with 13-cis-retinoic acid, 3-[5-(methylsulfonylpiperazinemethyl)-indolyl]-quinolone, 4-hydroxytamoxifen, 5-deoxyuridine, 5'-deoxy-5-fluorouridine, 5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654, abiraterone acetate, Abraxane, ABT-578, acolbifene, ADS-100380, ALT-110, or α-dihydroxybenzoate. Lutretamine, amifostine, aminoglutethimide, amrubicin, amsacrine, anagrelide, anastrozole, angiostatin, AP-23573, ARQ-197, arzoxifene, AS-252424, AS-605240, asparaginase, AT-9263, atrasentan, axitinib, AZD1152, bacillus Calmette-Guérin (BCG) vaccine, batabulin, 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, cediranib, CG-1521, CG-781, chlamydocin, chlorambucil, chlorotoxin, cilengitide, cimitidine, cisplatin, cladribine, clodronate, COL-3, CP-724714, cyclophosphamide, cyproterone, cyproterone acetate, cytarabine, cytosine alanine Binoside, dacarbazine, dacinostat, dactinomycin, dalotuzumab, danusertib, dasatanib, daunorubicin, decatanib, deguelin, denileukin, deoxycoformycin, depsipeptide, diallylpropionitrile, diethylstilbestrol, diftitox, docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin, yttrium-90 labeled edotreotide, edotreotide, EKB-569, EMD121974, endostatin, enzalutamide,Enzastaurin, epirubicin, epithilone B, ERA-923, Erbitux, erlotinib, estradiol, estramustine, etoposide, everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol, floxuridine, fludarabine, fludrocortisone, fluoxymesterone, flutamide, FOLFOX regimen, fulvestrant, galeterone, gefitinib, gemcitabine, gimatecan, 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, levamisole, liposomal Paclitaxel, lomustine, lonafarnib, lucanton, LY292223, LY292696, LY293646, LY293684, LY294002, LY317615, marimastat, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, mithramycin, mitomycin, mitotane, mitoxantrone, tozasertib, MLN8054, neovastatin, neratinib, neuradiab, nilotinib, nilotinib Nilutimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide, ofatumumab, oregovomab, 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, raltitrexed, razoxin, ridaforolimus, rituximab, romidepsin, RTA744, rubitecan, scriptaid, Sdx102, seliciclib, selumetinib, semaxanib, SF1126, sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668, streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib, synthetic estrogen, talampanel, talimogene laherparepvec, tamoxifen, temozolomide, temsirolimus, teniposide, tesmilifen, testosterone, tetrandrine, T GX-221, thalidomide, thioguanine, thiotepa, tremelimumab, tipifarnib, tivozanib, TKI-258, TLK286, topotecan, toremifene citrate, trabectedin, trastuzumab, tretinoin, trichostatin A, triciribine phosphate monohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproic acid, valproic acid In combination with one or more of the following: vicine, vandetanib, vatalanib, VEGF trap, vinblastine, vincristine, vindesine, vinorelbine, vitaxin, vitespan, vorinostat, VX-745, wortmannin, Xr311, zanolimumab, ZK186619, ZK-304709, ZM336372, ZSTK474.

[0301] Non-limiting examples of suitable anti-cancer agents that may be used in combination with the anti-SIRPα antibodies, or antigen-binding fragments thereof, of the present invention include cytostatic agents, immunomodulatory agents, cytotoxic agents, targeted therapeutic agents (small molecules, biologics, siRNAs and microRNAs) for cancer and neoplastic diseases; 1) Antimetabolites (methotrexate, 5-fluorouracil, gemcitabine, fludarabine, capecitabine, etc.), 2) alkylating agents, such as temozolomide and cyclophosphamide; 3) DNA interacting and DNA damaging agents, such as cisplatin, oxaliplatin, doxorubicin, 4) ionizing radiation, e.g., radiotherapy; 5) Topoisomerase II inhibitors, such as etoposide and doxorubicin; 6) Topoisomerase I inhibitors, such as irinotecan, topotecan, 7) Tubulin-interacting agents, such as paclitaxel, docetaxel, abraxane, epothilones, 8) Kinesin spindle protein inhibitors, 9) spindle checkpoint inhibitors, 10) Poly(ADP-ribose) polymerase (PARP) inhibitors, such as 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, such as bortezomib; 14) activators of mutant p53 that restore wild-type p53 activity; 15) Adenoviral-p53, 16) Bcl-2 inhibitors, such as ABT-263; 17) Heat shock protein (HSP) regulators, such as geldanamycin and 17-AAG; 18) Histone deacetylase (HDAC) inhibitors, such as vorinostat (SAHA); 19) Sex hormone regulators, a. Antiestrogens, e.g., tamoxifen, fulvestrant, b. Selective estrogen receptor modulators (SERMs), such as raloxifene; c. Antiandrogens, such as bicalutamide, flutamide, d. LHRH agonists, e.g., leuprolide; e. 5α-reductase inhibitors, such as finasteride, f. Cytochrome P450 C17 lyase (CYP450c17, also known as 17αC), g. Aromatase inhibitors, such as letrozole, anastrozole, exemestane, 20) EGFR kinase inhibitors, such as geftinib, erlotinib, and raptinib; 21) Dual erbB1 and erbB2 inhibitors, e.g., lapatinib; 22) Multi-targeted kinase (serine / threonine and / or tyrosine kinase) inhibitors, ABL kinase inhibitors, imatinib and nilotinib, dasatinib, b. VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK and ERK inhibitors, such as 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, such as GDC-0941, BEZ-235, BKM-120 and AZD-8055; i. Rapamycin and its analogs, such as temsirolimus, everolimus, and deforolimus; 23) and other anti-cancer drugs (also known as anti-tumor drugs), such as, but not limited to, ara-C, adriamycin, cytoxan, carboplatin, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-methyl-2-propanol, 2 ... Lucaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, vinblastine, vincristine, vindesine, vinorelbine, navelbine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, teniposide, cytarabine, pemetrexed, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide, ethinyl estradiol ol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, flutamide, medroxyprogesterone acetate, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, drolloxafine, hexamethylmelamine, Bexxar, Zevalin, Trisenox, Profimer, thiotepa, altretamine, Doxil, Ontak, Depocyt, Aranesp, Neupogen, Neulasta, Kepivance 24) Farnesyl protein transferase inhibitors, such as SARASAR™ (4-(2-(4-((11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoethyl]-piperidinecarboxamide), tipifarnib, 25) Interferons, such as 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, such as gemtuzumab ozogamicin; 31) Anti-VEGF antibodies, e.g., Avastin; 32) TRIAL ligands, such as 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, such as dalotuzumab (MK-0646) and lobatumumab (SCH717454); Examples include:

