Anti-sirp-α antibodies and methods of use thereof

Abstract

To provide anti-signal regulatory protein-α (SIRPA) antibodies, methods of generating such antibodies, and therapeutic uses and methods employing the antibodies.SOLUTION: There is provided an isolated anti-signal regulatory protein α (SIRPA) antibody that selectively binds SIRPA and down-regulates SIRPA expressed on the cell surface.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 432,503, filed December 9, 2016, which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listing as an ASCII text file The following ASCII text file submission is incorporated herein by reference in its entirety: Sequence Listing Computer Readable Form (CRF), filename 099061-1069197_SL.TXT, created December 7, 2017.

[0003] The present invention relates to anti-SIRPA antibodies and therapeutic uses of such antibodies. [Background technology]

[0004] Phagocytes, such as macrophages (MΦ) and dendritic cells (DCs), distinguish healthy from abnormal cells through a complex array of cell surface receptors that regulate cell activation state, proliferation, and / or effector function. Many of these receptors recognize diverse ligands that either mark unwanted cells for elimination (so-called "eat-me" signals) or protect normal cells from destruction (so-called "don't-eat-me" signals). In recent years, the SIRPα-CD47 axis has emerged as a critical determinant of programmed cell elimination by macrophages in various clinical settings, ranging from cancer cell survival to successful engraftment of hematopoietic cell transplants. Therapeutics that affect this pathway may fulfill a relevant medical need for disease amelioration, with particular relevance in various human cancers.

[0005] SIRPα (signal regulatory protein-α, SIRPA) belongs to the SIRP family of transmembrane receptors, primarily expressed in myeloid cell lineages (including MΦ, DC, and granulocytes). It is characterized by an extracellular domain containing two membrane-proximal IgC domains and a distal IgV domain. Unique to this family, SIRPA contains an intracellular cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). Upon receptor crosslinking, the tyrosine-phosphorylated ITIM site recruits and activates SHP phosphatases, negatively regulating cellular functions such as phagocytosis or proinflammatory cytokine release. CD47 serves as the primary ligand for SIRPA, and its widespread expression in most cell types, including endothelial / epithelial cells, leukocytes, and erythrocytes, suggests that it mediates a "don't-eat-me" signal to protect healthy cells from phagocyte-dependent clearance. Supporting this view, several studies have shown that adoptive transfer of red or white blood cells from CD47 knockout mice to wild-type recipients results in rapid elimination of CD47-deficient cells. Conversely, positional genetic analysis of multiple strains of immunocompromised mice receiving human hematopoietic cells identified the Sirpα allele in NOD mice as a causative factor for successful engraftment in xenotransplantation models. Subsequent studies demonstrated that allelic variants of SIRPA expressed exclusively in NOD mice retain the ability to bind human CD47 expressed on human hematopoietic stem cells, thus suppressing macrophage-dependent graft rejection.

[0006] Regulated expression of SIRPA and CD47 establishes a homeostatic control mechanism for regulating phagocytic activity. For example, apoptotic cells downregulate CD47 expression to promote engulfment by resident macrophages, while viable cells remain intact. Similarly, inflammatory stimuli such as LPS decrease SIRPA expression in MΦs and DCs, enhancing their activation during inflammation. However, dysregulation of SIRPA and CD47 expression contributes to immune-mediated diseases, such as cancer. Some tumors significantly increase CD47 expression compared with non-cancerous cells to evade immune surveillance mechanisms that normally eliminate malignant cells. Preclinical studies have demonstrated that genetic knockdown of CD47 in syngeneic tumor models, such as B16F10 melanoma, is sufficient to inhibit tumor growth in immunocompetent mice. Similar results have also been observed with CD47-knockdown human cancer cell lines implanted in immunocompromised mice. Alternatively, biological agents that disrupt the SIRPA-CD47 interaction, such as anti-CD47 antibodies, also enhance tumor elimination in mouse models. When combined with commercially available anti-tumor antigen antibodies, such as trastuzumab or rituximab, anti-CD47 antibodies promote a synergistic increase in anti-tumor responses compared with standard monotherapy. Yet, given the ubiquitous expression of CD47, anti-CD47 antibodies risk severe toxicity due to off-target effects that limit their therapeutic efficacy. Nevertheless, these studies establish a critical role for the SIRPA-CD47 pathway in regulating myeloid cells, with potential applications in cancer immunotherapy. Summary of the Invention

[0007] In certain aspects, the present disclosure provides agents that downregulate SIRPA, such as anti-SIRPA antibodies. Such agents can be used to treat, prevent, or reduce the risk of diseases or pathologies associated with SIRPA expression, activity, or signaling. In some aspects, the present disclosure relates to anti-SIRPA antibodies that can downregulate, i.e., reduce the levels of, SIRPA on human macrophages and dendritic cells, as well as the identification of SIRPA-expressing cell lines. In some aspects, the present disclosure relates to anti-SIRPA antibodies that antagonize the immunosuppressive SIRPA-CD47 interaction and promote phagocytosis of CD47-expressing tumor cells. In further aspects, the present disclosure provides unique SIRPA-specific antibodies that interfere with CD47 binding via non-competitive inhibition.

[0008] Thus, in one aspect, the present disclosure relates to a SIRPA antibody that selectively binds to SIRPA and downregulates SIRPA expressed on the cell surface. In some embodiments, the anti-SIRPA antibody reduces the cell surface level of SIRPA, reduces the intracellular level of SIRPA, reduces the total level of SIRPA, or any combination thereof. In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody induces SIRPA degradation, SIRPA cleavage, SIRPA internalization, SIRPA shedding, downregulation of SIRPA expression, or any combination thereof. In some embodiments that can be combined with any of the preceding embodiments, the antibody reduces the cellular level of SIRPA in vivo. In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody inhibits cell surface clustering of SIRPA. In further embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody inhibits or antagonizes one or more SIRPA activities, which SIRPA activities may be selected from the group consisting of: (a) SIRPA binding to one or more SIRPA ligands, optionally wherein the one or more SIRPA ligands are selected from the group consisting of CD47, surfactant proteins A and D, and any combination thereof; (b) binding to dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 reducing the proliferation of one or more cells selected from the group consisting of NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia;(c) inhibiting the migration of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia; (d) inhibiting the migration of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 inhibiting one or more functions of one or more cells selected from the group consisting of NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia;(e) inhibition of the removal of one or more selected from the group consisting of removal of apoptotic neurons, removal of neural tissue debris, removal of dysfunctional synapses, removal of non-neural tissue debris, removal of bacteria, removal of other foreign bodies, removal of disease-causing proteins, removal of disease-causing peptides, and removal of tumor cells, optionally wherein the disease-causing proteins are amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9 orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelial the tumor cell is selected from the group consisting of bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lung cancer, leukemia ... (f) inhibiting tumor cell killing of one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; (g) inhibiting the anti-tumor cell proliferation activity of one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells;(h) modulating the expression of one or more inflammatory receptors, optionally the one or more inflammatory receptors include CD86, and the one or more inflammatory receptors are expressed on one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, myeloid-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; (i) promoting or rescuing the functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and regulatory T cells; (j) promoting or rescuing the functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and regulatory T cells; (k) increasing the number of tumor-promoting myeloid / granulocytic immune suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells in tumors, peripheral blood, or other lymphoid organs; (l) enhancing the tumor-promoting activity of myeloid-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; (m) enhancing the survival of non-tumorigenic myeloid-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; (n) decreasing the activation of tumor-killing tumor-specific T lymphocytes; (o) decreasing the infiltration of tumor-killing tumor-specific NK cells; (p) increasing tumor volume; (q) increasing tumor growth rate.and (r) reducing the effectiveness of one or more immunotherapies that modulate anti-tumor T cell responses, optionally wherein the one or more immunotherapies are immunotherapies targeting one or more target proteins selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and any combination thereof, or one or more cancer vaccines. In some embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody induces one or more activities selected from the group consisting of: (a) increasing the number of tumor-infiltrating CD3+ T cells; (b) decreasing cellular levels of SIRPA in non-tumorigenic CD14+ myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally wherein the non-tumorigenic CD14+ myeloid cells are present in the blood; (c) decreasing the number of non-tumorigenic CD14+ myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally wherein the non-tumorigenic CD14+ myeloid cells are present in the blood. (d) reducing PD-L1 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (e) reducing PD-L2 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (f) reducing B7-H2 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC);(g) reducing B7-H3 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (h) reducing CD200R levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (i) reducing CD163 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC). (j) reducing CD206 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (k) reducing tumor growth rate of solid tumors; (l) reducing tumor volume; (m) increasing the efficacy of one or more PD-1 inhibitors; (n) increasing the efficacy of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies, optionally (o) increasing the efficacy of one or more chemotherapeutic agents, optionally wherein the one or more chemotherapeutic agents are gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, (p) increasing the proliferation of T cells in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs); (l) inhibiting the differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs);and (r) killing CD33-expressing immune suppressor non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells in solid tumors and associated vasculature when conjugated with a chemical or radiotoxin;

[0009] In some embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody inhibits the interaction between SIRPA and one or more SIRPA ligands. In some embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody reduces intracellular levels of SIRPA and inhibits the interaction between SIRPA and one or more SIRPA ligands. In some embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody blocks the binding of CD47 to human SIRPA.

[0010] In some embodiments that may be combined with any of the preceding embodiments, the antibody selectively binds to human SIRPA and does not substantially block CD47 binding to human SIRPA expressed on cells, and further, binding to human SIRPA reduces SIRPA levels on the cell surface. In some embodiments, the antibody binds to the D1 domain of SIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D2 domain of SIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D3 domain of SIRPA, e.g., human SIRPA. In some embodiments, such anti-SIRPA antibodies comprise a V comprising the amino acid sequence of SEQ ID NO:2. H V comprising the amino acid sequence of SEQ ID NO: 3 L In some embodiments, such anti-SIRPA antibodies compete with antibodies comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 10. HIn some embodiments, the anti-SIRPA antibody comprises a V region comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR1 comprising the amino acid sequence of SEQ ID NO: 9 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 9; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 10; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a CDR3 comprising the amino acid sequence of SEQ ID NO: 11 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 11. H In some embodiments, anti-SIRPA comprises a V region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 9 with no more than one amino acid substitution; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 with no more than one amino acid substitution; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 11 with no more than one amino acid substitution. H In some embodiments, the anti-SIRPA antibody comprises a V region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:9, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11. H In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a V region as shown in FIG. H V containing the amino acid sequence of the region H region or V in FIG. 14A H V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region HIn some embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody comprises a V region that includes a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 7. L In some embodiments, the V L The region comprises (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 with no more than two amino acid substitutions, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 6; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 7; (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, V L The region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 with no more than one amino acid substitution; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 7 with no more than one amino acid substitution; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 with no more than one amino acid substitution. L The region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, V L The region is V shown in FIG. L or V in Figure 14B LV having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region L In some embodiments, the antibody comprises an Fc region that reduces the level of FcγR expressed on the surface of a cell. In some embodiments, the antibody comprises an Fc region that reduces the level of FcγRBII on the surface of a cell.

[0011] In some embodiments that may be combined with any of the preceding embodiments, the antibody selectively binds to human SIRPA, but not to mouse SIRPA, and does not substantially block CD47 binding to human SIRPA expressed on cells. Furthermore, binding to human SIRPA reduces the level of SIRPA on the cell surface. In some embodiments, such an anti-SIRPA antibody is a V comprising the amino acid sequence of SEQ ID NO:2. H V comprising the amino acid sequence of SEQ ID NO: 3 L In some embodiments, the antibody binds to the D1 domain of SIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D2 domain of SIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D3 domain of SIRPA, e.g., human SIRPA. In some embodiments, the anti-SIRPA antibody has a V domain comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 16. H In some embodiments, the V HThe region comprises: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 15; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 16; (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR3 comprising the amino acid sequence of SEQ ID NO: 17 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, V H The region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 with no more than one amino acid substitution; a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 with no more than one amino acid substitution; a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 16 with no more than one amino acid substitution. H The region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17 in V of FIG. H V containing the amino acid sequence of the region H region or V in FIG. 14C H V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region H In some embodiments that may be combined with any of the preceding embodiments, V L The region comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 13.L The region comprises (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 12; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 13; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a CDR3 comprising the amino acid sequence of SEQ ID NO: 14 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, V L The region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 with no more than one amino acid substitution; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 with no more than one amino acid substitution; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 14 with no more than one amino acid substitution. L The region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, V L The region is V in Figure 14D. L or the amino acid sequence of the V region of FIG. L V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region L In some embodiments, the antibody comprises an Fc region that reduces the level of FcγR on the cell surface. In some embodiments, the antibody comprises an Fc region that reduces the level of FcγRBII on the cell surface.

[0012] In further aspects that may be combined with any of the preceding embodiments, the isolated anti-SIRPA of the present disclosure competes with one or more antibodies selected from the group consisting of 3F9, 9C2, 8A9, 8F4, 1E2, 7H9, and 4D8 for binding to SIRPA. In some embodiments, the antibody binds to the D1 domain of SIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D2 domain of mSIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D3 domain of SIRPA, e.g., human SIRPA. In some embodiments, the isolated anti-SIRPA antibody binds to essentially the same epitope as one or more antibodies selected from the group consisting of 3F9, 9C2, 8A9, 8F4, 1E2, 7H9, and 4D8. In some embodiments, the isolated anti-SIRPA antibody is V H Area and V L Includes the area, V H area, V L The region, or both, comprises at least 1, 2, 3, 4, 5, or 6 CDRs of a monoclonal selected from the group consisting of 3F9, 9C2, 8A9, 8F4, 1E2, 7H9, and 4D8.

[0013] In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody is a monoclonal antibody. In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody is a humanized antibody. In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody is a Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragment; or a multivalent antibody, and the antibody is of the IgG class, IgM class, or IgA class.

[0014] In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody is of the IgG class, IgM class, or IgA class. In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments that can be combined with any of the preceding embodiments, the antibody binds to an inhibitory Fc receptor. In some embodiments that can be combined with any of the preceding embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγRIIB). In some embodiments, the antibody reduces FcγRIIB levels on the cell surface. In some embodiments that may be combined with any of the preceding embodiments, (a) the anti-SIRPA antibody has a human or mouse IgG1 isotype and is selected from the group consisting of N297A, D265A, D270A, L234A, L235A, G237A, P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267 one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof, wherein residue numbering is according to EU or Kabat numbering, or an amino acid deletion in the Fc region at a position corresponding to glycine 236;(b) the anti-SIRPA antibody has an IgG1 isotype and comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and hinge region, optionally the IgG2 isotype CH1 and hinge region comprises: ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 34), and optionally the antibody Fc region comprises a S267E amino acid substitution, a L328F amino acid substitution, or both, and / or a N297A or N297Q amino acid substitution, where residue numbering is according to EU numbering; (c) the anti-SIRPA antibody has an IgG2 isotype and is selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, V309L, A330S, P331S, C214S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, H268E, N297A, N297Q, A330L, and any combination thereof. (d) the anti-SIRPA antibody has a human or mouse IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, E233P, F234V, L234A / F234A, S228P, S241P, L248E, T394D, N297A, N297Q, L235E, and any combination thereof, wherein residue numbering is according to EU or Kabat numbering;or (e) the anti-SIRPA antibody has a hybrid IgG2 / 4 isotype, optionally wherein the antibody comprises an amino acid sequence including amino acids 118-260 of human IgG2 and amino acids 261-447 of human IgG4, where residue numbering is according to EU or Kabat numbering. In some embodiments that may be combined with any of the preceding embodiments, (a) the anti-SIRPA antibody has a human or murine IgG1 isotype and contains any of the following residues: N297A, N297Q, D270A, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E , P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering is according to EU or Kabat numbering; (b) the anti-SIRPA antibody has an IgG2 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering is according to EU or Kabat numbering; or (c) the anti-SIRPA antibody has an IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of P233P, P233S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering is according to EU or Kabat numbering; , F234V, L234A / F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, wherein residue numbering is according to EU or Kabat numbering. In some embodiments that may be combined with any of the preceding embodiments, (a) the Fc region further comprises one or more additional amino acid substitutions at positions selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, wherein residue numbering is according to EU or Kabat numbering;(b) the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, where the residue numbering is according to EU or Kabat numbering; or (c) the Fc region further comprises a S228P amino acid substitution according to EU or Kabat numbering. In some embodiments that can be combined with any of the preceding embodiments, the antibody has an IgG4 isotype. In some embodiments that can be combined with any of the preceding embodiments, the anti-SIRPA antibody comprises a S228P amino acid substitution at residue position 228, a F234A amino acid substitution at residue position 234, and a L235A amino acid substitution at residue position 235, where the residue positions are numbered according to EU or Kabat numbering.

[0015] In some embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody is a bispecific antibody. In some embodiments, the anti-SIRPA antibody recognizes a first and a second antigen, wherein the first antigen is SIRPA and the second antigen is (a) an antigen that promotes transport across the blood-brain barrier; (b) an antigen that promotes transport across the blood-brain barrier selected from the group consisting of transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, llama single-domain antibody, TMEM30(A), protein transduction domain, TAT, Syn-B, penetratin, polyarginine peptides, angiopep peptides, and ANG1005;(c) a disease-causing agent selected from the group consisting of a disease-causing peptide or protein, or a disease-causing nucleic acid, wherein the disease-causing nucleic acid is an antisense GGCCCC (G2C4) repeat expansion RNA, and the disease-causing protein is amyloid beta, oligomeric amyloid beta, amyloid beta plaque, amyloid precursor protein or a fragment thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame), 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin an agent selected from the group consisting of purine light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine (PA) repeat peptide, ubiquitin, and proline-arginine (PR) repeat peptide; and (d) a ligand and / or protein expressed on immune cells, such as PD1 / PDL1, CD40, OX40, a ligand and / or protein selected from the group consisting of ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, and phosphatidylserine; and a protein, lipid, polysaccharide, or glycolipid expressed on one or more tumor cells;

[0016] In certain embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody is a conjugated antibody. For example, the anti-SIRPA antibody can be conjugated to a detectable marker, a toxin, or a therapeutic agent. In some embodiments, the anti-SIRPA antibody is conjugated to a toxin selected from the group consisting of ricin, ricin A chain, doxorubicin, daunorubicin, maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alphasarcin, gelonin, mitogellin, letostrictocin, phenomycin, enomycin, chrysin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycin, dolastatin, cc1065, and cisplatin.

[0017] In further embodiments that may be combined with any of the preceding embodiments, the anti-SIRPA antibody is selected from the group consisting of amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, and ataxin receptor 1 (AR1). ataxin-3, ataxin-7, ataxin-8, ataxin-10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation products, dipeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) in combination with one or more antibodies that specifically bind to a disease-causing protein selected from the group consisting of repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine (PR) repeat peptides, and any combination thereof; or PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, B The antibody or antibodies may be used in combination with one or more antibodies that bind to an immunomodulatory protein selected from the group consisting of TLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, TREM1, TREM2, CD33, Siglec-5, Siglec-7, Siglec-9, Siglec-11, phosphatidylserine, a disease-causing nucleic acid, an antisense GGCCCC (G2C4) repeat expansion RNA, and any combination thereof.

[0018] In a further aspect, the disclosure provides a method for reducing the activity, functionality, or viability of regulatory T cells, tumor-enclosed immunosuppressor dendritic cells, tumor-enclosed immunosuppressor macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, or chronic myeloid leukemia (CML) cells in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an agent that binds to or interacts with SIRPA, e.g., an antibody of any of the above embodiments.

[0019] In a further aspect, the disclosure provides a method of inducing or promoting the survival, maturation, functionality, migration, or proliferation of one or more immune cells in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an agent, e.g., an antibody of any of the above embodiments, that reduces cellular levels of SIRPA, inhibits the interaction between SIRPA and one or more SIRPA ligands, or both. In some embodiments, the one or more immune cells are selected from the group consisting of dendritic cells, macrophages, neutrophils, NK cells, microglia, T cells, T helper cells, cytotoxic T cells, and any combination thereof.

[0020] In another aspect, the disclosure provides a method of treating cancer, comprising administering to a patient having a tumor that expresses CD47 a therapeutically effective amount of an anti-SIRPA antibody of any of the above embodiments.

[0021] In a further aspect, the invention provides methods of treating cancer comprising administering a therapeutically effective amount of an agent that reduces cellular levels of SIRPA, e.g., an anti-SIRPA antibody of any of the above embodiments. In some embodiments, the method further comprises administering a therapeutic agent that inhibits PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73. In some embodiments, the therapeutic agent is an antibody that inhibits PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73.

[0022] In a further aspect, the present invention provides methods of treating cancer, comprising administering a therapeutically effective amount of an agent that reduces cellular levels of SIRPA, e.g., an anti-SIRPA antibody of any of the above embodiments. In some embodiments, the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule and / or one or more standard or investigational anti-cancer therapies. In some embodiments, the at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with an anti-SIRPA antibody. In some embodiments, the at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-B and T lymphocyte attenuator (BTLA) antibody, an anti-killer inhibitory receptor (KIR) antibody, an anti-GAL9 antibody, an anti-TIM-1 antibody, an anti-TIM3 antibody, an anti-TIM-4 antibody, an anti-A2AR antibody, an anti-CD3 In some embodiments that may be combined with any of the preceding embodiments, the one or more standard or investigational anti-cancer therapies are selected from the group consisting of radiation therapy, cytotoxic chemotherapy, targeted therapy, imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer (ACT) therapy, chimeric antigen receptor T cell transfer (CAR-T) therapy, vaccine therapy, and cytokine therapy.

[0023] In some embodiments that may be combined with any of the preceding method embodiments, the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory cytokine. In some embodiments, the at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with the anti-SIRPA antibody of any one of the preceding embodiments. In some embodiments, the at least one antibody that specifically binds to an inhibitory cytokine is selected from the group consisting of an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises administering to the individual at least one agonist antibody that specifically binds to a stimulatory checkpoint protein. In some embodiments, the at least one agonist antibody that specifically binds to a stimulatory checkpoint protein is administered in combination with the anti-SIRPA antibody of any of the preceding embodiments. In some embodiments, the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is selected from the group consisting of an agonistic anti-CD40 antibody, an agonistic anti-OX40 antibody, an agonistic anti-ICOS antibody, an agonistic anti-CD28 antibody, an agonistic anti-TREM1 antibody, an agonistic anti-TREM2 antibody, an agonistic anti-CD137 / 4-1BB antibody, an agonistic anti-CD27 antibody, an agonistic anti-glucocorticoid-inducible TNFR-related protein GITR antibody, an agonistic anti-CD30 antibody, an agonistic anti-BTLA antibody, an agonistic anti-HVEM antibody, an agonistic anti-CD2 antibody, an agonistic anti-CD5 antibody, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises administering to the individual at least one stimulatory cytokine. In some embodiments, the stimulatory cytokine is selected from the group consisting of IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, an IL-20 family member, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-beta, and any combination thereof.

[0024] In a further aspect, the present disclosure provides a method of treating cancer, comprising administering to a subject having cancer cells of the myeloid lineage that express SIRPA a therapeutically effective amount of an anti-SIRPA antibody of any one of the preceding embodiments.

[0025] In another aspect, the disclosure provides a method of treating cancer, the method comprising administering a therapeutically effective amount of the anti-SIRPA antibody of any one of the preceding embodiments to a subject having cancer, wherein the cancer is selected from the group consisting of sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma; or the cancer is selected from the group consisting of glioblastoma multiforme, renal clear cell carcinoma, adrenocortical carcinoma, bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma, pancreatic adenocarcinoma, renal cell carcinoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, and fibrosarcoma. In some embodiments, the anti-SIRPα antibody is selected from the group consisting of: allergic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, invasive breast carcinoma, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal carcinoma, head and neck squamous cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, hepatocellular carcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germinoma, thyroid carcinoma, thymoma, endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma. In some embodiments, the anti-SIRPα antibody is conjugated to a cytotoxic agent and / or induces ADCC.

[0026] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the anti-SIRPA antibody of any one of the preceding embodiments and a physiologically acceptable carrier. In some embodiments, the present disclosure provides the anti-SIRPA antibody of any one of the preceding embodiments for use in the treatment of cancer; and / or for use in a method of preparing a medicament for the treatment of cancer.

[0027] In a further aspect, the present disclosure provides a method of preventing, reducing the risk of, or treating a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, taupathy, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, stroke, frontotemporal dementia, spinal cord injury, Huntington's disease, infection, and cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an agent that reduces cellular levels of SIRPA, inhibits the interaction of SIRPA with one or more SIRPA ligands, or both. In some embodiments, the disease, disorder, or injury is cancer, and the agent is selected from the group consisting of: (a) promoting the proliferation, maturation, migration, differentiation, and / or functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated suppressor neutrophils, tumor-associated suppressor NK cells, non-tumorigenic CD14+ myeloid cells, and regulatory T cells; (b) increasing the infiltration of tumors by one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated suppressor neutrophils, tumor-associated suppressor NK cells, and regulatory T cells; and (c) increasing the infiltration of tumors by one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated suppressor neutrophils, tumor-associated suppressor NK cells, and regulatory T cells. (d) increasing the number of tumor-promoting myeloid / granulocytic immunosuppressive cells and / or non-tumorigenic CD14+ myeloid cells in tumors, peripheral blood, or other lymphoid organs; (d) increasing the tumor-promoting activity of myeloid-derived suppressor cells (MDSCs) and / or non-tumorigenic CD14+ myeloid cells; (e) increasing the expression of tumor-promoting cytokines in tumors or peripheral blood, optionally wherein the tumor-promoting cytokine is TGF-beta or IL-10; (f) increasing the tumor infiltration of tumor-promoting FoxP3+ regulatory T lymphocytes; (g) decreasing the activation of tumor-specific T lymphocytes with tumor-killing ability; (h) decreasing the infiltration of tumor-specific T lymphocytes with tumor-killing ability; (i) decreasing the infiltration of tumor-specific NK cells with tumor-killing ability;(j) reducing the tumor-killing ability of NK cells; (k) reducing the infiltration of tumor-specific B lymphocytes, which have the potential to enhance immune responses; (l) increasing tumor volume; (m) increasing the rate of tumor growth; (n) increasing metastasis; (o) increasing the rate of tumor recurrence; (p) reducing the effectiveness of one or more immunotherapies that modulate anti-tumor T cell responses, optionally the one or more immunotherapies are selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, P (q) inhibition of PLCγ / PKC / calcium mobilization; and (r) inhibition of PI3K / Akt, Ras / MAPK signaling, which is an immunotherapy targeting one or more target proteins selected from the group consisting of: D-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, and any combination thereof, or one or more cancer vaccines. In some embodiments that may be combined with any of the preceding embodiments, the disease, disorder, or injury is cancer, and the agent (a) increases the number of tumor-infiltrating CD3+ T cells; (b) decreases cellular levels of CD33 in non-tumorigenic CD14+ myeloid cells, optionally where the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally where the non-tumorigenic CD14+ myeloid cells are present in the blood; (c) decreases the number of non-tumorigenic CD14+ myeloid cells, optionally where the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally where the non-tumorigenic CD14+ myeloid cells are present in the blood; (d) decreases PD-L1 levels in one or more cells, optionally where the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs);(e) reducing PD-L2 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (f) reducing B7-H2 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (g) reducing B7-H3 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (h) reducing CD200R levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (i) reducing CD163 levels in one or more cells. (j) reducing CD206 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (k) reducing tumor growth rate of solid tumors; (l) reducing tumor volume; (m) increasing the effectiveness of one or more PD-1 inhibitors; (n) increasing the effectiveness of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies, optionally wherein the one or more checkpoint inhibitor therapies and / or immunomodulatory therapies target one or more of CTLA4, adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof;(o) increasing the effectiveness of one or more chemotherapeutic agents, optionally the one or more chemotherapeutic agents are gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and (p) increasing T cell proliferation in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs), any combination thereof; (q) inhibiting differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs); and (r) killing CD33-expressing immune suppressor myeloid cells and / or CD14-expressing cells in solid tumors and associated vasculature when conjugated with a chemical or radiotoxin. In some embodiments, the cancer expresses SIRPA or one or more SIRPA ligands.

