Anti-sirpa antibodies and methods of use thereof

Abstract

To provide therapeutic anti-SIRPA antibodies to treat diseases, disorders, and conditions associated with undesired SIRPA activity.SOLUTION: The present disclosure is generally directed to compositions that include antibodies, e.g., monoclonal antibodies, antibody fragments, etc., that specifically bind a SIRPA polypeptide, e.g., a mammalian SIRPA or human SIRPA, and use of such compositions in preventing, reducing risk, or treating an individual in need thereof.SELECTED DRAWING: Figure 10A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 676,813, filed May 25, 2018, which is incorporated herein by reference for any purpose.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 14, 2019, is named "40004-PCT_SL.txt" and is 99,798 bytes in size.

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

[0004] Phagocytic cells, such as macrophages (MΦ) and dendritic cells (DCs), distinguish healthy from diseased cells through a complex array of cell surface receptors that modulate the cell's activation state, proliferation, and / or effector function. Many of these receptors recognize diverse ligands that either mark unwanted cells for elimination (so-called "phagocytic" signals) or protect normal cells from destruction (so-called "antiphagocytic" signals). In recent years, the SIRPα-CD47 axis has emerged as a crucial determinant of programmed cell elimination by macrophages in a variety of clinical settings, ranging from cancer cell survival to successful hematopoietic cell transplantation. Therapeutics that affect this pathway may fill an important medical need, particularly in the context of multiple human cancers, potentially ameliorating disease.

[0005] SIRPα (signal-regulatory protein α) belongs to the SIRP family of transmembrane receptors, primarily expressed in myeloid cell lineages (including MΦ, DC, and granulocytes) and characterized by an extracellular region containing two membrane-proximal IgC and two membrane-distal IgV domains. SIRPα is unique among its family in that it contains an intracellular, cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). Upon receptor crosslinking, the tyrosine-phosphorylated TIM site recruits and activates SHP phosphatases, negatively regulating cellular functions such as phagocytosis or the release of inflammatory cytokines. CD47 serves as the primary ligand for SIRPα, and its widespread expression in most cell types, including endothelial / epithelial cells, leukocytes, and erythrocytes, suggests that it mediates a "stop-phagocytosis" signal to protect healthy cells from phagocyte-dependent elimination. Several studies support this view, demonstrating 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, gene location analysis in 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 confirmed that allelic variants of SIRPα expressed only in NOD mice retain the ability to bind human CD47, which is expressed on human hematopoietic stem cells, and thus suppress macrophage-dependent graft rejection.

[0006] Regulation of SIRPα and CD47 expression establishes a homeostatic control mechanism that modulates phagocyte activity. For example, apoptotic cells downregulate CD47 expression to facilitate engulfment by resident macrophages, while viable cells remain intact. Similarly, inflammatory stimuli such as LPS reduce SIRPα expression in macrophages and dendritic cells (DCs), enhancing their activation during inflammation. However, dysregulation of SIRPα and CD47 expression contributes to immune-related diseases, as seen in cancer. Some tumors significantly increase CD47 expression compared with noncancerous cells to evade immune surveillance, which normally eliminates 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 transplanted into immunocompromised mice. Alternatively, biologic agents that disrupt the SIRPα-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 facilitate synergistic enhancement of anti-tumor responses compared with standard monotherapy. However, given the ubiquitous expression of CD47, anti-CD47 antibodies carry the risk of severe toxicities due to off-target effects, limiting their therapeutic efficacy. Nevertheless, these studies establish a crucial role for the SIRPα-CD47 pathway in regulating myeloid cells, with potential applications in cancer immunotherapy.

[0007] Anti-SIRPA antibodies are described, for example, in International Patent Application Publication Nos. WO2018 / 057669, WO2018 / 026600, WO2017 / 178653, WO2017 / 068164, WO2016 / 063233, WO2016 / 205042, WO2015 / 138600, WO2013 / 0956352, WO2009 / 091547, WO2009 / 131453, and WO2009 / 046541. Summary of the Invention

[0008] Thus, there is a need for therapeutic anti-SIRPA antibodies to treat diseases, disorders, and conditions associated with unwanted SIRPA activity.

[0009] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0010] overview The present disclosure is generally directed to compositions comprising antibodies, eg, monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, etc., that specifically bind to human SIRPA, and methods of using such compositions.

[0011] Certain embodiments of the present disclosure relate, at least in part, to anti-SIRPA antibodies with improved and / or enhanced functional properties (e.g., compared to an anti-SIRPA antibody with a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, including, for example, an improved and / or enhanced ability to reduce cell surface levels of SIRPA on cells), and / or to anti-SIRPA antibodies with improved and / or enhanced binding kinetics values, and / or improved and / or enhanced KD values, and / or improved and / or enhanced EC50 values. In some embodiments, the anti-SIRPA antibodies of the present disclosure have a KD for human SIRPA that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower than that of an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-SIRPA antibodies of the present disclosure have a KD for human SIRPA of less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM (nm), or less than 0.5 nM.

[0012] In some embodiments, anti-SIRPA antibodies of the present disclosure reduce in vitro cell surface expression levels of SIRPA with an EC50 that is at least about 20%, at least about 30%, at least about 40%, or at least about 50% lower than an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. Advantageously, anti-SIRPA antibodies of the present disclosure reduce in vitro cellular levels of SIRPA with a 50% effective concentration (EC50) in the range of about 0.4 nM to about 0.5 nM. Advantageously, anti-SIRPA antibodies of the present disclosure have a dissociation constant (KD) for human SIRPA in the range of about 0.6 nM to 0.7 nM.

[0013] In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure binds to human SIRPA v1. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure binds to human SIRPA v2. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure binds to human SIRPB (SIRP beta) v3. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure does not bind to human SIRPB v1. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure does not bind to mouse SIRPA. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure does not bind to human SIRPγ. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present disclosure binds to cynomolgus monkey SIRPA. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the present disclosure do not bind to cynomolgus monkey SIRPB1. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the present disclosure bind to marmoset SIRPA.

[0014] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody binds to human SIRPA, human SIRPA v1, human SIRPA v2, cynomolgus monkey SIRPA, marmoset SIRPA, and human SIRPβ3.

[0015] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24.

[0016] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0017] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0018] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, or at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35.

[0019] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the light chain variable region comprises an amino acid sequence that is at least 90%, or at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0020] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises one, two, three, or four framework regions selected from VH FR1 comprising the amino acid sequence of SEQ ID NO: 25, VH FR2 comprising the amino acid sequence of SEQ ID NO: 26, VH FR3 comprising the amino acid sequence of SEQ ID NO: 27, and VH FR4 comprising the amino acid sequence of SEQ ID NO: 28.

[0021] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the light chain variable region comprises one, two, three, or four framework regions selected from VL FR1 comprising the amino acid sequence of SEQ ID NO: 29, VL FR2 comprising the amino acid sequence of SEQ ID NO: 30, VL FR3 comprising the amino acid sequence of SEQ ID NO: 31, and VL FR4 comprising the amino acid sequence of SEQ ID NO: 32.

[0022] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises one, two, three, or four framework regions selected from VH FR1 comprising the amino acid sequence of SEQ ID NO: 25, VH FR2 comprising the amino acid sequence of SEQ ID NO: 26, VH FR3 comprising the amino acid sequence of SEQ ID NO: 27, and VH FR4 comprising the amino acid sequence of SEQ ID NO: 28; and the light chain variable region comprises one, two, three, or four framework regions selected from VL FR1 comprising the amino acid sequence of SEQ ID NO: 29, VL FR2 comprising the amino acid sequence of SEQ ID NO: 30, VL FR3 comprising the amino acid sequence of SEQ ID NO: 31, and VL FR4 comprising the amino acid sequence of SEQ ID NO: 32.

[0023] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35.

[0024] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0025] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0026] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, 3F9-26, 3F9-27, 3F9-28, 3F9-29, 3F9-30, 3F9-31, 3F9-32, 3F9-33, 3F9-34, 3F9-35, 3F9-36, 3F9-37, 3F9-38, 3F9-39 ...9, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25, comprising HVR-H1, HVR-H2, and HVR-H3 (as shown in Table 8).

[0027] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region is selected from the group consisting of antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, 3F9-26, 3F9-27, 3F9-28, 3F9-29, 3F9-30, 3F9-31, 3F9-32, 3F9-33, 3F9-34, 3F9-35, 3F9-36, 3F9-37, 3F9-38, 3F9-39 ...9, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9-39, 3F9 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25, comprising HVR-L1, HVR-L2, and HVR-L3 (as shown in Table 7).

[0028] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25. and the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3 (shown in Table 7) of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

[0029] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, and a light chain variable region comprising HVR-L1, HVR-L2, and HVR-L3; and is selected from the group consisting of antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, 3F9-26, 3F9-27, 3F9-28, 3F9-29, 3F9-30, 3F9-31, 3F9-32, 3F9-33, 3F9-34, 3F9-35, 3F9-36, 3F9-37, 3F9-38, 3F9-39, 3F9-39 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25 (as shown in Tables 7 and 8).

[0030] In some embodiments that may be combined with any of the embodiments herein, the present disclosure relates to an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises one, two, three, or four framework regions selected from VH FR1, VH FR2, VH FR3 (VH,Fr3), and VH FR4, wherein VH FR1 comprises the amino acid sequence of SEQ ID NO: 25; VH FR2 comprises the amino acid sequence of SEQ ID NO: 26; VH FR3 comprises the amino acid sequence of SEQ ID NO: 27; VH FR4 comprises the amino acid sequence of SEQ ID NO: 28; and / or the light chain variable region comprises one, two, three, or four framework regions selected from VL FR1, VL FR2, VL FR3, and VL FR4, wherein VL FR1 comprises the amino acid sequence of SEQ ID NO: 29; VL FR2 comprises the amino acid sequence of SEQ ID NO: 30; An antibody is presented, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 31; and VL FR4 comprises the amino acid sequence of SEQ ID NO: 32.

[0031] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 49 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0032] In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated anti-SIRPA antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 47 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0033] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 48 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0034] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 49 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0035] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 53 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0036] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 54 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0037] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 51 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0038] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 52 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0039] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 55 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0040] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 56 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0041] In some embodiments, which may be combined with any of the embodiments herein, the invention provides an isolated anti-SIRPA antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 57 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0042] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody is a monoclonal antibody.

[0043] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody is a humanized antibody.

[0044] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment.

[0045] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody is a multivalent antibody.

[0046] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, the antibody being of the IgG class, the IgM class, or the IgA class.

[0047] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, the antibody being an IgG class antibody and being an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype.

[0048] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody binds to an inhibitory Fc receptor. In some embodiments that may be combined with any of the embodiments herein, the inhibitory Fc receptor is inhibitory Fc gamma receptor IIB (FcγRIIB).

[0049] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cellular levels of FcγRIIB.

[0050] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein 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, Antibodies are provided which comprise one or more amino acid substitutions at amino acid residues in the Fc region selected from the group consisting of P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof (residue numbering according to EU numbering), or which comprise an amino acid deletion at a position corresponding to glycine 236 in the Fc region.

[0051] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises one or more amino acid substitutions at residue positions in the Fc region selected from the group consisting of C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof (wherein the numbering of the amino acid residues is according to EU numbering or Kabat numbering).

[0052] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody reduces cell surface levels of SIRPA, reduces intracellular levels of SIRPA, reduces total cellular levels of SIRPA, or any combination thereof.

[0053] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody induces degradation of SIRPA, induces cleavage of SIRPA, induces internalization of SIRPA, induces release of SIRPA, induces downregulation of expression of SIRPA, or any combination thereof.

[0054] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA in vitro.

[0055] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA in vivo.

[0056] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates the expression of SIRPA in human monocytes.

[0057] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates the expression of SIRPA in human macrophages.

[0058] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates SIRPA expression in human macrophages by about 70-95%.

[0059] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and has an affinity (KD) for human SIRPA of less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.

[0060] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, the antibody having an affinity (KD) for human SIRPA of about 0.1 nM to 2 nM.

[0061] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, having an affinity for human SIRPA with a KD that is at least half, at least three, at least four, at least five, at least six, at least seven, at least eighth, at least ninth, or at least tenth of the affinity for human SIRPA of an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.

[0062] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA v1 in vitro with a 50% effective concentration (EC50) of about 0.05 nM to 2 nM or about 0.4 nM to about 0.5 nM, as measured by flow cytometry.

[0063] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA in vitro with a 50% effective concentration (EC50) of about 0.05-0.20 nM for human SIRPA v1, about 0.05-0.10 nM for human SIRPA v2, and / or about 0.05-1 nM for cynomolgus monkey SIRPA, as measured by flow cytometry.

[0064] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody binds to the D3 domain of human SIRPA v1 of SEQ ID NO: 1.

[0065] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody binds to amino acid residues R282, Q284, and G337 of human SIRPA v1 of SEQ ID NO: 1.

[0066] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody increases phagocytosis of tumor cells in macrophages, increases phagocytosis of tumor cells in M1 macrophages, increases phagocytosis of tumor cells in M2 macrophages, downregulates CD14 expression in macrophages, and / or any combination thereof.

[0067] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody enhances T cell proliferation.

[0068] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody blocks the interaction or binding of SIRPA with surfactant protein D (SP-D).

[0069] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody does not block the interaction or binding of SIRPA with CD47.

[0070] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates expression of CD32A / B on the cell surface.

[0071] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates CD14 expression on the cell surface.

[0072] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody enhances T cell proliferation without blocking the interaction of SIRPγ with CD47.

[0073] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody stimulates the production of ROS in monocytes and / or increases the expression of IL-8 in monocytes.

[0074] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody inhibits tumor growth in vivo.

[0075] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody reduces the number of CD14+ myeloid cells in peripheral blood and / or increases the number of CD14+ myeloid cells in a tumor.

[0076] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody binds to human SIRPA but does not substantially block binding of CD47 to SIRPA.

[0077] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody recognizes a first antigen and a second antigen, wherein the first antigen is SIRPA and the second antigen is (a) antigens that facilitate transport across the blood-brain barrier; (b) an antigen that facilitates transport across the blood-brain barrier selected from 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), and diphtheria toxin receptor; (c) a disease-causing agent selected from a disease-causing peptide or protein, and 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, α-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, or ataxin 1; a disease-causing agent selected from 0, 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 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 an immune cell, the ligand and / or protein being selected from 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. The antibody is presented.

[0078] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is conjugated to a peptide that facilitates transport across the blood-brain barrier. In some embodiments, the peptide is selected from CRM197, a protein transduction domain, TAT, Syn-B, penetratin, a polyarginine peptide, an angiopep peptide, and ANG1005.

[0079] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is an opsonizing antibody.

[0080] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is a conjugated antibody.

[0081] In some embodiments, which may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is conjugated to a detectable marker, a toxin, or a therapeutic agent.

[0082] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is 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, mitogenin, restrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin, Saponaria officinalis inhibitors, glucocorticoids, auristatins, auromycins, yttrium, bismuth, combrestatins, duocarmycins, dolastatins, cc1065, and cisplatin.

[0083] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein 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, α-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 natriuretic factor, islet amyloid polypeptide, and the like. one or more antibodies that specifically bind to a disease-causing protein selected from: i) tide, 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 any combination thereof;or an antibody used in combination with one or more antibodies that bind to an immunomodulatory 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, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, TREM1, TREM2, CD33, Siglec-5, Siglec-7, Siglec-9, Siglec-11, phosphatidylserine, disease-causing nucleic acids, antisense GGCCCC(G2C4) repeat-expanded RNA, and any combination thereof;

[0084] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of preventing, reducing the risk of, or treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, thereby treating the cancer. In some embodiments that may be combined with any of the embodiments herein, the present invention provides a method of preventing, reducing the risk of, or treating an individual having a disease, disorder, or injury that is cancer, comprising administering to the individual a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, thereby preventing, reducing the risk of, or treating the individual.

[0085] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, 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, small cell lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma, glioblastoma multiforme; renal clear cell carcinoma; adrenocortical carcinoma; bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma; pancreatic adenocarcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, indolent B-cell lymphoma, aggressive B-cell lymphoma, T-cell lymphoma, and leukemia. In one embodiment, the tumor is selected from the group consisting of lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, renal chromophobe, renal papillary 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 germ cell tumors, thyroid carcinoma, thymoma, endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

[0086] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, and 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. In some embodiments that may be combined with any of the embodiments herein, 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.

[0087] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, and 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 that may be combined with any of the embodiments herein, 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 that may be combined with any of the embodiments herein, the at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with an anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, anti-PD-1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, and anti-HVEM antibody, anti-BTLA (B- and T-lymphocyte attenuator) antibody, anti-KIR (Killer inhibitory receptor), anti-GAL9 antibody, anti-TIM-1 antibody, anti-TIM3 antibody, anti-TIM-4 antibody, anti-A2AR antibody, anti-CD39 antibody, anti-CD73 antibody, anti-LAG-3 antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-CD30 antibody, anti-TNFα antibody, anti-CD33 antibody, anti-Siglec-5 antibody, anti-Siglec-7 antibody, anti-Siglec-9 antibody, anti-Siglec-11 antibody, antagonistic anti-TREM1 antibody, antagonistic anti-TREM2 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-CD2 antibody, anti-CD5 antibody, and any combination thereof.

[0088] In some embodiments that may be combined with any of the embodiments herein, the one or more standard or investigational anti-cancer therapies are selected from 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.

[0089] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, and further comprising administering to the individual at least one antibody that specifically binds to an inhibitory cytokine. In some embodiments that may be combined with any of the embodiments herein, the at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with an anti-SIRPA antibody. In some embodiments that may be combined with any of the embodiments herein, the at least one antibody that specifically binds to an inhibitory cytokine is selected from an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof.

[0090] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, and further comprising administering to the individual at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein. In some embodiments that may be combined with any of the embodiments herein, the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is administered in combination with the anti-SIRPA antibody. In some embodiments that may be combined with any of the embodiments herein, the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is selected from 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.

[0091] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present disclosure, and further comprising administering to the individual at least one stimulatory cytokine. In some embodiments, the at least one stimulatory cytokine is selected from IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 family members, 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.

[0092] In some embodiments, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the present disclosure. In some embodiments, the present invention provides a vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the present disclosure. In some embodiments, the present invention provides an isolated host cell comprising a vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the present disclosure.

[0093] In some embodiments, the invention provides a method of producing an antibody that binds to human SIRPA, the method comprising culturing a host cell comprising a vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the disclosure, such that the antibody is produced. The invention further provides recovering the antibody produced by the cell.

