EA044628B1 - ANTI-SIRPA ANTIBODIES AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross reference to related applications

The invention claims benefit to U.S. Provisional Patent Application No. 62/676,813, filed May 25, 2018, which is incorporated herein by reference for all purposes.

List of sequences

The present invention contains a sequence listing that has been provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on May 14, 2019, is called 40004-PCT_SL.txt and is 99798 bytes in size.

Field of technology to which the invention relates

The present invention relates to anti-SIRPA antibodies and the therapeutic use of such antibodies.

State of the art

Phagocytes, such as macrophages (MPs) and dendritic cells (DCs), distinguish healthy from abnormal cells through a complex array of cell surface receptors that modulate cellular activation status, proliferation, and/or effector functions. Many of these receptors recognize various ligands that either mark unwanted cells for removal (called eat me signals) or protect normal cells from destruction (called don't eat me signals). In recent years, the SIRPa-CD47 axis has emerged as a critical determinant in programmed cell elimination by macrophages in a variety of clinical settings, ranging from cancer cell survival to successful engraftment of hematopoietic cell transplantation. Therapeutic agents targeting this pathway may address a relevant medical need for disease relief, which is particularly relevant in many types of human cancer.

SIRPa (signal regulatory protein-a) belongs to the SIRP family of transmembrane receptors, which are predominantly expressed in the myeloid cell line (including MF, DC, granulocytes, etc.) and are characterized by an extracellular region containing 2 membrane-proximal IgC domains and a distal IgV domain. Unique among this family, SIRPa contains an intracellular cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM). Upon receptor cross-linking, tyrosine-phosphorylated ITIM sites recruit and activate SHP phosphatases to negatively regulate cellular functions such as phagocytosis or inflammatory cytokine release. CD47 serves as a major ligand for SIRPa, and its widespread expression in most cell types, including endothelial/epithelial cells, leukocytes, and erythrocytes, suggests that it mediates the don't eat me signal to protect healthy cells from phagocyte-dependent clearance. In support of this view, several studies show that adoptive transfer of red blood cells or leukocytes from CD47-knockout mice to wild-type recipients results in rapid clearance of CD47-deficient cells. In contrast, positional genetic analysis of multiple strains of immunocompromised mice receiving human hematopoietic cells identified the Sirpa allele in NOD mice as a causative factor for successful engraftment in xenotransplantation models. Subsequent studies showed that the SIRPa allelic variant expressed only in NOD mice retained the ability to bind human CD47 expressed on human hematopoietic stem cells and thereby suppress macrophage-dependent graft rejection.

The regulated expression of SIRPa and CD47 establishes a homeostatic control mechanism to modulate phagocyte activity. For example, apoptotic cells downregulate CD47 expression to promote engulfment by resident macrophages while living cells remain intact. Also, inflammatory stimuli such as LPS downregulate SIRPa expression in macrophages and dendritic cells (DCs) to potentiate their activation during inflammation. However, dysregulation of SIRPa and CD47 expression contributes to immunopathological diseases, as seen in cancer. Some tumors significantly upregulate CD47 expression relative to non-cancerous cells in order to evade immunosurveillance mechanisms that normally eliminate malignant cells. Preclinical studies show that genetic knockdown of CD47 in syngeneic tumor models such as B16F10 melanoma is sufficient to inhibit tumor growth in immunocompetent mice. Similar results were observed for colonies of CD47 knockdown human cancer cells transplanted into immunocompromised mice. Alternatively, biological agents that disrupt the SIRPa-CD47 interaction, such as anti-CD47 antibodies, also improve tumor clearance in mouse models. When combined with commercial antitumor antigen antibodies such as trastuzumab or rituximab, anti-CD47 antibodies synergistically enhance the antitumor response compared with standard monotherapy. Additionally, given the ubiquitous expression of CD47, anti-CD47 antibodies risk a heavy toxic burden due to side effects that limit their therapeutic efficacy. However, these studies have established the critical importance of the SIRPa-CD47 pathway in regulating myeloid cells with potential applications in cancer immunotherapy.

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Anti-SIRPA antibodies have previously been described in, for example, International Patent Application Publications No.: WO 2018/057669, WO 2018/026600, WO 2017/178653, WO 2017/068164, WO 2016/063233,

WO 2016/205042, WO 2015/138600, WO 2013/0956352, WO 2009/091547, WO 2009/131453 and WO

2009/046541.

Accordingly, there is a need for therapeutic anti-SIRPA antibodies to treat diseases, disorders and conditions associated with undesirable SIRPA activity.

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

The essence of the invention

The present invention is generally directed to compositions that include antibodies, eg, monoclonal, chimeric, humanized antibodies, antibody fragments, etc., that specifically bind human SIRPA, and methods of using such compositions.

Certain aspects of the present invention are based, at least in part, on the identification of anti-SIRPA antibodies with improved and/or enhanced functional characteristics (e.g., relative to an anti-SIRPA antibody with a heavy chain variable region containing 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 SIRPA levels, and/or have improved and/or enhanced binding kinetics, and/or have improved and /or enhanced KD, and/or have improved and/or enhanced EC50 values In some embodiments, the anti-SIRPA antibodies of the present invention have a KD for human SIRPA that is at least 2-fold, at least 3-fold multiple, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or is 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 antibody of the present invention has a CD for human SIRPA of less than 6, less than 5, less than 4, less than 3, less than 2, less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6 or less than 0.5 nM.

In some embodiments, the anti-SIRPA antibodies of the present invention reduce cell surface levels of SIRPA expression in vitro with an EC50 of at least about 20, at least about 30, at least about 40, or at least about 40 , about 50% lower than an anti-SIRPA antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID No.: 2 and a light chain variable region containing the amino acid sequence of SEQ ID No.: 5. Preferably, the anti-SIRPA antibody of the present invention reduces cellular levels of SIRPA in vitro with half the maximum effective concentration (EC50), which ranges from about 0.4 to about 0.5 nM. Preferably, the anti-SIRPA antibody of the present invention has a dissociation constant (CD) for human SIRPA that ranges from about 0.6 to 0.7 nM.

In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention binds human SIRPA v1. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention binds human SIRPA v2. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention binds human SIRPB (SIRPbeta) v3. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention does not bind human SIRPB v1. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention does not bind murine SIRPA. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention does not bind human SIRPy. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention binds cynomolgus SIRPA. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention does not bind cynomolgus SIRPB 1. In some embodiments, which may be combined with any of the embodiments herein, the anti-SIRPA antibody of the present invention binds marmoset SIRPA.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody binds human SIRPA, human SIRPA v1, human SIRPA v2, cynomolgus SIRPA, marmoset SIRPA, and human SIRPe3.

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In some embodiments, which may be combined with any of the embodiments herein, the present 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 containing the amino acid sequence SEQ ID No: 20; HVR-H2 containing the amino acid sequence SEQ ID No: 21; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 22, 23 and 24.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 containing the amino acid sequence SEQ ID No: 9; HVR-L2 containing the amino acid sequence SEQ ID NO: 10; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 containing the amino acid sequence SEQ ID No: 20; HVR-H2 containing the amino acid sequence SEQ ID No: 21; HVR-H3 containing an amino acid sequence selected from SEQ ID NO: 22, 23 and 24; HVR-L1 containing the amino acid sequence SEQ ID NO: 9; HVR-L2 containing the amino acid sequence SEQ ID NO: 10; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 a sequence of amino acids, at least 90%, or at least 95%, or at least 99% identical to the amino acid sequence selected from SEQ ID NO: 33, 34 and 35.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 a sequence of amino acids, at least 90%, or at least 95%, or at least 99% identical to the amino acid sequence selected from SEQ ID NO: 36, 37, 38, 39, 40, 41, 42 , 43 and 44.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 contains one, two , three or four framework regions selected from VH FR1 containing the amino acid sequence SEQ ID No: 25, VH FR2 containing the amino acid sequence SEQ ID No: 26, VH FR3 containing the amino acid sequence SEQ ID No: 27 and VH FR4 containing the sequence amino acids SEQ ID No: 28.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 contains one, two , three or four framework regions selected from VL FR1 containing the amino acid sequence SEQ ID No: 29, VL FR2 containing the amino acid sequence SEQ ID No: 30, VL FR3 containing the amino acid sequence SEQ ID No: 31 and VL FR4 containing the sequence amino acids SEQ ID No: 32.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 contains one, two , three or four framework regions selected from VH FR1 containing the amino acid sequence SEQ ID No: 25, VH FR2 containing the amino acid sequence SEQ ID No: 26, VH FR3 containing the amino acid sequence SEQ ID No: 27 and VH FR4 containing the sequence amino acids SEQ ID No: 28; and wherein the light chain variable region comprises one, two, three or four framework regions selected from VL FR1 containing the amino acid sequence of SEQ ID No: 29, VL FR2 containing the amino acid sequence of SEQ ID No: 30, VL FR3 containing the amino acid sequence of SEQ ID No: 31 and VL FR4 containing the amino acid sequence SEQ ID No: 32.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds

- 3 044628 human SIRPA, wherein the antibody contains a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains an amino acid sequence selected from SEQ ID Nos: 33, 34 and 35.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 a sequence of amino acids, selected from SEQ ID Nos: 36, 37, 38, 39, 40, 41, 42, 43 and 44.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 a sequence of amino acids, selected from SEQ ID Nos: 33, 34 and 35, and wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID Nos: 36, 37, 38, 39, 40, 41, 42, 43 and 44.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 from antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9- 23, 3F9-24 or 3F9-25 (as shown in Table 8).

In some embodiments, which may be combined with any of the embodiments herein, the present 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 from antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9- 23, 3F9-24 or 3F9-25 (as shown in Table 7).

In some embodiments, which may be combined with any of the embodiments herein, the present 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 from antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9- 23, 3F9-24 or 3F9-25 (as shown in Table 8); and wherein the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3 from 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).

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region containing HVRH1, HVR-H2, and HVR-H3, and a variable light chain region containing HVR-L1, HVR-L2 and HVR-L3, where the antibody contains HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 from antibody 3F9-1, 3F92 , 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 and 8).

In some embodiments, which may be combined with any of the embodiments herein, the present 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 contains one, two , three or four framework regions selected from VH FR1, VH FR2, VH, Fr3 and VH FR4, where: VH FR1 contains the amino acid sequence of SEQ ID No: 25; VH FR2 contains the amino acid sequence SEQ ID No: 26; VH FR3 contains the amino acid sequence SEQ ID No: 27; and VH FR4 contains the amino acid sequence SEQ ID NO: 28; and/or the light chain variable region contains one, two, three or four framework regions selected from VL FR1, VL FR2, VL FR3 and VL FR4, wherein VL FR1 contains the amino acid sequence of SEQ ID No: 29; VL FR2 contains the amino acid sequence SEQ ID No: 30; VL FR3 contains the amino acid sequence SEQ ID No: 31; and VL FR4 contains the amino acid sequence SEQ ID NO: 32.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 a light chain the chain contains the amino acid sequence SEQ ID No: 50.

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

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 sequence amino acids SEQ ID No: 50.

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

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

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

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

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

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds

- 5 044628 human SIRPA, where the antibody binds to the inhibitory Fc receptor. In some embodiments, which may be combined with any of the embodiments herein, the Fc inhibitory receptor is Fc receptor gamma inhibitory receptor IIB (FcyRIIB).

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is of a human or murine IgG1 isotype and contains one or more amino acid substitutions in the Fc region on 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, where the numbering of amino acid residues is carried out according to the EU numbering, or contains a deletion of an amino acid in the Fc region at position , corresponding to glycine 236.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody contains one or more amino acid substitutions in the Fc region at an amino acid residue selected from the group consisting of: C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y and any combination thereof, where the numbering of amino acid residues is carried out according to the EU numbering.

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody causes degradation of SIRPA, causes degradation of SIRPA, causes internalization of SIRPA, causes shedding of SIRPA, causes negative regulation of SIRPA expression or any combination thereof.

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

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody negatively regulates SIRPA expression in human monocytes.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody negatively regulates SIRPA expression in human macrophages.

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody has an affinity (A.D.) for human SIRPA of less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 nM.

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody has an affinity for human SIRPA with a CD that is at least 2-fold at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9-fold or at least 10-fold lower than the affinity for human SIRPA of an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 2, and a light chain variable region containing the amino acid sequence

SEQ ID NO: 5.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 to 2 nM or from about 0.4 to about 0.5 nM, as determined by flow cytometry.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 SIRPA, as determined by flow cytometry.

In some embodiments, which may be combined with any of the embodiments herein, the present 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.

In some embodiments, which may be combined with any of the embodiments herein, the present 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.

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

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

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

In some embodiments, which may be combined with any of the embodiments herein, the present 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.

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody negatively regulates cell surface expression of CD14.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody improves T cell proliferation without blocking the interaction of SIRPy and CD47.

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

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

In some embodiments, which may be combined with any of the embodiments herein, the present 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.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds

- 7 044628 human SIRPA, where the antibody binds human SIRPA, but does not substantially block the binding of CD47 to SIRPA.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody recognizes a first and a second antigen, wherein the first antigen is SIRPA and the second antigen is:

(a) an antigen that promotes transport across the blood-brain barrier;

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

(c) a disease-causing agent selected from disease-causing peptides or disease-causing nucleic acid proteins, wherein the disease-causing nucleic acids are antisense GGCCCC (G2C4) RNA repeat expansions, the disease-causing proteins are selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques , amyloid precursor protein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, peroxide 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, medina, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, non-ATG repeat-associated (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptides , glycine-arginine repeat (GR) peptides, proline-alanine (PA) repeat peptides, ubiquitin and proline-arginine (PR) repeat peptides; and (d) ligands and/or proteins expressed on immune cells, where 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 a protein, lipid, polysaccharide or glycolipid expressed on one or more tumor cells.

In some embodiments, which may be combined with any of the embodiments herein, the present 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, protein transduction domain, TAT, Syn-B, penetratin, polyarginine peptide, angiopep peptide, and ANG1005.

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

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

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the anti-SIRPA antibody is conjugated to a toxin selected from ricin, ricin A chain, doxorubicin, daunorubicin, maytansinoid , taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracinedione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modezzan A chain, alpha sarcin, gelonin, mitogellin , restrictictocin, phenomycin, enomycin, chicken, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycin, dolastatin, CC1065 and cisplatin.

In some embodiments, which may be combined with any of the embodiments herein, the present 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 a disease-causing protein selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein , PrPSc, huntingtin, calcitonin, peroxide dismutase, ataxin,

- 8 044628 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, medina, prolactin, transthyretin, lysozyme, beta 2 microglobulins, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, non-ATG repeat-associated (RAN) translation product, dipeptide repeat (DPR) peptide, glycine alanine (GA) repeat peptide, glycine-proline peptides ( GP) repeat, glycine-arginine repeat (GR) peptides, proline-alanine (PA) repeat peptides, ubiquitin and proline-arginine (PR) repeat peptides and any combination thereof; or with one or more antibodies that bind an immunomodulatory protein selected from the group consisting of: PD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2 , PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73 , TREM1, TREM2, CD33, Siglec-5, Siglec-7, Siglec-9, Siglec-11, phosphatidylserine, disease-causing nucleic acids, antisense GGCCCC (G2C4) repeat expansion RNAs, and any combination thereof.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method for preventing, reducing the risk of, or treating cancer, wherein the method includes administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, thereby thereby providing cancer treatment. In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method of preventing, reducing the risk of, or treating an individual having a disease, disorder, or injury, wherein the disease, disorder, or injury is cancer, wherein the method includes administering to the individual a therapeutic an effective amount of the anti-SIRPA antibody of the present invention, thereby preventing, reducing the risk of, or providing treatment to the individual.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method includes administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, wherein the cancer is selected from sarcoma, cancer bladder, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, small cell lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer and fibrosarcoma, glioblastoma multiforme; renal clear cell carcinoma; adrenocortical carcinoma; urothelial carcinoma of the bladder, diffuse large B-cell lymphoma, lung adenocarcinoma; pancreatic adenocarcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, slow-growing B-cell lymphoma, aggressive B-cell lymphoma, T-cell lymphoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia ( Slymphocytic leukemia) chronic myeloid leukemia (CML), multiple myeloma, myelodysplastic syndromes, myeloproliferative neoplasms, invasive breast carcinoma, squamous cell carcinoma of the cervix, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, squamous cell carcinoma of the neck, diffuse B-large cell lymphoma, esophageal carcinoma, squamous cell carcinoma of the head and neck, chromophobe kidney cancer, papillary kidney cancer, low-grade glioma, hepatocellular carcinoma, squamous cell carcinoma of the lung, mesothelioma, serous cystadenocarcinoma of the ovary, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma , cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumors, thyroid carcinoma, thymoma, endometrial carcinoma of the uterine body, uterine carcinosarcoma and uveal melanoma.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method includes administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, wherein the method further includes administering a therapeutic agent , which inhibits PD1, PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, CTLA4, PDL2, 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, which 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.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method includes administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, wherein the method includes administering to an individual in need of at least one antibody that specifically binds to an inhibitory immune checkpoint molecule,

- 9 044628 and/or one or more standard or experimental anticancer therapies. In some embodiments, which may be combined with any of the embodiments herein, at least one antibody that specifically binds to an inhibitory immune checkpoint molecule is administered in combination with an anti-SIRPA antibody. In some embodiments, which may be combined with any of the embodiments herein, at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, L2 antibodies, anti-PD-1 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies and anti-HVEM antibodies, anti-B and T lymphocyte attenuator (BTLA) antibodies, anti-killer inhibitory receptor (KIR ) antibodies, anti-GAL9 antibodies, anti-T1M-1 antibodies, anti-TIM3 antibodies, anti-TIM-4 antibodies, anti-A2AR antibodies, anti-CD39 antibodies, anti-CD73 antibodies, anti-LAG-3 antibodies, anti -phosphatidylserine antibody, anti-CD27 antibody, anti-CD30 antibody, anti-TNFa antibody, anti-CD33 antibody, anti-Siglec-5 antibody, anti-Siglec7 antibody, anti-Siglec-9 antibody, anti-Siglec-11 antibody, antagonistic anti-TREM1 antibody, antagonistic anti-TREM2 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-CD2 antibody, anti-CD5 antibody, and any combination thereof.

