Anti-SIRPA antibodies and methods of use thereof

Abstract

The present disclosure generally relates to compositions comprising antibodies (eg, monoclonal antibodies, antibody fragments, etc.) that specifically bind to a SIRPA polypeptide (eg, mammalian SIRPA or human SIRPA), and the use of such compositions in preventing, reducing risk, or treating an individual in need thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 676,813, filed May 25, 2018, which is incorporated herein by reference for any purpose.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on May 14, 2019, is named 40004-PCT_SL.txt and is 99,798 bytes in size. Field of the Invention

[0005] The present disclosure relates to anti-SIRPA antibodies and therapeutic uses of such antibodies. Background of the Invention

[0007] Phagocytic cells, such as macrophages (MΦ) and dendritic cells (DC), distinguish healthy cells from abnormal cells through a complex array of cell surface receptors that regulate the cell's activation state, proliferation and / or effector function. Many of these receptors recognize different ligands that either mark unwanted cells for removal (so-called "eat-me" signals) or protect normal cells from destruction (so-called "don't-eat-me" signals). In recent years, the SIRPα-CD47 axis has become a key determinant of programmed cell removal by macrophages in a variety of clinical settings ranging from cancer cell survival to successful engraftment of hematopoietic cell transplants. Therapeutics that affect this pathway may meet the relevant medical needs of ameliorating diseases that have particular relevance in many types of human cancer.

[0008] Signal regulatory protein-α (SIRPα) belongs to the SIRP family of transmembrane receptors, which are mainly expressed in myeloid cell lineages (including MΦ, DC, granulocytes, etc.) and are characterized by an extracellular region containing two membrane-proximal IgC domains and a remote IgV domain. SIRPα is unique in this family and contains an intracellular cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). After receptor cross-linking, the tyrosine-phosphorylated ITIM site recruits and activates SHP phosphatases to negatively regulate cell functions, such as phagocytosis or inflammatory cytokine release. CD47 serves as the main ligand for SIRPα, and its widespread expression in most cell types, including endothelial cells / epithelial cells, leukocytes and erythrocytes, indicates that it mediates "don't eat me" signals to protect healthy cells from phagocyte-dependent clearance. In support of this notion, several studies have shown that adoptive transfer of red blood cells or leukocytes from CD47 knockout mice into wild-type recipients results in rapid clearance of CD47-deficient cells. Conversely, positional genetic analysis of multiple lines of immunocompromised mice receiving human hematopoietic cells identified the Sirpα allele in the NOD mouse as a causal factor for successful engraftment in a xenograft model. Subsequent studies have shown that allelic variants of SIRPα expressed only in NOD mice retain the ability to bind to human CD47 expressed on human hematopoietic stem cells and, therefore, suppress macrophage-dependent transplant rejection.

[0009] Regulated expression of SIRPα and CD47 establishes a homeostatic control mechanism to regulate phagocyte activity. For example, apoptotic cells downregulate the expression of CD47 to promote phagocytosis of resident macrophages, while living cells remain unharmed. Similarly, inflammatory stimuli such as LPS reduce SIRPα expression in macrophages and dendritic cells (DCs) to enhance their activation during inflammation. However, as seen in cancer, dysregulation of SIRPα and CD47 expression leads to immune-related diseases. Several tumors significantly increase the expression of CD47 relative to non-cancerous cells in order to circumvent the immune surveillance mechanisms that usually eliminate malignant cells. Preclinical studies have revealed that genetic knockdown of CD47 in syngeneic tumor models such as B16F10 melanoma is sufficient to inhibit tumor growth in immunocompetent mice. Similar results were observed in the case of human cancer cell lines with CD47 knockdown transplanted into immunocompromised mice. Alternatively, biological agents that disrupt the SIRPα-CD47 interaction, such as anti-CD47 antibodies, also enhance tumor clearance in mouse models. When combined with commercial anti-tumor antigen antibodies such as trastuzumab or rituximab, anti-CD47 antibodies promote a synergistic increase in anti-tumor responses compared to standard monotherapy. However, given the ubiquitous expression of CD47, anti-CD47 antibodies risk a significant toxicity burden due to off-target effects, limiting their therapeutic efficacy. Nevertheless, these studies identify a key role for the SIRPα-CD47 pathway in regulating myeloid cells, with potential applications in cancer immunotherapy.

[0010] Anti-SIRPA antibodies have been previously described, for example, in the following 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.

[0011] Therefore, there is a need for therapeutic anti-SIRPA antibodies to treat diseases, disorders and conditions associated with inappropriate SIRPA activity.

[0012] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0014] The present invention generally relates to compositions comprising antibodies, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, etc., that specifically bind to human SIRPA, and methods of using such compositions.

[0015] Certain aspects of the invention are based, at least in part, on the identification of anti-SIRPA antibodies having improved and / or enhanced functional characteristics (e.g., relative 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, for example, an improved and / or enhanced ability to reduce cell surface levels of SIRPA on cells, and / or having improved and / or enhanced binding kinetics, and / or having an improved and / or enhanced KD, and / or having an improved and / or enhanced EC50 value. In some embodiments, an anti-SIRPA antibody of the invention has a KD for human SIRPA that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold 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. In some embodiments, the anti-SIRPA antibodies of the invention 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, or less than 0.5 nM.

[0016] In some embodiments, the anti-SIRPA antibodies of the invention reduce cell surface expression of SIRPA in vitro 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, the anti-SIRPA antibodies of the invention reduce cellular levels of SIRPA in vitro at a half-maximal effective concentration (EC50) in the range of about 0.4 nM to about 0.5 nM. Advantageously, the anti-SIRPA antibodies of the invention have a dissociation constant (KD) for human SIRPA in the range of about 0.6 nM to 0.7 nM.

[0017] In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention bind to human SIRPA v1. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention bind to human SIRPA v2. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention bind to human SIRPB (SIRPβ) v3. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention do not bind to human SIRPB v1. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention do not bind to murine SIRPA. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention do not bind to human SIRPγ. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention bind to cynomolgus monkey SIRPA. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibodies of the invention do not bind to cynomolgus monkey SIRPB1. In some embodiments that may be combined with any of the embodiments herein, the anti-SIRPA antibody of the invention binds marmoset SIRPA.

[0018] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0019] In some embodiments that may be combined with any of the embodiments herein, the invention 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 the group consisting of SEQ ID NOs: 22, 23, and 24.

[0020] In some embodiments that may be combined with any of the embodiments herein, the invention 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 the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0021] In some embodiments that may be combined with any of the embodiments herein, the invention 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 the group consisting of 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 the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0022] In some embodiments that may be combined with any of the embodiments herein, the invention 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 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.

[0023] In some embodiments that may be combined with any of the embodiments herein, the invention 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 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.

[0024] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0025] In some embodiments that may be combined with any of the embodiments herein, the invention 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 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.

[0026] In some embodiments that may be combined with any of the embodiments herein, the invention 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 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 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.

[0027] In some embodiments that may be combined with any of the embodiments herein, the invention 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 the group consisting of SEQ ID NOs: 33, 34, and 35.

[0028] In some embodiments that may be combined with any of the embodiments herein, the invention 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0029] In some embodiments that may be combined with any of the embodiments herein, the invention 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 the group consisting of SEQ ID NOs: 33, 34, and 35, and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0030] In some embodiments that may be combined with any of the embodiments herein, the invention 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, HVR-H2, and HVR-H3 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 (as shown in Table 8).

[0031] In some embodiments that may be combined with any of the embodiments herein, the invention 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 comprises HVR-L1, HVR-L2, and HVR-L3 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 (as shown in Table 7).

[0032] In some embodiments that may be combined with any of the embodiments herein, the invention 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 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- 9-25 (as shown in Table 8); and wherein the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3 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 (as shown in Table 7).

[0033] In some embodiments that may be combined with any of the embodiments herein, the invention 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, wherein the antibody comprises antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 of 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).

[0034] In some embodiments that may be combined with any of the embodiments herein, the invention 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, 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; and 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.

[0035] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0036] In some embodiments that may be combined with any of the embodiments herein, the 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.

[0037] In some embodiments that 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.

[0038] In some embodiments that 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.

[0039] In some embodiments that 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.

[0040] In some embodiments that 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.

[0041] In some embodiments that 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.

[0042] In some embodiments that 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.

[0043] In some embodiments that 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.

[0044] In some embodiments that 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.

[0045] In some embodiments that 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.

[0046] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody is a monoclonal antibody.

[0047] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody is a humanized antibody.

[0048] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody is a Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragment.

[0049] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody is multivalent.

[0050] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody is of the IgG, IgM, or IgA class.

[0051] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody is of the IgG class and is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0052] In some embodiments that may be combined with any of the embodiments herein, the invention 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).

[0053] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cellular levels of FcyRIIB.

[0054] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody has a human or mouse IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at an amino acid residue 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, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof, wherein the numbering of the residues is according to EU numbering, or comprises a deletion of an amino acid at a position corresponding to glycine 236 in the Fc region.

[0055] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises one or more amino acid substitutions in the Fc region at a residue position 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 amino acid residues is according to EU numbering.

[0056] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0057] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody induces SIRPA degradation, induces SIRPA cleavage, induces SIRPA internalization, induces SIRPA shedding, induces downregulation of SIRPA expression, or any combination thereof.

[0058] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro.

[0059] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vivo.

[0060] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates the expression of SIRPA in human monocytes.

[0061] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates the expression of SIRPA in human macrophages.

[0062] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates the expression of SIRPA in human macrophages by about 70-95%.

[0063] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody 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.

[0064] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody has an affinity (KD) for human SIRPA of about 0.1 nM to 2 nM.

[0065] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody has an affinity for human SIRPA that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold lower than the affinity 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 for human SIRPA.

[0066] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA v1 in vitro with a half maximal 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.

[0067] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with a half maximal effective concentration (EC50) of about 0.05 to 0.20 nM for human SIRPA v1, about 0.05 to 0.10 nM for human SIRPA v2, and / or about 0.05 to 1 nM for cynomolgus monkey SIRPA, as measured by flow cytometry.

[0068] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0069] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0070] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody increases tumor cell phagocytosis in macrophages, increases tumor cell phagocytosis in M1 macrophages, increases tumor cell phagocytosis in M2 macrophages, downregulates CD14 expression in macrophages, and / or any combination thereof.

[0071] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody enhances T cell proliferation.

[0072] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody blocks the interaction or binding of SIRPA to surfactant protein D (SP-D).

[0073] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody does not block the interaction or binding of SIRPA to CD47.

[0074] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates cell surface expression of CD32A / B.

[0075] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody downregulates cell surface expression of CD14.

[0076] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody enhances T cell proliferation without blocking the interaction of SIRPγ and CD47.

[0077] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody stimulates ROS production in monocytes and / or increases IL-8 expression in monocytes.

[0078] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody inhibits tumor growth in vivo.

[0079] In some embodiments that may be combined with any of the embodiments herein, the invention 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 tumors.

[0080] In some embodiments that may be combined with any of the embodiments herein, the invention 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.

[0081] In some embodiments that may be combined with any of the embodiments herein, the invention 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:

[0082] (a) Antigens that promote transport across the blood-brain barrier;

[0083] (b) an antigen that promotes transport across the blood-brain barrier selected from the group consisting of transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), and diphtheria toxin receptor;

[0084] (c) a pathogen selected from a pathogenic peptide or a pathogenic protein, or a pathogenic nucleic acid, wherein the pathogenic nucleic acid is an antisense GGCCCC (G2C4) repeat sequence expanded RNA, and the pathogenic protein is selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques, 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, ataxin 10, Lewy body body), atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, β2-microglobulin, gelsolin, corneal epithelial protein, 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

[0085] (d) ligands and / or proteins expressed on immune cells, wherein the ligands and / or proteins are 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 proteins, lipids, polysaccharides or glycolipids expressed on one or more tumor cells.

[0086] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is conjugated to a peptide that promotes transport across the blood-brain barrier. In some embodiments, the peptide is selected from CRM197, a protein transduction domain, TAT, Syn-B, a penetratin, a polyarginine peptide, angiopep peptide, and ANG1005.

[0087] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is an opsonizing antibody.

[0088] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is a conjugated antibody.

[0089] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is conjugated to a detectable label, toxin, or therapeutic agent.

[0090] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is conjugated to a toxin selected from the group consisting of ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, daunorubic ...daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorub exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, CC1065, and cisplatin.

[0091] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is used in combination with one or more antibodies that specifically bind to a pathogenic protein selected from the group consisting of amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, tau protein, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), prion protein, PrPSc, huntingtin, depressor protein, leukocyte antigen 2 (LEA), leukocyte antigen 2 (LEA), thym ... 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, corneal epithelial protein, cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation product, 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 used in combination with one or more antibodies that bind to immunomodulatory proteins selected from the group consisting of PD1 / PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, TREM1, TREM2, CD33, Siglec-5, Siglec-7, Siglec-9, Siglec-11, phosphatidylserine, pathogenic nucleic acids, antisense GGCCCC(G2C4) repeat-expanded RNA, and any combination thereof.

[0092] In some embodiments that may be combined with any of the embodiments herein, the invention provides a method of preventing, reducing risk, or treating cancer, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the invention, thereby treating the cancer. In some embodiments that may be combined with any of the embodiments herein, the invention provides a method of preventing, reducing risk, or treating an individual having a disease, disorder, or injury, wherein the disease, disorder, or injury is cancer, the method comprising administering to the individual a therapeutically effective amount of an anti-SIRPA antibody of the invention, thereby preventing, reducing risk, or treating the individual.

[0093] In some embodiments that may be combined with any of the embodiments herein, the invention provides a method of treating cancer, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the invention, wherein the cancer is selected from 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; urothelial carcinoma of the bladder, 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, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, invasive breast carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, refractory renal cell carcinoma, renal papillary cell carcinoma, lower-grade gliomas, hepatocellular carcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumors, thyroid cancer, thymoma, uterine corpus endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

[0094] In some embodiments that may be combined with any of the embodiments herein, the invention provides a method of treating cancer, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the invention, the method 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.

[0095] In some embodiments that may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, the method comprising administering to the subject 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 the 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 selected from anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-PD-L2 antibodies, anti-PD-1 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies and anti-HVEM antibodies, anti-B- and T-lymphocyte attenuating factor (BTLA) antibodies, anti-killer cell inhibitory receptor (KIR) antibodies, anti-GAL9 antibodies, anti-TIM-1 antibodies, anti-TIM3 antibodies, anti-TIM-4 antibodies, 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, antagonist anti-TREM1 antibody, antagonist anti-TREM2 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-CD2 antibody, anti-CD5 antibody, and any combination thereof.

[0096] 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.

[0097] In some embodiments that may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, wherein the method further comprises administering to the subject 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.

[0098] In some embodiments that may be combined with any of the embodiments herein, the invention provides a method of treating cancer, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the invention, wherein the method further comprises administering to the individual at least one synergistic 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 synergistic 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 potentiator antibody that specifically binds to a stimulatory checkpoint protein is selected from a potentiator anti-CD40 antibody, a potentiator anti-OX40 antibody, a potentiator anti-ICOS antibody, a potentiator anti-CD28 antibody, a potentiator anti-TREM1 antibody, a potentiator anti-TREM2 antibody, a potentiator anti-CD137 / 4-1BB antibody, a potentiator anti-CD27 antibody, a potentiator anti-glucocorticoid-induced TNFR-related protein GITR antibody, a potentiator anti-CD30 antibody, a potentiator anti-BTLA antibody, a potentiator anti-HVEM antibody, a potentiator anti-CD2 antibody, a potentiator anti-CD5 antibody, and any combination thereof.

[0099] In some embodiments that may be combined with any of the embodiments herein, the invention provides a method of treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the invention, the method further comprising administering to the subject 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-β, and any combination thereof.

[0100] In some embodiments, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the present invention. 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 invention. 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 invention.

[0101] In some embodiments, the present invention provides a method for producing an antibody that binds to human SIRPA, wherein the method comprises culturing a host cell comprising a vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the present invention to allow the antibody to be produced. The present invention further provides for recovering the antibody produced by the cell.

[0102] In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-SIRPA antibody of the present invention and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 Shown is an amino acid sequence alignment of human SIRPA variant 1 and the extracellular domain of SIRPA proteins from various species. The reported percent identity demonstrates high homology within the extracellular region. Accession numbers are P78324 (human SIRPA variant 1), XP015313153 (cynomolgus macaque), JAB51896 (marmoset), G1U0I5 (rabbit), and XP005634938 (dog). Figure 1 SEQ ID NOs 59-63 are disclosed respectively in order of appearance.