[0302] " Estrogen receptor modulator " refers to the compound that interacts with or inhibits the binding of estrogen to receptor, regardless of mechanism.Examples of estrogen receptor modulator include but are not limited to tamoxifen, raloxifene, idoxifene, 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" refers to compounds that interact with or inhibit androgen binding to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

[0304] "Retinoid receptor modulators" refer to compounds that interact with or inhibit retinoid binding to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators 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] "Cytotoxic / cytostatic agents" refers to compounds that induce cell death or inhibit cell proliferation primarily by directly interfering with cell functioning or by inhibiting or disrupting cell mitosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activated compounds, microtubule inhibitors / microtubule stabilizers, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signaling pathways, antimetabolites, biological response modifiers, hormone / antihormonal therapeutics, hematopoietic growth factors, monoclonal antibody targeted therapeutics, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and Aurora kinase inhibitors.

[0306] Examples of cytotoxic / cytostatic agents include platinum coordination compounds, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosylate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profilomycin, cisplatin, irofulven, dexfosfamide, cis-aminedichloro(2-methylpyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans,trans,trans)bis-mu-(hexane-4-yl)-4-methyl-2-propanol, tetrachloro(2-methyl-2-propanol), ... These include, but are not limited to, 1,6-diamine)-mu-[diamineplatinum(II)]bis[diamine(chloro)platinum(II)], diarylididinyl spermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulfonyl-daunorubicin (see WO 00 / 50032).

[0307] An example of a hypoxia activated 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'-norvincaleukoblastine, docetaxol (Taxotere®), rhizoxin, dolastatin, mibobulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, and 2,3,4 , 5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, TDX258, epothilones (see, e.g., U.S. Pat. Nos. 6,284,781 and 6,288,237), and BMS188797.

[0310] Some examples of topoisomerase inhibitors are topotecan, hicaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-O-exobenzylidene-caltroeucin, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-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]-indolizino[1,2b]quinoline-10, 13(9H,15H)dione, lurtotecan, 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, asulaculin, (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-hexohydrofuro(3',4':6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione, 5-(3-aminopropyl)benzo[g]isoquinoline-5,10-dione N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one, and dimesna.

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

[0312] Examples of " histone deacetylase inhibitors " include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. 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] "Inhibitors of kinases involved in mitotic progression" include, but are not limited to, inhibitors of Aurora kinases, inhibitors of Polo-like kinases (PLKs), particularly inhibitors of PLK-1, inhibitors of bub-1, and inhibitors of bub-R1. An example of an "Aurora kinase inhibitor" is VX-680.

[0314] "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, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fostearin sodium hydrate, and raltitrexate. Xed, Partitrexed, Emitefur, Tiazofurin, Decitabine, Nolatrexed, Pemetrexed, Nelzarabine, 2'-Deoxy-2'-methylidenecytidine, 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, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]-thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-fluorouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl Examples of suitable antimicrobial agents include 2'-cyano-2'-deoxy-N4-palmitoyl-1-BD-arabinofuranosylcytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 2'-aminopyridine-2-carboxy ...

[0315] Examples of monoclonal antibody-targeted therapeutic agents include those that have a cytotoxic agent or radioisotope attached to a cancer cell-specific or target cell-specific monoclonal antibody. An example is Bexxar.

[0316] "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. Patent Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510, 5,589,485, and 5,616,616. 02,098, European Patent Publication Nos. 0618221, 0675112, 0604181, 0696593, WO94 / 19357, WO95 / 08542, WO95 / 11917, WO95 / 12612, WO95 / 12572, WO95 / 10514, U.S. Patent Nos. 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, U.S. Patent No. 5,571,792, WO96 / 17861, WO96 / 33159, WO96 / 34850, WO96 / 34851, WO96 / 30017, WO96 / 30018, W Nos. WO96 / 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. Pat. 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" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2), inhibitors of epithelial-derived, fibroblast-derived, or platelet-derived growth factors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, interferon-α, interleukin-12, pentosan polysulfate, nonsteroidal anti-inflammatory drugs (NSAIDs) such as 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 agents (corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone, etc.), carboxamidotriazoles, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (Fernandez et al., J. Lab. Clin. Med.105: 141-145 (1985)), and antibodies against VEGF (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, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,

[0039] Antioxidants include, but are not limited to, methylparaben, 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-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

[0320] Other therapeutic agents that modulate or inhibit angiogenesis and can be used in combination with the compounds of the present invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis 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 the activated form of thrombin-activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). TAFIa inhibitors are described in U.S. Patent Application Nos. 60 / 310,927 (filed August 8, 2001) and 60 / 349,925 (filed January 18, 2002).

[0321] "Agents that interfere with cell cycle checkpoints" refer to compounds that inhibit protein kinases that transmit cell cycle checkpoint signals, thereby enabling cancer cells to detect 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, flavopiridol, CYC202 (Cyclacel), and BMS-387032.