[0094] In some embodiments, the invention provides a pharmaceutical composition comprising an anti-SIRPA antibody of the present disclosure and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0095] [Figure 1]

[0023] Figure 1 shows an alignment of the amino acid sequences of human SIRPA variant 1 and the extracellular domains of SIRPA proteins from various species. The reported percent identity confirms the high homology within the extracellular region. The accession numbers are P78324 (human SIRPA variant 1), XP015313153 (cynomolgus monkey), JAB51896 (marmoset), G1U0I5 (rabbit), and XP005634938 (dog). SEQ ID NOs: 59-63 are disclosed in order of appearance, respectively. [Figure 2A]

[0023] Figure 1 lists potential humanized sequences of the heavy chain variable domain of the anti-SIRPA antibody, 3F9. The humanized sequences are based on the IGHV3-23*01 acceptor framework and the IGHJ4*01 junction region. SEQ ID NOs: 64-65, 2, 64, and 3-4, respectively, are disclosed in order of appearance. [Figure 2B]

[0023] Figure 1 lists potential humanized sequences of the light chain variable domain of the anti-SIRPA antibody, 3F9. The humanized sequences are based on the IGHV3-23*01 acceptor framework and the IGHJ4*01 junction region. SEQ ID NOs: 66-67, 5, 66, and 68-70 are disclosed in order of appearance, respectively. [Figure 3] FIG. 10 presents data demonstrating antibody-mediated down-regulation of SIRPA on primary human macrophages by murine and humanized anti-SIRPA3F9 antibodies. [Figure 4A] FIG. 1 presents data demonstrating that antibody-mediated receptor down-regulation is dependent on Fc N-linked glycans. [Figure 4B] We present data demonstrating that antibody-mediated enhancement of phagocytosis of tumor cells is dependent on N-linked glycans of Fc. [Figure 5A] FIG. 10 presents data showing that humanized anti-SIRPA 3F9 IgG4 antibody mutants were unable to induce phagocytosis of tumor cells. [Figure 5B] FIG. 10 presents data showing that humanized anti-SIRPA 3F9 IgG4 antibody mutants are unable to downregulate CD14 expression on macrophages. [Figure 6A] FIG. 1 shows that treatment with anti-SIRPA antibody, 3F9 mIgG1, down-regulates CD32A / B expression on primary human macrophages. [Figure 6B] FIG. 1 shows that treatment with anti-SIRPA antibody, 3F9, down-regulates CD32A and CD32B expression on primary human macrophages. [Figure 7A] FIG. 10 presents data showing that blockade of CD32A / B inhibits anti-SIRPA antibody, 3F9, mediated down-regulation of SIRPA on primary human macrophages. [Figure 7B] FIG. 10 presents data showing that blockade of CD32A / B inhibits anti-SIRPA antibody, 3F9-mediated enhancement of phagocytosis of tumor cells. [Figure 8A]

[0023] Figure 10 presents data showing that CD32A allotype influenced anti-SIRPA antibody 3F9-mediated downregulation of SIRPA. Donors 507 and 508 were CD32A-R / H131 heterozygous. [Figure 8B]

[0023] Figure 10 presents data showing that the CD32A allotype influenced anti-SIRPA antibody 3F9-mediated downregulation of SIRPA. Donors 516 and 517 were CD32A-H / H131 homozygous. [Figure 9A-9B] Figure 9 presents data demonstrating that antibody-mediated receptor downregulation leads to SIRPA degradation. In Figure 9A, 5 μg, 10 μg, and 20 μg of whole cell lysate were loaded onto SDS-PAGE and immunoblotted with anti-SIRPA cytoplasmic domain (a-SIRPAct). In Figure 9B, 20 μg of whole cell lysate was loaded onto SDS-PAGE and probed with a-SIRPAct and anti-SHP2. [Figure 10A]

[0023] Figure 10 presents data comparing the enhanced phagocytic activity of different Fc variants of the anti-SIRPA antibody, 3F9-22. Primary human macrophages were treated with the anti-SIRPA 3F9-22 antibody on a wild-type human IgG1 backbone, or on a human IgG1 bearing the S267E / L328F Fc mutant antibody, or an isotype control antibody. [Figure 10B] 10 presents data comparing the enhanced phagocytic activity of different Fc variants of the anti-SIRPA 3F9-22 antibody. Primary human macrophages were treated with the anti-SIRPA 3F9-22 antibody on a human IgG1 backbone bearing the S267E / L328F or A330S / P331S Fc mutant antibodies, or an isotype control antibody. [Figures 11A-11B] FIG. 11 presents data comparing the phagocytic activity of different Fc variants of the anti-SIRPA 3F9-22 antibody compared to anti-CD47 on M1 macrophages (FIG. 11B) and M2 macrophages (FIG. 11A). [Figures 12A-12B]FIG. 1 presents data showing that macrophages treated with the anti-SIRPA antibody, 3F9-22, reduced solid tumor cell viability in vitro. [Figures 13A-13B] Figure 13A presents data comparing the enhancement of T cell proliferation by different Fc variants of anti-SIRPA antibody 3F9-22 in a two-way MLR assay. Figure 13B presents data comparing the enhancement of T cell proliferation by different Fc variants of anti-SIRPA antibody 3F9-22 and anti-SIRPA antibody 3F9-14 compared to anti-CD47 IgG4 in a two-way MLR assay. [Figure 14A] FIG. 10 presents data comparing the enhancement of T cell proliferation by different Fc variants of the anti-SIRPA 3F9-22 antibody compared to anti-CD47 IgG4 in a one-way MLR assay. [Figure 14B] FIG. 1 shows representative FACS plots from a one-way MLR assay. [Figures 15A-15B] FIG. 10 presents data comparing the enhancement of TNF release by LPS-primed macrophages by different Fc variants of the anti-SIRPA antibody, 3F9-22. [Figure 16] FIG. 1 presents data showing the identification and visualization of "critical" residues for anti-SIRPA antibodies in a model of human SIRPA. [Figure 17A] Figure 10 presents data showing the effect of an anti-SIRPA antibody, 3F9-22, in combination with an anti-PD-L1 antibody on tumor growth inhibition in a syngeneic mouse colon cancer model.Figure 10 presents FACS histograms showing the expression of mouse CD47 (upper panel) and human CD47 (lower panel) in wild-type and engineered MC38 cell lines. [Figure 17B]Figure 10 presents data showing the effect of an anti-SIRPA antibody, 3F9-22, in combination with an anti-PD-L1 antibody on tumor growth inhibition in a syngeneic mouse colon cancer model. Figure 10 presents data showing the mean tumor growth curves for huSIRPA / huCD47 BAC transgenic mice implanted subcutaneously with MC38 cells engineered to lack expression of mouse CD47 and overexpress human CD47 (MC38-mCD47KO / huCD47+). [Figure 17C-1] Figure 17B presents data showing the effect of an anti-SIRPA antibody, 3F9-22, in combination with an anti-PD-L1 antibody on tumor growth inhibition in a syngeneic mouse colon cancer model. Spaghetti plots are presented for each individual animal within each treatment group in Figure 17B. [Figure 17C-2] Figure 17B presents data showing the effect of an anti-SIRPA antibody, 3F9-22, in combination with an anti-PD-L1 antibody on tumor growth inhibition in a syngeneic mouse colon cancer model. Spaghetti plots are presented for each individual animal within each treatment group in Figure 17B. [Figure 18A]

[0023] Figure 10 presents data showing the kinetics of antibody-mediated downregulation of SIRPA in bone marrow cells of tumor-bearing BAC transgenic mice treated with increasing doses of the anti-SIRPA antibody of the present disclosure.

[0024] Figure 10 presents data showing the relative changes in SIRPA expression on tumor-infiltrating bone marrow cells over time. [Figure 18B] Figure 10 presents data showing the kinetics of antibody-mediated downregulation of SIRPA in bone marrow cells of tumor-bearing BAC transgenic mice treated with increasing doses of the anti-SIRPA antibody of the present disclosure. Figure 10 presents data showing the relative changes in SIRPA expression on splenic bone marrow cells over time. [Figure 19A] FIG. 1 shows the treatment conditions for anti-SIRPA antibody-mediated downregulation of SIRPA studies in A375 tumor-bearing humanized NOG-EXL mice. [Figure 19B]FIG. 10 presents data showing the expression levels of SIRPA on human tumor macrophages, as MFI values ​​(left panel) or normalized values ​​(right panel), associated with treatment with anti-SIRPA antibodies of the present disclosure. [Figure 20A] 1 shows the expression levels of HLA-DR / MHC class 2, a marker of M1 macrophages, on human tumor macrophages isolated from humanized NOG-EXL mice bearing A375 tumors after treatment with the anti-SIRPA antibody of the present invention. Data are presented showing the expression of HLA-DR / MHC class 2 and CD86 as MFI values ​​(left panel) or normalized values ​​(right panel), respectively. [Figure 20B] 1 shows the expression levels of CD86, a marker of M1 macrophages, on human tumor macrophages isolated from humanized NOG-EXL mice bearing A375 tumors after treatment with an anti-SIRPA antibody of the present invention. Data are presented showing the expression of HLA-DR / MHC class 2 and CD86 as MFI values ​​(left panel) or normalized values ​​(right panel), respectively. [Figure 21A] 1 shows the downregulation of human SIRPA in splenic monocytes isolated from humanized NOG-EXL mice bearing A375 tumors, associated with administration of an anti-SIRPA antibody of the present invention. Data showing the expression of human SIRPA as MFI values ​​(left panel) or normalized values ​​(right panel) are presented. [Figure 21B] 1 shows the downregulation of human SIRPA in peripheral blood monocytes isolated from humanized NOG-EXL mice bearing A375 tumors, associated with administration of an anti-SIRPA antibody of the present invention. Data showing the expression of human SIRPA as MFI values ​​(left panel) or normalized values ​​(right panel) are presented. [Figure 22] FIG. 10 presents data demonstrating antibody-mediated down-regulation of SIRPA on primary human macrophages by different Fc variants of the anti-SIRPA 3F9-22 antibody. [Figure 23]FIG. 10 presents data comparing the enhancement of phagocytic activity of different Fc variants of the anti-SIRPA antibody, 3F9-22, compared to an anti-CD47 blocking antibody. [Figure 24] FIG. 10 presents data showing red blood cell and platelet counts in cynomolgus monkeys administered a control IgG antibody, the anti-SIRPA antibody 3F9-22 PS, the anti-SIRPA antibody 3F9-22 IgG4, the anti-SIRPA antibody 3F9-IgG2, and the anti-SIRPA antibody 3F9-22 NSLF. DETAILED DESCRIPTION OF THE INVENTION

[0096] It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the Detailed Description that follows.

[0097] The present disclosure relates to anti-SIRPA antibodies (e.g., monoclonal antibodies); methods of making and using such antibodies; pharmaceutical compositions comprising such antibodies; nucleic acids encoding such antibodies; and host cells comprising nucleic acids encoding such antibodies.

[0098] The techniques and procedures described or referred to herein are generally similar to those described in, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd ed. (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; "Current Protocols in Molecular Biology," (FMAusubel et al., eds., (2003)); "Methods in Enzymology" series (Academic Press, Inc.): "PCR2: A Practical Approach" (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988); "Antibodies, A Laboratory Manual," and "Animal Cell Culture" (RI Freshney, ed. (1987)); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Methods in Molecular Biology," Humana Press; "Cell Biology: A Laboratory Manual," vol. "Notebook" (ed. J.E.Cellis, 1998), Academic Press; "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Introduction to Cell and Tissue Culture" (J.P. Mather and P.E. Roberts, 1998), Plenum Press; "Cell and Tissue Culture: Laboratory Procedures" (eds. A. Doyle, J.B. Griffiths, and D.G. Newell, August 1993), J. Wiley and Sons; "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P.Calos (eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al. (eds., 1994); "Current Protocols in Immunology" (JE Coligan et al. (eds., 1991); "Short Protocols in Molecular Biology" (Wiley and Sons, 1999); "Immunobiology" (C.A. Janeway and P. Travers, 1997); "Antibodies" (P. Finch, 1997); "Antibodies: A Practical Approach" (D. Catty (ed., IRL Press, 1988-1989); "Monoclonal Antibodies: A Practical Approach" (P. Shepherd and C. Dean (eds., Oxford University Press, 2000); "Using Antibodies: A Laboratory Manual" (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); "The Antibodies" (M. Zanetti and J.D. Capra (eds., Harwood Academic Press, Publishers, 1995); and the widely used methods described in "Cancer: Principles and Practice of Oncology" (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993), are well understood and commonly employed by those skilled in the art using routine methods.

[0099] I. Definition As used herein, the terms "SIRPα" or "SIRPα polypeptide" or "SIRPA" or "SIRPA polypeptide" are used interchangeably to refer to any naturally occurring SIRPA from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. In some embodiments, the term encompasses both wild-type sequences and naturally occurring mutant sequences, e.g., splice variants or allelic variants. In some embodiments, the term encompasses "full-length," unprocessed SIRPA, as well as any form of SIRPA that results from intracellular processing. In some embodiments, the SIRPA is human SIRPA. In some embodiments, the amino acid sequence of an exemplary SIRPA is Uniprot Accession Number: P78324, as of April 25, 2018. In some embodiments, the amino acid sequence of an exemplary human SIRPA v1 is SEQ ID NO: 1. In some embodiments, the amino acid sequence of an exemplary human SIRPA v2 is GenBank: CAA71403.

[0100] The terms "anti-SIRPA antibody," "antibody that binds to SIRPA," and "antibody that specifically binds to SIRPA" refer to an antibody that is capable of binding to SIRPA with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting SIRPA. In one embodiment, the extent of binding of an anti-SIRPA antibody to an unrelated, non-SIRPA polypeptide is less than about 10% of the antibody's binding to SIRPA, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to SIRPA has an affinity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13M) In certain embodiments, the anti-SIRPA antibody binds to an epitope of SIRPA that is conserved among SIRPA from different species.

[0101] With respect to the binding of an antibody to a target molecule, the terms "specific binding to" or "specifically binds to" or "being specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the terms "specific binding to" or "specifically binds to" or "being specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions, e.g., about 10 -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 KD of M or less, or 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9Affinity can be exhibited by molecules with a KD in the range of M. As will be appreciated by those skilled in the art, affinity and KD values ​​are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding when a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0102] As used herein, the term "immunoglobulin" (Ig) is used interchangeably with "antibody." The term "antibody" is used herein in the broadest sense and covers, inter alia, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), including multispecific antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[0103] "Native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light ("L") chains and two identical heavy ("H") chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V) at one end. L ) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0104] For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0105] Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domain. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and mu ("μ"), respectively. The gamma and alpha classes are further divided into subclasses (isotypes) based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are described generally in, for example, Abbas et al., Cellular and Molecular Immunology, 4th ed. (WB Saunders Co., 2000).

[0106] The "variable region" or "variable domain" of an antibody, such as the anti-SIRPA antibody of the present disclosure, refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains, respectively, are referred to as "V H " and "V L These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding sites.

[0107] The term "variable" refers to the fact that the sequences of certain segments of the variable domains vary widely among antibodies, such as the anti-SIRPA antibodies of the present disclosure. The variable domains mediate antigen binding and define the specificity of a particular antibody for that particular antigen. However, variability is not evenly distributed throughout the variable domains. Instead, variability is concentrated in three segments called hypervariable regions (HVRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy-chain and light-chain variable domains each contain four FR regions that primarily adopt a beta-sheet conformation, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity with the HVRs from the other chain by the FR regions and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., "Sequences of Immunological Interest," 5th ed., National Institutes of Health, Bethesda, MD (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions of the antibody, such as participation in antibody-dependent cell-mediated cytotoxicity.

[0108] As used herein, the term "monoclonal antibody" refers to an antibody, such as the anti-SIRPA monoclonal antibody of the present disclosure, obtained from a population of substantially homogeneous antibodies; i.e., the population comprising individual antibodies is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, etc.), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences. For example, monoclonal antibodies for use in accordance with the present disclosure can be produced using, for example, phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1992); Sidhu et al., J. Mol. Biol., 338(2):299-310 (2004); Lee et al., J. Mol. Biol., 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci. USA, 101(34):12467-472 (2004); and Lee et al., J. Immunol. Methods, 284(1-2):119-132 (2004)), hybridoma methods (e.g., Kohler and Milstein,, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., "Monoclonal Antibodies and T Cell Hybridomas", 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), yeast display technology (see, e.g., WO2009 / 036379A2; WO2010105256; WO2012009568, and Xu et al., Protein Eng. Des. Sel., 26(10):663-70 (2013), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Nat'l Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnol., 14:845-851 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol.13:65-93 (1995).

[0109] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody, such as an anti-SIRPA antibody of the present disclosure, in its substantially intact form, in contrast to an antibody fragment. Specifically, a whole antibody includes an antibody with heavy and light chains, including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or variants of this amino acid sequence. In some cases, an intact antibody may have one or more effector functions.

[0110] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0111] Papain digestion of antibodies, such as the anti-SIRPA antibodies of the present disclosure, yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting its ability to readily crystallize. The Fab fragment contains the heavy chain (V H ) variable region domains, and one heavy chain's first constant domain (C H Fab fragments consist of the entire light chain, together with the Fab fragment (F(ab')2 fragment). Each Fab fragment is monovalent with respect to binding to antigen; i.e., each Fab fragment has a single antigen-binding site. Pepsin treatment of an antibody yields a single, large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment contains one or more cysteines from the hinge region of the antibody, called C(ab'). HF(ab')2 antibody fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of one domain. As used herein, Fab'-SH is the designation for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally came about as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0112] The Fc fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of an antibody are determined by sequences within the Fc region, the region that is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0113] A "functional fragment" of an antibody, such as an anti-SIRPA antibody of the present disclosure, generally comprises a portion of the intact antibody, including the antigen-binding or variable region of the intact antibody, or the Fc region of the antibody that retains or has been modified to bind to FcR. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0114] The term "diabody" refers to a V diabody in which inter-chain, but not intra-chain, pairing of variable domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domain and V L Bispecific diabodies refer to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (approximately 5-10 residues) between the V domains. H Domain and V L It is a heterodimer of two "crossover" sFv fragments in which the Fv domains are present on different polypeptide chains.

[0115] As used herein, a "chimeric antibody" refers to an antibody (immunoglobulin), such as a chimeric anti-SIRPA antibody of the present disclosure, in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from, for example, an antibody produced by immunizing macaque monkeys with an antigen of interest. As used herein, "humanized antibody" is used as a subset of "chimeric antibody."

[0116] "Humanized" forms of non-human (e.g., murine) antibodies, such as humanized forms of the anti-SIRPA antibodies of the present disclosure, are chimeric antibodies comprising amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to HVRs of a non-human antibody and all or substantially all of the FRs corresponding to FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0117] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to that of an antibody, such as an anti-SIRPA antibody of the present disclosure, produced by a human and / or made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries and yeast display libraries. Human antibodies can be prepared by administering antigen to transgenic animals, such as immunized xeno-mouse, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous genetic loci have been disabled, or can be generated through human B-cell hybridoma technology. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology), which have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled. See also, e.g., Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies produced via human B cell hybridoma technology.Alternatively, human antibodies can also be prepared by employing yeast libraries and methods disclosed, for example, in WO2009 / 036379A2; WO2010105256; WO2012009568; and Xu et al., Protein Eng. Des. Sel., 26(10):663-70 (2013).

[0118] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to regions of an antibody variable domain, such as regions of an anti-SIRPA antibody of the present disclosure, that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain V H Three in (H1, H2, H3), and V L (L1, L2, L3). In natural antibodies, H3 and L3 represent the greatest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring subtle specificity to antibodies. Naturally occurring camelid antibodies, consisting only of heavy chains, are functional and stable in the absence of light chains.

[0119] Numerous HVR delineations are used herein and are encompassed herein. In some embodiments, the HVRs can be Kabat complementarity-determining regions (CDRs), which are based on sequence variability and are the most commonly used (Kabat et al., supra). In some embodiments, the HVRs can be Chothia CDRs. Alternatively, Cothia refers to the location of structural loops (Cothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). In some embodiments, the HVRs can be AbM HVRs. AbM HVRs represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. In some embodiments, the HVRs can be "contact" HVRs. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are noted below. TIFF2025032076000002.tif56170

[0120] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0121] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0122] The phrases "numbering of variable domain residues as in EU or Kabat" or "numbering of amino acid positions as in EU or Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the collection of antibodies in EU or Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and insertion of residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The EU or Kabat numbering of residues may be determined for a given antibody by alignment with the "standard" Kabat numbering sequence in regions of homology of the antibody sequence.