[0105] Figure 2AThe potential humanized sequences of the heavy chain variable domain of the anti-SIRPA antibody 3F9 are listed. The humanized sequences are based on the IGHV3-23*01 acceptor framework and the IGHJ4*01 joining region. Figure 2A SEQ ID NOs 64-65, 2, 64, and 3-4 are disclosed, respectively, in order of appearance. Figure 2B Listed are potential humanized sequences of the light chain variable domain of the anti-SIRPA antibody 3F9. Figure 2B SEQ ID NOs 66-67, 5, 66, and 68-70 are disclosed, respectively, in order of appearance.

[0106] Figure 3 Data are presented showing antibody-mediated downregulation of SIRPA by murine and humanized anti-SIRPA 3F9 antibodies on primary human macrophages.

[0107] Figure 4A Data are presented showing that antibody-mediated receptor downregulation is dependent on N-linked Fc glycans. Figure 4B Data are presented showing that antibody-mediated enhancement of tumor cell phagocytosis is dependent on N-linked Fc glycans.

[0108] Figure 5A Describes data showing that humanized anti-SIRPA 3F9 IgG4 antibody variants fail to induce tumor cell phagocytosis. Figure 5B Describes data showing that humanized anti-SIRPA 3F9 IgG4 antibody variants fail to downregulate CD14 expression on macrophages.

[0109] Figure 6A We show that treatment with the anti-SIRPA antibody 3F9 mIgG1 downregulates CD32A / B expression on primary human macrophages. Figure 6B We show that treatment with the anti-SIRPA antibody 3F9 downregulates the expression of CD32A and CD32B on primary human macrophages.

[0110] Figure 7A Presents data showing that CD32A / B blockade inhibits anti-SIRPA antibody 3F9-mediated downregulation of SIRPA on primary human macrophages. Figure 7B Data are presented showing that CD32A / B blockade inhibits the enhancement of tumor cell phagocytosis mediated by the anti-SIRPA antibody 3F9.

[0111] Figure 8 sets forth data showing that CD32A allotype influences SIRPA downregulation mediated by the anti-SIRPA antibody 3F9. Figure 8A Donors 507 and 508 were shown to be heterozygous for CD32A-R / H131; Figure 8B Donors 516 and 517 were shown to be homozygous for CD32A-H / H131.

[0112] Figures 9A to 9B Describe data showing that antibody-mediated downregulation of the receptor leads to degradation of SIRPA. Figure 9A 5 μg, 10 μg and 20 μg of whole cell lysate were loaded on SDS-PAGE and immunoblotted with anti-SIRPA cytoplasmic domain (a-SIRPAct). Figure 9B 20 μg of whole cell lysate were loaded on SDS-PAGE and probed with a-SIRPAct and anti-SHP2.

[0113] FIG. 10A to FIG. 10B The present invention describes data comparing the enhancement of phagocytic activity of different Fc variants of the anti-SIRPA antibody 3F9-22. Figure 10A Primary human macrophages were treated with anti-SIRPA 3F9-22 antibody on a wild-type human IgG1 backbone or on a human IgG1 with S267E / L328F Fc mutations, or an isotype control antibody. Figure 10B Primary human macrophages were treated with anti-SIRPA 3F9-22 antibodies on a human IgG1 backbone with S267E / L328F or A330S / P331S Fc mutations or an isotype control antibody.

[0114] Figures 11A to 11B Data are presented comparing different Fc variants of the anti-SIRPA 3F9-22 antibody relative to anti-CD47 in M1 ( Figure 11B ) and M2( Figure 11A ) Phagocytic activity on macrophages.

[0115] Figure 12A and Figure 12B Presents data showing that macrophages treated with the anti-SIRPA antibody 3F9-22 reduced solid tumor cell viability in vitro.

[0116] Figure 13A Data are presented comparing T cell proliferation enhancement of different Fc variants of the anti-SIRPA antibody 3F9-22 in a two-way MLR assay. Figure 13B Data are presented comparing the enhancement of T cell proliferation by different Fc variants of the anti-SIRPA antibody 3F9-22 and the anti-SIRPA antibody 3F9-14 relative to anti-CD47 IgG4 in a two-way MLR assay.

[0117] Figure 14A Data are presented comparing the enhancement of T cell proliferation by different Fc variants of the anti-SIRPA antibody 3F9-22 relative to anti-CD47 IgG4 in a one-way MLR assay. Figure 14BRepresentative FACS plots of one-way MLR analysis are shown.

[0118] Figure 15A and Figure 15B Data are presented comparing different Fc variants of the anti-SIRPA antibody 3F9-22 for enhancing TNF release by LPS-primed macrophages.

[0119] Figure 16 Data are presented showing the identification and observation of "critical" residues of anti-SIRPA antibodies in a human SIRPA model.

[0120] 17A to 17C Data are presented showing the effect of the combination of the anti-SIRPA antibody 3F9-22 and an anti-PD-L1 antibody on tumor growth inhibition in a syngeneic mouse colon cancer model. Figure 17A Depicted are 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 Presented are data showing mean tumor growth curves of huSIRPA / huCD47 BAC transgenic mice subcutaneously implanted with MC38 cells engineered to lack mouse CD47 expression and overexpress human CD47 (MC38-mCD47KO / huCD47+). Figure 17C Explanation Figure 17B Spaghetti plot of each individual animal in each treatment group.

[0121] 18A to 18B Data are presented showing the kinetics of antibody-mediated downregulation of SIRPA in myeloid cells of tumor-bearing BAC transgenic mice treated with increasing doses of the anti-SIRPA antibodies of the present disclosure. Figure 18A Depicted are data showing relative changes in SIRPA expression on tumor-infiltrating myeloid cells over time. Figure 18B Depicted are data showing relative changes in SIRPA expression on splenic myeloid cells over time.

[0122] Figure 19A Treatment conditions showing anti-SIRPA antibody-mediated downregulation of SIRPA studied in humanized NOG-EXL mice bearing A375 tumors. Figure 19B Depicted are data showing the expression levels of SIRPA on human tumor macrophages in relation to treatment with the anti-SIRPA antibodies of the present disclosure, as MFI values ​​(left panel) or normalized values ​​(right panel).

[0123] FIG. 20A to FIG. 20B Showing M1 macrophage markers HLA-DR / MHC class 2 ( Figure 20A) and CD86( Figure 20B ) Expression levels on human tumor macrophages isolated from humanized NOG-EXL mice bearing A375 tumors after treatment with the anti-SIRPA antibodies of the present invention. Figure 20A and Figure 20B Data are illustrated showing the expression of HLA-DR / MHC class 2 and CD86, respectively, as MFI values ​​(left panel) or normalized values ​​(right panel).

[0124] Figures 21A to 21B Splenic mononuclear cells isolated from humanized NOG-EXL mice bearing A375 tumors ( Figure 21A ) and peripheral blood mononuclear cells ( Figure 21B ) in association with administration of the anti-SIRPA antibodies of the present invention. Figure 21A and Figure 21B Data are illustrated showing the expression of human SIRPA as MFI values ​​(left panel) or normalized values ​​(right panel).

[0125] Figure 22 Presents data showing antibody-mediated downregulation of SIRPA by different Fc variants of the anti-SIRPA 3F9-22 antibody on primary human macrophages.

[0126] Figure 23 Data are presented comparing the enhancement of phagocytic activity of different Fc variants of the anti-SIRPA antibody 3F9-22 compared to an anti-CD47 blocking antibody.

[0127] Figure 24 Described are data showing red blood cell and platelet counts in cynomolgus monkeys administered with a control IgG antibody, anti-SIRPA antibody 3F9-22 PS, anti-SIRPA antibody 3F9-22 IgG4, anti-SIRPA antibody 3F9-IgG2, and anti-SIRPA antibody 3F9-22 NSLF.

[0128] It should 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 become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the detailed description below. Detailed Description of the Invention

[0130] The present invention relates to anti-SIRPA antibodies (eg, 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.

[0131] The techniques and procedures described or referred to herein are generally well understood and are generally employed by those skilled in the art using conventional methods, such as the widely used methods described in 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 (FM Ausubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: 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, ed. Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RIFreshney), 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and TissueCulture: Laboratory Procedures (A.Doyle, JBGriffiths and DGNewell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wileyand Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, 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 JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0132] definition

[0133] Unless otherwise indicated, the terms "SIRPα" or "SIRPα polypeptide" or "SIRPA" or "SIRPA polypeptide" are used interchangeably herein to refer to any naturally occurring SIRPA from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus macaques) and rodents (e.g., mice and rats). In some embodiments, the term encompasses wild-type sequences and naturally occurring variant sequences, such as splice variants or allelic variants. In some embodiments, the term encompasses "full-length," unprocessed SIRPA, as well as any form of SIRPA produced by processing in a cell. In some embodiments, the SIRPA is human SIRPA. In some embodiments, the amino acid sequence of an exemplary SIRPA is Uniprot Accession No. 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.

[0134] 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 to render the antibody useful as a diagnostic and / or therapeutic agent when targeting SIRPA. In one embodiment, the extent of binding of the anti-SIRPA antibody to an unrelated, non-SIRPA polypeptide is less than about 10% of the binding of the antibody to SIRPA, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody binds to SIRPA with 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 smaller, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 In certain embodiments, the anti-SIRPA antibody binds to an epitope of SIRPA that is conserved among SIRPA from different species.

[0135] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from nonspecific binding. Specific binding can be measured, for example, by comparing the binding of an assay molecule 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, such as an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specifically binds" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target can be exhibited, for example, by a molecule that has about 10 binding affinity to the target. -4 M or lower, 10 -5 M or lower, 10 -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, or in the range of 10 -4 M to 10 -6 M or 10 -6 M to 10 -10 M or 10 -7 M to 10 -9 As will be appreciated by those skilled in the art, affinity is inversely proportional to the KD value. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0136] As used herein, the term "immunoglobulin" (Ig) is used interchangeably with "antibody." The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), including antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[0137] "Native antibodies" are usually 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, while the number of disulfide bridges varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H), followed by multiple constant domains. Each light chain has a variable domain (V L ) and has a constant domain at its other end; the constant domain of the light chain 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. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains.

[0138] For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0139] Light chains from any vertebrate species can be assigned to one of two distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain (CH) of an immunoglobulin, they 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 generally described in, for example, Abbas et al., Cellular and Molecular Immunology, 4th ed. (WB Saunders Co., 2000).

[0140] The "variable region" or "variable domain" of an antibody, such as an anti-SIRPA antibody of the present invention, refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0141] The term "variable" refers to the fact that in antibodies, such as the anti-SIRPA antibodies of the present invention, certain segments of the variable domain vary extensively in sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions that primarily adopt a β-sheet configuration, connected by three HVRs that form loops connecting and, in some cases, forming part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0142] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, etc.) that may be present in minor amounts, such as the monoclonal anti-SIRPA antibodies of the present invention. 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 hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic 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, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, and techniques for producing human antibodies or human-like antibodies in animals that possess some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences. For example, the monoclonal antibodies to be used according to the present invention can be made by a variety of techniques, including, 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. Patent 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 antibodies or human-like antibodies in animals that have human immunoglobulin loci or genes encoding part or all of human immunoglobulin sequences (see, 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).

[0143] The terms "full-length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to an antibody, such as the anti-SIRPA antibodies of the present invention, in its substantially intact form, rather than an antibody fragment. Specifically, intact antibodies include antibodies having heavy and light chains that include an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0144] "Antibody fragments" are molecules that are different from intact antibodies and contain a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules formed from antibody fragments; and multispecific antibodies.

[0145] Papain digestion of antibodies, such as the anti-SIRPA antibodies of the present invention, produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. The Fab fragment consists of an entire light chain together with the variable region (V) of the heavy chain. H ) and the first constant domain of a heavy chain (C H 1) together. Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two Fab fragments with different antigen binding activities linked by disulfide bonds and still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment in that the Fab' fragment has a C H The carboxyl terminus of the constant domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0146] The Fc fragment contains the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0147] "Functional fragments" of antibodies, such as the anti-SIRPA antibodies of the present invention, comprise a portion of an intact antibody, generally including the antigen-binding or variable region of the intact antibody or an Fc region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0148] The term "diabody" refers to a H With V LSmall antibody fragments prepared by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the domains, allowing interchain but not intrachain pairing of the variable domains, thereby generating bivalent fragments, that is, fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies are H and V L Domains are present on different polypeptide chains.

[0149] As used herein, "chimeric antibody" refers to an antibody (immunoglobulin), such as the chimeric anti-SIRPA antibody of the present invention, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody 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 Antibodies, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing macaques with an antigen of interest. "Humanized antibodies" as used herein are used as a subset of "chimeric antibodies."

[0150] "Humanized" forms of non-human (e.g., murine) antibodies, such as humanized forms of the anti-SIRPA antibodies of the present invention, are chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody and all or substantially all of the FRs correspond to FRs of a human antibody. A humanized antibody optionally may 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.

[0151] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human, such as the anti-SIRPA antibodies of the present invention, and / or an antibody produced using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries and yeast display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci are disabled, such as immunized xenogeneic mice, and by human B-cell hybridoma technology. Human antibodies can be produced using various 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). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); The methods described in 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 an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci are disabled, such as immunized xenogeneic mice (for XENOMOUSE™ technology, see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584). For human antibodies produced via human B cell hybridoma technology, see also, e.g., Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006). Alternatively, human antibodies can also be prepared by using yeast libraries and methods as disclosed in, for example, WO2009 / 036379A2; WO2010105256; WO2012009568; and Xu et al., Protein Eng. Des. Sel., 26(10):663-70 (2013).

[0152] The terms "hypervariable region," "HVR," or "HV" when used herein refer to regions of an antibody variable domain, such as the anti-SIRPA antibodies of the invention, that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the V H(H1, H2, H3), and three in V L In natural antibodies, H3 and L3 represent the majority of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. Naturally occurring camelid antibodies composed solely of heavy chains are functional and stable even in the absence of light chains.

[0153] A variety of HVR descriptions have been used and are encompassed herein. In some embodiments, HVRs may be the Kabat complementarity determining regions (CDRs) based on sequence variability and are the most commonly used (Kabat et al., supra). In some embodiments, HVRs may be Chothia CDRs. Chothia refers to the positions of structural loops instead (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, HVRs may be AbM HVRs. AbM HVRs represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. In some embodiments, HVRs may be "contact" HVRs. "Contact" HVRs are based on analysis of available composite crystal structures. The residues from each of these HVRs are annotated as follows.

[0154]

[0155] The HVRs may comprise the following "extended HVRs": 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 embodiments) (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.

[0156] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0157] The phrase "variable domain residue numbering as in EU or Kabat" or "amino acid position numbering as in EU or Kabat" and variations thereof refer to the numbering system used in EU or Kabat et al., supra, for heavy chain variable domains or light chain variable domains compiled for antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of a FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). For a given antibody, the EU or Kabat numbering of the residues can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence.

[0158] The EU or Kabat numbering system is generally used when referring to residues in the

[0077] variable domain (approximately residues 1-107 of the light chain and residues 1-113 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 or Kabat numbering system" or "EU index" is generally used when referring to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index as reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise stated herein, reference to residue numbering in an antibody variable domain means residue numbering according to the Kabat numbering system. Unless otherwise stated herein, reference to residue numbering in an antibody constant domain means residue numbering according to the EU or Kabat numbering system (e.g., see U.S. Patent Publication No. 2010-280227).

[0159] As used herein, an "acceptor human framework" is a human immunoglobulin framework comprising a V L or V HThe acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence therefrom, or it may comprise 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, preferably those changes occur at only three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and / or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to the VH. L The framework sequences are identical to those of human immunoglobulins or human consensus framework sequences.

[0160] "Human common framework" refers to human immunoglobulin V L or V H The framework of the most frequently occurring amino acid residues in the framework sequence selection. L or V H The sequence selection is a subset of variable domain sequences. Typically, the subset of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include sequences for V L , the subgroup may be subgroup κI, κII, κIII or κIV as described in Kabat et al., supra. H , the subgroup may be subgroup I, subgroup II or subgroup III as in Kabat et al., supra.

[0161] An "amino acid modification" at a designated position of, for example, an anti-SIRPA antibody of the invention refers to a substitution or deletion of the designated residue, or an insertion of at least one amino acid residue adjacent to the designated residue. An insertion "adjacent" to a designated residue means an insertion within one to two residues thereof. The insertion may be at the N-terminus or C-terminus of the designated residue. Preferred amino acid modifications herein are substitutions.