In some embodiments, which may be combined with any of the embodiments herein, one or more standard or investigational anticancer therapies are selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer therapy (ACT) , chimeric antigen receptor T cell transfer (CAP-T) therapy, vaccine therapy and cytokine therapy.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method includes administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, wherein the method further includes administering to an individual, at least one antibody that specifically binds to the inhibitory cytokine. In some embodiments, which may be combined with any of the embodiments herein, at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with an anti-SIRPA antibody. In some embodiments, which may be combined with any of the embodiments herein, 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 their combinations.

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

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides a method of treating cancer, wherein the method includes administering to an individual in need thereof a therapeutically effective amount of an anti-SIRPA antibody of the present invention, wherein the method further includes administering to an individual, at least one stimulatory cytokine. In some embodiments, the at least one stimulatory cytokine is selected from IFN-a4, IFN-β, ΓΕ-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, IL33, MCP-1, MIP-1-beta and any of them combination.

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

In some embodiments, the present invention provides a method of producing an antibody that binds to human SIRPA, where the method includes culturing a host cell containing a vector containing an isolated nucleic acid containing a nucleic acid sequence encoding an anti-SIRPA antibody of the present invention, so that the antibody is obtained . The present invention also provides recovery of an antibody produced by a cell.

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 drawings

In fig. 1 - aligned section of the amino acid sequence of the extracellular domains of human SIRPA variant 1 and SIRPA proteins from different species. The recorded percent identity shows high homology in the extracellular region. Accession numbers: P78324 (human SIRPA variant 1), XP015313153 (cynomolgus macaque), JAB51896 (monkey), G1U0I5 (rabbit) and XP005634938 (dog). In fig. 1, SEQ ID Nos. 59-63 are described, respectively, in order of appearance.

In fig. 2A lists potential humanized sequences of the heavy chain variable domain of the anti-SIRPA antibody 3F9. The humanized sequence is based on the IGHV3-23*01 acceptor framework region and the IGHJ4*01 junction region. In fig. 2A discloses SEQ ID Nos. 64-65, 2, 64 and 3-4, respectively, in order of appearance. In fig. 2B lists potential humanized light chain variable domain sequences of the anti-SIRPA antibody 3F9. In fig. 2B discloses SEQ ID Nos. 66-67, 5, 66, and 68-70, respectively, in order of appearance.

In fig. 3 presents data showing antibody-mediated down-regulation of SIRPA on primary human macrophages by murine and humanized anti-SIRPA 3F9 antibodies.

In fig. 4A presents data showing the dependence of antibody-mediated receptor down-regulation on N-linked Fc glycan. In fig. 4B presents data showing the dependence of antibody-mediated improvement in tumor cell phagocytosis on N-linked Fc glycan.

In fig. 5A presents data showing humanized anti-SIRPA 3F9 IgG4 antibody variants that failed to induce phagocytosis of tumor cells. In fig. 5B presents data showing humanized anti-SIRPA 3F9 IgG4 antibody variants that failed to down-regulate CD14 expression on macrophages.

In fig. 6A shows that treatment with the anti-SIRPA antibody 3F9 mIgG1 negatively regulates CD32A/B expression on primary human macrophages. In fig. 6B shows that treatment with the anti-SIRPA antibody 3F9 negatively regulates the expression of CD32A and CD32B on primary human macrophages.

In fig. 7A presents data showing that CD32A/B blockade inhibits anti-SIRPA antibody 3F9-mediated down-regulation of SIRPA on primary human macrophages. In fig. 7B presents data showing that blockade of CD32A/B inhibits the anti-SIRPA antibody 3F9-induced improvement in tumor cell phagocytosis.

In fig. 8 presents data showing that the CD32A allotype influences anti-SIRPA antibody 3F9-mediated down-regulation of SIRPA. In fig. 8A shows that donors 507 and 508 are CD32A-R/H131 heterozygous; in fig. 8B shows that donors 516 and 517 are CD32AH/H131 homozygous.

In fig. 9A, 9B provide data showing that antibody-mediated down-regulation of the receptor leads to degradation of SIRPA. In fig. 9A, 5, 10 and 20 μg of whole cell lysate were loaded onto SDS-PAGE and immunoblotted with anti-SIRPA cytoplasmic domain (aSIRPAct). In fig. 9B 20 μg of whole cell lysate was loaded onto SDS-PAGE, probed with a-SIRPAct and anti-SHP2.

In fig. 10A, 10B present data comparing the improvement in phagocyte activity of different Fc variants of the anti-SIRPA antibody 3F9-22. In fig. 10A, primary human macrophages are treated with anti-SIRPA 3F9-22 antibody on wild-type human IgG1 backbone or human IgG1 carrying the S267E/L328F Fc mutation or isotype control antibody. In fig. 10B, primary human macrophages are treated with anti-SIRPA 3F9-22 antibody on a human IgG1 backbone carrying the S267E/L328F or A330S/P331S Fc mutation on an isotype control antibody.

In fig. 11A, 11B present data comparing the activity of phagocytes of different Fc variants of the anti-SIRPA 3F9-22 antibody in relation to anti-CD47 on M1 (Fig. 11B) and M2 (Fig. 11A) macrophages.

In fig. 12A and FIG. 12B presents data showing that anti-SIRPA treated macrophages with anti-11 044628 body 3F9-22 reduce the viability of solid tumor cells in vitro.

In fig. 13A shows data comparing the improvement of T cell proliferation by different Fc variants of the anti-SIRPA antibody 3F9-22 in a 2-factor MLR analysis. In fig. 13B shows data comparing the improvement of T cell proliferation by different Fc variants of anti-SIRPA antibody 3F9-22 and anti-SIRPA antibody 3F9-14 compared to anti-CD47 IgG4 in a 2-factor MLR assay.

In fig. 14A shows data comparing the improvement of T cell proliferation by different Fc variants of anti-SIRPA antibody 3F9-22 relative to anti-CD47 IgG4 in a 1-factor MLR assay. In fig. 14B shows a typical SCAF plot from a 1-factor MLR analysis.

In fig. 15A and FIG. 15B shows data comparing the improvement in TNF release by different Fc variants of the anti-SIRPA antibody 3F9-22 in LPS-primed macrophages.

In fig. 16 presents data showing the identification and visualization of critical residues for an anti-SIRPA antibody in a model of human SIRPA.

In fig. 17A-17C show data showing the effect of anti-SIRPA antibody 3F9-22 on tumor growth inhibition in combination with anti-PD-L1 antibody in a synergistic mouse colon carcinoma model. In fig. 17A shows SCAF histograms showing the expression of mouse CD47 (top panel) and human CD47 (bottom panel) in wild-type and engineered MC38 cell colonies. In fig. 17B shows data showing average tumor growth curves in huSIRPA/huCD47 BAC transgenic mice subcutaneously implanted with MC38 cells engineered to deplete murine CD47 expression and overexpressing human CD47 (MC38-mCD47KO/huCD47+). In fig. 17C shows line graphs of each individual animal in each treatment group from FIG. 17V.

In fig. 18A, 18B provide data showing the kinetics of antibody-mediated down-regulation of SIRPA in myeloid cells from BAC tumor transgenic mice treated with increasing doses of the anti-SIRPA antibody of the present invention. In fig. 18A presents data showing the relative change in SIRPA expression over time on tumor-infiltrating myeloid cells. In fig. 18B presents data showing the relative change in SIRPA expression over time on spleen myeloid cells.

In fig. 19A shows treatment conditions in an anti-SIRPA antibody-mediated down-regulation of SIRPA study in humanized NOG-EXL mice bearing A375 tumors. In fig. 19B presents data showing the expression level of SIRPA on human tumor macrophages as MPC values (left panel) or normalized values (right panel) associated with treatment with anti-SIRPA antibodies of the present invention.

In fig. 20A, 20B show the expression levels of the M1 macrophage markers HLA-DR/MHC class 2 (FIG. 20A) and CD86 (FIG. 20B) on human tumor macrophages isolated from humanized NOG-EXL mice bearing A375 tumors after treatment with anti-SIRPA antibodies of the present invention. In fig. 20A and FIG. 20B presents data showing the expression of HLA-DR/MHC class 2 and CD86, respectively, as UPC values (left panels) or normalized values (right panels).

In fig. 21A, 21B show down-regulation of human SIRPA in splenic monocytes (FIG. 21A) and peripheral blood monocytes (FIG. 21B) isolated from humanized NOGEXL mice bearing A375 tumors associated with administration of the anti-SIRPA antibodies of the present invention. In fig. 21A and FIG. 21B presents data showing human SIRPA expression as UPC values (left panels) or normalized values (right panels).

In fig. 22 presents data showing antibody-mediated down-regulation of SIRPA on primary human macrophages by various Fc variants of anti-SIRPA 3F9-22 antibodies.

In fig. 23 presents data comparing the improvement in phagocyte activity of various Fc variants of the anti-SIRPA antibody 3F9-22 compared with anti-CD47 blocking antibodies.

In fig. 24 shows data showing red blood cell and platelet counts in cynomolgus monkeys administered control IgG antibody, anti-SIRPA antibody 3F9-22 PS, anti-SIRPA antibody 3F9-22 IgG4, anti-SIRPA antibody 3F9-IgG2, and anti-SIRPA antibody 3F9-22 NSLF.

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 invention will become apparent to one skilled in the art. These and other embodiments of the invention are further described in the detailed description that follows.

Detailed description

This invention relates to anti-SIRPA antibodies (eg, monoclonal antibodies); methods for obtaining and using such antibodies; pharmaceutical compositions containing such antibodies; nucleic acids encoding such antibodies; and host cells containing nucleic acids encoding such antibodies.

The methods and procedures described or referred to in this document are generally well understood and

- 12 044628 are typically used by those skilled in the art using traditional methodology, such as, for example, the widely used methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); Series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

I. Definitions.

The terms SIRPa or SIRPa polypeptide or SIRPA or SIRPA polypeptide, used interchangeably herein, refer to any naturally occurring SIRPA from any vertebrate source, including mammals such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rats) ), unless otherwise stated. In some embodiments, the term includes both wild-type sequences and naturally occurring variant sequences, such as splice variants or allelic variants. In some embodiments, the term includes full-length unprocessed SIRPA as well as any form of SIRPA that is produced by in-cell processing. In some embodiments, the SIRPA is a human SIRPA. In some embodiments, the amino acid sequence of the generic SIRPA is Uniprot accession No. P78324 dated April 25, 2018. In some embodiments, the amino acid sequence of the generic human SIRPA v1 is SEQ ID No: 1. In some embodiments, the amino acid sequence of the generic human SIRPA v2 is GenBank CAA71403.

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

With respect to the binding of an antibody to a target molecule, the term specific binding either specifically binds or is specific for a particular polypeptide or epitope on a particular polypeptide target means binding which is distinctly different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding may be determined by competition with a control molecule that is similar to the target, for example, an excess of unlabeled target. In this case, specific binding is observed if the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled target. The term specific binding or specifically binds to or is specific for a particular polypeptide or epitope on a particular polypeptide target, as used herein, may be indicated, for example, by a molecule having a CD for the target of about any 10 ^-4 or lower, 10 ^-5 or lower , 10 ^-6 or lower, 10 ^-7 or lower, 10 ^-8 or lower, 10 ^-9 or lower, 10 ^-10 or lower, 10 ^-11 or lower, 10 ^-12 M or lower, or CD in the range of 10 ^-4 to 10 ^-6 M or from 10 ^-6 to 10 ^-10 M or from 10 ^-7 to 10 ^-9 M. As will be clear to those skilled in the art, affinity and CD values are inversely related. High affinity for an antigen is measured by a low CD value. In one embodiment, the term specific binding refers to binding where the molecule binds to a specific polypeptide or epitope of a specific polypeptide without significantly binding to any other polypeptide or polypeptide epitope.

The term immunoglobulin (Ig) is used interchangeably with antibody throughout this document.

- 13 044628

The term antibody as used herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), including those derived from at least two intact antibodies and antibody fragments to the extent that they exhibit the desired biological activity.

Native antibodies are typically heterotetrameric glycoproteins of about 150,000 Daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while multiple disulfide bonds vary among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has evenly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; the light chain constant domain is the same as the first heavy chain constant domain, and the light chain variable domain is the same as the heavy chain variable domain. It is believed that specific amino acid residues form the interface between the light chain and heavy chain variable domains.

The structure and properties of different classes of antibodies are presented, for example, in Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and chapter 6.

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

The variable region or variable domain of an antibody, such as the anti-SIRPA antibody of the present invention, refers to the amino-terminal domains of the heavy and light chain of the antibody. The heavy chain and light chain variable domains may be designated VH and VL, respectively. These domains are usually the most variable parts of the antibody (relative to other antibodies of the same class) and contain antigen-binding sites.

The term variable refers to the fact that certain segments of the variable domains vary significantly in sequence among antibodies, such as the anti-SIRPA antibodies of the present invention. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, variability is not distributed evenly across the entire length of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in the variable domains of both the light chain and the heavy chain. The more conserved portions of variable domains are called framework regions (FRs). Each of the native heavy and light chain variable domains contains four KO regions, generally adopting a beta sheet configuration, connected by three HVRs that form loops connecting, and in some cases, forming part of the beta sheet structure. HVRs on each strand are held together in close proximity by KO regions and, with HVRs from the other strand, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991) )). The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cellular toxicity.

The term monoclonal antibody as used herein refers to an antibody, such as the anti-SIRPA monoclonal antibody of the present invention, derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or or post-translational modifications (eg, isomerizations, amidations, etc.), which may be present in minor quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by a hybridoma culture, without the admixture of other immunoglobulins. The modifier monoclonal denotes the nature of the antibody as being derived from an essentially homogeneous population of antibodies, and should not be construed as requiring

- 14 044628 obtaining an antibody in any specific way. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of methods, including, for example, the hybridoma method, recombinant DNA methods, and methods for producing human or human-like antibodies in animals that have part or all of the human immunoglobulin loci or genes. encoding the sequence of human immunoglobulin. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, for example, phage display technologies (see, for example, 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 method (eg, Kohler and Milstein., Nature, 256:495-97 (1975); Hongoet al., Hybridoma, 14 (3):253-260 (1995) , Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)) , recombinant DNA techniques (see, for example, US Patent No. 4,816,567), yeast presentation technologies (see, for example, WO 2009/036379A2; WO 2010105256; WO 2012009568 and Xu et al., Protein Eng. Des. Sel., 26(10): 663-70 (2013) and techniques for producing human and human-like antibodies in animals that have parts or all of human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, WO 1998/24893 ; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 ( 1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US 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 10:7797 83 (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).

The terms full-length antibody, intact antibody, or whole antibody are used interchangeably to refer to an antibody, such as the anti-SIRPA antibody of the present invention, in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include antibodies with heavy and light chains including the Fc region. The constant domains may be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

Antibody fragments refer to a molecule, other than the intact antibody, that contains a portion of the intact antibody that binds the antigen to which the intact antibody is bound. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; diabodies; linear antibodies (see US patent No. 5641870, example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules and polyspecific antibodies formed from antibody fragments.

Papain digest 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, where the designation reflects the ability to readily crystallize. The Fab fragment consists of a complete light chain together with a heavy chain variable domain ( _VH ) and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has one antigen binding site. Pepsin treatment of the antibody produces one large F(ab') ₂ fragment, which roughly corresponds to two disulfide-linked Fab fragments that have different antigen-binding activities and are also capable of antigen cross-linking. Fab' fragments differ from Fab fragments in that they have several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a designation for Fab' in which the cysteine residues of the constant domains bear three thiol groups. F(ab')2 antibody fragments are initially produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical combinations of antibody fragments are also known.

The Fc fragment contains the carboxy-terminal portions of both heavy chains, which are held together by disulfides. Antibody effector functions are determined by sequences in the Fc region, a region that is also recognized by Fc receptors (FcRs) found on certain cell types.