[0123] The EU numbering or Kabat numbering system is generally used when referring to residues in the variable domain (near residues 1 to 107 of the light chain and residues 1 to 113 of the heavy chain) (e.g., Kabat et al., "Sequences of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering or Kabat numbering system" or "EU index" is generally used when referring to residues in the heavy chain constant region of an immunoglobulin (e.g., the EU index as reported in Kabat et al., supra). The "EU index as per Kabat" refers to the residue numbering of a human IgG1 antibody according to EU. Unless otherwise specified herein, reference to residue numbers in the variable domain of an antibody means residue numbering according to the Kabat numbering system. Unless otherwise specified, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system or the Kabat numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0124] As used herein, an "acceptor human framework" refers to a V-type nucleotide sequence derived from a human immunoglobulin framework or a human consensus framework. L Framework or V HThe acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. When pre-existing amino acid changes are present in VH, these preferred changes occur at only three, two, or one of positions 71H, 73H, and 78H; for example, the amino acid residues at these positions can be 71A, 73T, and / or 78A. In one embodiment, the acceptor human framework for VL has the following amino acid sequence: V L or a human consensus framework sequence.

[0125] "Human consensus framework" refers to the V L Framework sequence or V H In selecting framework sequences, the framework represents the most commonly occurring amino acid residues. L Array or V H The sequence selection is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). L For V, the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV in Kabat et al., supra. H For , the subgroup can be subgroup I, subgroup II, or subgroup III.

[0126] For example, an "amino acid modification" at a specified position of an anti-SIRPA antibody of the present disclosure refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a specified residue means an insertion within 1 to 2 residues of that residue. The insertion may be on the N-terminal or C-terminal side of the specified residue. A preferred amino acid modification herein is a substitution.

[0127] An "affinity matured" antibody, such as an affinity matured anti-SIRPA antibody, of the present disclosure is an affinity matured antibody with one or more alterations within one or more HVRs thereof that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not possess those alteration(s). In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology, 10:779-783 (1992) describe the affinity matured antibody V. H Domain and V L Affinity maturation by shuffling with domains is described. Random mutagenesis of HVR and / or framework residues is described, for example, by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992).

[0128] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, noncovalent association. The folding of these two domains results in six hypervariable loops (three loops from each of the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) still has the ability to recognize and bind to antigen, although with lower affinity than the entire binding site.

[0129] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected into a single polypeptide chain. Preferably, the sFv polypeptide comprises a VH antibody domain and a VL antibody domain connected into a single polypeptide chain. H Domain and V L It further comprises a polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding.

[0130] Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or variant amino acid sequence Fc region), and vary with the antibody isotype.

[0131] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is typically defined as stretching from the amino acid residue at Cys226, or Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of 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.

[0132] 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.

[0133] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, relative to a native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein preferably possesses at least about 80% homology with a native-sequence Fc region and / or the Fc region of a parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0134] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Additionally, a preferred FcR is an FcR (gamma receptor) that binds IgG antibodies, including receptors of 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 within their cytoplasmic domains. The activating receptor, FcγRIIA, contains an immunoreceptor tyrosine-based activation motif ("ITAM") within its cytoplasmic domain. The inhibitory receptor, FcγRIIB, contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") within its cytoplasmic domain. As used herein, other FcRs, including those identified in the future, are encompassed by the term "FcR." FcRs may also extend the serum half-life of antibodies.

[0135] In vivo binding to FcRn and serum half-life of polypeptides that bind to human FcRn with high affinity can be assayed, for example, in transgenic mice expressing human FcRn, or in transfected human cell lines, or in primates to which polypeptides with mutant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody mutants with improved or reduced FcR binding. See also, for example, Shields et al., J. Biol. Chem., 9(2):6591-6604 (2001).

[0136] As used herein, "percent amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST software, BLAST-2 software, ALIGN software, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm known in the art, required to achieve maximum alignment over the entire length of the sequences being compared.

[0137] The term "compete" when used in the context of antibodies competing for the same epitope (e.g., neutralizing antibodies) refers to competition between the antibodies as determined by an assay in which the test antibody prevents or inhibits (e.g., reduces) specific binding of a reference molecule (e.g., a ligand or reference antibody) to a common antigen (e.g., SIRPA, or a fragment thereof). There are many types of competitive binding assays, such as solid-phase, direct or indirect radioimmunoassays (RIAs), solid-phase, direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology, 9:242-253); solid-phase, direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol., 137:3614-3619); solid-phase, direct labeled assays; solid-phase, direct labeled sandwich assays (see, e.g., Harlow and Lane, 1988, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press, 1996). Press); solid-phase direct RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol., 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology, 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol., 32:77-82) can be used to determine whether an antibody competes with another antibody. Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antibody or a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope that is sufficiently close to the epitope bound by the reference antibody that steric hindrance occurs.Further details regarding methods for determining competitive binding are provided herein. Typically, a competing antibody, when present in excess, inhibits (e.g., reduces) specific binding of a reference antibody to a common antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or nearly 100%.

[0138] As used herein, "interaction" between a SIRPA polypeptide and a second polypeptide encompasses, without limitation, protein-protein interactions, physical interactions, chemical interactions, bonds, covalent bonds, and ionic bonds. As used herein, an antibody "inhibits interaction" between two polypeptides if the antibody disrupts, reduces, or completely eliminates the interaction between the two polypeptides. An antibody of the present disclosure "inhibits interaction" between two polypeptides if the antibody binds to one of the two polypeptides. In some embodiments, the interaction may be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or nearly 100%.

[0139] The term "epitope" includes any determinant capable of binding by an antibody. An epitope is a region of an antigen to which an antibody targeting that antigen binds, and, if the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Epitopes are more often found on polypeptides, but in some cases can also be found on other types of molecules, such as nucleic acids. Epitopes can include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0140] An "agonist" or "activating" antibody is an antibody that induces (e.g., increases) one or more activities or functions of an antigen after the antibody binds to the antigen.

[0141] An "antagonist" or "blocking" or "inhibitory" antibody is one that reduces, inhibits, and / or eliminates (e.g., reduces) binding of an antigen to one or more ligands after the antibody has bound to the antigen, and / or reduces, inhibits, and / or eliminates (e.g., reduces) one or more activities or functions of the antigen after the antibody has bound to the antigen. In some embodiments, an antagonist, blocking, or inhibitory antibody substantially or completely inhibits binding of an antigen to one or more ligands and / or one or more activities or functions of the antigen.

[0142] An "isolated" antibody, such as an isolated anti-SIRPA antibody of the present disclosure, is an antibody (e.g., natural or recombinant) that has been identified, separated, and / or recovered from components of its production environment. Preferably, an isolated antibody is free from association with all other contaminant components from its production environment. Contaminant components from its production environment, such as those arising from recombinant transfected cells, are typically materials that would interfere with research, diagnostic, or therapeutic uses for the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be (1) purified to greater than 95% antibody by weight, and in some embodiments greater than 99% antibody by weight, as determined, for example, by the Lowry method; (2) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue staining or, preferably, silver staining. Isolated antibody includes the antibody in situ, within recombinant T cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or isolated antibody will be prepared by at least one purification step.

[0143] An "isolated" nucleic acid molecule encoding an antibody, such as the anti-SIRPA antibody of the present disclosure, is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule that it is normally associated with in the environment in which it is produced.Preferably, isolated nucleic acid is free from association with all components associated with the production environment.The isolated nucleic acid molecule encoding the polypeptide and antibody herein is in a form other than the form or situation in which it is found in nature.Therefore, isolated nucleic acid molecule is distinguished from the nucleic acid encoding the polypeptide and antibody herein that naturally exists in cells.

[0144] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Herein, such vectors are referred to as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0145] As used interchangeably herein, "polynucleotide" or "nucleic acid" refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or a synthetic reaction.

[0146] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, which progeny may not be completely identical (in shape or genomic DNA complementarity) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the invention.

[0147] As used herein, "carrier" refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the dosages and concentrations used. Physiologically acceptable carriers are often aqueous pH-buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0148] As used herein, the term "preventing" includes providing prophylaxis against the occurrence or recurrence of a particular disease, disorder, or condition in an individual who may be predisposed to, susceptible to, or at risk of developing such a disease, disorder, or condition, but who has not yet been diagnosed with the disease, disorder, or condition.

[0149] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms before the treatment methods described herein. "At risk" refers to an individual having one or more risk factors, which are measurable parameters known in the art that correlate with the occurrence of a particular disease, disorder, or condition. Individuals who have one or more of these risk factors have a higher probability of developing a particular disease, disorder, or condition than individuals who do not have one or more of these risk factors.

[0150] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of clinical pathology in the treated individual. Desired treatment effects include slowing the rate of progression of a particular disease, disorder, or condition, improving or alleviating these pathological conditions, and improving their remission or prognosis. An individual is successfully "treated," for example, if one or more symptoms associated with a particular disease, disorder, or condition are suppressed or eliminated.

[0151] An "effective amount" refers to the minimum amount required to achieve the desired therapeutic or preventive result, and for the period of time necessary to achieve this. An effective amount may be administered in a single dose or multiple doses. The effective amount herein may vary according to factors such as the individual's condition, age, sex, and weight, as well as the ability of the treatment to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of disease, alleviating the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, disease complications, and intermediate pathological phenotypes present at the time of disease onset. In therapeutic use, beneficial or desired results include clinical results such as alleviating one or more symptoms resulting from a disease, improving the quality of life of a person suffering from a disease, reducing the dosage of other medications required to treat the disease, enhancing the effect of another medication, such as through targeting, slowing disease progression, and / or prolonging survival. An effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may also be considered in the context of administering one or more therapeutic agents; a single agent may be considered to be administered in an effective amount if the desired result can or is achieved in combination with one or more other agents.

[0152] An "individual," for purposes of treatment, prevention, or risk reduction, refers to any animal classified as a mammal, including domestic and farm animals such as humans, dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc., as well as zoo, sport, or pet animals. In some embodiments, the individual is a human.

[0153] As used herein, "administration in combination with" another compound or composition includes simultaneous administration and / or administration at different times. Administration in combination also encompasses administration as a co-formulation or as separate compositions, including administration using the same or different routes of administration, at different dosing frequencies or intervals. In some embodiments, administration in combination is administration as part of the same therapeutic regimen.

[0154] The term "about" as used herein refers to the customary error range for each value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself.

[0155] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to an "antibody" is a reference to one to many antibodies, including molar amounts, and includes equivalents thereof known to those skilled in the art.

[0156] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0157] Anti-SIRPA antibody Anti-SIRPA antibody binding region In some embodiments, the anti-SIRPA antibodies of the present disclosure may bind to a conformational epitope. In some embodiments, the anti-SIRPA antibodies of the present disclosure may bind to a discontinuous SIRPA epitope. In some embodiments, the discontinuous SIRPA epitope comprises two or more peptides, three or more peptides, four or more peptides, five or more peptides, six or more peptides, seven or more peptides, eight or more peptides, nine or more peptides, or ten or more peptides. In some embodiments, the anti-SIRPA antibodies of the present disclosure may bind to a SIRPA epitope comprising one or more peptides. As disclosed herein, a SIRPA epitope is an epitope that is comprised of 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more amino acid residues of the amino acid sequence of SEQ ID NO: 1, or or more than 20 amino acid residues on a mammalian SIRPA protein corresponding to the amino acid sequence of

[0158] In some embodiments, the anti-SIRPA antibodies of the present disclosure include an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:7; or an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8, which binds to an epitope of human SIRPA that is the same as or overlaps with the SIRPA epitope bound by the anti-SIRPA antibody comprising: In some embodiments, the anti-SIRPA antibody of the present disclosure binds to essentially the same SIRPA epitope as an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;

[0159] In some embodiments, the anti-SIRPA antibody of the present disclosure competitively inhibits the binding of an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; In some embodiments, the anti-SIRPA antibody of the present disclosure competes for binding to SIRPA with an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;

[0160] In some embodiments, the anti-SIRPA antibodies of the present disclosure competitively inhibit the binding of at least one antibody selected from any of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the present disclosure competitively inhibit the binding of at least one antibody selected from any of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the present disclosure competitively inhibit the binding of at least one antibody selected from any of the antibodies listed in Tables 4, 5, 7, 8, and 10. H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. In some embodiments, the anti-SIRPA antibody of the disclosure is selected from the group consisting of m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof, is reduced by an amount ranging from about 50% to 100% compared to binding to SIRPA in the absence of an anti-SIRPA antibody. H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. In some embodiments, the anti-SIRPA antibody of the disclosure is selected from the group consisting of m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.3), m3F9 H1 / L3, h3F9 H2 / L1(14.70.4), m3F9 H1 / L3, h3F9 H2 / L1(14.70.5), m3F9 H1 / L3, h3F9 H2 / L1(14.70.6), m3F9 H1 / L3, h3F9 H2 / L1(14.70.7), m3F9 H1 / L3, h3F9 H2 / L1(14.70.8), m3F9 H1 / L3, h3F9 H2 / L1(14.70.9), m3F9 H1 / L3, h3F9 H2 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.1), m3F9 H1 / L3, h3F9 H2 / L1(14.70.1), m3F9 H1 / L3, h3F9 H2 / L1(14.70.2), m3F9 H1 / L3, h3F9 H2 / L1(14.70.3), m3F9 H1 / L3, h3F9 H2 / L1(14.70.1 ...2), m3F9 H1 / L3, h3F9 H2 / L1(14.70.1), m3F94), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. In some embodiments, m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 The anti-SIRPA antibody of the present disclosure reduces by 100% the binding to SIRPA of one or more antibodies selected from m3F9, h3F9 H1 / L1(14.70.1), h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof, to SIRPA. In some embodiments, the anti-SIRPA antibody is combined with m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, or h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof, and the one or more antibodies are selected from the group consisting of anti-SIRPA antibodies m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12 , 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9 10:1 ratio, 9:1 ratio, 8:1 ratio, 7:1 ratio, 6:1 ratio, 5:1 ratio, 4:1 ratio, 3:1 ratio, 2:1 ratio, 1:1 ratio, 0.75:1 ratio, 0.5:1 ratio, 0.25:1 ratio, 0.1:1 ratio, 0.075:1 ratio, 0.050:1 ratio to one or more antibodies selected from 3F9-24, 3F9-25, and any combination thereof; 0.025:1 ratio, 0.01:1 ratio, 0.0075:1 ratio, 0.0050:1 ratio, 0.0025:1 ratio, 0.001:1 ratio, 0.00075:1 ratio, 0.00050:1 ratio, 0.00025:1 ratio, 0.0001:1 ratio, 1:10 ratio, 1:9 ratio, 1:8 ratio, 1:7 ratio, 1:6 ratio, 1:5 ratio, 1:4 ratio, 1:3 ratio, 1:2 Ratios of 1:0.75, 1:0.5, 1:0.25, 1:0.1, 1:0.075, 1:0.050, 1:0.025, 1:0.01, 1:0.0075, 1:0.0050, 1:0.0025, 1:0.001, 1:0.00075, 1:0.00050, 1:0.00025, or 1:0.In some embodiments, the anti-SIRPA antibody is present in an amount corresponding to a ratio of m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 and H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. In some embodiments, the anti-SIRPA antibody is selected from the group consisting of m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, and h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3 F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24 , 3F9-25, and any combination thereof.

[0161] In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to an epitope of human SIRPA that is the same as or overlaps with a SIRPA epitope bound by at least one antibody selected from any of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the present disclosure are selected from m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, and 3F9-25 bind to an epitope of human SIRPA that is the same as or overlaps with the SIRPA epitope bound by at least one antibody selected from the group consisting of 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, and 3F9-25.

[0162] In some embodiments, the anti-SIRPA antibodies of the disclosure bind to essentially the same SIRPA epitope as at least one antibody selected from any of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the disclosure bind to essentially the same SIRPA epitope as at least one antibody selected from any of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the disclosure bind to m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and bind to essentially the same SIRPA epitope as bound by at least one antibody selected from the group consisting of 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, and 3F9-25. Detailed exemplary methods for mapping antibody-binding epitopes are presented in Morris (1996), "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0163] In some embodiments, the anti-SIRPA antibodies of the disclosure are selected from the group consisting of m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof.

[0164] Any suitable competitive or SIRPA binding assay known in the art, such as Biacore analysis, ELISA assay, or flow cytometry, may be used to assay the anti-SIRPA antibodies m3F9, h3F9 H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9 H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof. In an exemplary competitive assay, immobilized SIRPA or cells expressing SIRPA on the cell surface are incubated in a solution containing a first labeled antibody (e.g., a human antibody or a non-human primate antibody) that binds to SIRPA and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to SIRPA. The second antibody can be present in hybridoma supernatant. As a control, immobilized SIRPA or cells expressing SIRPA are incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to SIRPA, excess unbound antibody is removed, and the amount of label associated with immobilized SIRPA or cells expressing SIRPA is measured. If the amount of label associated with immobilized SIRPA or cells expressing SIRPA is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to SIRPA.See Harlow and Lane (1988), Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0165] In some embodiments, the present specification provides anti-SIRPA antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0166] In some embodiments, provided herein are: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22 (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 13; (a) sequence (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9 (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10 (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 19; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 13 (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21 (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 19; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) sequence (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 13; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 HVR-H3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 17;Anti-SIRPA antibodies are provided, comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18; and (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 19.

[0167] In some embodiments, the present disclosure provides a method for identifying a HVR-H1 comprising administering to a patient a HVR-H2 antigen-binding protein comprising administering to a patient a HVR-H1 antigen-binding protein comprising an HVR-H1 antigen-binding protein comprising an HVR-H2 antigen-binding protein comprising an HVR-H1 antigen-binding protein comprising an HVR-H2 antigen-binding protein comprising an HVR-H3 antigen-binding protein comprising an HVR-H4 antigen-binding protein comprising an HVR-H5 antigen-binding protein comprising an HVR-H6 antigen-binding protein comprising an HVR-H7 antigen-binding protein comprising an HVR-H8 antigen-binding protein comprising an HVR-H9 antigen-binding protein comprising an HVR-H1 antigen-binding protein comprising an HVR-H1 antigen-binding protein comprising an HVR-H2 ...3 antigen-binding protein comprising an HVR-H4 antigen-binding protein comprising an HVR-H5 H Anti-SIRPA antibodies containing HVR sequences are presented.

[0168] In some embodiments, provided herein is an HVR-L1 comprising at least one, at least two, or all three VHs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. L Anti-SIRPA antibodies containing HVR sequences are presented.

[0169] In some embodiments, provided herein is a VH HVR comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24. H and (b) at least one, at least two, or all three V domains selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. L V, containing the HVR sequence L domains are presented.

[0170] In some embodiments, provided herein are: (a) HVR-H1 comprising (i) an HVR-H1 having the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24. H and (b) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) an HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. L domains are presented.

[0171] In another embodiment, the anti-SIRPA antibody comprises a heavy chain variable domain (V) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35. HIn certain embodiments, a V has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35. H The sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an anti-SIRPA antibody comprising this sequence retains the ability to bind to SIRPA. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, an anti-SIRPA antibody comprises a V-type nucleotide sequence of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35, including post-translational modifications of this sequence. H In certain embodiments, V H comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24.