[0162] "Affinity matured" antibodies, such as the affinity matured anti-SIRPA antibodies of the present invention, are antibodies with one or more alterations in one or more HVRs that result in an improvement in the affinity of the antibody for the antigen relative to a parent antibody that does not have those alterations. In one embodiment, affinity matured antibodies have nanomolar or even picomolar affinities 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 affinity matured antibodies produced by V H and V L Affinity maturation by domain shuffling. 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).

[0163] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region and a light chain variable region in tight, non-covalent association. The folding of these two domains produces six hypervariable loops (3 loops each from the H chain and the L chain), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv comprising only three HVRs specific for an antigen) still has the ability to recognize and bind to an antigen, although its affinity is lower than that of the entire binding site.

[0164] "Single-chain Fv" also referred to as "sFv" or "scFv" is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a VH domain and a VL domain. H With V L A polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding.

[0165] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.

[0166] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the Fc region boundaries of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the amino acid residue at position Cys226 or the extension from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447, according to the EU numbering system) in the Fc region can be removed, for example, during manufacture or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of a complete antibody can include antibody populations in which all K447 residues are removed, antibody populations in which no K447 residue is removed, and antibody populations with a mixture of antibodies containing and not containing the K447 residue. The native sequence Fc region suitable for use in the antibodies of the present invention includes human IgG1, IgG2, IgG3, and IgG4.

[0167] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence 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.

[0168] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or compared to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide, and preferably from about one to about five amino acid substitutions. The variant Fc region herein preferably has at least about 80% homology to a native sequence Fc region and / or to the Fc region of a parent polypeptide, and most preferably has at least about 90% homology thereto, and more preferably has at least about 95% homology thereto.

[0169] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are receptors that bind to IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. The term "FcR" herein encompasses other FcRs, including those identified in the future. FcRs can also increase the serum half-life of antibodies.

[0170] Human FcRn high-affinity binding polypeptides can be analyzed for in vivo binding to FcRn and serum half-life, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered polypeptides with variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with improved or diminished FcR binding. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0171] 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 with the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0172] The term "compete" when used in the context of antibodies (e.g., neutralizing antibodies) that compete for the same epitope means competition between the antibodies as determined by an assay in which the antibody being tested 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). Many types of competitive binding assays can be used to determine whether an antibody competes with another antibody, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), 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 label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using an I-125 label (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 label RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such analyses involve the use of purified antigen bound to a solid surface or cell with either an unlabeled test antibody and a labeled reference antibody. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the test antibody. Typically, the test antibody is present in excess. Antibodies identified by competition analysis (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to produce steric hindrance. Additional details on methods for determining competitive binding are provided herein. Typically, when a competing antibody is present in excess, it will inhibit (e.g., reduce) 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 approaching 100%.

[0173] As used herein, an "interaction" between a SIRPA polypeptide and a second polypeptide encompasses, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits" an "interaction" between two polypeptides when it disrupts, reduces, or completely eliminates the interaction between the two polypeptides. An antibody of the invention "inhibits" an "interaction" between two polypeptides when it binds to one of the two polypeptides. In some embodiments, the interaction can be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or nearly 100%.

[0174] The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting that antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Epitopes are most commonly found on polypeptides, but in some cases can be found on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Typically, 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.

[0175] A "potentiator" antibody or "activating" antibody is an antibody that induces (eg, increases) one or more activities or functions of the antigen after the antibody binds to the antigen.

[0176] An "antagonist" antibody or "blocking" antibody or "inhibitory" antibody is an antibody that reduces, inhibits, and / or eliminates (e.g., reduces) antigen binding to one or more ligands after the antibody binds 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 binds to the antigen. In some embodiments, an antagonist antibody or blocking antibody or inhibitory antibody substantially or completely inhibits antigen binding to one or more ligands and / or one or more activities or functions of the antigen.

[0177] An "isolated" antibody, such as an isolated anti-SIRPA antibody of the present invention, is one that has been identified, separated, and / or recovered (e.g., naturally or recombinantly) from a component of the environment in which it is produced. Preferably, the isolated antibody is free of all other contaminant components from the environment in which it is produced. Contaminant components from the environment in which it is produced, such as components produced by recombinant transfected cells, are substances that typically interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified: (1) to greater than 95% by weight of the antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) 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) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes the antibody in situ within a recombinant T cell, as at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0178] An "isolated" nucleic acid molecule encoding an antibody, such as an anti-SIRPA antibody of the present invention, is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule normally associated with the environment in which it is produced. Preferably, the isolated nucleic acid is free from all components associated with the environment in which it is produced. The isolated nucleic acid molecules encoding polypeptides and antibodies herein are in a form that is different from the form or environment in which they are found in nature. Thus, isolated nucleic acid molecules are distinguished from the nucleic acids encoding polypeptides and antibodies herein that are naturally present in cells.

[0179] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid," which refers to a circular double-stranded DNA to which an additional DNA segment may be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which an additional DNA segment may be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thereby replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" or, in short, "expression vectors." In general, expression vectors used in recombinant DNA technology are often in the form of plasmids. In this specification, since plasmids are the most commonly used vector forms, "plasmids" and "vectors" are used interchangeably.

[0180] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0181] "Host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0182] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cells or mammals to which they are exposed at the dosages and concentrations used. Typically, a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, 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 dextrins; 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®. TM , polyethylene glycol (PEG) and PLURONICS TM .

[0183] As used herein, the term "prevent" includes providing for prevention of the occurrence or recurrence of a particular disease, disorder, or condition in an individual. An individual may be predisposed to, suspected of having, or at risk of having a particular disease, disorder, or condition, but has not yet been diagnosed with the disease, disorder, or condition.

[0184] 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 have displayed detectable disease or disease symptoms prior to treatment as described herein. "At risk" means that the individual has one or more risk factors, which are measurable parameters associated with developing a particular disease, disorder, or condition as known in the art. An individual who has one or more of these risk factors has a higher chance of developing the particular disease, disorder, or condition than an individual who does not have one or more of these risk factors.

[0185] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of clinical pathology in the individual being treated. Desired therapeutic effects include reducing the rate of progression of a particular disease, disorder, or condition, ameliorating or alleviating its pathological state, and alleviating or improving its prognosis. For example, a subject is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0186] An "effective amount" refers to an amount that is effective at least to achieve the desired therapeutic or preventive outcome at the desired dosage and time period. An effective amount can be provided in one or more administrations. The effective amount herein can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the treatment to elicit the desired response in the individual. An effective amount is also an amount in which the beneficial effects of the treatment outweigh any toxic or deleterious effects of the treatment. For prophylactic uses, beneficial or desired outcomes include outcomes such as eliminating or reducing the risk of disease, lessening the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during disease progression. For therapeutic uses, beneficial or desired outcomes include clinical outcomes such as reducing one or more symptoms caused by the disease, improving the patient's quality of life, reducing the dose of other drugs needed to treat the disease, enhancing the effect of another drug (such as through targeting), delaying disease progression, and / or prolonging survival. An effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly accomplish the prophylactic or therapeutic treatment. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be administered in an effective amount if the desired result can be achieved or is achieved in combination with one or more other agents.

[0187] For purposes of treatment, prevention, or risk reduction, a "subject" is any animal classified as a mammal, including humans, domestic and farm animals, zoo animals, sports animals, or pets, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the subject is a human.

[0188] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or as separate compositions, including at different dosing frequencies or time intervals, and using the same route of administration or different routes of administration. In some embodiments, administration in conjunction is administration as part of the same treatment regimen.

[0189] The term "about" as used herein refers to the common error range of the corresponding value that is easily known to those skilled in the art. When "about" a value or parameter is mentioned herein, it includes (and describes) embodiments for the value or parameter itself.

[0190] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, reference to an "antibody" is a reference to one or more antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[0191] It should be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0192] anti-SIRPA antibodies

[0193] Anti-SIRPA antibody binding region

[0194] In some embodiments, the anti-SIRPA antibodies of the invention may bind to a conformational epitope. In some embodiments, the anti-SIRPA antibodies of the invention 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 invention may bind to a SIRPA epitope comprising one or more peptides. As disclosed herein, a SIRPA epitope can comprise one or more peptides comprising five or more, six or more, seven or more, eight or more, nine 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 five or more, six or more, seven or more, eight or more, nine 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 a mammalian SIRPA protein corresponding to the amino acid sequence of SEQ ID NO: 1.

[0195] In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope of human SIRPA that is identical to or overlaps with the SIRPA epitope bound by the following anti-SIRPA antibodies: 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: An anti-SIRPA antibody comprising 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.In some embodiments, the anti-SIRPA antibodies of the invention bind to substantially the same SIRPA epitope as the following anti-SIRPA antibodies: 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: An anti-SIRPA antibody comprising 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.

[0196] In some embodiments, the anti-SIRPA antibodies of the present invention competitively inhibit the binding of the following anti-SIRPA antibodies: 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: An anti-SIRPA antibody comprising 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. In some embodiments, the anti-SIRPA antibodies of the present invention compete for SIRPA binding with the following anti-SIRPA antibodies: 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: An anti-SIRPA antibody comprising 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.

[0197] In some embodiments, the anti-SIRPA antibodies of the invention competitively inhibit the binding of at least one antibody selected from any one of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the invention competitively inhibit the binding of at least one antibody 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, 3 F9-14, 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, when the anti-SIRPA antibodies of the invention utilize one or more of the following: 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, when binding to SIRPA is reduced by an amount ranging from about 50% to 100% compared to binding to SIRPA in the absence of the 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, compete for binding to SIRPA. In some embodiments, when the anti-SIRPA antibodies of the invention utilize one or more antibodies selected from m3F9, h3F9 H1 / L1 (14.70.1), h3F9 H1 / L2 (14.70.2), h3F9H1 / L3, h3F9 H2 / L1 (14.70.4), h3F9 H2 / L2, h3F9H2 / 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, When the binding of the antibody 3F9-23, 3F9-24, 3F9-25, and any combination thereof to SIRPA is reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to binding to SIRPA in the absence of the anti-SIRPA antibody, the anti-SIRPA antibody binds to one or more of the following: 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 3F9-25, 3F9-26, 3F9-27, 3F9-28, 3F9-29, 3F9-30, 3F9-31, 3F9-32, 3F9-33, 3F9-34, 3F9-35, and any combination thereof, compete for binding to SIRPA. In some embodiments, one or more selected from m3F9, h3F9 H1 / L1 (14.70.1), h3F9 H1 / L2 (14.70.2), h3F9 H1 / L3, h3F9 H2 / L1 (14.70.3),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. An anti-SIRPA antibody of the invention that substantially completely blocks the binding of one or more antibodies selected from m3F9, h3F9, or any combination thereof to SIRPA indicates that the anti-SIRPA antibody substantially completely blocks the binding of one or more antibodies selected from m3F9, h3F9, or any combination thereof to SIRPA. H1 / L1(14.70.1), h3F9 H1 / L2(14.70.2), h3F9 H1 / L3, h3F9 H2 / L1(14.70.4), h3F9 H2 / L2, h3F9

[00155] In some embodiments, the present invention relates to antibodies that bind to SIRPA, such as 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 and one or more antibodies 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, 3F9- 25 and any combination thereof are present in amounts corresponding to a 10:1 ratio, a 9:1 ratio, an 8:1 ratio, a 7:1 ratio, a 6:1 ratio, a 5:1 ratio, a 4:1 ratio, a 3:1 ratio, a 2:1 ratio, a 1:1 ratio, a 0.75:1 ratio, a 0.5:1 ratio, a 0.25:1 ratio, a 0.1:1 ratio, a 0.075:1 ratio, a 0.050:1 ratio, a 0.025:1 ratio, a 0.01:1 ratio, a 0.0075:1 ratio, a 0.0.050:1 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 ratio, 1:0.75 ratio, 1:0.5 ratio, 1:0.25 ratio, 1: 0.1 ratio, 1:0.075 ratio, 1:0.050 ratio, 1:0.025 ratio, 1:0.01 ratio, 1:0.0075 ratio, 1:0.0050 ratio, 1:0.0025 ratio, 1:0.001 ratio, 1:0.00075 ratio, 1:0.00050 ratio, 1:0.00025 ratio or 1:0.0001 ratio of an anti-SIRPA antibody: one or more 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 9-1, 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, one or more 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 In some embodiments, the anti-SIRPA antibody is present in an excess amount ranging from about 1.5-fold to 100-fold, or greater, compared to the amount of the anti-SIRPA antibody that is present in the presence of 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, one or more selected from m3F9, h3F9 H1 / L1 (14.70.1), h3F9 H1 / L2 (14.70.2), h3F9 H1 / L3, h3F9 H2 / L1 (14.70.3), h3F9 H1 / L3, h3F9 H2 / L1 (14.70.4), h3F9 H1 / L3 (14.70.5), h3F9 H2 / L1 (14.70.6), h3F9 H1 / L3 (14.70.7), h3F9 H2 / L1 (14.70.8), h3F9 H1 / L3 (14.70.9), h3F9 H2 / L1 (14.70.1), h3F9 H1 / L2 (14.70.1), h3F9 H1 / L3 (14.70.1), h3F9 H70.4), 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, 3F 9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3 The anti-SIRPA antibody is present in an amount that is about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess compared to the amount of the antibody produced by the methods of claim 1 .

[0198] In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope of human SIRPA that is identical to or overlaps with an epitope of SIRPA bound by at least one antibody selected from any one of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope of human SIRPA that is identical to or overlaps with an epitope of SIRPA bound by at least one antibody selected from any one of the antibodies listed in Tables 4, 5, 7, 8, and 10. The SIRPA epitopes bound by the antibodies 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 are identical or overlapping.

[0199] In some embodiments, the anti-SIRPA antibodies of the invention bind to substantially the same SIRPA epitope bound by at least one antibody selected from any one of the antibodies listed in Tables 4, 5, 7, 8, and 10. In some embodiments, the anti-SIRPA antibodies of the invention bind to substantially the same SIRPA epitope bound by at least one antibody 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

[00155] The present invention relates to antibodies that bind to substantially the same SIRPA epitope as antibodies to 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. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ).

[0200] In some embodiments, the anti-SIRPA antibodies of the invention are combined with one or more of the following: m3F9, h3F9 H1 / L1 (14.70.1), h3F9 H1 / L2 (14.70.2), h3F9 H1 / L3, h3F9 H2 / L1 (14.70.4), h3F9

[00155] Antibodies that compete for binding to SIRPA include, but are not limited to, antibodies against H2 / L2, h3F9H2 / 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.

[0201] Any suitable competition assay or SIRPA binding assay known in the art, such as BIAcore analysis, ELISA analysis, or flow cytometry, can be used to determine whether an anti-SIRPA antibody binds to one or more of the antibodies selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / L2, h3F9 3F9-25, 3F9-26, 3F9-27, 3F9-28, 3F9-29, 3F9-30, 3F9-31, 3F9-32, 3F9-33, 3F9-34, 3F9-35, and any combination thereof, compete for binding to SIRPA. In an exemplary competition assay, immobilized SIRPA or cells expressing SIRPA on the cell surface are incubated in a solution comprising a first labeled antibody that binds to SIRPA (e.g., human or non-human primate) and a second unlabeled antibody to test the ability of the second unlabeled antibody to compete with the first antibody for binding to SIRPA. The second antibody may be present in the hybridoma supernatant. As a control, immobilized SIRPA or cells expressing SIRPA are incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. Following incubation under conditions permissive for binding of the first antibody 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 in the test sample is substantially reduced relative to that in the control sample, this indicates that the second antibody is competing 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).

[0202] In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, two, three, four, five, or six HVRs selected from the group consisting of: (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 the group consisting of 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 the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19.

[0203] In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, two, three, four, five, or six HVRs selected from the group consisting of: (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. 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) 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: 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-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: 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: 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-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: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:18 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: 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 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: 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; 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: 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) 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: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 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; (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:16 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; (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.

[0204] In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, at least two, or all three V H HVR sequence, the V H The HVR sequence is 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 NO: 22, 23 and 24.

[0205] In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, at least two, or all three V L HVR sequence, the V L The HVR sequence is 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 NO:11, 12, 13, 14, 15, 16, 17, 18 and 19.

[0206] In some embodiments, provided herein are anti-SIRPA antibodies comprising (a) V H domain, the V H The domain comprises 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; and (b) V L domain, the V LThe domain contains at least one, at least two, or all three V L HVR sequence, the V L The HVR sequence is 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 NO: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

[0207] In some embodiments, provided herein are anti-SIRPA antibodies comprising (a) V H domain, the V H The domain comprises (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; and (b) V L domain, the V L The domain comprises (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.