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

The term diabody refers to a small antibody fragment obtained by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the _VH and VL domains so that interchain rather than intrachain pairing of the variable domains is achieved, thereby obtaining a divalent fragment, i.e. fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two crossover sFv fragments, in which the VH and _VL domains of the two antibodies are present on different polypeptide chains.

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

Humanized forms of non-human (eg, murine) antibodies, such as the humanized forms of anti-SIRPA antibodies of the present invention, are chimeric antibodies containing amino acid residues from non-human HVRs and amino acid residues from human KOs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the HVRs (e.g., HVRs) correspond to the same of the non-human antibody, and all or essentially all KOs correspond to the same human antibodies. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A humanized form of an antibody, for example, a non-human antibody, refers to an antibody that has been humanized.

A human antibody is one that has an amino acid sequence corresponding to that of an antibody, such as the anti-SIRPA antibody of the present invention, produced by a human and/or produced using any technique for producing a human antibody as disclosed herein. This definition of a human antibody intentionally excludes a humanized antibody containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries and yeast display libraries. Human antibodies can be produced by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge but whose endogenous loci have been disabled, such as an immunized xenomouse, and also generated through human B-cell hybridoma technology. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be produced by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, for example, an immunized xenomouse (see, for example, US Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology ). See also, for example, Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated through human B-cell hybridoma technology. Alternatively, human antibodies can also be produced using yeast libraries and methods described in, for example, WO 2009/036379A2; WO 2010105256; WO 2012009568; and Xu et al., Protein Eng. Des. Sel., 26(10): 663-70 (2013).

The term hypervariable region, HVR or HV as used herein refers to regions of the variable domain of an antibody, such as the anti-SIRPA antibody of the present invention, that are hypervariable in sequence and/or form structurally defined loops. Antibodies typically contain six HVRs; three in V _H (H1, H2, H3) and three in V _L (L1, L2, L3). In native antibodies, H3 and L3 show the greatest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring high specificity to antibodies. Naturally occurring camelide antibodies, consisting of only the heavy chain, are functional and stable in the absence of the light chain.

A variety of HVR transdifferentiations are used and covered here. In some embodiments, HVRs may be Cabot complementarity determining regions (CDDs) based on sequence variability, and are most commonly used (Kabat et al., supra). In some embodiments, the HVRs may be Chotia OKO. Chothia instead refers to the arrangement of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, the HVRs may be AbM HVRs. AbM HVR represent a compromise between Cabot OKO and

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Chotia structural loops and are used in Oxford Molecular's AbM antibody modeling program. In some embodiments, the HVRs may be contact HVRs. Contact HVRs are based on the analysis of available complex crystal structures. Residues from each of these HVRs are listed below.

A loop Cabot ABM Chotia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 Н31-Н35В Н26-Н35В H26-H32 H3O-H35B (Cabot numbering) H1 H31-H35 H26-H35 H26-H32 H3O-H35 (Chotia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 FROM N95-N102 N95-N102 N96-N101 N93-N101

HVRs may contain stretched HVRs as follows: 24-36 or 24-34 (L1), 46-56 or 5056 (L2) and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50- 65 or 49-65 (preferred embodiment) (H2) and 93-102, 94-102 or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al., supra for each of these stretched HVR definitions.

Framework or KO residues are those variable domain residues that differ from HVR residues as defined herein.

The phrase EU or Cabot variable domain residue numbering or [0076] EU or Cabot amino acid position numbering and variants thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains of 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 the reduction or insertion into the KO or HVR of the variable domain. For example, a variable domain heavy chain may include one amino acid insertion (Cabot residue 52a) after H2 residue 52 and inserted residues (eg, Cabot residues 82a, 82b, and 82c, etc.) after heavy chain residue 82 KO. EU or Cabot residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with standard Cabot sequence numbering.

The EU and Kabat numbering system is typically used when referring to a residue in the [0077] variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The EU or Kabot numbering system or EU index is usually applied to a residue in the immunoglobulin heavy chain constant region (eg, EU index described by Kabat et al., supra). Cabot's EU index refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in the antibody variable domain indicate residue numbering using the Cabot numbering system. Unless otherwise noted herein, references to residue numbers in an antibody constant domain refer to residue numbering using the EU or Cabot numbering system (eg, see US Patent Publication No. 2010-280227).

An acceptor human framework region, as used herein, is a framework region comprising a V _L or V _H amino acid sequence framework region derived from a human immunoglobulin framework region or a human consensus framework region. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence thereof, or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. If pre-existing amino acid changes are present in VH, preferably such changes occur only at three, two or one positions 71H, 73H and 78H; for example, the amino acid residues at these positions may be 71A, 73T and/or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to the _VL human immunoglobulin framework sequence or the human consensus framework sequence.

A human consensus framework region is a framework region that represents the most commonly existing amino acid residues in a selection of human V _L or V _H immunoglobulin framework sequences. In general, selection of V _L or V _H human immunoglobulin sequences occurs from a subset of variable domain sequences. In general, a 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). For example, for V _L , the subgroup could be kappa I, kappa II, kappa III, or kappa

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IV as in Kabat et al., supra. Additionally, for VH, the subgroup may be subgroup I, subgroup II or subgroup III as in Kabat et al., supra.

Modification of an amino acid at a specific position, for example, the anti-SIRPA antibody of the present invention, refers to the substitution or deletion of a specific residue or the insertion of at least one amino acid residue adjacent to a specific residue. An insertion adjacent to a specific residue means an insertion within one or two residues. The insertion may be N-terminal or C-terminal to a specific residue. The preferred amino acid modification here is substitution.

An affinity mature antibody, such as an affinity mature anti-SIRPA antibody of the present invention, is an antibody with one or more changes in its HVR that provide an improvement in the affinity of the antibody for an antigen, compared to a parent antibody that does not have such changes. In one embodiment, the affinity mature antibody has nanomolar or even picomolar affinities for the target antigen. Affinity mature antibodies are prepared by techniques known in the art. For example, Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation through _VH and VL domain rearrangement. Random mutagenesis of HVR and/or framework residues is described in, for example: 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).

Fv is the minimal antibody fragment that contains the complete antigenic recognition and binding site. This fragment consists of a dimer of one heavy and light chain variable region domain in close non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops from each, H and L chains) are obtained that use amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, although with lower affinity than the full binding site.

Single-chain Fvs, also called sFvs or scFvs, are antibody fragments that contain the VH and VL domains of the antibody linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V _H and V _L domains, which allows the sFv to form the desired structure for antigen binding.

The effector functions of an antibody relate to the biological activity inherent in the Fc region (native Fc region sequence or variant Fc region amino acid sequence) of the antibody and vary depending on the isotype of the antibody.

The term Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the heavy chain of the Fc region of human IgG is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during antibody production or purification or by recombinant construction of the nucleic acid encoding the antibody heavy chain. Therefore, an intact antibody composition may contain antibody populations with all K447 residues removed, antibody populations lacking the K447 residues, and antibody populations having a mixture of antibodies with or without the K447 residue. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2, IgG3 and IgG4.

The native Fc region sequence contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The native sequence of human Fc regions includes the native sequence of the Fc region of human IgG1 (non-A and A allotypes); the native sequence of the Fc region of human IgG2; the native sequence of the Fc region of human IgG3; and the native sequence of the Fc region of human IgG4, as well as their naturally occurring variants.

A variant Fc region contains an amino acid sequence that differs from the native Fc region sequence due to 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 Fc region sequence or the Fc region of the parent polypeptide, for example, from about one to about ten amino acid substitutions and, preferably, from about one to about five amino acid substitutions in the native sequence of the Fc region, or in the Fc region of the original polypeptide. The variant Fc region herein preferably has at least about 80% homology to the native sequence Fc region and/or the Fc region of the parent polypeptide, and most preferably at least about 90% homology thereto, more preferably in at least about 95% homology with them.

Fc receptor or FcR describes a receptor that binds to the Fc region of an antibody. The preferred FcR is the native human FcR sequence. Moreover, a preferred FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor) , which have identical amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain. Other FcRs, including those that will be identified in the future, are encompassed by the term FcRs herein. FcRs can also increase the half-life of antibodies in serum.

The in vivo FcRn binding and serum half-life of human FcRn high-affinity binding polypeptides can be analyzed, for example, in transgenic mice or transfected human cell colonies expressing human FcRn, or in primates injected with polypeptides having a variant Fc region. WO 2004/42072 (Presta) describes antibody variants with improved or impaired binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591–6604 (2001).

As used herein, percent (%) amino acid sequence identity and homology to a peptide, polypeptide, or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after sequence alignment and introducing gaps , where necessary, to achieve the maximum percentage of sequence identity and without 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 a variety of ways that are within the skill of the art, for example, using publicly available computer programs such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR). One skilled in the art will be able to determine appropriate parameters for measuring alignment, including any algorithms known in the art, necessary to achieve maximum alignment over the entire length of the sequences being compared.

The term compete, when used in the context of antibodies (e.g., neutralizing antibodies) that compete for the same epitope, means competition between antibodies in an assay in which the test antibody prevents or inhibits (e.g., reduces) specific binding of a reference molecule (e.g., ligand or reference antibody) with a common antigen (eg, SIRPA or a fragment thereof). Many types of competitive binding assays can be used to determine whether an antibody competes with another, for example: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (FIA), competitive sandwich assay (see, for example, Stahli et al. al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin FIA (see, for example, Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase RIA with direct labeling using 1-125 tags (see, for example, Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin FIA (see, for example, Cheung, et al., 1990, Virology 176:546-552); and direct-labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically such an assay involves the use of purified antigen coupled to a solid surface or cells bearing either of an unlabeled test antibody and a labeled reference antibody. Competitive inhibition is measured by determining the amount of labels bound to the solid surface or cells in the presence of the test antibody. Typically the antibody being tested 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 close enough to the epitope bound by the reference antibody to cause steric hindrance. Additional details regarding methods for determining competitive binding are presented herein. Typically, if a competing antibody is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antibody to the common antigen by at least 20, 30, 40, 50, 60, 70, 80, 85, 90, 95. 97.5 and/or about 100%.

As used herein, the interaction between a SIRPA polypeptide and a second polypeptide includes, without limitation, protein-protein interaction, physical interaction, chemical interaction, binding, covalent binding, and ionic binding. As used herein, an antibody inhibits the interaction between two polypeptides if the antibody disrupts, reduces, or completely eliminates the interaction between the two polypeptides. The antibody of the present invention inhibits the interaction between two polypeptides if the antibody binds to one of the two polypeptides. In some embodiments, the interaction may be inhibited by at least about any of 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 97.5 and/or about

100%.

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 that attacks that antigen, and if the antigen is a polypeptide, includes certain amino acids that directly contact the antibody. Most often, epitopes are located on polypeptides, but in some cases, they can be located on other types of molecules, such as nucleic acids. Epitope determinants may include reactive surface moieties of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have certain three-dimensional structural characteristics, and/or certain charge characteristics. Typically, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of polypeptides and/or macromolecules.

An agonistic antibody or activating antibody is an antibody that causes (eg, enhances) one or more activities or functions of an antigen upon binding of the antibody to the antigen.

An antagonistic antibody or a blocking antibody or an inhibitory antibody is an antibody that reduces, inhibits, and/or eliminates (e.g., reduces) antigen binding of one or more ligands following binding of the antibody to an antigen, and/or reduces, inhibits, and/or eliminates (e.g., reduces) one or more activities or functions of an antigen after the antibody binds to the antigen. In some embodiments, antagonistic antibodies or blocking antibodies or inhibitory antibodies essentially or completely inhibit antigen binding to one or more ligands and/or one or more antigen activities or functions.

An isolated antibody, such as an isolated anti-SIRPA antibody of the present invention, is one that has been identified, separated and/or recovered from a component of its production medium (eg, naturally occurring or recombinantly). Preferably, the isolated antibody is free of association with all other contaminants from its production environment. Contaminant components from its production environment are products that will typically interfere with the research, diagnostic or therapeutic use of the antibody, and may include enzymes, hormones and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified: (1) to greater than 95% by weight of the antibody according to, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spin-beaker sequencer, or (3) homogeneity using SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue or, preferably, silverfish. The isolated antibody includes the antibody in situ within the recombinant T cells since at least one component of the antibody's natural environment will not be present. Typically, however, the isolated polypeptide or antibody will be obtained using at least one purification step.

An isolated nucleic acid molecule encoding an antibody, such as the anti-SIRPA antibody of the present invention, is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is typically associated in the environment in which it is receive. Preferably, the isolated nucleic acid is not associated with all components associated with the production medium. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form different from the form or array in which they occur in nature. The isolated nucleic acid molecules are therefore different from the nucleic acids encoding the polypeptides and antibodies herein that exist in cells in nature.

The term vector as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. One type of vector is a plasmid, which is a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial replication origin and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome and thereby replicate along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as recombinant expression vectors or simply expression vectors. In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. As used herein, plasmid and vector may be used interchangeably, since plasmid is the most commonly used form of vector.

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

A host cell includes a single cell or cell culture that can be or is the recipient for vector(s) for introducing polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) of the present invention.

Carriers as used herein include pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cell or mammal exposed to them at the dosages and concentrations used. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; 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 non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™.

As used herein, the term prevention includes providing prevention against the occurrence or recurrence of a specific disease, disorder or condition in an individual. An individual may be predisposed to, susceptible to, or at risk of developing such a disease, disorder or condition, but not yet have a diagnosed disease, disorder or condition.

As used herein, an individual at risk for developing a particular disease, disorder, or condition may or may not have an detectable disease or symptoms of a disease, and may or may not exhibit an detectable disease or symptoms of a disease prior to the treatment methods described herein. At risk means that a person has one or more risk factors, which are measurable parameters that correlate 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 likelihood of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.

As used herein, the term treatment refers to a clinical intervention designed to alter the natural course of the individual being treated over the course of a clinical pathology. Desirable effects of treatment include a reduction in the rate of progression, improvement or mitigation of the pathological condition, and remission or improvement in the prognosis of a particular disease, disorder or condition. An individual is successfully treated, for example, if one or more symptoms associated with a particular disease, disorder or condition are alleviated or eliminated.

An effective amount refers to at least an amount effective in doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result. An effective amount may be provided in one or more administrations. The effective amount herein may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the treatment to produce the desired response in the individual. An effective amount is also an amount in which any toxic or harmful effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing risk, reducing severity or delaying the onset of disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms caused by a disease, improving the quality of life of people suffering from the disease, reducing the dose of other drugs needed to treat the disease, enhancing the effect of another drug, e.g. through targeting, slowing disease progression and/or prolonging survival. Effective amount of drug

- 21 044628 agent, compound or pharmaceutical composition is an amount sufficient to provide prophylactic or therapeutic treatment directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an effective amount can be considered in the context of the administration of one or more therapeutic agents, and one agent can be considered to be administered in an effective amount if, in combination with one or more other agents, the desired result can be achieved or has already been achieved.

Individual for the purposes of treatment, prevention or risk reduction refers to any animal classified as a mammal, including humans, domestic, farm and zoo animals, sporting or pet animals such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats and the like. In some embodiments, the individual is a human.

As used herein, administration in combination with another compound or composition includes simultaneous administration and/or administration at different times. Administration in combination also includes administration as a joint formulation or administration as separate compositions, including at different dosing frequencies or intervals and using the same route of administration or different routes of administration. In some embodiments, administration in combination is administration as part of the same treatment regimen.

The term about as used herein refers to the typical error range for the corresponding value, well known to one skilled in the art. Reference to a value or parameter in this document includes (and describes) options that are associated with that value or parameter per se.

As used herein and in the accompanying claims, the singular forms a, an and the include the plural reference unless the context clearly indicates otherwise. For example, a reference to an antibody is a reference to one to many antibodies, such as molar amounts and includes equivalents thereof known to those skilled in the art, and so on.

It is understood that the aspect and embodiments of the present invention described herein include comprising, consisting of, and essentially consisting of aspects and embodiments.

Anti-SIRPA antibodies.

Anti-SIRPA antibody binding regions.

In some embodiments, the anti-SIRPA antibodies of the present invention can bind a conformational epitope. In some embodiments, the anti-SIRPA antibodies of the present invention can bind a discontinuous SIRPA epitope. In some embodiments, the discontinuous SIRPA epitope contains 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 present invention can bind a SIRPA epitope containing one or more peptides. As disclosed herein, SIRPA epitopes may comprise one or more peptides containing 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 than 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 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 the mammalian SIRPA protein corresponding to the amino acid sequence SEQ ID No: 1.

In some embodiments, the anti-SIRPA antibodies of the present invention bind to an epitope of human SIRPA that is the same as or overlaps with the SIRPA epitope bound by an anti-SIRPA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 2, and a variable a light chain region containing the amino acid sequence of SEQ ID No: 5, an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No: 6, anti- SIRPA antibody containing a heavy chain variable region containing the amino acid sequence SEQ ID No: 3, and a light chain variable region containing the amino acid sequence SEQ ID No: 7, anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence SEQ ID No: 3 , and a light chain variable region containing the amino acid sequence of SEQ ID No: 8, an anti-SIRPA antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No: 6 , an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No: 7, or an anti-SIRPA antibody

- 22 044628 lo, containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No: 8. In some embodiments, the anti-SIRPA antibodies of the present invention bind substantially the same same SIRPA epitope bound by an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No.: 2 and a light chain variable region containing the amino acid sequence of SEQ ID No.: 5, an anti-SIRPA antibody containing the heavy chain variable region , containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No: 6, an anti-SIRPA antibody containing a heavy chain variable region, containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region , containing the amino acid sequence of SEQ ID No: 7, an anti-SIRPA antibody, containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region, containing the amino acid sequence of SEQ ID No: 8, an anti-SIRPA antibody, containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No: 6, an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID No: 7, or an anti-SIRPA antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4 and a light chain variable region containing the amino acid sequence of SEQ ID No: 8.