[0172] In another embodiment, a light chain variable domain (V) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44. L In certain embodiments, an anti-SIRPA antibody is provided that comprises a V having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44. LThe sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an anti-SIRPA antibody comprising this sequence retains the ability to bind to SIRPA. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVRs (i.e., within the FRs). Optionally, the anti-SIRPA antibody comprises the V of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44, including post-translational modifications of this sequence. L In certain embodiments, V L comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0173] In some embodiments, V as in any of the embodiments presented above H and V as in any of the embodiments presented above. L In some embodiments, provided herein is an anti-SIRPA antibody comprising: H and V as in any of the embodiments presented above. LIn one embodiment, the antibody comprises a V in SEQ ID NO: 33, 34, or 35, and a V in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, or 44, respectively, including post-translational modifications of these sequences. H Array and V L Contains arrays.

[0174] In some embodiments, as used herein, a heavy chain variable domain (V H ) and the light chain variable domain (V L ), including V H and V L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 36 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 37 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 38 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 39 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 40 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 41. L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 42 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 43 L V comprising the amino acid sequence of SEQ ID NO: 33 H and V comprising the amino acid sequence of SEQ ID NO: 44 L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 36 L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 38L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 39 L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 40 L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 41. L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 42 L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 43 L V comprising the amino acid sequence of SEQ ID NO: 34 H and V comprising the amino acid sequence of SEQ ID NO: 44 L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 36 L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 38 L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 39 L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 40 L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 41. L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 42 L V comprising the amino acid sequence of SEQ ID NO: 35 H and V comprising the amino acid sequence of SEQ ID NO: 43 L and V comprising the amino acid sequence of SEQ ID NO: 35. H and V comprising the amino acid sequence of SEQ ID NO: 44 L An anti-SIRPA antibody is presented, wherein the antibody is selected from the group consisting of:

[0175] Further provided herein are: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24. H and (b) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) an HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. L In some embodiments, the antibody comprises the VH and VL sequences of SEQ ID NOs: 33, 34, or 35, and 36, 37, 38, 39, 40, 41, 42, 43, or 44, respectively.

[0176] Provided herein are anti-SIRPA antibodies that bind to an epitope of human SIRPA that is the same as or overlaps with an epitope bound by an anti-SIRPA antibody comprising: (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) an HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. In some embodiments, the antibody binds to an epitope of human SIRPA that is the same as or overlaps with an epitope bound by an anti-SIRPA antibody comprising: SEQ ID NO: 33, 34, or 35, and SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, or 44, respectively. H Array and V L In some embodiments, the epitope of human SIRPA is the same epitope to which the anti-SIRPA antibody binds.

[0177] In some embodiments, the anti-SIRPA antibody according to any of the above embodiments is a monoclonal antibody, including a humanized antibody and / or a human antibody. In some embodiments, the anti-SIRPA antibody is an antibody fragment, such as an Fv fragment, an Fab fragment, an Fab' fragment, an scFv fragment, a diabody fragment, or an F(ab')2 fragment. In some embodiments, the anti-SIRPA antibody is a substantially full-length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein.

[0178] In some embodiments, an anti-SIRPA antibody according to any of the above embodiments may incorporate any of the features described herein, either alone or in combination.

[0179] Anti-SIRPA antibodies are provided herein, which are useful, for example, for the diagnosis or treatment of SIRPA-mediated disorders.

[0180] The present disclosure is directed, in part, to, for example, anti-SIRPA antibodies capable of reducing cellular levels of SIRPA, anti-SIRPA antibodies capable of reducing cell surface levels of SIRPA, anti-SIRPA antibodies capable of degrading SIRPA, anti-SIRPA antibodies capable of reducing cellular levels of CD32A / B, anti-SIRPA antibodies capable of increasing or enhancing phagocytosis, anti-SIRPA antibodies capable of increasing or enhancing phagocytosis of tumor cells by macrophages, anti-SIRPA antibodies capable of increasing or enhancing the anti-tumor activity of anti-cancer therapy, anti-SIRPA antibodies capable of increasing or enhancing T cell proliferation, anti-SIRPA antibodies capable of increasing or enhancing the proliferation of macrophages, anti-SIRPA antibodies capable of increasing or enhancing the proliferation of tumor cells by ... The present invention relates to anti-SIRPA antibodies that exhibit improved and / or enhanced one or more functional properties (e.g., compared to an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5), including anti-SIRPA antibodies capable of increasing or enhancing the release of pro-inflammatory cytokines from the body and / or anti-SIRPA antibodies capable of binding to human SIRPA with improved / enhanced kinetics; methods of making and using such anti-SIRPA antibodies; pharmaceutical compositions containing such anti-SIRPA antibodies; nucleic acids encoding such anti-SIRPA antibodies; and host cells containing nucleic acids encoding such anti-SIRPA antibodies.

[0181] In some embodiments, the anti-SIRPA antibodies of the present disclosure may have one or more activities attributable, at least in part, to the antibody's ability to reduce cellular expression of SIRPA (e.g., cell surface expression) by inducing degradation, down-regulation, cleavage, receptor desensitization, and / or lysosomal targeting of SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure that reduce cellular levels of SIRPA are antibodies that exhibit one or more of the following properties: (1) inhibiting or reducing one or more SIRPA activities; (2) the ability to reduce SIRPA expression (such as at the mRNA level and / or protein level) in SIRPA-expressing cells; (3) the ability to interact with, bind to, or recognize SIRPA protein; (4) the ability to specifically interact with or specifically bind to SIRPA protein; and (5) the ability to treat, ameliorate, or prevent any aspect of a disease or disorder described or contemplated herein.

[0182] In some embodiments, the anti-SIRPA antibody has the following properties: a) a dissociation constant (KD) for human SIRPA that is lower than the KD of an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5; b) binds to human cells, such as human monocytes and human macrophages; c) reduces the cell surface level of SIRPA (e.g., reduces the cell surface level of SIRPA on human macrophage cells in vitro with a 50% effective concentration (EC50) that is lower than the EC50 of an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5); d) a dissociation constant (KD) for human SIRPA that may be in the range of about 0.6 nM to about 0.7 nM; and / or e) reduces the cell surface level of SIRPA (e.g., in and reducing cell surface levels of SIRPA on human macrophages in vitro at a 50% effective concentration (EC50), which may range from about 0.4 nM to about 0.5 nM. As disclosed herein, the 50% effective concentration (EC50) refers to the concentration at which an anti-SIRPA antibody of the present disclosure reduces the cellular level of SIRPA on or within a cell to half of the cellular level of SIRPA in untreated cells, or the concentration at which the antibody achieves half-maximal binding to SIRPA on a cell.

[0183] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA in vitro with a 50% effective concentration (EC50) for human SIRPA v1 of about 0.40 nM to about 0.5 nM, as measured by flow cytometry. In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA in vitro with a 50% effective concentration (EC50) for human SIRPA v1 of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, or about 0.1 nM, as measured by flow cytometry.In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody binds to cell surface levels of SIRPA in vitro at levels between about 5 nM and 1 nM, between about 1 nM and 0.9 nM, between about 1 nM and 0.8 nM, between about 1 nM and 0.7 nM, between about 1 nM and 0.6 nM, between about 1 nM and 0.5 nM, between about 1 nM and 0.4 nM, between about 1 nM and 0.3 nM, between about 1 nM and 0.5 nM, between about 1 nM and 0.6 nM, between about 1 nM and 0.5 nM, between about 1 nM and 0.4 nM, between about 1 nM and 0.5 nM, between about 1 nM and 0.6 nM, between about 1 nM and 0.5 ... .2nM, approximately 1nM~0.1nM, approximately 0.9nM~0.8nM, approximately 0.9nM~0.7nM, approximately 0.9nM~0.6nM, approximately 0.9nM~0.5nM, Approx. 0.9nM~0.4nM, Approx. 0.9nM~0.3nM, Approx. 0.9nM~0.2nM, Approx. 0.9nM~0.1nM, Approx. 0.8nM~0.6nM, Approx. 0. 8nM~0.5nM, approx. 0.8nM~0.4nM, approx. 0.8nM~0.3nM, approx. 0.8nM~0.2nM, approx. 0.8nM~0.1nM, approx. 0.7n M~0.5nM, approx. 0.7nM~0.4nM, approx. 0.7nM~0.3nM, approx. 0.7nM~0.2nM, approx. 0.7nM~0.1nM, approx. 0.6nM~0 The present invention provides antibodies that reduce human SIRPA v1 at a 50% effective concentration (EC50) in the range of about 0.4 nM, about 0.6 nM to 0.3 nM, about 0.6 nM to 0.2 nM, about 0.6 nM to 0.1 nM, about 0.5 nM to 0.3 nM, about 0.5 nM to 0.2 nM, about 0.5 nM to 0.1 nM, about 0.4 nM to 0.2 nM, about 0.4 nM to 0.1 nM, or about 0.3 nM to 0.1 nM.

[0184] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with a 50% effective concentration (EC50) of about 0.093 nM for human SIRPA v1, about 0.080 nM for human SIRPA v2, and / or about 0.879 nM for cynomolgus monkey SIRPA, as measured by flow cytometry. ... The cell surface levels of SIRPA in vitro were measured by flow cytometry at approximately 2nM to 0.05nM, approximately 1nM to 0.05nM, approximately 0.9nM to 0.05nM, approximately 0.8nM to 0.05nM, approximately 0.7nM to 0.05nM, approximately 0.6nM to 0.05nM, approximately 0.5nM to 0.05nM, approximately 0.4nM~0.05nM, approx. 0.3nM~0.05nM, approx. 0.20nM~0.05nM, approx. 0.15nM~0.05nM, approx. 0.10nM~0.05 nM, approximately 0.09nM~0.05nM, approximately 0.08nM~0.05nM, approximately 0.07nM~0.05nM, approximately 0.06nM~0.05nM, approximately 0.20nM ~0.06nM, approximately 0.15nM~0.06nM, approximately 0.10nM~0.06nM, approximately 0.09nM~0.06nM, approximately 0.08nM~0.06nM, Approx. 0.07nM~0.06nM, Approx. 0.20nM~0.07nM, Approx. 0.15nM~0.07nM, Approx. 0.10nM~Approx. 0.07nM, Approx. 0.09nM~ Antibodies are provided that reduce human SIRPA v1 or human SIRPA v2 at a 50% effective concentration (EC50) in the range of 0.07 nM, about 0.08 nM to 0.7 nM, about 0.20 nM to 0.08 nM, about 0.15 nM to 0.08 nM, about 0.10 nM to 0.08 nM, about 0.09 nM to 0.08 nM, about 0.20 nM to 0.09 nM, about 0.15 nM to 0.09 nM, or about 0.10 nM to 0.9 nM.

[0185] In some embodiments that may be combined with any of the embodiments herein, the present disclosure provides an isolated antibody that binds to human SIRPA and reduces cell surface levels of SIRPA in vitro with a 50% effective concentration (EC50) of about 0.107 nM for human SIRPA v1, about 0.082 nM for human SIRPA v2, and / or about 0.107 nM for cynomolgus monkey SIRPA, as measured by flow cytometry.

[0186] The anti-SIRPA antibodies of the present disclosure are advantageous because they reduce cell surface expression of SIRPA more potently (e.g., with a lower EC50) than a control anti-SIRPA antibody (e.g., a control anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5). Furthermore, the anti-SIRPA antibodies of the present disclosure are advantageous because they have a higher affinity for SIRPA (e.g., about 100-fold higher affinity) (e.g., a lower KD value as measured by surface plasmon resonance) than a control anti-SIRPA antibody (e.g., a control anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5).

[0187] Certain embodiments of the present disclosure are at least in part characterized by improved and / or enhanced functional properties (e.g., compared to an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5), including, but not limited to, an improved / enhanced ability to reduce cell surface levels of SIRPA on cells, including, but not limited to, reduced cell proliferation of monocytes, macrophages, T cells, dendritic cells, and / or microglia; reduced proliferation of dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia, activated M1 microglia, and the like. Decreased proliferation of T cells induced by neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, helper T cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; Decreased survival of ... Decreased proliferation of macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, helper T cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; Decreased proliferation of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, helper T cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia Inhibition of migration; Reduction in one or more functions of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, helper T cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; Reduction in proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils, and / or microglia; Reduction in overall functionality of monocytes, macrophages, T cells, dendritic cells, neutrophils, and / or microglia;Inhibition of beneficial immune responses against different types of cancer selected from 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, acute myeloid leukemia, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer; dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakola disease, stroke, acute trauma, chronic Inhibition of beneficial immune responses to different types of neurological disorders selected from trauma, idiopathic tremor, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, stroke, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, and multiple sclerosis; binding to SIRPA ligands on tumor cells; binding to SIRPA ligands on dendritic cells binding, bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils, and / or macrophages; inhibition of tumor cell killing by one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells; inhibition of anti-tumor cell proliferation activity of one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells; microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, Inhibiting the anti-tumor cell metastatic activity of one or more of T cells, helper T cells, or cytotoxic T cells; modulating the expression of one or more inflammatory receptors, such as CD86, expressed on one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells; enhancing the infiltration of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, immunosuppressive neutrophils, and regulatory T cells into tumors;increasing the number of tumor-promoting myeloid / granulocytic immunosuppressive cells in the peripheral blood or other lymphoid organs, or in tumors; enhancing the tumor-promoting activity of myeloid-derived suppressor cells; decreasing the activation of tumor-specific T lymphocytes with tumor-killing potential; decreasing the infiltration of tumor-specific T lymphocytes with tumor-killing potential; increasing the rate of tumor growth; increasing the rate of tumor recurrence; one or more immunotherapies that modulate anti-tumor T cell responses, optionally including one or more of the following: CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, VISTA, KIR, GAL 9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD39, CD70, TREM1, TREM2, Siglec-5, Siglec-7, Siglec-9, Siglec-11, SirpA, CD447, CSF-1 receptor, and any combination thereof, or one or more immunotherapies, or one or more chemotherapeutic agents and / or cancer vaccines;

[0188] In some embodiments, treating cancer with anti-SIRPA antibodies as described herein can (i) increase the number of tumor-infiltrating CD3+ T cells; (ii) optionally reduce the cellular levels of SIRPA in non-tumorigenic CD14+ myeloid cells that are tumor-infiltrating cells or, optionally, present in the blood; (iii) optionally reduce the number of non-tumorigenic CD14+ myeloid cells that are tumor-infiltrating cells or, optionally, present in the blood; and (iv) optionally reduce the number of non-tumorigenic myeloid-derived suppressor cells (MDSCs) in one or more cells that are non-tumorigenic. (v) capable of reducing the levels of PD-L1, PD-L2, B7-H7, B7-H3, CD200R, CD163, and / or CD206 in solid tumors; (vi) capable of reducing tumor growth rate; (vii) capable of reducing tumor volume; (viii) optionally capable of increasing the efficacy of one or more PD-1 inhibitors; and (viii) optionally capable of increasing the levels of CTL4, adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof. (ix) optionally, the efficacy of one or more chemotherapeutic agents is increased, wherein one or more of the chemotherapeutic agents are gemcitabine, capecitabine, an anthracycline, doxorubicin (Adriamycin®), epirubicin (Elence®), taxane, paclitaxel (Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (x) increased proliferation of T cells in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs); (xi) inhibited differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs);(xii) When conjugated to chemicals or radiotoxins, they can kill CD33-expressing immunosuppressive non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells within solid tumors and blood vessels;

[0189] In some embodiments, the bone marrow cells of the present disclosure include, but are not limited to, CD45+CD14+ bone marrow cells, CD14+ bone marrow cells, and myeloid-derived suppressor cells (MDSCs). In some embodiments, the bone marrow cells of the present disclosure are non-tumorigenic bone marrow cells. Immunosuppressive cells are also sometimes referred to as myeloid-derived suppressor cells (MDSCs). In humans, MDSCs express the following combinations of markers: (1) CD14+ HLA-DRlow / -, (2) CD14+ IL4Rα+, (3) CD14+ HLA-DR- IL4Rα+, (4) CD34+ CD14+ CD11b+ CD33+, (5) CD11b+ CD14+ CD33+, (6) CD33+ HLA-DR-, (7) Lin- HLA-DR-, (8) Lin- HLA-DR- CD33+, (9) Lin- HLA-DR- CD33+ CD11b+, (10) Lin- CD33+ CD11b+ CD15+, (11) Lin- HLA-DR- CD33+ CD11b+ CD14- CD15+, (12) CD11b+ CD14- CD33+, and (13) CD11b+ CD14- HLA-DR- CD33+ CD15+, (14) CD33+ HLA-DR- CD15+, (15) CD15+ IL4Rα+, (16) CD11b+ CD15+ CD66b+, (17) CD15+ FSClow SSChigh, (18) CD15high CD33+, (19) CD11b+ CD14- CD15+, (20) CD66b+ SSChigh, and (21) CD11b+ CD15+ (see also Solito S et al., Annals of NY Academy of Sciences, 2014). In mice, MDSCs can be defined by the expression of surface markers CD45+, CD11b+, Gr1+, and / or Il4Ra+. Further exemplary immunosuppressive monocyte lineages are CD45+, CD11b+, Gr1low; and CD45+, CD11c+.

[0190] In some embodiments, the anti-SIRPA antibodies of the present disclosure reduce cell surface levels of SIRPA, reduce intracellular levels of SIRPA, reduce total cellular levels of SIRPA, or any combination thereof, in the presence of only SIRPA's natural ligand or binding partner.

[0191] In some embodiments, the anti-SIRPA antibodies of the present disclosure prevent, reduce, or inhibit SIRPA-mediated activity of a ligand or binding partner of SIRPA, including, for example, CD47, surfactant protein A, and / or surfactant protein D, which are ligands or binding partners of SIRPA.

[0192] In some embodiments, the anti-SIRPA antibodies of the present disclosure selectively bind to human SIRPA, including human allelic variants, also referred to herein as "polymorphic" variants, including human SIRPA v1 and human SIRPA v2, bind to human SIRPβ3, bind to cynomolgus monkey SIRPA, and bind to marmoset SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure do not bind to mouse / mouse SIRPA, do not bind to SIRPB v1 (SIRPβ1), do not bind to rabbit SIRPA, and do not bind to rat SIRPA.

[0193] In some embodiments, the anti-SIRPA antibody of the present disclosure selectively binds to human SIRPA. In some embodiments, the anti-SIRPA antibody of the present disclosure selectively binds to human SIRPA and cynomolgus monkey SIRPA. In some embodiments, the anti-SIRPA antibody of the present disclosure binds to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA. In some embodiments, the anti-SIRPA antibody of the present disclosure binds to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA, but does not bind to mouse SIRPA. In some embodiments, the anti-SIRPA antibody of the present disclosure binds to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA, but does not bind to SIRPβ1. In some embodiments, the anti-SIRPA antibody of the present disclosure binds to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA, but does not bind to mouse SIRPA and does not bind to human SIRPβ1. In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to human SIRPA v1, human SIRPA v2, cynomolgus monkey SIRPA, and human SIRPAβ3, but do not bind to mouse SIRPA and do not bind to human SIRPβ1. In any combination of the above embodiments, the anti-SIRPA antibodies of the present invention do not block the binding or interaction of SIRPA with CD47.

[0194] 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, blocking, or reducing the interaction (e.g., binding) of SIRPA with one or more SIRPA ligands, such as CD47.

[0195] In some embodiments, the anti-SIRPA antibodies of the present disclosure may have one or more activities attributable, at least in part, to the ability of the antibody to reduce cellular levels (e.g., cell surface expression) of SIRPA by inducing degradation, down-regulation, cleavage, receptor desensitization, and / or lysosomal targeting of SIRPA.