[0208] In another aspect, the anti-SIRPA antibody comprises a heavy chain variable domain (V H ) 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. H In certain embodiments, V is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35. HThe sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-SIRPA antibody comprising the 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 in regions outside the HVR (i.e., in the FR). Optionally, the anti-SIRPA antibody comprises the V region of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. H Sequence, including post-translational modifications of the sequence. In a specific embodiment, V H comprising one, two or three HVRs selected from the following: (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 NO: 22, 23 and 24.

[0209] In another aspect, an anti-SIRPA antibody is provided, wherein the antibody comprises a light chain variable domain (V LLD) 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, V is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44. LSequences contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-SIRPA antibodies comprising such sequences retain 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 in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-SIRPA antibody comprises a V sequence 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. L Sequence, including post-translational modifications of the sequence. In a specific embodiment, V L comprising one, two or three HVRs selected from the following: (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 NO:11, 12, 13, 14, 15, 16, 17, 18 and 19.

[0210] In some embodiments, an anti-SIRPA antibody is provided, wherein the antibody comprises a V as in any of the embodiments provided above. H and V as in any of the embodiments provided above L In some embodiments, provided herein are anti-SIRPA antibodies, wherein the antibody comprises a V as in any one of the embodiments provided above. H and V as in any of the embodiments provided above L In one embodiment, the antibody comprises V in SEQ ID NO: 33, 34 or 35 and SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43 or 44, respectively.H and V L sequences, including post-translational modifications of those sequences.

[0211] In some embodiments, provided herein are anti-SIRPA antibodies comprising a heavy chain variable domain (V H ) and light chain variable domain (V L ), where V H and V L Selected from the group consisting of: 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: 38 L ; V comprising the amino acid sequence of SEQ ID NO: 34 Hand 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 .

[0212] Further provided herein are anti-SIRPA antibodies that competitively inhibit binding and / or compete with binding of an anti-SIRPA antibody comprising: (a) V H domain, the V H The domain comprises (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; and (b) V L domain, the V L The antibody comprises (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 the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. In some embodiments, the antibody comprises the VH and VL sequences in SEQ ID NOs: 33, 34, or 35 and SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, or 44, respectively.

[0213] Provided herein are anti-SIRPA antibodies that bind to an epitope of human SIRPA that is identical to or overlaps with an epitope bound by an anti-SIRPA antibody comprising: (a) a VH domain comprising (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 the group consisting of SEQ ID NOs: 22, 23, and 24; and (b) a VL domain comprising (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 the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, and 19. In some embodiments, the antibody comprises the V sequences in SEQ ID NOs: 33, 34, or 35 and SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44, respectively. H and V L In some embodiments, the epitope of human SIRPA is the same epitope bound by an anti-SIRPA antibody.

[0214] In some embodiments, the anti-SIRPA antibody according to any of the above embodiments is a monoclonal antibody, including humanized antibodies and / or human antibodies. In some embodiments, the anti-SIRPA antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or 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.

[0215] In some embodiments, an anti-SIRPA antibody according to any of the above embodiments may incorporate any of the features as described herein, alone or in combination.

[0216] Provided herein are anti-SIRPA antibodies. The provided antibodies are useful, for example, in diagnosing or treating SIRPA-mediated disorders.

[0217] The present invention relates in part to anti-SIRPA antibodies that exhibit one or more improved and / or enhanced functional characteristics (e.g., relative 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, for example, anti-SIRPA antibodies that can reduce cellular levels of SIRPA, anti-SIRPA antibodies that can reduce cell surface levels of SIRPA, anti-SIRPA antibodies that can degrade SIRPA, anti-SIRPA antibodies that can reduce cellular levels of CD32A / B, anti-SIRPA antibodies that can increase or enhance phagocytosis, anti-SIRPA antibodies that can increase or enhance phagocytosis of tumor cells by macrophages, anti-SIRPA antibodies that can enhance The present invention relates to anti-SIRPA antibodies that increase or enhance the anti-tumor activity of anti-cancer therapies, anti-SIRPA antibodies that increase or enhance T cell proliferation, anti-SIRPA antibodies that increase or enhance pro-inflammatory cytokine release from macrophages, and / or anti-SIRPA antibodies that bind 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.

[0218] In some embodiments, the anti-SIRPA antibodies of the invention may have one or more activities that are attributable, at least in part, to the ability of the antibody to reduce cellular expression (e.g., cell surface expression) of SIRPA by inducing degradation, downregulation, cleavage, receptor desensitization, and / or lysosomal targeting of SIRPA. In some embodiments, an anti-SIRPA antibody of the invention that reduces cellular levels of SIRPA is an antibody that exhibits one or more of the following characteristics: (1) inhibits or reduces one or more SIRPA activities; (2) reduces SIRPA expression (such as at the mRNA level and / or at the protein level) in SIRPA-expressing cells; (3) the ability to interact with, bind to, or recognize a SIRPA protein; (4) the ability to specifically interact with or bind to a SIRPA protein; and (5) the ability to treat, ameliorate, or prevent any aspect of a disease or disorder described or contemplated herein.

[0219] In some embodiments, the anti-SIRPA antibodies exhibit one or more of the following properties: a) have a lower dissociation constant (KD) for human SIRPA 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; b) bind to human cells, such as human monocytes and macrophages; c) reduce cell surface levels of SIRPA (e.g., reduce cell surface levels of SIRPA on human macrophages in vitro with a half maximal effective concentration (EC50) 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); 5) can have a half-maximal effective concentration of an anti-SIRPA antibody having a light chain variable region with the amino acid sequence of NO:5; d) a dissociation constant (KD) for human SIRPA in the range of about 0.6 nM to about 0.7 nM; and / or e) reduce cell surface levels of SIRPA (e.g., reducing cell surface levels of SIRPA on human macrophages in vitro with a half-maximal effective concentration (EC50) in the range of about 0.4 nM to about 0.5 nM). As disclosed herein, the half-maximal effective concentration (EC50) refers to the concentration of an anti-SIRPA antibody of the invention that reduces the cellular level of SIRPA on or in a cell to half that of untreated cells, or the concentration at which the antibody achieves half-maximal binding to SIRPA on a cell.

[0220] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with a half maximal effective concentration (EC50) for human SIRPAv1 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 invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with a half maximal effective concentration (EC50) for human SIRPAv1 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 invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with a half maximal effective concentration (EC50) for human SIRPA v1 in the range of about 5 nM to 1 nM, about 1 nM to 0.9 nM, about 1 nM to 0.8 nM, about 1 nM to 0.7 nM, about 1 nM to 0.6 nM, about 1 nM to 0.5 nM, about 1 nM to 0.4 nM, about 1 nM to 0.3 nM, about 1 nM to 0.2 nM, about 1 nM to 0.1 nM , about 0.9 nM to 0.8 nM, about 0.9 nM to 0.7 nM, about 0.9 nM to 0.6 nM, about 0.9 nM to 0.5 nM, about 0.9 nM to 0.4 nM, about 0.9 nM to 0.3 nM, about 0.9 nM to 0.2 nM, about 0.9 nM to 0.1 nM, about 0.8 nM to 0.6 nM, about 0.8 nM to 0.5 nM, about 0.8nM to 0.4nM, about 0.8nM to 0.3nM, about 0.8nM to 0.2nM, about 0.8nM to 0.1nM, about 0.7nM to 0.5nM, about 0.7nM to 0.4nM, about 0.7nM to 0.3nM, about 0.7nM to 0.2nM, about 0.7nM to 0.1nM, about 0.6nM to 0.4nM, 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, as measured by flow cytometry.

[0221] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with an 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. In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with an 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 half maximal effective concentration (EC50) of v2 is in the range of about 2 nM to 0.05 nM, about 1 nM to 0.05 nM, about 0.9 nM to 0.05 nM, about 0.8 nM to 0.05 nM, about 0.7 nM to 0.05 nM, about 0.6 nM to 0.05 nM, about 0.5 nM to 0.05 nM, about 0.4 nM to 0.05 nM, about 0 0.05nM, about 0.15nM to 0.05nM, about 0.10nM to 0.05nM, about 0.09nM to 0.05nM, about 0.08nM to 0.05nM, about 0.07nM to 0.05nM, about 0.06nM to 0.05nM, about 0.20nM to 0.06nM, about 0.08nM to 0.05nM, about 0.10nM to 0.05nM, about 0.15nM to 0.05nM, about 0.10nM to 0.05nM, about 0.09nM to 0.05nM, about 0.08nM to 0.05nM, about 0.09nM to 0.05nM, about 0.10nM to 0.05nM, about 0.11nM to 0.05nM, about 0.12nM to 0.05nM, about 0.13nM to 0.05nM 0.15nM to 0.06nM, about 0.10nM to 0.06nM, about 0.09nM to 0.06nM, about 0.08nM to about 0.06nM, about 0.07nM to 0.06nM, about 0.20nM to 0.07nM, about 0.15nM to 0.07nM, about 0.10nM to about 0.07nM, about 0.09nM to 0.07nM , 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, as measured by flow cytometry.

[0222] In some embodiments that may be combined with any of the embodiments herein, the invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro with a half maximal 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.

[0223] Advantageously, the anti-SIRPA antibodies of the present invention reduce cell surface expression of SIRPA more effectively (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, advantageously, the anti-SIRPA antibodies of the present invention have a higher affinity (e.g., up to about 100-fold higher affinity) for SIRPA (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).

[0224] Certain aspects of the present invention are based, at least in part, on the identification of anti-SIRPA antibodies that exhibit one or more improved and / or enhanced functional characteristics (e.g., relative 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 improved / enhanced ability to reduce cell surface levels of SIRPA on cells, resulting in reduction, neutralization, prevention or control of one or more SIRPA activities, including but not limited to, reduction of cell growth of monocytes, macrophages, T cells, dendritic cells and / or microglial cells; reduction of proliferation of dendritic cells, bone marrow-derived dendritic cells, monocytes, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells ... induced by T cells, M1 macrophages, activated M1 macrophages and / or M2 macrophages; reduced the survival of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and / or M2 microglia; reduced the survival of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 The invention also can inhibit the proliferation of macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and / or M2 microglia; inhibit the migration of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and / or M2 microglia; reduce the proliferation of ... 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, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; reducing the proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils, and / or microglia; reducing the overall functionality of monocytes, macrophages, T cells, dendritic cells, neutrophils, and / or microglia;Inhibits 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; inhibits beneficial immune responses against different types of cancer selected from dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy disease, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, essential tremor, Behcet's disease 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, diseases of aging, epilepsy, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration and multiple sclerosis; binds to SIRPA ligands on tumor cells; binds to SIRPA ligands on dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils and / or macrophages; inhibits tumor cell killing by one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells or cytotoxic T cells; inhibits microglia, macrophages, dendritic cells , bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; inhibit the anti-tumor cell metastasis activity of one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; modulate 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, T helper cells, or cytotoxic T cells; enhance the infiltration of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, bone marrow-derived suppressor cells, tumor-associated macrophages, immunosuppressive neutrophils, and regulatory T cells into tumors;Increase the number of tumor-promoting myeloid / granulocytic immunosuppressive cells in tumors, peripheral blood or other lymphoid organs; enhance the tumor-promoting activity of bone marrow-derived suppressor cells; reduce the activation of tumor-specific T lymphocytes with tumor-killing potential; reduce the infiltration of tumor-specific T lymphocytes with tumor-killing potential; increase tumor growth rate; increase tumor recurrence rate; reduce the efficacy of one or more immunotherapies that regulate anti-tumor T cell responses, optionally wherein the one or more immunotherapies are targeted to one or more selected from CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, Immunotherapy targeting proteins such as 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, Siglec-9, Siglec-11, SirpA, CD447, CSF-1 receptor, and any combination thereof, or reducing the efficacy of one or more chemotherapeutic agents and / or multiple cancer vaccines. ;

[0225] In some embodiments, treating cancer with an anti-SIRPA antibody as described herein can: (i) increase the number of tumor-infiltrating CD3+ T cells; (ii) reduce the cellular level of SIRPA in non-tumorigenic CD14+ myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells or optionally wherein the non-tumorigenic CD14+ myeloid cells are present in the blood; (iii) reduce the number of non-tumorigenic CD14+ myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloid cells are tumor-infiltrating cells or optionally wherein the non-tumorigenic CD14+ myeloid cells are present in the blood; (iv) reduce the level of PD-L1, PD-L2, B7-H7, B7-H3, CD200R, CD163 and / or CD206 in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); v) reducing the tumor growth rate of solid tumors; (vi) reducing tumor volume; (vii) increasing the efficacy of one or more PD-1 inhibitors; (viii) increasing the efficacy of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies, optionally wherein the one or more checkpoint inhibitor therapies and / or immunomodulatory therapies target one or more of CTL4, adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (ix) increasing the efficacy of one or more chemotherapeutic agents, optionally wherein one or more of the chemotherapeutic agents is gemcitabine, capecitabine, anthracycline, doxorubicin, or a combination thereof. Epirubicin Taxane, paclitaxel docetaxel 5-fluorouracil (5-FU), cyclophosphamide Carboplatin and any combination thereof; (x) increasing the proliferation of T cells 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) killing CD33-expressing immunosuppressive non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells in solid tumors and associated blood vessels when conjugated to chemical or radioactive toxins.

[0226] In some embodiments, myeloid cells of the present invention include, but are not limited to, CD45+CD14+ myeloid cells, CD14+ myeloid cells, and bone marrow-derived suppressor cells (MDSCs). In some embodiments, myeloid cells of the present invention are non-tumorigenic myeloid cells. Immunosuppressive cells are sometimes also referred to as bone marrow-derived suppressor cells (MDSCs). In humans, MDSCs can be defined by one of the following marker combinations: (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- LA-DR-CD33+CD11b+CD14-CD15+; (12) CD11b+CD14-CD33+; (13) CD11b+CD14-HLA-DR-CD33+CD15+; (14) CD33+HLA-DR-CD15+; (15) CD15+IL4Rα+; (16) CD11b+CD15+CD66b+; (17) CD15+FSC low SSC high; (18) CD15 highCD33+; (19) CD11b+CD14-CD15+; (20) CD66b+SSC high; and (21) CD11b+CD15+ (see also Solito S et al. Annals of the NY Academy of Sciences, 2014). In mice, MDSCs can be defined by the expression of the surface markers CD45+, CD11b+, Gr1+, and / or 114Ra+. Additional exemplary immunosuppressive monocyte lineages are CD45+, CD11b+, Gr1low; and CD45+, CD11c+.

[0227] In some embodiments, the anti-SIRPA antibodies of the invention reduce cell surface levels of SIRPA, reduce intracellular levels of SIRPA, reduce total cellular levels of SIRPA, or any combination thereof, only in the presence of a natural ligand or binding partner of SIRPA.

[0228] In some embodiments, the anti-SIRPA antibodies of the invention prevent, reduce or inhibit SIRPA-mediated activity of a SIRPA ligand or binding partner, including, for example, the SIRPA ligand or binding partner CD47, surfactant protein A and / or surfactant protein D.

[0229] In some embodiments, the anti-SIRPA antibodies of the invention selectively bind to human SIRPA, including human allelic variants, also referred to herein as "polymorphic" variants, including human SIRPA v1 and human SIRPAv2, bind to human SIRPβ3, bind to cynomolgus macaque SIRPA, and bind to marmoset SIRPA. In some embodiments, the anti-SIRPA antibodies of the invention do not bind to mouse / murine SIRPA, do not bind to SIRPB v1 (SIRPβ1), do not bind to rabbit SIRPA, and do not bind to rat SIRPA.

[0230] In some embodiments, the anti-SIRPA antibodies of the present invention selectively bind to human SIRPA. In some embodiments, the anti-SIRPA antibodies of the present invention selectively bind to human SIRPA and cynomolgus monkey SIRPA. In some embodiments, the anti-SIRPA antibodies of the present invention bind to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA. In some embodiments, the anti-SIRPA antibodies of the present invention bind to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA, but do not bind to murine SIRPA. In some embodiments, the anti-SIRPA antibodies of the present invention bind to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA, but do not bind to SIRPβ1. In some embodiments, the anti-SIRPA antibodies of the present invention bind to human SIRPA v1, human SIRPA v2, and cynomolgus monkey SIRPA, but do not bind to murine SIRPA and do not bind to human SIRPβ1. In some embodiments, the anti-SIRPA antibodies of the invention bind to human SIRPA v1, human SIRPA v2, cynomolgus monkey SIRPA, and human SIRPA β3, but do not bind to murine SIRPA and do not bind to human SIRP β 1. In any combination of the above embodiments, the anti-SIRPA antibodies of the invention do not block the binding or interaction of SIRPA and CD47.