[0322] "Agents that interfere with receptor tyrosine kinases (RTKs)" refer to compounds that inhibit RTKs, thereby inhibiting the mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Additional agents include the inhibitors of RTKs described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

[0323] "Inhibitors of cell proliferation and survival signaling pathways" refers to agents that inhibit signaling cascades downstream of cell surface receptors. Such agents include inhibitors of serine / threonine kinases (including, but not limited to, 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 005 / 43361, 60 / 734188, 60 / 652737, 60 / 670469), 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 The term also 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 particular integrins expressed on capillary endothelial cells. v β6, α v It refers to antagonists of β8, α1β1, α2β1, α5β1, α6β1, and α6β4 integrins. v β3, α v β5, αv It 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-dimethylpyrrol-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-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

[0326] Combinations of the 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 malignant diseases. PPAR-γ and PPAR-δ are nuclear peroxisome proliferator-activated receptors γ and δ. 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 the development of 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 (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-55, and others. 5, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-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 antibodies or antigen-binding fragments of the invention may also be useful in treating or preventing breast cancer in combination with aromatase inhibitors, including, but not limited to, anastrozole, letrozole, and exemestane.

[0328] The antibodies or antigen-binding fragments of the invention may also be useful in treating cancer in combination with the following chemotherapeutic agents: abarelix (Plenaxis depot®); aldesleukin (Prokine®); aldesleukin (Proleukin®); alemtuzumab (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (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®); Brefeldin A; Busulfan intravenous (Busulfex®); Busulfan oral (Myleran®); Calsterone (Methosarb®); Capecitabine (Xeloda®); Carboplatin (Paraplatin®); Carmustine (BCNU®, BiCNU®); Carmustine (Gliadel®); Implantable polipheprosan 20 carmustine (Gliadel®) Wafer®; celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®);Liposomal cytarabine (DepoCyt®); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection (Fragmin®); daratumumab (DARZALEX®); darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); liposomal daunorubicin (DanuoXome®); daunorubicin Daunorubicin, daunomycin (Daunorubicin®); Daunorubicin, daunomycin (Cerbidine®); Degarelix (Firmagon®); Denileukin-Difitox (Ontak®); Dexrazoxane (Zinecard®); Dexrazoxane Hydrochloride (Totect®); Didemnin B; 17-DMAG; Docetaxel (Taxotere®); Doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®); liposomal doxorubicin (Doxil®); dromostanolone propionate (Dromostanolone®); dromostanolone propionate (Masterone Injection®); eculizumab injection (Soliris®) Elliott's B solution Solution®); eltrombopag (Promacta®); epirubicin (Ellence®); epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); ethinyl estradiol; etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®); everolimus tablets (Afinitor®); exemestane (Aromasin®); ferumoxytol (Feraheme Injection®);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®); hydroxyurea (Hydrea®); ibritumomab tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alpha-2a (Roferon A®); interferon alpha-2b (Intron A®); iobenguane I 123 injection (AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinib tablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®, Leucovorin®); leuprolide acetate (Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); mechlorethamine, nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex®) tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®);Mitotane (Lysodren®); Mitoxantrone (Novantrone®); Mithramycin; Nandrolone phenpropionate (Durabolin-50®); Nelarabine (Arranon®); Nilotinib (Tasigna®); Nofetumomab (Verluma®); Ofatumumab (Arzerra®); Oprelvekin (Neumega®); Oxaliplatin (Eloxatin®); Paclitaxel (Paxene®) trademark); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanib tablets (Votrient™); pegademase (Adagen (bovine pegademase)®); pegaspargase (Oncaspar®); pegfilgrastim (Neulasta®); pemetrexed Rexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor (Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); pralatrexate injection (Folotyn®); procarbazine (Matulane®); quinacrine (Atabrine®); rapamycin; rasburicase (E litek®); raloxifene hydrochloride (Evista®); rituximab (Rituxan®); romidepsin (Istodax®); romiplostim (Nplate®); sargramostin (Leukine®); sargramostin (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®);Tamoxifen (Nolvadex®); Temozolomide (Temodar®); Temsirolimus (Torisel®); Teniposide, VM-26 (Vumon®); Testolactone (Teslac®); Thioguanine, 6-TG (Thioguanine®); Thiopurines; Thiotepa (Thioplex®); Topotecan (Hycamtin®); Toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab / I-131 Tositumomab (Bexxar®); Trans-Retinoic Acid; Trastuzumab (Herceptin®); Tretinoin, ATRA (Vesanoid®); Triethylenemelamine; Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin; and zoledronate (Zometa®).

[0329] In one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof of the invention is used in combination with other agents, including, but not limited to, casopitant (GlaxoSmithKline), netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, NJ), diphenhydramine (sold as Benadryl® by Pfizer; New York, NY), hydroxyzine (sold as Atarax® by Pfizer; New York, NY), metoclopramide (sold as Reglan® by A.H. Robins Co.; Richmond, VA), lorazepam (sold as Ativan® by Wyeth; Madison, NJ), alprazolam (sold as Xanax® by Pfizer; New York, NY), and others. New York, NY), haloperidol (sold as Haldol® by Ortho-McNeil; Raritan, NJ), droperidol (Inapsine®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, GA), dexamethasone (sold as Decadron® by Merck and Co.; Rahway, NJ), methylprednisolone (sold as Medrol® by Pfizer; New York, NY), prochlorperazine (sold as Compazine® by Glaxosmithkline; Research Triangle Park, NC), granisetron (sold as Kytril® by Hoffmann-La Roche Inc.; Nutley, NJ), ondansetron (marketed as Zofran® by Glaxosmithkline; Research Triangle Park, NC), dolasetron (marketed as Anzemet® by Sanofi-Aventis; New York, NY), or tropisetron (marketed as Navoban® by Novartis; East Hanover, NJ).