[0196] In some embodiments, the anti-SIRPA antibodies of the present disclosure down-regulate intracellular SIRPA expression. In some embodiments, the down-regulation of intracellular SIRPA expression is a reduction in cell surface expression of SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure down-regulate SIRPA expression in monocytes (e.g., human monocytes). In some embodiments, the anti-SIRPA antibodies of the present disclosure down-regulate SIRPA expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRPA antibodies of the present disclosure down-regulate SIRPA expression on the cell surface of macrophages by more than 70%, more than 75%, more than 80%, more than 85%, or more than 95%, compared to the level of SIRPA cell surface expression in macrophages not treated with the anti-SIRPA antibodies of the present disclosure. In some embodiments, an anti-SIRPA antibody of the present disclosure downregulates SIRPA expression on the cell surface of macrophages by about 70-90%, about 70-85%, about 70-80%, but not by about 70-75%, about 75-90%, about 75-85%, about 75-80%, about 80-90%, about 80-85%, or about 85-95%, compared to the level of SIRPA cell surface expression in macrophages not treated with an anti-SIRPA antibody of the present disclosure. In some embodiments, the macrophages are human macrophages, including, but not limited to, human M1 macrophages and human M2 macrophages.

[0197] The cellular level of SIRPA may refer, without limitation, to the cell surface level of SIRPA, the intracellular level of SIRPA, and the total level of SIRPA.In some embodiments, the reduction of the cellular level of SIRPA comprises the reduction of the cell surface level of SIRPA.As used herein, an anti-SIRPA antibody reduces the cell surface level of SIRPA if it induces a 25% or greater reduction in the cell surface 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, using flow cytometry, such as fluorescence-activated cell sorting (FACS), to measure the cell surface level of SIRPA.In some embodiments, the reduction of the cellular level of SIRPA comprises the reduction of 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 reduction 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, immunostaining, Western blot analysis, co-immunoprecipitation, and flow cytometry. In some embodiments, a reduction in the cellular level of SIRPA includes a reduction in the total SIRPA level. As used herein, an anti-SIRPA antibody reduces the total SIRPA level if it induces a 25% or greater reduction in the total SIRPA level, as measured by any in vitro, cell-based assay, or suitable in vivo model described herein or known in the art, for example, immunostaining, Western blot analysis, co-immunoprecipitation, and flow cytometry. In some embodiments, the anti-SIRPA antibody induces degradation of SIRPA, cleavage of SIRPA, internalization of SIRPA, release of SIRPA, down-regulation of SIRPA expression, or any combination thereof.In some embodiments, cellular levels of SIRPA are measured on primary cells (e.g., dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, and macrophages) or on cell lines using a SIRPA cellular assay.

[0198] In some embodiments, down-regulation of an anti-SIRPA antibody is characterized by an IC50 of 200 nM or less, typically 100 nM or less, following exposure of human macrophages to the antibody for 4 hours at 37°C. 50 (50% of SIRPA expressed on the cell surface is downregulated). In some embodiments, SIRPA remains downregulated for at least 24 hours after exposure to an antibody of the invention. Cells can be analyzed for surface expression of SIRPA using any technique, for example, flow cytometry.

[0199] In some embodiments, the anti-SIRPA antibodies of the disclosure reduce 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%, 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.

[0200] In some embodiments, which may be combined with any of the down-regulating activities summarized in the preceding paragraph, the anti-SIRPA antibodies of the present disclosure inhibit clustering of SIRPA at the cell surface.

[0201] In some embodiments, the anti-SIRPA antibody of the present disclosure downregulates SIRPA but does not block, inhibit, or reduce the binding of CD47 to SIRPA ligands, such as SIRPA. In the context of the present invention, an antibody against SIRPA that does not block the binding of CD47 to SIRPA refers to an antibody that does not result in a significant reduction in the binding of CD47 to SIRPA when the antibody is incubated with cells expressing CD47 and SIRPA. In the context of CD47 binding to SIRPA, a "significant reduction" refers to a reduction in binding of 30% or less, typically at least 25%, at least 20%, at least 15%, or at least 10% or less, compared to the binding of CD47 to SIRPA in the presence of an isotype-matched control antibody that does not bind to SIRPA. Exemplary assays for assessing blocking activity are set forth in the examples herein. For example, cells expressing human SIRPA, such as human macrophages or CHO cells modified to recombinantly express human SIRPA, are plated in a 96-well plate at 10 cells per well. 5 Cells were seeded at 100 μl, 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.

[0202] In some embodiments, the anti-SIRPA antibodies of the present disclosure down-regulate the expression of CD32A / B (i.e., FcγRIIA / FcγRIIB) in cells. In some embodiments, the down-regulation of CD32A / B (i.e., FcγRIIA / FcγRIIB) expression in cells is a reduction in cell surface expression of CD32A / B (i.e., FcγRIIA / FcγRIIB). In some embodiments, the anti-SIRPA antibodies of the present disclosure down-regulate the expression of CD32A / B in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRP antibodies of the present disclosure down-regulate the expression of CD32A (i.e., FcγRIIA) in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRP antibodies of the present disclosure down-regulate the expression of CD32B (i.e., FcγRIIB) in macrophages (e.g., human macrophages). In some embodiments, anti-SIRPA antibodies of the present disclosure downregulate cell surface expression of CD32A (i.e., FcγRIIA) in human macrophages by about 75%, about 80%, or about 85%, compared to the level of cell surface expression of CD32A in human macrophages not treated with an anti-SIRPA antibody of the present disclosure. In some embodiments, anti-SIRPA antibodies of the present disclosure downregulate cell surface expression of CD32A in human macrophages by about 70-85%, compared to the level of cell surface expression of CD32A in human macrophages not treated with an anti-SIRPA antibody of the present disclosure. In some embodiments, anti-SIRPA antibodies of the present disclosure downregulate cell surface expression of CD32B (i.e., FcγRIIB) in human macrophages to undetectable levels.

[0203] In one aspect, the present disclosure provides antibodies, such as isolated (e.g., monoclonal) antibodies, that interact with or otherwise bind to a region, such as an epitope, within a SIRPA protein of the present disclosure. In some embodiments, the antibody interacts with or otherwise binds to a region, such as an epitope, within a SIRPA protein of the present disclosure with improved / enhanced kinetics (e.g., compared to an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5). In some embodiments, the antibody has an EC of less than or equal to that of a control antibody. 50 a lower 50% effective concentration (EC 50 ), which interacts with or otherwise binds to a region, such as an epitope, within a SIRPA protein on human cells, such as dendritic cells, myeloid cells, monocytes, and macrophages. In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to a SIRPA protein and, after binding to the SIRPA protein, modulate one or more SIRPA activities, e.g., activities associated with SIRPA expression, on the cell. SIRPA proteins of the present disclosure include, but are not limited to, mammalian SIRPA proteins, human SIRPA proteins, mouse SIRPA proteins, cynomolgus monkey SIRPA proteins, and rat SIRPA proteins.

[0204] In some embodiments, antibodies of the present disclosure may bind to SIRPA in a pH-dependent manner. In some embodiments, antibodies of the present disclosure may bind to SIRPA at neutral pH and may be internalized without dissociating from the SIRPA protein. Alternatively, at acidic pH, antibodies of the present disclosure may dissociate from SIRPA upon internalization and then be degraded via the endosomal / lysosomal pathway. In certain embodiments, anti-SIRPA antibodies bind to SIRPA at pH ranges of 5.5 to 8.0, 5.5 to 7.5, 5.5 to 7.0, 5.5 to 6.5, 5.5 to 6.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0, 7.5 to 8.0, 6.0 to 7.5, 6.0 to 7.0, or 6.5 to 7.5. In certain embodiments, the anti-SIRPA antibody dissociates from SIRPA at a pH below 6.0, below 5.5, below 5.0, below 4.5, below 4.0, below 3.5, below 3.0, below 2.5, or below 2.0.

[0205] SIRPA is a type I single-pass transmembrane protein. Within the amino acid sequence of human SIRPA (SEQ ID NO: 1), the extracellular domain is located at amino acid residues 31-373; the transmembrane domain is located at amino acid residues 374-394; and the intracellular domain is located at amino acid residues 395-504. Human SIRPA contains a single V set and two C1 sets of Ig superfamily (IgSF) domains, designated the D1 domain, D2 domain, and D3 domain, 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. As one skilled in the art will appreciate, the start and end residues of the domains of the present disclosure may vary depending on the computer modeling program used or the method used to determine the domains.

[0206] In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to the D3 domain of SIRPA. In some embodiments, the anti-SIRPA antibodies of the present disclosure bind to the D3 domain of human SIRPA comprising amino acid residues 254 to 348 of the amino acid sequence of human SIRPA set forth in SEQ ID NO: 1. In some embodiments, the 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 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 amino acid sequence of human SIRPA set forth in SEQ ID NO: 1. In some embodiments, an anti-SIRPA antibody of the present disclosure binds to an epitope within the D3 domain of human SIRPA, where the epitope includes amino acid residues R282, Q284, and G337 of the amino acid sequence of human SIRPA v1 (SEQ ID NO: 1). In some embodiments, an anti-SIRPA antibody of the present disclosure binds to an epitope within the D3 domain of human SIRPA, where the epitope includes amino acid residues Q281, R282, Q284, L285, W287, R295, E297, V302, and W315 of the amino acid sequence of human SIRPA v1 (SEQ ID NO: 1).

[0207] In some embodiments, the antibody binds to the D3 domain of SIRPA, for example, 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, as bound by an antibody having the CDRs of the antibody designated m3F9, as described herein. Thus, in some embodiments, the antibody of the present disclosure binds to the same SIRPA epitope, or a portion of the SIRPA epitope, as bound by an antibody having the CDRs of the antibody designated m3F9, as described herein.

[0208] Certain embodiments of the present disclosure are at least in part characterized by improved and / or enhanced functional properties (e.g., compared to an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5), including, but not limited to, an improved / enhanced ability to reduce cell surface levels of SIRPA on cells, including, but not limited to, reduced cell proliferation of monocytes, macrophages, T cells, dendritic cells, and / or microglia; reduced proliferation of T cells induced by dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia, activated M1 microglia, M2 microglia, macrophages, M1 macrophages, activated M1 macrophages, and / or M2 macrophages; reduced proliferation of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 Decreased survival of NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; Decreased or reduced proliferation of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, 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, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; Decreased or reduced proliferation of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, NK cells, M1 macrophages, M1 neutrophils, M1 Inhibition of migration of NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, helper T cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia;Reduction or inhibition of one or more functions of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, 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, helper T cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; reduction in proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils, and / or microglia; reduction in overall functionality of monocytes, macrophages, T cells, dendritic cells, neutrophils, and / or microglia; bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, uterine cancer Inhibition of beneficial immune responses against different types of cancer selected from endometrial cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, acute myeloid leukemia, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer; dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, and idiopathic Inhibition of beneficial immune responses to different types of neurological disorders selected from tremor, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, stroke, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, and multiple sclerosis; binding to SIRPA ligands on tumor cells; dendritic cells binding of SIRPA ligands to bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils, and / or macrophages; inhibition of tumor cell killing by one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells; inhibition of anti-tumor cell proliferation activity of one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells;Inhibiting the anti-tumor cell metastatic activity of one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells; modulating the expression of one or more inflammatory receptors, such as CD86, expressed on one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, helper T cells, or cytotoxic T cells; immunosuppressive dendritic cells, immunosuppressive macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, immunosuppressive neutrophils. and regulatory T cells; promoting or rescuing the functionality of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, immunosuppressive neutrophils, immunosuppressive NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and regulatory T cells; and promoting or rescuing the functionality of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, immunosuppressive neutrophils, immunosuppressive NK cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, and non-tumorigenic CD45+ Increased infiltration of tumors by CD14+ myeloid cells and / or regulatory T cells; increased numbers of tumor-promoting myeloid / granulocytic immunosuppressive cells in peripheral blood or other lymphoid organs or within tumors; enhanced tumor-promoting activity of myeloid-derived suppressor cells; decreased activation of tumor-specific T lymphocytes with tumor-killing potential; prolonged survival of non-tumorigenic myeloid-derived suppressor cells and / or non-tumorigenic CD45+ CD14+ myeloid cells; decreased infiltration and / or activation of tumor-specific T lymphocytes with tumor-killing potential; increased rate of tumor growth; increased rate of tumor recurrence;one or more immunotherapies that modulate anti-tumor T cell responses, optionally including one or more of: CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD39, CD70, TREM1, TREM2, Siglec-5, Siglec-7, S based on the identification of anti-SIRPA antibodies that exhibit amelioration and / or enhancement of one or more SIRPA activities, resulting in reduction, neutralization, prevention, or inhibition, including a decrease in the effectiveness of one or more immunotherapies, or one or more chemotherapeutic agents and / or cancer vaccines, that are immunotherapies targeting one or more proteins selected from iglec-9, Siglec-11, SIRPA, CD447, CSF-1 receptor, and any combination thereof;

[0209] In some embodiments, which may be combined with any of the other embodiments described above, the anti-SIRPA antibody of the present disclosure inhibits (i) tumor-infiltrating CD3 + (ii) increasing the number of T cells; and (iii) increasing the number of non-tumorigenic CD14 T cells, which are optionally tumor-infiltrating cells or, optionally, present in the blood. + (iii) reducing cellular levels of SIRPA in bone marrow cells; and (iv) reducing non-tumorigenic CD14, optionally tumor-infiltrating cells, or optionally present in the blood. +(iv) reducing the levels of PD-L1, PD-L2, B7-H2, B7-H3, CD200R, CD163, and / or CD206 in one or more cells, which are optionally non-tumorigenic myeloid-derived suppressor cells (MDSCs); (v) reducing tumor growth rate of solid tumors; (vi) reducing tumor volume; (vii) increasing the effectiveness of one or more PD-1 inhibitors; (viii) optionally increasing the effectiveness of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies targeting one or more of CTL4, the adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (ix) optionally, wherein one or more of the chemotherapeutic agents is selected from the group consisting of gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), ethambutol, acetaminophen, phenytoin ... (x) increasing the efficacy of one or more chemotherapeutic agents, including pirubicin (Elence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (x) increasing T-cell proliferation in the presence of non-tumorigenic myeloid-derived suppressor cells (MDSCs); (xi) inhibiting the differentiation, survival, and / or one or more functions of non-tumorigenic myeloid-derived suppressor cells (MDSCs); and (xii) when conjugated to a chemical or radiotoxin, inducing, enhancing, or augmenting one or more activities, including killing CD33-expressing immunosuppressive non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells within solid tumors and within blood vessels.

[0210] In some embodiments, the anti-SIRPA antibodies of the present disclosure reduce the activity, functionality, or survival of regulatory T cells, tumor-embedded immunosuppressive dendritic cells, tumor-embedded immunosuppressive macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, or chronic myeloid leukemia (CML).

[0211] In some embodiments, the anti-SIRPA antibodies of the present 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 selected from dendritic cells, macrophages, neutrophils, NK cells, microglia, T cells, helper T cells, cytotoxic T cells, and any combination thereof, in an individual.

[0212] Binding affinity of anti-SIRPA antibodies In some embodiments of any of the antibodies presented herein, the antibody has a cytotoxicity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13The antibody has a dissociation constant (Kd) of M (M). The dissociation constant can be determined via any analytical method, including any biochemical or biophysical technique, such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, e.g., the Octet System from ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analysis. In one embodiment, the Kd is measured by a radiolabeled antigen binding assay (RIA). In some embodiments, the RIA is performed with the Fab form of the antibody of interest and its antigen, as described, for example, in Chen et al., J. Mol. Biol., 293:865-881 (1999)). In some embodiments, Kd is measured using a Biacore surface plasmon resonance assay, e.g., an assay using a BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscataway, NJ) performed at 25°C on a CM5 chip with immobilized antigen at approximately 10 response units (RU). D is determined using a monovalent antibody (e.g., a Fab) or a full-length antibody. D is determined using the full-length antibody in a monovalent form.

[0213] antibody fragment In some embodiments of any of the antibodies presented herein, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med., 9:129-134 (2003). For a review of scFv fragments, see, e.g., WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0214] Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP 404097; WO 1993 / 01161; Hudson et al., Nat. Med., 9:129-134 (2003). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med., 9:129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (see, for example, U.S. Patent No. 6,248,516).

[0215] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0216] Chimeric and humanized antibodies In some embodiments of any of the antibodies presented herein, the antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0217] In some embodiments of any of the antibodies presented herein, the antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. In certain embodiments, humanized antibodies are substantially non-immunogenic in humans. In certain embodiments, humanized antibodies have substantially the same affinity for a target as an antibody derived from another species from which the humanized antibody is derived. See, for example, U.S. Patent Nos. 5,530,101, 5,693,761, 5,693,762, and 5,585,089. In certain embodiments, amino acids in the variable domain of an antibody are identified that can be modified to reduce its immunogenicity without reducing the natural affinity of the antigen-binding domain. See, for example, U.S. Patent Nos. 5,766,886 and 5,869,619. Generally, a humanized antibody comprises one or more variable domains in which the HVRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody will also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0218] Humanized antibodies, and methods for making them, are reviewed, for example, in Almagro et al., Front. Biosci., 13:1619-1633 (2008), and are further described, for example, in U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409. Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, for example, Sims et al., J. Immunol., 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light chain or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); These include, but are not limited to, 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)).

[0219] Human antibodies In some embodiments of any of the antibodies provided herein, the antibody is a human antibody. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk et al., Curr. Opin. Pharmacol., 5:368-74 (2001); and Lonberg, Curr. Opin. Immunol., 20:450-459 (2008).

[0220] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Mouse strains deficient in mouse antibody production and containing large fragments of the human Ig loci can be engineered with the expectation that such mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the great diversity of variable genes and the proper regulation of antibody production and expression. By taking advantage of the mouse machinery for antibody diversification and selection and the lack of immune tolerance to human proteins, the human antibody repertoire replicated in these mouse strains can yield high-affinity, fully human antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity can be generated and selected. Certain exemplary methods are described in U.S. Patent No. 5,545,807 and EP 546,073. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0221] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor, J. Immunol., 133:3001 (1984); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826, which describes the production of human IgM monoclonal antibodies from hybridoma cell lines. Human hybridoma technology (trioma technology) is also described in Vollmers et al., Histology and Histopathology, 20(3):927-937 (2005); and Vollmers et al., Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be produced by isolating variable domain sequences of Fv clones selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0222] In some embodiments of any of the antibodies presented herein, the antibody is a human antibody isolated by in vitro methods and / or by screening combinatorial libraries for antibodies with one or more desired activities. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display (Adimab), and the like. As described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994), in one particular phage display method, repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing desired binding properties. See also Sidhu et al., J. Mol. Biol., 338(2):299-310, 2004; Lee et al., J. Mol. Biol., 340(5):1073-1093, 2004; Fellowes Proc. Natl. Acad. Sci. USA, 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods, 284(1-2):119-132 (2004). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources yield high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to yield a single source of antibodies against a broad range of non-self and also self antigens, as described by Griffiths et al., EMBO J. 12:725-734 (1993).Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable HVR3 region and induce rearrangement in vitro, as described by Hoogenboom et al., J. Mol. Biol., 227:381-388, 1992. Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Publications Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are human antibodies or human antibody fragments, as contemplated herein.

[0223] Constant region including the Fc region In some embodiments of any of the anti-SIRPA antibodies provided herein, the antibody comprises an Fc. In some embodiments, the Fc is a human IgG1, IgG2, IgG3, and / or IgG4 isotype. In some embodiments, the antibody is an IgG, IgM, or IgA class antibody.

[0224] In certain embodiments of any of the anti-SIRPA antibodies presented herein, the antibody has an IgG2 isotype. In some embodiments, the anti-SIRPA antibody contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region comprises an Fc region. In some embodiments, the anti-SIRPA antibody induces one or more SIRPA activities, or induces them independently of binding to an Fc receptor. In some embodiments, the anti-SIRPA antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc gamma receptor IIB (FcγIIB).