In some embodiments, the anti-SIRPA antibodies of the present invention competitively inhibit the binding of an anti-SIRPA antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID No: 2 and a light chain variable region containing the amino acid sequence of SEQ ID No: 5, anti -SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No: 6, an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No: 7, an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No.: 8, an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No.: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No.: 6, an anti-SIRPA antibody containing a heavy chain variable region, containing the amino acid sequence of SEQ ID No.: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No.: 7, or an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No.: 4, and a light chain variable region, containing the amino acid sequence of SEQ ID No: 8. In some embodiments, the anti-SIRPA antibodies of the present invention compete with an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 2, and a light chain variable region containing the sequence amino acids SEQ ID No.: 5, an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No.: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No.: 6, an anti-SIRPA antibody containing the variable region heavy chain containing the amino acid sequence of SEQ ID No: 3, and a light chain variable region containing the amino acid sequence of SEQ ID No: 7, with an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 3, and the light chain variable region containing the amino acid sequence of SEQ ID No: 8, by an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No: 6, by an anti-SIRPA antibody, containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4, and a light chain variable region containing the amino acid sequence of SEQ ID No: 7, or an anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No: 4 , and a light chain variable region containing the amino acid sequence SEQ ID No: 8 for binding to SIRPA.

In some embodiments, the anti-SIRPA antibodies of the present invention competitively inhibit the binding of at least one antibody selected from any of the antibodies listed in table. 4, 5, 7, 8 and 10. In some embodiments, the anti-SIRPA antibodies of the present invention competitively inhibit the binding of at least one antibody selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1 /L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9 -4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F914, 3F9-15, 3F9-16, 3F9 -17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any of them

- 23 044628 readings. In some embodiments, the anti-SIRPA antibody of the present invention competes with one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3 , h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F97, 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, for binding to SIRPA when the anti-SIRPA antibody reduces the binding of one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1 , 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9 -14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof , with SIRPA at an amount ranging from about 50 to 100%, compared to binding to SIRPA in the absence of anti-SIRPA antibody. In some embodiments, the anti-SIRPA antibody of the present invention competes with one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3 , h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7 , 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9 -20, 3F921, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof for binding to SIRPA when the anti-SIRPA antibody reduces the binding of one or more antibodies selected from m3F9, h3F9 H1/L1 ( 14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9- 14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof with SIRPA by at least 50, at least 55, by at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or 100% compared to binding to SIRPA in the absence of anti-SIRPA antibody. In some embodiments, an anti-SIRPA antibody of the present invention that reduces the binding of one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1 /L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9 -7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19 , 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof, with SIRPA 100% indicates that the anti-SIRPA antibody almost completely blocks the binding of one or more antibodies, selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9 -12, 3F9-13, 3F9-14, 3F9-15, 3F916, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9 -25 and any combination thereof, with SIRPA. In some embodiments, an anti-SIRPA antibody and one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2 /L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9- 8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F914, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9- 21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof, are present in an amount that corresponds to a 10:1 ratio, 9:1 ratio, 8:1 ratio, 7:1 ratio, 6: 1 ratio, 5:1 ratio, 4:1 ratio, 3:1 ratio, 2:1 ratio, 1:1 ratio, 0.75:1 ratio, 0.5:1 ratio, 0.25:1 ratio, 0 ,1:1 ratio, 0.075:1 ratio, 0.050:1 ratio, 0.025:1 ratio, 0.01:1 ratio, 0.0075: ratio, 0.0050:1 ratio, 0.0025:1 ratio, 0.001: ratio, 0.00075:1 ratio, 0.00050:1 ratio, 0.00025:1 ratio, 0.0001: 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 anti-SIRPA antibody to one or more antibodies selected from m3F9, h3F9 H1/L1 (14.70, 1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9- 15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof. In some embodiments, the anti-SIRPA antibody is present in excess in an amount that ranges from about 1.5-fold to 100-fold or greater than 100-fold compared to the amount of one or more antibodies selected from m3F9, h3F9 H1/ L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9- 1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F99, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9- 14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof. In some embodiments, the anti-SIRPA antibody is 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. multiple, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, An 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess over the amount of one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70.1), h3F9 H1/L2 ( 14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9- 17, 3F9-18,

- 24 044628

3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25 and any combination thereof.

In some embodiments, the anti-SIRPA antibodies of the present invention bind to an epitope of human SIRPA that is the same as or overlaps with a SIRPA epitope bound by at least one antibody selected from any of the antibodies listed in table. 4, 5, 7, 8 and 10. In some embodiments, the anti-SIRPA antibodies of the present invention bind to an epitope of human SIRPA that is the same as or overlaps with the SIRPA epitope associated with at least one antibody selected from m3F9 , h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/ L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-

12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25.

In some embodiments, the anti-SIRPA antibodies of the present invention bind predominantly the same SIRPA epitope bound by at least one antibody selected from any of the antibodies listed in table. 4, 5, 7, 8 and 10. In some embodiments, the anti-SIRPA antibodies of the present invention bind predominantly to the same SIRPA epitope bound by at least one antibody selected from m3F9, h3F9 H1/L1 (14,70,1 ), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9 -3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15 , 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24, 3F9-25. Detailed exemplary methods for mapping the epitope to which an antibody binds are presented in Morris (1996) Epitope Mapping Protocols, Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In some embodiments, the anti-SIRPA antibodies of the present invention compete with one or more antibodies selected from m3F9, h3F9 H1/L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3 , h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F97, 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 for binding to SIRPA.

Any suitable competition 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 competes with one or more antibodies selected from m3F9, h3F9 H1 /L1 (14,70,1), h3F9 H1/L2 (14,70,2), h3F9 H1/L3, h3F9 H2/L1 (14,70,4), h3F9 H2/L2, h3F9 H2/L3, 3F9 -1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13 , 3F914, 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 bind to SIRPA. In a typical competition assay, immobilized SIRPA or cells expressing SIRPA on the cell surface are incubated in a solution containing a first labeled antibody that binds to SIRPA (eg, human or non-human primate) and a second unlabeled antibody that is tested for ability 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 containing the first labeled antibody but not the second, unlabeled antibody. After incubation under conditions allowing binding of the first antibody to SIRPA, excess unbound antibody is removed and the amount of label associated with the immobilized SIRPA or SIRPA-expressing cells is measured. If the amount of labels associated with immobilized SIRPA or cells expressing SIRPA is substantially reduced in the test sample compared to the control sample, this means that the second antibody is competing with the first antibody for binding to SIRPA. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch, 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

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 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing an amino acid sequence selected from SEQ ID Nos: 22, 23 and 24; (d) HVR-L1 containing the amino acid sequence SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID Nos: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

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 SEQ ID No.: 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing the amino acid sequence SEQ ID No: 22; (d) HVR-L1 containing the amino acid sequence of SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 containing the amino acid sequence of SEQ ID No: 11; (a) HVR-H1 containing the amino acid sequence SEQ ID No: 20; (b) HVR-H2 containing

- 25 044628 amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No.; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing

- 26 044628 amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. amino acid sequence SEQ ID No. (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing ; (a) HVR-H1 containing; (b) HVR-H2 containing; (c) HVR-H3 containing; (d) HVR-L1 containing (e) HVR-L2 containing ; and (f) HVR-L3 containing

- 27 044628 amino acid sequence SEQ ID No: 14; (a) HVR-H1 containing the amino acid sequence SEQ ID No: 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing the amino acid sequence SEQ ID No: 24; (d) HVR-L1 containing the amino acid sequence of SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 containing the amino acid sequence of SEQ ID No: 15; (a) HVR-H1 containing the amino acid sequence SEQ ID No: 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing the amino acid sequence SEQ ID No: 24; (d) HVR-L1 containing the amino acid sequence of SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 containing the amino acid sequence of SEQ ID No: 16; (a) HVR-H1 containing the amino acid sequence SEQ ID No: 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing the amino acid sequence SEQ ID No: 24; (d) HVR-L1 containing the amino acid sequence of SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 containing the amino acid sequence of SEQ ID No: 17; (a) HVR-H1 containing the amino acid sequence SEQ ID No: 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing the amino acid sequence SEQ ID No: 24; (d) HVR-L1 containing the amino acid sequence of SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 containing the amino acid sequence of SEQ ID No: 18; and (a) HVR-H1 containing the amino acid sequence of SEQ ID No: 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; (c) HVR-H3 containing the amino acid sequence SEQ ID No: 24; (d) HVR-L1 containing the amino acid sequence of SEQ ID No: 9; (f) HVR-L2 containing the amino acid sequence SEQ ID No: 10; and (f) HVR-L3 containing the amino acid sequence of SEQ ID No: 19.

In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, at least two, or all three V _H HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID No. : 20; (b) HVR-H2 containing the amino acid sequence SEQ ID No: 21; and (c) HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 22, 23 and 24.

In some embodiments, provided herein are anti-SIRPA antibodies comprising at least one, at least two, or all three V _L HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID No. : 9; (b) HVR-L2 containing the amino acid sequence 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.

In some embodiments, provided herein are anti-SIRPA antibodies comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, containing the amino acid sequence of SEQ ID No: 20, (ii) HVR-H2 containing the amino acid sequence of SEQ ID No: 21 and (iii) HVR-H3 containing the amino acid sequence selected from SEQ ID Nos: 22, 23 and 24 and ( b) a V _L domain containing at least one, at least two or all three VL HVR sequences selected from (i) HVR-L1 containing the amino acid sequence SEQ ID No: 9, (ii) HVR-L2 , containing the amino acid sequence of SEQ ID No: 10 and (c) HVR-L3, containing the amino acid sequence selected from SEQ ID Nos: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

In some embodiments, provided herein are anti-SIRPA antibodies comprising (a) a V _H 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 containing the amino acid sequence selected from SEQ ID Nos.: 22, 23 and 24 and (b) VL domain containing (i) HVR-L1 containing the amino acid sequence SEQ ID No.: 9, (ii) HVR-L2 containing the amino acid sequence of SEQ ID No: 10 and (c) HVR-L3 containing the amino acid sequence selected from SEQ ID Nos: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

In another aspect, the anti-SIRPA antibody comprises a heavy chain variable domain (Vh) sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the sequence amino acids selected from SEQ ID Nos: 33, 34, and 35. In certain embodiments, a _VH sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence selected from SEQ ID NOs:

- 28 044628

33, 34 and 35, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-SIRPA antibody containing this sequence retains the ability to bind to SIRPA. In certain embodiments, a total of 1 to 10 amino acids are 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 are substituted, inserted and/or deleted in SEQ ID No: 33, SEQ ID No: 34 or SEQ ID No: 35. In certain embodiments, substitutions, insertions and deletions occur in regions outside the HVR (ie, in the KO). Optionally, the anti-SIRPA antibody contains the V _H sequence of SEQ ID No: 33, SEQ ID No: 34 or SEQ ID No: 35, including post-translational modifications of this sequence. In a specific embodiment, V _H contains one, two or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID No: 20, (b) HVR-H2 containing the amino acid sequence of SEQ ID No: 21 and (c) HVRH3 containing an amino acid sequence selected from SEQ ID NOs: 22, 23 and 24.

In another aspect, an anti-SIRPA antibody is provided, wherein the antibody comprises a light chain variable domain (V _L ) 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. In certain embodiments, a V _L sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to the amino acid sequence selected from SEQ ID Nos: 36, 37, 38, 39, 40, 41, 42, 43 and 44, contains substitutions (e.g. , conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-SIRPA antibody containing this sequence retains the ability to bind to SIRPA. In some embodiments, a total of 1 to 10 amino acids are 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 are 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 In certain embodiments, substitutions, insertions and deletions occur in regions outside the HVR (ie, in the KO). Optionally, the anti-SIRPA antibody contains the V _L sequence SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 41, SEQ ID No: 42, SEQ ID No: 43 or SEQ ID No: 44, including post-translational modifications to this sequence. In a specific embodiment, V _L contains one, two or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID No: 9, (b) HVR-L2 containing the amino acid sequence of SEQ ID No: 10 and ( c) HVR-L3 containing an amino acid sequence selected from SEQ ID Nos: 11, 12, 13, 14, 15, 16, 17, 18 and 19.

In some embodiments, an anti-SIRPA antibody is provided, wherein the antibody comprises VH as in any of the embodiments presented above, and VL as in any of the embodiments presented above. In some embodiments, provided herein are anti-SIRPA antibodies, wherein the antibody comprises VH as in any of the embodiments presented above, and V _L as in any of the embodiments presented above. In one embodiment, the antibody contains 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, including post-translational modifications of these sequences.

In some embodiments, provided herein are anti-SIRPA antibodies comprising a heavy chain variable domain (VH) and a variable light chain domain (Vl), wherein VH and _VL are selected from the group consisting of: VH comprising the amino acid sequence of SEQ ID No.: 33 and V _L containing the amino acid sequence SEQ ID No.: 36; V _H containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 37; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 38; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 39; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 40; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 41; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 42; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 43; VH containing the amino acid sequence of SEQ ID No: 33 and V _L containing the amino acid sequence of SEQ ID No: 44; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 36; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 38; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 39; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 40; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 41; VH containing the sequence

- 29 044628 amino acids SEQ ID No: 34 and V _L containing the amino acid sequence SEQ ID No: 42; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 43; VH containing the amino acid sequence of SEQ ID No: 34 and V _L containing the amino acid sequence of SEQ ID No: 44; VH containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 36; VH containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 38; VH containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 39; Vh containing the amino acid sequence SEQ ID No: 35 and V _L containing the amino acid sequence SEQ ID No: 40; VH containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 41; VH containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 42; VH containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 43; and V _H containing the amino acid sequence of SEQ ID No: 35 and V _L containing the amino acid sequence of SEQ ID No: 44.

Additionally, provided herein are anti-SIRPA antibodies that competitively inhibit binding of, and/or compete for binding with, an anti-SIRPA antibody comprising (a) a V _H domain containing (i) HVR-H1 containing the amino acid sequence SEQ ID No.: 20, (ii) HVRH2 containing the amino acid sequence of SEQ ID No: 21 and (iii) HVR-H3 containing the amino acid sequence selected from SEQ ID Nos: 22, 23 and 24 and (b) and V _L domain containing ( i) HVRL1 containing the amino acid sequence SEQ ID No: 9, (ii) HVR-L2 containing the amino acid sequence SEQ ID No: 10 and (c) HVR-L3 containing the 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 of SEQ ID Nos: 33, 34, or 35 and SeQ ID Nos: 36, 37, 38, 39, 40, 41, 42, 43 or 44, respectively.

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

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

In some embodiments, an anti-SIRPA antibody according to any of the above embodiments may adopt any of the characteristics, alone or in combination, as described herein.

Anti-SIRPA antibodies are presented herein. The present antibodies are useful, for example, for the diagnosis or treatment of SIRPA-mediated disorders.

This invention relates, in part, to anti-SIRPA antibodies that exhibit one or more improved and/or enhanced functional characteristics (e.g., 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 chain containing the amino acid sequence SEQ ID No: 5), including, for example, anti-SIRPA antibodies capable of reducing cellular levels of SIRPA, anti-SIRPA antibodies capable of reducing cell surface levels of SIRPA, anti-SIRPA antibodies capable of degrading SIRPA, anti-SIRPA antibodies capable of reducing cellular levels of CD32A/B, anti-SIRPA antibodies capable of increasing or improving phagocytosis, anti-SIRPA antibodies capable of increasing or improving phagocytosis of tumor cells by macrophages, anti-SIRPA antibodies capable of increasing or improving the antitumor activity of anticancer therapies, anti-SIRPA antibodies capable of increasing or improving T cell proliferation, anti-SIRPA antibodies capable of increasing or improving pro-inflammatory cytokine release from macrophages, and/or anti-SIRPA antibodies capable of binding human SIRPA with improved/enhanced kinetics; methods for producing 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.

- 30 044628

In some embodiments, anti-SIRPA antibodies of the present invention may have one or more activities that exist due, at least in part, to the ability of the antibodies to reduce cellular expression (e.g., cell surface expression) of SIRPA, causing destruction, down-regulation, cleavage, desensitization receptor and/or lysosomal damage to SIRPA. In some embodiments, an anti-SIRPA antibody of the present invention that reduces cellular levels of SIRPA is an antibody that exhibits one or more of the following characteristics: (1) inhibits or reduces one or more SIRPA activities; (2) the ability to reduce SIRPA expression (such as at the mRNA level and/or protein level) in SIRPA-expressing cells; (3) the ability to interact with, bind to, or recognize the SIRPA protein; (4) the ability to specifically interact with or bind to the SIRPA protein; and (5) the ability to treat, alleviate, or prevent any aspect of the disease or disorder described or discussed herein.