[0330] Other side effects of cancer treatment include red and white blood cell deficiencies. Thus, in one embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof is associated with an agent 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 antigen-binding fragment thereof of the present invention is administered in conjunction with anti-cancer radiation therapy. For example, in one embodiment of the present invention, the radiation therapy is external beam radiation therapy (EBT), a method for delivering high-energy X-ray beams to the tumor location. The beams are generated outside the patient's body (e.g., by a linear accelerator) and targeted to the tumor site. These X-rays can destroy cancer cells, and with careful treatment planning, can spare surrounding normal tissue. The radiation source is not placed inside the patient's body. In one embodiment of the present invention, the radiation therapy is proton beam therapy, a type of conformal radiation therapy in which protons instead of X-rays are bombarded to diseased tissue. In one embodiment of the present invention, the radiation therapy is conformal external beam radiation therapy, a procedure that utilizes advanced technology to tailor radiation therapy to individual body structures. In one embodiment of the present invention, the radiation therapy is brachytherapy, a temporary placement of radioactive material inside the body, usually used to deliver an overdose or high dose of radiation to a certain area.

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

[0333] Experimental and diagnostic uses The anti-SIRPα antibodies and antigen-binding fragments thereof disclosed herein may be used as affinity purification agents. In this process, the anti-SIRPα antibodies and antigen-binding fragments thereof 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 contacted with a sample containing the SIRPα protein to be purified, and the support is then washed with an appropriate 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] Further provided are antigens for generating secondary antibodies that are useful, for example, for performing Western blots and other immunoassays discussed herein.

[0335] Anti-SIRPα antibodies (e.g., humanized antibodies) and antigen-binding fragments thereof may also be useful in diagnostic assays for SIRPα protein, for example, detecting expression in specific cells, tissues, or serum, for example, myeloid cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, and dendritic cells. Such diagnostic methods may be useful in diagnosing various diseases.

[0336] The present invention encompasses ELISA assays (enzyme-linked immunosorbent assays) that incorporate the use of the anti-SIRPα antibodies or antigen-binding fragments thereof disclosed herein.

[0337] For example, such a method may include the following steps: (a) coating a substrate (e.g., a well of a microtiter plate, e.g., the surface of a plastic plate) with an anti-SIRPα antibody or an antigen-binding fragment thereof; (b) applying to said substrate a sample to be tested for the presence of SIRPα; (c) washing the plate to remove any unbound material in the sample; (d) applying a detectably labeled antibody (e.g., an antibody conjugated to an enzyme) that is also specific for the SIRPα antigen; (e) washing the substrate to remove unbound labeled antibody; (f) once the labeled antibody is bound to the enzyme, applying a chemical that converts it into a fluorescent signal via the enzyme; and (g) detecting the presence of the labeled antibody.

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

[0339] In further embodiments, the labeled antibody or antigen-binding fragment thereof is labeled with 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 3H, it can be detected by scintillation counter in the presence of a scintillant.

[0340] The anti-SIRPα antibodies or antigen-binding fragments thereof of the present invention may be used in Western blot or immuno-protein blot procedures. Such procedures form part of the present invention and are described, for example, in (1) optionally transferring proteins from a sample to be tested for the presence of SIRPα (e.g., from a PAGE or SDS-PAGE electrophoretic separation of proteins in the sample) to a membrane or other solid support, using methods known in the art (e.g., semi-dry blotting or tank blotting); contacting the membrane or other solid substrate to be tested for the presence of bound SIRPα or a fragment thereof with an anti-SIRPα antibody or antigen-binding fragment thereof of the invention; (2) washing the membrane one or more times to remove unbound anti-SIRPα antibody or fragment and other unbound substances; and (3) detecting the bound 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 onto which proteins to be tested for the presence of SIRPα in non-denaturing PAGE (polyacrylamide gel electrophoresis) or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels have been transferred (e.g., after electrophoretic separation in the gel). Prior to contacting the membrane with the anti-SIRPα antibody or fragment, the membrane is optionally blocked, e.g., with non-fat dry milk, which binds to nonspecific protein-binding sites on the membrane.

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

[0343] The anti-SIRPα antibodies and antigen-binding fragments thereof disclosed herein may be used in immunohistochemical assays. Such methods form part of the present invention, e.g. (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 an anti-SIRPα antibody or antigen-binding fragment thereof of the present invention; and (2) detecting the antibody or fragment on or in the cell; Includes.

[0344] If the antibody or fragment thereof is detectably labeled, it can be detected directly, or the antibody or fragment may be bound by a detectably labeled secondary antibody that is detected.

[0345] Certain anti-SIRPα antibodies and antigen-binding fragments thereof disclosed herein may be used for in vivo tumor imaging. Such methods may include injecting a radiolabeled anti-SIRPα antibody or antigen-binding fragment thereof into a patient being 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 locations containing high concentrations of the antibody or fragment that binds to the tumor, e.g., tumor. Detection of the location may include radiolabeling of SIRPα. + Indicates the presence of tumor and tumor cells.

[0346] Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography). Labels include, for example, iodine-123 ( 123 I) and technetium-99m ( 99m Tc), or in combination with, for example, PET imaging or indium-111 11 C. 13 N, 15 O or 18 F (see, e.g., Gordon et al., (2005) International Rev. Neurobiol. 67:385-440).

[0347] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions of the anti-SIRPα antibodies and antigen-binding fragments of the present invention, the antibodies or antigen-binding fragments thereof are mixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0348] Formulations of therapeutic and diagnostic agents may be prepared by mixing acceptable carriers, excipients, or stabilizers, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension (see, 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 invention administered alone or in combination with another therapeutic agent may be measured, for example, by LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose at which toxic and therapeutic effects occur in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD 50 / ED 50 The data obtained from these cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably within the ED 50 The dosage may vary within this range depending upon the dosage form and route of administration used.