[0028] In a further aspect, the disclosure provides a method of treating, preventing, or reducing the risk of a disease, disorder, or injury, comprising an agent that downregulates SIRPA, wherein the disease, disorder, or injury is selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, tauopathy, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, stroke, frontotemporal dementia, spinal cord injury, and Huntington's disease. In some embodiments, the agent downregulates SIRPA, e.g., an anti-SIRPA antibody of any one of the preceding embodiments.

[0029] In a further aspect, the present disclosure provides a method for the treatment of a rhodopsin-related inflammatory disease (HDG) associated with an inflammatory bowel disease (IGD) associated with an inflammatory bowel disease (IGE ... H a polynucleotide comprising a nucleic acid sequence encoding the V region of the anti-SIRPA antibody of any one of the embodiments described herein; LIn a further embodiment, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the V H an expression vector comprising a polynucleotide comprising a nucleic acid sequence encoding a region, or L In some embodiments, the present disclosure provides an expression vector comprising a polynucleotide comprising a nucleic acid sequence encoding a V H Polynucleotides encoding the regions and V L In a further aspect, the present disclosure provides an expression vector comprising a polynucleotide encoding a V H a host cell comprising a polynucleotide comprising a nucleic acid sequence encoding a V region; L In some embodiments, the present disclosure provides a host cell comprising a polynucleotide comprising a nucleic acid sequence encoding a V H Polynucleotides encoding the regions and V L In some embodiments, the present disclosure provides a host cell comprising a polynucleotide encoding the region. In some embodiments, the present disclosure provides a host cell comprising the expression vector of any one of the preceding embodiments. In a further aspect, the present disclosure provides a method of producing an anti-SIRPA antibody, the method comprising culturing a host cell of any of the preceding embodiments under conditions in which the antibody is expressed. In some embodiments, the host cell is a mammalian host cell. [Brief explanation of the drawings]

[0030] [Figure 1A]

[0023] Figure 1 shows an amino acid sequence alignment between the two most common alleles of the human SIRPA protein (v1 (SEQ ID NO: 1) and v2 (SEQ ID NO: 45)), describing the different residues within the ligand-binding domain. Accession numbers are NP542970 and CAA71403, respectively. [Figure 1B] 1 shows an amino acid sequence alignment between human SIRPA v1 protein (SEQ ID NO: 1) and human SIRPB1 protein (SEQ ID NO: 46), describing the homology between the two proteins. Accession numbers are NP542970 and O00241, respectively. [Figure 2]

[0023] Figure 1 shows an amino acid sequence alignment between human SIRPA protein (SEQ ID NO: 1) and mouse SIRPA protein (SEQ ID NO: 47), describing the homology between the two proteins. Accession numbers are NP542970 and Q6P6I8, respectively. [Figure 3A] The left panel shows FACS histograms of selected SIRPA antibodies binding to rodent Chinese hamster ovary cell lines (CHO) expressing either human SIRPA (HuSIRPA) or mouse SIRPA (MuSIRPA). The shaded histograms represent CHO-MuSIRPA cells. The open and closed histograms represent CHO-HuSIRPA cells. The right panel shows the relative MFI values of SIRPA antibodies binding to HuSIRPA compared to MuSIRPA. Results are expressed as fold over background. Background levels are set to 1 on the y-axis. Antibody mIgG is an isotype negative control. [Figure 3B] FACS histograms of selected SIRPA antibodies binding to primary human macrophages are shown. Antibody mIgG represents a negative isotype control. Shaded histograms represent cells stained with anti-mouse IgG secondary antibody alone. Open and black histograms represent SIRPA-positive cell populations. [Figure 3C] Figure 1 shows surface plasmon resonance sensorgrams of the indicated anti-SIRPA antibodies binding to recombinant soluble HuSIRPA protein. Anti-mouse IgG antibody immobilized on a CM5 chip captured the anti-SIRPA antibody, and serial dilutions of His-tagged soluble HuSIRPA protein were run over the antibody. Kd values were determined by curve-fitting analysis. [Figure 3D] Figure 1 shows the binding of increasing concentrations of anti-SIRPA antibodies to human SIRPA overexpressed on CHO cells. EC50 values were calculated by fitting the data to a sigmoidal curve using Graph Pad Prism. [Figure 4A]FACS histograms of recombinant soluble human CD47 (HuCD47) binding to CHO-HuSIRPA cells in the presence of either anti-SIRPA antibody (dashed histogram) or mouse IgG1 isotype control (solid black histogram). His-tagged HuCD47 was detected with a PE-labeled anti-HIS-tag secondary antibody. As a negative control (shaded histogram), CHO-HuSIRPA cells were stained with an anti-HIS-tagged PE secondary antibody in the absence of HuCD47. [Figure 4B] Figure 1 shows the relative MFI values of HuCD47 binding to CHO-HuSIRPA cells in the presence of the indicated anti-SIRPA antibodies or mouse IgG1 isotype control. Results are expressed as fold over background by dividing the MFI values of HuCD47 and antibody-treated samples by the MFI value of cells stained with anti-HIS-tagged PE in the absence of HuCD47. [Figure 5A] Figure 1 shows the induction of human SIRPA-dependent luciferase expression in a cell-based reporter assay. BWZ / NFAT-luciferase reporter cells (BWZ) were engineered to stably express the human SIRPA-DAP12 chimera (BWZ-HuSIRPA). Cells were stimulated with increasing concentrations of plate-bound recombinant HuCD47. Only cells expressing the HuSIRPA chimera induced luciferase expression in a dose-dependent manner, as measured by luminescence signal. Results are expressed as fold over background. Background levels are set at 1 on the y-axis. [Figure 5B]This figure shows the ability of CD47-blocking and non-CD47-blocking anti-SIRPA antibodies to affect HuSIRPA-dependent luciferase expression in a cell-based reporter assay. BWZ-HuSIRPA cells were seeded into wells with or without plate-bound CD47 protein. All CD47-blocking antibodies (1B3, 12D6, 1H11, 5F7) strongly suppressed the luminescence signal. Two non-CD47-blocking anti-SIRPA antibodies did not reduce luciferase expression. Results are expressed as fold over background. Background level is set at 1 on the y-axis. [Figure 6A] Figure 1 shows human SIRPA-dependent or human SIRPB1-dependent induction of luciferase expression in a cell-based reporter assay. BWZ-HuSIRPA and BWZ-HuSIRPB1 reporter cells were stimulated with plate-bound full-length anti-SIRPA antibodies or mIgG1 isotype control. CD47-blocking anti-SIRPA antibodies activated both SIRPA-expressing and SIRPB1-expressing reporter cells, whereas non-CD47-blocking anti-SIRPA antibodies (3F9 and 9C2) specifically activated only BWZ-HuSIRPA cells. [Figure 6B] Figure 6B shows surface plasmon resonance sensorgrams of the indicated anti-SIRPA antibodies binding to recombinant soluble HuSIRPA or HuSIRPB1 antigens. Anti-mouse IgG antibodies immobilized on a CM5 chip captured the anti-SIRPA antibodies, and equimolar concentrations of antigen were flowed over the captured antibodies. [Figure 7A] Figure 1 shows downregulation of SIRPA receptors in primary human macrophages in response to antibody stimulation. Cells were treated with either a soluble full-length isotype control or a soluble full-length anti-SIRPA antibody, followed by staining with a DyLight650-conjugated anti-SIRPA reference antibody (SA56-DyL650) that binds to a distinct epitope bin. [Figure 7B]Figure 1 shows downregulation of SIRPA receptors in primary human macrophages treated with a non-CD47 blocking antibody. For comparison, macrophages were also treated with two CD47 blocking antibodies (12D6 and 5F7). Results are presented as a percentage of reference antibody binding, calculated by dividing the MFI value of the anti-SIRPA antibody-treated sample by the MFI value of the isotype control-treated sample. [Figure 8A] A live-cell phagocytosis assay was established using macrophages as effector cells and pHrode-labeled tumor cells as targets. Biotinylated lentil agglutinin (LCA), a mannose-binding lectin, was complexed with avidin-conjugated pHrodo Red dye. The LCA-pHrodo complex was then mixed with Raji cells (a human B-cell lymphoma line) to coat the cell surface with pHrodo through LCA-bound carbohydrate structures on the cell membrane. Labeled Raji cells (Raji-Red) alone or opsonized with anti-CD20 antibody were mixed with macrophages at a 2:1 ratio and incubated for 2 hours to allow cells to phagocytose. Phagocytic activity was measured by counting the percentage of CD14-APC+ / PE+ macrophages by FACS analysis. [Figure 8B] Enhanced phagocytic activity of macrophages treated with a non-CD47-blocking anti-SIRPA antibody. Macrophages were cultured overnight in 2.5% FBS RPMI medium containing 5 μg / mL of 3F9, 9C2, or 1B3 (CD47-blockers), or an isotype control. Raji-Red cells alone or opsonized with an anti-CD20 antibody were mixed with macrophages at a 2:1 ratio, and phagocytic activity was determined as described above. [Figure 8C] Figure 1 shows enhanced phagocytic activity of macrophages treated with CD47-blocking anti-SIRPA antibodies. Macrophages were cultured overnight in 2.5% FBS RPMI medium containing 5 μg / mL of 12D6, 9C5, 1H11, 5F7, 1B3, 3F9 (non-CD47 blocker), or isotype control. Phagocytic activity was measured as described above. [Figure 9A]Figure 1 shows downregulation of SIRPA receptors in primary human monocytes in response to antibody stimulation. Cells were treated with either a soluble full-length isotype control or the anti-SIRPA antibody 3F9, followed by staining with a DyLight650-conjugated anti-SIRPA reference antibody (SA56-DyL650) that binds to a distinct epitope bin. [Figure 9B] Respiratory burst from primary human monocytes isolated from two healthy donors (HD) is shown. Cells were stimulated with soluble full-length mouse IgG1 isotype control or anti-SIRPA antibodies 3F9 and 9C2. In all experiments, reactive oxygen species (ROS) production was monitored by labeling cells with 2 μM of the fluorescent indicator CM-H2DCFDA. [Figure 9C] Figure 1 shows IL-8 secretion from primary human monocytes stimulated overnight with a non-CD47 blocking antibody. Supernatants were collected and cytokine concentrations were determined by standard ELISA protocol according to the manufacturer's instructions (eBioscience). [Figure 10A] Expression of mouse and human SIRPA in peripheral blood monocytes (solid line) and granulocytes (dashed line) in huSIRPA-tg mice by FACS staining is shown. Human SIRPA was detected with anti-hSIRPα / β-APC (clone SE5A5, Biolegend). Mouse SIRPA was detected with anti-mSIRPα-APC (clone p84, Biolegend). Isotype staining is shown as a shaded histogram. [Figure 10B] Tumor volume measurements are shown for huSIRPA-tg mice subcutaneously implanted with Raji B-cell lymphoma cells. Three mice per group received either 5 x 10 or 1 x 10 Raji cells. Solid tumor formation was determined by caliper measurements twice weekly. [Figure 10C]Figure 10C shows huSIRPA expression in peripheral blood cells from mice administered either 10 mg / kg of 3F9 (solid histogram) or isotype control (shaded histogram) antibody. The top panel of Figure 10C shows detection of huSIRPA using commercially available anti-hSIRPα / β-APC (clone SE5A5, Biolegend), an antibody that binds to a different epitope than 3F9. The bottom panel of Figure 10C shows detection of huSIRPA with internally generated anti-hSIRPα-DyLight650 (clone 9C2), an antibody that binds to the same epitope as 3F9. [Figure 10D] Figure 10D shows downregulation of huSIRPA expression in splenocytes after antibody treatment in vivo. The top panel of Figure 10D shows the gating strategy for single cell suspensions from mouse spleens stained with anti-mouse F4 / 80 FITC and anti-mouse CD11b Pacific Blue. The bottom panel of Figure 10D shows huSIRPA expression from two splenic myeloid populations (F4 / 80LoCD11bLo and F4 / 80HiCD11bHi). The solid histogram represents huSIRPA expression in mice administered an isotype control antibody, and the dashed histogram represents huSIRPA expression in mice administered 3F9. [Figure 11A] Figure 11A shows downregulation of huSIRPA expression in tumor-associated myeloid cells after antibody treatment in vivo. The top panel of Figure 11A shows the gating strategy for single cell suspensions from tumors stained with anti-mouse F4 / 80 FITC and anti-mouse CD11b Pacific Blue. The bottom panel of Figure 11A shows huSIRPA expression from two splenic myeloid populations (F4 / 80+ and CD11b+). The solid histogram represents huSIRPA expression in mice administered an isotype control antibody, and the dashed histogram represents huSIRPA expression in mice administered 3F9. [Figure 11B]Radiance values of Raji-luciferase lymphoma cells injected subcutaneously into huSIRPA-tg mice are shown. On day 10, mice were randomized into treatment or control groups based on their radiance values and were dosed every 3–4 days with 10 mg / kg of 3F9 or mouse IgG1 antibody via ip injection until study termination. Tumor luminescence values after the start of dosing were normalized to the luminescence value on the day of randomization and analyzed for significance by linear regression. [Figure 12A] Figure 12A shows downregulation of huSIRPA-expressing huCD45+huCD14+ cells recovered from humanized mice bearing MDA-MB-231 tumors after in vivo antibody treatment. The top panel of Figure 12A shows huSIRPA expression levels in peripheral blood huCD45+huCD14+ cells from mice administered an ip injection of either an isotype control, 3F9, or Keytruda (pembrolizumab, Merck). The bottom panel of Figure 11A shows huSIRPA expression levels in tumor-infiltrating huCD45+huCD14+ cells from mice administered an ip injection of either an isotype control, 3F9, or Keytruda (pembrolizumab, Merck). [Figure 12B] Shown are the percentages of huCD45+huCD14+ cells present in the peripheral blood (FIG. 12B, top panel) or within tumors (FIG. 12B, bottom panel) from mice administered an ip injection of either isotype control, 3F9, or Keytruda (pembrolizumab, Merck). [Figure 12C] This provides data showing that the percentage of human CD45+ cells in the blood of humanized mice is reduced after dosing with SIRPA antibody 3F9. Data are adjusted for donor, initial blood parameters (CD45, CD33, CD3), initial animal weight, and initial tumor volume. ***p<0.002 by multiple linear regression (Rlm() function) versus the control group (muIgG1). [Figure 13A]Figure 1 plots the mean tumor volume in NSG mice transplanted with human immune stem cells derived from various umbilical cord blood donors (donors 5031, 5048, and 129). Humanized mice were subcutaneously implanted with the human breast cancer cell line MDA-MB-231 and randomized to treatment or control groups based on day -1 tumor volume, huCD34+ stem cell donor, pre-randomization body weight, and pre-randomization huC45+ engraftment rate. Mice were dosed with either 40 mg / kg mouse IgG1 or 3F9 by i.p. injection every 4 days, or 10 mg / kg Keytruda every 5 days. The solid gray line represents the mean tumor volume in isotype control-treated mice, the solid black line represents the mean tumor volume in Keytruda-treated mice, and the dashed black line represents the mean tumor volume in 3F9-treated mice. [Figure 13B] The mean tumor volumes in humanized NSG mice from huCD34+ stem cell donors are plotted. The top panel of Figure 13B shows the mean tumor volumes from treated and control mice transplanted with stem cells from donors 5031 and 5048. The bottom panel of Figure 13B shows the mean tumor volumes from treated and control mice transplanted with stem cells from donor 129. The solid gray line represents the mean tumor volume of isotype control-treated mice, the solid black line represents the mean tumor volume of Keytruda-treated mice, and the dashed black line represents the mean tumor volume of 3F9-treated mice. [Figure 14A] Potential humanized sequences for the heavy chain variable domain of 3F9 are listed below. The humanized sequences are based on the IGHV3-23*01 acceptor framework and the IGHJ4*01 joining region. Figure 14A discloses SEQ ID NOs: 48 to 53, respectively, in order of appearance. [Figure 14B] Potential humanized sequences for the light chain variable domain of 3F9 are listed. The humanized sequences are based on the IGKV3-11*01 acceptor framework and the IGKJ2*01 connecting region. Figure 14B discloses SEQ ID NOs: 54-60, respectively, in order of appearance. [Figure 14C]Potential humanized sequences for the heavy chain variable domain of 9C2 are listed. The humanized sequences are based on the IGHV1-46*01 acceptor framework and the IGHJ4*01 binding region. Figure 14C discloses SEQ ID NOs: 61, 49, and 62-67, respectively, in order of appearance. [Figure 14D] Potential humanized sequences for the light chain variable domain of 9C2 are listed below. The humanized sequences are based on the IGKV3-11*01 acceptor framework and the IGKJ2*01 connecting region. Figure 14D discloses SEQ ID NOS: 68, 55, and 69-74, respectively, in order of appearance. CDR sequences are in bold. CDR definitions are AbM from the website www.bioinf.org.uk / abs / . "b" denotes buried side chains. "p" denotes partially buried. "i" denotes side chains at the interface between the VH and VL domains. Sequence differences between human and mouse germline are indicated with an asterisk (*). Potential additional mutations in the framework are noted below the sequences. Potential changes in the CDR sequences are noted below each CDR sequence. These may prevent asparagine (N) deamidation. [Figure 15A] Deglycosylation of 3F9 by treatment with EndoS (16A) was shown, and deglycosylation did not affect antigen recognition (16B). [Figure 15B] Deglycosylation of 3F9 by treatment with EndoS (16A) was shown, and deglycosylation did not affect antigen recognition (16B). [Figure 16] We provide data showing that both glycoforms of 3F9 significantly downregulated the surface expression of SIRPA compared with isotype control-treated macrophages, but the deglycosylated form showed partially reduced activity compared with the glycosylated form. [Figure 17A]Data are provided showing the surface expression levels of FcγRIIIA (Panel 18A, CD16) and FcγRIIA / B (Panel 18B, CD32A / B) in macrophages treated with control or 3F9 antibody. The antibody used to detect FcγRII for this analysis does not distinguish between activating receptors (FcγRIIA) and inhibitory receptors (FcγRIIB). [Figure 17B] Data are provided showing the surface expression levels of FcγRIIIA (Panel 18A, CD16) and FcγRIIA / B (Panel 18B, CD32A / B) in macrophages treated with control or 3F9 antibody. The antibody used to detect FcγRII for this analysis does not distinguish between activating receptors (FcγRIIA) and inhibitory receptors (FcγRIIB). [Figure 18] 1 provides data showing cell surface levels of FcγRIIA (left panel) and FcγRIIB (right panel) using receptor-specific antibodies in macrophages treated with glycosylated and deglycosylated forms of 3F9. DETAILED DESCRIPTION OF THE INVENTION

[0031] term As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "antibody" optionally includes combinations of two or more such molecules, and the like.

[0032] As used herein, the term "about" refers to a normal range of error for the respective value, which is readily known to one of ordinary skill in the art.

[0033] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, e.g., bispecific antibodies, so long as they exhibit the desired antigen-binding activity.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for, for example, naturally occurring mutations or variant antibodies that may arise during the production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0035] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules, such as scFv molecules; and multispecific antibodies formed from antibody fragments.

[0036] An antibody that "binds to the same epitope" or "has the same binding specificity" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An antibody that binds to the same epitope as the reference antibody may bind to the same epitope as the reference antibody, or may bind to a portion of the epitope. Exemplary competition assays are provided herein.

[0037] As used herein, "V region" refers to an antibody variable region domain comprising the segments framework 1, CDR1, framework 2, CDR2, and framework 3, including CDR3 and framework 4, which are added to the V segment as a result of rearrangement of heavy and light chain V region genes during B cell differentiation.

[0038] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions (HVRs) in each chain that interrupt the four "framework" regions established by the light and heavy chain variable regions. CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each chain are designated CDR1, CDR2, and CDR3, and are numbered consecutively starting from the N-terminus and are also identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. The term "CDR" may be used interchangeably with "HVR."

[0039] The amino acid sequences of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, Structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)).The definition of an antigen-binding site is also found in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol., 203, 121-153, (1991); Pedersen et al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, pp. 141-172 1996. References to CDRs determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0040] "Epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more commonly at least five, or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0041] "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, excluding the first constant region of a native immunoglobulin. This term refers to native and variant Fc regions. Thus, "Fc region" in the context of a native immunoglobulin typically refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, as well as the flexible hinge at the N-terminus of these domains. For IgA and IgM, the Fc may include the J chain. For IgG, the native Fc includes immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ. Although it is understood in the art that the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined as comprising residues C226 or P230 at its carboxyl terminus, using EU index numbering, such as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The C-terminal lysine of the Fc region (residue 447 according to the EU, or Kabat, numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, intact antibody compositions can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4. The term "Fc region" encompasses naturally occurring allelic variants of the Fc region, as well as modifications that modulate effector function. The Fc region also includes variants that do not result in a change in biological function, for example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function.

[0042] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. FcRs suitable for use in the present invention are typically native human FcRs or variants.

[0043] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0044] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide, for example, about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein preferably has at least about 80% identity, most preferably at least about 90% identity, and more preferably at least about 95% identity to the native-sequence Fc region and / or the Fc region of a parent polypeptide.

[0045] An "antagonist" or "inhibitory" antibody is an antibody, such as an anti-SIRPA antibody of the present disclosure, that inhibits or reduces (e.g., decreases) one or more activities or functions of an antigen after the antibody binds to the antigen. In some embodiments, an antagonist antibody may block the binding of one or more ligands to the antigen. In some embodiments, an antagonist or inhibitory antibody substantially or completely inhibits one or more activities or functions of the antigen and / or binding of the ligand to the antigen.

[0046] The term “equilibrium dissociation constant” (K D ) is the association rate constant (k a ,time-1 M -1 ) divided by the dissociation rate constant (k d ,time -1 ) The equilibrium dissociation constant can be measured using any method. Thus, in some embodiments, the antibodies of the disclosure have a K of less than about 50 nM, typically less than about 25 nM, or less than 10 nM, e.g., less than about 5 nM or less than about 1 nM, and often less than about 100 pM, as determined by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C. D In some embodiments, the antibodies of the present disclosure have a mAb content of 5 x 10 when measured as a bivalent antibody. -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 Below is M's K D In the context of the present invention, "improved" K D is lower than K D Refers to...

[0047] As used herein, the term "bivalent molecule" refers to a molecule having two antigen-binding sites. In some embodiments, a bivalent molecule of the present invention is a bivalent antibody or a bivalent fragment thereof. In some embodiments, a bivalent molecule of the present invention is a bivalent antibody. In some embodiments, a bivalent molecule of the present invention is an IgG. Generally, monoclonal antibodies have a bivalent basic structure. IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (2 and 5, respectively) and therefore have a higher valency. This bivalency increases the avidity of the antibody for the antigen.

[0048] As used herein, the terms "bivalent binding" or "bivalently bind" refer to the binding of both antigen-binding sites of a bivalent molecule to its antigen. Preferably, both antigen-binding sites of a bivalent molecule share the same antigen specificity.

[0049] As used herein, the term "valency" refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody contains one binding site for an antigen. A bivalent antibody contains two binding sites for the same antigen.

[0050] The phrases "specifically (or selectively) bind" or "specifically (or selectively) immunoreact with" an antigen or target, when referring to a protein or peptide, refer to a binding reaction in which an antibody binds to the antigen or target of interest. In the context of the present invention, an antibody typically has a K that is at least 100 times greater than its affinity for other antigens. D In some embodiments, the antibody binds to SIRPA with a K that is at least 100 times greater than its affinity for other antigens. D In some embodiments, the antibody binds to human SIRPA. Thus, as used herein, "specific binding" or "selective binding" does not necessarily require (although it can include) exclusive binding. An antibody that specifically binds to a target has a binding affinity of at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M-1 or 10 6 M -1 , and in other cases about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 ~10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 or higher. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0051] The anti-SIRPA antibodies of the present invention "downregulate" the level of SIRPA present on the cell surface of SIRPA-expressing cells. Thus, as used in this disclosure, "downregulation" refers to the ability of an antibody to reduce the level of SIRPA present on the cell surface of SIRPA-expressing cells, such as human macrophages. The anti-SIRPA antibodies of the present invention are considered to downregulate SIRPA if the level of SIRPA detected on the cell surface is reduced by at least 75%, at least 80%, at least 85%, or at least 90% compared to an isotype-matched control antibody.

