Anti-SIRPA antibodies and methods of use thereof

Abstract

The present disclosure generally relates to compositions comprising antibodies (e.g., monoclonal antibodies, antibody fragments, etc.) that specifically bind to a SIRPA polypeptide (e.g., 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 is a divisional application of an invention application with a filing date of May 24, 2019, a Chinese application number of 201980034624.6, and an invention name of “Anti-SIRPA Antibodies and Methods of Using Them”. This application claims priority to U.S. Provisional Application No. 62 / 676,813 filed on 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

[0006] 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 cell activation state, proliferation and / or effector function. Many of these receptors recognize different ligands that mark undesirable 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 can meet the relevant medical needs of improving diseases that have specific relevance in many types of human cancers.

[0007] 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 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, tyrosine-phosphorylated ITIM sites recruit and activate SHP phosphatases to negatively regulate cell functions, such as phagocytosis or inflammatory cytokine release. CD47 serves as the main ligand of 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 strains 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.

[0008] Regulated expression of SIRPα and CD47 establishes a homeostatic control mechanism to regulate phagocytic activity. For example, apoptotic cells downregulate the expression of CD47 to promote phagocytosis of resident macrophages, while live 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 immune-competent mice. Similar results were observed in human cancer cell lines with CD47 knockdown transplanted into immunocompromised mice. Alternatively, biological agents that destroy 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 run the risk of a severe 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.

[0009] Anti-SIRPA antibodies have been previously described, for example, in the following international patent application publications: WO2018 / 057669, WO2018 / 026600, WO2017 / 178653, WO2017 / 068164, WO2016 / 063233, WO2016 / 205042, WO2015 / 138600, WO2013 / 0956352, WO2009 / 091547, WO2009 / 131453, and WO2009 / 046541.

[0010] Therefore, there is a need for therapeutic anti-SIRPA antibodies to treat diseases, disorders and conditions associated with inappropriate SIRPA activity.

[0011] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0012] The present invention generally relates to compositions comprising antibodies, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, etc., that specifically bind human SIRPA, and methods of using such compositions.

[0013] 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, 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.

[0014] In some embodiments, the anti-SIRPA antibodies of the invention reduce the 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 the 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.

[0015] 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 antibodies of the invention bind marmoset SIRPA.

[0016] 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.

[0017] 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 SEQ ID NOs: 22, 23, and 24.

[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 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; HVR-L2 comprising the amino acid sequence of SEQ ID NO:10; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs:11, 12, 13, 14, 15, 16, 17, 18, and 19.

[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; 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 33, 34, and 35.

[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 light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[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, 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.

[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 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).

[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 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).

[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 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. 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).

[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 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).

[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 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.

[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 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.

[0034] 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.

[0035] 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.

[0036] 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.

[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:53 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:54 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:51 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:52 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:55 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:56 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:57 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 antibody that binds to human SIRPA, wherein the antibody is a monoclonal antibody.

[0045] 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.

[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 Fab, Fab', Fab'-SH, F(ab') 2 , Fv or scFv fragments.

[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 multivalent 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 of the IgG class, the IgM class, or the IgA class.

[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 of the IgG class and is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[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 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).

[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 reduces cellular levels of FcγRIIB.

[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 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 the EU numbering, or comprises a deletion of an amino acid at a position corresponding to glycine 236 in the Fc region.

[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 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 the amino acid residues is according to EU numbering.

[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 reduces cell surface levels of SIRPA, reduces intracellular levels of SIRPA, reduces total cellular levels of SIRPA, or any combination thereof.

[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 anti-SIRPA antibody induces SIRPA degradation, induces SIRPA cleavage, induces SIRPA internalization, induces SIRPA shedding, induces downregulation of SIRPA expression, or any combination thereof.

[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 antibody reduces cell surface levels of SIRPA in vitro.

[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 antibody reduces cell surface levels of SIRPA in vivo.

[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 anti-SIRPA antibody downregulates expression of SIRPA in human monocytes.

[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 anti-SIRPA antibody downregulates expression of SIRPA in human macrophages.

[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 macrophages by about 70-95%.

[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 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.

[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 antibody has an affinity (KD) for human SIRPA of about 0.1 nM to 2 nM.

[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 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.

[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 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.

[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 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.

[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 binds to the D3 domain of human SIRPA v1 of SEQ ID NO:1.

[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 binds to amino acid residues R282, Q284, and G337 of human SIRPA v1 of SEQ ID NO:1.

[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 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.

[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 anti-SIRPA antibody enhances T cell proliferation.

[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 blocks the interaction or binding of SIRPA to surfactant protein D (SP-D).

[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 does not block the interaction or binding of SIRPA to CD47.

[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 downregulates cell surface expression of CD32A / B.

[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 downregulates cell surface expression of CD14.

[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 enhances T cell proliferation without blocking the interaction of SIRPγ and CD47.

[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 stimulates ROS production in monocytes and / or increases IL-8 expression in monocytes.

[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 inhibits tumor growth in vivo.

[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 reduces the number of CD14+ myeloid cells in peripheral blood and / or increases the number of CD14+ myeloid cells in tumors.

[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 antibody binds to human SIRPA but does not substantially block the binding of CD47 to SIRPA.

[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 antibody recognizes a first antigen and a second antigen, wherein the first antigen is SIRPA and the second antigen is:

[0080] (a) Antigens that promote transport across the blood-brain barrier;

[0081] (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;

[0082] (c) a pathogen selected from a pathogenic peptide or a pathogenic protein, a pathogenic nucleic acid, wherein the pathogenic nucleic acid is an antisense GGCCCC (G2C4) repeat sequence expansion RNA, and the pathogenic protein is selected from amyloid β, oligomeric amyloid β, amyloid β plaques, amyloid precursor protein or its fragment, 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 (Lewy body) body), 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 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

[0083] (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.

[0084] 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.

[0085] 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.

[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 a conjugated antibody.

[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 conjugated to a detectable label, toxin, or therapeutic agent.

[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 conjugated to a toxin selected from the group consisting of ricin, ricin A chain, doxorubicin, daunorubicin, maytansinoids, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, exotoxin, PE A, PE40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoids, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, cc1065 and cisplatin.

[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 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 protein, depressor protein, leukocyte antigen, leukocyte antigen, thymocyte antigen, leukocyte antigen, thymocyte antigen, thymocyte antigen 2, leukocyte antigen 3, leukocyte antigen 4, leukocyte antigen 5, leukocyte antigen 6, leukocyte antigen 7, thymocyte antigen 8, leukocyte antigen 9, thymocyte antigen 20, thymocyte antigen 12, thymocyte antigen 13, thymocyte antigen 14, thymocyte antigen 15, thymocyte antigen 16, thymocyte antigen 17, thymocyte antigen 18, thymocyte antigen 22, thymocyte antigen 23, thymocyte antigen 24, thymocyte antigen 25, thymocyte antigen 27, thymocyte antigen 28, thymocyte antigen 29, thymocyte antigen 30, thymocyte antigen 31, thymocyte antigen 32, thymocyte antigen 33, thymocyte antigen 34, thymocyte antigen 35, thymocyte antigen 36, thymocyte 37, thymocyte 38, thymocyte 39, thymocyte 39, thymocyte 39 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 expansion RNA, and any combination thereof.

[0090] 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.

[0091] 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; bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma; pancreatic adenocarcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, indolent B-cell lymphoma, aggressive B-cell lymphoma, T-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL), leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, invasive carcinoma of the breast, squamous cell carcinoma of the cervix, endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, squamous cell carcinoma of the head and neck, refractory renal cell, renal papillary cell carcinoma, lower grade glioma, hepatocellular carcinoma, squamous cell carcinoma of the lung, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

[0092] 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.

[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, the method comprising administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule and / or one or more standard or investigational anti-cancer therapies. In some embodiments that may be combined with any of the embodiments herein, at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with an anti-SIRPA antibody. In some embodiments that may be combined with any of the embodiments herein, the at least one antibody that specifically binds to an inhibitory checkpoint molecule is 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.