In some embodiments, anti-SIRPA antibodies exhibit one or more of the following properties: a) have a dissociation constant (CD) for human SIRPA that is lower than that of an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID No. : 2, and a light chain variable region containing the amino acid sequence SEQ ID NO: 5; b) bind to human cells such as human monocytes and macrophages; c) reduce cell surface levels of SIRPA (eg, reduce cell surface levels of SIRPA on human macrophages in vitro to a half-maximal effective concentration (EC50) that is lower than that of an anti-SIRPA antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID No: 2, and a light chain variable region containing the amino acid sequence of SEQ ID No: 5; d) have a dissociation constant (CD) for human SIRPA that may vary from about 0.6 to about 0.7 nM; and/or e) reduce cell surface levels of SIRPA (eg, reduce cell surface levels of SIRPA on human macrophages in vitro to a half-maximal effective concentration (EC50) that may vary from about 0.4 nM to about 0.5 nM. As described herein, half-maximal effective concentration (EC50) refers to the concentration at which the anti-SIRPA antibody of the present invention reduces cellular levels of SIRPA on or in a cell to half the level of untreated cells, or the concentration at which the antibody achieves half-maximal binding to SIRPA on the cell.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro at a half-maximal effective concentration (EC50) to human SIRPA v1 from about 0. 40 nM to about 0.5 nM by flow cytometry. In some embodiments, which may be combined with any of the embodiments herein, the present 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) to human SIRPA v1 of about 5. about 4, about 3, about 2, about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0, 2 or about 0.1 nM by flow cytometry. In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro at a half-maximal effective concentration (EC50) to human SIRPA v1 in the range of about 5 to 1 nM, about 1 to 0.9 nM, about 1 to 0.8 nM, about 1 to 0.7 nM, about 1 to 0.6 nM, about 1 to 0.5 nM , from about 1 to 0.4 nM, from about 1 to 0.3 nM, from about 1 to 0.2 nM, from about 1 to 0.1 nM, from about 0.9 to 0.8 nM, from about 0.9 to 0.7 nM, about 0.9 to 0.6 nM, about 0.9 to 0.5 nM, about 0.9 to 0.4 nM, about 0.9 to 0, 3 nM, about 0.9 to 0.2 nM, about 0.9 to 0.1 nM, about 0.8 to 0.6 nM, about 0.8 to 0.5 nM, about 0 .8 to 0.4 nM, from about 0.8 nM to 0.3 nM, from about 0.8 nM to 0.2 nM, from about 0.8 to 0.1 nM, from about 0.7 to 0 .5 nM, from about 0.7 to 0.4 nM, from about 0.7 to 0.3 nM, from about 0.7 to 0.2 nM, from about 0.7 to 0.1 nM, from about 0.6 to 0.4 nM, about 0.6 to 0.3 nM, about 0.6 to 0.2 nM, about 0.6 to 0.1 nM, about 0.5 to 0, 3 nM, about 0.5 to 0.2 nM, about 0.5 to 0.1 nM, about 0.4 to 0.2 nM, about 0.4 to 0.1 nM, or about 0 .3 to 0.1 nM according to flow cytometry.

In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro from a half-maximal effective concentration (EC50) to human SIRPA v1 of about 0.093 nM , to human SIRPA v2 about 0.080 nM, and/or to cynomolgus SIRPA about 0.879 nM by flow cytometry. In some embodiments, which may be combined with any of the embodiments herein, the present invention provides an isolated antibody that binds to human SIRPA, wherein the antibody reduces cell surface levels of SIRPA in vitro to a half-maximal effective concentration (EC50) to human SIRPA v1 or to human SIRPA v2

- 31 044628 in the range from about 2 to 0.05 nM, from about 1 to 0.05 nM, from about 0.9 to 0.05 nM, from about 0.8 to 0.05 nM, from about 0.7 up to 0.05 nM, from about 0.6 to 0.05 nM, from about 0.5 to 0.05 nM, from about 0.4 to 0.05 nM, from about 0.3 to 0.05 nM, from about 0.20 to 0.05 nM, from about 0.15 to 0.05 nM, from about 0.10 to 0.05 nM, from about 0.09 to 0.05 nM, from about 0.08 nM to 0.05 nM, from about 0.07 to 0.05 nM, from about 0.06 to 0.05 nM, from about 0.20 to 0.06 nM, from about 0.15 to 0.06 nM, from about 0.10 to 0.06 nM, from about 0.09 to 0.06 nM, from about 0.08 to about 0.06 nM, from about 0.07 to 0.06 nM, from about 0.20 to 0.07 nM, from about 0.15 to 0.07 nM, from about 0.10 to about 0.07 nM, from about 0.09 to 0.07 nM, from about 0.08 to 0.7 nM , from about 0.20 to 0.08 nM, from about 0.15 to 0.08 nM, from about 0.10 to 0.08 nM, from about 0.09 to 0.08 nM, from about 0.20 to 0.09 nM, from about 0.15 to 0.09 nM, or from about 0.10 to 0.9 nM by flow cytometry.

In some embodiments, which may be combined with any of the embodiments herein, the present 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) to human SIRPA v1 of about 0.107 nM , to human SIRPA v2 about 0.082 nM, and/or to cynomolgus SIRPA about 0.107 nM by flow cytometry.

Preferably, the anti-SIRPA antibodies of the present invention reduce cell surface expression of SIRPA more effectively (eg, with a lower EC50) compared to a control anti-SIRPA antibody (eg, a control anti-SIRPA antibody having a heavy chain variable region containing the amino acid sequence SEQ ID No: 2 , and a light chain variable region containing the amino acid sequence of SEQ ID No: 5). Moreover, preferably, the anti-SIRPA antibodies of the present invention have higher affinity (e.g., up to about 100-fold higher affinity) for SIRPA (e.g., lower surface plasmon resonance CD value) compared to the control anti-SIRPA antibody ( for example, 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 containing the amino acid sequence of SEQ ID No: 5.

Certain aspects of the present invention are based at least in part on the identification of anti-SIRPA antibodies that exhibit one or more improved and/or enhanced functional characteristics (for example, relative to an anti-SIRPA antibody having a heavy chain variable region containing the sequence of amino acids SEQ ID No: 2, and a light chain variable region comprising the amino acid sequence SEQ ID No: 5), including an improved/enhanced ability to reduce cell surface levels of SIRPA on cells, resulting in the reduction, neutralization, prevention or control of one or more SIRPA activities, including without limitation, decreased growth of monocytes, macrophages, T cells, dendritic cells and/or microglia; decreased T cell proliferation caused by dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia, activated M1 microglia, M2 microglia, macrophages, M1 macrophages, activated M1 macrophages and/or M2 macrophages; decreased survival of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and/or M2 microglia; decreased proliferation 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; inhibition of 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; decrease in one or more functions of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and/or M2 microglia; decreased proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils and/or microglia; decreased overall functionality of monocytes, macrophages, T cells, dendritic cells, neutrophils and/or microglia; inhibition of positive immune response to various types of cancer selected from bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma , non-Hodgkin's lymphoma, acute myeloid leukemia, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma and thyroid cancer; inhibition of positive immune response to various types of neurological disorders 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

- 32 044628

Nasu-Hakola, stroke, acute trauma, chronic trauma, essential tremor, Behçet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, ShyDrager syndrome, progressive supranuclear palsy, cortical-basal ganglion degeneration, acute disseminated encephalomyelitis, granulomatous diseases, sarcoidosis, diseases of old age, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration and multiple sclerosis; binding to the SIRPA ligand on tumor cells; binding to the SIRPA ligand on dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils and/or macrophages; inhibiting tumor cell killing 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 antitumor 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 antitumor 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; modulated 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; improving the infiltration of one or more immunosuppressive dendritic cells, immunosuppressive macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, immunosuppressive neutrophils and regulatory T cells into the tumor; an increase in the number of tumor-promoting myeloid/granulocyte immunosuppressive cells in the tumor, in the peripheral blood or other lymphoid organ; improving tumor-promoting activity of myeloid-derived suppressor cells; decreased activation of tumor-specific T lymphocytes with the potential to destroy the tumor; decreased infiltration of tumor-specific T lymphocytes with the potential to destroy the tumor; increasing the rate of tumor growth; increased tumor recurrence rate; optionally reducing the effectiveness of one or more immunotherapies that modulate antitumor T cell responses, wherein the one or more immunotherapies are immunotherapies targeting one or more proteins selected from CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27. GITR, PD-L1, CTLA4, PD-L2, PD-1, В7-Н3, В7-Н4, HVEM, LIGHT, BTLA, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD39, CD70, TREM1, TREM2, Siglec-5, Siglec-7, Siglec-9, Siglec11, SirpA, CD447, CSF-1 receptor and any combination thereof, or one or more chemotherapeutic agents and/or cancer vaccines.

In some embodiments, treating cancer with anti-SIRPA antibodies as described herein can: (i) increase the number of tumor-infiltrating CD3+ T cells; (ii) reduce cellular levels of SIRPA in non-malignant CD14+ myeloid cells, optionally where non-malignant CD14+ myeloid cells are tumor infiltrating cells or, optionally, where non-malignant CD14+ myeloid cells are present in the blood; (iii) reduce the number of non-malignant CD14+ myeloid cells, optionally where non-malignant CD14+ myeloid cells are tumor infiltrating cells or, optionally, where non-malignant CD14+ myeloid cells are present in the blood; (iv) reduce the levels of PD-L1, PD-L2, B7-H7, B7-H3, CD200R, CD163 and/or CD206 in one or more cells, optionally where one or more cells are non-malignant myeloid suppressor cells (MLSCs) ; (v) reduce tumor growth rate in solid tumors; (vi) reduce tumor volume; (vii) increase the effectiveness of one or more PD-1 inhibitors; (viii) enhance the effectiveness of one or more immune checkpoint inhibitory and/or immunomodulatory therapies, wherein the one or more immune checkpoint inhibitory and/or immunomodulatory therapies target one or more of CTL4, adenosine pathway, PD-L1, PD-L2, OX40, TIM3, LAG3 or any combination thereof; (ix) enhance the effectiveness of one or more chemotherapeutic agents, optionally wherein the one or more chemotherapeutic agents are gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®) , 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (x) increase T cell proliferation in the presence of non-malignant myeloid suppressor cells (MLSC); (xi) inhibit the differentiation, survival and/or one or more functions of non-malignant myeloid suppressor cells (MLSC); and (xii) destroy CD33-expressing immunosuppressive non-malignant myeloid cells and/or non-malignant CD14-expressing cells in solid tumors and associated blood vessels upon conjugation with a chemical or radioactive toxin.

In some embodiments, the myeloid cells of the present invention include, but are not limited to, CD45+CD14+ myeloid cells, CD14+ myeloid cells, and myeloid suppressor cells.

- 33 044628 cells (MLSC). In some embodiments, the myeloid cells of the present invention are non-malignant myeloid cells. Immunosuppressive cells are sometimes also called myeloid suppressor cells (MSCs). In humans, MLSC can be defined by one of the following combinations of markers: (1) CD14+ HLA-DRlow/-, (2) CD14+ IL4Ra+, (3) CD14+ HLADR- IL4Ra+, (4) CD34+ CD14+ CD11b+ CD33+, (5) CD11b+ CD14+ CD33+, (6) CD33+ HLA-DR-, (7) LinHLA-DR-, (8) Lin- HLA-DR-CD33+, (9) Lin- HLA-DR- CD33+ CD11b+, (10) Lin- CD33+ CD11b+ CD15+ , (11) Lin-HLA-DR- CD33+ CD11b+ CD14- CD15+, (12) CD11b+ CD14- CD33+, (13) CD11b+ CD14- HLADR- CD33+ CD15+, (14) CD33+ HLA-DR- CD15+, (15) CD15+ IL4Ra+ , (16) CD11b+ CD15+ CD66b+, (17) CD15+ FSClow.SSChigh, (18) CD15high CD33+, (19) CD11b+ CD14- CD15+, (20) CD66b+ SShigh and (21) CD11b+ CD15+ (see also Solito S et al Annals of the NY Academy of Sciences, 2014). In mice, MLSCs can be defined by the expression of surface markers CD45+, CD11b+, Gr1+ and/or Il4Ra+. Additional typical immunosuppressive monocyte colonies include CD45+, CD11b+, Grllow; and CD45+, CD11c+.

In some embodiments, an anti-SIRPA antibody of the present invention reduces cell surface levels of SIRPA, reduces intracellular levels of SIRPA, reduces total cellular levels of SIRPA, or any combination thereof, only in the presence of natural SIRPA ligands or binding partners.

In some embodiments, an anti-SIRPA antibody of the present invention prevents, reduces, or inhibits the SIRPA-mediated activity of a SIRPA ligand or binding partner, including, for example, a SIRPA ligand or binding partner CD47, surfactant protein A, and/or surfactant protein D.

In some embodiments, the anti-SIRPA antibody of the present invention selectively binds to human SIRPA, including human allelic variants, also referred to herein as polymorphic variants, including human SIRPA v1 and human SIRPA v2, binds human SIRPe3, binds cynomolgus SIRPA, and binds marmoset SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention does not bind mouse SIRPA, does not bind SIRPB v1 (SIRPe 1), does not bind rabbit SIRPA, and does not bind rat SIRPA.

In some embodiments, the anti-SIRPA antibody of the present invention selectively binds to human SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention selectively binds to human SIRPA and cynomolgus SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to human SIRPA v1, human SIRPA v2, and cynomolgus SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to human SIRPA v1, human SIRPA v2, and cynomolgus SIRPA, but does not bind to mouse SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to human SIRPA v1, human SIRPA v2, and cynomolgus SIRPA, but does not bind to SIRPe 1. In some embodiments, the anti-SIRPA antibody of the present invention binds to human SIRPA v1, human SIRPA v2 and cynomolgus SIRPA, but does not bind to mouse SIRPA and does not bind to human SIRPe 1. In some embodiments, the anti-SIRPA antibody of the present invention binds to human SIRPA v1, human SIRPA v2, cynomolgus SIRPA, and human SIRPAe3, but does not bind murine SIRPA and does not bind human SIRPe 1. In any combination of the above embodiments, the anti-SIRPA antibody of the present invention does not block the binding or interaction of SIRPA and CD47.

Certain aspects of the present invention relate to anti-SIRPA antibodies that negatively regulate, i.e. reduce cellular levels of SIRPA. In some embodiments, an anti-SIRPA antibody reduces cellular levels of SIRPA without inhibiting, blocking, or reducing the interaction (eg, binding) between SIRPA and one or more SIRPA ligands, such as CD47.

In some embodiments, the anti-SIRPA antibodies of the present invention may have one or more activities that arise at least in part due to the ability of the antibodies to reduce cellular levels (e.g., cell surface expression) of SIRPA through disruption, downregulation, cleavage, receptor desensitization, and /or lysosomal targeting SIRPA.

In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the expression of SIRPA in cells. In some embodiments, down-regulation of SIRPA expression in cells reduces cell surface expression of SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the expression of SIRPA in monocytes (eg, human monocytes). In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the expression of SIRPA in macrophages (eg, human macrophages). In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the cell surface expression of SIRPA on

- 34 044628 macrophage by more than 70%, more than 75%, more than 80%, more than 85% or more than 95% compared to the level of cell surface expression of SIRPA in macrophages not treated with the anti-SIRPA antibody of the present inventions. In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates cell surface expression of SIRPA on a macrophage by about 70-90%, by about 70-85%, by about 70-80%, but by about 70-75%, by about 75- 90%, about 75-85%, about 75-80%, about 80-90%, about 80-85%, or about 85-95% compared to the level of cell surface expression of SIRPA in macrophages not treated with anti- SIRPA antibody of the present invention. In some embodiments, the macrophages are human macrophages, including, but not limited to, human M1 macrophages and human M2 macrophages.

Cellular SIRPA levels can refer to, but are not limited to, cell surface SIRPA levels, intracellular SIRPA levels, and total SIRPA levels. In some embodiments, reducing cellular levels of SIRPA includes reducing cell surface levels of SIRPA. As used herein, an anti-SIRPA antibody reduces cell surface levels of SIRPA if it causes a reduction of 25% or more in cell surface levels of SIRPA as determined by any in vitro cell assay or suitable in vivo model described herein or known in the art, for example, by flow cytometry such as fluorescence activated cell sorting (FCAS) to measure cell surface levels of SIRPA. In some embodiments, reducing cellular levels of SIRPA includes reducing intracellular levels of SIRPA. As used herein, an anti-SIRPA antibody reduces intracellular levels of Siglec-9 if it causes a reduction of 25% or more in intracellular levels of SIRPA as determined by any in vitro cell assay or suitable in vivo model described herein or known in the art. techniques, for example, according to immunostaining, Western blotting, co-immunoprecipitation and cellular cytometry. In some embodiments, reducing cellular levels of SIRPA includes reducing overall levels of SIRPA. As used herein, an anti-SIRPA antibody reduces total SIRPA levels if it causes a 25% or greater reduction in total SIRPA levels as determined by any in vitro cell assay or suitable in vivo model described herein or known in the art, for example, immunostaining, Western blotting, co-immunoprecipitation and cellular cytometry. In some embodiments, anti-SIRPA antibodies cause SIRPA destruction, SIRPA cleavage, SIRPA internalization, SIRPA shedding, down-regulation of SIRPA expression, or any combination thereof. In some embodiments, cellular levels of SIRPA are measured on primary cells (eg, dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, and macrophages) or on colonies of cells using a cellular SIRPA assay.