[0231] Certain aspects of the present invention relate to anti-SIRPA antibodies that downregulate, i.e., reduce, the cellular levels of SIRPA. In some embodiments, the anti-SIRPA antibodies reduce the cellular levels of SIRPA without inhibiting, blocking, or reducing the interaction (e.g., binding) between SIRPA and one or more SIRPA ligands (e.g., CD47).

[0232] In some embodiments, an anti-SIRPA antibody of the invention may have one or more activities that are 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, downregulation, cleavage, receptor desensitization, and / or lysosomal targeting of SIRPA.

[0233] In some embodiments, the anti-SIRPA antibodies of the invention downregulate SIRPA expression in cells. In some embodiments, downregulation of SIRPA expression in cells is reduced cell surface expression of SIRPA. In some embodiments, the anti-SIRPA antibodies of the invention downregulate SIRPA expression in monocytes (e.g., human monocytes). In some embodiments, the anti-SIRPA antibodies of the invention downregulate SIRPA expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRPA antibodies of the invention downregulate macrophage cell surface expression of SIRPA by greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 95%, compared to the level of SIRPA cell surface expression in macrophages not treated with the anti-SIRPA antibodies of the invention. In some embodiments, the anti-SIRPA antibodies of the invention downregulate macrophage cell surface expression of SIRPA by about 70-90%, about 70-85%, about 70-80%, 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 the anti-SIRPA antibodies of the invention. In some embodiments, the macrophages are human macrophages, including but not limited to human M1 macrophages and human M2 macrophages.

[0234] Cellular levels of SIRPA can refer to, but are not limited to, cell surface levels of SIRPA, intracellular levels of SIRPA, and total levels of SIRPA. In some embodiments, a reduction in cellular levels of SIRPA comprises a reduction in cell surface levels of SIRPA. As used herein, an anti-SIRPA antibody reduces cell surface levels of SIRPA if it induces a 25% or greater reduction in cell surface levels of SIRPA, as measured by any in vitro cell-based assay described herein or known in the art, or a suitable in vivo model, for example, flow cytometry, such as fluorescence activated cell sorting (FACS), to measure cell surface levels of SIRPA. In some embodiments, a reduction in cellular levels of SIRPA comprises a reduction in intracellular levels of SIRPA. As used herein, an anti-SIRPA antibody reduces the intracellular level of Siglec-9 if it induces a 25% or greater decrease in the intracellular level of SIRPA, as measured by any in vitro cell-based assay or suitable in vivo model described herein or known in the art, such as immunostaining, Western blot analysis, immunoprecipitation, and cell cytometry. In some embodiments, the decrease in the cellular level of SIRPA comprises a decrease in the total level of SIRPA. As used herein, an anti-SIRPA antibody reduces the total level of SIRPA if it induces a 25% or greater decrease in the total level of SIRPA, as measured by any in vitro cell-based assay or suitable in vivo model described herein or known in the art, such as immunostaining, Western blot analysis, immunoprecipitation, and cell cytometry. In some embodiments, the anti-SIRPA antibody induces SIRPA degradation, SIRPA cleavage, SIRPA internalization, SIRPA shedding, downregulation of SIRPA expression, or any combination thereof. In some embodiments, the SIRPA cellular assay is used to measure cellular levels of SIRPA on primary cells (eg, dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, and macrophages) or on cell lines.

[0235] In some embodiments, the downregulating anti-SIRPA antibody has an IC of 200 nM or less, typically 100 nM or less, after exposure of human macrophages to the antibody for 4 hours at 37°C. 50 (50% downregulation of SIRPA expressed on the cell surface.) In some embodiments, SIRPA remains downregulated for at least 24 hours after exposure to an antibody of the invention. Cells can be analyzed for SIRPA surface expression using any technique, such as flow cytometry.

[0236] In some embodiments, an anti-SIRPA antibody of the invention reduces the cellular level 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%, 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 %, 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.

[0237] In some embodiments, which may be combined with any of the down-regulating activities outlined in the above paragraphs, the anti-SIRPA antibodies of the invention inhibit cell surface clustering of SIRPA.

[0238] In some embodiments, the anti-SIRPA antibodies of the present invention downregulate SIRPA but do not block, inhibit or reduce the binding of a SIRPA ligand (e.g., CD47) to 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 cause a significant reduction in CD47 binding to SIRPA when the antibody is incubated with CD47 and cells expressing SIRPA. "Significantly reduced" in the context of CD47 binding to SIRPA means 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 CD47 binding to SIRPA in the presence of an isotype-matched control antibody that does not bind SIRPA. Illustrative assays for assessing blocking activity are described in the Examples herein. For example, cells expressing human SIRPA, such as human macrophages, or cells modified to recombinantly express human SIRPA, such as CHO cells, are cultured at 10 5Cells were seeded per well in a 96-well plate, washed, and incubated in 100 μl of buffer containing 1.0 μg / ml of monoclonal antibody or isotype control for fluorescence-activated cell sorting. The cells were then washed and incubated on ice for 30 minutes in soluble human CD47. The cells were then analyzed for surface-bound CD47.

[0239] In some embodiments, the anti-SIRPA antibodies of the present invention downregulate CD32A / B expression (i.e., FcγRIIA / FcγRIIB) in cells. In some embodiments, downregulation of CD32A / B (i.e., FcγRIIA / FcγRIIB) expression in cells is decreased cell surface expression of CD32A / B (i.e., FcγRIIA / FcγRIIB). In some embodiments, the anti-SIRPA antibodies of the present invention downregulate CD32A / B expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRP antibodies of the present invention downregulate CD32A (i.e., FcγRIIA) expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRPA antibodies of the present invention downregulate CD32B (i.e., FcγRIIB) expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRPA antibodies of the invention downregulate the cell surface expression of CD32A (i.e., FcγRIIA) in human macrophages by about 75%, about 80%, or about 85%, compared to the level of CD32A cell surface expression in human macrophages not treated with the anti-SIRPA antibodies of the invention. In some embodiments, the anti-SIRPA antibodies of the invention downregulate the cell surface expression of CD32A in human macrophages by about 70-85%, compared to the level of CD32A cell surface expression in human macrophages not treated with the anti-SIRPA antibodies of the invention. In some embodiments, the anti-SIRPA antibodies of the invention downregulate the cell surface expression of CD32B (i.e., FcγRIIB) in human macrophages to undetectable levels.

[0240] In one aspect, the present invention 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 invention. In some embodiments, the antibodies interact with or otherwise bind to a region, such as an epitope, within a SIRPA protein of the invention with improved / enhanced kinetics (e.g., relative 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 antibodies interact with or otherwise bind to a region, such as an epitope, within a SIRPA protein on human cells, such as dendritic cells, myeloid cells, monocytes, macrophages, etc., at a half maximal effective concentration (EC 50 ) is less than the half-maximal effective concentration of a control antibody (e.g., relative 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 anti-SIRPA antibodies of the invention bind to a SIRPA protein and, upon binding to the SIRPA protein, modulate one or more SIRPA activities, such as an activity associated with SIRPA expression on a cell. The SIRPA proteins of the invention include, but are not limited to, mammalian SIRPA proteins, human SIRPA proteins, mouse SIRPA proteins, cynomolgus monkey SIRPA proteins, and rat SIRPA proteins.

[0241] In some embodiments, the antibodies of the present invention can bind to SIRPA in a pH-dependent manner. In some embodiments, the antibodies of the present invention can bind to SIRPA at neutral pH and be internalized without dissociating from the SIRPA protein. Alternatively, at acidic pH, after the antibodies of the present invention are internalized and subsequently degraded through the endosomal / lysosomal pathway, the antibodies can dissociate from SIRPA. In certain embodiments, the anti-SIRPA antibodies bind to SIRPA at a pH within the following ranges: 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, 6.5 to 7.5. In certain embodiments, the anti-SIRPA antibody dissociates from SIRPA at a pH of less than 6.0, less than 5.5, less than 5.0, less than 4.5, less than 4.0, less than 3.5, less than 3.0, less than 2.5, or less than 2.0.

[0242] SIRPA is a single-pass type I membrane 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 comprises a single V group and two C1 group Ig superfamily (IgSF) domains, referred to as the D1 domain, the D2 domain, and the D3 domain, respectively. The D1 domain comprises amino acid residues 32-137 of human SIRPA; the D2 domain comprises amino acid residues 148-247 of human SIRPA; and the D3 domain comprises amino acid residues 254-348 of human SIRPA. As will be appreciated by those skilled in the art, the start and end residues of the domains of the present invention may vary depending on the computer modeling program or method used to define the domains.

[0243] In some embodiments, the anti-SIRPA antibodies of the invention bind to the D3 domain of SIRPA. In some embodiments, the anti-SIRPA antibodies of the invention bind to the D3 domain of human SIRPA, which domain comprises amino acid residues 254-348 of the human SIRPA amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope within the D3 domain of human SIRPA. In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope within the D3 domain of human SIRPA, wherein the epitope comprises an amino acid sequence selected from the group consisting of amino acid residues 254-348, amino acid residues 254-274, amino acid residues 264-279, amino acid residues 274-289, amino acid residues 273-331, amino acid residues 281-315, amino acid residues 281-337, amino acid residues 284-299, amino acid residues 294-309, amino acid residues 304-319, amino acid residues 314-329, amino acid residues 324-339, and amino acid residues 334-348 of the human SIRPA amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope within the D3 domain of human SIRPA, wherein the epitope comprises amino acid residues R282, Q284, and G337 of the human SIRPA v1 amino acid sequence (SEQ ID NO: 1). In some embodiments, the anti-SIRPA antibodies of the invention bind to an epitope within the D3 domain of human SIRPA, wherein the epitope includes amino acid residues Q281, R282, Q284, L285, W287, R295, E297, V302, and W315 of the human SIRPA v1 amino acid sequence (SEQ ID NO: 1).

[0244] In some embodiments, the antibodies bind to the D3 domain of SIRPA, e.g., human SIRPA. In some embodiments, the anti-SIRPA antibodies of the invention bind to the same SIRPA epitope, or a portion of a SIRPA epitope, as bound by an antibody having the CDRs of the antibody designated m3F9 as described herein. Thus, in some embodiments, the antibodies of the invention bind to the same SIRPA epitope, or a portion of a SIRPA epitope, as bound by an antibody having the CDRs of the antibody designated m3F9 as described herein.

[0245] Certain aspects of the present invention are based, at least in part, on the ability to exhibit one or more improved and / or enhanced functional characteristics (e.g., relative to a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 3). NO:5) wherein the functional characteristics include improved / enhanced ability to reduce cell surface levels of SIRPA on cells, resulting in reduction, neutralization, prevention or control of one or more SIRPA activities, including but not limited to reducing cell growth of monocytes, macrophages, T cells, dendritic cells and / or microglial cells; reducing T cell proliferation induced by dendritic cells, bone marrow-derived dendritic cells, monocytes, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells, macrophages, M1 macrophages, activated M1 macrophages and / or M2 macrophages; reducing 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 survival rate; reduce or decrease 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 proliferation of NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia, and / or M2 microglia; inhibition of migration 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;Reduce or inhibit 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, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and / or M2 microglia; reduce the proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils and / or microglia; reduce the overall functionality of monocytes, macrophages, T cells, dendritic cells, neutrophils and / or microglia; inhibit the proliferation of bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, Kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, acute myeloid leukemia, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma and thyroid cancer; inhibiting the beneficial immune response to different types of cancer selected from dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tau protein disease, Nahar's disease, stroke, acute trauma, chronic trauma, essential tremor, Behcet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, summer disease - beneficial immune responses in different types of neurological disorders such as Choi syndrome, progressive supranuclear palsy, corticobasal degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, diseases of aging, epilepsy, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration and multiple sclerosis; binds to SIRPA ligands on tumor cells; binds to SIRPA ligands on dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils and / or macrophages; inhibits microglia, Tumor cell killing by one or more of macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; inhibition of the anti-tumor cell proliferation activity of one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; inhibition of the anti-tumor cell metastasis activity of one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells;Modulating the expression of one or more inflammatory receptors (such as CD86) expressed on one or more of microglia, macrophages, dendritic cells, bone marrow-derived dendritic cells, neutrophils, T cells, T helper cells, or cytotoxic T cells; enhancing the infiltration of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, bone marrow-derived suppressor cells, tumor-associated macrophages, immunosuppressive neutrophils, and regulatory T cells into tumors; promoting or rescuing immunosuppressive dendritic cells, immunosuppressive macrophages, immunosuppressive neutrophils, immunosuppressive NK cells, bone marrow-derived suppressor cells, Increase the functionality of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, immunosuppressive neutrophils, immunosuppressive NK cells, bone marrow-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, tumor-associated NK cells, non-tumorigenic CD45+CD14+ myeloid cells and regulatory T cells into tumors; increase the number of pro-tumorigenic myeloid / granulocytic immunosuppressive cells in tumors, peripheral blood or other lymphoid organs; enhance bone marrow-derived The invention relates to a method for inhibiting the tumor-promoting activity of suppressor cells derived from the bone marrow of a patient with tumor-killing potential; reducing the activation of tumor-specific T lymphocytes with tumor-killing potential; enhancing the survival of non-tumorigenic bone marrow-derived suppressor cells and / or non-tumorigenic CD45+CD14+ myeloid cells; reducing the infiltration and / or activation of tumor-specific T lymphocytes with tumor-killing potential; increasing the tumor growth rate; increasing the tumor recurrence rate; reducing the efficacy of one or more immunotherapies that regulate anti-tumor T cell responses, optionally wherein the one or more immunotherapies are targeted to 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, GAL9, 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 reduce the efficacy of one or more chemotherapeutic agents and / or multiple cancer vaccines. ;

[0246] In some embodiments that may be combined with any of the other embodiments above, the anti-SIRPA antibodies of the invention induce, enhance, or increase one or more activities, including: (i) increasing tumor infiltrating CD3 + (ii) reducing the number of non-tumorigenic CD14 +Cellular levels of SIRPA in myeloid cells, optionally including non-tumorigenic CD14 + Myeloid cells are tumor infiltrating cells or, optionally, non-tumorigenic CD14 + Myeloid cells are present in the blood; (iii) reduce non-tumorigenic CD14 + The number of myeloid cells, optionally including non-tumorigenic CD14 + Myeloid cells are tumor infiltrating cells or, optionally, non-tumorigenic CD14 + myeloid cells present in the blood; (iv) reducing the level of PD-L1, PD-L2, B7-H2, B7-H3, CD200R, CD163 and / or CD206 in one or more cells, optionally wherein the one or more cells are non-tumorigenic myeloid-derived suppressor cells (MDSCs); (v) reducing the tumor growth rate of solid tumors; (vi) reducing tumor volume; (vii) increasing the efficacy of one or more PD-1 inhibitors; (viii) increasing the efficacy of one or more checkpoint inhibitor therapies and / or immunomodulatory therapies, optionally wherein the one or more checkpoint inhibitor therapies and / or immunomodulatory therapies target one or more of CTL4, adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3 or any combination thereof; (ix) increasing the efficacy of one or more chemotherapeutic agents, optionally wherein one or more of the chemotherapeutic agents is gemcitabine, capecitabine, anthracycline, doxorubicin, or any combination thereof. Epirubicin Taxanes, paclitaxel Docetaxel 5-fluorouracil (5-FU), cyclophosphamide Carboplatin and any combination thereof; (x) increasing the proliferation of T cells 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) killing CD33-expressing immunosuppressive non-tumorigenic myeloid cells and / or non-tumorigenic CD14-expressing cells in solid tumors and associated blood vessels when conjugated to chemical or radioactive toxins.

[0247] In some embodiments, the anti-SIRPA antibodies of the invention 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).

[0248] In some embodiments, the anti-SIRPA antibodies of the invention induce or promote the survival, maturation, functionality, migration, or proliferation of one or more immune cells in a subject, e.g., one or more immune cells selected from dendritic cells, macrophages, neutrophils, NK cells, microglial cells, T cells, T helper cells, cytotoxic T cells, and any combination thereof.

[0249] Anti-SIRPA antibody binding affinity

[0250] In some embodiments of any of the antibodies provided herein, the antibody has <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or lower, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 - 13 The dissociation constant (Kd) of an antibody is determined by any analytical technique, including any biochemical or biophysical technique, such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, for example, ForteBio's Octet system), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analysis. In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In some embodiments, an RIA is performed using an antibody of interest in Fab form and its antigen, for example, as described in Chen et al. J. Mol. Biol. 293:865-881 (1999). In some embodiments, Kd is measured using BIACORE surface plasmon resonance analysis, e.g., at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU) using a BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscataway, NJ). In some embodiments, Kd is determined using monovalent antibodies (e.g., Fab) or full-length antibodies. D In some embodiments, K is determined using full-length antibodies in a monovalent format. D .