[0094] 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.

[0095] 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 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.

[0096] 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 an anti-SIRPA antibody. In some embodiments that may be combined with any of the embodiments herein, 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.

[0097] 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 to the individual at least one stimulatory cytokine. In some embodiments, the at least one stimulatory cytokine is selected from IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-β, and any combination thereof.

[0098] 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.

[0099] In some embodiments, the present invention provides a method of making an antibody that binds to human SIRPA, wherein the method comprises culturing a host cell comprising a vector so that the antibody is produced, the vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-SIRPA antibody of the present invention. The present invention further provides for recovering the antibody produced by the cell.

[0100] 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

[0101] 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 recorded percent identity demonstrates high homology within the extracellular region. Accession numbers are P78324 (human SIRPA variant 1), XP015313153 (cynomolgus monkey), JAB51896 (marmoset), G1U0I5 (rabbit), and XP005634938 (dog). Figure 1 SEQ ID NOs 59-63 are disclosed respectively in the order of appearance.

[0102] 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 in order of appearance, respectively. 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 in order of appearance, respectively.

[0103] Figure 3 Data are presented showing antibody-mediated downregulation of SIRPA by murine and humanized anti-SIRPA 3F9 antibodies on primary human macrophages.

[0104] 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.

[0105] Figure 5A Data are presented showing that humanized anti-SIRPA 3F9 IgG4 antibody variants fail to induce tumor cell phagocytosis. Figure 5B Data are presented showing that humanized anti-SIRPA 3F9 IgG4 antibody variants fail to downregulate CD14 expression on macrophages.

[0106] Fig. 6A It was shown that anti-SIRPA antibody 3F9mIgG1 treatment downregulated CD32A / B expression on primary human macrophages. Figure 6B It was shown that anti-SIRPA antibody 3F9 treatment downregulated CD32A and CD32B expression on primary human macrophages.

[0107] Fig. 7A Data are presented 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.

[0108] FIG. 8A to FIG. 8B Data are presented showing that CD32A allotype affects SIRPA downregulation mediated by the anti-SIRPA antibody 3F9. Fig. 8A Donors 507 and 508 were shown to be heterozygous for CD32A-R / H131; Figure 8BDonors 516 and 517 were shown to be homozygous for CD32A-H / H131.

[0109] FIG. 9A to FIG. 9B Describe data showing that antibody-mediated downregulation of the receptor leads to degradation of SIRPA. Fig.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). Fig. 9B 20 μg of whole cell lysate was loaded on SDS-PAGE and probed with a-SIRPAct and anti-SHP2.

[0110] FIG. 10A to FIG. 10B Data are presented comparing the enhancement of phagocytic activity of different Fc variants of the anti-SIRPA antibody 3F9-22. Fig. 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. Fig. 10B Primary human macrophages were treated with anti-SIRPA 3F9-22 antibody on a human IgG1 backbone with S267E / L328F or A330S / P331S Fc mutations or an isotype control antibody.

[0111] FIG. 11A to FIG. 11B Data are presented comparing different Fc variants of the anti-SIRPA 3F9-22 antibody relative to anti-CD47 in M1 ( Fig. 11B ) and M2( Fig.11A ) Phagocytic activity on macrophages.

[0112] Fig. 12A and Fig. 12B Data are presented showing that macrophages treated with the anti-SIRPA antibody 3F9-22 reduced solid tumor cell viability in vitro.

[0113] Fig.13A Data are presented comparing different Fc variants of the anti-SIRPA antibody 3F9-22 for T cell proliferation enhancement in a two-way MLR assay. Fig. 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.

[0114] Fig.14AData 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. Fig. 14B Representative FACS plots of one-way MLR analysis are shown.

[0115] Fig.15A and Fig. 15B Data are presented comparing different Fc variants of the anti-SIRPA antibody 3F9-22 for enhancement of TNF release by LPS-primed macrophages.

[0116] Fig.16 Data are presented showing the identification and observation of "critical" residues of anti-SIRPA antibodies in the human SIRPA model.

[0117] FIG. 17A to FIG. 17C Data are presented showing the effect of the combination of anti-SIRPA antibody 3F9-22 and anti-PD-L1 antibody on tumor growth inhibition in a syngeneic mouse colon cancer model. Fig.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. Fig. 17B Depicted are data showing mean tumor growth curves of huSIRPA / huCD47BAC transgenic mice implanted subcutaneously with MC38 cells engineered to lack mouse CD47 expression and to overexpress human CD47 (MC38-mCD47KO / huCD47+). Fig. 17C Explanation Fig. 17B Spaghetti plot of each individual animal in each treatment group.

[0118] FIG. 18A to FIG. 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 disclosure. Fig.18A Depicted are data showing relative changes in SIRPA expression on tumor-infiltrating myeloid cells over time. Fig.18B Depicted are data showing relative changes in SIRPA expression on splenic myeloid cells over time.

[0119] Fig.19A Treatment conditions showing anti-SIRPA antibody-mediated downregulation of SIRPA studied in humanized NOG-EXL mice bearing A375 tumors. Fig.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 graph) or normalized values ​​(right graph).

[0120] FIG. 20A to FIG. 20B Displays M1 macrophage markers HLA-DR / MHC class 2 ( Fig. 20A ) and CD86( Fig. 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. Fig. 20A and Fig. 20B Data are illustrated showing the expression of HLA-DR / MHC class 2 and CD86, respectively, as MFI values ​​(left graph) or normalized values ​​(right graph).

[0121] FIG. 21A to FIG. 21B Splenic mononuclear cells isolated from humanized NOG-EXL mice bearing A375 tumors are shown. Fig.21A ) and peripheral blood mononuclear cells ( Fig.21B ) in association with administration of the anti-SIRPA antibodies of the invention. Fig.21A and Fig.21B Data are illustrated showing the expression of human SIRPA as MFI values ​​(left panel) or normalized values ​​(right panel).

[0122] Fig. 22 Data are presented showing antibody-mediated downregulation of SIRPA by different Fc variants of the anti-SIRPA 3F9-22 antibody on primary human macrophages.

[0123] Fig.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.

[0124] Fig.24 Described are data showing red blood cell and platelet counts in cynomolgus monkeys administered with a control IgG antibody, anti-SIRPA antibody 3F9-22PS, anti-SIRPA antibody 3F9-22IgG4, anti-SIRPA antibody 3F9-IgG2, and anti-SIRPA antibody 3F9-22NSLF.

[0125] 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

[0126] 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.

[0127] 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 the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (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: APractical 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: ALaboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., JS Lippincott Company, 1993).

[0128] definition

[0129] Unless otherwise indicated, the terms "SIRPα" or "SIRPα polypeptide" or "SIRPA" or "SIRPA polypeptide" are used interchangeably herein and refer herein to any native SIRPA from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys) 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, 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.

[0130] 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 that binds to SIRPA has a binding 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, e.g. 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.

[0131] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "binding specifically 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 a test molecule to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule 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 "specific binding" or "binding specifically 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 at 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 an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0132] Herein, the term "immunoglobulin" (Ig) is used interchangeably with "antibody". The term "antibody" is used herein in the broadest sense and specifically covers 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.

[0133] "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 bonds varies in 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 at its other end has a constant domain; 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 the heavy chain variable domain.

[0134] 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, p. 71 and Chapter 6.

[0135] The light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain (CH) of an immunoglobulin, it 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. Based on relatively minor differences in CH sequence and function, the γ and α classes are further divided into subclasses (isotypes), 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 edition (WB Saunders Co., 2000).

[0136] 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.

[0137] 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 domains are widely different in sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, the variability is not evenly distributed throughout the span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each include four FR regions, which mainly adopt a β-sheet configuration and are connected by three HVRs, which form loops connecting the β-sheet structure and in some cases form part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FR region and, together with the HVRs of the other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0138] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting 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 culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be prepared 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 having 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. Pat. No. 4,816,567), yeast display technology (see, e.g., WO2009 / 036379A2; WO2010105256; WO2012009568; and Xu et al., Protein Eng. Des. Sel., 26(10):663-70 (2013)), and techniques for producing human antibodies or human-like antibodies in animals having 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 2008:1151-1163; 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)).