[0052] An "isolated" antibody, such as an anti-SIRPa antibody of the present disclosure, is one that has been identified, separated, and / or recovered (e.g., naturally or recombinantly) from a component of its production environment. Preferably, an isolated polypeptide is free of association with all other contaminating components from its production environment. Contaminating components from its production environment, such as those arising from recombinantly transfected cells, are substances that would typically interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody, as determined, for example, by the Lowry method; (2) to an extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue, preferably silver staining. Since at least one component of the antibody's natural environment will not be present, an isolated antibody includes the antibody in situ within recombinant T cells. Ordinarily, however, isolated polypeptide or antibody will be prepared by at least one purification step.

[0053] The term "identical" or "percent identity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a comparison window or designated region, are the same or have a specified percentage of the same type of amino acid residues (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) over a specified region. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways, including using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). Thus, for purposes of the present invention, BLAST 2.0 can be used with the default parameters described to determine percent sequence identity for nucleic acid or polypeptide sequences.

[0054] Summary of Certain Aspects of the Invention The present disclosure relates to agents (e.g., anti-SIRPA antibodies) that inhibit the interaction between SIRPA and one or more SIRPA ligands; methods of making and using such agents (e.g., anti-SIRPA antibodies); pharmaceutical compositions comprising such agents (e.g., anti-SIRPA antibodies); nucleic acids encoding such agents (e.g., anti-SIRPA antibodies); and host cells comprising nucleic acids encoding such agents (e.g., anti-SIRPA antibodies).

[0055] Agents of the present disclosure that reduce cellular levels of SIRPA and / or inhibit the interaction between SIRPA and one or more SIRPA ligands are molecules that have one or more of the following characteristics: (1) inhibit or reduce one or more SIRPA activities; (2) the ability to inhibit or reduce the binding of SIRPA to one or more of its ligands; (3) the ability to reduce SIRPA expression (such as at the mRNA level and / or protein level) in SIRPA-expressing cells; (4) the ability to interact with, bind to, or recognize SIRPA protein; (5) the ability to specifically interact with or bind to SIRPA protein; and (6) the ability to treat, ameliorate, or prevent any aspect of a disease or disorder described or contemplated herein.

[0056] Exemplary agents that inhibit the production of SIRPA include, but are not limited to, compounds that specifically inhibit SIRPA synthesis and / or release, antisense molecules directed against SIRPA, or small interfering RNA (siRNA) molecules directed against nucleic acids encoding SIRPA. Further exemplary agents that inhibit one or more SIRPA activities include, but are not limited to, anti-SIRPA antibodies that specifically bind to SIRPA proteins, compounds that specifically inhibit one or more SIRPA activities, such as small molecule inhibitors and / or peptide inhibitors, compounds that specifically inhibit SIRPA by binding to one or more ligands, SIRPA structural analogs, or RNA or DNA aptamers that bind to SIRPA. In some embodiments, an agent that reduces the cellular level of SIRPA and / or inhibits the interaction between SIRPA and one or more SIRPA ligands is an allosteric inhibitor. In some embodiments, an agent that reduces the cellular level of SIRPA and / or inhibits the interaction between SIRPA and one or more SIRPA ligands is an orthosteric inhibitor.

[0057] In certain embodiments, agents that reduce cellular levels of SIRPA and / or inhibit the interaction between SIRPA and one or more SIRPA ligands are small molecule inhibitors, including, but not limited to, small peptides or peptide-like molecules, soluble peptides, and synthetic non-peptidyl organic or inorganic compounds. Small molecule inhibitors can have a molecular weight of about 100 to about 20,000 daltons (Da), about 500 to about 15,000 Da, or about 1,000 to about 10,000 Da. Methods for making and testing the inhibitory effect of small molecules on one or more SIRPA activities are well known in the art, and such methods can be used to evaluate the effect of small molecule inhibitors on SIRPA activity. For example, any of the methods and assays disclosed herein can be used to screen for small molecule inhibitors that reduce cellular levels of SIRPA and / or inhibit the interaction between SIRPA and one or more SIRPA ligands.

[0058] In certain embodiments, an agent that reduces the cellular level of SIRPA and / or inhibits the interaction between SIRPA and one or more SIRPA ligands comprises at least one antisense molecule that can block or reduce the expression of functional SIRPA by targeting a nucleic acid encoding SIRPA. The nucleic acid sequence of SIRPA is known in the art. For example, human SIRPA can have the nucleic acid sequence set forth in NCBI accession number NM_080792 or Y10375.1, and mouse SIRPA can have the nucleic acid sequence set forth in NCBI accession number BC062197. Methods for preparing antisense oligonucleotide molecules are known, and such methods can be used to prepare antisense oligonucleotides that specifically bind to one or more SIRPA mRNAs without cross-reacting with other polynucleotides. Exemplary targeting sites include, but are not limited to, the initiation codon, the 5' regulatory region, the coding sequence including any conserved consensus regions, and the 3' untranslated region. In certain embodiments, antisense oligonucleotides are about 10 to about 100 nucleotides in length, about 15 to about 50 nucleotides in length, about 18 to about 25 nucleotides in length, or longer. In certain embodiments, the oligonucleotides further comprise chemical modifications to increase nuclease resistance, etc., such as phosphorothioate linkages and 2'-O-sugar modifications, which are known to those of skill in the art.

[0059] In certain embodiments, the agent that reduces the cellular level of SIRPA and / or inhibits the interaction between SIRPA and one or more SIRPA ligands comprises at least one siRNA molecule that can block or reduce the expression of functional SIRPA by targeting a nucleic acid encoding SIRPA. Methods for preparing siRNA molecules are well known in the art, and such methods can be used to prepare siRNA molecules that specifically target SIRPA mRNA without cross-reacting with other polynucleotides. siRNA molecules can be produced by methods such as typical solid-phase oligonucleotide synthesis, and often incorporate chemical modifications to increase the half-life and / or efficacy of the siRNA agent and / or enable a more robust delivery formulation. Alternatively, siRNA molecules can be delivered using a vector encoding an expression cassette for intracellular transcription of the siRNA.

[0060] In certain embodiments, the agent that reduces the cellular levels of SIRPA and / or inhibits the interaction between SIRPA and one or more SIRPA ligands is an RNA or DNA aptamer that binds to or physically interacts with SIRPA and blocks the interaction of SIRPA with one or more of its ligands. In certain embodiments, the aptamer comprises at least one RNA or DNA aptamer that binds to the mature form of SIRPA.

[0061] In certain embodiments, an agent that reduces cellular levels of SIRPA and / or inhibits the interaction between SIRPA and one or more SIRPA ligands comprises at least one Siglec-9 structural analog. The term "SIRPA structural analog" refers to a compound that has a three-dimensional structure similar to that of a portion of SIRPA and binds to one or more CD3 ligands under physiological conditions in vitro or in vivo, where the binding at least partially inhibits the biological activity of SIRPA. Suitable SIRPA structural analogs can be designed and synthesized through molecular modeling of SIRPA binding to a ligand, such as the SIRPA ligands disclosed herein. SIRPA structural analogs can be monomers, dimers, or higher-order multimers in any desired combination of identical or different structures to achieve improved affinity and biological effect. In some embodiments, the agent binds to or interacts with the amino acid sequence of SIRPA.

[0062] In certain embodiments, agents that reduce cellular levels of SIRPA and / or inhibit the interaction between SIRPA and one or more SIRPA ligands include soluble SIRPA receptor proteins, soluble SIRPA-Fc fusion proteins, etc. In certain embodiments, such agents bind to one or more SIRPA ligands, thereby preventing the interaction between the SIRPA ligands and the SIRPA receptor.

[0063] Assay Agents that reduce the cellular levels of SIRPA and / or inhibit the interaction between SIRPA and one or more SIRPA ligands can be identified and / or characterized using methods well known in the art, for example, using radiolabeled inhibitor assays, optical assays, protein binding assays, biochemical screening assays, immunoassays, mass shift measurement assays, fluorescence assays, and / or fluorescent peptide cleavage assays.

[0064] Binding and Other Assays In certain embodiments, agents that reduce the cellular levels of SIRPA and / or inhibit the interaction between SIRPA and one or more SIRPA ligands can be identified by techniques well known in the art for detecting the presence of an interaction and / or the binding affinity of a SIRPA agent candidate to SIRPA.

[0065] In certain embodiments, drugs that interact with SIRPA can be identified using a radiolabeled inhibitor assay. For example, a known amount of a radiolabeled drug candidate can be incubated with a known amount of immobilized SIRPA and a buffer solution. The immobilized SIRPA can then be washed with a buffer solution, and the immobilized SIRPA can be measured for the remaining radiolabeled SIRPA drug candidate using techniques known in the art, such as a gamma counter. A measurement indicating the presence of a radiolabeled substance can indicate that the radiolabeled drug candidate can interact with and / or bind to SIRPA.

[0066] In certain embodiments, drugs that interact with SIRPA can be identified using optical techniques. An exemplary optical technique for detecting SIRPA-interacting agents can include, for example, attaching SIRPA to a colorimetric resonant graft surface, thereby shifting the wavelength of reflected light due to a change in the optical path that the light must take, and then measuring an additional change in the wavelength of reflected light when a candidate drug is allowed to interact with SIRPA. For example, no change in the wavelength of the measured reflected light when a drug is incubated with SIRPA can indicate that the drug candidate is unable to interact with SIRPA. A change in the wavelength of the measured reflected light when a drug candidate is incubated with SIRPA can indicate that the drug candidate can bind to and / or interact with SIRPA.

[0067] In certain embodiments, drugs that interact with SIRPA can be identified using protein binding assays. An exemplary protein binding assay for detecting SIRPA binders can include, for example, co-immunoprecipitation of SIRPA in the presence of a drug candidate. For example, SIRPA can be incubated with a drug candidate in a buffer, and then SIRPA can be captured in the presence of the drug candidate using an immobilized molecule specific for capturing SIRPA, such as an anti-SIRPA antibody, and potentially bind to SIRPA with the potential interacting agent during a washing procedure known in the art. SIRPA can then be released together with the potential interacting agent, and the presence of the drug candidate can be detected based on the characteristics of the drug candidate, for example, by techniques such as mass spectrometry and / or Western blotting.

[0068] In certain embodiments, drugs that interact with SIRPA can be identified using biochemical and / or immunoassay assays well known in the art. Exemplary techniques can include assays for quantitatively measuring changes in SIRPA concentration and / or protein half-life using techniques such as Western blot, immunostaining, and co-immunoprecipitation. For example, a drug candidate can be incubated with a sample containing SIRPA, such as cells expressing SIRPA, and then the amount and / or cellular level of SIRPA protein can be measured at time points during a time course study. Changes in protein amount, cellular level, and / or protein half-life compared to a control treatment can indicate that the SIRPA drug candidate may be able to alter SIRPA half-life and / or activity.

[0069] In certain embodiments, mass shift measurement assays can be used to identify drugs that interact with SIRPA. An exemplary mass shift measurement assay can include detecting the presence of a strongly and / or covalently bound SIRPA agent by measuring a change in SIRPA mass when the drug candidate interacts with SIRPA, for example, by using an instrument such as, but not limited to, a mass spectrometer. For example, mass shift assays can be performed in whole protein and / or peptide-based assays, depending on the nature of the drug candidate interaction. Detection of a mass shift that correlates with the addition of the drug candidate to SIRPA can indicate that the drug candidate can interact with or otherwise inhibit SIRPA. Furthermore, an exemplary mass shift measurement assay can include detecting the addition of a mass to SIRPA that correlates with the respective drug candidate mass when the drug candidate interacts with SIRPA, using a technique such as surface plasmon resonance. For example, changes in the refractive index of light can be measured and correlated with changes in the mass of SIRPA attached to the sensor surface.

[0070] In certain embodiments, chemical cross-linking assay can be used to identify SIRPA agents that interact with SIRPA.For example, drug candidates can be incubated with SIRPA in vivo or in vitro together with molecular cross-linking agents that can covalently link SIRPA-interacting drug candidates to the SIRPA molecule.Then, techniques such as but not limited to mass spectrometry and / or Western blotting can be used to identify drug candidates that can interact with SIRPA or otherwise inhibit SIRPA.For example, detecting SIRPA that is covalently cross-linked with drug candidates can indicate that drug candidates can interact with SIRPA or otherwise inhibit SIRPA.

[0071] In certain embodiments, agents that interact with SIRPA can be identified using a fluorescence assay. For example, a known amount of a fluorescent agent candidate can be incubated with a known amount of immobilized SIRPA and a buffer solution. The immobilized SIRPA can then be washed with the buffer solution, and the immobilized SIRPA can be measured for the remaining fluorescent SIRPA agent candidate using techniques known in the art, such as, but not limited to, fluorescence detection. A measurement indicating the presence of a fluorescent substance can indicate that the fluorescent agent candidate can interact with and / or bind to SIRPA.

[0072] Assays known in the art and described herein (e.g., Examples 2-11) can be used to identify and test the biological activity of SIRPA agents of the disclosure. In some embodiments, assays are provided that test the ability of a SIRPA agent to modulate one or more Siglec-9 activities.

[0073] Anti-SIRP-alpha (SIRPA) antibodies BRIEF SUMMARY OF CERTAIN ANTI-SIRPA ANTIBODY EMBODIMENTS OF THE DISCLOSURE In some embodiments, the anti-SIRPA antibodies of the present disclosure have one or more antagonistic activities due, at least in part, to the antibody's ability to downregulate cellular SIRPA. In some embodiments, the isolated SIRPA antibodies of the present disclosure selectively bind to SIRPA and downregulate SIRPA. In some embodiments, the antibody does not block the binding of a SIRPA ligand, such as CD47, to SIRPA expressed on cells. In alternative embodiments, the antibody blocks the binding of a SIRPA ligand, such as CD47, to SIRPA. In some embodiments, the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody. Exemplary descriptions of such antibodies are found throughout this disclosure. In some embodiments, the antibody is a bispecific antibody that recognizes a first antigen and a second antigen.

[0074] In some embodiments, anti-SIRPA antibodies of the present disclosure selectively bind to human SIRPA, e.g., human allelic variants, but not to SIRPB, although these are also referred to herein as "polymorphic" variants. Figure 1A shows an amino acid sequence alignment between the two most common alleles of the human SIRPA protein (v1 and v2, respectively, with accession numbers NP542970 and CAA71403, respectively), noting the different residues within the ligand-binding domain. Thus, in some embodiments, anti-SIRPA antibodies of the present disclosure bind to a linear or conformational epitope present in allelic variants of human SIRPA but absent in SIRPB or mouse SIRPA. Figure 1B shows an amino acid sequence alignment between the human SIRPAv1 protein and the human SIRPB1 protein, with accession numbers NP542970 and O00241, respectively, noting the homology between the two proteins. Figure 2 shows an amino acid sequence alignment between human and mouse SIRPA proteins, accession numbers NP542970 and Q6P6I8, respectively, noting the homology between the two proteins. In some embodiments, the antibodies of the present disclosure selectively bind to human and mouse SIRPA and do not bind to SIRPB.

[0075] SIRPA is a type I single-pass transmembrane protein. In the amino acid sequence of human SIRPA (SEQ ID NO: 1), the extracellular domain is located at amino acid residues 31 to 373; the transmembrane domain is located at amino acid residues 374 to 394; and the intracellular domain is located at amino acid residues 395 to 504.

[0076] Human SIRPA contains one V set and two C1 sets of Ig superfamily (IgSF) domains, designated the D1, D2, and D3 domains, respectively. The D1 domain contains amino acid residues 32-137 of human SIRPA; the D2 domain contains amino acid residues 148-247 of human SIRPA; and the D3 domain contains amino acid residues 254-348 of human SIRPA.

[0077] In some embodiments, anti-SIRPA antibodies of the present disclosure bind to the D1 domain of SIRPA. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to the D1 domain of human SIRPA comprising amino acid residues 32-137 of the human SIRPA amino acid sequence of SEQ ID NO: 1. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to an epitope within the D1 domain of human SIRPA. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to an epitope within the D1 domain of human SIRPA, wherein the epitope comprises an amino acid sequence selected from the group consisting of amino acid residues 32-137, amino acid residues 32-52, amino acid residues 55-121, amino acid residues 58-73, amino acid residues 68-83, amino acid residues 78-93, amino acid residues 88-103, amino acid residues 98-113, amino acid residues 108-123, and amino acid residues 118-133 of the human SIRPA amino acid sequence of SEQ ID NO: 1.

[0078] In some embodiments, anti-SIRPA antibodies of the present disclosure bind to the D2 domain of SIRPA. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to the D2 domain of human SIRPA comprising amino acid residues 148-247 of the human SIRPA amino acid sequence of SEQ ID NO: 1. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to an epitope within the D2 domain of human SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to an epitope within the D2 domain of human SIRPA, wherein the epitope comprises an amino acid sequence selected from the group consisting of amino acid residues 148-247, amino acid residues 148-168, amino acid residues 158-173, amino acid residues 168-183, amino acid residues 170-228, amino acid residues 178-193, amino acid residues 188-203, amino acid residues 198-213, amino acid residues 208-223, amino acid residues 218-233, and amino acid residues 228-243 of the human SIRPA amino acid sequence of SEQ ID NO: 1.

[0079] In some embodiments, anti-SIRPA antibodies of the present disclosure bind to the D3 domain of SIRPA. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to the D3 domain of human SIRPA comprising amino acid residues 254-348 of the human SIRPA amino acid sequence of SEQ ID NO: 1. In some embodiments, anti-SIRPA antibodies of the present disclosure bind to an epitope within the D3 domain of human SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to an epitope within the D3 domain of human SIRPA, wherein the epitope comprises an amino acid sequence selected from the group consisting of amino acid residues 254-348, amino acid residues 254-274, amino acid residues 264-279, amino acid residues 274-289, amino acid residues 273-331, amino acid residues 281-315, amino acid residues 281-337, amino acid residues 284-299, amino acid residues 294-309, amino acid residues 304-319, amino acid residues 314-329, amino acid residues 324-339, and amino acid residues 334-348 of the human SIRPA amino acid sequence of SEQ ID NO: 1.

[0080] In some embodiments, the antibody binds to the D1 domain of SIRPA, e.g., human SIRPA. In some embodiments, the antibody binds to the D2 domain of SIRPA, e.g., human SIRPA. In some aspects, the antibody binds to the D3 domain of SIRPA, e.g., human SIRPA. In some embodiments, the anti-SIRPA antibody of the present disclosure binds to the same SIRPA epitope or a portion of the SIRPA epitope bound by an antibody having the CDRs of the antibody designated 3F9 in Table 2. In some embodiments, the anti-SIRPA antibody of the present disclosure competes with 3F9 for binding to SIRPA and binds to all of the same portion of the epitope as 3F9. In some embodiments, the antibody binds to the same SIRPA epitope or a portion of the SIRPA epitope bound by an antibody having the CDRs of the antibody designated 9C2 in Table 2. Thus, in some embodiments, an antibody of the present disclosure binds to the same SIRPA epitope or portion of a SIRPA epitope bound by an antibody having the CDRs of the antibody designated 3F9 in Table 2, and binds to the same SIRPA epitope or portion of a SIRPA epitope bound by an antibody having the CDRs of the antibody designated 9C2 in Table 2.

[0081] In some embodiments, the anti-SIRPA antibodies of the disclosure compete with 3F9 and 9C2 for binding to human SIRPA.

[0082] In a preferred embodiment, the antibody of each of the preceding three paragraphs does not block the binding of CD47 to SIRPA.

[0083] SIRPA downregulation Certain aspects of the present disclosure relate to anti-SIRPA antibodies that downregulate, i.e., reduce, cellular levels of SIRPA. In some embodiments, the anti-SIRPA antibody reduces cellular levels of SIRPA without inhibiting the interaction (e.g., binding) between SIRPA and one or more SIRPA ligands (e.g., CD47). In some embodiments, the anti-SIRPA antibody reduces cellular levels of SIRPA and inhibits the interaction (e.g., binding) between SIRPA and one or more SIRPA ligands (e.g., CD47).

[0084] The cellular level of SIRPA refers to, but is not limited to, the cell surface level of SIRPA, the intracellular level of SIRPA, and the total level of SIRPA. In some embodiments, a decrease in the cellular level of SIRPA includes a decrease in the cell surface level of SIRPA. As used herein, an anti-SIRPA antibody decreases the cell surface level of SIRPA if it induces a 25% or greater decrease in the cell surface level of SIRPA, as measured by any in vitro cell-based assay described herein or known in the art or a suitable in vivo model, for example, by measuring the cell surface level of SIRPA using flow cytometry, such as fluorescence-activated cell sorting (FACS). In some embodiments, a decrease in the cellular level of SIRPA includes a decrease in the intracellular level of SIRPA. As used herein, an anti-SIRPA antibody reduces the intracellular level of Siglec-9 if it induces a 25% or greater decrease in the intracellular level of SIRPA as measured by any in vitro cell-based assay or suitable in vivo model described herein or known in the art, for example, by immunostaining, Western blot analysis, co-immunoprecipitation, and cell cytometry. In some embodiments, a decrease in the intracellular level of SIRPA includes a decrease in the total level of SIRPA. As used herein, an anti-SIRPA antibody reduces the total level of SIRPA if it induces a 25% or greater decrease in the total level of SIRPA as measured by any in vitro cell-based assay or suitable in vivo model described herein or known in the art, for example, by immunostaining, Western blot analysis, co-immunoprecipitation, and cell cytometry. In some embodiments, an anti-SIRPA antibody induces SIRPA degradation, SIRPA cleavage, SIRPA internalization, SIRPA shedding, downregulation of SIRPA expression, or any combination thereof. In some embodiments, cellular levels of SIRPA are measured in primary cells (eg, dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, and macrophages) or cell lines using a SIRPA cellular assay.

[0085] In some embodiments, downregulation of anti-SIRPA antibodies is demonstrated by an IC of 200 nM or less, typically 100 nM or less (50% of cell surface expressed SIRPA is downregulated) after 4 hours of exposure of human macrophages to the antibody at 37°C. 50 In some embodiments, SIRPA remains downregulated for at least 24 hours of exposure to an antibody of the invention. Cells can be analyzed for SIRPA surface expression using any technique, for example, flow cytometry.

[0086] In some embodiments, the anti-SIRPA antibodies of the disclosure increase cellular levels of SIRPA by at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, or at least 59%, compared to cellular levels of SIRPA in the absence of the anti-SIRPA antibody. at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more.

[0087] In some embodiments, which may be combined with any of the down-regulating activities summarized in the previous paragraph, the anti-SIRPA antibodies of the present disclosure inhibit cell surface clustering of SIRPA.

[0088] In some embodiments, the anti-SIRPA antibodies of the present disclosure downregulate SIRPA but do not block the binding of a SIRPA ligand, e.g., CD47, to SIRPA. In the context of the present invention, an antibody that does not block the binding of CD47 to SIRPA refers to an antibody that does not result in a significant reduction in CD47 binding to SIRPA when the antibody is incubated with CD47 and cells expressing SIRPA. A "significant reduction" in the context of CD47 binding to SIRPA refers to a reduction of 30% or less, typically at least 25%, at least 20%, at least 15%, or at least 10% or less, compared to CD47 binding to SIRPA in the presence of an isotype-matched control antibody that does not bind to SIRPA. Exemplary assays for assessing blocking activity are described in the Examples. For example, cells expressing human SIRPA, e.g., human macrophages, such as CHO cells modified to express human SIRPA, are cultured in a 96-well plate at 10 5 Cells were seeded per well, washed, and incubated in 100 μl of fluorescence-activated cell sorting buffer containing 1.0 μg / ml of monoclonal antibody or isotype control. Cells were then washed and incubated with soluble human CD47 for 30 minutes on ice. Cells were then analyzed for surface-bound CD47.

[0089] Alternatively, in some embodiments, the anti-SIRPA antibodies of the present disclosure downregulate SIRPA but block CD47 binding to SIRPA. Antibodies that block CD47 binding typically block CD47 binding by 50% or more, typically 75%, or 90% or more.

[0090] Inhibition of SIRPA activity In some embodiments, the anti-SIRPA antibodies of the present disclosure inhibit one or more activities of SIRPA, including but not limited to: SIRPA binding to one or more SIRPA ligands, optionally wherein the one or more SIRPA ligands are selected from the group consisting of CD47, surfactant proteins A and D, and any combination thereof; SIRPA binding to dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 Reducing the proliferation of one or more cells selected from the group consisting of NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia; inhibiting the migration of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia; inhibiting the migration of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 inhibiting one or more functions of one or more cells selected from the group consisting of NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia;Inhibition of the removal of one or more selected from the group consisting of removal of apoptotic neurons, removal of neural tissue debris, removal of dysfunctional synapses, removal of non-neural tissue debris, removal of bacteria, removal of other foreign bodies, removal of disease-causing proteins, removal of disease-causing peptides, and removal of tumor cells, optionally wherein the disease-causing proteins are amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9or f72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, The protein is selected from the group consisting of cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine (PA) repeat peptide, ubiquitin, and proline-arginine (PR) repeat peptide, and the tumor cell is selected from the group consisting of bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lung cancer, inhibiting tumor cell killing by one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; inhibiting the anti-tumor cell proliferation activity of one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells;modulating the expression of one or more inflammatory receptors, optionally the one or more inflammatory receptors include CD86, and the one or more inflammatory receptors are expressed on one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, myeloid-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; promoting or rescuing the functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and regulatory T cells; promoting or rescuing the functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and regulatory T cells; increasing the number of tumor-promoting myeloid / granulocytic immune suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells in tumors, peripheral blood, or other lymphoid organs; enhancing the tumor-promoting activity of myeloid-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; enhancing the survival of non-tumorigenic myeloid-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; decreasing the activation of tumor-killing tumor-specific T lymphocytes; decreasing the infiltration of tumor-killing tumor-specific NK cells; increasing tumor volume; increasing tumor growth rate;and reducing the effectiveness of one or more immunotherapies that modulate anti-tumor T cell responses, optionally wherein the one or more immunotherapies are immunotherapies targeting one or more target proteins selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and any combination thereof, or one or more cancer vaccines;

[0091] In some embodiments that may be combined with any of the other embodiments described above, the anti-SIRPA antibodies of the present disclosure induce one or more activities selected from the group consisting of: increasing the number of tumor-infiltrating CD3+ T cells; decreasing cellular levels of SIRPA in non-tumorigenic CD14+ myeloid cells, where optionally the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells or, optionally, the non-tumorigenic CD14+ myeloid cells are present in the blood; decreasing the number of non-tumorigenic CD14+ myeloid cells. reducing non-tumorigenic CD14+ myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally wherein the non-tumorigenic CD14+ myeloid cells are present in the blood; reducing PD-L1 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); reducing PD-L2 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC). reducing B7-H2 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); reducing B7-H3 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); reducing CD200R levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC). reducing CD163 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); reducing CD206 levels in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSC); reducing tumor growth rate of solid tumors; reducing tumor volume; increasing the efficacy of one or more PD-1 inhibitors;increasing the effectiveness of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies, optionally the one or more checkpoint inhibitor therapies and / or immunomodulatory therapies targeting one or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; increasing the effectiveness of one or more chemotherapeutic agents, optionally the one or more chemotherapeutic agents being gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®), increasing the proliferation of T cells in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs); inhibiting the differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs); and killing CD33-expressing immune suppressor non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells in solid tumors and associated vasculature when conjugated with chemical or radiotoxins.