[0251] Antibody fragments

[0252] In some embodiments of any of the antibodies provided herein, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv 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, for example, 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 comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0253] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP404097; 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 comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6,248,516).

[0254] 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 (eg, E. coli or phage), as described herein.

[0255] Chimeric and humanized antibodies

[0256] In some embodiments of any of the antibodies provided 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 another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0257] In some embodiments of any of the antibodies provided herein, the antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to 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 the target as antibodies from another species from which the humanized antibody was derived. See, for example, U.S. Patents 5,530,101, 5,693,761, 5,693,762, and 5,585,089. In certain embodiments, amino acids of antibody variable domains that can be modified without reducing the natural affinity of the antigen-binding domain while reducing its immunogenicity are identified. See, for example, U.S. Patents 5,766,886 and 5,869,619. Typically, a humanized antibody comprises one or more variable domains, wherein HVR (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody optionally also comprises 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 a non-human antibody (eg, the antibody from which the HVR residues are derived), eg, to restore or improve antibody specificity or affinity.

[0258] Humanized antibodies and methods for making them are reviewed in, for example, Almagro et al. Front. Biosci. 13:1619-1633 (2008), and further described in, for example, 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, but are not limited to, framework regions selected using the "best-fit" method (see, for example, 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, for example, Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening 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)).

[0259] human antibodies

[0260] In some embodiments of any of the antibodies provided herein, the antibody is a human antibody. Human antibodies can be produced using various 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).

[0261] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Large fragments of human Ig loci can be used to engineer mouse strains that lack mouse antibody production, and it is expected that such mice will produce human antibodies without mouse antibodies. Large human Ig fragments can maintain large variable gene diversity and appropriately regulate antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection and lacking immune tolerance to human proteins, the regenerated human antibody repertoires in these mouse strains can produce 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 produced and selected. Some exemplary methods are described in U.S. Patent No. 5,545,807, EP 546,073, and EP 546,073. In addition, see, for example, description of XENOMOUSE TM U.S. Patent Nos. 6,075,181 and 6,150,584 describe the technology; US Patent No. 5,770,429 describes KM U.S. Patent No. 7,041,870; and description of the technology Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0262] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol. 133:3001 (1984); and Boerner et al. J. Immunol. 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 1 03:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM 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 generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0263] In some embodiments of any of the antibodies provided herein, the antibodies are human antibodies isolated by in vitro methods and / or screening of combinatorial libraries for antibodies with the desired one or more 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 similar methods. In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen binding phage, as described in Winter et al. Ann. Rev. Immunol. 12:433-455 (1994). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. See also Sidhu et al. J. Mol. Biol. 338(2):299-310, 2004; Lee et al. J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(-2):119-132 (2004). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a primary repertoire (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, a primary library can also be made synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers comprising a random sequence encoding a highly variable HVR3 region and for achieving in vitro rearrangement, 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. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered herein to be human antibodies or human antibody fragments.

[0264] Constant region including Fc region

[0265] 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 of the IgG, IgM, or IgA class.

[0266] In certain embodiments of any of the anti-SIRPA antibodies provided 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 includes an Fc region. In some embodiments, the anti-SIRPA antibody induces one or more SIRPA activities independently of or independent 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).

[0267] In certain embodiments of any of the antibodies provided 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 includes 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).

[0268] In certain embodiments of any of the anti-SIRPA antibodies provided 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 includes 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).

[0269] In certain embodiments of any of the anti-SIRPA antibodies provided 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 of human IgG2 according to EU numbering and amino acids 261-447 of human IgG4 according to EU numbering (WO 1997 / 11971; WO 2007 / 106585).

[0270] In some embodiments, the Fc region increases clustering without activating complement compared to a corresponding antibody comprising an Fc region without the amino acid substitutions. In some embodiments, the antibody induces one or more activities of the target to which the antibody specifically binds. In some embodiments, the antibody binds to SIRPA.

[0271] It may also be desirable to modify the anti-SIRPA antibodies of the invention to modify effector function and / or increase the serum half-life of the antibody. For example, the Fc receptor binding sites on the constant region can be modified or mutated to remove or reduce binding affinity for certain Fc receptors such as FcγRI, FcγRII, and / or FcγRIII to reduce antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is reduced by removing N-glycosylation from the Fc region of the antibody (e.g., in the CH2 domain of IgG). In some embodiments, effector function is reduced by modifying regions such as 233-236, 297, and / or 327-331 of human IgG, as described in WO 99 / 58572 and Armour et al., Molecular Immunology 40:585-593 (2003); Reddy et al., J. Immunology 164:1925-1933 (2000). In other embodiments, it may also be desirable to modify the anti-SIRPA antibodies of the invention to modify effector function, thereby increasing selectivity for targeting ITIM-containing FcgRIIb (CD32b) to increase clustering of SIRPA antibodies on adjacent cells without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis.

[0272] To increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into an antibody (particularly an 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 of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life of the IgG molecule in vivo.

[0273] Multispecific antibodies

[0274] Multispecific antibodies are antibodies that have binding specificity for at least two different epitopes, including epitopes on the same or another polypeptide (e.g., one or more SIRPA polypeptides of the present invention). In some embodiments, the multispecific antibody may be a bispecific antibody. In some embodiments, the multispecific antibody may be a trispecific antibody. In some embodiments, the multispecific antibody may be a tetraspecific antibody. Such antibodies may be derived from full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). In some embodiments, the multispecific antibody comprises 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, the multispecific antibody comprises a first antigen-binding region that binds to SIRPA and a second antigen-binding region that binds to a second polypeptide.

[0275] Provided herein are multispecific antibodies comprising a first antigen-binding region, wherein the first antigen-binding region comprises six HVRs of an antibody described herein that binds to SIRPA; and a second antigen-binding region that binds to a second polypeptide. In some embodiments, the first antigen-binding region comprises V H or V L .

[0276] In some embodiments of any of the multispecific antibodies, the second polypeptide is an antigen that promotes transport across the blood-brain barrier. A variety of antigens and peptides that promote 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 transduction domains (such as TAT, Syn-B or transmembrane peptides), 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)).

[0277] In some embodiments of any of the multispecific antibodies, the second polypeptide is a pathogenic protein selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, tau protein, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin protein, calcitonin, superoxide dismutase, ataxia, ataxia 1, ataxia 2, ataxia 3, ataxia 7, ataxia 8, ataxia 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta2 microglobulin, gelsolin, corneal epithelial protein, cystatin, immunoglobulin light chain (d) a ligand and / or protein expressed on an immune cell, wherein the ligand and / or protein is selected from the group consisting of 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 protein, lipid, polysaccharide, or glycolipid expressed on one or more tumor cells; and any combination thereof.

[0278] The multivalent antibody may recognize SIRPA antigens as well as, but not limited to, additional antigens such as Aβ peptide antigens, α-synuclein protein antigens, tau protein antigens, TDP-43 protein antigens, prion protein antigens, huntingtin protein antigens, RAN translation product antigens (including dipeptide repeats (DPR peptides) consisting 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 antibody transfer across the blood-brain barrier. In some embodiments, the second polypeptide is transferrin. In some embodiments, the second polypeptide is tau protein. 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.

[0279] A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, a polypeptide chain may comprise VD1-(X1) n -VD2-(X2) n -Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, the polypeptide chain may comprise V H -C H 1-Flexible joint-V H -C H 1-Fc region chain; or V H -C H 1-V H -C H 1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for example, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides encompassed herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0280] 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); WO 93 / 08829; and Traunecker et al. EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). See also WO 2013 / 026833 (CrossMab). Multispecific antibodies can also be made by engineering electrostatic manipulation effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); cross-linking two or more antibodies (see, e.g., U.S. Patent No. 4,676,980); using leucine; using "diabody" technology for making 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 trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147:60 (1991).

[0281] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576). Antibodies also include herein "Dual Acting FAbs" or "DAFs" comprising antigen-binding sites that bind to multiple SIRPAs (see, e.g., US 2008 / 0069820).

[0282] Antibody variants

[0283] In some embodiments of any of the antibodies provided herein, amino acid sequence variants of the antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibodies.

[0284] Substitution, insertion, and deletion variants

[0285] In some embodiments of any of the antibodies provided herein, antibody variants having one or more amino acid substitutions are provided. 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 and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody.

[0286] Table 1: Amino acid substitutions

[0287]

[0288]

[0289] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties:

[0290] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0291] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0292] (3) Acidic: Asp, Glu;

[0293] (4) Basic: His, Lys, Arg;

[0294] (5) Residues that affect chain orientation: Gly, Pro; and

[0295] (6) Aromatic: Trp, Tyr, Phe.

[0296] For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class. Such substituted residues may, for example, be introduced into regions of human antibodies that are homologous to non-human antibodies, or into non-homologous regions of the molecule.

[0297] When making changes to the polypeptides or antibodies described herein, according to certain embodiments, the hydropathic index of the amino acids can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. They 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0298] The importance of the hydropathic amino acid index in conferring interactive biological function on proteins 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 for other amino acids with similar hydropathic indices or scores and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, substitutions of amino acids with a hydropathic index within ±2 are included. In certain embodiments, substitutions of amino acids with a hydropathic index within ±1 are included, and in certain embodiments, substitutions of amino acids with a hydropathic index within ±0.5 are included.

[0299] It is also understood in the art that substitutions of similar amino acids can be made effectively based on hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as in the case of the present invention. In certain embodiments, the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., the biological properties of the protein.

[0300] The following hydropathicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0±1); aspartic acid (+3.0±1); glutamic acid (+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 making changes based on similar hydrophilicity values, in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included, in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±1 are included, and in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±0.5 are included. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions."

[0301] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in the HVRs. Such changes may, for example, be outside the antigen contact residues in the HVRs. 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.

[0302] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0303] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may also be substituted with serine to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bond(s) may be added to the antibody to increase its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0304] Glycosylation variants

[0305] In some embodiments of any of the antibodies provided herein, the antibody is altered to increase or decrease the degree of antibody glycosylation. Adding glycosylation sites to an antibody or deleting glycosylation sites from an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0306] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are 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 may also be used.

[0307] Preferably, the addition of glycosylation sites to the antibody is achieved by altering the amino acid sequence to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alternatively, the alteration may be made by adding or substituting one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

[0308] In the case where the antibody comprises an Fc district, the carbohydrate connected thereto can be changed. The natural antibody produced by mammalian cells generally comprises branched biantennary oligosaccharides, which are generally connected to the Asn297 of the CH2 domain in the Fc district by an N bond according to Kabat numbering. Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In certain embodiments, the oligosaccharides in the antibodies of the present invention may be modified to produce antibody variants with some improved properties.

[0309] In one embodiment, antibody variants are provided that have a carbohydrate structure lacking 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 related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 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 that lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004) and Kanda et al. Biotechnol. Bioeng. 94(4):680-688 (2006)).

[0310] Modified constant region

[0311] In some embodiments of any of the anti-SIRPA antibodies provided herein, the antibody Fc is an antibody Fc isotype and / or modification. In some embodiments, the antibody Fc isotype and / or modification is capable of binding to an Fcγ receptor.

[0312] Exemplary antibody Fc isotypes and modifications are provided below in Table 2. In some embodiments, the anti-SIRPA antibodies of the invention are capable of binding to an Fcγ receptor and have an Fc isotype listed in Table 2 below.

[0313] Table 2: Exemplary anti-SIRPA antibody Fc isotypes capable of binding to Fcγ receptors

[0314]

[0315]

[0316] In addition to the isotypes described in Table 2, and without wishing to be bound by theory, it is believed that antibodies with the human IgG1 or IgG3 isotype and mutants thereof that bind to Fcg receptors I, IIA, IIC, IIIA, IIIB in humans and / or Fcg receptors I, III and IV in mice (e.g., Strohl (2009) Current Opinion in Biotechnology 2009, 20:685-691) can also be used as transient potentiator antibodies.

[0317] In some embodiments, the antibody that binds to Fcγ receptors belongs to IgG class, IgM class or IgA class. In some embodiments, the antibody that binds to Fcγ receptors has 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, 10 or more, 11 or more, 12 or more or all thirteen) amino acid substitutions at residue positions selected from the group consisting of in the Fc region: C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, in any combination (residue positions are according to EU or Kabat numbering). 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. In some embodiments, the Fc region comprises an amino acid substitution at positions E345R, E430G, and S440Y.

[0318] In certain embodiments, antibodies that bind to Fcγ receptors have an IgG2 isotype. In some embodiments, antibodies that bind to Fcγ receptors contain a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, antibodies that bind to Fcγ receptors bind to inhibitory Fc receptors. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-γ 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., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from 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 (US 2007 / 0148167; Armour et al. (1999) Eur J Immunol 29:2613-2624; Armour et al. (2000) The Haematology Journal 1(Suppl 1):27; Armour et al. (2000) The Haematology Journal 1(Suppl 1):27), C232S and / or C233S (White et al. (2015) Cancer Cell 27, 138-148), S267E, L328F (Chu et al., (2008) Mol Immunol, 45:3926-3933), M252Y, S254T and / or T256E, where amino acid positions are according to the EU or Kabat numbering convention.

[0319] In some embodiments, the antibody that binds to an Fcγ receptor is of the IgG2 isotype, having a heavy chain constant domain comprising a C127S amino acid substitution, wherein amino acid positions are according to the EU or Kabat numbering convention (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO2008079246).

[0320] In some embodiments, the antibody that binds to an Fcγ receptor is of the IgG2 isotype, having a kappa light chain constant domain comprising a C214S amino acid substitution, wherein amino acid positions are according to the EU or Kabat numbering convention (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO2008079246).

[0321] In certain embodiments, antibodies that bind to Fcγ receptors have an IgG1 isotype. In some embodiments, antibodies that bind to Fcγ receptors contain a mouse IgG1 constant region. In some embodiments, antibodies that bind to Fcγ receptors contain a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments, antibodies that bind to Fcγ receptors bind to inhibitory Fc receptors. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-γ 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., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) RJ Biol. Chem. 276, 6591-6604), D270A, L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92: 11980-11984; Alegre et al. (1994) Transplantation 57: 1537-1543.31; Xu et al. (2000) Cell Immunol, 200: 16-26), G237A (Alegre et al. (1994) Transplantation 57: 1537-1543.31; Xu et al. (2000) Cell Immunol, 200: 16-26), Immunol, 200: 16-26), P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109: 1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A and / or T394D, wherein amino acid positions are according to the EU or Kabat numbering convention.

[0322] In some embodiments, the antibody comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and hinge region (White et al., (2015) Cancer Cell 27, 138-148). In certain embodiments, the IgG2 isotype CH1 and hinge region comprises the amino acid sequence of ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 58). In some embodiments, the antibody Fc region comprises an S267E amino acid substitution, an L328F amino acid substitution, or both, and / or an N297A or N297Q amino acid substitution, wherein amino acid positions are according to the EU or Kabat numbering conventions.

[0323] In certain embodiments, the antibody that binds to an Fcγ receptor has an IgG4 isotype. In some embodiments, the antibody that binds to an Fcγ receptor contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. In some embodiments, the antibody that binds to an Fcγ receptor 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., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from L235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol, 164:1925-1933), S267E, E318A, L328F, M252Y, S254T, and / or T256E, where amino acid positions are numbered according to EU or Kabat conventions.

[0324] In certain embodiments, the antibody that binds to an Fcγ receptor has a hybrid IgG2 / 4 isotype. In some embodiments, the antibody that binds to an Fcγ receptor comprises an amino acid sequence comprising amino acids 118 to 260 of human IgG2 according to EU or Kabat numbering and amino acids 261-447 of human IgG4 according to EU or Kabat numbering (WO 1997 / 11971; WO 2007 / 106585).

[0325] In certain embodiments, the antibodies contain a mouse IgG4 constant region (Bartholomaeus et al. (2014). J. Immunol. 192, 2091-2098).

[0326] 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 or Kabat numbering, and any combination thereof.

[0327] In certain embodiments, the antibodies contain one or more amino acid substitutions in the Fc region at a residue position selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof, wherein the residue numbering is according to EU or Kabat numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, L243A, L235A, and P331S, wherein the residue position numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G and P331S, wherein the residue position numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G and K322A, wherein the residue position numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, A330S, and P331S, wherein 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, wherein 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, wherein 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, wherein 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, wherein the numbering of the residue positions is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at position C127S, wherein 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 residue positions is according to EU numbering.