[0225] In certain embodiments of any of the antibodies presented herein, the antibody has an IgG1 isotype. In some embodiments, the anti-SIRPA antibody contains a mouse IgG1 constant region. In some embodiments, the anti-SIRPA antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region comprises an Fc region. In some embodiments, the anti-SIRPA antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc gamma receptor IIB (FcγIIB).

[0226] In certain embodiments of any of the anti-SIRPA antibodies presented herein, the antibody has an IgG4 isotype. In some embodiments, the anti-SIRPA antibody contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. In some embodiments, the anti-SIRPA antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc gamma receptor IIB (FcγIIB).

[0227] In certain embodiments of any of the anti-SIRPA antibodies presented herein, the antibody has a hybrid IgG2 / 4 isotype. In some embodiments, the anti-SIRPA antibody comprises an amino acid sequence comprising amino acids 118 to 260, according to EU numbering, of human IgG2 and amino acids 261 to 447, according to EU numbering, of human IgG4 (WO1997 / 11971; WO2007 / 106585).

[0228] In some embodiments, the Fc region increases clustering without activating complement compared to a corresponding antibody comprising an Fc region that does not contain amino acid substitutions. In some embodiments, the antibody induces one or more activities of a target to which the antibody specifically binds. In some embodiments, the antibody binds to SIRPA.

[0229] It may also be desirable to modify the anti-SIRPA antibodies of the present disclosure to modify effector function and / or extend the serum half-life of the antibody. For example, the binding site for an Fc receptor on the constant region may be modified or mutated to remove or reduce binding affinity to certain Fc receptors, such as FcγRI, FcγRII, and / or FcγRIII, thereby reducing antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is impaired by removing N-glycosylation of the Fc region of the antibody (e.g., within the CH2 domain of IgG). In some embodiments, effector function is impaired by modifying regions such as 233-236, 297, and / or 327-331 of human IgG, as described in WO 99 / 58572, Armour et al., Molecular Immunology, 40:585-593 (2003); Reddy et al., J. Immunology, 164:1925-1933 (2000). In other embodiments, it may also be desirable to modify effector function by altering the anti-SIRPA antibodies of the present disclosure to increase selectivity for ITIM-containing FcgRIIb (CD32b), thereby increasing clustering of SIRPA antibodies on neighboring cells without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis.

[0230] To extend the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into the antibody (e.g., antibody fragment), 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 contributes to the extended serum half-life of the IgG molecule in vivo.

[0231] multispecific antibodies Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes, including epitopes on the same or different polypeptides (e.g., one or more SIRPA polypeptides of the present disclosure). In some embodiments, multispecific antibodies can be bispecific antibodies. In some embodiments, multispecific antibodies can be trispecific antibodies. In some embodiments, multispecific antibodies can be tetraspecific antibodies. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). In some embodiments, multispecific antibodies comprise a first antigen-binding region that binds to a first site on SIRPA and a second antigen-binding region that binds to a second site on SIRPA. In some embodiments, multispecific antibodies comprise a first antigen-binding region that binds to SIRPA and a second antigen-binding region that binds to a second polypeptide.

[0232] Provided herein is a multispecific antibody comprising a first antigen-binding region that binds to SIRPA and comprises six HVRs of the antibodies described herein, and a second antigen-binding region that binds to a second polypeptide. In some embodiments, the first antigen-binding region comprises six HVRs of the antibodies described herein. H or V L Includes:

[0233] In some embodiments of any of the multispecific antibodies, the second polypeptide is an antigen that facilitates transport across the blood-brain barrier. Numerous antigens and peptides that facilitate transport across the blood-brain barrier are known in the art (see, e.g., Gabathuler R., Neurobiol. Dis., 37:48-57 (2010)). Such second antigens and peptides include, but are not limited to, 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, including CRM197 (a non-toxic mutant of diphtheria toxin), TMEM 30(A) (flippase), protein import domains such as TAT, Syn-B, or penetratin, polyarginine, or generally positively charged peptides, Angiopep peptides such as ANG1005 (see, e.g., Gabathuler, 2010), and other cell surface proteins enriched on blood-brain barrier endothelial cells (see, e.g., Daneman et al., PLoS One, 5(10):e13741 (2010)).

[0234] In some embodiments of any of the multispecific antibodies, the second polypeptide 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), 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 chain AL, S-IBM protein, repeat-associated non-AT G(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; (d) ligands and / or proteins expressed on immune cells, such as CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA-4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG-3, and phosphatidylserine; and / or (e) a disease-causing protein selected from a protein, lipid, polysaccharide, or glycolipid, and any combination thereof, expressed on one or more tumor cells.

[0235] In addition to the SIRPA antigen, the multivalent antibody may recognize additional antigens, such as, but not limited to, Aβ peptide antigen, α-synuclein protein antigen, tau protein antigen, TDP-43 protein antigen, prion protein antigen, huntingtin protein antigen, RAN translation product antigen (containing dipeptide repeats (DPR peptides) composed of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR)), insulin receptor, insulin-like growth factor receptor, or transferrin receptor, or any other antigen that facilitates transport of the antibody across the blood-brain barrier. In some embodiments, the second polypeptide is transferrin. In some embodiments, the second polypeptide is tau. In some embodiments, the second polypeptide is Aβ. In some embodiments, the second polypeptide is TREM2. In some embodiments, the second polypeptide is α-synuclein.

[0236] A multivalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), where one or more polypeptide chains comprise two or more variable domains. For example, one or more polypeptide chains may comprise VD1-(X1) n -VD2-(X2) n -Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, one or more polypeptide chains may comprise a V H -C H 1-Flexible Linker-V H -C H 1 - Fc region chain; or V H -C H 1-V H -C HThe multivalent antibody herein may comprise one or more Fc region chains. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0237] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). See also WO2013 / 026833 (CrossMab). Multispecific antibodies can also be made by manipulating electrostatic steering effects to create heterodimeric antibody Fc molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies (see, e.g., U.S. Pat. No. 4,676,980); using leucine; using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing triabodies as described, for example, in Tutt et al., J. Immunol., 147:60 (1991).

[0238] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, e.g., US2006 / 0025576). Antibodies herein also include "dual-acting FAbs" or "DABs" (see, e.g., US2008 / 0069820), which contain antigen-binding sites that bind to multiple SIRPAs.

[0239] Antibody variants For any of the antibodies presented herein, in some embodiments, amino acid sequence variants of the antibody are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.

[0240] Substitution, insertion, and deletion mutants In some embodiments of any of the antibodies provided herein, antibody variants are provided that have one or more amino acid substitutions. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. TIFF2025032076000003.tif178170

[0241] Substantial alteration of antibody biological properties is achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone, e.g., as a sheet or helix, in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chains. Naturally occurring residues are grouped based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe It can be divided into:

[0242] For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member from another class. Such substituted residues may be introduced, for example, into regions of a human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.

[0243] According to certain embodiments, when making changes to the polypeptides or antibodies described herein, the hydrophobicity index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. The hydrophobicity indexes are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0244] The importance of the hydrophobicity index of amino acids in conferring interactive biological function to a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted with other amino acids having a similar hydrophobicity index or hydrophobicity score and still retain similar biological activity. When making changes based on hydrophobicity index, certain embodiments include amino acid substitutions with hydrophobicity indices within ±2. Certain embodiments include substitutions with hydrophobicity indices within ±1, and certain embodiments include substitutions with hydrophobicity indices within ±0.5.

[0245] It is also understood in the art that substitutions with similar amino acids can be made effectively on the basis of hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as is the case with the present invention. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0246] These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0±1); aspartic acid (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5), and tryptophan (-3.4). When changes are made based on similar hydrophilicity values, certain embodiments include amino acid substitutions within ±2 of their hydrophilicity values, certain embodiments include substitutions within ±1 of their hydrophilicity values, and certain embodiments include substitutions within ±0.5 of their hydrophilicity values. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions."

[0247] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made within an HVR that do not substantially reduce binding affinity. Such changes may, for example, be outside of antigen-contacting residues within the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0248] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to 100 or more residues for polypeptides, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0249] Any cysteine ​​residue 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 the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0250] Glycosylation variants In some embodiments of any of the antibodies presented herein, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0251] Glycosylation of antibodies is typically 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 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.

[0252] Addition of glycosylation sites to an 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 of, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0253] If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide attached, generally via an N-linkage, to Asn297, according to the Kabat numbering, of the CH2 domain of the Fc region. The oligosaccharide may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to create antibody variants with specific, improved properties.

[0254] In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. See, e.g., U.S. Patent Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng., 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys., 249:533-545 (1986); US2003 / 0157108), and knockout cell lines such as CHO cells in which the alpha-1,6-fucosyltransferase gene, FUT8, has been knocked out (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng., 87:614 (2004); and Kanda et al., Biotechnol. Bioeng., 94(4):680-688 (2006)).

[0255] Modified constant region In some embodiments of any of the anti-SIRPA antibodies provided herein, the Fc of the antibody is an isotype and / or modification of the antibody Fc. In some embodiments, the isotype and / or modification of the antibody Fc is capable of binding to an Fc gamma receptor.

[0256] Exemplary antibody Fc isotypes and modifications are provided below in Table 2. In some embodiments, an anti-SIRPA antibody of the disclosure capable of binding to an Fc gamma receptor has an Fc isotype listed in Table 2 below. TIFF2025032076000004.tif255170TIFF2025032076000005.tif48170

[0257] In addition to the isotypes listed in Table 2, and without being bound by theory, it is believed that antibodies with human IgG1 or IgG3 isotypes and mutants thereof that bind to Fcg receptor I, Fcg receptor IIA, Fcg receptor IIC, Fcg receptor IIIA, Fcg receptor IIIB in humans, and / or Fcg receptor I, Fcg receptor III, and Fcg receptor IV in mice (e.g., Strohl (2009), Current Opinion in Biotechnology, 2009, 20:685-691) may also act as transient agonist antibodies.

[0258] In some embodiments, the Fc gamma receptor binding antibody is an antibody of the IgG class, IgM class, or IgA class. In some embodiments, the Fc gamma receptor binding antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or all thirteen) amino acid substitutions in the Fc region at residue positions selected from the group consisting of C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y (residue positions according to EU numbering or Kabat numbering), in any combination. In some embodiments, the Fc region comprises an amino acid substitution at position E430G. In some embodiments, the Fc region comprises amino acid substitutions at positions L243A, L235A, and P331A. In some embodiments, the Fc region comprises amino acid substitutions at positions L243A, L235A, and P331A. In some embodiments, the Fc region comprises amino acid substitutions at positions K322A and E430G. In some embodiments, the Fc region comprises amino acid substitutions at positions P331S and E430G. In some embodiments, the Fc region comprises amino acid substitutions at positions A330S, P331S, and E430G. In some embodiments, the Fc region comprises amino acid substitutions at positions K322A, A330S, and P331S. In some embodiments, the Fc region comprises amino acid substitutions at positions K322A, P331S, and E430G. In some embodiments, the Fc region comprises amino acid substitutions at positions A330S, P331S, and E430G. In some embodiments, the Fc region comprises amino acid substitutions at positions S267E and L328F.In some embodiments, the Fc region comprises an amino acid substitution at position C127S, hi some embodiments, the Fc region comprises amino acid substitutions at positions E345R, E430G, and S440Y.

[0259] In certain embodiments, the Fc gamma receptor-binding antibody has an IgG2 isotype. In some embodiments, the Fc gamma receptor-binding antibody contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region comprises an Fc region. 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 Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are 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 (US2007 / 0148167; Armour et al. (1999), Eur J Immunol, 29:2613-2624; 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 follow the EU numbering or Kabat numbering convention.

[0260] In some embodiments, the Fc gamma receptor binding antibody has an IgG2 isotype with a heavy chain constant domain containing a C127S amino acid substitution, where the amino acid positions follow the EU numbering or Kabat numbering convention (White et al. (2015), Cancer Cell, 27, 138-148; Lightle et al. (2010), PROTEIN SCIENCE, 19:753-762; and WO2008079246).

[0261] In some embodiments, the Fc gamma receptor binding antibody has an IgG2 isotype with a kappa light chain constant domain containing a C214S amino acid substitution, where the amino acid positions follow the EU numbering or Kabat numbering convention (White et al. (2015), Cancer Cell, 27, 138-148; Lightle et al. (2010), PROTEIN SCIENCE, 19:753-762; and WO2008079246).

[0262] In certain embodiments, the Fc gamma receptor-binding antibody has an IgG1 isotype. In some embodiments, the Fc gamma receptor-binding antibody contains a mouse IgG1 constant region. In some embodiments, the Fc gamma receptor-binding antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region comprises an Fc region. 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 Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype).In some embodiments, the one or more amino acid substitutions are 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; 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). Immunol, 200:16~26), P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L2 34V, 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 follow EU numbering or Kabat numbering conventions.

[0263] In some embodiments, the antibody comprises a heavy chain constant domain 1 (CH1) and hinge region of an IgG2 isotype (White et al. (2015), Cancer Cell, 27, 138-148). In certain embodiments, the CH1 and hinge regions of an IgG2 isotype contain the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 58). In some embodiments, the Fc region of the antibody contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and / or an N297A or N297Q amino acid substitution, where the amino acid positions are according to EU numbering or Kabat numbering conventions.

[0264] In certain embodiments, the Fc gamma receptor-binding antibody has an IgG4 isotype. In some embodiments, the Fc gamma receptor-binding antibody contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. 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 Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., compared 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, and / or T256E, where the amino acid positions are according to EU numbering or Kabat numbering conventions.

[0265] In certain embodiments, the Fc gamma receptor-binding antibody has a hybrid IgG2 / 4 isotype. In some embodiments, the Fc gamma receptor-binding antibody comprises an amino acid sequence containing amino acids 118 to 260, according to EU or Kabat numbering, of human IgG2 and amino acids 261 to 447, according to EU or Kabat numbering, of human IgG4 (WO1997 / 11971; WO2007 / 106585).

[0266] In certain embodiments, the antibody contains a murine IgG4 constant region (Bartholomaeus et al. (2014), J. Immunol., 192, 2091-2098).

[0267] In some embodiments, the Fc region further contains one or more additional amino acid substitutions selected from A330L, L234F; L235E, and P331S, according to EU numbering or Kabat numbering; and any combination thereof.

[0268] In certain embodiments, the antibody contains one or more amino acid substitutions in the Fc region at residue positions selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof, where the numbering of the residues is according to EU numbering or Kabat numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, L243A, L235A, and P331S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G and P331S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G and K322A, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, A330S, and P331S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, K322A, A330S, and P331S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, K322A, and A330S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, K322A, and P331S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions S267E and L328F, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at position C127S, where the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E345R, E430G, and S440Y, where the numbering of the residue positions is according to EU numbering.

[0269] In some embodiments, an antibody that binds to a SIRPA protein may include an antibody that reduces the cellular level of SIRPA (e.g., cell surface level of SIRPA), inhibits the interaction (e.g., binding) between SIRPA and / or one or more SIRPA ligands, and inhibits one or more activities of the SIRPA protein. Such an antibody inhibits one or more activities of the SIRPA protein by preventing the interaction (e.g., binding) of SIRPA with one or more SIRPA ligands, or by preventing signal transduction from the extracellular domain of SIRPA to the cytoplasm in the presence of one or more SIRPA ligands. The antibody may also inhibit one or more activities of the SIRPA protein by reducing the cell surface level of SIRPA through inducing SIRPA degradation, SIRPA desensitization, SIRPA cleavage, SIRPA internalization, SIRPA release, down-regulation of SIRPA expression, and / or lysosomal degradation of SIRPA. In some embodiments, such anti-SIRPA antibodies may not transiently activate SIRPA.

[0270] In some embodiments, the anti-SIRPA antibodies of the present disclosure may have the epitope specificity of the transient agonist anti-SIRPA antibodies of the present disclosure, but have an Fc domain that is not capable of binding to an Fcg receptor and therefore is unable to, for example, transiently cluster and activate SIRPA.

[0271] In some embodiments, the anti-SIRPA antibodies of the disclosure have, but are not limited to, the following activities: the ability to decrease the binding of a SIRPA protein to one or more SIRPA ligands, such as sialic acid-containing glycolipids or sialic acid-containing glycoproteins; the ability to decrease the binding of inhibitors of cytokine signaling (SOCS) proteins (e.g., SOCS3 proteins) to a SIRPA protein; the ability to increase degradation of a SIRPA protein by the proteasome; the ability to increase the degradation of a SIRPA protein by circulating dendritic cells, macrophages, monocytes, T cells, and / or microglia. the ability to reduce the functional expression of SIRPA on the surface of cells; the ability to reduce phosphorylation of Tyr-340 and Tyr-358 by Src family tyrosine kinases, such as LCK and FYN; the ability to reduce the recruitment and binding of the tyrosine-specific protein phosphatase β-glucan; the ability to reduce the recruitment and binding of SHP1 and SHP2, the guanine nucleotide-acting PLC-g1; the ability to reduce the recruitment and binding of Crkl, an exchange factor for dynamin 1; and the recruitment of the spleen tyrosine kinase Syk. and its binding to SH3-SH2-SH3 growth factor receptor-bound protein 2 (Grb2); its recruitment and binding to multiple SH2-containing proteins; its ability to increase intracellular calcium mobilization; its ability to modulate the production of pro-inflammatory cytokines IL-1β, IL-8, and TNF-α; its ability to decrease activation of phosphoinositide 3 kinase; its ability to increase proliferation of monocytes, macrophages, dendritic cells, T cells, and / or microglia. ability to increase cell proliferation in monocytes, macrophages, dendritic cells, T cells, and / or microglia; ability to increase tyrosine phosphorylation on multiple cellular proteins; ability to increase phagocytic activity in monocytes, macrophages, dendritic cells, and / or microglia; ability to increase cell proliferation in monocytes, macrophages, dendritic cells, T cells, and / or microglia; ability to increase phosphorylation of signaling molecules that mediate ITAM signaling; ability to increase function of pattern recognition receptors; ability to increase function of Toll-like receptors;The compounds have one or more of the following abilities: increasing the function of damage-associated molecular pattern (DAMP) receptors; modulating the expression of CC chemokine receptor 7 (CCR7); and increasing the clearance of cellular and protein debris.

[0272] In some embodiments, the anti-SIRPA antibodies of the present disclosure have an Fc region that exhibits reduced binding to one or more Fcγ receptors. Examples of such Fc regions and modifications are provided in Table 3 below. In some embodiments, the antibodies have an Fc isotype listed in Table 3 below.

[0273] In some embodiments, the anti-SIRPA antibodies of the disclosure that have reduced binding to Fc gamma receptors have an Fc isotype listed in Table 3 below. TIFF2025032076000006.tif194170

[0274] In certain embodiments, the anti-SIRPA antibody has an IgG1 isotype. In some embodiments, the antibody contains a mouse IgG1 constant region. In some embodiments, the antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region comprises an Fc region. 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., compared to a wild-type Fc region of the same isotype).