[0092] In some embodiments, the anti-SIRPA antibodies of the present disclosure decrease the activity, functionality, or viability of regulatory T cells, tumor-enclosed immunosuppressor dendritic cells, tumor-enclosed immunosuppressor macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, or chronic myeloid leukemia (CML).

[0093] In some embodiments, the anti-SIRPA antibodies of the disclosure induce or promote the survival, maturation, functionality, migration, or proliferation of one or more immune cells, for example, the one or more immune cells in the individual are selected from the group consisting of dendritic cells, macrophages, neutrophils, NK cells, microglia, T cells, T helper cells, cytotoxic T cells, and any combination thereof.

[0094] As used herein, the level of SIRPA can refer to the expression level of the gene encoding SIRPA; the expression level of one or more transcripts encoding SIRPA; the expression level of SIRPA protein; and / or the amount of SIRPA protein present in a cell and / or on the cell surface. Any method known in the art for measuring levels of gene expression, transcription, translation, and / or protein abundance or localization can be used to determine the level of SIRPA.

[0095] In some embodiments, the isolated anti-SIRPA antibody of the present disclosure is a murine antibody. In some embodiments, the isolated anti-SIRPA antibody of the present disclosure is a human antibody, a humanized antibody, a bispecific antibody, a monoclonal antibody, a multivalent antibody, or a chimeric antibody. Exemplary descriptions of such antibodies are found throughout this disclosure.

[0096] In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to human SIRPA, including human allelic variants ( FIG. 1A , Accession Nos. NP542970 and CAA71403). In some embodiments, the anti-SIRPA antibodies specifically bind to primate SIRPA, including human SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure specifically bind to both human SIRPA and primate SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure specifically bind to human SIRPA and cross-react with mouse SIRPA.

[0097] HVR sequences of SIRPA-downregulating antibodies that do not block CD47 binding In some embodiments, the anti-SIRPA antibodies of the present disclosure downregulate SIRPA and do not block CD47 binding to SIRPA. In some embodiments, such antibodies comprise a heavy chain variable region comprising HVR3 of antibody 3F9 set forth in SEQ ID NO: 11. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 11 with 1, 2, 3, 4, or 5 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 11 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 11 with 1, 2, or 3 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 has 1 or 2 amino acid substitutions compared to the sequence set forth in SEQ ID NO: 11. In some embodiments, 1 or 2 amino acids are deleted relative to SEQ ID NO: 11. In some embodiments, HVR3 has at least 65% identity or at least 75% identity to the amino acid sequence of SEQ ID NO:11.

[0098] In some embodiments, the heavy chain variable region of an anti-SRPA antibody of the invention comprises HVR3 described in the previous paragraph and HVR1 and / or HVR2 of antibody 3F9 described in Table 3. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 9 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 9 with 1, 2, or 3 amino acid substitutions, or with 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 has at least 70%, at least 80%, or at least 90% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, HVR2 comprises the sequence of SEQ ID NO: 10 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR2 comprises the sequence of SEQ ID NO: 10 with 1, 2, or 3 amino acid substitutions, or with 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR2 has at least 70%, at least 80%, or at least 90% identity to the amino acid sequence of SEQ ID NO:10.

[0099] In some embodiments, the anti-SIRPA antibody comprises a heavy chain variable region comprising HVR3 of SEQ ID NO:11, HVR1 of SEQ ID NO:9, and HVR2 of SEQ ID NO:10.

[0100] In some embodiments, the anti-SIRPA antibody comprises a light chain variable region comprising HVR3 of antibody 3F9 described in Table 2. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO:8 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO:8 with 1, 2, or 3 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 has 1 or 2 amino acid substitutions compared to the sequence set forth in SEQ ID NO:8. In some embodiments, 1 or 2 amino acids have been deleted relative to SEQ ID NO:8. In some embodiments, HVR3 has at least 65% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, HVR3 has at least 85% identity to the amino acid sequence of SEQ ID NO:8.

[0101] In some embodiments, the light chain variable region of an anti-SRPA antibody of the invention comprises HVR3 described in the previous paragraph and HVR1 and / or HVR2 of antibody 3F9 described in Table 2. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 6 with 1, 2, 3, 4, 5, or 6, or 1, 2, 3, 4, or 5 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 6 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 6 with 1, 2, or 3 amino acids, or with 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 has at least 70%, at least 80%, or at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, HVR2 comprises the sequence of SEQ ID NO: 7 with 1, 2, or 3 amino acids; or with 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR2 has at least 70%, at least 85% identity to the amino acid sequence of SEQ ID NO:7.

[0102] In some embodiments, the anti-SIRPA antibody comprises a light chain variable region having an HVR3 of SEQ ID NO:8, an HVR1 of SEQ ID NO:6, and an HVR2 of SEQ ID NO:7.

[0103] In some embodiments, an anti-SIRPA antibody of the disclosure comprises a heavy chain variable region comprising HVR3, HVR2, and HVR1 of antibody 3F9 as described in Table 3, and a light chain variable region comprising HVR3, HVR2, and HVR1 of antibody 3F9 as described in Table 2. In some embodiments, an anti-SIRPA antibody comprises the six CDRs of 3F9, and at least one HVR differs from the HVR of 3F9 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVR of 3F9. In some embodiments, such an antibody comprises two HVRs that differ from the corresponding HVR of 3F9 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVR of 3F9. In some embodiments, an antibody comprises three HVRs that differ from the corresponding HVR of 3F9 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVR of 3F9. In some embodiments, the antibody comprises four HVRs that differ from the corresponding HVRs of 3F9 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVRs of 3F9. In some embodiments, the antibody comprises five HVRs that differ from the corresponding HVRs of 3F9 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVRs of 3F9. In some embodiments, the antibody comprises one, two, or three amino acid changes, or one or two amino acid changes in each HVR compared to the corresponding HVRs of 3F9.

[0104] In some embodiments, the anti-SIRPA antibody comprises a heavy chain variable region comprising HVR3 of antibody 9C2 set forth in SEQ ID NO: 17. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 17 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 7 with 1, 2, or 3 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 has 1 or 2 amino acid substitutions compared to the sequence set forth in SEQ ID NO: 17. In some embodiments, 1 or 2 amino acids are deleted relative to SEQ ID NO: 17. In some embodiments, HVR3 has at least 65% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, HVR3 has at least 85% identity to the amino acid sequence of SEQ ID NO: 17.

[0105] In some embodiments, the heavy chain variable region of an anti-SRPA antibody of the invention comprises HVR3 described in the previous paragraph and HVR1 and / or HVR2 of antibody 9C2 described in Table 3. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 15 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 15 with 1, 2, or 3 amino acids, or with 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 has at least 70%, at least 80%, or at least 90% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, HVR2 comprises the sequence of SEQ ID NO: 16 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR2 comprises the sequence of SEQ ID NO: 16 with 1, 2, or 3 amino acids, or with 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, the HVR has at least 70%, at least 80%, or at least 90% identity to the amino acid sequence of SEQ ID NO:16.

[0106] In some embodiments, the anti-SIRPA antibody comprises a heavy chain variable region comprising HVR3 of SEQ ID NO:17, HVR1 of SEQ ID NO:15, and HVR2 of SEQ ID NO:16.

[0107] In some embodiments, the anti-SIRPA antibody comprises a light chain variable region comprising HVR3 of antibody 9C2 described in Table 2. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 14 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 comprises the sequence set forth in SEQ ID NO: 4 with 1, 2, or 3 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR3 has 1 or 2 amino acid substitutions compared to the sequence set forth in SEQ ID NO: 14. In some embodiments, 1 or 2 amino acids are deleted relative to SEQ ID NO: 14. In some embodiments, HVR3 has at least 65% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, HVR3 has at least 85% identity to the amino acid sequence of SEQ ID NO: 14.

[0108] In some embodiments, the light chain variable region of an anti-SIRPA antibody of the invention comprises HVR3 described in the previous paragraph and HVR1 and / or HVR2 of antibody 9C2 described in Table 2. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 12 with 1, 2, 3, or 4 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 comprises the sequence of SEQ ID NO: 12 with 1, 2, or 3 amino acid substitutions, or 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR1 has at least 70% identity, at least 80% identity, or at least 90% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, HVR2 comprises the sequence of SEQ ID NO: 13 with 1, 2, or 3 amino acid substitutions, or 1 or 2 amino acid substitutions, e.g., conservative substitutions. In some embodiments, HVR2 has at least 70%, at least 85% identity to the amino acid sequence of SEQ ID NO: 13.

[0109] In some embodiments, the anti-SIRPA antibody comprises a light chain variable region having an HVR3 of SEQ ID NO:14, an HVR1 of SEQ ID NO:12, and an HVR2 of SEQ ID NO:13.

[0110] In some embodiments, an anti-SIRPA antibody of the disclosure comprises a heavy chain variable region comprising HVR3, HVR2, and HVR1 of antibody 9C2 as set forth in Table 3, and a light chain variable region comprising HVR3, HVR2, and HVR1 of antibody 9C2 as set forth in Table 2. In some embodiments, an anti-SIRPA antibody comprises at least one HVR that differs from the corresponding HVR of 9C2 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVR of 9C2. In some embodiments, such an antibody comprises two HVRs that differ from the corresponding HVR of 9C2 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVR of 9C2. In some embodiments, an antibody comprises three HVRs that differ from the corresponding HVR of 9C2 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVR of 9C2. In some embodiments, the antibody comprises four HVRs that differ from the corresponding HVRs of 9C2 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVRs of 3F9. In some embodiments, the antibody comprises five HVRs that differ from the corresponding HVRs of 9C2 by one, two, or three amino acids, or one or two amino acids, compared to the corresponding HVRs of 9C2. In some embodiments, the antibody comprises one, two, or three amino acid changes, or one or two amino acid changes, in each HVR compared to the corresponding HVRs of 9C2.

[0111] In some embodiments, the N residue present in light chain CDR2 from 3F9 (SEQ ID NO: 7) may be substituted with Q, S, A, or D. In some embodiments, the N residue in light chain CDR3 from 3F9 (SEQ ID NO: 8) of Table 2 may be substituted with Q, S, A, or D. In some cases, the N residue in both light chain CDR2 and CDR3 is substituted with Q, S, A, or D. In some embodiments, the C in light chain CDR3 from 3F9 (SEQ ID NO: 8) may be substituted with A, S, or L.

[0112] In some embodiments, the N residue present in the heavy chain CDR1 from 9C2 (SEQ ID NO: 15) may be substituted with Q, S, or A. In some embodiments, one or both N residues present in the heavy chain CDR2 from 9C2 (SEQ ID NO: 16) may be substituted with Q, S, or A. In some embodiments, the D of the heavy chain CDR3 residues Asp-Gly (DG) of SEQ ID NO: 17 may be substituted with A, S, or E. In some embodiments, the N residue in the light chain CDR2 from 9C2 (SEQ ID NO: 13) may be substituted with Q, S, D, or A. In some embodiments, the N residue in the light chain CDR3 from 9C2 (SEQ ID NO: 14) may be substituted with Q, S, D, or A. The light chain CDR3 of SEQ ID NO: 14 may also contain an H, Y, or F residue substituted in place of the Trp residue of the 9C2 light chain CDR3.

[0113] Antibody Framework Any of the antibodies described herein further comprise a framework, preferably a human immunoglobulin framework. For example, in some embodiments, an antibody comprises an HVR of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. The human immunoglobulin framework can be part of a human antibody, or a non-human antibody can be humanized by replacing one or more endogenous framework regions with a human framework region(s). Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0114] In some embodiments, an antibody of the present disclosure has the binding specificity of 3F9 and comprises heavy chain HVR1, HVR2, and HVR3 sequences as described above, and further comprises at least one heavy chain framework as shown in Figure 14A. Examples include the sequences of hSB-3F9-H1 or hSB-3F9-H2 in Figure 14A. In this context, "framework" refers to the FR1, FR2, FR3, and FR4 sequences, excluding the CDR sequences. In some embodiments, an anti-SIRPA antibody has a framework having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework shown in Figure 14A, where the percent identity is determined based on the FR1, FR2, FR3, and FR4 sequences excluding the CDRs.

[0115] In some embodiments, an antibody of the present disclosure has the binding specificity of 3F9 and comprises the light chain HVR1, HVR2, and HVR3 sequences as described above, and further comprises at least one light chain framework as shown in Figure 14B. Examples include the sequences of hSB-3F9-L1, hSB-3F9-L2, or hSB-3F9-L3 in Figure 14B. In this context, "framework" refers to the FR1, FR2, FR3, and FR4 sequences, excluding the CDR sequences. In some embodiments, an anti-SIRPA antibody has a framework having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework shown in Figure 14B, where the percent identity is determined based on the FR1, FR2, FR3, and FR4 sequences excluding the CDRs.

[0116] In some embodiments, the anti-SIRPA antibodies of the disclosure are selected from the group consisting of V, VB, VC, VD, VE, VF ... H Area and V L Includes the area.

[0117] In some embodiments, an antibody of the present disclosure has the binding specificity of 9C2 and comprises heavy chain HVR1, HVR2, and HVR3 sequences as described above, and further comprises at least one heavy chain framework as shown in Figure 14C. For example, the sequences include hSB-9C2-H1, hSB-9C2-H2, hSB-9C2-H3, or hSB-9C2-H4 in Figure 14C. In this context, "framework" refers to the FR1, FR2, FR3, and FR4 sequences, excluding the CDR sequences. In some embodiments, an anti-SIRPA antibody has a framework having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework shown in Figure 14C, where the percent identity is determined based on the FR1, FR2, FR3, and FR4 sequences excluding the CDRs.

[0118] In some embodiments, an antibody of the present disclosure has the binding specificity of 3F9 and comprises light chain HVR1, HVR2, and HVR3 sequences as described above, and further comprises at least one light chain framework as shown in Figure 14D. For example, the sequences include hSB-9C2-L1, hSB-9C2-L2, hSB-9C2-L3, or hSB-9C2-L in Figure 14D. "Framework" in this context refers to the FR1, FR2, FR3, and FR4 sequences, excluding the CDR sequences. In some embodiments, an anti-SIRPA antibody has a framework having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework shown in Figure 14D, where the percent identity is determined based on the FR1, FR2, FR3, and FR4 sequences excluding the CDRs.

[0119] In some embodiments, the anti-SIRPA antibodies of the disclosure are selected from the group consisting of V, VB, VC, VD, VE, VF ... H Area and V L Includes the area.

[0120] In some embodiments, the anti-SIRPA antibodies of the invention comprise one or more substitutions relative to the sequences of the CDR and framework regions shown in Figures 10A-10D. In some embodiments, the substitutions are conservative substitutions. Exemplary substitutions are provided below. JPEG2025118630000002.jpg199170

[0121] In some embodiments, substitutions may be non-conservative. Naturally occurring residues can be divided into groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) Aromatics: trp, tyr, phe. In some embodiments, non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0122] Any cysteine residues not involved in maintaining the proper conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to an antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment. In some embodiments, the anti-SIRPA antibody comprises a substitution at one or more C residues in the sequences shown in Figures 14A-14D.

[0123] In some embodiments, anti-SIRPA antibodies may also contain substitutions at N residues that are potential deamidation sites, hi some embodiments, anti-SIRPA antibodies of the invention contain substitutions at one or more N residues of the sequences shown in Figures 14A-14D.

[0124] In some embodiments, the anti-SIRPA antibody comprises a substitution at a W residue, as W residues may be susceptible to oxidation, hi some embodiments, the substitution is at one or more W residues of the sequences shown in Figures 14A-14D.

[0125] In some embodiments, anti-SIRPA antibodies containing an Asp-Gly (DG) sequence that may be susceptible to isoaspartic acid formation may have A or S substituted for Gly, or E substituted for Asp.

[0126] Anti-SIRPA1 antibody binding affinity Anti-SIRPA of the present disclosure may have nanomolar or even picomolar affinity for SIRPA. In certain embodiments, the dissociation constant (K D ) is about 0.05 to about 100 nM. For example, the K D is about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 900 pM, about 800 pM, about 790 pM, about 780 pM, about 770 pM, about 760 pM, about 750 pM, about 740 pM, about 730 pM, about 720 pM, about 710 pM, about 700 pM, about 650 pM, about 600 pM, about 590 pM, about 580 pM, about 570 pM, about 560 pM, about 550 pM, about 540 pM, about 530 pM, about 520 pM, about 5 The range is from about 10 pM, about 500 pM, about 450 pM, about 400 pM, about 350 pM, about 300 pM, about 290 pM, about 280 pM, about 270 pM, about 260 pM, about 250 pM, about 240 pM, about 230 pM, about 220 pM, about 210 pM, about 200 pM, about 150 pM, about 100 pM, or about 50 pM to about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about 40 pM.

[0127] In some embodiments, the K of an anti-SIRPA for binding to human SIRPA DThe Kd of an anti-SIRPA antibody against human SIRPA may be about 200 nM or less, about 100 nM or less, about 50 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less. In some embodiments, the Kd of an anti-SIRPA antibody against human SIRPA is about 100 pM or less, or about 50 pM or less, less than about 10 pM, or less than about 1 pM. In some embodiments, the binding affinity is in the range of about 1 pM to about 200 nM. In some embodiments, the Kd of an anti-SIRPA antibody against human SIRPA may be about 200 nM or less, about 200 nM or less, about 100 pM or less, or about 50 pM or less, less than about 10 pM, or less than about 1 pM. In some embodiments, the binding affinity is in the range of about 1 pM to about 200 nM. In some embodiments, the Kd of an anti-SIRPA antibody against human SIRPA may be about 200 nM or less, or about 100 pM or less, or less than about 10 p ... less D is in the range of about 1 pM to about 100 nM.

[0128] In some embodiments, the K of an anti-SIRPA antibody to human SIRPA D is less than 15nM, less than 14.5nM, less than 14nM, less than 13.5nM, less than 13nM, less than 12.9nM, less than 12.8nM, less than 12.7nM, less than 12.6nM, less than 12.5nM, less than 12.4nM, less than 12.3nM, less than 12.2nM, less than 12.1nM, less than 12nM, less than 11.5nM, less than 11nM, less than 10.9nM Less than 10.8nM, Less than 10.7nM, Less than 10.6nM, Less than 10.5nM, Less than 10.4nM, Less than 10.3nM, Less than 10.2nM, Less than 10.1nM, Less than 10nM, Less than 9.5nM, Less than 9nM, Less than 8.5nM, Less than 8nM, Less than 7.5nM, Less than 7nM, Less than 6.9nM, Less than 6.8nM, Less than 6.7nM, 6 Less than 0.6nM, Less than 6.5nM, Less than 6.4nM, Less than 6.3nM, Less than 6.2nM, Less than 6.1nM, Less than 6nM, Less than 5.5nM, Less than 5nM, Less than 4.5nM, Less than 4nM, Less than 3.5nM, Less than 3.4nM, Less than 3.3nM, Less than 3.2nM, Less than 3.1nM, Less than 3nM, Less than 2.9nM, Less than 2.8nM, Less than 2.7nM , less than 2.6 nM, less than 2.5 nM, less than 2.4 nM, less than 2.3 nM, less than 2.2 nM, 2.1 nM, less than 2 nM, less than 1.9 nM, less than 1.8 nM, less than 1.7 nM, less than 1.6 nM, less than 1.5 nM, less than 1.4 nM, less than 1.3 nM, less than 1.2 nM, less than 1.1 nM, less than 1 nM, 0.95 nM, or less than 0.9 nM. In some embodiments, the dissociation constant is in the range of about 50 nM to about 100 pM.

[0129] The dissociation constant can be determined by any analytical technique, including any biochemical or biophysical technique, such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, for example, the Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analysis.

[0130] antibody fragment Certain aspects of the present disclosure relate to fragments of the SIRPA antibodies described herein, which fragments retain SIRPA binding activity. In some embodiments, the antibody fragments are Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragments. In some embodiments, the antibody fragments are provided in a multivalent format.

[0131] Multivalent antibodies In some embodiments, the anti-SIRPA antibodies of the present invention may be in a multivalent format that is internalized faster than bivalent antibodies by cells expressing the antigen to which the antibody binds. The anti-SIRPA antibodies or antibody fragments thereof of the present disclosure may be multivalent antibodies (other than IgM class) having three or more antigen-binding sites (e.g., tetravalent antibodies), which can be easily produced by recombinant expression of nucleic acids encoding the antibody polypeptide chains. The multivalent antibody may comprise a dimerization domain and three or more antigen-binding sites. In a typical embodiment, the dimerization domain comprises an Fc region or hinge region. In this scenario, the antibody comprises an Fc region and three or more antigen-binding sites amino-terminal to the Fc region. In some embodiments, the multivalent antibody contains three to eight, e.g., four, antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), and the polypeptide chain or chains comprise two or more variable domains.

[0132] Bispecific and multispecific antibodies Certain aspects of the present disclosure relate to bispecific or multispecific antibodies comprising an anti-SIRPA antibody described herein and an antibody that binds to a second antigen or a second SIRPA epitope. Bispecific and multispecific antibodies can be made using any method.

[0133] In some embodiments, the antibody is a bispecific antibody comprising the variable region of an anti-SIRPA antibody described in the present disclosure and an antibody that binds to a second antigen. In some embodiments, the second antigen is a protein selected from the group consisting of PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73. In some embodiments, the second antigen is an antigen that promotes transport across the blood-brain barrier; an antigen that promotes transport across the blood-brain barrier selected from the group consisting of transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, llama single domain antibody, TMEM30(A), protein transduction domain, TAT, Syn-B, penetratin, polyarginine peptides, angiopep peptides, and ANG1005;A disease-causing agent selected from the group consisting of a disease-causing peptide or protein, or a disease-causing nucleic acid, wherein the disease-causing nucleic acid is an antisense GGCCCC (G2C4) repeat expanded RNA, and the disease-causing protein is amyloid beta, oligomeric amyloid beta, amyloid beta plaque, amyloid precursor protein or a fragment thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (open reading frame 72 of chromosome 9), or a protein encoding amyloid precursor protein (AFP) or a fragment thereof. ), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light an agent selected from the group consisting of chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine (PA) repeat peptide, ubiquitin, and proline-arginine (PR) repeat peptide; or a ligand and / or protein expressed on immune cells, such as PD1 / PDL1, CD40, OX40, IC a ligand and / or protein selected from the group consisting of OS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, and phosphatidylserine; and a protein, lipid, polysaccharide, or glycolipid expressed on one or more tumor cells;

[0134] Fc area In some embodiments, the antibodies of the present disclosure comprise an Fc region. For example, the antibodies can be of the IgG, IgM, or IgA class. In some embodiments, they have an IgG1, IgG2, IgG3, or IgG4 isotype. Typically, the Fc region is a native human Fc region or a variant thereof.

[0135] In some embodiments, the anti-SIRPA antibody of the present disclosure retains the ability to bind to Fc gamma receptors. In some embodiments, such antibodies may have the characteristic of causing SIRPA clustering and transient stimulation. Such antibodies may then act as long-term inhibitors of SIRPA expression and / or one or more activities of SIRPA by inducing SIRPA degradation, SIRPA desensitization, SIRPA cleavage, SIRPA internalization, SIRPA shedding, lysosomal degradation of SIRPA, or otherwise downregulating SIRPA. In some embodiments, the anti-SIRPA antibody reduces the level of Fc gamma receptors on the cell surface.

[0136] In some embodiments, the Fc region is an Fc region that binds to receptors such as the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain (see, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). FcRs can also increase the serum half-life of antibodies.

[0137] The Fc region can include one or more mutations that affect the activity of the Fc region, for example, in binding to an Fc receptor.

[0138] In some embodiments, the antibodies of the present disclosure bind to an inhibitory Fc receptor. In a specific embodiment, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB). In some embodiments, the antibodies of the present disclosure reduce the expression level of inhibitory Fc-gamma receptor IIB on the cell surface. In some embodiments, the Fc region comprises one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from the group consisting of V234A (Alegre et al., (1994) Transplantation 57:1537-1543. 31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Cole et al., (1999) Transplantation, 68:563-571), H268Q, V309L, A330S, P331S (U.S. Patent Application Publication No. 2007 / 0148167; Armour et al., (1999) Eur J Immunol 29: 2613-2624; Armour et al., (2000) The Haematology Journal 1(Suppl. 1):27; Armour et al., (2000) The Haematology Journal 1(Suppl.1):27), C232S, and / or C233S (White et al. (2015) Cancer Cell 27, 138-148), S267E, L328F (Chu et al. (2008) Mol Immunol, 45:3926-3933), M252Y, S254T, and / or T256E, where the amino acid positions are according to EU or Kabat numbering rules.

[0139] In some embodiments, the antibodies of the invention have an IgG2 isotype with heavy chain constant domains containing the amino acid substitutions C127S or C2214S, where the amino acid positions are according to the EU or Kabat numbering conventions (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO 2008 / 079246).

[0140] In certain embodiments, the antibodies of the present disclosure have an IgG1 isotype. In some embodiments, the Fc gamma receptor-binding antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB). In some embodiments, the antibodies of the present disclosure reduce the level of inhibitory Fc gamma receptor IIB expressed on the surface of a cell. In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) RJ Biol. Chem. 276, 6591-6604), D270A, L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al. (1994) Transplantation 57:1537-1543. 31; Xu et al. (2000) Cell Immunol, 200:16-26), G237A (Alegre et al. (1994) Transplantation 57:1537-1543. 31; Xu et al. (2000) Cell Immunol, 200:16-26), P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, and / or T394D, where the amino acid positions are according to EU or Kabat numbering conventions.