[0328] In some embodiments, antibodies that bind to a SIRPA protein may include antibodies that reduce the cellular level of SIRPA (e.g., cell surface levels of SIRPA), inhibit the interaction (e.g., binding) between SIRPA and / or one or more SIRPA ligands, and inhibit one or more activities of a SIRPA protein. Such antibodies inhibit one or more activities of a SIRPA protein by preventing the interaction (e.g., binding) between SIRPA and 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 antibodies may also inhibit one or more activities of a SIRPA protein by reducing the cell surface level of SIRPA by inducing SIRPA degradation, SIRPA desensitization, SIRPA cleavage, SIRPA internalization, SIRPA shedding, downregulation of SIRPA expression, and / or lysosomal degradation of SIRPA. In some embodiments, such anti-SIRPA antibodies may not transiently activate SIRPA.

[0329] In some embodiments, an anti-SIRPA antibody of the invention may have the epitope specificity of a transient potentiator anti-SIRPA antibody of the invention, but have an Fc domain that is unable to bind Fcg receptors and therefore cannot, for example, transiently cluster and activate SIRPA.

[0330] In some embodiments, the anti-SIRPA antibodies of the invention have one or more of the following activities, but are not limited to: the ability to reduce 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 reduce the binding of a suppressor of cytokine signaling (SOCS) protein (e.g., SOCS3 protein) to a SIRPA protein; the ability to increase the proteasomal degradation of a SIRPA protein; the ability to reduce the expression of SIRPA on circulating dendritic cells, macrophages, monocytes, T cells, and / or microglial cells; The ability of the phosphodiesterase to inhibit the functional expression of the surface of the mitochondria; reducing the ability of Src family tyrosine kinases such as LCK and FYN to phosphorylate Tyr-340 and Tyr-358; reducing the recruitment and binding ability of tyrosine-specific protein phosphatases SHP1 and SHP2; reducing the recruitment and binding ability of PLC-g1, which acts as a guanine nucleotide exchange factor for dynamin-1; reducing the recruitment and binding ability of Crkl; reducing the recruitment and binding ability of spleen tyrosine kinase Syk; reducing the SH3-SH2-SH3 growth factor receptor binding protein 2 (G rb2) recruitment and binding ability; reduce the recruitment and binding ability of multiple SH2-containing proteins; increase the ability of intracellular calcium mobilization; regulate the production of proinflammatory cytokines IL-1β, IL-8 and TNF-α; reduce the activation of phosphoinositide 3-kinase; increase the growth of monocytes, macrophages, dendritic cells, T cells and / or microglial cells; increase the survival of monocytes, macrophages, dendritic cells, T cells and / or microglial cells; increase the tyrosine phosphorylation on multiple cellular proteins; increase the expression of monocytes, macrophages, dendritic cells, T cells and / or microglial cells. The invention also provides the invention claims an ability to increase the phagocytic activity of monocytes, macrophages, dendritic cells, T cells and / or microglial cells; the ability to increase the cell proliferation of monocytes, macrophages, dendritic cells, T cells and / or microglial cells; the ability to increase the phosphorylation of signaling molecules that mediate ITAM signaling; the ability to increase the function of pattern recognition receptors; the ability to increase the function of Toll-like receptors; the ability to increase the function of damage-associated molecular pattern (DAMP) receptors; the ability to regulate the expression of CC chemokine receptor 7 (CCR7); and the ability to increase the clearance of cellular and protein debris.

[0331] In some embodiments, the anti-SIRPA antibodies of the invention 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.

[0332] In some embodiments, the anti-SIRPA antibodies of the invention with reduced binding to Fcγ receptors have an Fc isotype listed in Table 3 below.

[0333] Table 3: Exemplary anti-SIRPA antibody Fc isotypes with reduced binding to Fcγ receptors

[0334]

[0335]

[0336] In certain embodiments, the anti-SIRPA antibody has an IgG1 isotype. In some embodiments, the antibody comprises a mouse IgG1 constant region. In some embodiments, the antibody comprises a human IgG1 constant region. In some embodiments, the human IgG1 constant region comprises an Fc region. In some embodiments, the Fc region comprises one or more modifications. For example, in some embodiments, the Fc region comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).

[0337] In some embodiments, the one or more amino acid substitutions are selected from 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, wherein 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.

[0338] In some embodiments, the anti-SIRPA antibody has an IgG1 isotype, wherein the heavy chain constant region contains a C220S amino acid substitution according to the EU 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 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., relative 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, wherein amino acid positions are according to the EU or Kabat numbering convention (Vafa O. et al., (2014) Methods 65: 114-126).

[0339] 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 includes 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., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from 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; US 8614299 B2; Vafa O. et al., (2014) Methods 65: 114-126), T394D, M252Y, S254T, T256E, N297A and / or N297Q, wherein amino acid positions are according to the EU or Kabat numbering convention. In some embodiments, the antibody has an IgG4 isotype and comprises an S228P amino acid substitution at residue position 228, an F234A amino acid substitution at residue position 234, and an L235A amino acid substitution at residue position 235 (residue positions are according to EU numbering).

[0340] In some embodiments, the Fc region further contains one or more additional amino acid substitutions selected from M252Y, S254T and / or T256E, wherein amino acid positions are according to the EU or Kabat numbering convention.

[0341] In some embodiments of any of the antibodies provided herein, the modified antibody Fc is an IgG1 modified Fc. In some embodiments, the IgG1 modified Fc comprises one or more modifications. For example, in some embodiments, the IgG1 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from 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: 1537-1543.31; Xu et al. (2000) Cell Immunol, 200: 16-26), G237A (Alegre et al. (1994) Transplantation 57: 1537-1543.31; Xu et al. (2000) Cell Immunol, 200: 16-26). 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, wherein amino acid positions are according to the EU numbering convention.

[0342] In some embodiments of any of the IgG1 modified Fcs, the Fc comprises an N297A mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises a D265A and N297A mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises a D270A mutation according to EU numbering. In some embodiments, the IgG1 modified Fc comprises an L234A and L235A mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises an L234A and G237A mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises an L234A, L235A, and G237A mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises one or more (including all) of the P238D, L328E, E233, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises one or more of the S267E / L328F mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises P238D, L328E, E233D, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises P238D, L328E, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises P238D, S267E, L328E, E233D, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises P238D, S267E, L328E, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises C226S, C229S, E233P, L234V, and L235A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises L234F, L235E, and P331S mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc, the Fc comprises S267E and L328F mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc, the Fc comprises an S267E mutation according to EU numbering.In some embodiments of any of the IgG1 modified Fcs, the Fc comprises substitution of the constant heavy chain 1 (CH1) and hinge region of IgG1 with the CH1 and hinge region of IgG2 containing a kappa light chain (amino acids 118-230 of IgG2 according to EU numbering).

[0343] In some embodiments of any of the IgG1 modified Fcs, the Fc comprises two or more amino acid substitutions that increase antibody clustering without complement activation compared to a corresponding antibody having an Fc region that does not comprise the two or more amino acid substitutions. Thus, in some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc is an antibody comprising an Fc region, wherein the antibody comprises an amino acid substitution at position E430G and one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, and any combination thereof, according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S, according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G and P331S, according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G, A330S, and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid substitutions at positions E430G, K322A, and P331S according to EU numbering.

[0344] In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc herein may further comprise an A330L mutation (Lazar et al. Proc Natl Acad Sci USA, 103:4005-4010 (2006)), or one or more of L234F, L235E, and / or P331S mutations (Sazinsky et al. Proc Natl Acad Sci USA, 105:20167-20172 (2008)) according to the EU numbering convention to eliminate complement activation. In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise one or more of A330L, A330S, L234F, L235E, and / or P331S according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise one or more mutations to enhance antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S254T, and T256E mutations according to the EU numbering convention). In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise one or more of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and / or S440W according to EU numbering.

[0345] Other aspects of the present invention relate to antibodies with modified constant regions (i.e., Fc regions). Antibodies that rely on binding to FcgR receptors to activate their targeted receptors lose their potentiator activity if 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)). Therefore, it is believed that when anti-SIRPA antibodies of the present invention with appropriate epitope specificity have an Fc domain (CH1 and hinge region) derived from the human IgG2 isotype or another type of Fc domain or variant thereof that preferentially binds to the inhibitory FcgRIIB r receptor, the antibodies can activate the target antigen with minimal side effects.

[0346] In some embodiments of any of the antibodies provided herein, the modified antibody Fc is an IgG2 modified Fc. In some embodiments, the IgG2 modified Fc comprises one or more modifications. For example, in some embodiments, the IgG2 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments of any of the IgG2 modified Fc, the one or more amino acid substitutions are selected from V234A according to the EU numbering convention (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. The Haematology Journal 1 (Suppl 1): 27 (2000); Armour et al. The 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 IgG2 modified Fcs, the Fc comprises an amino acid substitution at position V234A and G237A according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises an amino acid substitution at position C219S or C220S according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises an amino acid substitution at position A330S and P331S according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering.

[0347] In some embodiments of any of the IgG2 modified Fcs, 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 WO 2008 / 079246). In some embodiments of any of the IgG2 modified Fcs, the antibody has an IgG2 isotype, wherein the kappa light chain constant domain comprises 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 WO 2008 / 079246).

[0348] In some embodiments of any of the IgG2 modified Fcs, the Fc comprises a C220S amino acid substitution according to the EU numbering convention. In some embodiments of any of the IgG2 modified Fcs, the antibody is of an IgG2 isotype wherein the kappa light chain constant domain comprises a C214S amino acid substitution according to the EU numbering convention.

[0349] In some embodiments of any of the IgG2 modified Fcs, the Fc comprises a C219S amino acid substitution according to the EU numbering convention. In some embodiments of any of the IgG2 modified Fcs, the antibody is of an IgG2 isotype wherein the kappa light chain constant domain comprises a C214S amino acid substitution according to the EU numbering convention.

[0350] In some embodiments of any of the IgG2 modified Fcs, the Fc comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and a hinge region (White et al. Cancer Cell 27:138-148 (2015)). In certain embodiments of any of the IgG2 modified Fcs, the IgG2 isotype CH1 and hinge region comprise an amino acid sequence according to EU numbering 118-230. In some embodiments of any of the IgG2 modified Fcs, the antibody Fc region comprises an S267E amino acid substitution, an L328F amino acid substitution, or both, and / or an N297A or N297Q amino acid substitution according to the EU numbering convention.

[0351] In some embodiments of any of the IgG2 modified Fcs, 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 IgG2 modified Fcs, the Fc may further comprise one or more mutations to enhance antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S254T, and T256E mutations according to EU numbering convention). In some embodiments of any of the IgG2 modified Fcs, the Fc may further comprise A330S and P331S.

[0352] In some embodiments of any of the IgG2 modified Fcs, the Fc is an IgG2 / 4 hybrid Fc. In some embodiments, the IgG2 / 4 hybrid Fc comprises IgG2 aa 118 to 260 and IgG4 aa 261 to 447. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises one or more amino acid substitutions at positions H268Q, V309L, A330S, and P331S according to EU numbering.

[0353] In some embodiments of either of the IgG1 and / or IgG2 modified Fc, 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.

[0354] In certain embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises one or more amino acid substitutions at a residue position 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 IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, A330S, and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, K322A, and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions C127S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E345R, E430G, and S440Y according to EU numbering.

[0355] In some embodiments of any one of the antibodies provided herein, the modified antibody Fc is an IgG4 modified Fc. In some embodiments, the IgG4 modified Fc comprises one or more modifications. For example, in some embodiments, the IgG4 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments of any one of the IgG4 modified Fc, 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 one of the IgG4 modified Fc, Fc may further comprise L235A, G237A, and E318A according to the EU numbering convention. In some embodiments of any of the IgG4 modified Fcs, the Fc may further comprise S228P and L235E according to the EU numbering convention. In some embodiments of any of the IgG4 modified Fcs, the IgG4 modified Fc may further comprise S267E and L328F according to the EU numbering convention.

[0356] In some embodiments of any of the IgG4 modified Fcs, the IgG4 modified Fc comprises an S228P mutation according to the EU numbering convention (Angal et al. Mol Immunol. 30: 105-108 (1993)) and / or with one or more mutations described in (Peters et al. J Biol Chem. 287(29): 24525-33 (2012)) to enhance antibody stabilization.

[0357] In some embodiments of any of the IgG4 modified Fcs, the IgG4 modified Fc may further comprise one or more mutations to enhance antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S254T, and T256E mutations according to the EU numbering convention).

[0358] In some embodiments of any of the IgG4 modified Fcs, the Fc comprises L235E according to EU numbering. In certain embodiments of any of the IgG4 modified Fcs, the Fc comprises one or more amino acid substitutions at a residue position 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 IgG4 modified 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 IgG4 modified Fcs, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises an amino acid substitution at position E430 according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc region comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises amino acid substitutions at positions E345R, E430G, and S440Y according to EU numbering.

[0359] Other antibody modifications

[0360] In some embodiments of any of the antibodies, the antibody is a derivative. The term "derivative" refers to a molecule that is chemically modified except for the insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, the derivative comprises a covalent modification, including but not limited to chemical bonding to a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, the chemically modified antigen-binding protein may have a greater circulation half-life than an antigen-binding protein that is not chemically modified. In certain embodiments, the chemically modified antigen-binding protein may have improved targeting capabilities for desired cells, tissues, and / or organs. In some embodiments, the derived antigen-binding protein is covalently modified to include one or more water-soluble polymers, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patents No. 4,640,835, No. 4,496,689, No. 4,301,144, No. 4,670,417, No. 4,791,192, and No. 4,179,337. In certain embodiments, the derivatized antigen binding protein comprises one or more polymers including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol, and mixtures of such polymers.

[0361] In certain embodiments, the derivatives are covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are bonded to one or more specific positions of the derivatives, such as the amino terminus. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivatives. In certain embodiments, PEG is used to improve the therapeutic ability of antigen-binding proteins. In certain embodiments, PEG is used to improve the therapeutic ability of humanized antibodies. Some such methods are discussed, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0362] Peptide analogs are commonly used in the medical industry as non-peptide drugs, and their properties are 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. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or preventive effects. Typically, a peptide mimetic is structurally similar to an exemplary polypeptide (i.e., a polypeptide having biochemical properties or pharmacological activity), such as a human antibody, but has one or more peptide linkages, optionally replaced by linkages selected from the group consisting of: -CH2NH-, -CH2S-, -CH2=CH2-, -CH-CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, 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 instead of L-lysine) can be used to generate more stable peptides. Additionally, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variant 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 forming intramolecular disulfide bridges that cyclize the peptide.

[0363] Drug conjugation involves the coupling of a bioactive cytotoxic (anticancer) payload or drug to an antibody that specifically targets certain tumor markers (e.g., ideally, a polypeptide found only in or on tumor cells). The antibody tracks these proteins throughout the body and attaches itself to the surface of the cancer cell. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then takes up or internalizes the antibody and the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cancer. Because of this targeting, the drug ideally has fewer side effects than other chemotherapeutic agents and provides a wider therapeutic window. The technology for conjugating antibodies is disclosed or known in the art (see, for example, Jane de Lartigue OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al. Bioconjugate Chemistry 21(1):5-13 (2010)).

[0364] Antibody framework

[0365] 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 HVRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human common framework. A human immunoglobulin framework can be part of a human antibody, or a non-human antibody can be humanized by replacing one or more endogenous frameworks with human framework regions. Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from 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)). 993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening 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)).

[0366] In some embodiments, the anti-SIRPA antibodies of the invention comprise 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 (as shown in Table 9). In some embodiments, the anti-SIRPA antibodies of the invention comprise a heavy chain variable region comprising VH FR1, wherein VH FR1 comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-SIRPA antibodies of the invention comprise a heavy chain variable region comprising VH FR2, wherein VH FR2 comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-SIRPA antibodies of the invention comprise a heavy chain variable region comprising VH FR3, wherein VH FR3 comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-SIRPA antibodies of the invention comprise a heavy chain variable region comprising VH FR4, wherein VH FR4 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, an anti-SIRPA antibody of the invention 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.

[0367] In some embodiments, an anti-SIRPA antibody of the invention comprises a heavy chain variable region comprising 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.

[0368] In some embodiments, the anti-SIRPA antibodies of the invention comprise 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 (as shown in Table 9). In some embodiments, the anti-SIRPA antibodies of the invention comprise a light chain variable region comprising VL FR1, wherein VL FR1 comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-SIRPA antibodies of the invention comprise a light chain variable region comprising VL FR2, wherein VL FR2 comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-SIRPA antibodies of the invention comprise a light chain variable region comprising VL FR3, wherein VL FR3 comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-SIRPA antibodies of the invention comprise a light chain variable region comprising VL FR4, wherein VL FR4 comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-SIRPA antibody of the invention 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.