[0139] The terms "full length antibody", "intact antibody" or "complete antibody" are used interchangeably and refer to an antibody, such as an anti-SIRPA antibody of the present invention, in its substantially intact form, not an antibody fragment. Specifically, intact antibodies include antibodies having heavy and light chains including an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0140] "Antibody fragment" refers to a molecule that is different from an intact antibody and contains 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'), 2and Fv fragments; diabodies; linear antibodies (see US Pat. No. 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.

[0141] 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 H ) and the first constant domain of one 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 the antibody produces a single large F(ab') 2 The Fab' fragment is a fragment that roughly corresponds to two Fab fragments with different antigen binding activities linked by disulfide bonds and is still able to cross-link antigen. The Fab' fragment is different 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 have a free thiol group. F(ab') 2 Antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

[0142] The Fc fragment contains the carboxyl-terminal portions of two heavy chains held together by disulfides. 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 types of cells.

[0143] "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 region or variable region of an 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.

[0144] The term "diabody" refers to a H With V L Small 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 to produce 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 areH and V L Domains are present on different polypeptide chains.

[0145] 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 chain 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, and 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, as 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."

[0146] A "humanized" form of a non-human (e.g., murine) antibody, such as a humanized form of an anti-SIRPA antibody of the invention, is a chimeric antibody 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, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the 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.

[0147] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human, such as an anti-SIRPA antibody of the present invention, and / or an antibody made 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 produced via 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); Boerner et al., J. Immunol., 147 (1): 86-95 (1991) methods described 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 antigens to transgenic animals that have 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. Pat. Nos. 6,075,181 and 6,150,584). For human antibodies produced via human B cell hybridoma technology, see, 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).

[0148] The term "hypervariable region," "HVR," or "HV" when used herein refers to regions of an antibody variable domain, such as regions of 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 V H(H1, H2, H3), and three in V L In natural antibodies, H3 and L3 represent most of the diversity 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 only of heavy chains are functional and stable in the absence of light chains.

[0149] A variety of HVR descriptions have been used and are encompassed herein. In some embodiments, HVRs may be 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 position 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.

[0150]

[0151] The HVR may comprise an "extended HVR" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (preferred 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.

[0152] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0153] The phrase "variable domain residue numbering as in EU or Kabat" or "amino acid position numbering as in EU or Kabat" and variants thereof refer to the numbering system for heavy chain variable domains or light chain variable domains used for antibody compilation in EU or Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of the 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, etc. according to Kabat). 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.

[0154] The EU or Kabat numbering system is usually 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 Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU or Kabat numbering system" or "EU index" is usually used when referring to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in the same document of Kabat et al.). The "EU index as in Kabat" refers to the residue numbering of human IgG1EU antibodies. Unless otherwise stated herein, when referring to the residue numbering in the antibody variable domain, it means the residue numbering according to the Kabat numbering system. Unless otherwise stated herein, when referring to the residue numbering in the antibody constant domain, it means the residue numbering according to the EU or Kabat numbering system (e.g., see U.S. Patent Publication No. 2010-280227).

[0155] As used herein, an "acceptor human framework" is a V sequence comprising a V sequence derived from a human immunoglobulin framework or a human common framework. 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 human immunoglobulin framework sequence or the human common framework sequence is identical.

[0156] "Human common framework" refers to human immunoglobulin V L or V H The framework sequence is the most frequently occurring amino acid residue 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 a subset as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for V L , the subgroup may be subgroup κI, κII, κIII or κIV as described in the previous literature of Kabat et al. In addition, for V H The subgroup may be subgroup I, subgroup II or subgroup III as in Kabat et al., supra.

[0157] An "amino acid modification" at a specified position of, for example, an anti-SIRPA antibody of the invention refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to a specified residue means an insertion within one to two residues thereof. The insertion may be at the N-terminus or C-terminus of the specified residue. Preferred amino acid modifications herein are substitutions.

[0158] "Affinity matured" antibodies, such as the affinity matured anti-SIRPA antibodies of the invention, are antibodies with one or more alterations in one or more of their 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, the affinity matured antibody has 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).

[0159] "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. Six hypervariable loops (3 loops each in the H chain and L chain) are generated by the folding of these two domains, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half an Fv containing 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.

[0160] "Single-chain Fv" is also referred to as "sFv" or "scFv", and is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a V H With V L A polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding.

[0161] 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.

[0162] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the Fc region boundaries of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as an amino acid residue at position Cys226 or an extension from Pro230 to its carboxyl end. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may 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 may include an antibody population in which all K447 residues are removed, an antibody population in which no K447 residue is removed, and an antibody population 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.

[0163] 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.

[0164] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of a parent polypeptide, for example from about one to about ten amino acid substitutions in the 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 with the native sequence Fc region and / or with the Fc region of a parent polypeptide, and most preferably has at least about 90% homology therewith, more preferably has at least about 95% homology therewith.

[0165] "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 alternative splicing 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 FcRs identified in the future. FcRs can also increase the serum half-life of antibodies.

[0166] 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 to which polypeptides with variant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0167] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to 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 of 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.

[0168] The term "compete" when used in the context of antibodies that compete for the same epitope (e.g., neutralizing antibodies) means competition between 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 a 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 radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (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 assay, solid phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 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 determining 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 an adjacent epitope that is 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%.

[0169] 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 may be inhibited by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or close to 100%.

[0170] 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 the 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 may be found on other types of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Typically, antibodies specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0171] 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.

[0172] 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.

[0173] An "isolated" antibody, such as an isolated anti-SIRPA antibody of the 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 was produced. Preferably, an isolated antibody is free of all other contaminant components from the environment in which it was produced. Contaminant components from the environment in which it was produced, such as components produced by recombinant transfected cells, are substances that typically interfere with the research, diagnostic, or therapeutic use 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 dye. An isolated antibody includes the antibody in situ within a recombinant T cell, since 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.

[0174] 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 that is normally associated with it in 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 different from the nucleic acids encoding polypeptides and antibodies herein that are naturally present in cells.

[0175] The term "vector" as used herein 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) may be integrated into the genome of the host cell when introduced into the host cell, and thereby replicated 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.

[0176] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer 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.

[0177] "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 progeny of a single host cell, and 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.

[0178] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto 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 .

[0179] As used herein, the term "preventing" includes providing prevention with respect to 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.

[0180] 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 methods described herein. "At risk" means that the individual has one or more risk factors, which are measurable parameters associated with the development of 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 a particular disease, disorder, or condition than an individual who does not have one or more of these risk factors.

[0181] As used herein, the term "treatment" refers to clinical intervention intended to alter the natural course of clinical pathology in the individual being treated. The desired therapeutic effect includes reducing the rate of progression of a particular disease, disorder, or condition, ameliorating or alleviating its pathological state, and relieving 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.

[0182] "Effective amount" refers to an amount that can at least effectively achieve the desired treatment or prevention results at the desired dosage and time period. The effective amount can be provided in one or more administrations. The effective amount herein can vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the treatment to induce the desired response in the individual. The effective amount is also the amount in which the beneficial effects of the treatment exceed any toxic or harmful effects of the treatment. For preventive use, the beneficial or desired results include results such as: eliminating or reducing the risk of the disease, reducing the severity of the disease, or delaying the onset of the disease, including the biochemical, histological and / or behavioral symptoms of the disease, its complications and the intermediate pathological phenotypes presented during the development of the disease. For therapeutic use, the beneficial or desired results include the following clinical results: such as reducing one or more symptoms caused by the disease, improving the quality of life of the patient, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug (such as via targeting), delaying disease progression and / or prolonging survival time. The effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly complete preventive 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" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or is achieved in conjunction with one or more other agents.

[0183] "Subject" for purposes of treatment, prevention or risk reduction refers to any animal classified as a mammal, including humans, domestic and farm animals and 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.