[0275] In some embodiments, the one or more amino acid substitutions are N297A, N297Q (Bolt S et al. (1993), Eur J Immunol, 23:403-411), D265A, D270A, L234A, L235A (McEarchern et al. (2007), Blood, 109:1185-1192), C226S, C229S (McEarchern et al. (2007), Blood, 109:1185-1192), P238S (Davis et al. (2007), J Rheumatol, 34:2204~2210), E233P, L234V (McEarchern et al. (2007), Blood, 109:1185~1192), P238A, A327Q, A327G, P329A (Shields RL et al. (2001), J Biol Chem., 276(9):6591~604), K322A, L234F, L235E (Hezareh et al. (2001), J Virol, 75, 12161~12168; Oganesyan et al. (2008), Acta Crystallographica, 64, 700~704), P331S (Oganesyan et al. (2008) Acta Crystallographica, 64, 700-704), T394D (Wilkinson et al. (2013), MAbs, 5(3):406-417), A330L, M252Y, S254T, and / or T256E, where amino acid positions are according to the EU or Kabat numbering convention. In certain embodiments, the Fc region further comprises an amino acid deletion at a position corresponding to glycine 236 according to the EU or Kabat numbering convention.

[0276] In some embodiments, the anti-SIRPA antibody has an IgG1 isotype with a heavy chain constant region containing an amino acid substitution of C220S according to the EU numbering or Kabat numbering convention. In some embodiments, the Fc region further contains one or more additional amino acid substitutions selected from A330L, L234F; L235E, and / or P331S according to the EU numbering or Kabat numbering convention. In certain embodiments, the anti-SIRPA antibody has an IgG2 isotype. In some embodiments, the anti-SIRPA antibody contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region comprises an Fc region. 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., compared to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, V309L, A330S, P331S, C232S, C233S, M252Y, S254T, and / or T256E, where the amino acid positions follow EU numbering or Kabat numbering conventions (Vafa O. et al. (2014), Methods, 65:114-126).

[0277] In certain embodiments, the anti-SIRPA antibody has an IgG4 isotype. In some embodiments, the anti-SIRPA antibody contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. 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., compared to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are E233P, F234V, L235A, G237A, E318A (Hutchins et al. (1995), Proc Natl Acad Sci USA, 92:11980-11984), S228P, L234A / F234A, L236E, S241P, L248E (Reddy et al. (2000), J Immunol, 164:1925-1933; Angal et al. (1993), Mol Immunol., 30(1):105-8; US8614299B2; Vafa (2014), Methods, 65:114-126), T394D, M252Y, S254T, T256E, N297A, and / or N297Q, where amino acid positions are according to the EU numbering or Kabat numbering convention. In some embodiments, the antibody has an IgG4 isotype and contains 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 (residue positions according to EU numbering).

[0278] In some embodiments, the Fc region further contains one or more additional amino acid substitutions selected from M252Y, S254T, and / or T256E, where the amino acid positions are according to the EU numbering or Kabat numbering convention.

[0279] In some embodiments of any of the antibodies presented herein, the Fc of the modified antibody is a modified Fc of IgG1. In some embodiments, the modified Fc of IgG1 comprises one or more modifications. For example, in some embodiments, the modified Fc of IgG1 comprises one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions include N297A (Bolt S et al. (1993), Eur J Immunol, 23:403-411), D265A (Shields et al. (2001), RJ Biol. Chem., 276, 6591-6604), L234A, L235A (Hutchins et al. (1995), Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al. (1994), Transplantation, 57:1537-1543; Xu et al. (2000), Cell Immunol, 200:16-26), G237A (Alegre et al. (1994), Transplantation, 57:1537-1543; Xu et al. (2000), Cell Immunol, 200:16-26), or GA (Alegre et al. (1994), Transplantation, 57:1537-1543; Xu et al. (2000), Cell Immunol, 200:16-26). Immunol, 200:16-26), 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, and / or T256E, where the amino acid positions follow the EU numbering convention.

[0280] In some embodiments of any of the modified Fc of IgG1, the Fc comprises an N297A mutation according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises D265A and N297A mutations according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises a D270A mutation according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises L234A and L235A mutations according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises L234A and G237A mutations according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises L234A, L235A, and G237A mutations according to EU numbering. In some embodiments of any of the modified Fcs of IgG1, the Fc comprises one or more (including all) mutations of P238D, L328E, E233, G237D, H268D, P271G, and A330R according to EU numbering. In some embodiments of any of the modified Fcs of IgG1, the Fc comprises one or more mutations of S267E / L328F according to EU numbering. In some embodiments of any of the modified Fcs of IgG1, the Fc comprises mutations of P238D, L328E, E233D, G237D, H268D, P271G, and A330R according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises the following mutations according to EU numbering: P238D, L328E, G237D, H268D, P271G, and A330R. In some embodiments of any of the modified Fc of IgG1, the Fc comprises the following mutations according to EU numbering: P238D, S267E, L328E, E233D, G237D, H268D, P271G, and A330R. In some embodiments of any of the modified Fc of IgG1, the Fc comprises the following mutations according to EU numbering: P238D, S267E, L328E, G237D, H268D, P271G, and A330R.In some embodiments of any of the modified Fc of IgG1, the Fc comprises the C226S, C229S, E233P, L234V, and L235A mutations according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises the L234F, L235E, and P331S mutations according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises the S267E and L328F mutations according to EU numbering. In some embodiments of any of the modified Fc of IgG1, the Fc comprises the S267E mutation according to EU numbering. In some embodiments of any of the modified Fcs of IgG1, the Fc comprises a substitution of the heavy chain constant region 1 (CH1) and hinge region of IgG1 with the CH1 and hinge region of IgG2 (amino acids 118-230 of IgG2 according to EU numbering), along with a kappa light chain.

[0281] In some embodiments of any of the modified IgG1 Fcs, the Fc comprises two or more amino acid substitutions that increase antibody clustering without activating complement, compared to a corresponding antibody having an Fc region that does not comprise these two or more amino acid substitutions. Thus, in some embodiments of any of the modified IgG1 Fcs, the modified IgG1 Fc is an antibody comprising an Fc region, wherein the antibody comprises an amino acid substitution at position E430G according to EU numbering, and one or more amino acid substitutions within the Fc region at residue positions selected from L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, and any combination thereof. In some embodiments, the modified IgG1 Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G, A330S, and P331S according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments, the modified Fc of IgG1 comprises amino acid substitutions at positions E430G, K322A, and P331S according to EU numbering.

[0282] In some embodiments of any of the modified IgG1 Fc's, the modified IgG1 Fc's herein may be combined with an A330L mutation (Lazar et al., Proc Natl Acad Sci USA, 103:4005-4010 (2006)), or one or more of an L234F, L235E, and / or P331S mutation (Sazinsky et al., Proc Natl Acad Sci USA, 105:20167-20172 (2008)), according to the EU numbering convention, to abolish complement activation. In some embodiments of any of the modified IgG1 Fc's, the modified IgG1 Fc may further include one or more of an A330L, A330S, L234F, L235E, and / or P331S mutation according to the EU numbering convention. In some embodiments of any of the modified Fc of IgG1, the modified Fc of IgG1 may further comprise one or more mutations that extend the half-life of the antibody in human serum (e.g., one or more (including all) of M252Y, S254T, and T256E mutations according to the EU numbering convention). In some embodiments of any of the modified Fc of IgG1, the modified Fc of IgG1 may further comprise one or more of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and / or S440W according to EU numbering.

[0283] Another aspect of the present disclosure relates to antibodies with modified constant regions (i.e., Fc regions). Antibodies that rely on binding to FcgR receptors to activate target receptors can lose their agonistic activity when engineered to eliminate FcgR binding (see, e.g., Wilson et al., Cancer Cell, 19:101-113 (2011); Armour et al., Immunology, 40:585-593 (2003); and White et al., Cancer Cell, 27:138-148 (2015)). Thus, it is believed that anti-SIRPA antibodies of the present disclosure with the appropriate epitope specificity can activate target antigens with minimal adverse effects if the antibodies have an Fc domain (CH1 region and hinge region) derived from the human IgG2 isotype or another type of Fc domain capable of preferentially binding to the inhibitory FcgRIIB receptor or a variation thereof.

[0284] In some embodiments of any of the antibodies presented herein, the Fc of the modified antibody is a modified Fc of IgG2. In some embodiments, the modified Fc of IgG2 comprises one or more modifications. For example, in some embodiments, the modified Fc of IgG2 comprises one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype). In some embodiments of any of the modified Fc of IgG2, the one or more amino acid substitutions are V234A (Alegre et al., Transplantation, 57:1537-1543 (1994); Xu et al., Cell Immunol, 200:16-26 (2000)); G237A (Cole et al., Transplantation, 68:563-571 (1999)); H268Q, V309L, A330S, P331S (US2007 / 0148167; Armour et al., Eur J Immunol, 29:2613-2624 (1999); Armour et al., Haematology Journal, 1 (Suppl. 1):27 (2000)), C219S, and / or C220S (White et al., Cancer Cell, 27, 138-148 (2015)); S267E, L328F (Chu et al., Mol Immunol, 45:3926-3933 (2008)); and M252Y, S254T, and / or T256E. In some embodiments of any of the modified Fcs of IgG2, the Fc comprises amino acid substitutions at positions V234A and G237A according to EU numbering. In some embodiments of any of the modified Fcs of IgG2, the Fc comprises amino acid substitutions at positions C219S or C220S according to EU numbering. In some embodiments of any of the modified Fcs of IgG2, the Fc comprises amino acid substitutions at positions A330S and P331S according to EU numbering. In some embodiments of any of the modified Fcs of IgG2, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering.

[0285] In some embodiments of any of the modified Fc of IgG2, the Fc comprises a C127S amino acid substitution according to the EU numbering convention (White et al. (2015), Cancer Cell, 27, 138-148; Lightle et al., Protein Sci., 19:753-762 (2010); and WO2008 / 079246). In some embodiments of any of the modified Fc of IgG2, the antibody has an IgG2 isotype with a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention (White et al., Cancer Cell, 27:138-148 (2015); Lightle et al., Protein Sci., 19:753-762 (2010); and WO2008 / 079246).

[0286] In some embodiments of any of the modified Fc of IgG2, the Fc comprises a C220S amino acid substitution according to the EU numbering convention. In some embodiments of any of the modified Fc of IgG2, the antibody has an IgG2 isotype with a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention.

[0287] In some embodiments of any of the modified Fc of IgG2, the Fc comprises a C219S amino acid substitution according to the EU numbering convention. In some embodiments of any of the modified Fc of IgG2, the antibody has an IgG2 isotype with a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention.

[0288] In some embodiments of any of the modified IgG2 Fcs, the Fc comprises the heavy chain constant domain 1 (CH1) and hinge region of the IgG2 isotype (White et al., Cancer Cell, 27:138-148 (2015)). In certain embodiments of any of the modified IgG2 Fcs, the CH1 and hinge region of the IgG2 isotype comprise the amino acid sequence of 118-230 according to EU numbering. In some embodiments of any of the modified IgG2 Fcs, the Fc region of the antibody comprises an amino acid substitution of S267E, an amino acid substitution of L328F, or both, and / or an amino acid substitution of N297A or N297Q according to the EU numbering convention.

[0289] In some embodiments of any of the modified Fcs of IgG2, the Fc further comprises one or more amino acid substitutions at positions E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W according to EU numbering. In some embodiments of any of the modified Fcs of IgG2, the Fc may further comprise one or more mutations that extend the half-life of the antibody in human serum (e.g., one or more (including all) of the following mutations according to the EU numbering convention: M252Y, S254T, and T256E). In some embodiments of any of the modified Fcs of IgG2, the Fc may further comprise A330S and P331S.

[0290] In some embodiments of any of the modified Fcs of IgG2, the Fc is an IgG2 / 4 hybrid Fc. In some embodiments, the IgG2 / 4 hybrid Fc comprises amino acids 118-260 of IgG2 and amino acids 261-447 of IgG4. In some embodiments of any modified Fc of IgG2, the Fc comprises one or more amino acid substitutions at positions H268Q, V309L, A330S, and P331S according to EU numbering.

[0291] In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises one or more additional amino acid substitutions selected from A330L, L234F; L235E, or P331S according to EU numbering; and any combination thereof.

[0292] In certain embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises one or more amino acid substitutions at residue positions selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof, according to EU numbering. In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S, according to EU numbering. In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G and P331S, according to EU numbering. In some embodiments for any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments for any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G, A330S, and P331S according to EU numbering. In some embodiments for any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments for any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments for any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E430G, K322A, and P331S according to EU numbering. In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering.In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some embodiments of any of the modified Fcs of IgG1 and / or IgG2, the Fc comprises amino acid substitutions at positions E345R, E430G, and S440Y according to EU numbering.

[0293] In some embodiments of any of the antibodies presented herein, the Fc of the modified antibody is a modified IgG4 Fc. In some embodiments, the modified IgG4 Fc comprises one or more modifications. For example, in some embodiments, the modified IgG4 Fc comprises one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype). In some embodiments of any of the modified IgG4 Fcs, the one or more amino acid substitutions are selected from L235A, G237A, S229P, L236E (Reddy et al., J Immunol, 164:1925-1933 (2000)), S267E, E318A, L328F, M252Y, S254T, and / or T256E, according to the EU numbering convention. In some embodiments of any of the modified IgG4 Fc's, the Fc may further comprise L235A, G237A, and E318A, according to the EU numbering convention. In some embodiments of any of the modified IgG4 Fc's, the Fc may further comprise S228P and L235E, according to the EU numbering convention. In some embodiments of any of the modified IgG4 Fc's, the IgG4 modified Fc may further comprise S267E and L328F, according to the EU numbering convention.

[0294] In some embodiments of any of the modified Fc of IgG4, the modified Fc of IgG4 may be combined with the S228P mutation (Angal et al., Mol Immunol., 30:105-108 (1993)), and / or one or more mutations described in (Peters et al., J Biol Chem., 287(29):24525-33 (2012)), according to the EU numbering convention, to enhance antibody stabilization.

[0295] In some embodiments of any of the modified Fc of IgG4, the modified Fc of IgG4 may further comprise one or more mutations that extend the half-life of the antibody in human serum (e.g., one or more (including all) of the following mutations, according to the EU numbering convention: M252Y, S254T, and T256E).

[0296] In some embodiments of any of the modified IgG4 Fcs, the Fc comprises L235E according to EU numbering. In certain embodiments of any of the modified IgG4 Fcs, the Fc comprises one or more amino acid substitutions at residue positions selected from C127S, F234A, L235A, L235E, S267E, K322A, L328F, E345R, E430G, S440Y, and any combination thereof according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises an amino acid substitution at position E430 according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc region comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some embodiments of any of the modified IgG4 Fcs, the Fc comprises amino acid substitutions at positions E345R, E430G, and S440Y according to EU numbering.

[0297] Other antibody modifications In some embodiments of any of the antibodies, the antibody is a derivative. The term "derivative" refers to a molecule that includes a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative includes a covalent modification, including, but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified antigen-binding protein may have a longer circulating half-life than an antigen-binding protein that is not chemically modified. In certain embodiments, a chemically modified antigen-binding protein may have improved targeting ability to desired cells, tissues, and / or organs. In some embodiments, a derivative antigen-binding protein is covalently modified to include the conjugation of one or more water-soluble polymers, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In certain embodiments, the derivative antigen-binding proteins comprise one or more polymers, including, but not limited to, monomethoxypolyethylene glycol, dextran, cellulose, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), poly(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers.

[0298] In certain embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are attached at one or more specific positions, for example, at the amino terminus of the derivative. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In certain embodiments, PEG is used to improve the therapeutic potential of antigen-binding proteins. In certain embodiments, PEG is used to improve the therapeutic potential of humanized antibodies. Certain such methods are discussed, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0299] In the pharmaceutical industry, peptide analogs are commonly used as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics." Fauchere, J. Adv. Drug Res., 15:29 (1986); and Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated herein by reference for any purpose. Such compounds are often developed with the aid of computerized molecular modeling. Structurally similar peptides and therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. In general, a peptidomimetic is structurally similar to a paradigm polypeptide (i.e., a polypeptide having biochemical properties or pharmacological activity), such as a human antibody, but optionally has one or more peptide bonds replaced with a bond selected from -CHNH-, -CHS-, -CH-CH-, -CH=CH- (cis and trans), -COCH-, -CH(OH)CH-, and -CHSO-, by methods well known in the art. In certain embodiments, systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Additionally, constrained peptides containing consensus sequences, or variations on substantially identical consensus sequences, can be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by reference for any purpose); for example, by adding internal cysteine ​​residues capable of cyclizing the peptide or forming intramolecular disulfide bridges.

[0300] Drug conjugation involves coupling a biologically active, cytotoxic (anticancer) payload or drug to an antibody that specifically targets a particular tumor marker (e.g., a polypeptide ideally found only in or on tumor cells). The antibody tracks these proteins in the body and attaches itself to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal within the tumor cell, which then absorbs or internalizes the antibody, along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cancer. Because of this targeting, the drug ideally has low side effects and offers a broader therapeutic window than other chemotherapeutic agents. Techniques for conjugating antibodies have been disclosed and are known in the art (see, e.g., Jane de Lartigue, OncLive, July 5, 2012; "ADC Review on antibody-drug conjugates"; and Ducry et al., Bioconjugate Chemistry, 21(1):5-13 (2010)).

[0301] Antibody Framework Any of the anti-SIRPA antibodies described herein further comprises a framework (FR). In some embodiments, the framework is a human immunoglobulin framework. For example, in some embodiments, an antibody (e.g., an anti-SIRPA antibody) comprises an HVR as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. A human immunoglobulin framework may be part of a human antibody, or a non-human antibody may be humanized by replacing one or more endogenous frameworks with a human framework region(s). Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence 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)); and human mature framework regions. These include, but are not limited to, (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)).

[0302] In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising one or more (e.g., one or more, two or more, three or more, or all four) framework regions selected from VH FR1, VH FR2, VH FR3, and VH FR4 (shown in Table 9). In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising a VH FR1 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising a VH FR2 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising a VH FR3 comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising a VH FR4 comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising a VH FR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 27, and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 28.

[0303] In some embodiments, an anti-SIRPA antibody of the disclosure comprises a heavy chain variable region, including VH FR1, VH FR2, VH FR3, and VH FR4, of anti-SIRPA antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

[0304] In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a light chain variable region comprising one or more (e.g., one or more, two or more, three or more, or all four) framework regions selected from VL FR1, VL FR2, VL FR3, and VL FR4 (shown in Table 9). In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a light chain variable region comprising a VL FR1 comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a light chain variable region comprising a VL FR2 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a light chain variable region comprising a VL FR3 comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a light chain variable region comprising a VL FR4 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a light chain variable region comprising a VL FR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 30, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 31, and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 32.

[0305] In some embodiments, an anti-SIRPA antibody of the disclosure comprises a light chain variable region, including VL FR1, VL FR2, VL FR3, and VL FR4, of anti-SIRPA antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

[0306] In some embodiments, an anti-SIRPA antibody of the present disclosure comprises a heavy chain variable region comprising a VH FR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 27, and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising a VL FR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 30, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 31, and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-SIRPA antibody of the disclosure comprises VH FR1, VH FR2, VH FR3, and VH FR4 of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25. and a light chain variable region comprising VL FR1, VL FR2, VL FR3, and VL FR4 of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

[0307] In some embodiments, the invention provides an anti-SIRPA antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:47 and the light chain comprises the amino acid sequence of SEQ ID NO:50.

[0308] In some embodiments, the invention provides an anti-SIRPA antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:48 and the light chain comprises the amino acid sequence of SEQ ID NO:50.

[0309] In some embodiments, the invention provides an anti-SIRPA antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:49 and the light chain comprises the amino acid sequence of SEQ ID NO:50.

[0310] In some embodiments, the invention provides an anti-SIRPA antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:51 and the light chain comprises the amino acid sequence of SEQ ID NO:50.