[0350] In a further embodiment, an additional therapeutic agent is administered to a subject in conjunction with the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention according to the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

[0351] The mode of administration can be varied and includes oral, rectal, transmucosal, enteral, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial.

[0352] In a specific embodiment, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention can be administered by an invasive route such as injection. In a further embodiment of the present invention, the anti-SIRPα antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation or aerosol delivery. Administration by a non-invasive route (e.g., orally, for example, in a pill, capsule, or tablet) is also within the scope of the present invention.

[0353] The present invention provides containers (e.g., plastic or glass vials, e.g., with caps, or chromatography columns, hollow needles, or cylindrical syringes) containing an antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition thereof. The present invention also provides injection devices containing an antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition thereof. An injection device is a device for introducing a substance into a patient's body via a parenteral route, e.g., intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an autoinjector) comprising, for example, a syringe barrel or barrel for holding the fluid to be injected (e.g., an antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof), a needle for puncturing the skin and / or a blood vessel for injection of the fluid, and a plunger for forcing the fluid from the syringe barrel through the needle bore. In one embodiment of the present invention, the injection device containing an antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition thereof, is an intravenous (IV) injection device. Such devices contain the antibody or fragment, or a pharmaceutical composition thereof, in a cannula or trocar / needle that can be attached to tubing, which can be attached to a bag or reservoir for holding a fluid (e.g., saline or lactated Ringer's solution containing NaCl, sodium lactate, KCl, CaCl, and optionally glucose) that is introduced into the patient's body through the cannula or trocar / needle. In one embodiment of the present invention, the antibody or fragment, or a pharmaceutical composition thereof, can be introduced into the device and removed from the cannula through which the trocar is inserted when the trocar and cannula are inserted into a subject's vein. The IV device can be inserted, for example, into a peripheral vein (e.g., in the hand or arm); into the superior or inferior vena cava, or right atrium (e.g., central vein); or into the subclavian, internal jugular, or femoral vein and advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., central venous line). In one embodiment of the invention, the injection device is an autoinjector, a jet injector or an extracorporeal infusion pump.Jet injectors use a high-pressure narrow jet of liquid to penetrate the epidermis and introduce the antibody or fragment, or pharmaceutical composition thereof, into the patient's body. Extracorporeal infusion pumps are medical devices that deliver the antibody or fragment, or pharmaceutical composition thereof, into the patient's body in controlled amounts. Extracorporeal infusion pumps may be electrically or mechanically actuated. Different pumps operate in different ways, for example, syringe pumps hold fluid in a syringe reservoir and a movable piston controls fluid delivery, while elastomeric pumps hold fluid in a stretchable balloon reservoir and pressure from the balloon's elastic wall drives fluid delivery. In peristaltic pumps, a set of rollers squeezes along the length of a flexible tubing structure to force fluid forward. In multi-channel pumps, fluid can be delivered from multiple reservoirs at multiple rates.

[0354] The pharmaceutical compositions disclosed herein can also be administered by needleless hypodermic injection device, such as the device disclosed in U.S. Patent No. 6,620,135; U.S. Patent No. 6,096,002; U.S. Patent No. 5,399,163; U.S. Patent No. 5,383,851; U.S. Patent No. 5,312,335; U.S. Patent No. 5,064,413; U.S. Patent No. 4,941,880; U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556.Such needleless device containing the pharmaceutical composition also constitutes part of the present invention.The pharmaceutical compositions disclosed herein can 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 drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates; U.S. Patent No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems, and modules are known to those skilled in the art, and those that include the pharmaceutical composition of the present invention are 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 a tumor. Furthermore, the antibody or fragment may be administered in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody and targeted to, for example, a tumor. The liposome will selectively target and be taken up by 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 achieve improvement of the target disease state while minimizing undesirable side effects. Therefore, the amount of biologic 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 a 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 believed in the art to affect treatment.Generally, the dose is started at a dose slightly lower than the optimal dose, and then increased by small increments until a desired or optimal effect is achieved relative to any negative side effects.Important diagnostic measures include, for example, measures of inflammation, 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 that is the target of treatment, thereby minimizing the immune response to the reagent.For human subjects, humanized antibodies and fully human antibodies may be desirable.

[0358] The antibodies or antigen-binding fragments thereof disclosed herein may be provided by continuous infusion or by administered doses, e.g., once daily, 1 to 7 times per week, once weekly, twice weekly, monthly, twice monthly, quarterly, semi-annually, yearly, etc. Doses 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 typically 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. 52:151-144). Doses may be provided to achieve a predetermined target concentration of anti-SIRPα antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / mL or more. In other embodiments, anti-SIRPα antibodies of the invention are administered at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg / subject, e.g., subcutaneously or intravenously, once a week, twice a week, "every four weeks," monthly, twice a month, or quarterly.

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

[0360] kit Further provided are kits containing one or more components, including, but not limited to, the anti-SIRPα antibodies or antigen-binding fragments discussed herein, in association with one or more additional components, including, but not limited to, a pharmaceutically acceptable carrier and / or a therapeutic agent discussed herein. The antibodies or fragments, and / or the therapeutic agents may be formulated as pure compositions or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

[0361] In one embodiment, the kit comprises an anti-SIRPα antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention 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 invention comprising an anti-SIRPα antibody or antigen-binding fragment thereof of the invention together with a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agents formulated together, optionally in a pharmaceutical composition in one common container.

[0363] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration, for example, the kit may include one or more of the hypodermic needleless or other injection devices discussed above.