[0184] 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 administration 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.

[0185] 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.

[0186] Unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references. 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 on.

[0187] It should be understood that aspects and embodiments of the present invention described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments.

[0188] Anti-SIRPA antibody

[0189] Anti-SIRPA antibody binding region

[0190] 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 10 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 on a mammalian SIRPA protein corresponding to the amino acid sequence of SEQ ID NO: 1.

[0191] 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 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.

[0192] In some embodiments, the anti-SIRPA antibodies of the 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 invention compete for binding to SIRPA 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.

[0193] 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 following: m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / 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 F9-4, 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, when the anti-SIRPA antibodies of the invention make one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / 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- 9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof, when the binding of an antibody to SIRPA is reduced by about 50% to 100% compared to the binding to SIRPA in the absence of the anti-SIRPA antibody, the anti-SIRPA antibody is combined with one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.3), h3F9H1 / L4, h3F9H2 / L2 (14.70.4), h3F9H1 / L5, h3F9H2 / L3 (14.70.5), h3F9H2 / L4 (14.70.6), h3F9H2 / L4 (14.70.7), h3F9H2 / L4 (14.70.8), h3F9H2 / L4 (14.70.9), h3F9H2 / L4 (14.70.10), h3F9H2 / L4 (14.70.11), h3F9H2 / L4 (14.70.4), h3F9H2 / 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 compete for binding to SIRPA. In some embodiments, when the anti-SIRPA antibodies of the invention make one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / 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, reduces binding to SIRPA by at least 50%, at least 55%, or at least 60% compared to binding to SIRPA in the absence of the anti-SIRPA antibody. 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%, the anti-SIRPA antibody binds to one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / 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, compete for binding to SIRPA. In some embodiments, one or more antibodies selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.3),4), h3F9H2 / 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, to SIRPA. An anti-SIRPA antibody of the invention that substantially completely blocks the binding of one or more antibodies selected from m3F9, h3F9H1 / 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 to SIRPA L1(14.70.1), h3F9H1 / L2(14.70.2), h3F9H1 / L3, h3F9H2 / L1(14.70.4), h3F9H2 / 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, binding to SIRPA. In some embodiments, the anti-SIRPA antibody and one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / 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 The antibodies of 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 0.0050:1 ratio, a 0.0025:1 ratio, a 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 anti-SIR PA antibody: one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / L2, h3F9H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, Antibodies of 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, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / L2, h3F9H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3 F9-10, 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, the anti-SIRPA antibody is present in an excess amount ranging from about 1.5-fold to 100-fold or greater than 100-fold. In some embodiments, the anti-SIRPA antibody is present in an excess amount ranging from about 1.5-fold to 100-fold or greater than 100-fold compared to the amount of the antibody selected from m3F9, h3F9H1 / L1(14.70.1), h3F9H1 / L2(14.70.2), h3F9H1 / L3, h3F9H2 / L1(14.70.3), h3F9H1 / L4, h3F9H2 / L2(14.70.4), h3F9H1 / L5, h3F9H2 / L3(14.70.5), h3F9H2 / L1(14.70.6), h3F9H2 / L2(14.70.7), h3F9H2 / L1(14.70.8), h3F9H2 / L2(14.70.9), h3F9H2 / L1(14.70.1), h3F9H2 / L2(14.70.1), h3F9H2 / L2(14.70.4), h3F9H2 / 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 The anti-SIRPA antibody is present in an amount that is about 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 of 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25, and any combination thereof.

[0194] 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. , 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 antibodies bind to the same or overlapping SIRPA epitope.

[0195] 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 at least one epitope selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / 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, 3F9-26 Detailed exemplary methods for locating epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0196] In some embodiments, the anti-SIRPA antibodies of the invention are combined with one or more selected from m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / L2, h3F9H2 / L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-21, 3F9-32, 3F9-43, 3F9-54, 3F9-65, 3F9-76, 3F9-87, 3F9-98, 3F9-11, 3F9-22, 3F9-33, 3F9-44, 3F9-55, 3F9-66, 3F9-87, 3F9-98, 3F9-12, 3F9-23, 3F9-34, 3F9-45, 3F9-67, 3F9-87, 3F9-98, 3F9-13, 3F9-24, 3F9-36, 3F9-87, 3F9-98, 3F9-14 9-6, 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.

[0197] Any suitable competition assay or SIRPA binding assay known in the art, such as a BIAcore assay, an ELISA assay, or flow cytometry, can be used to determine whether an anti-SIRPA antibody binds to one or more of m3F9, h3F9H1 / L1 (14.70.1), h3F9H1 / L2 (14.70.2), h3F9H1 / L3, h3F9H2 / L1 (14.70.4), h3F9H2 / L2, h3F9H2 / L3, 3F9-1, Antibodies to 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 an exemplary competition assay, SIRPA or cells expressing SIRPA on the cell surface are immobilized 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. After incubation under conditions that allow the first antibody to bind to SIRPA, excess unbound antibody is removed and the amount of label associated with the immobilized SIRPA or cells expressing SIRPA is measured. If the amount of label associated with the immobilized SIRPA or cells expressing SIRPA in the test sample is significantly reduced relative to that in the control sample, it indicates that the second antibody competes with the first antibody for binding to SIRPA. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0198] In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence selected from SEQ ID NO: 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 the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

[0199] In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-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, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:12; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:22; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:13; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:22 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:10 NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:17; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:22; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:18; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:22; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:18; NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:19; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:23; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises 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, which comprises the amino acid sequence of SEQ ID NO:16; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:23; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:17; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:16; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:17; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:23; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:18; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:23; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises 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, which comprises the amino acid sequence of SEQ ID NO:24; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:15; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:24; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:16; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:24; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:16 NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:24; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:9; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:17; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; (c) HVR-H3, which comprises 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. ;

[0200] 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, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; and (c) HVR-H3, which comprises an amino acid sequence selected from SEQ ID NO:22, 23 and 24.

[0201] 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, which comprises the amino acid sequence of SEQ ID NO:9; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:10; and (c) HVR-L3, which comprises the amino acid sequence selected from SEQ ID NO:11, 12, 13, 14, 15, 16, 17, 18 and 19.

[0202] 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.

[0203] 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.

[0204] 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 ) sequence. In certain embodiments, a V sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NOs: 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, which comprises the amino acid sequence of SEQ ID NO:20; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:21; and (c) HVR-H3, which comprises the amino acid sequence selected from SEQ ID NO:22, 23 and 24.

[0205] 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, a V sequence that 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. LThe 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 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.

[0206] 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 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.

[0207] 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 .

[0208] Further provided herein are anti-SIRPA antibodies that competitively inhibit the binding of and / or compete with the 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 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.

[0209] 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 NO: 33, 34 or 35 and SEQ ID NO: 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.

[0210] In some embodiments, the anti-SIRPA antibody according to any of the above embodiments is a monoclonal antibody, including a humanized antibody and / or a human antibody. In some embodiments, the anti-SIRPA antibody is an antibody fragment, such as a 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 classes or isotypes as defined herein.

[0211] 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.

[0212] Anti-SIRPA antibodies are provided herein. The antibodies provided are useful, for example, in diagnosing or treating SIRPA-mediated disorders.

[0213] 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 Anti-SIRPA antibodies that increase or enhance the anti-tumor activity of anti-cancer therapy, anti-SIRPA antibodies that can increase or enhance T cell proliferation, anti-SIRPA antibodies that can increase or enhance the release of pro-inflammatory cytokines from macrophages, and / or anti-SIRPA antibodies that can 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.

[0214] 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) inhibiting or reducing one or more SIRPA activities; (2) the ability to reduce SIRPA expression in SIRPA-expressing cells (such as at the mRNA level and / or at the protein level); (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.