In some embodiments, the down-regulated anti-SIRPA antibody has an IC ₅₀ of 200 nM or less, typically 100 nM or less (down-regulated by 50% of SIRPA expressed on the cell surface), after 4 hours of treatment of human macrophages with the antibody at 37°C. In some embodiments, SIRPA remains down-regulated for at least 24 hours of treatment with an antibody of the present invention. Cells can be analyzed for surface expression of SIRPA using any technique, such as flow cytometry.

In some embodiments, the anti-SIRPA antibodies of the present invention reduce cellular levels of SIRPA by at least 25, at least 26, at least 27, at least 28, at least 29, at least , 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38 , at least 39, at least 40, at least 41%, at least 42%, at least 43%, at least 44%, at least 45, at least , 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54 , at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, according at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least , 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79 , at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, according 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 compared to cellular SIRPA levels in the absence of anti-SIRPA antibody.

In some embodiments, which may be combined with any of the negative regulatory activities summarized in the preceding paragraphs, an anti-SIRPA antibody

- 35 044628 of the present invention inhibits the surface cell accumulation of SIRPA.

In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates SIRPA but does not block, inhibit, or reduce SIRPA ligand binding, e.g.

CD47, with SIRPA. In the context of the present invention, an antibody directed against SIRPA that does not block binding

CD47 with SIRPA refers to an antibody that does not cause a significant reduction in the binding of CD47 to SIRPA when the antibody is incubated with CD47 and cells expressing SIRPA. A significant reduction in the context of CD47 binding to SIRPA refers to a reduction in binding of 30% or less, typically at least 25, at least 20, at least 15, or at least 10% or less compared to binding CD47 with SIRPA in the presence of an isotype-matched control antibody that does not bind SIRPA. An exemplary assay for assessing blocking activity is presented in the examples herein. For example, cells that express human SIRPA, such as human macrophages, or cells, such as CHO cells, that are modified to recombinantly express human SIRPA are seeded at 10 cells/well in a 96-well plate, washed and incubated in 100 μl buffer. for fluorescence-activated cell sorting containing 1.0 μg/ml monoclonal antibody or isotype control. Cells are then washed and incubated with soluble human CD47 for 30 minutes on ice. The cells are then analyzed for surface bound CD47.

In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the expression of CD32A/B in cells (ie, FcyRIIA/FcyRIIB). In some embodiments, down-regulation of CD32A/B (ie, FcyRIIA/FcyRIIB) expression in cells reduces cell surface expression of CD32A/B (ie, FcyRIIA/FcyRIIB). In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates CD32A/B expression in macrophages (eg, human macrophages). In some embodiments, the anti-SIRP antibody of the present invention negatively regulates the expression of CD32A (ie, FcyRIIA) in macrophages (eg, human macrophages). In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the expression of CD32B (ie, FcyRIIB) in macrophages (eg, human macrophages). In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates the cell surface expression of CD32A (i.e., FcyRIIA) in human macrophages by about 75, about 80, or about 85% relative to the level of CD32A cell surface expression in human macrophages, not treated with the anti-SIRPA antibody of the present invention. In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates 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 antibody of the present invention. In some embodiments, the anti-SIRPA antibody of the present invention negatively regulates cell surface expression of CD32B (ie, FcyRIIB) in human macrophages to undetectable levels.

In one aspect, the present invention provides antibodies, such as isolated (eg, monoclonal) antibodies, that react with or otherwise bind to a region, such as an epitope, in the SIRPA protein of the present invention. In some embodiments, antibodies react with or otherwise bind to a region, such as an epitope, in the SIRPA protein of the present invention with improved/enhanced kinetics (e.g., relative to an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence SEQ ID No. : 2, and a light chain variable region containing the amino acid sequence SEQ ID No: 5). In some embodiments, the antibodies interact with or otherwise bind to a region, such as an epitope, in the SIRPA protein on human cells, such as dendritic cells, myeloid cells, monocytes, macrophages, etc. with a half-maximal effective concentration (EC ₅₀ ) that is lower than that of a control antibody (eg, 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 containing the amino acid sequence of SEQ ID No: 5). In some embodiments, the anti-SIRPA antibodies of the present invention bind to the SIRPA protein and modulate one or more SIRPA activities upon binding to the SIRPA protein, for example, an activity associated with the expression of SIRPA on a cell. The SIRPA proteins of the present invention include, but are not limited to, mammalian SIRPA protein, human SIRPA protein, mouse SIRPA protein, cynomolgus SIRPA protein, and rat SIRPA protein.

In some embodiments, the antibodies of the present invention can bind SIRPA pH in an independent manner. In some embodiments, the antibodies of the present invention can bind to SIRPA at neutral pH and be internalized without being separated from the SIRPA protein. Alternatively, at acidic pH, the antibodies of the present invention may be separated from SIRPA once they have been internalized and then degraded by the endosomal/lysosomal route. In certain embodiments, the anti-SIRPA antibody binds SIRPA at pH ranges that range from 5.5 to 8.0, 5.5 to 7.5, 5.5 to 7.0, 5 .5 to 6.5, from 5.5 to 6.0, from 6.0 to 8.0, from 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 is released 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.

SIRPA is a single spanning membrane protein, type I. In the amino acid sequence of human SIRPA (SEQ ID No: 1), the extracellular domain is located at amino acid residues 31-373; the transmembrane domain is located at amino acid residues 374-394; and the intracellular domain is located at amino acid residues 395-504. Human SIRPA contains one V set and two d sets of Ig superfamily (IgSF) domains, named D1 domain, D2 domain and D3 domain, respectively. The D1 domain contains amino acid residues 32-137 of human SIRPA; The D2 domain contains amino acid residues 148-247 of human SIRPA; and the D3 domain contains amino acid residues 254-348 of human SIRPA. One skilled in the art will appreciate that the starting and ending residues of the domains of the present invention may vary when using a computer modeling program or method used to determine the domain.

In some embodiments, the anti-SIRPA antibody of the present invention binds to the D3 domain of SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to the D3 domain of human SIRPA containing amino acid residues 254-348 of amino acid sequence SEQ ID NO: 1 of human SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to an epitope in the D3 domain of human SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to an epitope in the D3 domain of human SIRPA, wherein the epitope comprises an amino acid sequence selected from amino acid residues 254-348, amino acid residues 254-274, amino acid residues 264-279, amino acid residues 274- 289, amino acid residues 273-331, amino acid residues 281-315, amino acid residues 281-337, amino acid residues 284299, 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 amino acid sequence SEQ ID No: 1 of human SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to an epitope in the D3 domain of human SIRPA, where the epitope includes amino acid residues R282, Q284 and G337 of the amino acid sequence (SEQ ID No: 1) of human SIRPA v1. In some embodiments, the anti-SIRPA antibody of the present invention binds to an epitope in the D3 domain of human SIRPA, where the epitope includes amino acid residues Q281, R282, Q284, L285, W287, R295, E297, V302 and W315 of the amino acid sequence (SEQ ID No: 1 ) human SIRPA v1.

In some embodiments, the antibody binds to the D3 domain of SIRPA, for example, human SIRPA. In some embodiments, the anti-SIRPA antibody of the present invention binds to the same SIRPA epitope or a portion of a SIRPA epitope bound by an antibody having a CCA antibody designated m3F9, as described herein. Therefore, in some embodiments, an antibody of the present invention binds to the same SIRPA epitope or a portion of a SIRPA epitope bound by an antibody having a CCA antibody designated m3F9, as described herein.

Certain aspects of the present invention are based at least in part on the identification of anti-SIRPA antibodies that exhibit one or more improved and/or enhanced functional characteristics (e.g., relative to an anti-SIRPA antibody having a heavy chain variable region comprising the amino acid sequence SEQ ID No.: 2, and a light chain variable region comprising the amino acid sequence SEQ ID No.: 5), including an improved/enhanced ability to reduce cell surface levels of SIRPA on cells causing reduction, neutralization, prevention or control of one or more SIRPA activities including, but not limited to, decreased growth of monocytes, macrophages, T cells, dendritic cells and/or microglia; decreased T cell proliferation caused by dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia, activated M1 microglia, M2 microglia, macrophages, M1 macrophages, activated M1 macrophages and/or M2 macrophages; decreased survival of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and/or M2 microglia; decreased proliferation 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; inhibition of 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-37 044628 cells, granulocytes, microglia , M1 microglia, activated M1 microglia and/or M2 microglia; decrease in one or more functions of neutrophils, dendritic cells, bone marrow-derived dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes, microglia, M1 microglia, activated M1 microglia and/or M2 microglia; decreased proliferation of monocytes, macrophages, T cells, dendritic cells, neutrophils and/or microglia; decreased overall functionality of monocytes, macrophages, T cells, dendritic cells, neutrophils and/or microglia; inhibition of positive immune response to various types of cancer selected from bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma , non-Hodgkin's lymphoma, acute myeloid leukemia, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma and thyroid cancer; inhibition of the positive immune response to various types of neurological disorders 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, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, essential tremor, Behçet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, ShyDrager syndrome, progressive supranuclear palsy, corticobasal ganglion degeneration, acute disseminated encephalomyelitis, granulomatous diseases, sarcoidosis, diseases of the elderly age, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration and multiple sclerosis; binding to the SIRPA ligand on tumor cells; binding to the SIRPA ligand on dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, T cells, neutrophils and/or macrophages; inhibiting tumor cell killing 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 antitumor 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 antitumor 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; modulated 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; improving the infiltration of one or more immunosuppressive dendritic cells, immunosuppressive macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, immunosuppressive neutrophils and regulatory T cells into the tumor; an increase in the number of tumor-promoting myeloid/granulocyte immunosuppressive cells in the tumor, in the peripheral blood or other lymphoid organ; improving tumor-promoting activity of myeloid-derived suppressor cells; decreased activation of tumor-specific T lymphocytes with the potential to destroy the tumor; decreased infiltration of tumor-specific T lymphocytes with the potential to destroy the tumor; increasing the rate of tumor growth; increased tumor recurrence rate; optionally reducing the effectiveness of one or more immunotherapies that modulate antitumor T cell responses, wherein the one or more immunotherapies are immunotherapies targeting one or more proteins selected from CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27. GITR, PD-L1, CTLA4, PD-L2, PD-1, В7-Н3, В7-Н4, HVEM, LIGHT, BTLA, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD39, CD70, TREM1, TREM2, Siglec-5, Siglec-7, Siglec-9, Siglec11, SirpA, CD447, CSF-1 receptor and any combination thereof, or one or more chemotherapeutic agents and/or cancer vaccines.

In some embodiments, which may be combined with any of the other embodiments above, the anti-SIRPA antibody of the present invention causes, improves, or enhances one or more activities including: (i) increasing the number of tumor-infiltrating CD3+ T cells; (ii) reducing cellular levels of SIRPA in non-malignant CD14+ myeloid cells, optionally where non-malignant CD14+ myeloid cells are tumor infiltrating cells or, optionally, where non-malignant CD14+ myeloid cells are present in the blood; (iii) reducing the number of non-malignant CD14+ myeloid cells, optionally where non-malignant CD14+ myeloid cells are tumor infiltrating cells or, optionally, where non-malignant CD14+ myeloid cells are present in the blood; (iv) reducing the levels of PD-L1, PD-L2, B7-H7, B7-H3, CD200R, CD163 and/or CD206 in one or more cells, optionally where one or more cells are non-malignant myeloid suppressor cells (MLSCs) ; (v) decreased tumor growth rate in solid tumors; (vi) reduction in tumor volume; (vii) increasing the effectiveness of one or more PD-1 inhibitors; (viii) by

- 38 044628 increasing the effectiveness of one or more immune checkpoint inhibitory and/or immunomodulatory therapies, where one or more immune checkpoint inhibitory 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 effectiveness of one or more chemotherapeutic agents, optionally wherein the one or more chemotherapeutic agents are gemcitabine, capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®) , 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (x) increased proliferation of T cells in the presence of non-malignant myeloid suppressor cells (MLSC); (xi) inhibition of differentiation, survival and/or one or more functions of non-malignant myeloid suppressor cells (MLSC); and (xii) killing CD33-expressing immunosuppressive non-malignant myeloid cells and/or non-malignant CD14-expressing cells in solid tumors and associated blood vessels by conjugation with a chemical or radioactive toxin.

In some embodiments, an anti-SIRPA antibody of the present invention reduces the activity, functionality, or survival of regulatory T cells, tumor-associated immunosuppressive dendritic cells, tumor-associated immunosuppressive macrophages, myeloid suppressor cells, tumor-associated macrophages, acute myeloid leukemia cells ( AML), chronic lymphocytic leukemia (CLL) or chronic myeloid leukemia (CML) cells.

In some embodiments, the anti-SIRPA antibody of the present invention induces or promotes the survival, maturation, functionality, migration or proliferation of one or more immune cells, for example, one or more immune cells selected from dendritic cells, macrophages, neutrophils, NK cells, microglia , T cells, T helper cells, cytotoxic T cells, and any combination thereof in an individual.

Binding affinity of anti-SIRPA antibody.

In some embodiments of any of the antibodies provided herein, the antibody has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g. 10 ^-8 M or less, for example from 10 ^-8 M to 10 ^-13 M, for example from 10 ^-9 M to 10 ^-13 M). Dissociation constants can be determined by any analytical method, including any biochemical or biophysical method such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, for example, Octet System from ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and calorimetric or fluorescence protein melting assays. In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In some embodiments, RIA is performed with a Fab version of the antibody of interest and its antigen, for example, as described by Chen et al. J. Mol. Biol. 293:865-881(1999)). In some embodiments, the Kd is modified using a BIACORE surface plasmon resonance assay, e.g., a BIACORE-2000 or BIACORE-3000 assay (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). In some embodiments, CD is determined using a monovalent antibody (eg, Fab) or a full-length antibody. In some embodiments, the CD is determined using a full-length antibody in monovalent form.

Antibody fragments.

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. A review of specific antibody fragments is provided in Hudson et al. Nat. Med. 9:129-134 (2003). An overview of scFv fragments is presented in, for example, WO 93/16185; and US Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having an increased in vivo half-life, see US Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific. See, for example, EP404097; WO 1993/01161; Hudson et al. Nat. Med. 9:129-134 (2003). Tribodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003). Single-domain antibodies are antibody fragments containing all or part of the heavy chain of the variable domain of all or part of the light chain of the variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (see, for example, US Pat. No. 6,248,516).

Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of the intact antibody, as well as production by recombinant host cells (eg, E. coli or phage), as described herein.

- 39 044628

Chimeric and humanized antibodies.

In some embodiments of any of the antibodies provided herein, the antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567. In one example, the chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant area. In another example, a chimeric antibody is a class switched antibody in which the class or subclass has been changed from the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments of any of the antibodies provided herein, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the original non-human antibody. In certain embodiments, the humanized antibody is substantially non-immunogenic in humans. In certain embodiments, the humanized antibody has substantially the same affinity for the target as an antibody from the other species from which the humanized antibody is derived. See, for example, US patent No. 5530101, 5693761; 5693762; and 5,585,089. In certain embodiments, amino acids of an antibody variable domain that can be modified without reducing the natural affinity of the antigen binding domain while reducing its immunogenicity are identified. See, for example, US Patent Nos. 5,766,886 and 5,869,619. In general, a humanized antibody contains one or more variable domains in which the HVR (or portions thereof) are derived from a non-human antibody and the KO (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally also contains at least a portion of the human constant region. In some embodiments, some of the KO residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which the HVR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

Humanized antibodies and methods of preparation are discussed, for example, in Almagro et al. Front. Biosci. 13:161 9-1633 (2008) and are also described, for example, in US Patent Nos. 5821337, 7527791, 6982321 and 7087409. Human framework regions that can be used for humanization include, but are not limited to: framework regions selected from using an optimal fitting method (see, for example, Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from a human antibody consensus sequence of a particular subset of light or heavy chain variable regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J Immunol 151:2623 (1993) human mature (somatically mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening KO libraries (see, for example, Vasa et al. J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al. J. Biol. Chem. 271:22611-22618 (1996 )).

Human antibodies.