[0141] In some embodiments, an antibody of the present disclosure has an IgG1 isotype and comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and hinge region (White et al., (2015) Cancer Cell 27, 138-148). In certain embodiments, the IgG2 isotype CH1 and hinge region comprises the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 34). In some embodiments, the antibody Fc region contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and / or an N297A or N297Q amino acid substitution, wherein the amino acid positions are according to EU or Kabat numbering conventions.

[0142] In some embodiments, the anti-SIRPA antibody has an IgG2 isotype and comprises one or more amino acid substitutions in the Fc region at residues selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, V309L, A330S, P331S, C214S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, H268E, N297A, N297Q, A330L, and any combination thereof, wherein residue numbering is according to EU numbering rules.

[0143] In certain embodiments, antibodies of the present disclosure have an IgG4 isotype. Some embodiments comprise an Fc region comprising a human IgG4 constant region and containing one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from L235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol, 164:1925-1933), S267E, E318A, L328F, M252Y, S254T, T256E, E233P, F234V, L234A / F234A, S228P, S241P, L248E, T394D, N297A, N297Q, L235E, and any combination thereof, where residue numbering is according to EU numbering.

[0144] In some embodiments, the anti-SIRPA antibodies of the present disclosure have a hybrid IgG2 / 4 isotype. In certain embodiments, the antibodies comprise an amino acid sequence comprising amino acids 118-260 of human IgG2 and amino acids 261-447 of human IgG4, where residue numbering is according to EU numbering.

[0145] In some embodiments, the anti-SIRPA antibodies of the disclosure have a human or murine IgG1 isotype and comprise one or more amino acid substitutions in the Fc region at residues selected from the group consisting of N297A, N297Q, D270A, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, wherein residue numbering is according to EU numbering.

[0146] In some embodiments, the anti-SIRPA antibodies of the disclosure have an IgG2 isotype and comprise one or more amino acid substitutions in the Fc region at residues selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, wherein residue numbering is according to EU numbering.

[0147] In some embodiments, the anti-SIRPA antibodies of the disclosure have an IgG4 isotype and comprise one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of E233P, F234V, L234A / F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, wherein residue numbering is according to EU numbering.

[0148] In some embodiments, the anti-SIRPA antibodies of the disclosure comprise an Fc region further comprising one or more additional amino acid substitutions at positions selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, wherein residue numbering is according to EU numbering.

[0149] In some embodiments, the anti-SIRPA antibodies of the disclosure comprise an Fc region further comprising one or more additional amino acid substitutions at positions selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein residue numbering is according to EU numbering.

[0150] In some embodiments, the anti-SIRPA antibodies of the disclosure comprise an Fc region further comprising an amino acid substitution of S228P according to EU numbering.

[0151] In some embodiments, the anti-SIRPA antibodies of the disclosure have an IgG4 isotype and comprise an amino acid substitution of S228P at residue 228, an amino acid substitution of F234A at residue 234, and an amino acid substitution of L235A at residue 235, wherein the numbering of the residue positions is according to EU numbering.

[0152] In some embodiments, the anti-SIRPA antibodies of the present disclosure may be modified to modulate effector function and / or increase the serum half-life of the antibody. For example, the Fc receptor binding site on the constant region may be modified or mutated to remove or reduce binding affinity to specific Fc receptors, such as FcγRI, FcγRII, and / or FcγRIII, to reduce antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is inhibited by removing N-glycosylation in the Fc region of the antibody (e.g., in the CH2 domain of IgG). In some embodiments, effector function is inhibited by modifying regions such as 233-236, 297, and / or 327-331 of human IgG, as described in PCT Publication WO 99 / 58572 and Armour et al., Molecular Immunology 40: 585-593 (2003); Reddy et al., J. Immunology 164:1925-1933 (2000). In other embodiments, it may be desirable to modify the anti-SIRPA antibodies of the disclosure to increase binding selectivity for ITIM-containing FcγRIIb (CD32b) to modify effector function such as increasing clustering of SIRPA antibodies on neighboring cells without activating effector functions such as ADCC.

[0153] In some embodiments, salvage receptor binding epitopes can be incorporated into antibodies (particularly antibody fragments) to increase the serum half-life of the antibody, as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0154] Other amino acid sequence modifications Amino acid sequence modifications of the anti-SIRPA antibodies, or antibody fragments thereof, of the present disclosure are also contemplated. For example, they may be desirable to improve the binding affinity and / or other biological properties of the antibody or antibody fragment.

[0155] In some embodiments, additional amino acid sequences can be fused to the amino or carboxy terminus of an anti-SIRPA antibody, including, but not limited to, antibodies with an N-terminal methionyl residue, fusion to a cytotoxic polypeptide, or fusion to an enzyme or polypeptide that increases the serum half-life of the antibody.

[0156] In some embodiments, antibodies of the invention may be mutated to alter the original glycosylation pattern of the antibody, e.g., by deleting one or more mutation sites to prevent glycosylation with particular carbohydrate moieties, and / or by adding one or more glycosylation sites to introduce desired carbohydrate moieties.

[0157] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0158] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

[0159] Other antibody modifications The anti-SIRPA antibodies of the present disclosure, or antibody fragments thereof, can be further modified to include additional moieties, such as moieties for antibody derivatization, drug moieties to be conjugated to the antibody, etc. Examples of moieties suitable for antibody derivatization include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used therapeutically under defined conditions. Such techniques and other suitable formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th ed., edited by Alfonso Gennaro, Philadelphia College of Pharmacy and Science (2000).

[0160] In some embodiments, cytotoxic agents or drugs may be conjugated to the anti-SIRPA antibodies of the present invention, for example, for the treatment of cancers such as multiple myeloma or other cancers that express SIRPA on the cell surface. Techniques for conjugating antibodies have been described and are known in the art (see Jane de Lartigue, OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al., (2010). Bioconjugate Chemistry 21 (1): 5-13). In some embodiments, the anti-SIRPA antibody is conjugated to a toxin selected from the group consisting of ricin, ricin A chain, doxorubicin, daunorubicin, maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alphasarcin, gelonin, mitogellin, lexstrictocin, phenomycin, enomycin, chrysin, crotin, calicheamicin, Saponaria officinali inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycin, dolastatin, cc1065, and cisplatin.

[0161] Nucleic acids, vectors, and host cells The anti-SIRPA antibodies of the present disclosure are typically produced using recombinant methods. Thus, in some aspects, the invention provides isolated nucleic acids comprising nucleic acid sequences encoding any of the anti-SIRPA antibodies described herein; vectors comprising such nucleic acids, and host cells into which nucleic acids are introduced that are used to replicate and / or express the nucleic acids encoding the antibodies. Such nucleic acids may be used to encode the V of an anti-SIRPA antibody. L and / or V HIn some embodiments, the host cell may encode an amino acid sequence (e.g., an antibody light chain and / or heavy chain) containing: (1) V L Polynucleotides encoding amino acid sequences and V H (2) a vector containing a polynucleotide encoding an amino acid sequence; or L a first vector containing a polynucleotide encoding an amino acid sequence; and V H and a second vector containing a polynucleotide encoding the amino acid sequence. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell; or a human cell. In some embodiments, the host cell is a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

[0162] In a further aspect, the invention provides methods of making the anti-SIRPA antibodies described herein. In some embodiments, the methods comprise culturing a host cell described in the preceding paragraph under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0163] Suitable vectors containing a polynucleotide encoding an antibody of the present disclosure, or a fragment thereof, include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended for use, useful cloning vectors will generally be capable of autonomous replication, may have a single target for a particular restriction endonuclease, and / or may carry a marker gene that can be used to select clones containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0164] An expression vector is generally a replicable polynucleotide construct containing a nucleic acid of the present disclosure. Expression vectors can be replicable in host cells as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, such as adenoviruses, adeno-associated viruses, retroviruses, and any other vectors.

[0165] Suitable host cells for expressing the anti-SIRPA antibodies described herein include both prokaryotic and eukaryotic cells. For example, anti-SIRPA antibodies can be produced in bacteria, particularly if glycosylation and Fc effector function are not required. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified. Alternatively, the host cell can be a eukaryotic host cell, e.g., a filamentous fungus or yeast cell in which the glycosylation pathway has been "humanized" to produce antibodies with partial or fully human glycosylation patterns, a vertebrate, an invertebrate, or a plant cell. Examples of invertebrate cells include insect cells. Numerous baculovirus strains have been identified that can be used with insect cells. Plant cell cultures can also be used as host cells.

[0166] In some embodiments, vertebrate host cells are used to produce the anti-SIRPA antibodies of the present disclosure. For example, mammalian cell lines, such as the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells can be used to express anti-SIRPA antibodies. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, such as DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0167] Pharmaceutical compositions and treatments using anti-SIRPA antibodies Pharmaceutical Composition Anti-SIRPA antibodies can be incorporated into various formulations for therapeutic administration by combining the antibody with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into solid, semi-solid, liquid, or gaseous preparations. Examples of such formulations include, but are not limited to, tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, gels, microspheres, and aerosols. Depending on the desired formulation, pharmaceutical compositions can contain pharmaceutically acceptable, non-toxic carriers for diluents, which are vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include, but are not limited to, distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical compositions or formulations of the present disclosure can further contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and surfactants.

[0168] The pharmaceutical compositions of the present disclosure can also include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increasing the polypeptide's half-life, reducing its toxicity, and enhancing solubility or uptake). Examples of such modifying or complexing agents include, but are not limited to, sulfate, gluconate, citrate, and phosphate. The polypeptides of the composition can also be complexed with molecules that enhance their in vivo properties. Such molecules include, but are not limited to, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0169] Further examples of formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 22nd Edition (2012).

[0170] For oral administration, active ingredients can be administered in solid dosage forms such as capsules, tablets, and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. The active ingredient(s) can be enclosed in a gelatin capsule along with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, and magnesium carbonate. Examples of additional inactive ingredients that can be added to provide desired color, taste, stability, buffering capacity, dispersion, or other known desirable characteristics include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be prepared as sustained-release products to provide continuous release of medication over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0171] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0172] Ingredients used to formulate pharmaceutical compositions are preferably highly pure and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic, and made under GMP conditions.

[0173] Formulations can be optimized for retention and stabilization in the brain or central nervous system. When administered to the cranial compartment, it is desirable for the drug to be retained within the compartment and not diffuse or otherwise cross the blood-brain barrier. Stabilization techniques include cross-linking, multimerization, or linking to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. to achieve increased molecular weight.

[0174] Other strategies for increasing retention include entrapment of antibodies, such as the anti-SIRPA antibodies of the present disclosure, in biodegradable or bioerodible implants. The release rate of the therapeutically active agent is controlled by the transport rate through the polymer matrix and the biodegradability of the implant. Drug transport through the polymer barrier is also affected by the solubility of the compound, polymer hydrophilicity, the degree of polymer crosslinking, the swelling of the polymer upon hydration to make the polymer barrier more permeable to the drug, the implant geometry, and the like. The implant is dimensioned to match the size and shape of the area selected as the implant site. The implant can be a particle, sheet, patch, plaque, fiber, microcapsule, or the like, and can be of any size or shape that fits the selected insertion site.

[0175] Implants may distribute or encapsulate the active agent throughout the polymer matrix, with the active agent reservoir being encapsulated by the polymer matrix. The choice of polymer composition used will depend on the site of administration, desired duration of treatment, patient tolerance, the nature of the disease being treated, etc. Polymer characteristics include biodegradability at the implantation site, compatibility with the drug of interest, ease of encapsulation, and half-life in a physiological environment.

[0176] Biodegradable polymer compositions that can be used include organic esters or ethers that, upon degradation, yield physiologically acceptable degradation products containing the monomer. Anhydrides, amides, orthoesters, etc., can be used by themselves or in combination with other monomers. The polymers are condensation polymers. The polymers can be crosslinked or non-crosslinked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids, either homopolymers or copolymers, and polysaccharides. Polyesters of interest include polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof. By using the L-lactate or D-lactate salts, slowly biodegrading polymers are achieved, while degradation is substantially enhanced with the racemate. Copolymers of glycolic acid and lactic acid are of particular interest, with the biodegradation rate being controlled by the ratio of glycolic acid to lactic acid. The most rapidly degrading copolymers have approximately equal amounts of glycolic acid and lactic acid, while either homopolymer is more resistant to degradation. The ratio of glycolic acid to lactic acid also affects the brittleness of the implant, with more flexible implants being desirable for larger geometries. Polysaccharides of interest include calcium alginate and functionalized celluloses, particularly carboxymethylcellulose esters, characterized by their water-insolubility and molecular weights of approximately 5 kD to 500 kD. Biodegradable hydrogels can also be used in the implants of the present invention. Hydrogels are typically copolymer materials characterized by their ability to absorb fluid. Exemplary biodegradable hydrogels that can be used are described in Heller in: Hydrogels in Medicine and Pharmacy, edited by N.A. Peppes, Vol. III, CRC Press, Boca Raton, Fla., 1987, pp. 137-149.

[0177] Pharmaceutical compositions of the present disclosure containing anti-SIRPA antibodies of the present disclosure can be administered to an individual, preferably a human, in need of treatment with an anti-SIRPA antibody according to known methods, such as intravenous administration as a bolus, or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.

[0178] The dosage and desired drug concentration of the pharmaceutical composition of the present disclosure may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is well within the skill of one of ordinary skill in the art. Animal experiments provide reliable guidance for determining effective amounts for human treatment. Interspecies scaling of effective amounts can be performed according to the principles described in Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, eds., Yacobi et al., Pergamon Press, New York, 1989, pp. 42-46.

[0179] For in vivo administration of any of the anti-SIRPA antibodies of the present disclosure, typical dosages can vary from about 10 ng to up to about 100 mg / kg of individual body weight per day, depending on the route of administration, and preferably from about 1 mg / kg / day to 10 mg / kg / day. Depending on the severity of the disease, disorder, or condition being treated, treatment may be continued for repeated administrations over several days or longer until a desired suppression of symptoms is achieved.

[0180] An exemplary dosing regimen may involve administering an initial dose of about 2 mg / kg of anti-SIRPA antibody, followed by a maintenance dose of about 1 mg / kg every other week. Other dosing regimens may be useful, depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, dosing an individual from 1 to 21 times per week is contemplated herein. In certain embodiments, dosages ranging from about 3 μg / kg to about 2 mg / kg (e.g., about 3 μg / kg, about 10 μg / kg, about 30 μg / kg, about 100 μg / kg, about 300 μg / kg, about 1 mg / kg, and about 2 mg / kg) may be used. In certain embodiments, the dosing frequency is 3 times a day, 2 times a day, 1 time a day, 2 days a day, 1 time a week ...

[0181] The dosage of a particular anti-SIRPA antibody can be empirically determined in individuals who have received one or more doses of the anti-SIRPA antibody. Individuals are given increasing doses of the anti-SIRPA antibody. Clinical symptoms of the disease, disorder, or condition (e.g., cancer) of the disclosure can be monitored to assess the effectiveness of the anti-SIRPA antibody.

[0182] Administration of the anti-SIRPA antibodies of the present disclosure can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the anti-SIRPA antibodies can be essentially continuous over a preselected period of time, or can be in a series of spaced doses.

[0183] It is within the scope of this disclosure that different formulations are effective for different treatments and different disorders, and that administration intended to treat a particular organ or tissue may require delivery in a different manner than to other organs or tissues. Moreover, dosages may be administered by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0184] In one aspect of the present invention, an agent that downregulates SIRPA, such as an anti-SIRPA antibody, is used as a therapeutic agent. Such an agent is administered to treat, alleviate, and / or prevent a disease or pathology associated with SIRPA expression, activity, and / or signaling in a subject. The treatment regimen is carried out by identifying a subject, e.g., a human patient, suffering from (or at risk of developing) a disease or disorder associated with SIRPA expression, activity, and / or signaling, such as cancer or other neoplastic disorders, using standard methods. In some embodiments, cells with pathology associated with SIRPA expression, activity, and / or signaling express a SIRPA ligand, e.g., CD47. In some embodiments, cells with pathology associated with SIRPA expression, activity, and / or signaling express SIRPA.

[0185] As described in further detail below, an agent that downregulates SIRPA, such as an anti-SIRPA antibody, can be used in combination with an additional therapeutic agent used to treat a disease or pathology associated with SIRPA expression, activity, or signaling. The terms "in combination" and "together" are used interchangeably in this disclosure. The additional therapeutic agent can be administered before, after, or simultaneously with the agent that downregulates SIRPA, such as an anti-SIRPA antibody.

[0186] In one embodiment of the present disclosure, for example, an anti-SIRPA antibody preparation is administered to a human subject, including an anti-SIRPA antibody that reduces the expression of SIRPA on the cell surface but does not substantially block the binding of a ligand, e.g., CD47, to SIRPA. The administration of the antibody can neutralize, inhibit, or interfere with the expression, activity, and / or signaling function of SIRPA mediated by ligand binding, e.g., CD47 binding. In one embodiment, the disease or disorder associated with SIRPA expression is cancer. In some embodiments, the anti-SIRPA antibody is administered to a patient with a cancer, such as a hematoproliferative disorder of myeloid cells that express SIRPA. In a typical embodiment, the anti-SIRPA antibody is administered to a patient with a cancer that expresses CD47.

[0187] In certain embodiments, the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer cancer), germ cell tumors, childhood sarcoma, sinonasal natural killer, melanoma (e.g., metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis cancer, childhood solid tumors, cancer of the urinary tract, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (producing B, T, NK, and plasma cells), e.g., all types of leukemia, lymphoma, and myeloma, e.g., acute myeloid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML); acute, chronic, lymphocytic and / or myeloid leukemias, such as undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma with Waldenstrom's hypergammaglobulinemia (LP) L); myelomas, e.g., IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nerves, including seminoma, teratocarcinoma, astrocytoma, schwannoma; fibrosarcoma, rhabdomyosarcoma tumors of mesenchymal origin, including rhabdomyoscarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example, T-cell and B-cell tumors, including T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including but not limited to the small cell and cerebriform cell types; preferably, large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma;Head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. The anti-SIRPA antibodies of the present invention can also be used to treat metastatic cancer.

[0188] In some embodiments, the cancer is selected from the group consisting of sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma.

[0189] In some embodiments, the cancer is selected from the group consisting of glioblastoma multiforme, renal clear cell carcinoma, adrenocortical carcinoma, bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma, pancreatic adenocarcinoma, renal cell carcinoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma, invasive breast carcinoma, cervical squamous cell carcinoma, cervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, hepatocellular carcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

[0190] In some embodiments, the anti-SIRPA antibodies of the present disclosure may be administered in combination with a therapeutic agent that acts as a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor targets PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, and CD73. In exemplary embodiments, the therapeutic agent is an antibody against a checkpoint inhibitor selected from D1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73. In some embodiments, a combination of antibodies against checkpoint inhibitors is administered with the anti-SIRPA antibody of the invention.

[0191] In some embodiments, the anti-SIRPA antibodies of the present disclosure may be administered in combination with at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein, such as an agonistic anti-CD40 antibody, an agonistic anti-OX40 antibody, an agonistic anti-ICOS antibody, an agonistic anti-CD28 antibody, an agonistic anti-TREM1 antibody, an agonistic anti-TREM2 antibody, an agonistic anti-CD137 / 4-1BB antibody, an agonistic anti-CD27 antibody, an agonistic anti-glucocorticoid-inducible TNFR-related protein GITR antibody, an agonistic anti-CD30 antibody, an agonistic anti-BTLA antibody, an agonistic anti-HVEM antibody, an agonistic anti-CD2 antibody, an agonistic anti-CD5 antibody, and any combination thereof.

[0192] In some embodiments, the anti-SIRPA antibodies of the invention are administered in combination with radiation therapy and / or chemotherapeutic agents, including, but not limited to, the following groups: antimetabolites / anticancer drugs, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (methotrexate, pemetrexed, mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); Antiproliferative / antimitotic agents, e.g., natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (paclitaxel, dotaxel), vincristine, vinblastine, nocodazole, epothilones, eribulin, and navelbine; epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cycloheximide, cycloheximide); toxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP16); DNA methyltransferase inhibitors (azacytidine); antibiotics such as dactinomycin (actinomycin D), da unorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; an enzyme (L-asparaginase, which metabolizes L-asparagine systemically and depletes cells that do not have the ability to synthesize asparagine themselves); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine, and methylmelamine (hexamethyl antiproliferative / antimitotic antimetabolites such as melamine and thiotepa), alkylsulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes (dacarbazine (DTIC)); folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole);Anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); thrombolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, and abciximab; antimigratory agents; antisecretory agents (vleverdin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, and mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein, and pomalidomide) and growth factor inhibitors such as ziv-aflibercept (vascular endothelial growth factor (VEGF) inhibitors; fibroblast growth factor (FGF) inhibitors); inhibitors of apoptosis protein (IAP) antagonists (bilinapant); and histone deacetylase (HDAC) inhibitors (vorinostat). , romidepsin, chidamide, panobinostat, mocetinostat, abexinostat, belinostat, entinostat, resminostat, gibinostat, xinostat, SB939); proteasome inhibitors (ixazomib); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, panitumumab, pertuzumab, cerebrospinal fluid ... Tuximab, adalimumab, golimumab, infliximab, rituximab, ocrelizumab, ofatumumab, obinutuzumab, alemtuzumab, abciximab, atlizumab, daclizumab, denosumab, efalizumab, elotuzumab, rovelizumab, ruplizumab, ustekinumab, visilizumab, gemtuzumab ozogamicin, brentuximab vedotin (brentuximab vedotin); chimeric antigen receptors; cell cycle inhibitors (flavopiridol, roscovitine, bryostatin-1) and differentiation inducers (tretinoin); mTOR inhibitors; topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone);PARP inhibitors (niraparib, olaparib); focal adhesion kinase (FAK) inhibitors (defactinib (VS-6063), VS-4718, VS-6062, GSK2256098); growth factor signaling kinase inhibitors (cediranib, galunisertib, rociletinib, vandetanib, afatinib, EGF816, AZD4547); c-Met inhibitors (capmatinib, INC280); ALK inhibitors (ceritinib, crizotinib); mitochondrial dysfunction inducers; toxins, such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin and caspase activators; and chromatin disruptors. In some embodiments, the chemotherapeutic agent is a B-Raf inhibitor, a MEK inhibitor, a VEGF inhibitor, a VEGFR inhibitor, a tyrosine kinase inhibitor, an antimitotic agent, or any combination thereof;

[0193] In some embodiments, the anti-SIRPA antibodies of the present disclosure are administered in combination with adoptive cell transfer (ACT) therapy, chimeric antigen receptor T cell transfer (CAR-T) therapy, vaccine therapy, and / or cytokine therapy.

[0194] In some embodiments, the anti-SIRPA antibodies of the present disclosure are administered in combination with at least one antibody that specifically binds to an inhibitory cytokine, such as an anti-CCL2 antibody, an anti-CSF-1 antibody, or an anti-IL-2 antibody.

[0195] In some embodiments, the anti-SIRPA antibodies of the present disclosure are administered in combination with at least one stimulatory cytokine. In some embodiments that may be combined with any of the preceding embodiments, the at least one stimulatory cytokine is selected from the group consisting of IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, an IL-20 family member, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-beta, and any combination thereof.

[0196] In some embodiments, the agent that downregulates SIRPA, such as anti-SIRPA antibody, is administered to a patient with neurological disorder, or is administered to reduce the risk of, delay the onset of, or prevent neurological disorder.In some embodiments, the neurological disorder is dementia, including frontotemporal dementia, Alzheimer's disease, or vascular dementia.In some embodiments, the patient has mild cognitive impairment.

[0197] In some embodiments, an agent that downregulates SIRPA, such as an anti-SIRPA antibody, is administered to a patient with Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, a tauopathy, or multiple sclerosis. In some embodiments, the agent is administered to a patient with Creutzfeldt-Jakob disease, normal pressure hydrocephalus, Nasu-Hakola disease, stroke, infection, traumatic brain injury, progressive supranuclear palsy, dementia pugilistica (chronic traumatic encephalopathy), Parkinsonism linked to chromosome 17, Lytico-Bodig disease (Parkinson-Dementia Complex of Guam), tangle-predominant dementia, gliomas and gliocytomas, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticotemporal degeneration, argyrophilic grain disease (AGD), frontotemporal lobar degeneration, dementia with Lewy bodies, multiple system atrophy, Scheid-Drager syndrome, progressive supranuclear palsy, or corticobasal ganglia degeneration. [Example]

[0198] The following examples are offered by way of illustration only, not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially similar results.

[0199] Example 1: Generation of anti-SIRPA antibodies The amino acid sequence of human SIRPA preprotein is set forth below in SEQ ID NO: 1. Human SIRPA contains a signal peptide located at amino acid residues 1 to 30 of SEQ ID NO: 1. Human SIRPA contains an extracellular immunoglobulin-like variable (IgV) domain located at amino acid residues 32 to 137 of SEQ ID NO: 1; additional extracellular immunoglobulin-like constant (IgC) domain sequences located at amino acid residues 148 to 247 and 254 to 348 of SEQ ID NO: 1; a transmembrane domain located at amino acid residues 374 to 394 of SEQ ID NO: 1; and an intracellular domain located at amino acid residues 395 to 504 of SEQ ID NO: 1. SIRPAv1 amino acid sequence (SEQ ID NO: 1): 10 20 30 40 50 MEPAGPAPGR LGPLLCLLLA ASCAWSGVAG EEELQVIQPD KSVLVAAGET 60 70 80 90 100 ATLRCTATSL IPVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN 110 120 130 140 150 NMDFSIRIGN ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPSA 160 170 180 190 200 PVVSGPAARA TPQHTVSFTC ESHGFSPRDI TLKWFKNGNE LSDFQTNVDP 210 220 230 240 250 VGESVSYSIH STAKVVLTRE DVHSQVICEV AHVTLQGDPL RGTANLSETI 260 270 280 290 300 RVPPTLEVTQ QPVRAENQVN VTCQVRKFYP QRLQLTWLEN GNVSRTETAS 310 320 330 340 350 TVTENKDGTY NWMSWLLVNV SAHRDDVKLT CQVEHDGQPA VSKSHDLKVS 360 370 380 390 400 AHPKEQGSNT AAENTGSNER NIYIVVGVVC TLLVALLMAA LYLVRIRQKK 410 420 430 440 450 AQGSTSSTRL HEPEKNAREI TQDTNDITYA DLNLPKGKKP APQAAEPNNH 460 470 480 490 500 TEYASIQTSP QPASEDTLTY ADLDMVHLNR TPKQPAPKPE PSFSEYASVQ VPRK

[0200] Crystal structure analysis of the SIRPA-CD47 complex resolves the ligand-binding site to a variable loop connecting the β-sheet strands in the IgV domain of SIRPA. The CD47-binding interface consists of amino acid residues S59–P65, L96–F104, and K123–D130.