[0215] 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 lower half maximal effective concentration (EC50) 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); NO:5; d) the dissociation constant (KD) for human SIRPA may be in the range of about 0.6 nM to about 0.7 nM; and / or e) reduce the cell surface level of SIRPA (e.g., reduce the cell surface level of SIRPA on human macrophages in vitro, and its half maximal effective concentration (EC50) may be 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 at which the anti-SIRPA antibody of the present invention reduces the cellular level of SIRPA on or in a cell to half of that of an untreated cell, or the concentration at which the antibody achieves half maximal binding to SIRPA on a cell.

[0216] 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 0.9nM to 0.8nM, about 0.9nM to 0.7nM, about 0.9nM to 0.6nM, about 0.9nM to 0.5nM, about 0.9nM to 0.4nM, about 0.9nM to 0.3nM, about 0.9nM to 0.2nM, about 0.9nM to 0.1nM, about 0.8nM to 0.6nM, about 0.8nM to 0.5nM, 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.

[0217] 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, wherein the half maximal effective concentration (EC50) for human SIRPA v1 is about 0.093 nM, the half maximal effective concentration for human SIRPA v2 is about 0.080 nM, and / or the half maximal effective concentration for cynomolgus monkey SIRPA is about 0.879 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, wherein the half maximal effective concentration (EC50) for human SIRPA v1 or for human SIRPA v2 is about 0.080 nM, and / or the half maximal effective concentration for cynomolgus monkey SIRPA is about 0.879 nM, 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. 0.3nM to 0.05nM, about 0.20nM 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 0.07nM 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 0.11nM to 0.05nM, about 0.12nM to 0.05nM, about 0.13nM to 0.05nM, about 0.14nM to 0.05nM, about 0.15nM to 0.05nM, about 0.1 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.

[0218] 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.

[0219] Advantageously, the anti-SIRPA antibodies of the 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). In addition, advantageously, the anti-SIRPA antibodies of the 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).

[0220] Certain aspects of the 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 microglia; reduction of proliferation of dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia, activated M1 microglia, M2 microglia, macrophages, T cells, dendritic cells and / or microglia; reduction of proliferation of dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M2 microglia, M1 microglia, activated M1 microglia, M2 ... cells, M1 macrophages, activated M1 macrophages and / or M2 macrophages-induced T cell proliferation; reduce the survival rate 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 survival rate of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 The invention relates to the invention to 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; bind to SIRPA ligands on tumor cells; bind to SIRPA ligands on dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils and / or macrophages; inhibit 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; inhibit 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 of 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. ;

[0221] 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 (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 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.

[0222] 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 (MDSC). 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 (MDSC). In humans, MDSCs can be defined by one of the following combinations of markers: (1) CD14+HLA-DRlow / -; (2) CD14+IL4Rα+; (3) CD14+HLA-DR-IL4Rα+; (4) CD34+CD14+CD11b+CD33+; (5) CD11b+CD14+CD33+; (6) CD33+HLA-DR-; (7) Lin-HLA-DR-; (8) Lin-HLA-DR-CD33+; (9) Lin-HLA-DR-CD33+CD11b+; (10) Lin-CD33+CD11b+CD15+; (11) Lin-HLA-DR-CD33+CD11b+CD15+; (12) Lin-HLA-DR-CD33+CD11b+CD15+; (13) Lin-HLA-DR-CD33+CD11b+CD15+; (14) Lin-HLA-DR-CD33+CD11b+CD15+; (15) Lin-HLA-DR-CD33+CD11b+CD15+; (16) Lin-HLA-DR-CD33+CD11b+CD15+; (17) Lin-HLA-DR-CD33+CD11b+CD15+; (18) Lin-HLA-DR-CD33+CD11b+CD15+; (19) Lin-HLA-DR-CD33+CD11b+CD15+; (20) Lin-HLA-DR-CD33+CD11b+CD15+; (21) Lin-HLA-DR-CD33+CD11b+CD15+; (22) Lin-HLA-DR-CD33+CD11b+CD15+; (23) Lin-HLA-DR-CD33+CD11b+CD15+; (24 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 high CD33+; (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 expression of the surface markers CD45+, CD11b+, Gr1+, and / or 114Ra+. Additional exemplary immunosuppressive monocyte lineages are CD45+, CD11b+, Gr1 low; and CD45+, CD11c+.

[0223] 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.

[0224] 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, SIRPA ligand or binding partner CD47, surfactant protein A and / or surfactant protein D.

[0225] 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 monkey 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.

[0226] 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.

[0227] Certain aspects of the invention relate to anti-SIRPA antibodies that down-regulate, 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).

[0228] 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.

[0229] 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.

[0230] The cellular level of SIRPA may refer to, but is not limited to, the cell surface level of SIRPA, the intracellular level of SIRPA, and the total level of SIRPA. In some embodiments, the reduction in the cellular level of SIRPA comprises a reduction in the cell surface level of SIRPA. As used herein, an anti-SIRPA antibody reduces the cell surface level of SIRPA if it induces a reduction in the cell surface level of SIRPA by 25% or more, as measured by any in vitro cell-based assay described herein or known in the art or a suitable in vivo model, for example, using flow cytometry, such as fluorescence activated cell sorting (FACS) to measure the cell surface level of SIRPA. In some embodiments, the reduction in the cellular level of SIRPA comprises a reduction in the intracellular level of SIRPA. As used herein, an anti-SIRPA antibody reduces the intracellular level of Siglec-9 if it induces a decrease in the intracellular level of SIRPA by 25% or more, 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, immunocoprecipitation, and cell cytometry. In some embodiments, the reduction in the cellular level of SIRPA comprises a reduction in the total level of SIRPA. As used herein, an anti-SIRPA antibody reduces the total level of SIRPA if it induces a decrease in the total level of SIRPA by 25% or more, 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, immunocoprecipitation, 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.

[0231] In some embodiments, the down-regulating 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 of exposure to an antibody of the invention. Cells may be analyzed for SIRPA surface expression using any technique, such as flow cytometry.

[0232] 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 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%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more.

[0233] In some embodiments that 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.

[0234] In some embodiments, the anti-SIRPA antibodies of the 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 case of CD47 binding to SIRPA refers to a reduction in binding of 30% or less, typically at least 25%, at least 20%, at least 15%, or at least 10% or less, compared to 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 incubated with 10 5Cells / well were seeded in 96-well plates, washed, and incubated in 100 μl buffer containing 1.0 μg / ml of monoclonal antibody or isotype control for fluorescence activated cell sorting. Cells were then washed and incubated on ice for 30 minutes in soluble human CD47. Cells were then analyzed for surface-bound CD47.

[0235] In some embodiments, the anti-SIRPA antibodies of the 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 invention downregulate CD32A / B expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRP antibodies of the invention downregulate CD32A (i.e., FcγRIIA) expression in macrophages (e.g., human macrophages). In some embodiments, the anti-SIRPA antibodies of the 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.

[0236] In one aspect, the 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 a human cell, such as a dendritic cell, a myeloid cell, a monocyte, a macrophage, etc., at a half maximal effective concentration (EC 50 ) is less than half the 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 activities 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.

[0237] In some embodiments, the antibodies of the invention can bind to SIRPA in a pH-dependent manner. In some embodiments, the antibodies of the 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 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 in the following range: 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.

[0238] 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 determine the domains.

[0239] 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).

[0240] 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 that is bound by an antibody having the CDRs of an 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 that is bound by an antibody having the CDRs of an antibody designated m3F9 as described herein.