[0369] In some embodiments, an anti-SIRPA antibody of the invention comprises a light chain variable region comprising 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.

[0370] In some embodiments, an anti-SIRPA antibody of the invention 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 VLFR2 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 VLFR4 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-SIRPA antibody of the invention comprises a heavy chain variable region comprising 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 VLFR1, 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.

[0371] In some embodiments, the present invention provides an 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: 47 and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0372] In some embodiments, the present invention provides an 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.

[0373] In some embodiments, the present invention provides an 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.

[0374] In some embodiments, the present invention provides an 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.

[0375] In some embodiments, the present invention provides an 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.

[0376] Nucleic acids, vectors and host cells

[0377] The anti-SIRPA antibodies of the present invention can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids having a nucleotide sequence encoding any of the anti-SIRPA antibodies of the present invention are provided. Such nucleic acids can encode V sequences comprising the anti-SIRPA antibodies. L The amino acid sequence and / or the V H In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In some embodiments, host cells comprising such nucleic acids are also provided. In some embodiments, the host cells comprise (e.g., have been transduced with): (1) a vector comprising a V encoding an antibody; L The amino acid sequence of the antibody V H or (2) a vector comprising a nucleic acid encoding an antibody V L The first vector of the nucleic acid containing the amino acid sequence of the antibody V H 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). The host cells of the present invention also include (but are not limited to) isolated cells, cells cultured in vitro, and cells cultured in vitro.

[0378] Methods for producing anti-SIRPA antibodies of the present invention are provided. In some embodiments, the method comprises culturing a host cell of the present invention comprising a nucleic acid encoding an anti-SIRPA antibody under conditions suitable for expressing the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0379] For recombinant production of the anti-SIRPA antibodies of the present invention, 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 that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0380] Suitable vectors comprising a nucleic acid sequence encoding any of the anti-SIRPA antibodies or cell surface expressed fragments or polypeptides (including antibodies) of the present invention as described herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the cloning vector of choice can vary depending on the host cell to be used, suitable cloning vectors are generally capable of self-replication, may have a single target for a specific restriction endonuclease, and / or may carry a gene for a marker that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are commercially available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0381] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, the anti-SIRPA antibodies of the present invention can be produced in bacteria, particularly when glycosylation and Fc effector functions are not desired. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. Following expression, the antibodies can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0382] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns (e.g., Gerngross Nat. Biotech. 22:1409-1414 (2004); and Li et al. Nat. Biotech. 24:210-215 (2006)).

[0383] Host cells suitable for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (e.g., U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, describing plant cells for producing antibodies in transgenic plants). TM technology).

[0384] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be suitable. Other examples of suitable mammalian host cell lines are monkey kidney CV1 strain (COS-7) transformed with SV40; human embryonic kidney strain (293 or 293 cells, as described, for example, in Graham et al. J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, 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 liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT) 060562); TRI cells, as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other suitable 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 producing antibodies, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0385] Pharmaceutical compositions / formulations

[0386] Provided herein are pharmaceutical compositions and / or pharmaceutical formulations comprising an anti-SIRPA antibody of the invention and a pharmaceutically acceptable carrier.

[0387] In some embodiments, pharmaceutically acceptable carriers are preferably non-toxic to the recipient at the dosage and concentration used. The antibodies described herein can be formulated into preparations in solid, semi-solid, liquid or gaseous forms. 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 desired formulation, pharmaceutically acceptable carriers may include pharmaceutically acceptable non-toxic carriers of diluents, which are commonly used in the formulation of pharmaceutical compositions for animal or human administration. In certain embodiments, the pharmaceutical composition may include a formulation material for modifying, maintaining or maintaining, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition.

[0388] In certain embodiments, pharmaceutically acceptable carriers include, but are not limited to, 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 borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-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; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride). the pharmaceutical composition may also include, but is not limited to, hydroxybenzoic acid, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, 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, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. Other examples of formulations suitable for various types of administration can be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press 22nd ed. (2013). For a brief review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0389] Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

[0390] The formulation can be optimized for retention and stabilization in the brain or central nervous system. When the agent is administered to the cranial compartment, it is desirable to retain the agent in that compartment and not diffuse or otherwise cross the blood-brain barrier. Stabilization techniques include cross-linking, polymerizing, or attaching to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. to achieve an increase in molecular weight.

[0391] Other strategies for increasing retention include embedding antibodies, such as the anti-SIRPA antibodies of the present invention, in biodegradable or bioerodible implants. The rate of release of the therapeutic agent is controlled by the rate of transport through the polymer matrix and the rate of biodegradation of the implant. The implant can be a particle, sheet, patch, plaque, fiber, microcapsule, etc. and can have any size or shape compatible with the selected insertion site. The biodegradable polymer composition that can be used can be an organic ester or ether that produces physiologically acceptable degradation products upon degradation, including monomers. Anhydrides, amides, orthoesters, etc. can be used alone or in combination with other monomers. The polymer will be a condensation polymer. The polymer can be cross-linked or non-cross-linked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids, either homopolymers or copolymers, and polysaccharides. Among the polyesters of interest are polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof. Polysaccharides of interest include calcium alginate and functionalized cellulose, particularly carboxymethyl cellulose esters, which are characterized by being water-insoluble and having a molecular weight of about 5 kD to 500 kD. Biodegradable hydrogels may also be used in the implants of the present invention. Hydrogels are typically copolymer materials characterized by their ability to absorb liquids.

[0392] Therapeutic uses

[0393] As disclosed herein, the anti-SIRPA antibodies of the invention can be used to prevent, reduce the risk of, or treat diseases and disorders.

[0394] In one aspect of the invention, agents that downregulate SIRPA, such as anti-SIRPA antibodies, are used as therapeutic agents. Such agents are administered to treat, alleviate, and / or prevent a disease or pathology associated with SIRPA expression, activity, and / or signaling in a subject. The treatment regimen is performed by identifying a subject (e.g., a human patient) having (or at risk of developing) a disease or condition associated with SIRPA expression, activity, and / or signaling (e.g., cancer or other neoplastic disease) using standard methods. In some embodiments, cells having a pathology associated with SIRPA expression, activity, and / or signaling express a SIRPA ligand, such as CD47. In some embodiments, cells having a pathology associated with SIRPA expression, activity, and / or signaling express SIRPA.

[0395] As described in further detail below, agents that downregulate SIRPA, such as anti-SIRPA antibodies, can be used in combination with additional therapeutic agents for treating diseases or pathologies associated with SIRPA expression, activity, or signaling. The terms "combination" and "in conjunction with" are used interchangeably herein. The additional therapeutic agent administered in combination with the anti-SIRPA antibody can be administered before, after, or simultaneously with the agent that downregulates SIRPA, such as the anti-SIRPA antibody.

[0396] In one aspect of the present invention, an anti-SIRPA antibody formulation is administered to a human subject, for example 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 (e.g., CD47) to SIRPA. Administration of such an antibody can eliminate, inhibit, or interfere with the expression, activity, and / or signaling function of SIRPA mediated by ligand binding (e.g., CD47 binding).

[0397] In one embodiment, the disease or disorder associated with SIRPA expression is cancer. In some embodiments, an anti-SIRPA antibody is administered to a patient with a cancer that expresses SIRPA, such as a hematological proliferative disorder of myeloid cells. In a typical embodiment, an anti-SIRPA antibody is administered to a patient with a cancer that expresses CD47.

[0398] In certain embodiments, cancers to be prevented or treated by the methods of the present invention 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 carcinoma, non-squamous NSCLC, glioma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer / stomach cancer, cancer), including gastrointestinal and gastrointestinal stromal cancers, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer (or carcinomas), stomach cancer, germ cell tumors, pediatric sarcomas, sinonasal natural killers, melanoma (e.g., metastatic malignant melanoma, (e.g., malignant melanoma of the skin or eye), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, myelodysplastic syndromes, colorectal neoplasms, solid tumors, tumor angiogenesis, spinal cord tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma; environmentally induced cancers, including cancers induced by asbestos; virus-related cancers (e.g., human papillomavirus (HPV)-related tumors); and hematologic malignancies derived from either of the two major blood cell lineages, the myeloid lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias. diseases such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma;Lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), indolent lymphoma, large B-cell diffuse lymphoma, B-cell hematologic malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, polymorphic (e.g., Ki 1+) Large Cell Lymphoma, Adult T-Cell Lymphoma / Leukemia, Mantle Cell Lymphoma, Angioimmunoblastic T-Cell Lymphoma, Angiocentric Lymphoma, Intestinal T-Cell Lymphoma, Primary Mediastinal B-Cell Lymphoma, Precursor T-Lymphoblastic Lymphoma, T-Lymphoblastic; and Lymphoma / Leukemia (T-Lbly / T-ALL), Peripheral T-Cell Lymphoma, Lymphoblastic Lymphoma, Post-Transplant Lymphoproliferative Disorder, True Histiocytic Lymphoma, Primary Central Nervous System Lymphoma, Primary Effusion Lymphoma, Lymphoblastic Lymphoma The patients were selected for the treatment of patients with primary refractory DLBCL, rituximab-refractory DLBCL, rituximab / CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-refractory DLBCL, relapsed or refractory (R / R) DLBCL, and / or CAR-T-ineligible DLBCL, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary ...

Claims

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, which is set forth in the amino acid sequence of SEQ ID NO:20; HVR-H2, which is set forth in the amino acid sequence of SEQ ID NO:21; HVR-H3, which is set forth in the amino acid sequence selected from SEQ ID NO:22, 23 and 24; and the light chain variable region comprises: HVR-L1, which is set forth in the amino acid sequence of SEQ ID NO:9; HVR-L2, which is set forth in the amino acid sequence of SEQ ID NO:10; and HVR-L3, which is set forth in the amino acid sequence selected from SEQ ID NO:11, 12, 13, 14, 15, 16, 17, 18 and 19.

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

28.

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

32.

4. The antibody of claim 1, wherein the heavy chain variable region comprises one, two, three or four framework regions selected from VH FR1 as shown in the amino acid sequence of SEQ ID NO:25, VH FR2 as shown in the amino acid sequence of SEQ ID NO:26, VH FR3 as shown in the amino acid sequence of SEQ ID NO:27, and VH FR4 as shown in the amino acid sequence of SEQ ID NO:28; and wherein the light chain variable region comprises one, two, three or four framework regions selected from VLFR1 as shown in the amino acid sequence of SEQ ID NO:29, VL FR2 as shown in the amino acid sequence of SEQ ID NO:30, VLFR3 as shown in the amino acid sequence of SEQ ID NO:31, and VL FR4 as shown in 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% identical to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35, and wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

6. The antibody of claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35, and wherein the light chain variable region comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

7. The antibody of claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 33, 34, and 35, and wherein the light chain variable region comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

8. The antibody of claim 1, wherein (1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 36, (2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 36, (3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 36, (4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 37, (5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38, (6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38, (7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38, (8) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, (9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, (10) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, (11) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40, (12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40, (13) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40, (14) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 41, (15) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 41, (16) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 41, (17) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42, (18) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42, (19) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42, (20) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 43, (21) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 43, (22) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 43, (23) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 44, (24) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 44, or (25) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

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 is represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, and 35, and wherein the light chain variable region is represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

10. The antibody of any one of claims 1 to 9, wherein the antibody is a monoclonal antibody.

11. The antibody of any one of claims 1 to 9, wherein the antibody is a humanized antibody.

12. The antibody of any one of claims 1 to 9, wherein the antibody is a Fab, Fab', Fab'-SH, F(ab')2, Fv or scFv fragment.

13. The antibody of any one of claims 1 to 9, wherein the antibody is a multivalent antibody.

14. The antibody of any one of claims 1 to 9, wherein the antibody is of the IgG class, the IgM class or the IgA class.

15. The antibody of claim 14, wherein the antibody is of the IgG class and is of the IgG1, IgG2, IgG3 or IgG4 isotype.

16. The antibody of any one of claims 1 to 9 and 15, wherein the antibody binds to an inhibitory Fc receptor.

17. The antibody of claim 16, wherein the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcgRIIB).

18. The antibody of claim 17, wherein the antibody reduces cellular levels of FcgRIIB.

19. The antibody of any one of claims 1 to 9, 15 and 17, wherein the antibody has a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region selected from the group consisting of: N297A, D265A, D270A, L234A, L235A, G237A, P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E23 3P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof, wherein the numbering of positions is according to EU numbering, or comprises a deletion of an amino acid at the position corresponding to glycine 236 in the Fc region.

20. The antibody of any one of claims 1 to 9, 15 and 17, wherein the antibody has a human IgG1 isotype and comprises one or more amino acid substitutions 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 positions is according to EU or Kabat numbering.

21. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:47 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

22. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:48 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

23. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:49 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

24. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:53 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

25. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:54 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

26. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:51 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

27. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:52 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

28. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:55 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

29. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:56 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

30. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:57 and the light chain has the amino acid sequence shown in SEQ ID NO:

50.

31. Use of an antibody according to any one of claims 1 to 30 for the preparation of a medicament for treating breast cancer, colon cancer, rectal cancer, kidney cancer, leukemia, lung cancer, melanoma, lymphoma, pancreatic cancer, ovarian cancer, esophageal cancer or mesothelioma in an individual.

32. Use of an antibody according to any one of claims 1 to 30 for the preparation of a medicament for treating non-small cell lung cancer, pancreatic adenocarcinoma, renal cell carcinoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), myelodysplastic syndrome, head and neck squamous cell carcinoma or hepatocellular carcinoma in an individual.

33. The use of claim 31 or 32, wherein a therapeutic agent is administered to the individual that inhibits or enhances 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.

34. The use of claim 31 or 32, wherein at least one antibody that specifically binds to an inhibitory checkpoint molecule and / or one or more standard or investigational anti-cancer therapies are administered to the individual.

35. The use of claim 34, wherein at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered to the individual.

36. The use of claim 35, wherein the at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-PD-L2 antibodies, anti-PD-1 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies and anti-HVEM antibodies, anti-B- and T-lymphocyte attenuating factor (BTLA) antibodies, anti-killer cell inhibitory receptor (KIR) antibodies, anti-GAL9 antibodies, anti-TIM-1 antibodies, anti-TIM3 antibodies, anti-TIM-4 antibodies, anti-A2AR antibodies , anti-CD39 antibodies, anti-CD73 antibodies, anti-LAG-3 antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-CD30 antibodies, anti-TNFα antibodies, anti-CD33 antibodies, anti-Siglec-5 antibodies, anti-Siglec-7 antibodies, anti-Siglec-9 antibodies, anti-Siglec-11 antibodies, antagonist anti-TREM1 antibodies, antagonist anti-TREM2 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-CD2 antibodies, anti-CD5 antibodies, and any combination thereof.

37. The use of claim 36, 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.

38. The use of claim 31 or 32, wherein at least one antibody that specifically binds to an inhibitory cytokine is administered to the individual.

39. The use of claim 38, wherein the at least one antibody that specifically binds to an inhibitory cytokine is selected from the group consisting of an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof.

40. The use of claim 31 or 32, wherein at least one potentiating antibody that specifically binds to a stimulatory checkpoint protein is administered to the individual.

41. The use of claim 40, wherein the at least one enhancing antibody that specifically binds to a stimulatory checkpoint protein is selected from an enhancing anti-CD40 antibody, an enhancing anti-OX40 antibody, an enhancing anti-ICOS antibody, an enhancing anti-CD28 antibody, an enhancing anti-TREM1 antibody, an enhancing anti-TREM2 antibody, an enhancing anti-CD137 / 4-1BB antibody, an enhancing anti-CD27 antibody, an enhancing anti-glucocorticoid-induced TNFR-related protein GITR antibody, an enhancing anti-CD30 antibody, an enhancing anti-BTLA antibody, an enhancing anti-HVEM antibody, an enhancing anti-CD2 antibody, an enhancing anti-CD5 antibody, and any combination thereof.

42. The use of claim 31 or 32, wherein at least one stimulatory cytokine is administered to the individual.

43. The use of claim 42, wherein 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-β, and any combination thereof.

44. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody of any one of claims 1 to 30.

45. A vector comprising the nucleic acid of claim 44.

46. ​​An isolated host cell comprising the nucleic acid of claim 44 or the vector of claim 45.

47. An isolated host cell expressing the antibody of any one of claims 1 to 30.

48. A method of producing an antibody that binds to human SIRPA, comprising culturing the cell of claim 46 or 47 such that the antibody is produced.

49. The method of claim 48, further comprising recovering antibodies produced by the cells.

50. A pharmaceutical composition comprising the antibody of any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

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