[0201] Multiple polymorphisms of SIRPA have been identified in humans. The amino acid sequence alignment of the two most common variants, designated SIRPA v1 and v2, was generated by 2-way blast (Figure 1A). Both SIRPA variants have been reported to bind to CD47 with similar affinity, as most of the sequence variations are located across the ligand-binding site. Alternatively, SIRPB1, another member of the SIRP family, shares high sequence identity with SIRPA but does not bind to CD47. The amino acid sequence alignment of SIRPAv1 and SIRPB1, generated by 2-way blast (Figure 1B), shows that the extracellular domains of both proteins (excluding the leader sequence) share approximately 90% identity. However, a single A57M substitution is sufficient to rearrange the S59-P65 ligand-binding interface to prevent SIRPB1 from binding to CD47. Furthermore, CD47 binding is highly species-specific, with human CD47 recognizing a single allelic variant of mouse SIRPA expressed only by NOD mice. An amino acid sequence alignment of human SIRPAv1 and C57BL6 SIRPA, generated by two-way blasting (Figure 2), shows that the extracellular domains of both proteins (excluding the leader sequence) share approximately 60% identity.

[0202] Anti-SIRPA antibody generation Immunization Procedure Rapid Prime Method: Four 50-day-old female BALB / c mice were immunized using the following procedure: a series of subcutaneous aqueous injections containing human SIRPA antigen but no adjuvant were given over a 19-day period. Mice were housed in a ventilated rack system from Lab Products. All four mice were euthanized on day 19, and lymphocytes were harvested for hybridoma cell line generation.

[0203] Standard Method: Four 50-day-old female BALB / c or NZB / W mice were immunized using the following procedure. Mice were housed in a ventilated rack system from Lab Products. Mice were intraperitoneally injected every three weeks with 25 μg of protein antigen per mouse (total volume of 125 μL per mouse) of human SIRPA antigen mixed with CpG-ODN adjuvant. Test bleeds were performed by saphenous vein puncture 7 days after the second boost. Test bleeds (immune serum) were tested by indirect ELISA assay to determine the two mice that best responded to the fusion. Mice may require a third and fourth boost and another test bleed 7 days after the boost to assess pre-fusion titers. If antibody titers were sufficiently high, the two best responding mice were given a final intravenous boost via the lateral tail vein. Four days after the IV boost, the mice were euthanized for fusion. The spleens were harvested and lymphocytes isolated from the spleens were used in a fusion process to generate hybridomas.

[0204] Hybridoma Development Lymphocytes were isolated and fused with murine SP2 / 0 myeloma cells in the presence of polyethylene glycol (PEG 1500) according to standard Roche protocols. Fused cells were cultured using a one-step cloning method (HAT selection). This method uses semi-solid methylcellulose-based HAT selection medium to combine hybridoma selection and cloning into a single step. Single cell-derived hybridomas grow on the semi-solid medium to form monoclonal colonies. Ten days after the fusion event, 948 resulting hybridoma clones were transferred to 96-well tissue culture plates and grown in HT-containing medium until they reached mid-logarithmic growth (5 days).

[0205] Hybridoma screening Tissue culture supernatants from 948 hybridomas were tested by indirect ELISA (primary screening) to screen for antigen, probed for both IgG and IgM antibodies using a goat anti-IgG / IgM (H&L)-HRP secondary, and developed with TMB substrate. Clones with an OD >0.2 in this assay were used for the next round of testing. Positive cultures were screened for antigen to confirm secretion and to eliminate nonspecific or "sticky" mAbs, and retested on an irrelevant antigen (human transferrin) to rule out false positives. All clones of interest were isotyped by antibody capture ELISA to determine whether they were IgG or IgM isotypes.

[0206] Hybridoma cell culture The hybridoma cell lines of interest were maintained in culture in 24-well culture plates for 32 days after transfer to 96-well plates. This is called the stability period, and clones are tested for stable secretion. During this stability period, temporary frozen cell line backups are made of all clones of interest for storage at -80°C (6-month viability). Hybridomas were periodically tested for secretion and specificity during this period.

[0207] Subcloning To ensure monoclonality, the top hybridoma cell lines (clones) were subcloned. Subcloning was performed by replating the parent clones using a single-step cloning system. 24 to 90 subclones were transferred to 96-well culture plates. The subclones were screened by indirect ELISA and antibody capture ELISA. The top subclones for each parent were picked for expansion in culture. Any parent clones that were less than 50% clonal underwent a second round of subcloning.

[0208] The antibodies were then screened for SIRPA binding. Antibodies that tested positive for binding to human SIRPA were tested for their ability to block ligand binding and inhibit ligand-induced SIRPA activity in multiple cell types. The isotype and bin category of each antibody are listed in Table 1. In Table 1, "ND" indicates antibodies for which the bin category has not been determined. JPEG2025118630000003.jpg63170

[0209] Antibody heavy and light chain variable domain sequences The amino acid sequences encoding the light and heavy chain variable domains of the generated antibodies were determined using standard techniques. The EU or Kabat light chain HVR sequences of the antibodies are listed in Tables 2-5. The EU or Kabat light chain HVR sequences of the antibodies are listed in Table 2. The EU or Kabat heavy chain HVR sequences of the antibodies are listed in Table 3. The EU or Kabat light chain framework (FR) sequences of the antibodies are listed in Table 4. The EU or Kabat heavy chain framework (FR) sequences of the antibodies are shown in Table 5. 3F9 : Heavy chain variable domain sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQT PEKRLEWVATISDYGGSYTYYPDSVKGRFTISRDNAKYTLYLQMSSLRSEDTAL YYCARPPYDDYYGGFAYWGQGTLVTVSA (SEQ ID NO: 2) 3F9 : Light chain variable domain sequence DIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWY QQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQH NRELPCTFGGGTKLEIK (SEQ ID NO: 3) 9C2 : Heavy chain variable domain sequence EFQLQQSGAELVKPGASVKISCKASGYSLTGYNMNWVKQS RGKSLEWIGNINPHYGSSTYNQNFKDKATLTVDKSSSAAYMQFNSLTSEDSAVY YCAREGYDGVFDYWGQGTTLTVSS (SEQ ID NO: 4) 9C2 : Light chain variable domain sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPG SSPKPWIYVTSNLASGVPTRFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNP RTFGGGTKLEIK (SEQ ID NO: 5) 8A9 : Heavy chain variable domain sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWMHWVKQR PGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVY YCTRSGYGKYDFDYWGQGTTLTVSS (SEQ ID NO: 35) 8A9 : Light chain variable domain sequence DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWY QQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQH NWEIPWTFGGGTKLEIK (SEQ ID NO: 36) 8F4 : Heavy chain variable domain sequence QIQLVQSGPELKKPGETVKISCKASDYTFTDYSMHWVKQA PGKDLKWMGWINTETGEPTYADDFKGRFAFSLEASASTAYLQINNLKNEDTATY FCARHGYPHYYFDYWGQGTTLTVSS (SEQ ID NO: 37) 8F4 : Light chain variable domain sequence DIVMTQSQKFMSTSVGDRVSITCKASQNVPTAVAWYQQKP GQSPKALIYLASNRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCLQHWNY PRTFGGGTKLEIK (SEQ ID NO: 38) 1E2 : Heavy chain variable domain sequence EVQLVESGGDLVKPGGSLKLSCAASGFSFSSYAMSWVRQT PAKRLEWVATISGSGGYTYYPDSMKGRFTISRDNAKDILYLQMSSLRSEDTAMY YCARDPRYTTLYAMDYWGQGTSVTVSS (SEQ ID NO: 39) 1E2 : Light chain variable domain sequence NIMMTQSPSFLAVSAGEKVTMSCKSSQSIFSGSNQKNYLA WYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC HQHLSSCTFGGGTKLEIK (SEQ ID NO: 40) 7H9 : Heavy chain variable domain sequence DVQLQESGPGLVKPSQSLSLTCTVTGFSISRGYDWHWIRH FPGNILEWMGYITYSGISNYNPSLKSRISITHDTSKNHFFLRLNSVTAEDTATY YCARGGGAWFTYWGQGTLVTVSA (SEQ ID NO: 41) 7H9 : Light chain variable domain sequence DIVMTQSPATLSVTPGDRVSLSCRASQSISDSLHWYHQKS HESPRLLIKYASQSISGIPSRFSAGGSGSDFTLTINSVEPEDVGVYYCQNGHSL PWTFGGGTKLEIK (SEQ ID NO: 42) 4D8 : Heavy chain variable domain sequence EVKLEESGGGLVKPGGSMKLSCAASGFTFSDAWMDWVRQS PEKGLEWVAEIRGKTTNYATYYAESVKGRFTISRDDSKSSVYLQMNSFSTEDTG IYYCTRRNWGFAYWGQGTLVTVSA (SEQ ID NO: 43) 4D8 : Light chain variable domain sequence DILLTQSPAILSVSPGERVSFSCRASQTIGTSIHWYQQRT NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQTNSW PLTFGAGTKLELK (SEQ ID NO: 44) JPEG2025118630000004.jpg41170JPEG2025118630000005.jpg41170JPEG2025118630000006.jpg49170JPEG2025118630000007.jpg59170

[0210] Example 2: Characterization of anti-SIRPA antibodies The initial characterization of SIRPA antibodies involved screening for their ability to bind to the human receptor ectopically expressed on a rodent Chinese hamster ovary cell line, henceforth referred to as CHO-huSIRPA, followed by screening on primary human macrophages. Cells were harvested and plated in 96-well plates at 10 5 Cells were plated at 100 μl per well, washed, and incubated in 100 μl of FACS buffer containing Fc blocking reagent and 1.0 μg / ml of the indicated monoclonal antibody. Cells were then washed twice and incubated on ice for 30 minutes in FACS buffer containing a 1:200 dilution of APC-conjugated secondary antibody. Cells were washed twice with cold FACS buffer and acquired on a BD FACS Canto. Data analysis and calculation of mean fluorescence intensity (MFI) values or % positive cells were performed using FlowJo (TreeStar) software version 10.0.7.

[0211] Some antibodies, such as 3F9 and 9C2, demonstrated binding to CHO-huSIRPA as indicated by positive SIRPA antibody staining detected by FACS analysis (black and white histogram) (Figure 3A). A negative isotype control (not shown) did not bind to the cells. Similarly, 3F9 and 9C2 did not bind to CHO cells highly overexpressing mouse SIRPA (CHO-mSIRPA) (Figure 3A, shaded histogram). This confirmed the specificity of the antibodies for the human antigen. Importantly, 3F9 and 9C2 also bound to primary human macrophages, the primary target cell population for in vivo efficacy (Figure 3B). MFI values for the cell lines to which the SIRPA antibodies bind are graphed in Figure 3A and listed in Table 6, typically showing MFI values greater than 100-fold above background levels. JPEG2025118630000008.jpg63170

[0212] Antigen affinity measurements for 3F9 and 9C2 were obtained using standard surface plasmon resonance (SPR) techniques (Figure 3C). Binding studies were performed using a Biacore T200 (GE). An anti-mouse IgG capture antibody was amine-coupled to a CM5 sensor chip using standard NHS / EDC activation. SIRPA antibody was diluted to 50 nM in 1x HBS-EP + running buffer and captured on the sensor chip surface. To record sensorgram traces, serial dilutions of recombinant soluble human SIRPA antigen were injected over the captured SIRPA antibody. As with buffer injections, data were processed by subtracting RU values from the reference cell. Binding curves were globally fitted to a 1:1 interaction model to obtain the rate constants listed in Table 7. 3F9 and 9C2 each exhibited a 1.0 x 10 -8 and 8.0 x 10 -8 K of M D It bound to the human monomeric SIRPA antigen. JPEG2025118630000009.jpg31170

[0213] Cell-based affinity assays were also performed to confirm the apparent affinity of 3F9 and 9C2 for cell surface antigens. Serial dilutions of the monoclonal antibodies were performed at 10 5 After the addition of fluorescently labeled secondary antibodies, which were added to CHO-huSIRPA cells and allowed to reach binding equilibrium at 4°C, and a brief washing step, MFI values as a function of titrated antibody concentration were recorded by FACS analysis (Figure 3D). Curves were fitted using nonlinear regression analysis with Graphpad Prism 6 software. Cell-based titration experiments with 3F9 and 9C2 yielded EC50 values of 2.6 nM and 1.6 nM, respectively.

[0214] Example 3: Identification of CD47-blocking and non-blocking SIRPA antibodies Given the role of the SIRPA-CD47 pathway in suppressing phagocytic cell effector function, all previously described antagonistic therapies rely on competitive inhibition to block the receptor-ligand interaction. Similarly, the SIRPA antibodies in this application were screened for their ability to block CD47 binding to CHO-huSIRPA. Cells were harvested and plated at 10 ng / well in a 96-well plate. 5 Cells were plated at 100 μl per well, washed, and incubated in 100 μl of FACS buffer containing 1.0 μg / ml of the indicated monoclonal antibody or isotype control. Cells were then washed and incubated on ice for 30 minutes in FACS buffer containing 250 nM His-tagged soluble human CD47. Cells were washed again and stained with a PE-conjugated anti-His tag monoclonal antibody to detect surface-bound CD47. Data analysis and calculation of MFI values or % positive cells were performed using FlowJo (TreeStar) software version 10.0.7.

[0215] As shown in Figure 4A, soluble CD47 specifically bound to CHO-huSIRPA cells, as indicated by positive PE staining by FACS analysis (open histogram). In the absence of CD47-His, anti-His tag antibodies failed to bind to the cells (shaded histogram). When CHO-huSIRPA cells were preincubated with the indicated SIRPA antibodies, some clones, such as 12D6 and 1B3, showed almost complete blockade of soluble CD47 binding (dashed histogram). However, 3F9 and 9C2 represent two unique clones that do not inhibit soluble CD47 binding to CHO-huSIRPA cells. The MFI values of soluble CD47-bound cells are graphed as fold over background in Figure 4B, confirming that 3F9 and 9C2 do not interfere with CD47 interaction.

[0216] Example 4: SIRPA antibodies regulate SIRPA-dependent gene expression In addition to ligand blocking, SIRPA antibodies were also screened for their ability to inhibit CD47-induced gene expression using a luciferase reporter gene under the control of the NFAT (nuclear factor of activated T cells) promoter. The cell line BW5147.G.1.4 (ATCC® TIB48™), derived from murine thymic lymphoma T lymphocytes, was infected with Cignal Lenti NFAT-luciferase virus (Qiagen) and a lentivirus expressing a human SIRPA-DAP12 chimera in which the intracellular ITIM motif of SIRPA was replaced with the intracellular ITAM motif of DAP12. Soluble human CD47 protein was serially diluted in PBS and adsorbed onto tissue culture plates. After washing, 10 cells expressing the huSIRPA / DAP12 chimera (BWZ-huSIRPA) were cultured. 5NFAT-luciferase reporter cells were seeded onto plates and incubated overnight at 37°C. Luciferase activity was measured by adding OneGlo reagent (Promega) to each well and incubating the samples for 3 minutes at room temperature on a plate shaker. Luminescence signals were quantified using a BioTek Synergy™ microplate reader with GEN5™ 2.04 software.

[0217] As shown in Figure 5A, plate-bound human CD47 induced luciferase activity in a dose-dependent manner in reporter cells expressing chimeric human SIRPA / DAP12. Importantly, parental BWZ reporter cells lacking SIRPA / DAP12 expression did not emit a luminescent signal in response to CD47. This demonstrated that the chimeric receptor mimics signaling events initiated through ligand binding. Next, anti-SIRPA antibodies were evaluated for their ability to block CD47-dependent luciferase activity in BWZ-huSIRPA reporter cells. As described above, soluble human CD47 protein was diluted in PBS and adsorbed onto 96-well tissue culture plates. After washing, 10 5 BWZ-huSIRPA reporter cells were plated with either an isotype control antibody or the indicated anti-SIRPA antibody and incubated overnight at 37°C. Figure 5B demonstrates that, according to the CD47 binding assay described above, anti-SIRPA antibodies that block CD47 binding to CHO-huSIRPA cells, such as 12D6 and 5F7, also inhibit CD47-dependent luciferase activity in reporter cells. Similarly, anti-SIRPA antibodies that do not block CD47 binding to CHO-huSIRPA cells, such as 9C2 and 3F9, also do not inhibit CD47-dependent luciferase activity in BWZ-huSIRPA cells. Furthermore, anti-SIRPA antibodies do not induce signaling in solution, as reporter cells incubated with soluble SIRPA antibodies do not emit a luminescent signal in the absence of plate-bound CD47.

[0218] Example 5: Identification of SIRPA-specific antibodies Initial characterization of SIRPA antibodies identified a class of CD47-blocking and non-blocking antibodies capable of binding to primary human myeloid cells. However, given the high sequence homology between SIRPα and SIRPβ1 (approximately 90% identity), SIRPA-specific binding remains a critical feature of an ideal anti-SIRPA lead antibody. To screen SIRPA antibodies for SIRPβ1 cross-reactivity, BWZ-NFAT / luciferase reporter cells were transduced with lentivirus expressing human SIRPβ1. Unlike SIRPα, SIRPβ1 requires coexpression of the DAP12 adaptor for global cell surface localization. Consequently, BWZ-huSIRPβ1 cells were also transduced with a lentivirus expressing human DAP12 separately. To test for luciferase activation, selected SIRPA antibodies or isotype controls were diluted to 10 μg / mL in PBS and adsorbed onto tissue culture plates. After washing, 10 cells expressing either huSIRPA / DAP12 chimera (BWZ-huSIRPA) or huSIRPβ1 + DAP12 (BWZ-huSIRPβ1) were cultured. 5 NFAT-luciferase reporter cells were seeded onto plates and incubated overnight at 37°C. Luciferase activity was measured by adding OneGlo reagent (Promega) to each well and incubating the samples for 3 minutes at room temperature on a plate shaker. Luminescence signals were quantified using a BioTek Synergy™ microplate reader with GEN5™ 2.04 software.

[0219] As shown in Figure 6A, plate-bound SIRPA antibodies induced luciferase activity in reporter cells expressing chimeric human SIRPA / DAP12 to the same extent as previously observed with plate-bound CD47. However, most SIRPA antibodies also induced luciferase activity in BWZ-huSIRPβ1 reporter cells, indicating that these antibodies cross-react with both SIRPα and SIRPβ1. Interestingly, two antibody clones, 3F9 and 9C2, specifically activated BWZ-huSIRPA cells but not BWZ-huSIRPβ1, suggesting that these two clones represent unique SIRPA-specific antibodies. To confirm this observation, we performed SPR-based binding studies using a Biacore T200 (GE). Anti-mouse IgG capture antibodies were amine-coupled to a CM5 sensor chip using standard NHS / EDC activation. Either the 3F9 or 9C2 SIRPA antibody was diluted to 50 nM in 1x HBS-EP+ running buffer and captured on the sensor chip surface. Equimolar concentrations of recombinant soluble human SIRPA antigen or human SIRPB1 antigen were injected over the captured SIRPA antibody, and sensorgram traces were recorded. Data were processed by subtracting RU values from the reference cell and buffer injections. The sensorgrams in Figure 6B clearly show an increase in response units after injection of SIRPA antigen for the capture antibodies 3F9 and 9C2. In contrast, when SIRPB1 antigen was injected over the captured antibody, almost no binding response above background was recorded. Thus, the results from Figures 6A and 6B identify clones 3F9 and 9C2 as SIRPA-specific antibodies.

[0220] Example 6: SIRPA-specific antibodies reduce cell surface expression of SIRPα in human macrophages It is often observed that antibodies targeting certain ITIM / ITAM receptors expressed on the surface of immune cells can reduce the surface levels of these receptors on monocytes, macrophages, dendritic cells, neutrophils, and / or microglia.

[0221] The ability of anti-SIRPA antibodies to reduce cell surface expression of SIRPα was evaluated in primary human macrophages (huMacs). Human monocytes were isolated from peripheral blood of healthy donors and differentiated into macrophages in vitro. After differentiation, 10 5 huMacs were harvested and seeded into 96-well tissue culture plates with either isotype control or soluble anti-SIRPA antibody at 1–5 μg / ml. After 4 hours of treatment or overnight incubation, cells were analyzed for SIRPα surface expression by flow cytometry. SIRPα expression was detected using a DyLight650-conjugated anti-human SIRPA antibody, which belongs to a distinct epitope bin from 9C2 and 3F9.

[0222] As shown in Figure 7A, the SIRPA-specific antibodies, 3F9 and 9C2, significantly reduced SIRPα expression by approximately 90% compared to isotype control-treated macrophages. FACS analysis reveals that receptor downregulation occurred within hours of antibody addition and persisted throughout overnight treatment. This contrasts with CD47-blocking antibodies, such as 1B3 or 3D2, which reduced receptor expression by less than 50%. Because antibody clones 3F9 and 9C2 are also non-CD47 blocking antibodies, other non-CD47 blocking antibodies were screened for receptor downregulation. Figure 7B shows that in most cases, non-CD47 blocking antibodies as a class significantly reduced SIRPα expression by approximately 90% or more. Again, consistent with previous observations, CD47 blocking antibodies, in this example, 5F7 and 12D6, were less effective at receptor downregulation in comparison. Thus, Figures 7A and 7B establish SIRPα downregulation as a distinct feature of non-ligand-blocking SIRPA antibodies. By reducing receptor expression, these antibodies may antagonize the SIRPα-CD47 signaling pathway through noncompetitive inhibition, a novel mechanism not previously explored in this field.

[0223] Example 7: Downregulation of SIRPα enhances phagocytosis of tumor cells by human macrophages Tumor cells evade immune surveillance through upregulation of CD47, thereby transmitting inhibitory signals to phagocytes. Therefore, antagonistic antibodies counteract this inhibition and enhance tumor cell phagocytosis. To determine whether SIRPA antibodies effectively inhibit SIRPα signaling by downregulating the receptor, a tumor cell phagocytosis assay was developed based on pH cell fluorescence acquisition. Red avidin (Invitrogen) is a streptavidin molecule conjugated with pHrodo Red dye, a fluorescent marker that acquires fluorescence in acidic environments such as phagosomes. To label target tumor cells, 500 nM red avidin was mixed with 15 nM biotinylated Lens Culinaris Agglutinin (LCA; Vector Labs). The red avidin-LCA complex was then mixed with 250,000 Raji cells at a 1:1 volume ratio in serum-free RPMI medium on ice. The carbohydrate-binding properties of LCA link red avidin to carbohydrate structures on the tumor cell surface. After a brief washing step, red avidin-LCA-labeled Raji cells were mixed with monocyte-derived human macrophages in serum-free RPMI medium and incubated for 2 hours at 37°C. Macrophages were then harvested and stained with anti-CD14 APC in FACS buffer containing FcγR-blocking antibodies on ice. Phagocytic activity was measured by counting the percentage of APC / pHrodo-double-positive macrophages. As a control, unlabeled Raji cells were mixed with macrophages to establish background fluorescence.

[0224] Figure 8A(i-ii) establishes the validity of this assay. Monocyte-derived macrophages were cultured at 10 5Cells / well were seeded into 96-well tissue culture plates and treated overnight with an isotype control antibody. The following day, 250,000 red avidin-labeled or unlabeled Raji cells were mixed with macrophages for 2 hours and then analyzed by flow cytometry. The histogram in Figure 8A(i) demonstrates the shift in pH fluorescence observed when macrophages were co-cultured with red avidin-labeled Raji cells (filled and open histogram) compared to unlabeled cells (shaded histogram). However, this shifted population was not associated with total CD14 + Opsonization of red avidin-labeled Raji cells with anti-CD20 antibody (rituximab) resulted in pHrodo + The addition of rituximab resulted in an even further shift in macrophage population (dashed histogram), consistent with antibody-dependent phagocytosis enhancing tumor cell clearance. + Macrophages are total CD14 + They represent approximately 20% of the macrophages and represent an approximately four-fold increase in phagocytic activity.

[0225] To test SIRPA antibodies, macrophages were treated overnight with the indicated candidate antibodies or isotype control. The next day, labeled Raji cells were added to the treated macrophages, followed by quantification of phagocytic activity. As shown in Figure 8B, both 3F9 and 9C2 significantly increased CD14 expression compared to isotype-treated macrophages. + / pHrodo +- macrophage populations increased 2.5-fold and 1.5-fold, respectively. Combination therapy, in which rituximab-opsonized Raji cells were added to 3F9- or 9C2-treated macrophages, further enhanced tumor cell phagocytosis compared to isotype-treated macrophages. Rituximab alone increased phagocytic activity approximately fourfold relative to untreated cells, whereas rituximab + 3F9 or rituximab + 9C2 treatment increased phagocytosis by sevenfold and sixfold, respectively. Because 3F9 and 9C2 are SIRPA-specific antibodies that do not competitively inhibit CD47 binding, Figure 8C compares the phagocytic activity of macrophages treated with CD47-blocking antibodies versus non-CD47-blocking antibodies. Of the CD47-blocking antibodies, only 12D6 and 5F7 significantly increased tumor cell uptake by approximately 30-40% compared to isotype-treated macrophages. In comparison, the phagocytic activity of 3F9-treated macrophages increased twofold. Thus, the results from Figure 8A-C establish that antibody-mediated downregulation of SIRPα on macrophages enhances the phagocytic uptake of tumor cells. Combining SIRPA antibodies with anti-tumor antigen antibodies further enhances tumor cell elimination by effector cells. Finally, compared with anti-SIRPA antibodies that competitively inhibit CD47 interaction, antibodies that noncompetitively inhibit CD47 binding by reducing SIRPα expression demonstrate superior ability to stimulate tumor cell phagocytosis by macrophages.