[0241] Certain aspects of the 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 an improved / enhanced ability to reduce cell surface levels of SIRPA on cells, to cause 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 microglia; reduction of proliferation of dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia, activated M1 microglia, M2 microglia, M3 microglia, M4 microglia, M5 microglia, M6 microglia, M7 microglia, M8 microglia, M9 microglia, M10 microglia, M11 microglia, M12 microglia, M13 microglia, M14 microglia, M15 microglia, M16 microglia, M17 microglia, M18 microglia, M19 microglia, M20 microglia, M21 microglia, M22 microglia, M37 microglia, M18 microglia, M22 microglia, M37 microglia, M19 microglia, M22 microglia, M37 microglia, M4 microglia, M5 microglia, M6 microglia, M7 microglia, M8 microglia, M9 microglia, M18 microglia, M19 microglia, M22 microglia, M23 microglia, M24 microglia, M25 microglia, M26 microglia, M37 microglia, M4 microglia, M5 cells, M2 microglial cells, macrophages, M1 macrophages, activated M1 macrophages and / or M2 macrophage-induced T cell proliferation; reduce neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglial cells, M1 microglial cells, glial cells, activated M1 microglial cells and / or M2 microglial cells; reduce or decrease the survival rate of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglial cells, M1 microglial cells, activated M1 microglial cells cells and / or M2 microglial cells; inhibit the proliferation of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages, activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglial cells, M1 microglial cells, activated M1 microglial cells and / or M2 microglial cells;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 proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils and / or microglia the overall functionality of glial cells, neutrophils and / or microglial cells; inhibiting beneficial immune responses against different types of cancers selected from bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma, renal pelvic cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, acute myeloid leukemia, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma and thyroid cancer; inhibiting beneficial immune responses against different types of cancers selected from dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathic disease, Nahar's disease, stroke, acute trauma, chronic trauma, essential tremor, Behcet's disease, Parkinson's disease, Lewy body dementia, multiple sclerosis, The invention relates to a novel novel anti-SIRPA inhibitor that is directed against a variety of cell types, including leukemia, myeloid leukemia, leukopenia ... inhibiting 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; inhibiting 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;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; promote or rescue 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, and regulatory T cells; increase the infiltration of one or more of immunosuppressive dendritic cells, immunosuppressive macrophages, immunosuppressive neutrophils, immunosuppressive ... Infiltration of one or more of suppressive 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 the pro-tumorigenic activity of bone marrow-derived suppressor cells; reduce the activation of tumor-specific T lymphocytes with tumor-killing potential; enhance non-tumorigenic The invention relates to a method for reducing the survival of 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 tumor growth rate; increasing tumor recurrence rate; reducing the efficacy of one or more immunotherapies that modulate 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, PD-L1, CTLA4, PD-L2, PD- 1. Immunotherapy of proteins of 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. ;

[0242] 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) reduction of 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 (MDSC); (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 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.

[0243] 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).

[0244] 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.

[0245] Anti-SIRPA antibody binding affinity

[0246] 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, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 - 13 The dissociation constant (Kd) of a target protein (M) can be determined by any analytical technique, including any biochemical or biophysical technique, such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, e.g., 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 a radiolabeled antigen binding assay (RIA). In some embodiments, an RIA is performed with an antibody of interest in Fab form and its antigen, e.g., 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 about 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 .

[0247] Antibody fragments

[0248] 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, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a review of Fab and F(ab') fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life 2 For a discussion of fragments, see U.S. Patent No. 5,869,046.

[0249] Diabodies are antibody fragments with two antigen binding sites, which may 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 contain 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. Pat. No. 6,248,516).

[0250] Antibody fragments can be made 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.

[0251] Chimeric and humanized antibodies

[0252] 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.

[0253] 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 a target as antibodies from another species from which humanized antibodies are 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. Humanized antibodies optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, the antibody from which the HVR residues are derived), eg, to restore or improve antibody specificity or affinity.

[0254] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro et al. Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., U.S. Pat. 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, 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)); 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)).

[0255] Human Antibodies

[0256] 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).

[0257] 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 repertoire in these mouse strains can produce high-affinity fully human antibodies for 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 US Patent Nos. 6,075,181 and 6,150,584 for the technology; description US Patent No. 5,770,429 describing KM U.S. Patent No. 7,041,870 for the technology; and Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0258] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol. 133:3001 (1984); and Boerner et al. J. Immunol. 147:86 (1991)). Human antibodies produced 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 methods described, e.g., in U.S. Pat. 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 desired human constant domains. The following describes techniques for selecting human antibodies from antibody libraries.

[0259] In some embodiments of any of the antibodies provided herein, the antibody is a human antibody isolated by in vitro methods and / or screening combinatorial libraries for antibodies having 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 having 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):1 19-132 (2004). Phages 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, the primary repertoire can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self antigens as well as self antigens without any immunization, as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, the primary library can also be made synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers comprising a random sequence for encoding the highly variable HVR3 region and for completing 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. Pat. No. 5,750,373 and U.S. Pat. Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0260] Constant region including Fc region

[0261] 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 class, the IgM class, or the IgA class.

[0262] 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 or is 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 an inhibitory Fc-gamma receptor IIB (FcγIIB).

[0263] 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 an inhibitory Fc-gamma receptor IIB (FcγIIB).

[0264] 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 an inhibitory Fc-gamma receptor IIB (FcγIIB).

[0265] 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).

[0266] 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 a target to which the antibody specifically binds. In some embodiments, the antibody binds to SIRPA.

[0267] 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 may 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 of 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 functions, thereby increasing the selectivity for detection against ITIM-containing FcgRIIb (CD32b) to increase clustering of SIRPA antibodies on neighboring cells without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis.

[0268] To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into an antibody (particularly an antibody fragment), as described, for example, in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG) that is responsible for increasing the serum half-life of an IgG molecule in vivo. 1 IgG 2 IgG 3 or IgG 4 )'s Fc region.

[0269] Multispecific Antibodies

[0270] A multispecific antibody is an antibody that has 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 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 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.

[0271] 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 .

[0272] 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 penetratin), polyarginine or generally positively charged peptides, Angiopep peptides (such as ANG1005) (see, e.g., Gabathuler, 2010), and other cell surface proteins enriched on blood-brain barrier endothelial cells (see, e.g., Daneman et al. PLoS One 5(10):e13741 (2010)).

[0273] 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 body, 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 (d) ligands and / or proteins expressed on immune cells, wherein the ligands and / or proteins are selected from 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) proteins, lipids, polysaccharides or glycolipids expressed on one or more tumor cells; and any combination thereof.

[0274] The multivalent antibody may recognize the SIRPA antigen 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) composed of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR), insulin receptor, insulin-like growth factor receptor, or transferrin receptor, or any other antigen that promotes 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.

[0275] The multivalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the 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 in the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, the polypeptide chain may include 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 preferably further comprises at least two (and preferably four) light chain variable domain polypeptides herein. The multivalent antibody may, for example, comprise about two to about eight light chain variable domain polypeptides herein. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0276] 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. Pat. 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-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies (see, e.g., U.S. Pat. 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).

[0277] Also included herein are engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US2006 / 0025576). Antibodies herein also include "Dual Acting FAbs" or "DAFs" comprising antigen binding sites that bind to multiple SIRPAs (see, e.g., US2008 / 0069820).

[0278] Antibody variants

[0279] 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.

[0280] Substitution, insertion and deletion variants

[0281] In some embodiments of any of the antibodies provided herein, antibody variants having one or more amino acid substitutions are provided. The amino acid sequence variants of the antibody 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.

[0282] Table 1: Amino Acid Substitutions

[0283]

[0284]

[0285] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their ability to maintain: (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a 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: (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0286] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0287] (3) Acidic: Asp, Glu;

[0288] (4) Basic: His, Lys, Arg;

[0289] (5) Residues that affect chain orientation: Gly, Pro; and

[0290] (6) Aromatic: Trp, Tyr, Phe.

[0291] 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.

[0292] 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).

[0293] 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 replace other amino acids with similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, substitutions of amino acids with hydropathic indices within ±2 are included. In certain embodiments, substitutions of amino acids with hydropathic indices within ±1 are included, and in certain embodiments, substitutions of amino acids with hydropathic indices within ±0.5 are included.

[0294] It is also understood in the art that substitution of similar amino acids can be effectively made 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 neighboring amino acids, correlates with its immunogenicity and antigenicity, i.e., the biological properties of the protein.

[0295] The following hydrophilicity 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, substitution of amino acids with hydrophilicity values ​​within ±2 is included, in certain embodiments, substitution of amino acids with hydrophilicity values ​​within ±1 is included, and in certain embodiments, substitution of amino acids with hydrophilicity values ​​within ±0.5 is 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".