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

[0365] The kit may also include a second therapeutic agent, such as an anti-CD47 antibody, an anti-APRIL antibody, an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PDL1 antibody, an anti-TIGIT 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-ILT4 antibody, an anti-ILT5 antibody, an anti-ILT6 antibody, an anti-ILT7 antibody, , 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 organic small molecule inhibitor of such targets; the antibody or antigen-binding fragment thereof may be an antibody against ... 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 an antigen selected from the group consisting of rituximab, ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab, obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, dinituximab, trastuzumab, cetuximab, rituximab-RLI, c.60C3-RLI, Hul4.18-IL2, KM2812, AFM13, and (CD20) 2xCD16, erlotinib (Tarceva), daratumumab, aretuzumab, pertuzumab, brentuximab, elotuzumab, ibritumumab, ifavotuzumab, farletuzumab, otreltuzumab, carotuximab, epratuzumab, inebilizumab, lumletuzumab, 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, ensyki cimab, vedotin (Adcetris), ibritumomab tiuxetan, ABBV-838, HuMax-AXL-ADC, and adotrastuzumab emtansine (Kadcyla); including but not limited to anthracyclines (doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone), oxaliplatin, bortezomib, cyclophosphamide, bleomycin, vorinostat, paclitaxel, 5-fluorouracil, Radiation or chemotherapy drugs including rasil, cytarabine, prednisolone, docetaxel, mitomycin C, topotecan / camptothecin, etoposide, zoledronic acid, methotrexate, ibrutinib, aflibercept, bevacizumab, toremifene, vinblastine, vincristine, idelalisib, mercaptopurine, thalidomide, sorafenib; and drugs that bind to cyclic dinucleotides or other STING pathway agonists.

[0366] Detection and Treatment Kits For convenience, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention can be provided in a kit, i.e., a packaged combination of predetermined amounts of reagents, together with instructions for use in performing a diagnostic or detection assay. If the antibody or fragment is labeled with an enzyme, the kit will contain substrates and cofactors required by the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), may be included. The relative amounts of various reagents may vary widely to provide concentrations in solution of the 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 with the appropriate concentration.

[0367] Also provided are diagnostic or detection reagents for use in various detection assays, such as immunoassays such as ELISA (sandwich or competitive), and kits containing one or more such reagents. The components of the kit may be pre-attached to a solid support or may be coated onto the surface of a solid support 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 prior to use or may require combination with one or more components, such as a buffer, an antibody-enzyme conjugate, an enzyme substrate, etc. The kit may also contain additional reagents, such as a blocking reagent to reduce nonspecific binding to the solid surface, a washing reagent, an enzyme substrate, etc. The solid surface may be in the form of a tube, bead, microtiter plate, microsphere, or other material suitable for immobilizing proteins, peptides, or polypeptides. In particular embodiments, 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 include any of the capture agents and detection reagents described herein. Optionally, the kit may also contain instructions for carrying out the methods of the invention.

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

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

[0370] As discussed above in the Combination Therapy section, co-administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the period during which the agents exert their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[0371] Treatment and detection kits as 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 of the composition as a detection reagent or therapeutic agent. Containers for use in such kits may typically include at least one vial, test tube, flask, bottle, syringe, or other suitable container into which one or more of the detection and / or treatment composition(s) may be placed, and preferably dispensed as appropriate. If a second therapeutic agent is also provided, the kit may also include a second, separate container into which the second detection and / or treatment composition may be placed. Alternatively, multiple compounds may be prepared in a single pharmaceutical composition or packaged within a single container means, such as a vial, flask, syringe, bottle, or other suitable single container. The kits disclosed herein also typically include a means for containing the sealed vial(s) for commercial sale, such as an injection- or blow-molded plastic container into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorescent, or other type of detectable label or detection means is included in the kit, the labeling agent may be provided in the same container as the single detection or therapeutic composition, or may be located in a second, separate container means into which the second composition may be placed and dispensed as appropriate. Alternatively, the detection reagent and label may be prepared in a single container means, and in most instances the kit will also typically include means for containing a sealed vial(s) for commercial sale and / or convenient packaging and delivery.

[0372] Also provided are devices or apparatuses for carrying out the detection or monitoring methods described herein. Such devices may include a chamber or tube into which a sample can be introduced, a fluid handling system optionally including a valve or pump to direct the flow of the sample through the device, a filter 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 detectable label bound to the capture agent's immunocomplex. Sample flow may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once the sample is applied), or active (e.g., by the application of forces generated through mechanical pumps, electroosmotic pumps, centrifugal force, or high air pressure), or by a combination of active and passive forces.

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

[0374] Preferred Embodiments Embodiment 1. a. a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:69, or an amino acid sequence that differs from SEQ ID NO:1 by one, two, or three conservative substitutions; b. a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:70, or an amino acid sequence that differs from SEQ ID NO:2 by one, two, or three conservative substitutions; c. a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that differs from SEQ ID NO:3 by one, two, or three conservative substitutions; d. a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that differs from SEQ ID NO:4 by one, two, or three conservative substitutions; e. a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:73, or an amino acid sequence that differs from SEQ ID NO:5 by one, two, or three conservative substitutions; and f. a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:74, or an amino acid sequence that differs from SEQ ID NO:6 by one, two, or three conservative substitutions; or g. a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that differs from SEQ ID NO:1 by one, two, or three conservative substitutions; h. a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that differs from SEQ ID NO:2 by one, two, or three conservative substitutions; i. a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that differs from SEQ ID NO:3 by one, two, or three conservative substitutions; j. a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that differs from SEQ ID NO:4 by one, two, or three conservative substitutions; k. a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that differs from SEQ ID NO:5 by one, two, or three conservative substitutions; and l. a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that differs from SEQ ID NO:6 by one, two, or three conservative substitutions; one or more of, and optionally including each of, An antibody or antigen-binding fragment thereof that binds to human SIRPα.

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

[0376] Embodiment 3. SEQ ID NO:75, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:78, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:80, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:82, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:84, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:86, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:88, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and SEQ ID NO: 102, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:76, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:90, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:92, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:94, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:96, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:98, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:100, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and SEQ ID NO: 104, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; a light chain variable region comprising an amino acid sequence selected from the group consisting of: or SEQ ID NO:7, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO: 12, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO: 16, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:18, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and SEQ ID NO:30, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:8, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:20, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:22, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:24, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:26, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; SEQ ID NO:28, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and SEQ ID NO:32, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; a light chain variable region comprising an amino acid sequence selected from the group consisting of: including one or both of the following: The antibody or antigen-binding fragment of embodiment 2.