[0226] Example 8: Downregulation of SIRPα activates primary human monocytes While macrophages may be the primary effector cell population driving tumor cell elimination in response to anti-SIRPA therapy, SIRPA antibodies engage multiple myeloid cell lineages that express SIRPα. Among these cells are monocytes, which populate peripheral blood and are therefore readily accessible for assay target engagement upon antibody administration in vivo. To identify potential biomarkers, primary monocytes were isolated from the peripheral blood of healthy donors and assayed for activation markers after antibody treatment.

[0227] The ability of anti-SIRPA antibodies to reduce the surface expression of SIRPα was tested on monocytes. 5 Monocytes were seeded onto 96-well tissue culture plates with 5 μg / ml of either an isotype control or a soluble anti-SIRPA antibody. After overnight incubation, cells were analyzed by flow cytometry for SIRPα surface expression. SIRPα expression was detected using DyLight650-conjugated anti-human SIRPA antibodies belonging to different epitope bins. Figure 9A shows that 3F9 reduces SIRPα surface expression by 50% compared to isotype control-treated cells. Although receptor downregulation appears less robust in monocytes than previously observed in macrophages, monocytes were assayed for the production of inflammatory mediators, such as reactive oxygen species (ROS) and proinflammatory cytokines. To detect ROS production, 10 5 Monocytes were seeded onto 96-well tissue culture plates with 10 μg / ml of either an isotype control or a soluble anti-SIRPA antibody. Subsequently, the cells were labeled with 2 μM of the fluorescent dye, CM-H2DCFDA. After 1 h of antibody-mediated stimulation at 37°C, the relative fluorescence units in the cells were measured at an excitation wavelength of 495 nm and an emission wavelength of 530 nm. The relative fluorescence index of stimulated cells was obtained by subtracting the background fluorescence of labeled cells incubated with medium alone and / or isotype control antibody. Plates were read using a BioTek Synergy™ microplate reader with GEN5™ 2.04 software. Figure 9B shows that SIRPA-specific antibodies, 3F9 and 9C2, stimulated ROS production in monocytes isolated from two healthy donors. Furthermore, Figure 9C shows the effect of 10 SIRPA-specific antibodies, 3F9 and 9C2, on ROS production in monocytes isolated from two healthy donors after overnight treatment with an antibody that downregulates SIRPα. 5 monocytes produce elevated amounts of IL-8. Thus, the results from Figures 9A-C suggest that in addition to reducing receptor surface expression, anti-SIRPA antibodies also bias cells toward a more active phenotype.

[0228] Example 9: SIRPA-specific antibodies reduce cell surface expression of SIRPα in vivo To determine whether anti-SIRPA antibodies reduce cell surface expression of the receptor in an in vivo model system, we generated human BAC transgenic mice encoding the human SIRPA gene in a RAG2-deficient and IL2Rγ-chain-deficient background. The expression levels of huSIRPA were analyzed in mouse bone marrow cells by flow cytometry. As shown in Figure 10A, monocytes and granulocytes isolated from mouse peripheral blood expressed human SIRPA as well as endogenous mouse SIRPA. Bone marrow-derived macrophages and dendritic cells also expressed huSIRPA. Thus, huSIRPA-tg mice faithfully reproduce the expression pattern of human SIRPA in mouse cells. To further determine whether huSIRPA retains its inhibitory function, huSIRPA-tg mice were transplanted with Raji cells, a human B-cell lymphoma cell line that overexpresses human CD47. As shown in Figure 10B, subcutaneous administration of Raji cells resulted in solid tumor formation, suggesting that huSIRPA-tg mice support the engraftment of CD47+ human cells.

[0229] To test antibody-mediated receptor downregulation in vivo, huSIRPA-tg mice received a single intraperitoneal (ip) injection of 10 mg / kg of 3F9 (anti-SIRPA antibody) or MOPC21 (mouse IgG1 isotype control). The following day, blood samples were collected from the mice into heparin-coated collection tubes and processed for FACS analysis. Additionally, spleens were harvested and processed for FACS analysis. Briefly, blood and splenocyte samples were incubated in ACK lysis buffer for 5 minutes to lyse red blood cells and then thoroughly washed with cold PBS. Cells were then resuspended in FACS buffer (PBS + 2% FBS + Fc receptor blocking solution). Peripheral blood myeloid cells were stained with anti-mouse CD11b-Pacific Blue and either anti-human SIRPα / β-APC (clone SE5A5) or DyLight 650-conjugated 9C2, a human SIRPA-specific antibody identified by hybridoma screening. Data were acquired on a BD FACS CANTO™ II cytometer (Becton Dickinson) and analyzed with FlowJo software. As shown in Figure 10C, gating of CD11b+ blood monocytes and granulocytes labeled with anti-human SIRPα / β-APC reveals that 3F9 treatment does not reduce the cell surface levels of huSIRPA on both cell types when compared to mice treated with an isotype control. However, 3F9 treatment blocks the binding of 9C2-DyLight 650 to huSIRPA on peripheral blood cells. Because 3F9 and 9C2 bind to the same epitope, this blockade demonstrates that 3F9 occupies the receptor on peripheral blood cells without downregulating expression.

[0230] Single cell suspensions from mouse spleens were also obtained from isotype control and 3F9-treated animals. Splenocytes were stained with anti-mouse CD11b-Pacific Blue, anti-mouse F4 / 80-FITC, and anti-human SIRPα / β-APC (clone SE5A5). Data were acquired on a BD FACS CANTO™ II cytometer (Becton Dickinson) and analyzed with FlowJo software. As shown in Figure 10D, two major myeloid cell populations were identified in the spleen based on the F4 / 80 and CD11b markers: F4 / 80 and CD11b. Lo CD11b + / - population (probably red pulp macrophages) and F4 / 80 Hi CD11b Hi As shown in control-treated mice, both populations express huSIRPA, but 3F9 treatment primarily expresses F4 / 80 Lo CD11b + / - Furthermore, F4 / 80 down-regulated huSIRPA expression in IL-17 cells. Lo CD11b - The population expands only in the spleens of 3F9-treated mice. Hi CD11b Hi Observed in the spleen population.

[0231] These results demonstrate that, using huSIRPA-tg mice, anti-SIRPA antibodies bind to huSIRPA in vivo and functionally downregulate the receptor on myeloid cells. The results further demonstrate that the huSIRPA antibody 3F9 engages huSIRPA on peripheral blood cells and splenic myeloid cells but internalizes the receptor in a cell type- or context-dependent manner.

[0232] Example 10: Anti-tumor effect of anti-SIRPA antibody in a BAC transgenic mouse model To evaluate the antitumor effect of anti-SIRPA antibodies, preliminary experiments were conducted using huSIRPA-tg mice. Twelve female huSIRPA-tg mice, approximately 8 to 12 weeks old, were unilaterally implanted with 500,000 Raji-luciferase cells mixed in Matrigel solution into the right flank. Tumor engraftment was monitored from day 7 post-implantation through day 10 by caliper measurement of tumor volume and bioluminescence imaging. On day 10, tumors grew to a volume of approximately 80 to 120 mm. 3 When the mice reached 1000kJ / s, they were administered D-luciferin substrate by i.p. injection and imaged with an in vivo imaging system. The mice were then imaged using an in vivo imaging system to measure the mean radiance (photons / sec / cm) of the luciferase signal from the Raji cells. 2 Mice were randomized into treatment or control groups (6 mice per group) based on the mean tumor volume (sr) of the control group. Starting on day 10, mice received intraperitoneal injections of either 10 mg / kg 3F9 (anti-SIRPA) or a mouse IgG1 control antibody twice weekly for the duration of the study. Mice were observed daily and weighed twice weekly using a digital scale. The mean tumor volume in the control group was 1500 mm 3 The study was terminated when tumors reached a median age of 100%. At the end of the study, tumors were harvested and processed for FACS analysis. Briefly, tumor samples were treated with collagenase for 30 minutes at 37°C. Samples were dissociated through a cell strainer and resuspended in 2% FBS in PBS. Red blood cells in the samples were lysed using ACK lysis buffer, and the cells were then washed in 2% FBS in PBS. Cells were counted using a hemocytometer, and one million cells were stained with fluorescent dye-conjugated antibodies for 30 minutes on ice and then washed with 2% FBS in PBS. Cells were fixed with 4% paraformaldehyde in PBS. All stained cells were analyzed using a FACS Canto (BD Biosciences), and data were analyzed using FlowJo software (TreeStar). Tumor-infiltrating myeloid cells were stained with anti-mouse CD11b-Pacific Blue, anti-mouse F4 / 80-FITC, and anti-human SIRPα / β-APC (clone SE5A5). As shown in Figure 11A, two major myeloid populations were identified based on F4 / 80 and CD11b markers: F4 / 80 and CD11b. + CD11b+ Group (F4 / 80 + cells) and F4 / 80 - CD11b + population (CD11b + As shown in the isotype control-treated mice, both populations express huSIRPA. However, 3F9 treatment did not result in F4 / 80 - CD11b + huSIRPA expression was downregulated only in F4 / 80 cells. + CD11b + huSIRPA expression was not reduced in the cells.

[0233] As shown in Figure 11B, administration of anti-SIRPA antibody 3F9 appeared to inhibit tumor growth in vivo compared to vehicle-control treated animals, as measured by bioluminescence imaging. Linear regression analysis of mean radiance values indicates that, when corrected for pretreatment radiance values at day 10, a near-significant trend for efficacy emerged at day 17 (p = 0.06). This trend continued in subsequent measurements (p values of 0.16, 0.77, and 0.18), but given the variability in tumor growth and the limited number of available huSIRPA-tg mice, this study lacked statistical power to reach the desired significance level.

[0234] Example 11: Antitumor effect of anti-SIRPA antibody in a humanized mouse model Immunodeficient female NSG mice (Jax) transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells to reconstitute human immune cell lineages, including the myeloid and lymphoid cell compartments, served as a platform for measuring the immunomodulatory potential of anti-SIRPA antibodies. Successful engraftment of mature human immune cells was defined as >25% huCD45+ cells in the peripheral blood at 12 weeks post-injection. Furthermore, humanized mice were screened for high numbers of human CD14+, CD11b+, and CD33+ cells in the peripheral blood.

[0235] For immuno-oncology efficacy studies, humanized mice were subcutaneously implanted in the right flank with MDA-MB-231 cells, a triple-negative human breast cancer cell line that responds to checkpoint inhibitor therapy in this model system. Pretreatment tumor volume was measured with digital calipers when tumors became palpable, and tumor volumes were between 60 and 120 mm on day -1. 3 Mice were randomized into treatment or control groups (12 mice per group) when the tumor volume reached 1000 mm. Starting on day 0, mice received intraperitoneal injections of either 40 mg / kg 3F9 (anti-SIRPA) or a murine IgG1 control antibody every 4 days for the duration of the study. A third group instead received intraperitoneal injections of 10 mg / kg pembrolizumab (Keytruda, Merck) every 5 days for the duration of the study. Body weights, clinical observations, and digital caliper measurements were recorded twice weekly after treatment initiation. When the mean tumor volume in the control group reached 2000 mm, the tumor volume was 1000 mm. 3 The study was terminated when the spleen and tumor reached 100%. At termination, blood, spleen, and tumor were collected and processed for FACS analysis. Briefly, tumor samples were treated with collagenase for 30 minutes at 37°C. Spleen and tumor samples were dissociated through a cell strainer and resuspended in 2% FBS in PBS. Red blood cells in the samples were lysed using ACK lysis buffer, and the cells were then washed with 2% FBS in PBS and stained with fluorescent dye-conjugated antibodies for 30 minutes on ice. The cells were fixed with 4% paraformaldehyde in PBS. All stained cells were analyzed using a FACS Canto (BD Biosciences), and data were analyzed using FlowJo software (TreeStar).

[0236] As shown in Figure 12A, treatment with the SIRPA antibody 3F9 significantly reduced peripheral blood huCD45 expression in tumor-bearing humanized mice compared to either isotype control-treated or Keytruda-treated mice. + huCD14 + The cell surface levels of SIRPA were reduced in myeloid cells. However, the cell surface expression level of SIRPA was significantly higher than that of intratumoral huCD45. + huCD14 +These results are similar to previous observations in huSIRPA-tg mice, in which antibody-mediated receptor downregulation occurred in a cell-type- and context-dependent manner.

[0237] As shown in Figure 12B, treatment with the SIRPA antibody 3F9 significantly reduced peripheral blood huCD45 expression in tumor-bearing humanized mice compared to either isotype control-treated or Keytruda-treated mice. + huCD14 + In contrast, both 3F9 and Keytruda reduced the percentage of intratumoral huCD45 + huCD14 + Furthermore, 3F9 treatment significantly increased the percentage of myeloid cells in the peripheral blood of tumor-bearing humanized mice compared to the isotype control group. + It reduced the overall percentage of white blood cells (Figure 12C).

[0238] To account for various factors other than treatment that influence tumor growth in this model system, multiple linear regression analysis using R's lm() function was used to adjust tumor volumes for differences in: 1) huCD34+ stem cell donor, 2) day -1 tumor volume, 3) animal weight before randomization, and 4) pre-randomization huCD45+ cell engraftment rate. Figure 13A plots the mean tumor volume per group for each time point. Both 3F9 and Keytruda treatment groups significantly reduced tumor volume at early and late time points compared to the isotype control group, with the effect primarily observed between days 22 and 28. Graphical representation of tumor volume measurements by huCD34+ stem cell donor reveals that mice transplanted with human immune cells from donors 5031 and 5048 had significantly inhibited tumor growth when treated with either 3F9 or Keytruda compared to the isotype control, as shown in Figure 13B. In contrast, mice transplanted with human immune cells from donor 129 did not record a significant reduction in tumor volume in any treatment group compared to the isotype control group. Note, however, that the mean tumor volume in the control group from donor 129 recipients was lower than the control groups from donors 5031 and 5048. Such inter-donor variability in tumor growth highlights the need for appropriate controls to properly interpret results in this platform.

[0239] The data presented above demonstrate that the SIRPA antibody, 3F9, engages the receptor in vivo and induces downregulation of SIRPA in specific cell populations. Analysis of both circulating and tumor-infiltrating immune cells revealed that 3F9 treatment reduced CD14+ myeloid cells in peripheral blood and concomitantly increased CD14+ cells in tumors. Unlike Keytruda, which reduced CD4+ and CD8+ T cells in blood and tumors, 3F9 did not significantly affect T cell numbers, suggesting that it primarily acts on the myeloid compartment. Importantly, receptor downregulation and changes in myeloid cell populations with 3F9 correlated with significant tumor growth inhibition comparable to Keytruda therapy. Collectively, these studies support the preclinical efficacy of anti-SIRPA antibodies as therapeutic agents for treating human cancers.

[0240] Example 12: In silico antibody humanization of 3F9 and 9C2 Antibody humanization is used to convert antibodies generated in different species to most closely resemble human antibodies through sequence and structural relationships to prevent immunogenicity upon human administration. Antibodies from different species share characteristic sequence and structural features that allow the specificity-determining regions (SDRs) of the nonhuman antibody to be grafted onto a human antibody framework. This results in the retention of the specificity of the nonhuman antibody. The humanization process involves identifying the sequence and characteristics of a nonhuman antibody, including the framework region and SDRs. Antibodies are humanized using the following criteria: 1) the percent similarity in the framework region between the nonhuman antibody and known human antibodies, 2) the length similarity in the SDRs between the nonhuman antibody and known human antibodies, 3) the genes used to create the framework region of the human antibody, and 4) previous use of human antibody frameworks in humanization and as therapeutics. The similarity of the framework region and the length of the SDRs are important because differences result in structural differences in the antibody that can alter the specificity of the antibody. Certain genes used to create the framework of a human antibody are known to be beneficial or detrimental to antibody stability or specificity and are therefore selectively used or avoided. Finally, previously successfully humanized frameworks, including those used in human therapeutics, that are well tolerated with good half-lives may be candidates for successful humanization in the future.

[0241] As shown in Figures 14A-D, humanized light and heavy chain variable region sequences were identified for the SIRPA antibodies, 3F9 and 9C2. The initial humanized sequence of the 3F9 heavy chain variable domain (hSB-3F9-H1; Figure 14A) is a "CDR-swap" with no changes to the human framework. The subsequent humanized heavy chain sequence (hSB-3F9-H2) has altered framework residues (changes shown in bold compared to the sequence above). In Figure 14B, hSB-3F9-L1 is a "CDR-swap" of the light chain variable domain with no changes to the human framework. The subsequent humanized light chain sequence has altered framework residues (changes shown in bold compared to the sequence above; gray boxed residues are from the previous version). The light chain CDRs from 3F9 also contain potential deamidation sites (marked with a #) that can be replaced with Q, S, A, or D. Additionally, the variable domain of 3F9 contains a free Cys at position 96, which could potentially cause problems during manufacturing. This site can be substituted with an A, S, or L residue, as long as antigen binding is not altered. In Figure 14C, hSB-9C2-H1 is a "CDR swap" of the heavy chain variable domain with no changes to the human framework. The subsequent humanized heavy chain sequence has altered framework residues (changes shown in bold compared to the sequence above; gray boxed residues are from the previous version). The heavy chain CDRs from 9C2 also contain potential deamidation sites (marked with #) that can be substituted with Q, S, or A. 9C2 also contains an Asp-Gly (DG) sequence (marked with @) in CDR-H3, which may be susceptible to isoaspartic acid formation. This site can be substituted with an A, S, or E residue, as long as antigen binding is not altered. In Figure 14D, hSB-9C2-L1 is a "CDR swap" of the light chain variable domain with no changes to the human framework. The subsequent humanized light chain sequence changes framework residues (changes shown in bold compared to the sequence above; gray boxed residues are from the previous version). The light chain CDRs from 9C2 contain potential deamidation sites (marked with #) that can be substituted with Q, S, D, or A.9C2 also contains a Trp residue (marked with a ^) in CDR-L3 that may be sensitive to oxidation. This site can be substituted with H, Y, or F residues as long as antigen binding is not altered.

[0242] Example 13: Epitope mapping of anti-SIRPA antibody binding site Epitope mapping of anti-SIRPA antibodies was performed using an alanine scanning library generated by shotgun mutagenesis of the human SIRPA cDNA sequence. A SIRPA expression construct encoding a C-terminal V5 epitope tag was subjected to high-throughput alanine scanning mutagenesis (reviewed in Davidson and Doranz, 2014 Immunology 143, 13-20) to generate a comprehensive mutation library. Each residue representing the SIRPA extracellular domain (amino acids 31-374) was mutated, mostly to alanine, with alanine codons mutated to serine.

[0243] SIRPA mutant library clones arranged in 384-well microplates were individually transfected into HEK-293T cells and allowed to express for 22 hours. The antibodies were digested to generate Fabs, and the cells were then incubated with the Fabs diluted in 10% normal goat serum (NGS) (Sigma-Aldrich, St. Louis, MO). Prior to library screening, the primary Fab concentration was determined using an independent immunofluorescence titration curve against cells expressing wild-type SIRPA to ensure the signal was within the linear range of detection. The Fabs were detected using 7.5 μg / ml AlexaFluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Westgrove, PA) in 10% NGS. The cells were washed twice with PBS and resuspended in Cellstripper (Cellgro, Manassas, VA) containing 0.1% BSA (Sigma-Aldrich, St. Louis, MO). In some cases, more stringent conditions are used, such as increasing pH, temperature, and dissociation time. Mean cellular fluorescence is detected using an Intellicyt high-throughput flow cytometer (HTFC, Intellicyt, Albuquerque, NM). Fab reactivity for each mutant clone is calculated relative to wild-type SIRPA protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type SIRPA-transfected controls.

[0244] Mutant residues within library clones are identified as "critical" for the Fab-binding epitope if they do not support reactivity of the test Fab but do support reactivity of a commercially available reference antibody, MAB4546 (R&D Systems), or an additional anti-SIRPA Fab. This counterscreen strategy facilitates the elimination of SIRPA mutants that are locally misfolded or have expression defects.

[0245] Example 14: FcγRIIB downregulation by anti-SIRPA antibodies In addition to the intended target antigen, myeloid lineage cells also express multiple Fc receptors that can bind the Fc domain of therapeutic antibodies. Fcγ receptors (FcγRs) constitute the best-characterized and most potent receptor class for mediating Fc-dependent effector functions. FcγRs include both ITAM-associated activating receptors (FcγRI, FcγRIIA, and FcγRIIIA) and ITIM-containing inhibitory receptors (FcγRIIB), and coexpression of activating / inhibitory receptors on the same cell establishes a threshold for cell activation. Generally, ligation of activating FcγRs by immune complexes initiates several signaling cascades that result in cell activation and the subsequent induction of effector functions. These activities vary between myeloid cell types but can include antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and upregulation of several proinflammatory cytokines and chemokines. In contrast, ligation of the inhibitory receptor FcγRIIB by immune complexes counteracts the immunostimulatory signals of activating FcγRs, which support the maintenance of tissue homeostasis. For example, several studies have established that genetic knockout of FcγRIIB results in increased proinflammatory macrophage activity in mouse models of immune complex-mediated inflammation. Because FcγRIIB is the only FcγR with inhibitory activity, it plays a central role in regulating FcγR-mediated inflammation by myeloid cells. In the context of the tumor microenvironment, FcγRIIB expression levels may determine the polarization state of tumor-associated macrophages and regulate macrophage effector function in vivo.

[0246] To assess whether FcγRs are involved in the in vitro activity of anti-SIRPA antibodies, antibody 3F9 was treated with EndoS (New England Biolabs) to remove Fc-linked glycans. The enzymatic reaction completely cleaved the carbohydrate structures, as shown by the LCA blot in Figure 15A, which detects mannose residues on the Fc glycans. Importantly, the deglycosylation reaction did not affect antigen recognition, as both 3F9 and deglycosylated 3F9 bound equally well to SIRPA in cell-based binding assays (Figure 15B). Next, the ability of deglycosylated 3F9 to reduce the cell surface expression of SIRPA on primary human macrophages (huMacs) was compared with glycosylated 3F9. Briefly, human monocytes were isolated from the peripheral blood of two healthy donors (HD1 and HD2) and differentiated into macrophages in vitro. After differentiation, 10 5 huMacs were harvested and seeded onto 96-well tissue culture plates with increasing concentrations of anti-SIRPA antibody. After overnight incubation, cells were analyzed by flow cytometry for SIRPA surface expression. Receptor expression was detected using a DyLight650-conjugated anti-human SIRPA antibody, which belongs to a different epitope bin than 9C2 and 3F9.

[0247] As shown in Figure 16, both glycoforms of 3F9 significantly downregulated surface expression of SIRPA compared with isotype control-treated macrophages. However, in both donors, the deglycosylated 3F9 variant exhibited partially reduced activity compared with the glycosylated antibody. For example, 3F9 downregulated SIRPA expression by approximately 90% and 85% in HD1 and HD2, respectively. In contrast, deglycosylated 3F9 achieved only 70% and 75% receptor downregulation, respectively, in the same donor macrophages. This finding suggests that anti-SIRPA antibodies such as 3F9 require FcγR engagement for maximal activity.

[0248] To determine which FcγR contributes to the in vitro activity of 3F9, monocyte-derived macrophages obtained from two healthy donors were treated overnight with either an isotype control antibody or the anti-SIRPA antibody 3F9 and assessed for surface expression levels of FcγRIIIA (CD16) and FcγRIIA / B (CD32A / B). As shown in Figure 17A, 3F9 treatment moderately reduced surface expression of FcγRIIIA compared to isotype control-treated macrophages. In contrast, a significant downregulation of FcγRIIA / B was evident in 3F9-treated macrophages compared to isotype control-treated cells (Figure 17B).

[0249] Because the detection antibody used to measure surface levels of FcγRII (clone FUN-2; Biolegend) does not distinguish between activating (FcγRIIA) and inhibitory (FcγRIIB) receptors, this assay was repeated using receptor-specific antibodies. As previously described, monocyte-derived macrophages obtained from two healthy donors were treated overnight with either an isotype control antibody or the indicated glycoforms of 3F9. Figure 18 shows that 3F9 significantly downregulated FcγRIIA in macrophages by approximately 70–85% compared to isotype control-treated cells. This effect was Fc domain-dependent, as deglycosylation of the antibody abolished receptor downregulation. However, when assessing surface expression of FcγRIIB, 3F9 treatment reduced expression of the inhibitory receptor to nearly undetectable levels compared to isotype control-treated macrophages (Figure 18). Even the deglycosylated form of 3F9 showed potent downregulation of FcγRIIB, suggesting that the murine IgG1 isoform of 3F9 may preferentially associate with human FcγRIIB. Without being bound by theory, by targeting two ITIM-bearing receptors (SIRPA and FcγRIIB) so that they are downregulated, 3F9 can bias macrophages toward an activated phenotype. Therefore, in the context of tumor biology, reprogramming tumor-associated macrophages in the tumor microenvironment from a pro-tumor phenotype toward an anti-tumor phenotype using anti-SIRPA antibodies represents a promising modality for cancer immunotherapy.

[0250] All patents, patent applications, accession numbers, and other published references cited herein are hereby incorporated by reference in their entirety for the disclosure of the subject matter to which they pertain and to which they are cited herein.

Claims

1. An isolated anti-SIRPA antibody that selectively binds to signal regulatory protein alpha (SIRPA) and downregulates SIRPA expressed on the cell surface.

2. 2. The isolated anti-SIRPA antibody of claim 1, which binds to one or more polymorphic variants of human SIRPA.

3. 3. The anti-SIRPA antibody of claim 1 or 2, which reduces cell surface levels of SIRPA, reduces intracellular levels of SIRPA, reduces total levels of SIRPA, or any combination thereof.

4. 4. The anti-SIRPA antibody of claim 1, wherein the antibody induces SIRPA degradation, SIRPA cleavage, SIRPA internalization, SIRPA shedding, downregulation of SIRPA expression, or any combination thereof.

5. 5. The anti-SIRPA antibody of claim 1, which reduces cellular levels of SIRPA in vivo.

6. 6. The anti-SIRPA antibody of claim 1, which inhibits cell surface clustering of SIRPA.

7. 7. The anti-SIRPA antibody of claim 1, which inhibits one or more SIRPA activities.