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

Human antibodies can be produced by administering an immunogen to a transgenic animal that is modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. It is possible to construct mouse strains deficient in mouse antibody production with large fragments of human Ig loci, with the expectation that such a mouse will produce human antibodies in the absence of mouse antibodies. Large fragments of human Ig can preserve a wide variety of variable genes, as well as proper regulation of antibody production and expression. Using the mouse mechanism for antibody diversification and selection, and the absence of immunological tolerance to human proteins, the reconstituted human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using hybridoma techniques, antigen-specific human mAbs with the desired specificity can be generated and selected. Certain exemplary methods are described in US Patent No. 5545807, EP 546073 and EP 546073. See also, for example, US Patent Nos. 6075181 and 6150584, describing XENOMOUSE™ technology; US Patent No. 5,770,429 describing HUMAB® technology; US Patent No. 7,041,870 describing K-M MOUSE® technology and US Patent Application Publication No. US 2007/0061900 describing VELOCIMOUSE® technology. The human variable regions of their intact antibody generated by such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be produced by hybridoma-based methods. Were described

- 40 044628 colonies of human myeloma and murine-human heteromyeloma cells for the production of human monoclonal antibodies. (See, for example, Kozbor J. Immunol. 133:3001 (1984) and Boerner et al. J. Immunol. 147:86 (1991)). Human antibodies generated through human Cell Hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 1 03:3557-3562 (2006). Additional methods include those described, for example, in US Patent No. 7189826 (describing the production of monoclonal human IgM antibodies from colonies of hybridoma cells). 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 to isolate an Fv clone of variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

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 with the desired activity or activities. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosomal display (CAT), yeast display (Adimab) and the like. In certain phage display methods, the VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and recombined randomly into phage libraries, which are then screened for antigen-binding phage as described in Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). For example, many methods are known in the art for creating phage display libraries and screening such libraries for antibodies having 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; Fellows Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(-2):1 19-132 (2004). Phage typically displays antibody fragments either as Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibody to immunogen without the requirement of hybridoma construction. Alternatively, the intact repertoire can be cloned (eg from humans) to provide a single source of antibodies to a wide range of non-self as well as self-antigens without any immunization, as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, intact libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable HVR3 regions and to complete the rearrangement in vitro, as described by Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patent publications describing human antibody phage libraries include, for example, US Patent No. 5,750,373 and US Patent Publications Nos. 2007/0292936 and 2009/0002360. Antibodies isolated from human antibody libraries are considered to be human antibodies or human antibody fragments in this document.

Constant regions including Fc regions.

In some embodiments of any of the anti-SIRPA antibodies provided herein, the antibody comprises an Fc. In some embodiments, the Fc is human IgG1, IgG2, IgG3 and/or IgG4 isotypes. In some embodiments, the antibody is an IgG class, an IgM class, or an IgA class.

In certain embodiments of any of the anti-SIRPA antibodies provided herein, the antibody is of the IgG2 isotype. In some embodiments, the anti-SIRPA antibody comprises a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, an anti-SIRPA antibody causes one or more SIRPA activities or, independently, Fc receptor binding. In some embodiments, the anti-SIRPA antibody binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc receptor gamma IIB (FcyIIB).

In certain embodiments of any of the antibodies provided herein, the antibody is of the IgG1 isotype. In some embodiments, the anti-SIRPA antibody comprises a mouse IgG1 constant region. In some embodiments, the anti-SIRPA antibody comprises 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 an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc receptor gamma IIB (FcyIIB).

In certain embodiments of any of the anti-SIRPA antibodies provided herein, the antibody is of the IgG4 isotype. In some embodiments, the anti-SIRPA antibody comprises a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments, an anti-SIRPA antibody

- 41 044628 binds the inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is the inhibitory Fc receptor gamma PV (FcyIIB).

In certain embodiments of any of the anti-SIRPA antibodies provided herein, the antibody is of a hybrid IgG2/4 isotype. In some embodiments, the anti-SIRPA antibody comprises an amino acid sequence comprising amino acids 118-260 according to the EU numbering of human IgG2, and amino acids 261-447 according to the EU numbering of human IgG4 (WO 1997/11971; WO 2007/106585).

In some embodiments, the Fc region causes accumulation without activating complement compared to a corresponding antibody containing an Fc region that does not contain amino acid substitutions. In some embodiments, the antibody causes one or more activities of a target specifically bound by the antibody. In some embodiments, the antibody binds to SIRPA.

It may also be desirable to modify the anti-SIRPA antibody of the present invention to modify effector function and/or to increase the serum half-life of the antibody. For example, the Fc receptor binding site on the constant region may be modified or mutated to eliminate or reduce binding affinity for certain Fc receptors, such as FcyRI, FcyRII and/or FcyRIII, to reduce antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is attenuated by removing the Nglycosylation of the Fc region (eg, in the CH2 domain of an IgG) of the antibody. In some embodiments, effector function is attenuated by modification of regions such as 233-236, 297 and/or 327-331 of human IgG, as described in WO 99/58572 and Armor 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 antibody of the present invention to modify the effector function to enhance selectivity for ITIM-containing FcgRIIb (CD32b) to increase the accumulation of SIRPA antibodies on neighboring cells without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis.

To increase the serum half-life of an antibody, a salvage receptor binding epitope can be introduced into an antibody (especially an antibody fragment) as described in US Pat. No. 5,739,277, for example. As used herein, the term salvage receptor binding epitope refers to an epitope in the Fc region of an IgG molecule (eg, IgG1, _IgG2 , _IgG3 , or _IgG4 ) that is responsible for increasing the in vivo half-life of the IgG molecule.

Polyspecific antibodies.

Polyspecific antibodies are those that have binding specificity for at least two different epitopes, including those on one or another polypeptide (eg, one or more SIRPA polypeptides of the present invention). In some embodiments, the multispecific antibody may be a bispecific antibody. In some embodiments, the multispecific antibody may be a trispecific antibody. In some embodiments, the multispecific antibody may be a tetraspecific antibody. Such antibodies can be obtained from full-length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies). In some embodiments, the multispecific antibody comprises a first antigen binding region that binds to a first SIRPA site and contains a second antigen binding region that binds to a second SIRPA site. In some embodiments, the polyspecific antibodies comprise a first antigen binding region that binds to SIRPA and a second antigen binding region that binds to a second polypeptide.

Provided herein are multispecific antibodies comprising a first antigen binding region, wherein the first antigen binding region comprises the six HVR antibodies described herein that bind to SIRPA, and a second antigen binding region that binds to a second polypeptide. In some embodiments, the first antigen binding region comprises a V _H or V _L antibody described herein.

In some embodiments of any of the polyspecific antibodies, the second polypeptide is an antigen that facilitates transport across the blood-brain barrier. A variety of antigens and peptides are known in the art that promote transport across the blood-brain barrier (see, for example, 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), receptor diphtheria toxin, 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, for example, Gabathuler, 2010) and other cell surface proteins,

- 42 044628 which are enriched in endothelial cells of the blood-brain barrier (see, for example, Daneman et al. PLoS One 5(10):e13741 (2010)).

In some embodiments of any of the polyspecific antibodies, the second polypeptide is a disease-causing protein selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, peroxide 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, medina, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepipelin, cystatin, immunoglobulin light chain AL, S-IBM protein, non-ATG repeat associated (RAN ) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptides, glycine-arginine repeat (GR) peptides, proline-alanine (PA) repeat peptides, ubiquitin and peptides proline-arginine (PR) repeat; (d) ligands and/or proteins expressed on immune cells, where 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 (f) a protein, lipid, polysaccharide or glycolipid expressed on one or more tumor cells, and any combination thereof.

Multivalent antibodies can recognize SIRPA antigen, as well as, without limitation, additional antigens such as Ae peptide antigen, α-synuclein protein antigen, Tau protein antigen, TDP-43 protein antigen, prion protein antigen, huntingtin protein antigen, RAN translation products antigen (including dipeptide repeats, (DPR peptides) consisting of glycine-alanine (GA), glycinaproline (GP), glycine-arginine (GR), proline-alanine (PA) or proline-arginine (PR)), insulin receptor, insulin-like receptor growth factor or transferrin receptor or any other antigen that facilitates the transfer of the antibody across the blood-brain barrier. In some embodiments, the second polypeptide is transferrin. In some embodiments, the second polypeptide is Tau. In some embodiments, the second polypeptide is Ae. In some embodiments, the second polypeptide is TREM2. In some embodiments, the second polypeptide is α-synuclein.

A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain or chains comprise two or more variable domains. For example, the polypeptide chain or chains may comprise VD1-(X1) _n -VD2-(X2) _n -Fc, where VD1 is the first variable domain, VD2 is the second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent an amino acid or a polypeptide and n is 0 or 1. Also, the polypeptide chain or chains may comprise a V _H -C _H 1-hybrid linker chain-VHC _H 1-Fc region; or chain V _H -C _H 1-V _H -C _H 1-Fc region. The multivalent antibody here preferably further comprises at least two (and preferably four) light chain variable domains of the polypeptides. A multivalent antibody herein may, for example, comprise from about two to about eight light chain polypeptide variable domains. The light chain variable domain of the polypeptides contemplated herein may comprise a light chain variable domain and optionally further comprises a CL domain.

Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello Nature 305: 537 (1983), WO 93/08829 and Traunecker et al. EMBO J. 10:3655 (1991)) and projection-to-recess design (see, for example, US Pat. No. 5,731,168). See also WO 2013/026833 (CrossMab). Multispecific antibodies can also be produced by engineering proteins using the effect of electrostatic interaction to produce Fc heterodimeric antibody molecules (WO 2009/089004A1); cross-linking two or more antibodies (see, for example, US patent No. 4676980); the use of leucine; the use of diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and the use of single chain Fv (scFv) dimers (see, for example, Gruber et al. J. Immunol. 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including octopus antibodies, are also included herein (see, for example, US 2006/0025576). As used herein, the antibody also includes a dual action FAb or DAF containing an antigen binding site that binds to a variety of SIRPAs (see US 2008/0069820, for example).

Antibody options.

In some embodiments of any of the antibodies presented herein, variant amino acid sequences of the antibodies are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.

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Variants of substitution, insertion and deletion.

In some embodiments of any of the antibodies provided herein, variants of the antibody are provided having one or more amino acid substitutions. Variants of antibody amino acid sequences can be obtained by introducing suitable modifications into the nucleotide sequences encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequences of the antibody.

Table 1

Amino acid substitutions

Original balance Typical substitutions Preferred Substitutions Ala (A) Val; Leu; lie Val Arg(R) Lys; Gin; Asn Lys Asn(N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His(H) Asn; Gin; Lys; Arg Arg He(I) Leu; Val; Met; Ala; Ph; Norleucine Leu Leu (L) Norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met(M) Leu; Ph; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Ph; Thr; Ser Phe Val(V) lie; Leu; Met; Ph; Ala; Norleucine Leu

Significant modifications of the biological properties of an antibody are made by selecting substitutions that differ significantly in their effect of preserving (a) the structure of the polypeptide backbone in the region of the substitution, for example, in the form of a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chains. Naturally occurring residues are divided into groups based on common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) alkaline His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

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

When making changes to a polypeptide or antibody described herein, according to certain embodiments, the amino acid hydrophobicity index may be taken into account. Each amino acid has a hydrophobicity index based on its hydrophobicity and charge characteristics. They include: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of an amino acid's hydrophobicity index in imparting interactive biological function to a protein is understood in the art. Kyte et al. J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be replaced by other amino acids having a similar hydrophobicity index or index and still retain similar biological activity. When making changes based on the hydrophobicity index, in certain embodiments, the implementation is included within ±2. In certain embodiments, those that are ±1 are included, and those that are ±0.5 are included.

It is also understood in the art that substitution of such amino acids can be effectively carried out on the basis of hydrophilicity, in particular where the biologically functional protein or peptide thus created is intended for use in immunological embodiments, as in this case. In certain embodiments, the greatest local average hydrophilicity of a protein, driven by the hydrophilicity of its neighboring amino acids, correlates with immunogenicity and antigenicity, i.e. with the biological properties of protein.

The following hydrophilicity values are assigned to these amino acid residues: arginine (+3.0); lysine (+3.0±1); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain embodiments, substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those that are included are within ±0.5. It is possible to identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also called epitope core regions.

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

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred residues or more, as well as intra-sequence insertions of one or multiple amino acid residues. Examples of terminal inserts include an antibody with an N-terminal methionine residue. Other options for inserting an antibody molecule include fusion at the N- or C-terminus of the antibody with an enzyme (eg, for ADEPT) or polypeptide, which increases the half-life of the antibody in serum.

Any cysteine residue not involved in maintaining the proper conformation of the antibody can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine linkages may be added to an antibody to improve its stability (particularly if the antibody is an antibody fragment such as an Fv fragment).

Glycosylation variants.

In some embodiments of any of the antibodies provided herein, the antibody is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites to an antibody may conveniently be accompanied by a change in the amino acid sequence such that one or more glycosylation sites are created or deleted.

Glycosylation of antibodies is usually either N-linked or O-linked. N-linking refers to the addition of a carbohydrate group to the side chain of an aspartic 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 group of 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 addition of one of the sugars N-acetylgalactosamine, galactose or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to an antibody is conveniently accomplished by changing the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding or substituting one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).

If the antibody contains an Fc region, the carbohydrate attached to it may be altered. Native antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide that is typically N-linked to Asn297 according to the Cabot CH2 numbering of the Fc domain. The oligosaccharide may include various carbohydrates, for example, mannose, Nacetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the biantenary stem of the oligosaccharide. In some embodiments, modifications to the oligosaccharide in the antibody of the present invention can be made to create variants of the antibody with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that does not have fucose attached (directly or indirectly) to the Fc region. See, for example, US Patent Publications Nos. 2003/0157108 and 2004/0093621. Examples of publications related to defucosylated or fucose-deficient antibody variants include: US 2003/0157108; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell colonies capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108) and knockout cell colonies such as alpha1,6-fucosyltransferase gene, FUT8, CHO knockout cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) and Kanda et al. Biotechnol. Bioeng. 94(4):680- 688 (2006)).

Modified permanent areas.

In some embodiments of any of the anti-SIRPA antibodies provided herein, the Fc antibody is isotypes and/or modifications of the Fc antibody. In some embodiments, the Fc antibody isotype and/or modification is capable of binding to the Fc gamma receptor.

Typical isotypes and modifications of Fc antibodies are presented in table. 2 below. In some embodiments, the anti-SIRPA antibody of the present invention is capable of binding an Fc gamma receptor having the Fc isotype listed in Table 1. 2 below.

table 2

Typical isotypes of anti-SIRPA Fc antibodies that are capable of binding the Fc gamma receptor

Fc Isotype Mutation (EU numbering scheme) IgGl N297A IgGl D265A and N297A IgGl D270A IgGl L234A and L235A L234A and G237A L234A and L235A and G237A IgGl D270A, and/or P238D, and/or L328E, and/or E233D, and/or G237D and/or H268D, and/or P271G, and/or A330R IgGl P238D and L328E and E233D and G237D and H268D and P271G and A330R IgGl P238D and L328E and G237D and H268D and P271G and A330R

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IgGl P238D and S267E and L328F and E233D and G237D and H268D and P271G A330R IgGl P238D and S267E and L328F and G237D and H268D and P271G and A33OR IgG2 V234A and G237A IgG4 L235A and G237A and E318A IgG4 S228P and E236E IgG2/4 hybrid IgG2 aa 118 to 260 and IgG4 aa 261 to 447 H268Q and V309L; and A33OS and P331S IgGl C226S and C229S and E233P and L234V and L235A IgGl L234F and L235E and P331S IgG2 C232S or C233S IgG2 A330S HP331S IgGl S267E and L328F S267E only IgG2 S267E and L328F IgG4 S267E and L328F IgG2 WT NS with Kappa (light chain) LC NS C127S with Kappa LC Kappa LC C214S Kappa LC C214S and HC C233S Kappa LC C214S and HC C232S Any of the above mutations together with P330S and P331S mutations F(ab’)2 fragment of WT IgGl and any of the above mutations IgGl Replacement of constant heavy 1 (CH1) and hinge region IgGl with CH1 and IGg2 hinge region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD HKPSNTKVDKTVER KCCVECPPCP (SEQ Ш No: 58) With Kappa LC IgGl Any of the above mutations together with A330L/A330S and/or L234F and/or L235E and/or P331S IgGl, IgG2 or IgG4 Any of the above mutations together with M252Y and/or S254T and/or T256E Mouse IgGl, mouse IgG2a, mouse IgG2b For mouse models of disease IgG4 W.T. IgGl Any of the above mutations together with E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, S440W and/or any combination thereof. IgG2 Any of the above mutations together with E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, S440W and/or any combination thereof.

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 their mutants (for example, Strohl (2009) Current Opinion in Biotechnology 2009, 20:685-691) that bind Fcg receptors I, IIA , IIC, IIIA, IIIB in human and/or Fcg receptors I, III and IV in mice, may also act as transient agonist antibodies.

In some embodiments, the Fc gamma receptor binding antibody is of the IgG class, the IgM class, or the IgA class. In some embodiments, the Fc gamma binding

- 47 044628 receptor antibody has an IgGl, 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 in the Fc region at residue positions selected from the group consisting of: C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y in any combination (residue position according to EU or Cabot numbering). In some embodiments, the Fc region contains an amino acid substitution at position E430G. In some embodiments, the Fc region contains an amino acid substitution at positions L243A, L235A, and P331A. In some embodiments, the Fc region contains an amino acid substitution at positions L243A, L235A, P331A. In some embodiments, the Fc region contains an amino acid substitution at positions K322A and E430G. In some embodiments, the Fc region contains an amino acid substitution at positions P331S and E430G. In some embodiments, the Fc region contains an amino acid substitution at positions A330S, P331S, and E430G. In some embodiments, the Fc region contains an amino acid substitution at positions K322A, A330S, and P331S. In some embodiments, the Fc region contains an amino acid substitution at positions K322A, P331S, and E430G. In some embodiments, the Fc region contains an amino acid substitution at positions A330S, P331S, and E430G. In some embodiments, the Fc region contains an amino acid substitution at positions S267E and L328F. In some embodiments, the Fc region contains an amino acid substitution at position C127S. In some embodiments, the Fc region contains an amino acid substitution at positions E345R, E430G, and S440Y.