[0377] Embodiment 4. The following features: An EC of less than 10 nM, preferably less than 5 nM, more preferably less than 1.5 nM, more preferably less than 1.0 nM, more preferably less than 0.5 nM, and most preferably about 0.3 nM or less 50 and binding to cells expressing human SIRPαV1 protein; An EC of less than 10 nM, preferably less than 5 nM, more preferably less than 1.5 nM, more preferably less than 1.0 nM, more preferably less than 0.5 nM, and most preferably about 0.3 nM or less 50and binding to cells expressing human SIRPαV2 protein; At an antibody concentration of 50 nM, preferably 67 nM, more preferably 100 nM, or the EC 50 does not appreciably bind to SIRPβ1 protein at concentrations 10-fold greater, preferably 50-fold greater, more preferably 100-fold greater, more preferably 200-fold greater than IC of less than 10.0 nM, more preferably less than 5.0 nM, more preferably less than 2.5 nM, and most preferably about 1.0 nM or less 50 inhibiting the binding of human SIRPα to CD47; and exhibiting a T20 "humanity" score of at least 79, more preferably 85; 4. The antibody or antigen-binding fragment of embodiment 3, having the following structure:

[0378] Embodiment 5. The following heavy chain sequence / light chain sequence combinations: SEQ ID NO:78 / SEQ ID NO:90 SEQ ID NO:78 / SEQ ID NO:92 SEQ ID NO:78 / SEQ ID NO:94 SEQ ID NO:78 / SEQ ID NO:96 SEQ ID NO:78 / SEQ ID NO:98 SEQ ID NO:78 / SEQ ID NO:100 SEQ ID NO:80 / SEQ ID NO:90 SEQ ID NO:80 / SEQ ID NO:92 SEQ ID NO:80 / SEQ ID NO:94 SEQ ID NO:80 / SEQ ID NO:96 SEQ ID NO:80 / SEQ ID NO:98 SEQ ID NO:80 / SEQ ID NO:100 SEQ ID NO:82 / SEQ ID NO:90 SEQ ID NO:82 / SEQ ID NO:92 SEQ ID NO:82 / SEQ ID NO:94 SEQ ID NO:82 / SEQ ID NO:96 SEQ ID NO:82 / SEQ ID NO:98 SEQ ID NO:82 / SEQ ID NO:100 SEQ ID NO:84 / SEQ ID NO:90 SEQ ID NO:84 / SEQ ID NO:92 SEQ ID NO:84 / SEQ ID NO:94 SEQ ID NO:84 / SEQ ID NO:96 SEQ ID NO:84 / SEQ ID NO:98 SEQ ID NO:84 / SEQ ID NO:100 SEQ ID NO:86 / SEQ ID NO:90 SEQ ID NO:86 / SEQ ID NO:92 SEQ ID NO:86 / SEQ ID NO:94 SEQ ID NO:86 / SEQ ID NO:96 SEQ ID NO:86 / SEQ ID NO:98 SEQ ID NO:86 / SEQ ID NO:100 SEQ ID NO:88 / SEQ ID NO:90 SEQ ID NO:88 / SEQ ID NO:92 SEQ ID NO:88 / SEQ ID NO:94 SEQ ID NO:88 / SEQ ID NO:96 SEQ ID NO:88 / SEQ ID NO:98 SEQ ID NO:88 / SEQ ID NO:100 SEQ ID NO:10 / SEQ ID NO:20 SEQ ID NO:10 / SEQ ID NO:22 SEQ ID NO:10 / SEQ ID NO:24 SEQ ID NO:10 / SEQ ID NO:26 SEQ ID NO:10 / SEQ ID NO:28 SEQ ID NO:12 / SEQ ID NO:20 SEQ ID NO:12 / SEQ ID NO:22 SEQ ID NO:12 / SEQ ID NO:24 SEQ ID NO:12 / SEQ ID NO:26 SEQ ID NO:12 / SEQ ID NO:28 SEQ ID NO:14 / SEQ ID NO:20 SEQ ID NO:14 / SEQ ID NO:22 SEQ ID NO:14 / SEQ ID NO:24 SEQ ID NO:14 / SEQ ID NO:26 SEQ ID NO:14 / SEQ ID NO:28 SEQ ID NO:16 / SEQ ID NO:20 SEQ ID NO:16 / SEQ ID NO:22 SEQ ID NO:16 / SEQ ID NO:24 SEQ ID NO:16 / SEQ ID NO:26 SEQ ID NO:16 / SEQ ID NO:28 SEQ ID NO:18 / SEQ ID NO:20 SEQ ID NO:18 / SEQ ID NO:22 SEQ ID NO:18 / SEQ ID NO:24 SEQ ID NO:18 / SEQ ID NO:26 SEQ ID NO:18 / SEQ ID NO:28 or in each instance at least 90%, 95%, 97%, 98% or 99% identical to each SEQ ID NO; 2. The antibody or antigen-binding fragment of embodiment 1, comprising one of:

[0379] Embodiment 6. The antibody or antigen-binding fragment of one of embodiments 1 to 5, wherein the antibody is an intact IgG.

[0380] Embodiment 7. The antibody or antigen-binding fragment of one of embodiments 1-6, wherein the antibody comprises a wild-type or mutated IgG2 Fc region.

[0381] Embodiment 8. The antibody or antigen-binding fragment of one of embodiments 1 to 6, wherein the antibody comprises a mutated IgG1 Fc region.

[0382] Embodiment 9. The antibody or antigen-binding fragment of one of embodiments 1 to 6, wherein the antibody comprises a wild-type or mutated IgG4 Fc region.