[0311] In some embodiments, the invention provides an anti-SIRPA antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:52 and the light chain comprises the amino acid sequence of SEQ ID NO:50.

[0312] Nucleic acids, vectors, and host cells The anti-SIRPA antibodies of the present disclosure can be made using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, an isolated nucleic acid is provided having a nucleotide sequence encoding any of the anti-SIRPA antibodies of the present disclosure. Such nucleic acids may encode the V, ... L and / or V H In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In some embodiments, host cells containing such nucleic acids are also provided. In some embodiments, the host cells are L and an amino acid sequence comprising the V of the antibody H or (2) a vector comprising a nucleic acid encoding an amino acid sequence comprising the V of an antibody. L a first vector comprising a nucleic acid encoding an amino acid sequence comprising: Hand a second vector (e.g., transduced with) that comprises a nucleic acid encoding an amino acid sequence comprising: In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or 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.

[0313] Methods for making an anti-SIRPA antibody of the present disclosure are provided. In some embodiments, the method comprises culturing a host cell of the present disclosure comprising a nucleic acid encoding an anti-SIRPA antibody under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

[0314] For recombinant production of the anti-SIRPA antibodies of the present disclosure, nucleic acids encoding the anti-SIRPA antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0315] Suitable vectors containing nucleic acid sequences encoding any of the anti-SIRPA antibodies of the present disclosure, or their cell surface expression fragments or polypeptides (including antibodies) described herein, include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors may be constructed according to standard techniques or may be selected from a large number of cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended for use, but useful cloning vectors generally have the ability to autonomously replicate, may possess a single target for a particular restriction endonuclease, and / or may carry a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+), and their derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These cloning vectors, and many others, are commercially available from sources such as BioRad, Strategene, and Invitrogen.

[0316] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, the anti-SIRPA antibodies of the present disclosure can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. Expression of antibody fragments and polypeptides in bacteria is described in, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0317] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns, are also suitable cloning or expression hosts for antibody-encoding vectors (e.g., Gerngross, Nat. Biotech., 22:1409-1414 (2004); and Li et al., Nat. Biotech., 24:210-215 (2006)).

[0318] Suitable host cells for the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. In particular, numerous baculovirus strains have been identified that can be used with insect cells for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0319] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can also be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV1 cell line; human embryonic kidney cell 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); see, e.g., Mather et al., Annals TRI cells; MRC5 cells; and FS4 cells, described in NYAcad. Sci., 383:44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including 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 certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (eds. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0320] Pharmaceutical Compositions / Formulations Provided herein are pharmaceutical compositions and / or pharmaceutical formulations comprising an anti-SIRPA antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0321] In some embodiments, pharmaceutically acceptable carriers are preferably non-toxic to recipients at the dosages and concentrations employed. The antibodies described herein can be formulated into preparations in solid, semi-solid, liquid, or gaseous form. Examples of such formulations include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Depending on the formulation, pharmaceutically acceptable carriers can include any desired pharmaceutically acceptable, non-toxic carrier, including diluents, which are vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. In certain embodiments, pharmaceutical compositions can include formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition.

[0322] In certain embodiments, the pharmaceutically acceptable carrier may be selected from the group consisting of amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides. preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. Further examples of formulations suitable for various types of administration can be found in "The Remington: Science and Practice of Pharmacy," Pharmaceutical Press, 22nd Edition (2013). For a brief review of methods for drug delivery, see Langer, Science, 249:1527-1533 (1990).

[0323] 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, as well as aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0324] Formulations can be optimized for retention and stabilization in the brain or central nervous system. When a drug is administered to the intracranial compartment, it is desirable for the drug to be retained within this compartment and not diffuse or otherwise cross the blood-brain barrier. Stabilization methods include cross-linking, multimerization, or linking to groups such as polyethylene glycol, polyacrylamide, or neutral protein carriers to achieve increased molecular weight.

[0325] Other strategies for increasing retention include encapsulating antibodies, such as the anti-SIRPA antibodies of the present disclosure, within biodegradable or bioerodible implants. The release rate of the therapeutically active agent is controlled by the rate of transport through the polymer matrix and the biodegradation of the implant. The implants can be particles, sheets, patches, plaques, fibers, microcapsules, etc., and can be of any size or shape compatible with the selected insertion site. Biodegradable polymer compositions that can be utilized can be organic esters or organic ethers that degrade to yield physiologically acceptable degradation products, including monomers. Anhydrides, amides, orthoesters, etc., can be used by themselves or in combination with other monomers. The polymers will be condensation polymers. The polymers may be crosslinked or non-crosslinked. Polymers 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. Among the polysaccharides of interest are calcium alginate and functionalized celluloses, particularly carboxymethylcellulose esters, characterized by insolubility in water and a molecular weight of about 5 kD to 500 kD. Biodegradable hydrogels may also be utilized within the implants of the subject invention. Hydrogels are typically copolymeric materials characterized by their ability to absorb fluid.

[0326] therapeutic use As disclosed herein, the anti-SIRPA antibodies of the present disclosure can be used to prevent, reduce the risk of, or treat diseases and disorders.

[0327] 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 the disease or pathology associated with SIRPA expression, activity, and / or signal transduction in a subject. The therapeutic regimen is carried out by using standard methods to identify a subject, such as a human patient, who suffers from (or is at risk of developing) a disease or disorder associated with SIRPA expression, activity, and / or signal transduction, such as cancer or other neoplastic disorders. In some embodiments, cells with pathology associated with SIRPA expression, activity, and / or signal transduction express SIRPA ligand, such as CD47. In some embodiments, cells with pathology associated with SIRPA expression, activity, and / or signal transduction express SIRPA.

[0328] 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 signal transduction. In the present disclosure, the terms "in combination" and "in combination" are used interchangeably. The additional therapeutic agent administered in combination with an anti-SIRPA antibody can be administered before, after, or simultaneously with the agent that downregulates SIRPA, such as an anti-SIRPA antibody.

[0329] In one embodiment of the present disclosure, for example, an anti-SIRPA antibody preparation is administered to a human subject, comprising an anti-SIRPA antibody that reduces the expression of SIRPA on the cell surface but does not substantially block the binding of a ligand, for example, 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 binding to a ligand, for example, binding to CD47.

[0330] In one embodiment, the disease or disorder associated with the expression of SIRPA is cancer.In some embodiments, anti-SIRPA antibody is administered to patients with cancer, such as hematoproliferative disease, of bone marrow cells that express SIRPA.In typical embodiments, anti-SIRPA antibody is administered to patients with cancer that expresses CD47.

[0331] In certain embodiments, cancers prevented or treated by the methods of the present disclosure include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, non-squamous NSCLC, glioma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastric (gastric or stomach) cancer including gastrointestinal cancer and gastrointestinal stromal 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, Cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or cancer), stomach cancer, germ cell tumors, pediatric sarcoma, sinonasal natural killer cell lymphoma, melanoma (e.g., metastatic malignant melanoma such as cutaneous malignant melanoma or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system Hematological tumors derived from two major blood cell lineages, i.e., myeloid cell lineages (producing granulo...

Claims

1. An isolated antibody that binds to human SIRPA, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23, and 24; the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

2. 2. The antibody of claim 1, wherein the heavy chain variable region comprises one, two, three or four framework regions selected from VH FR1 comprising the amino acid sequence of SEQ ID NO:25, VH FR2 comprising the amino acid sequence of SEQ ID NO:26, VH FR3 comprising the amino acid sequence of SEQ ID NO:27, and VH FR4 comprising the amino acid sequence of SEQ ID NO:

28.

3. 2. The antibody of claim 1, wherein the light chain variable region comprises one, two, three or four framework regions selected from a VL FR1 comprising the amino acid sequence of SEQ ID NO:29, a VL FR2 comprising the amino acid sequence of SEQ ID NO:30, a VL FR3 comprising the amino acid sequence of SEQ ID NO:31, and a VL FR4 comprising the amino acid sequence of SEQ ID NO:

32.

4. 2. The antibody of claim 1, wherein the heavy chain variable region comprises one, two, three or four framework regions selected from VH FR1 comprising the amino acid sequence of SEQ ID NO:25, VH FR2 comprising the amino acid sequence of SEQ ID NO:26, VH FR3 comprising the amino acid sequence of SEQ ID NO:27, and VH FR4 comprising the amino acid sequence of SEQ ID NO:28; and the light chain variable region comprises one, two, three or four framework regions selected from VL FR1 comprising the amino acid sequence of SEQ ID NO:29, VL FR2 comprising the amino acid sequence of SEQ ID NO:30, VL FR3 comprising the amino acid sequence of SEQ ID NO:31, and VL FR4 comprising the amino acid sequence of SEQ ID NO:

32.

5. The antibody of claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, or at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35.

6. 6. The antibody of claim 1 or claim 5, wherein the light chain variable region comprises an amino acid sequence that is at least 90%, or at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

7. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35.

8. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

9. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

10. 1. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3 (as shown in Table 8) of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

11. 1. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3 (as shown in Table 7) of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

12. 1. An isolated antibody that binds to human SIRPA, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HVR-H1, HVR-H2, HVR-H3, HVR-H4, HVR-H5, HVR-H6, ​​HVR-H7, HVR-H8, HVR-H9, HVR-H10, HVR-H21, HVR-H3, HVR-H4, HVR-H5, HVR-H6, ​​HVR-H7, HVR-H8, HVR-H9, HVR-H11, HVR-H12, HVR-H13, HVR-H14, HVR-H15, HVR-H16, HVR-H17, HVR-H18, HVR-H19, HVR-H20, HVR-H19, HVR-H21, HVR-H22, HVR-H23, HVR-H24, HVR-H25, HVR-H16, HVR-H17, HVR-H18, HVR-H19, HVR-H20, HVR-H19, HVR-H21, HVR-H22, HVR-H23, HVR-H24, HVR-H25, HVR-H26, HVR-H17, HVR-H18, HVR-H19 ... and HVR-H3 (shown in Table 8); and an antibody wherein the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3 (shown in Table 7) of antibody 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25.

13. An isolated antibody that binds to human SIRPA, the antibody comprising a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, and a light chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, and comprising antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-1 and HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of 3F9-1, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, or 3F9-25 (as shown in Tables 7 and 8).

14. 14. The antibody of claim 10, wherein the heavy chain variable region comprises one, two, three, or four framework regions selected from VH FR1, VH FR2, VH FR3, and VH FR4, wherein VH FR1 comprises the amino acid sequence of SEQ ID NO:25; VH FR2 comprises the amino acid sequence of SEQ ID NO:26; VH FR3 comprises the amino acid sequence of SEQ ID NO:27; VH FR4 comprises the amino acid sequence of SEQ ID NO:28; and / or the light chain variable region comprises one, two, three, or four framework regions selected from VL FR1, VL FR2, VL FR3, and VL FR4, wherein VL FR1 comprises the amino acid sequence of SEQ ID NO:29; VL FR2 comprises the amino acid sequence of SEQ ID NO:30; VL FR3 comprises the amino acid sequence of SEQ ID NO:31; and VL FR4 comprises the amino acid sequence of SEQ ID NO:

32.

15. 15. The antibody of any one of claims 1 to 14, which is a monoclonal antibody.

16. 16. The antibody of claim 1 , which is a humanized antibody.

17. Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab') ２ The antibody of claim 1 which is a fragment, an Fv fragment, or an scFv fragment.

18. 18. The antibody of claim 1 , which is a multivalent antibody.

19. 19. The antibody of any one of claims 1 to 18, which is an antibody of the IgG class, the IgM class, or the IgA class.

20. 20. The antibody of claim 19, which is an antibody of the IgG class and is an IgG1, IgG2, IgG3, or IgG4 isotype.

21. 21. An antibody according to any one of claims 1 to 20, which binds to an inhibitory Fc receptor.

22. 22. The antibody of claim 21 , wherein the inhibitory Fc receptor is inhibitory Fc gamma receptor IIB (FcgRIIB).

23. The antibody of claim 22, which reduces intracellular levels of FcgRIIB.

24. 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, M252Y, S254T, T256E 24. The antibody of any one of claims 1 to 23, comprising one or more amino acid substitutions at amino acid residues in the Fc region selected from the group consisting of: N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof (residue numbering according to EU numbering), or comprising an amino acid deletion at a position corresponding to glycine 236 in the Fc region.

25. 25. The antibody of any one of claims 1 to 24, comprising one or more amino acid substitutions at residue positions in the Fc region selected from the group consisting of 127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof (wherein the numbering of the amino acid residues is according to EU numbering or Kabat numbering).

26. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:47 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

27. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:48 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

28. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:49 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

29. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:53, and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

30. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:54 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

31. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:51, and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

32. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:52 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

33. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:55, and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

34. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:56 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

35. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:57 and the light chain comprising the amino acid sequence of SEQ ID NO:

50.

36. The antibody of any one of claims 1 to 35, wherein the anti-SIRPA antibody reduces cell surface levels of SIRPA, reduces intracellular levels of SIRPA, reduces total cellular levels of SIRPA, or any combination thereof.

37. The antibody of any one of claims 1 to 36, wherein the anti-SIRPA antibody induces degradation of SIRPA, induces cleavage of SIRPA, induces internalization of SIRPA, induces release of SIRPA, downregulates expression of SIRPA, or any combination thereof.

38. 38. An antibody described in any one of claims 1 to 37, which reduces cell surface levels of SIRPA in vitro.

39. 39. The antibody of any one of claims 1 to 38, which reduces cell surface levels of SIRPA in vivo.

40. 40. The antibody of any one of claims 1 to 39, which downregulates expression of SIRPA in human monocytes.

41. 41. The antibody of any one of claims 1 to 40, which downregulates expression of SIRPA in human macrophages.

42. The antibody of claim 41, which downregulates expression of SIRPA in human macrophages by about 70-95%.

43. Affinity for human SIRPA v1 (K) of less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. Ｄ 43. The antibody of any one of claims 1 to 42, having the following structure:

44. Affinity for human SIRPA v1 of approximately 0.1 nM to 2 nM (K Ｄ 44. The antibody of any one of claims 1 to 43, having the following structure:

45. A K that is at least half, at least a third, at least a fourth, at least a fifth, at least a sixth, at least a seventh, at least an eighth, at least a ninth, or at least a tenth of the affinity for human SIRPA of an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

5. Ｄ 45. The antibody of any one of claims 1 to 44, having affinity for human SIRPA v1 at

46. 46. ​​The antibody of any one of claims 1 to 45, which reduces cell surface levels of SIRPA v1 in vitro with a 50% effective concentration (EC50) of about 0.05 nM to 2 nM as measured by flow cytometry.

47. 46. ​​The antibody of any one of claims 1 to 45, which reduces cell surface levels of SIRPA in vitro with a 50% effective concentration (EC50) of about 0.05-0.20 nM for human SIRPA v1, about 0.05-0.10 nM for human SIRPA v2, and / or about 0.05-1 nM for cynomolgus SIRPA, as measured by flow cytometry.

48. 48. The antibody of any one of claims 1 to 47, which binds to human SIRPA, human SIRPA v1, human SIRPA v2, cynomolgus SIRPA, marmoset SIRPA, and human SIRPβ3.

49. 49. The antibody of any one of claims 1 to 48, which binds to the D3 domain of human SIRPA v1 of SEQ ID NO:

1.

50. 50. The antibody of any one of claims 1 to 49, which binds to amino acid residues R282, Q284, and G337 of human SIRPA v1 of SEQ ID NO:

1.

51. 51. The antibody of any one of claims 1 to 50, wherein the anti-SIRPA antibody increases phagocytosis of tumor cells in macrophages, increases phagocytosis of tumor cells in M1 macrophages, increases phagocytosis of tumor cells in M2 macrophages, downregulates expression of CD14 in macrophages, and / or any combination thereof.

52. 52. The antibody of any one of claims 1 to 51, wherein the anti-SIRPA antibody enhances T cell proliferation.

53. 53. The antibody of any one of claims 1 to 52, wherein the anti-SIRPA antibody enhances T cell proliferation without blocking the interaction of SIRPy with CD47.

54. The antibody of any one of claims 1 to 53, wherein the anti-SIRPA antibody stimulates the production of ROS in monocytes and / or increases the expression of IL-8 in monocytes.

55. The antibody of any one of claims 1 to 54, wherein the anti-SIRPA antibody inhibits tumor growth in vivo.

56. 56. The antibody of any one of claims 1 to 55, wherein the anti-SIRPA antibody reduces the number of CD14+ myeloid cells in peripheral blood and / or increases the number of CD14+ myeloid cells in a tumor.

57. 57. The antibody of any one of claims 1 to 56, which binds to human SIRPA but does not substantially block binding of CD47 to SIRPA.

58. The antibody recognizes a first antigen and a second antigen, the first antigen being SIRPA and the second antigen being (a) an antigen that facilitates transport across the blood-brain barrier; (b) an antigen that facilitates transport across the blood-brain barrier selected from 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), and diphtheria toxin receptor; (c) a disease-causing agent selected from a disease-causing peptide or protein, 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 fragments thereof, tau, IAPP, α-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 1 a disease-causing agent selected from: .OMEGA., 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 selected from 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.

58. The anti-SIRP antibody of any one of claims 1 to 57,

59. 11. A method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-SIRP antibody of any one of claims 1-58, thereby treating the cancer.

60. Cancers include sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, small cell lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma, glioblastoma multiforme; renal clear cell carcinoma; adrenal cortical carcinoma; bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma; pancreatic adenocarcinoma, renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, indolent B-cell lymphoma, aggressive B-cell lymphoma, T-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms (MPN), ...

60. The method of claim 59, wherein the organism is selected from invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal carcinoma, head and neck squamous cell carcinoma, renal chromophobe, renal papillary cell carcinoma, low-grade glioma, hepatocellular carcinoma, lung carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid carcinoma, thymoma, endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

61. 61. The method of claim 59 or claim 60, further comprising administering a therapeutic agent that inhibits or agonizes 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.

62. 62. The method of any one of claims 59 to 61, 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.

63. 63. The method of claim 62, further comprising administering at least one antibody that specifically binds to an inhibitory checkpoint molecule, in combination with the anti-SIRPA antibody.

64. The at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from the group consisting of anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, anti-PD-1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, and anti-HVEM antibody, anti-BTLA (B- and T-lymphocyte attenuator) antibody, anti-KIR (Killer inhibitory 64. The method of claim 62 or claim 63, wherein the antibody is selected from 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-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.

65. 65. The method of claim 64, wherein the one or more standard or investigational anti-cancer therapies are selected from 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.

66. 66. The method of any one of claims 59 to 65, further comprising administering to the individual at least one antibody that specifically binds to the inhibitory cytokine.

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

68. 68. The method of any one of claims 59 to 67, further comprising administering to the individual at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein.

69. 69. The method of claim 68, wherein the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is selected from 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-induced 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.

70. 70. The method of any one of claims 59 to 69, further comprising administering to the individual at least one stimulatory cytokine.

71. 71. The method of claim 70, wherein the at least one stimulatory cytokine is selected from 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.

72. 60. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody of any one of claims 1 to 58.

73. A vector comprising the nucleic acid of claim 72.

74. 74. An isolated host cell comprising the nucleic acid of claim 72 or the vector of claim 73.

75. 60. An isolated host cell expressing the antibody of any one of claims 1 to 58.

76. A method for producing an antibody that binds to human SIRPA, comprising culturing a cell of claim 74 or claim 75 such that the antibody is produced.

77. 77. The method of claim 76, further comprising recovering the antibody produced by the cells.

78. 60. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 58 and a pharma- ceutically acceptable carrier.

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