[0296] 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 antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may, for example, be outside the antigen contact residues in 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.

[0297] 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 insertion variants of the antibody molecule include fusion 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.

[0298] Any cysteine ​​residues not involved in maintaining the proper conformation of the antibody may also be substituted with serine in general to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to increase its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0299] Glycosylation variants

[0300] In some embodiments of any one 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 making the antibody lack glycosylation sites can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.

[0301] Glycosylation of antibodies is usually 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 any 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, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0302] Preferably, the addition of glycosylation sites to the antibody is achieved by changing the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also 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).

[0303] In the case where the antibody comprises an Fc region, the carbohydrates attached thereto may be changed. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically connected to the Asn297 of the CH2 domain of the Fc region 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 "trunk" 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 certain improved properties.

[0304] In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. See, e.g., U.S. Patent Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US 2004 / 0110704; US2004 / 0110282; US2004 / 0109865; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells lacking 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)).

[0305] Modified constant region

[0306] 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.

[0307] Exemplary antibody Fc isotypes and modifications are provided in Table 2 below. In some embodiments, the anti-SIRPA antibodies of the invention are capable of binding to an Fc gamma receptor and have an Fc isotype listed in Table 2 below.

[0308] Table 2: Exemplary anti-SIRPA antibody Fc isotypes capable of binding to Fcγ receptors

[0309]

[0310]

[0311] In addition to the isotypes described in Table 2, and without wishing to be bound by theory, it is believed that antibodies with human IgG1 or IgG3 isotypes 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) may also be used as transient potentiator antibodies.

[0312] In some embodiments, the antibody binding to Fcγ receptors belongs to IgG class, IgM class or IgA class. In some embodiments, the antibody binding 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 the following 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, 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 position E345R, E430G, and S440Y.

[0313] In certain embodiments, the antibody that binds to the Fcγ receptor has an IgG2 isotype. In some embodiments, the antibody that binds to the Fcγ receptor contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, the antibody that binds to the Fcγ receptor binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is an 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 (US2007 / 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, wherein amino acid positions are according to the EU or Kabat numbering convention.

[0314] In some embodiments, the antibody that binds to an Fcγ receptor is of the IgG2 isotype having a heavy chain constant domain containing a C127S amino acid substitution, wherein the 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).

[0315] In some embodiments, the antibody that binds to an Fcγ receptor is of the IgG2 isotype having a kappa light chain constant domain containing a C214S amino acid substitution, wherein the 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).

[0316] In certain embodiments, the antibody that binds to the Fcγ receptor has an IgG1 isotype. In some embodiments, the antibody that binds to the Fcγ receptor contains a mouse IgG1 constant region. In some embodiments, the antibody that binds to the Fcγ receptor contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments, the antibody that binds to the Fcγ receptor binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is an 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 the amino acid positions are according to the EU or Kabat numbering convention.

[0317] 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 contain the amino acid sequence of ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 58). In some embodiments, the antibody Fc region contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and / or an N297A or N297Q amino acid substitution, wherein the amino acid positions are according to the EU or Kabat numbering convention.

[0318] In certain embodiments, the antibody that binds to the Fcγ receptor has an IgG4 isotype. In some embodiments, the antibody that binds to the Fcγ receptor contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments, the antibody that binds to the Fcγ receptor binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is an 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 L235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol, 164: 1925-1933), S267E, E318A, L328F, M252Y, S254T and / or T256E, wherein the amino acid positions are according to EU or Kabat numbering conventions.

[0319] In certain embodiments, the antibody that binds to the Fcγ receptor has a hybrid IgG2 / 4 isotype. In some embodiments, the antibody that binds to the 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).

[0320] In certain embodiments, the antibody contains a mouse IgG4 constant region (Bartholomaeus et al. (2014). J. Immunol. 192, 2091-2098).

[0321] 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.

[0322] In certain embodiments, the antibody contains 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 in the Fc region, 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 positions 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, wherein the numbering of residue positions is according to EU numbering.

[0323] In some embodiments, antibodies that bind to SIRPA proteins 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 SIRPA proteins. Such antibodies inhibit one or more activities of SIRPA proteins 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. Antibodies may also inhibit one or more activities of SIRPA proteins 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.

[0324] 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 incapable of binding to Fcg receptors and therefore is incapable of, for example, transiently clustering and activating SIRPA.

[0325] 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 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 inhibitory factor of cytokine signaling (SOCS) proteins (e.g., SOCS3 proteins) to SIRPA proteins; the ability to increase the proteasomal degradation of SIRPA proteins; the ability to reduce the expression of SIRPA on circulating dendritic cells, macrophages, monocytes, T cells and / or microglial cells; The ability of the tyrosine kinases such as LCK and FYN to phosphorylate Tyr-340 and Tyr-358; reduce the recruitment and binding of tyrosine-specific protein phosphatases SHP1 and SHP2; reduce the recruitment and binding of PLC-g1, which acts as a guanine nucleotide exchange factor for dynein-1; reduce the recruitment and binding of Crk1; reduce the recruitment and binding of spleen tyrosine kinase Syk; reduce the SH3-SH2-SH3 growth factor receptor binding protein 2 (G rb2) recruitment and binding to it; reduce the recruitment and binding of multiple SH2-containing proteins; increase the ability of intracellular calcium movement; 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 ability to increase the phagocytic activity of monocytes, macrophages, dendritic 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 signal transduction molecules that mediate ITAM signal transduction; 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.

[0326] 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.

[0327] 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.

[0328] Table 3: Exemplary anti-SIRPA antibody Fc isotypes with reduced binding to Fcγ receptors

[0329]

[0330]

[0331] In certain embodiments, the anti-SIRPA antibody has an IgG1 isotype. In some embodiments, the antibody contains a mouse IgG1 constant region. In some embodiments, the antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region 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).

[0332] 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 the 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.

[0333] 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 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 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).

[0334] 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 8614299B2; Vafa O. et al., (2014) Methods 65: 114-126), T394D, M252Y, S254T, T256E, N297A and / or N297Q, wherein the 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, a 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).

[0335] 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.

[0336] 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 the amino acid positions are according to the EU numbering convention.

[0337] 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 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 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's, the Fc comprises S267E and L328F mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc's, the Fc comprises a S267E mutation according to EU numbering.In some embodiments of any of the IgG1 modified Fcs, the Fc comprises replacing 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).

[0338] In some embodiments of any of the IgG1 modified Fcs, the Fc comprises two or more amino acid substitutions that increase antibody clustering without activating complement compared to a corresponding antibody having an Fc region that does not comprise 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 at residue positions in the Fc region 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.

[0339] 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.

[0340] Other aspects of the invention relate to antibodies with modified constant regions (i.e., Fc regions). Antibodies that rely on binding to FcgR receptors to activate targeted receptors lose their potentiator activity if they are 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 the anti-SIRPA antibodies of the invention with appropriate epitope specificity have an Fc domain (CH1 and hinge region) from the human IgG2 isotype or another type of Fc domain or variant thereof that is capable of preferentially binding to an inhibitory FcgRIIBr receptor, the antibodies can activate the target antigen with minimal side effects.

[0341] 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.

[0342] 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).

[0343] 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 the IgG2 isotype, wherein the kappa light chain constant domain comprises a C214S amino acid substitution according to the EU numbering convention.

[0344] 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 the IgG2 isotype, wherein the kappa light chain constant domain comprises a C214S amino acid substitution according to the EU numbering convention.

[0345] 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.

[0346] 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 the EU numbering convention). In some embodiments of any of the IgG2 modified Fcs, the Fc may further comprise A330S and P331S.

[0347] 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 IgG2aa 118 to 260 and IgG4aa 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.

[0348] In some embodiments of any 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.

[0349] In certain embodiments of any of the IgG1 and / or IgG2 modified Fcs, 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 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 IgG1 and / or IgG2 modified Fcs, 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 Fcs, 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 Fcs, 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 Fcs, 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 Fcs, 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 Fcs, 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 Fcs, 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.