[0383] Embodiment 10. An antibody or antigen-binding fragment thereof that binds to the same epitope of human SIRPα as the antibody of embodiment 5.

[0384] Embodiment 11. The antibody or antigen-binding fragment of any of Embodiments 1 to 10, which is humanized.

[0385] Embodiment 12. The antibody or antigen-binding fragment of any of embodiments 1-11, wherein 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.

[0386] Embodiment 13. The antibody or antigen-binding fragment of any of embodiments 1 to 11, wherein the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises SEQ ID NO: 16 and each light chain comprises SEQ ID NO: 28.

[0387] Embodiment 14. The antibody or antigen-binding fragment of any of embodiments 1-11, wherein 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.

[0388] Embodiment 15. The antibody or antigen-binding fragment of any of embodiments 1-11, wherein the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises SEQ ID NO:80 and each light chain comprises SEQ ID NO:90.

[0389] Embodiment 16. The antibody or antigen-binding fragment of any of embodiments 1 to 11, wherein the antibody or antigen-binding fragment is a humanized antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises SEQ ID NO:80 and each light chain comprises SEQ ID NO:92.

[0390] Embodiment 17. The antibody or antigen-binding fragment of any of embodiments 1-11, wherein 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:95.

[0391] Embodiment 18. The antibody or antigen-binding fragment of any one of embodiments 1 to 17, which comprises a glycosylation pattern characteristic of expression by mammalian cells, and optionally is glycosylated by expression from a CHO cell.

[0392] Embodiment 19. An isolated polypeptide comprising the amino acid sequence of any on...

Claims

Claim 1: An isolated therapeutic antibody that binds to human SIRPα, having an EC50 activity of less than 10 nM on cells expressing human SIRPαV1 protein having the sequence of SEQ ID NO:

34. ５０ comprising a variable region that binds at The variable region is Pro of SEQ ID NO: 34 ７４ and a human SIRP isoform in which the proline corresponding to residue 74 of SEQ ID NO: 34 is replaced with alanine; The variable region comprises 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; Isolated therapeutic antibodies.

2. Binding of human SIRPα having the sequence of SEQ ID NO: 34 to human CD47 was evaluated using an IC of less than 10 nM. ５０ 2. The isolated therapeutic antibody of claim 1, wherein the antibody inhibits

3. EC<10 nM on cells expressing human SIRPαV2 protein ５０ 2. The isolated therapeutic antibody of claim 1, which binds to

4. EC50 of less than 1 nM in cells expressing human SIRPαV1 protein having the sequence of SEQ ID NO: 34 ５０ and binds to cells expressing human SIRPαV2 protein with an EC ５０ 4. The isolated therapeutic antibody of claim 3, which binds to

5. 2. The isolated therapeutic antibody of claim 1, which is an IgG.

6. Each of the two variable regions of the IgG has an EC20 activity of less than 10 nM on cells expressing a human SIRPαV1 protein having the sequence of SEQ ID NO:

34. ５０ and Pro of SEQ ID NO: 34 ７４ 6. The isolated therapeutic antibody of claim 5, wherein the antibody distinguishes between a human SIRP isoform comprising a proline in the SIRP IgV domain at a position corresponding to SEQ ID NO: 34 and a human SIRP isoform in which the proline corresponding to residue 74 of SEQ ID NO: 34 is replaced with an alanine.

7. 6. The isolated therapeutic antibody of claim 5, exhibiting a T20 "humanity" score of at least 79.

8. 7. The isolated therapeutic antibody of claim 6, which exhibits a T20 "humanity" score of at least 79.

9. 5. The isolated therapeutic antibody of claim 4, which is an IgG.

10. Each of the two variable regions of the IgG has an EC20 activity of less than 10 nM on cells expressing a human SIRPαV1 protein having the sequence of SEQ ID NO:

34. ５０ and Pro of SEQ ID NO: 34 ７４ 10. The isolated therapeutic antibody of claim 9, wherein the antibody distinguishes between a human SIRP isoform comprising a proline in the SIRP IgV domain at a position corresponding to SEQ ID NO: 34 and a human SIRP isoform in which the proline corresponding to residue 74 of SEQ ID NO: 34 is replaced with an alanine.

11. 10. The isolated therapeutic antibody of claim 9, which exhibits a T20 "humanity" score of at least 79.

12. 11. The isolated therapeutic antibody of claim 10, exhibiting a T20 "humanity" score of at least 79.

13. A composition comprising the therapeutic antibody of claim 1 and a pharmaceutically acceptable carrier or diluent.

14. A composition comprising the therapeutic antibody of claim 4 and a pharmaceutically acceptable carrier or diluent.

15. A composition comprising the therapeutic antibody of claim 5 and a pharmaceutically acceptable carrier or diluent.

16. A composition comprising the therapeutic antibody of claim 6 and a pharmaceutically acceptable carrier or diluent.

17. A composition comprising the therapeutic antibody of claim 7 and a pharmaceutically acceptable carrier or diluent.

18. A composition comprising the therapeutic antibody of claim 8 and a pharmaceutically acceptable carrier or diluent.

19. A composition comprising the therapeutic antibody of claim 9 and a pharmaceutically acceptable carrier or diluent.

20. A composition comprising the therapeutic antibody of claim 10 and a pharmaceutically acceptable carrier or diluent.

21. A composition comprising the therapeutic antibody of claim 11 and a pharmaceutically acceptable carrier or diluent.

22. A composition comprising the therapeutic antibody of claim 12 and a pharmaceutically acceptable carrier or diluent.

23. EC values ​​at 50 nM antibody concentration or against SIRPαV1 or SIRPαV2 ５０ 13. The isolated therapeutic antibody of any one of claims 1 to 12, which does not appreciably bind to SIRPβ1 protein at a concentration 10-fold greater than

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