8. (a) SIRPA binding to one or more SIRPA ligands, optionally wherein the one or more SIRPA ligands are selected from the group consisting of CD47, surfactant proteins A and D, and any combination thereof; (b) reducing the proliferation of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia; (c) inhibiting the migration of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia; (d) inhibiting one or more functions of one or more cells selected from the group consisting of dendritic cells, bone marrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1 microglia, activated M1 microglia, and M2 microglia; (e) inhibition of the removal of one or more selected from the group consisting of removal of apoptotic neurons, removal of neural tissue debris, removal of dysfunctional synapses, removal of non-neural tissue debris, removal of bacteria, removal of other foreign bodies, removal of disease-causing proteins, removal of disease-causing peptides, and removal of tumor cells, optionally wherein the disease-causing proteins are amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuretic factor, pancreatic islet amyloid amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation products, dipeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine the tumor cells are selected from the group consisting of bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer; (f) inhibition of tumor cell killing by one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; (g) inhibiting the anti-tumor cell proliferation activity of one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; (h) modulating the expression of one or more inflammatory receptors, optionally the one or more inflammatory receptors include CD86, and the one or more inflammatory receptors are expressed on one or more of microglia, macrophages, neutrophils, NK cells, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; (i) promoting or rescuing the functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and regulatory T cells; (j) increasing the infiltration of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, non-tumorigenic CD45+CD14+ myeloid cells, and regulatory T cells into tumors; (k) increasing the number of tumor-promoting myeloid / granulocytic immune suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells in tumors, peripheral blood, or other lymphoid organs; (l) enhancing the tumor-promoting activity of myeloid-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; (m) enhancing survival of non-tumorigenic myeloid-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; (n) reducing the activation of tumor-specific T lymphocytes with tumor-killing ability; (o) reducing the infiltration of tumor-specific NK cells with tumor-killing ability; (p) increasing tumor volume; (q) increasing the rate of tumor growth; and (r) reducing the efficacy of one or more immunotherapies that modulate anti-tumor T cell responses, optionally the one or more immunotherapies are PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VIST A, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and any combination thereof, or one or more cancer vaccines, to reduce the effectiveness of one or more immunotherapies that modulate anti-tumor T cell responses.

8. The anti-SIRPA antibody of claim 1 , which opposes one or more SIRPA activities selected from the group consisting of:

9. (a) increasing the number of tumor-infiltrating CD3+ T cells; (b) reducing cellular levels of SIRPA in non-tumorigenic CD14+ myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells or, optionally, wherein the non-tumorigenic CD14+ myeloid cells are present in the blood; (c) reducing the number of non-tumorigenic CD14+ myeloid cells, optionally the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally the non-tumorigenic CD14+ myeloid cells are present in the blood; (d) reducing PD-L1 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (e) reducing PD-L2 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (f) reducing B7-H2 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (g) reducing B7-H3 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (h) reducing CD200R levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (i) reducing CD163 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (j) reducing CD206 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (k) reducing the tumor growth rate of solid tumors; (l) reducing tumor volume; (m) increasing the efficacy of one or more PD-1 inhibitors; (n) increasing the efficacy of one or more checkpoint inhibitor therapy and / or immunomodulatory therapy, optionally the one or more checkpoint inhibitor therapy and / or immunomodulatory therapy targeting one or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (o) increasing the effectiveness of one or more chemotherapeutic agents, optionally wherein the one or more chemotherapeutic agents are gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (p) increasing T cell proliferation in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs); (l) inhibiting the differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs); and (r) killing CD33-expressing immune suppressor non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells in solid tumors and associated vasculature when conjugated with a chemical or radiotoxin; 9. The anti-SIRPA antibody of claim 1 , which induces one or more activities selected from the group consisting of:

10. 10. The anti-SIRPA antibody of claim 1, which inhibits the interaction between SIRPA and one or more SIRPA ligands.

11. 11. The anti-SIRPA antibody of claim 10, which reduces cellular levels of SIRPA and inhibits the interaction between SIRPA and one or more SIRPA ligands.

12. 10. The anti-SIRPA antibody of claim 1, which blocks binding of CD47 to human SIRPA.

13. 10. The anti-SIRPA antibody of any one of claims 1 to 9, wherein the antibody selectively binds to SIRPA and does not substantially block binding of CD47 to SIRPA expressed on cells, and further wherein binding of the antibody to SIRPA reduces SIRPA levels on the cell surface, and optionally wherein SIRPA is human SIRPA.

14. 14. The anti-SIRPA antibody of claim 13, which binds to the D1 domain of SIRPA, the D2 domain of SIRPA, or the D3 domain of SIRPA.

15. The antibody (a) comprises the amino acid sequence of SEQ ID NO: 2 Ｈ V comprising the amino acid sequence of SEQ ID NO:3 Ｌ 15. The anti-SIRPA antibody of claim 13 or 14, which competes with an antibody comprising the sequence

16. V comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

17. (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:9, a CDR1 comprising the amino acid sequence of SEQ ID NO:9 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO:9; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 10; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a CDR3 comprising the amino acid sequence of SEQ ID NO: 11 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 11 V including Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

18. V Ｈ 18. The anti-SIRPA antibody of claim 17, wherein the region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 9 with no more than one amino acid substitution, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 with no more than one amino acid substitution, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 11 with no more than one amino acid substitution.

19. V comprising CDR1 comprising the amino acid sequence of SEQ ID NO:9, CDR2 comprising the amino acid sequence of SEQ ID NO:10, and CDR3 comprising the amino acid sequence of SEQ ID NO:11 Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

20. V shown in FIG. Ｈ V containing the amino acid sequence of the region Ｈ region, or V in FIG. Ｈ V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

21. V comprising a CDR3 comprising the amino acid sequence of SEQ ID NO:8, a CDR1 comprising the amino acid sequence of SEQ ID NO:6, or a CDR2 comprising the amino acid sequence of SEQ ID NO:7 Ｌ 21. The anti-SIRPA antibody of claim 13, comprising the region:

22. (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 6; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:7, or a CDR2 comprising the amino acid sequence of SEQ ID NO:7 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO:7; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO:

8. V including Ｌ 21. The anti-SIRPA antibody of claim 13, comprising the region:

23. V Ｌ 23. The anti-SIRPA antibody of claim 22, wherein the region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 with no more than one amino acid substitution, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 7 with no more than one amino acid substitution, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 with no more than one amino acid substitution.

24. V Ｌ 21. The anti-SIRPA antibody of any one of claims 13 to 20, wherein the region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

25. V Ｌ The region is V shown in FIG. Ｌ or the amino acid sequence of the V region of FIG. Ｌ V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region Ｈ 21. The anti-SIRPA antibody of claim 13, comprising the region:

26. V comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

27. (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 15; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 16; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR3 comprising the amino acid sequence of SEQ ID NO: 17 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO:

17. V including Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

28. V Ｈ 28. The anti-SIRPA antibody of claim 27, wherein the region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 with no more than one amino acid substitution, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 with no more than one amino acid substitution, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 16 with no more than one amino acid substitution.

29. V comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 15, CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR3 comprising the amino acid sequence of SEQ ID NO: 17 Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

30. V shown in FIG. Ｈ V containing the amino acid sequence of the region Ｈ region, or V in FIG. Ｈ V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region Ｈ 16. The anti-SIRPA antibody of any one of claims 13 to 15, comprising the region:

31. V Ｌ 31. The anti-SIRPA antibody of any one of claims 26 to 30, wherein the region comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a CDR2 comprising the amino acid sequence of SEQ ID NO:

13.

32. (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 with no more than two amino acid substitutions, or a CDR1 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 12; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 with no more than two amino acid substitutions, or a CDR2 having at least about 90% identity to the amino acid sequence of SEQ ID NO: 13; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a CDR3 comprising the amino acid sequence of SEQ ID NO: 14 with no more than two amino acid substitutions, or a CDR3 having at least about 90% identity to the amino acid sequence of SEQ ID NO:

14. V including Ｌ 31. The anti-SIRPA antibody of any one of claims 26 to 30, comprising the region:

33. V Ｌ 33. The anti-SIRPA antibody of claim 32, wherein the region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 with no more than one amino acid substitution, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 with no more than one amino acid substitution, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4, or a CDR3 comprising the amino acid sequence of SEQ ID NO: 14 with no more than one amino acid substitution.

34. V comprising CDR1 comprising the amino acid sequence of SEQ ID NO:6, CDR2 comprising the amino acid sequence of SEQ ID NO:7, and CDR3 comprising the amino acid sequence of SEQ ID NO:8 Ｌ 21. The anti-SIRPA antibody of claim 13, comprising the region:

35. V Ｌ The region is V in FIG. Ｌ or V in FIG. 14D Ｈ V having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the region Ｌ 31. The anti-SIRPA antibody of any one of claims 26 to 30, comprising the region:

36. 15. The isolated anti-SIRPA antibody of claim 13 or 14, wherein the anti-SIRPA competes with one or more antibodies for binding to SIRPA, and the antibodies are selected from the group consisting of 8A9, 8F4, 1E2, 7H9, and 4D8.

37. 15. The isolated anti-SIRPA antibody of claim 13 or 14, wherein the anti-SIRPA binds to essentially the same epitope as one or more antibodies selected from the group consisting of 3F9, 9C2, 8A9, 8F4, 1E2, 7H9, and 4D8.

38. The antibody is V Ｈ Area and V Ｌ region, V Ｈ area, V Ｌ 38. The isolated anti-SIRPA antibody of claim 36 or 37, wherein the region, or both, comprises at least one, two, three, four, five, or six CDRs of a monoclonal antibody selected from the group consisting of 3F9, 9C2, 8A9, 8F4, 1E2, 7H9, and 4D8.

39. 39. The anti-SIRPA antibody of any one of claims 1 to 38, which is a monoclonal antibody.

40. 40. The anti-SIRPA antibody of any one of claims 1 to 39, which is a humanized antibody.

41. Fab, Fab', Fab'-SH, F(ab') ２ 41. The anti-SIRPA antibody of any one of claims 1 to 40, which is an Fv or scFv fragment.

42. 42. The anti-SIRPA antibody of any one of claims 1 to 41, which is a multivalent antibody.

43. 43. The anti-SIRPA antibody of any one of claims 1 to 42, which is of the IgG class, IgM class, or IgA class.

44. 44. The anti-SIRPA antibody of claim 43, having an IgG1, IgG2, IgG3, or IgG4 isotype.

45. 45. The anti-SIRPA antibody of claim 44, which binds to an inhibitory Fc receptor.

46. 46. The anti-SIRPa antibody of claim 45, wherein the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγRIIB).

47. 43. The anti-SIRPA antibody of any one of claims 1 to 42, which reduces cellular levels of FcγR.

48. 48. The anti-SIRPa antibody of claim 47, which reduces cellular levels of FcγRIIB.

49. (a) The anti-SIRPA antibody has a human or mouse IgG1 isotype and is selected from the group consisting of N297A, D265A, D270A, L234A, L235A, G237A, P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof, wherein residue numbering is according to EU numbering, or an amino acid deletion in the Fc region at a position corresponding to glycine 236; (b) the anti-SIRPA antibody has an IgG1 isotype and comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and hinge region, optionally the IgG2 isotype CH1 and hinge region comprises the amino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 34), and optionally the antibody Fc region comprises a S267E amino acid substitution, a L328F amino acid substitution, or both, and / or a N297A or N297Q amino acid substitution, wherein residue numbering is according to EU numbering; (c) the anti-SIRPA antibody has an IgG2 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, V309L, A330S, P331S, C214S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, H268E, N297A, N297Q, A330L, and any combination thereof, wherein residue numbering is according to EU numbering; (d) the anti-SIRPA antibody has a human or mouse IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, E233P, F234V, L234A / F234A, S228P, S241P, L248E, T394D, N297A, N297Q, L235E, and any combination thereof, wherein residue numbering is according to EU numbering; or (e) The anti-SIRPA antibody of claim 46, wherein the anti-SIRPA antibody has a hybrid IgG2 / 4 isotype, optionally wherein the antibody comprises an amino acid sequence comprising amino acids 118-260 of human IgG2 and amino acids 261-447 of human IgG4, wherein residue numbering is according to EU numbering.

50. (a) the anti-SIRPA antibody has a human or murine IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of N297A, N297Q, D270A, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, wherein residue numbering is according to EU numbering; (b) the anti-SIRPA antibody has an IgG2 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, wherein residue numbering is according to EU numbering; or (c) The anti-SIRPA antibody of claim 44, wherein the anti-SIRPA antibody has an IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of E233P, F234V, L234A / F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, wherein residue numbering is according to EU numbering.

51. (a) the Fc region further comprises one or more additional amino acid substitutions at positions selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, wherein residue numbering is according to EU numbering; (b) the Fc region further comprises one or more additional amino acid substitutions at positions selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein residue numbering is according to EU numbering; or 51. The anti-SIRPA antibody of claim 50, wherein (c) the Fc region further comprises a S228P amino acid substitution according to EU numbering.

52. 41. The anti-SIRPA antibody of any one of claims 1 to 40, having an IgG4 isotype.

53. 41. The anti-SIRPA antibody of claim 40, comprising an S228P amino acid substitution at residue position 228, an F234A amino acid substitution at residue position 234, and an L235A amino acid substitution at residue position 235, wherein the numbering of the residue positions is according to EU numbering.

54. 54. The anti-SIRPA antibody of any one of claims 1 to 53, which is a bispecific antibody.

55. The antibody recognizes a first and a second antigen, the first antigen being SIRPA and the second antigen being (a) an antigen that promotes transport across the blood-brain barrier; (b) an antigen that promotes transport across the blood-brain barrier selected from the group consisting of transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, llama single domain antibody, TMEM30(A), protein transduction domain, TAT, Syn-B, penetratin, polyarginine peptide, angiopep peptide, and ANG1005; (c) a disease-causing agent selected from the group consisting of a disease-causing peptide or protein, or a disease-causing nucleic acid, wherein the disease-causing nucleic acid is an antisense GGCCCC (G2C4) repeat expansion RNA, and the disease-causing protein is amyloid beta, oligomeric amyloid beta, amyloid beta plaque, amyloid precursor protein or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin an agent selected from the group consisting of: 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation products, dipeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine (PR) repeat peptides; and (d) a ligand and / or protein expressed on an immune cell, the ligand and / or protein being selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, and phosphatidylserine; and a protein, lipid, polysaccharide, or glycolipid expressed on one or more tumor cells.

55. The anti-SIRPA antibody of claim 54, wherein:

56. 56. The anti-SIRPA antibody of any one of claims 1 to 55, wherein the anti-SIRPA antibody is a conjugated antibody.

57. 57. The anti-SIRPA antibody of claim 56, conjugated to a detectable marker, toxin, or therapeutic agent.

58. 58. The anti-SIRPA antibody of claim 57, which is conjugated to a toxin selected from the group consisting of ricin, ricin A chain, doxorubicin, daunorubicin, maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alphasarcin, gelonin, mitogellin, letostrictocin, phenomycin, enomycin, chrysin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycin, dolastatin, cc1065, and cisplatin.

59. Amyloid beta, oligomeric amyloid beta, amyloid beta plaque, amyloid precursor protein or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy bodies, atrial natriuresis factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation products, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine in combination with one or more antibodies that specifically bind to a disease-causing protein selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TI 59. The anti-SIRPA antibody of any one of claims 1 to 58, used in combination with one or more antibodies that bind to an immunomodulatory protein selected from the group consisting of M1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, TREM1, TREM2, CD33, Siglec-5, Siglec-7, Siglec-9, Siglec-11, phosphatidylserine, a disease-causing nucleic acid, an antisense GGCCCC (G2C4) repeat expansion RNA, and any combination thereof.

60. A method for reducing the activity, functionality, or viability of regulatory T cells, tumor-enclosed immunosuppressor dendritic cells, tumor-enclosed immunosuppressor macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, or chronic myeloid leukemia (CML) cells in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an agent that binds to or interacts with SIRPA.

61. A method for inducing or promoting the survival, maturation, functionality, migration, or proliferation of one or more immune cells in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an agent that reduces cellular levels of SIRPA, inhibits the interaction of SIRPA with one or more SIRPA ligands, or both.

62. 62. The method of claim 61 , wherein the one or more immune cells are selected from the group consisting of dendritic cells, macrophages, neutrophils, NK cells, microglia, T cells, T helper cells, cytotoxic T cells, and any combination thereof.

63. 60. A method of treating cancer comprising administering to a patient having a tumor that expresses CD47 a therapeutically effective amount of an anti-SIRPA antibody of any one of claims 1-58.

64. A method of treating cancer comprising administering a therapeutically effective amount of an agent that reduces intracellular levels of SIRPA.

65. 65. The method of claim 64, wherein the agent is an anti-SIRPA antibody of any one of claims 1 to 58.

66. 66. The method of claim 63, 64, or 65, further comprising administering a therapeutic agent that inhibits PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73.

67. 67. The method of claim 66, wherein the therapeutic agent is an antibody that inhibits PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73.

68. 66. The method of claim 63, 64, or 65, further comprising administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule and / or one or more standard or investigational anti-cancer therapies.

69. 67. The method of claim 66, wherein at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with an anti-SIRPA antibody.

70. The at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, and an anti-HVEM antibody, an anti-B and T lymphocyte attenuator (BTLA) antibody, an anti-killer inhibitory receptor (KIR) antibody, an anti-GAL9 antibody, an anti-TIM-1 antibody, an anti-TIM3 antibody, an anti-TIM-4 antibody, an anti-A2AR antibody, an anti-CD39 antibody, an anti-CD73 antibody, an anti- 68. The method of claim 66 or 67, wherein the antibody is selected from the group consisting of an LAG-3 antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-CD30 antibody, an anti-TNFα antibody, an anti-CD33 antibody, an anti-Siglec-5 antibody, an anti-Siglec-7 antibody, an anti-Siglec-9 antibody, an anti-Siglec-11 antibody, an antagonistic anti-TREM1 antibody, an antagonistic anti-TREM2 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-CD2 antibody, an anti-CD5 antibody, and any combination thereof.

71. 67. The method of claim 66, wherein the one or more standard or investigational anti-cancer therapies are selected from the group consisting of radiation therapy, cytotoxic chemotherapy, targeted therapy, imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer (ACT) therapy, chimeric antigen receptor T cell transfer (CAR-T) therapy, vaccine therapy, and cytokine therapy.

72. 72. The method of any one of claims 63 to 71, further comprising administering to the individual at least one antibody that specifically binds to the inhibitory cytokine.

73. 73. The method of claim 72, wherein at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with an anti-SIRPA antibody.

74. 68. The method of claim 66 or 67, wherein the at least one antibody that specifically binds to an inhibitory cytokine is selected from the group consisting of an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof.

75. 75. The method of any one of claims 63 to 74, further comprising administering to the individual at least one agonist antibody that specifically binds to the stimulatory checkpoint protein.

76. 67. The method of claim 66, wherein at least one agonist antibody that specifically binds to a stimulatory checkpoint protein is administered in combination with the anti-SIRPA antibody.

77. 77. The method of claim 75 or 76, wherein the at least one agonist antibody that specifically binds to a stimulatory checkpoint protein is selected from the group consisting of an agonist anti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28 antibody, an agonist anti-TREM1 antibody, an agonist anti-TREM2 antibody, an agonist anti-CD137 / 4-1BB antibody, an agonist anti-CD27 antibody, an agonist anti-glucocorticoid-inducible TNFR-related protein GITR antibody, an agonist anti-CD30 antibody, an agonist anti-BTLA antibody, an agonist anti-HVEM antibody, an agonist anti-CD2 antibody, an agonist anti-CD5 antibody, and any combination thereof.

78. 78. The method of any one of claims 63 to 77, further comprising administering to the individual at least one stimulatory cytokine, optionally IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, an IL-20 family member, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-beta, and any combination thereof.

79. 60. A method of treating cancer comprising administering to a patient having SIRPA-expressing cancer cells of the myeloid lineage a therapeutically effective amount of an anti-SIRPA antibody of any one of claims 1 to 58.

80. 10. A method of treating cancer comprising administering to a subject having cancer a therapeutically effective amount of the anti-SIRPA antibody of any one of claims 1-58, wherein the cancer is selected from the group consisting of sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma.

81. 100. A method of treating cancer, comprising administering a therapeutically effective amount of the anti-SIRPA antibody of any one of claims 1 to 58 to a subject having cancer, wherein the cancer is selected from the group consisting of glioblastoma multiforme, renal clear cell carcinoma, adrenocortical carcinoma, bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma, pancreatic adenocarcinoma, renal cell carcinoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma, myeloma, leukemia, and leukemia. tumour, invasive carcinoma of the breast, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal carcinoma, head and neck squamous cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, hepatocellular carcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germinoma, thyroid carcinoma, thymoma, endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

82. 82. The method of claim 80 or 81, wherein the anti-SIRPa antibody is conjugated to a cytotoxic agent and / or induces ADCC.

83. 60. A pharmaceutical composition comprising the anti-SIRPA antibody of any one of claims 1 to 58 and a physiologically acceptable carrier.

84. 60. The anti-SIRPA antibody of any one of claims 1 to 58 for use in the treatment of cancer.

85. 60. An antibody described in any one of claims 1 to 58 for use in a method for preparing a medicament for the treatment of cancer.

86. 1. A method of preventing, reducing the risk of, or treating a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, taupathy, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, stroke, frontotemporal dementia, spinal cord injury, Huntington's disease, infection, and cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an agent that decreases cellular levels of SIRPA, inhibits the interaction of SIRPA with one or more SIRPA ligands, or both.

87. The disease, disorder, or injury is cancer and the agent is (a) promoting the proliferation, maturation, migration, differentiation, and / or functionality of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated suppressor neutrophils, tumor-associated suppressor NK cells, non-tumorigenic CD14+ myeloid cells, and regulatory T cells; (b) increasing the infiltration of one or more of immune suppressor dendritic cells, immune suppressor macrophages, immune suppressor neutrophils, immune suppressor NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated suppressor neutrophils, tumor-associated suppressor NK cells, and regulatory T cells into the tumor; (c) increasing the number of tumor-promoting myeloid / granulocytic immunosuppressive cells and / or non-tumorigenic CD14+ myeloid cells in tumors, peripheral blood, or other lymphoid organs; (d) increasing the tumor-promoting activity of myeloid-derived suppressor cells (MDSCs) and / or non-tumorigenic CD14+ myeloid cells; (e) increasing expression of a tumor-promoting cytokine in the tumor or peripheral blood, optionally wherein the tumor-promoting cytokine is TGF-beta or IL-10; (f) increasing tumor infiltration of tumor-promoting FoxP3+ regulatory T lymphocytes; (g) reducing the activation of tumor-specific T lymphocytes with tumor-killing ability; (h) reducing the infiltration of tumor-specific T lymphocytes with tumor-killing ability; (i) reducing the infiltration of tumor-specific NK cells with tumor-killing ability; (j) reducing the tumor-killing ability of NK cells; (k) reducing the infiltration of tumor-specific B lymphocytes, which may enhance the immune response; (l) increasing tumor volume; (m) increasing tumor growth rate; (n) increasing metastasis; (o) increasing the rate of tumor recurrence; (p) reducing the effectiveness of one or more immunotherapies that modulate an anti-tumor T-cell response, optionally the one or more immunotherapies are immunotherapies targeting one or more target proteins selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, and any combination thereof, or one or more cancer vaccines; (q) inhibition of PLCγ / PKC / calcium mobilization; and (r) Inhibition of PI3K / Akt and Ras / MAPK signaling 87. The method of claim 86, wherein the method inhibits one or more SIRPA activities selected from the group consisting of:

88. The disease, disorder, or injury is cancer and the agent is (a) increasing the number of tumor-infiltrating CD3+ T cells; (b) reducing cellular levels of CD33 in non-tumorigenic CD14+ myeloid cells, optionally the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally the non-tumorigenic CD14+ myeloid cells are present in the blood; (c) reducing the number of non-tumorigenic CD14+ myeloid cells, optionally the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells, or optionally the non-tumorigenic CD14+ myeloid cells are present in the blood; (d) reducing PD-L1 levels in one or more cells, optionally where the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (e) reducing PD-L2 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (f) reducing B7-H2 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (g) reducing B7-H3 levels in one or more cells, optionally the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (h) reducing CD200R levels in one or more cells, optionally where the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (i) reducing CD163 levels in one or more cells, optionally where the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (j) reducing CD206 levels in one or more cells, optionally where the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (k) reducing the tumor growth rate of solid tumors; (l) reducing tumor volume; (m) increasing the efficacy of one or more PD-1 inhibitors; (n) increasing the efficacy of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies, optionally wherein the one or more checkpoint inhibitor therapies and / or immunomodulatory therapies target one or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (o) increasing the effectiveness of one or more chemotherapeutic agents, optionally the one or more chemotherapeutic agents are gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (p) increasing T cell proliferation in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs); (q) inhibiting the differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs); and (r) when conjugated to a chemical or radiotoxin, exhibits one or more SIRPA activities selected from the group consisting of killing CD33-expressing immune suppressor myeloid cells and / or CD14-expressing cells in solid tumors and associated vasculature.

89. 89. The method of claim 87 or 88, wherein the cancer expresses SIRPA or one or more SIRPA ligands.

90. 86. The method of claim 85, wherein the disease, disorder, or injury is selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, taupathy, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, stroke, frontotemporal dementia, spinal cord injury, and Huntington's disease, and the agent is an anti-SIRPA antibody that downregulates SIRPA.

91. V of the anti-SIRPA antibody of any one of claims 1 to 55 Ｈ A polynucleotide comprising a nucleic acid sequence encoding a region.

92. V of the anti-SIRPA antibody of any one of claims 1 to 55 Ｌ A polynucleotide comprising a nucleic acid sequence encoding a region.

93. 93. An expression vector comprising the polynucleotide of claim 91 or the polynucleotide of claim 92.

94. 93. An expression vector comprising the polynucleotide of claim 91 and the polynucleotide of claim 92.

95. 93. A host cell comprising the polynucleotide of claim 91 or the polynucleotide of claim 92.

96. 93. A host cell comprising the polynucleotide of claim 91 and the polynucleotide of claim 92.

97. 95. A host cell comprising the expression vector of claim 93 or 94.

98. 98. A method of producing an anti-SIRPA antibody, comprising culturing a host cell of any one of claims 95-97 under conditions in which the antibody is expressed.

99. 99. The method of claim 98, wherein the host cell is a mammalian host cell.

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