[0350] 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 the 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.

[0351] 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.

[0352] 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).

[0353] 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 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 an amino acid substitution at position 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 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 an amino acid substitution at position E345R, E430G, and S440Y according to EU numbering.

[0354] Other Antibody Modifications

[0355] In some embodiments of any one of the antibodies, the antibody is a derivative. The term "derivative" refers to a chemically modified molecule including an insertion, deletion or substitution of an amino acid (or nucleic acid). In certain embodiments, the derivative comprises a covalent modification, including, but not limited to, chemical bonding with a polymer, a lipid or other organic or inorganic part. 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 polymer connections, including, but not limited to, polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. See, for example, U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. In certain embodiments, the 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.

[0356] In certain embodiments, the derivative is covalently modified with a polyethylene glycol (PEG) subunit. In certain embodiments, one or more water-soluble polymers are bonded to one or more specific positions of the derivative, such as the amino terminus. In certain embodiments, one or more water-soluble polymers are randomly connected to one or more side chains of the derivative. 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 in, for example, U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0357] Peptide analogs are commonly used in the medical industry as non-peptide drugs, and their properties are similar to those of the template peptide. Such types of non-peptide compounds are called "peptide mimetics" or "peptidomimetic". 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 usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically applicable peptides can be used to produce similar therapeutic or preventive effects. Typically, peptide mimetics are structurally similar to paradigm polypeptides (i.e., polypeptides with biochemical properties or pharmacological activity), such as human antibodies, but have one or more peptide connections that are optionally replaced by methods well known in the art through a connection selected from the following: -CH 2 NH-, -CH 2 S-、-CH 2 =CH 2 -、-CH-CH-(cis and trans), -COCH 2 -、-CH(OH)CH 2 - and - CH 2 SO-. In certain embodiments, systematic substitution of one or more amino acids of the common 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. In addition, constrained peptides comprising a common sequence or a substantially identical common 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.

[0358] Drug conjugation involves the coupling of a biologically active 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 all the way through the body and attaches itself to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs 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 lower side effects than other chemotherapeutic agents and gives a wider therapeutic window. The technology for conjugating antibodies is disclosed or known in the art (see, e.g., Jane de Lartigue OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al. Bioconjugate Chemistry 21(1):5-13 (2010)).

[0359] Antibody framework

[0360] 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. The human immunoglobulin framework may be part of a human antibody, or a non-human antibody may be humanized by replacing one or more endogenous frameworks with 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 common 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 (1 993)); 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)).

[0361] In some embodiments, an anti-SIRPA antibody of the invention comprises a heavy chain variable region comprising one or more (e.g., one or more, two or more, three or more, or all four) framework regions selected from VH FR1, VH FR2, VH FR3, and VH FR4 (as shown in Table 9). In some embodiments, an anti-SIRPA antibody of the invention comprises a heavy chain variable region comprising VH FR1, wherein VH FR1 comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, an anti-SIRPA antibody of the invention comprises a heavy chain variable region comprising VH FR2, wherein VH FR2 comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, an anti-SIRPA antibody of the invention comprises a heavy chain variable region comprising VH FR3, wherein VH FR3 comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, an anti-SIRPA antibody of the invention comprises 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] 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.

[0371] Nucleic acids, vectors and host cells

[0372] The anti-SIRPA antibodies of the invention can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids having a nucleotide sequence encoding any of the anti-SIRPA antibodies of the invention are provided. Such nucleic acids can encode V sequences comprising the anti-SIRPA antibodies. L The amino acid sequence and / or 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 cell comprises (e.g., has been transduced with): (1) a V encoding an antibody; L The amino acid sequence of the antibody V H or (2) a vector containing a nucleic acid encoding an amino acid sequence of an antibody; L A first vector containing a nucleic acid encoding an amino acid sequence of H In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (such as a Y0, NS0, 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.

[0373] Methods for making anti-SIRPA antibodies of the invention are provided. In some embodiments, the method comprises culturing a host cell of the 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).

[0374] For recombinant production of the anti-SIRPA antibodies of the 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 specifically binding to genes encoding the heavy and light chains of the antibody).

[0375] Suitable vectors comprising nucleic acid sequences 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 selected 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 genes for markers 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 can be purchased from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0376] 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, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, the antibodies can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

[0377] 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)).

[0378] 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. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0379] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be suitable. Other examples of suitable mammalian host cell lines are monkey kidney CV1 strain (COS-7) transformed by 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 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0380] Pharmaceutical compositions / formulations

[0381] Provided herein are pharmaceutical compositions and / or pharmaceutical formulations comprising an anti-SIRPA antibody of the invention and a pharmaceutically acceptable carrier.

[0382] 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, pharmaceutical compositions may include formulation materials such as pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration for modifying, maintaining or maintaining the composition.

[0383] 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 be any of a pharmaceutical composition comprising: ... Other examples of formulations suitable for various types of administration can be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press 22nd edition (2013). For a brief review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0384] 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.

[0385] The formulation can be optimized to be maintained and stabilized 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 linking to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. to achieve an increase in molecular weight.

[0386] Other strategies for increasing retention include embedding antibodies, such as the anti-SIRPA antibodies of the present invention, in biodegradable or bioerodible implants. The release rate of the therapeutically active agent is controlled by the transport rate through the polymer matrix and the biodegradation rate of the implant. The implant may be a particle, sheet, patch, plaque, fiber, microcapsule, etc. and may have any size or shape compatible with the selected insertion site. The biodegradable polymer composition that can be used may be an organic ester or ether that produces physiologically acceptable degradation products when degraded, including monomers. Anhydrides, amides, orthoesters, etc. may be used by themselves or in combination with other monomers. The polymer will be a condensation polymer. The polymer may be cross-linked or non-cross-linked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids, which may be 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 are calcium alginate and functionalized cellulose, particularly carboxymethyl cellulose esters, characterized by water insolubility, molecular weight of about 5 kD to 500 kD, etc. Biodegradable hydrogels may also be used in the implants of the present invention. Hydrogels are typically copolymer materials characterized by the ability to absorb liquids.

[0387] Therapeutic Uses

[0388] As disclosed herein, the anti-SIRPA antibodies of the invention can be used to prevent, reduce the risk of, or treat diseases and disorders.

[0389] 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) suffering from (or at risk of suffering from) 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.

[0390] As described in further detail below, an agent that downregulates SIRPA, such as an anti-SIRPA antibody, can be used in combination with an additional therapeutic agent for treating a disease or pathology associated with SIRPA expression, activity, or signaling. The terms "combination" and "in conjunction with" are used interchangeably in the present invention. 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.

[0391] In one aspect of the invention, an anti-SIRPA antibody preparation is administered to a human subject, which, for example, comprises 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 the antibody can eliminate or inhibit or interfere with the expression, activity, and / or signaling function of SIRPA mediated by ligand binding (e.g., CD47 binding).

[0392] 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 having 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 having a cancer that expresses CD47.

[0393] 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 cancer and gastrointestinal stromal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer (or carcinoma), gastric cancer, germ cell tumors, pediatric sarcomas, sinonasal natural killers, melanoma (e.g., metastatic malignant melanoma, etc. (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 syndrome, colorectal neoplasms, solid tumors, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma (Kaposi's 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), myel...

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 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; and the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 comprising the amino acid sequence of SEQ ID NO: 25, VH FR2 comprising the amino acid sequence of SEQ ID NO: 26, VH FR3 comprising the amino acid sequence of SEQ ID NO: 27, and VH FR4 comprising the amino acid sequence of SEQ ID NO:

28.

3. The antibody of claim 1, 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.

4. The antibody of claim 1, 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.

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

6. The antibody of claim 1 or 5, wherein the light chain variable region comprises an amino acid sequence that is at least 90% or at least 95% or at least 99% identical to an amino acid sequence selected from SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43 and 44.

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

8. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, 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.

9. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from 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.

10. 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).

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