In certain embodiments, the Fc gamma receptor binding antibody is of the IgG2 isotype. In some embodiments, the Fc gamma receptor binding antibody comprises a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, the Fc gamma receptor binding antibody binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc receptor gamma IIB (FcyIIB). 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 (eg, relative to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from V234A (Alegre et al., (1994) Transplantation 57:1537-1543. 31; Xu et al., (2000) Cell Immunol, 200:16-26). G237A (Cole et al. (1999) Transplantation, 68:563-571). H268Q, V309L, A330S, P331S (US 2007/0148167; Armour et al. (1999) Eur J Immunol 29: 2613-2624; Armour et al. (2000) The Haematology Journal l (Suppl,1):27; Armour et al. (2000) The Hematology Journal 1(Suppl.1):27), C232S, and/or C233S (White et al. (2015) Cancer Cell 27, 138-148). S267E, L328F (Chu et al., (2008) Mol Immunol, 45:3926-3933), M252Y, S254T, and/or T256E, where the amino acid position is indicated in accordance with the EU or Cabot numbering system.

In some embodiments, the Fc gamma receptor binding antibody is an IgG2 isotype with a heavy chain constant domain that contains a C127S amino acid substitution, where the amino acid position is indicated in accordance with the EU or Cabot numbering system (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO 2008079246).

In some embodiments, the Fc gamma receptor binding antibody has an IgG2 isotype with a kappa light chain constant domain that contains a C214S amino acid substitution, where the amino acid position is indicated in accordance with the EU or Cabot numbering system (White et al., (2015) Cancer Cell 27 , 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO 2008079246).

In certain embodiments, the Fc gamma receptor binding antibody is of the IgG1 isotype. In some embodiments, the Fc gamma receptor binding antibody comprises a murine IgG1 constant region. In some embodiments, the Fc gamma receptor binding antibody comprises a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments, the Fc gamma receptor binding antibody binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc receptor gamma IIB (FcyIIB). 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 (eg, relative to a wild-type Fc region of the same isotype). In some embodiments, 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) R. J. Biol. Chem. 276, 6591- 6604), D270A, L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al., (1994) Transplantation 57:1537-1543. 31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Alegre et al. (1994) Transplantation 57:1537-1543. 31; Xu et al. (2000) Cell Immunol, 200:16-26), P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192),

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P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L,

M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A and/or T394D, where the amino acid position is indicated according to the EU or Cabot numbering system.

In some embodiments, the antibody includes a heavy chain constant domain 1 (CH1) and an IgG2 isotype hinge region (White et al., (2015) Cancer Cell 27, 138-148). In certain embodiments, the CH1 and hinge region of the IgG2 isotype comprises the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID No: 58). In some embodiments, the Fc region of the antibody comprises an S267E amino acid substitution, an L328F amino acid substitution, or both, and/or an N297A or N297Q amino acid substitution, where the position of the amino acid is indicated in accordance with the EU or Cabot numbering system.

In certain embodiments, the Fc gamma receptor binding antibody is of the IgG4 isotype. In some embodiments, the Fc gamma receptor binding antibody comprises a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments, the Fc gamma receptor binding antibody binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc receptor gamma IIB (FcyIIB). 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 (eg, relative to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from L235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol, 164:1925-1933), S267E, E318A, L328F, M252Y, S254T and/or T256E, where the amino acid position is indicated according to the EU or Cabot numbering system.

In certain embodiments, the Fc gamma receptor binding antibody is of a hybrid IgG2/4 isotype. In some embodiments, the Fc gamma receptor binding antibody comprises an amino acid sequence comprising amino acids 118-260 according to the EU or Cabot numbering of human IgG2, and amino acids 261-447 according to the EU or Cabot numbering of human IgG4 (WO 1997/11971; WO 2007/ 106585).

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

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

In certain embodiments, the antibody contains one or more amino acid substitutions in the Fc region at a residue position selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, s440Y, and any combination thereof , where the numbering of residues is carried out according to EU or Cabot numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, L243A, L235A and P331S, where residue numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G and P331S, where residue numbering is based on EU numbering. In some embodiments, the Fc region contains an amino acid substitution at positions E430G and K322A, where residue numbering is based on EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, A330S, and P331S, where residue numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, K322A, A330S, and P331S, where residue numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, K322A, and A330S, where residue numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E430G, K322A, and P331S, where residue numbering is according to EU numbering. In some embodiments, the Fc region contains an amino acid substitution at positions S267E and L328F, where residue numbering is according to EU numbering. In some embodiments, the Fc region contains an amino acid substitution at position C127S, where the residue numbering is according to EU numbering. In some embodiments, the Fc region contains amino acid substitutions at positions E345R, E430G, and S440Y, where residue numbering is according to EU numbering.

In some embodiments, antibodies that bind SIRPA protein may include antibodies that reduce cellular levels of SIRPA (e.g., cell surface SIRPA levels), inhibit interaction (e.g., binding) between SIRPA and/or one or more SIRPA ligands, and inhibit one or more more SIRPA protein activities. Such antibodies inhibit one or more activities of the SIRPA protein either by preventing interaction (eg, binding) between SIRPA and one or more SIRPA ligands or by preventing signal transduction from the extracellular domain of SIRPA into the cell cytoplasm in the presence of one or more SIRPA ligands. Antibodies can also inhibit one or more SIRPA protein activities by reducing SIRPA cell surface levels, causing SIRPA degradation, SIRPA desensitization, SIRPA degradation, SIRPA internalization, SIRPA shedding, down-regulation of SIRPA expression, and/or lysosomal degradation of SIRPA. In some embodiments, such anti-SIRPA antibodies cannot transiently activate SIRPA.

In some embodiments, the anti-SIRPA antibodies of the present invention may have specificity for an epitope of the transient agonistic anti-SIRPA antibody of the present invention, but have an Fc domain that is unable to bind Fcg receptors and therefore is not capable of, for example, transiently accumulating and activating SIRPA .

In some embodiments, the anti-SIRPA antibodies of the present invention have, without limitation, one or more of the following activities: the ability to reduce the binding of a SIRPA protein to one or more SIRPA ligands, such as sialic acid-containing glycolipids or sialic acid-containing glycoproteins, the ability to reduce protein binding suppressor of cytokine signals (SOCS) (e.g. SOCS3 protein) with SIRPA protein, ability to increase proteasomal degradation of SIRPA protein, ability to reduce functional expression of SIRPA on the surface of circulating dendritic cells, macrophages, monocytes, T cells and/or microglia, ability to reduce Tyr phosphorylation -340 and Tyr-358 through Src family tyrosine kinases such as LCK and FYN, ability to reduce recruitment and binding to tyrosine-specific protein phosphatases SHP1 and SHP2, ability to reduce recruitment and binding to PLC-g1, which acts as a guanine nucleotide exchange factor for Dynamin- 1, ability to reduce recruitment and binding to Crk1, ability to reduce recruitment and binding to splenic tyrosine kinase Syk, ability to reduce recruitment and binding to SH3-SH2-SH3 growth factor receptor-associated protein 2 (Grb2), ability to reduce recruitment and binding to multiple SH2 containing proteins, the ability to increase intracellular calcium mobilization, the ability to modulate the formation of proinflammatory cytokines ΓΕ-1β, IL-8 and TNF-α, the ability to reduce the activation of phosphoinositide 3 kinase, the ability to increase the growth of monocytes, macrophages, dendritic cells, T cells and/or microglia , ability to increase the survival of monocytes, macrophages, dendritic cells, T cells and/or microglia, ability to increase tyrosine phosphorylation on a variety of cellular proteins, ability to increase phagocytic activity of monocytes, macrophages, dendritic cells and/or microglia, ability to increase cellular proliferation of monocytes, macrophages , dendritic cells, T cells and/or microglia, ability to increase phosphorylation of signaling molecules that mediate ITAM signaling, ability to increase pattern recognition receptor function, ability to increase toll-like receptor function, ability to increase damage-associated molecular moiety (DAMP) receptor function , the ability to modulate the expression of C-C chemokine receptor 7 (CCR7) and the ability to increase cellular and protein debris.

In some embodiments, the anti-SIRPA antibodies of the present invention have an Fc region that exhibits reduced binding to one or more Fcy receptors. Examples of such Fc regions and modifications are presented in table. 3 below. In some embodiments, the antibody has an Fc isotype listed in table. 3 below.

In some embodiments, the anti-SIRPA antibodies of the present invention with reduced binding to Fc gamma receptors have the Fc isotype listed in table. 3 below.

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Table 3

Typical Fc isotypes of anti-SIRPA antibodies with reduced binding to the Fc gamma receptor

Fc isotype Mutation (EU numbering scheme) IgGl N297A or N297Q and/or D270A IgGl D265A, D270A and/or N297A IgGl L234AhL235A IgG2 V234A and G237A IgG4 F235A and G237A and E318A E233P and/or F234V N297A or N297Q IgG4 S228P and L236E S241P S241PhL248E S228P and F234A and L235A IgG2 H268Q and V309L and A33OS and P331S IgGl C220S and C226S and C229S and P238S IgGl C226S and C229S and E233P and L234V and L235A IgGl E233P and L234V and L235A and 0236-removed P238A D265A N297A A327Q or A327G P329A IgGl K322A and L234A and L235A IgGl L234F and L235E and P331S IgGl or IgG4 T394D IgG2 C232S or C233S N297A or N297Q IgG2 V234A and G237A and P238S and H268A and V309L A33OS HP331S IgGl, IgG2 or IgG4 delta a, b, c, ab, ac, g modifications IgGl Any of the above mutations together with A330L or L234F and/or L235E and/or P331S IgGl, IgG2 or IgG4 Any of the above mutations together with M252Y and/or S254T and/or T256E

In certain embodiments, the anti-SIRPA antibody is of the IgG1 isotype. In some embodiments, the antibody comprises a murine IgG1 constant region. In some embodiments, the antibody comprises 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 (eg, relative to a wild-type Fc region of the same isotype).

In some embodiments, 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, 109:11851192), P238A, A327Q, A327G, P329A (Shields RL, et al., (2001) J Biol Chem. 276(9):6591-604) , K322A, L234F, L235E (Hezareh, et al., (2001) J Virol75, 12161-12168; Oganesyan et al., (2008). Acta Crystallographica 64, 700-704), P331S (Oganesyan et al., (2008) ) Acta Crystallographica 64, 700-704), T394D (Wilkinson et al. (2013) MAbs 5(3): 406-417), A330L, M252Y, S254T, and/or T256E, where the amino acid position is indicated in

- 51 044628 according to the EU or Cabot numbering system. In certain embodiments, the Fc region further includes a deletion of an amino acid at a position corresponding to glycine 236 according to the EU or Cabot numbering system.

In some embodiments, the anti-SIRPA antibody is an IgG1 isotype with a heavy chain constant region that contains a C220S amino acid substitution according to the EU or Cabot numbering system. In some embodiments, the Fc region further comprises one or more additional amino acid substitutions selected from A330L, L234F; L235E, and/or P331S according to the EU or Cabot numbering system. In certain embodiments, the anti-SIRPA antibody is of the IgG2 isotype. In some embodiments, the anti-SIRPA antibody comprises 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 (eg, 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, where position amino acids are indicated according to the EU or Cabot numbering system (Vafa O. et al., (2014) Methods 65:114-126).

In certain embodiments, the anti-SIRPA antibody is of the IgG4 isotype. In some embodiments, the anti-SIRPA antibody comprises 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 (eg, 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 (Reddv et al., (2000) J Immunol,164:1925-1933; Angal et al., (1993) Mol Immunol. 30(1):105-8; US 8614299 B2; Vafa O. et al., (2014) Methods 65:114-126), T394D, M252Y, S254T, T256E, N297A and/or N297Q, where the amino acid position is indicated according to the EU or Cabot numbering system. In some embodiments, the antibody is an IgG4 isotype and contains an S228P amino acid substitution at residue position 228, an F234A amino acid substitution at residue position 234, and an L235A amino acid substitution at residue position 235 (EU numbering residue position).

In some embodiments, the Fc region further comprises one or more additional amino acid substitutions selected from M252Y, S254T and/or T256E, where the amino acid position is indicated in accordance with the EU or Cabot numbering system.

In some embodiments of any of the antibodies provided herein, the modified Fc antibody is an IgG1 modified Fc. In some embodiments, the IgG1 modified Fc contains one or more modifications. For example, in some embodiments, the IgG1 modified Fc contains one or more amino acid substitutions (eg, relative to the wild-type Fc region of the same isotype). In some embodiments, 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) R. J. 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), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E , L328F, A330L, M252Y, S254T and/or T256E, where the amino acid position is indicated according to the EU numbering system.

In some embodiments, any of the IgG1 modified Fcs, the Fc contains the N297A mutation according to the EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the D265A and N297A mutations according to the EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the D270A mutation according to the EU numbering. In some embodiments, the modified Fc IgG1 contains L234A and L235A mutations according to EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the L234A and G237A mutations according to the EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the L234A, L235A, and G237A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc contains one or more (including all) of the P238D, L328E, E233, G237D, H268D, P271G, and A330R EU numbering mutations. In some embodiments, any of the IgG1 modified Fcs, the Fc contains one or more of the S267E/L328F mutations according to the EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc contains P238D, L328E, E233D, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments, any of the IgG1

- 52 044628 modified Fc, Fc contains P238D, L328E, G237D, H268D, P271G and A330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc contains P238D, S267E, L328E, e233D, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains P238D, S267E, L328E, G237D, H268D, P271G, and A330R mutations according to EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the C226S, C229S, E233P, L234V, and L235A mutations according to EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the L234F, L235E, and P331S mutations according to the EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the S267E and L328F mutations according to the EU numbering. In some embodiments, any of the IgG1 modified Fcs, the Fc contains the S267E mutation according to the EU numbering. In some embodiments of any of the IgG1 modified Fcs, the Fc comprises a constant heavy 1 (CH1) and IgG1 hinge region substituent with CH1 and an IgG2 hinge region (amino acids 118-230 of IgG2 according to EU numbering) with a kappa light chain.

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

In some embodiments, any of the IgG1 modified Fcs, the IgG1 modified Fcs may further comprise herein combined with the 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 system, to exclude complement activation. In some embodiments of any of the IgG1 modified Fc, 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, any of the IgG1 modified Fc, the IgG1 modified Fc may further comprise one or more mutations to improve the half-life of the antibody in human serum (e.g., one or more (including all) of the M252Y, S254T, and T256E mutations according to the EU numbering system) . In some embodiments of any of the IgG1 modified Fc, the IgG1 modified Fc may further comprise one or more of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and/or S440W, as defined by EU numbering.

Other aspects of the present invention relate to antibodies having modified constant regions (ie, Fc regions). Antibodies that rely on FcgR receptor binding to activate target receptors may lose their agonist effect if designed to exclude FcgR binding (see, for example, Wilson et al. Cancer Cell 19:101-113 (2011); Armor at al. Immunology 40:585-593(2003); and White et al. Cancer Cell 27:138-148(2015)). Thus, it is believed that an anti-SIRPA antibody of the present invention with the correct epitope specificity can activate the target antigen, with minimal side effects, if the antibody has an Fc domain from the human IgG2 isotype (CH1 and hinge region) or another type of Fc domain that is capable of preferentially binding inhibitory FcgRIIB r receptors or variants thereof.

In some embodiments of any of the antibodies provided herein, the modified Fc antibody is an IgG2 modified Fc. In some embodiments, the IgG2 modified Fc contains one or more modifications. For example, in some embodiments, the IgG2 modified Fc contains one or more amino acid substitutions (eg, relative to the wild-type Fc region of the same isotype). In some embodiments of any of the IgG2 modified Fcs, one or more amino acid substitutions are selected from V234A

- 53 044628 (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 (US 2007/0148167; Armour et al. Eur J Immunol 29: 2613-2624(1999); Armour et al. The Haematology Journal 1 (Suppl. 1):27(2000); Armour et al. 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 according to the EU numbering system. In some embodiments, any of the IgG2 modified Fcs, the Fc contains an amino acid substitution at positions V234A and G237A according to EU numbering. In some embodiments, any of the IgG2 modified Fcs, the Fc contains an amino acid substitution at positions C219S or C220S according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc contains an amino acid substitution at positions A330S and P331S according to EU numbering. In some embodiments, any of the IgG2 modified Fcs, the Fc contains an amino acid substitution at positions S267E and L328F according to EU numbering.

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

In some embodiments, any of the IgG2 modified Fcs, the Fc contains a C220S amino acid substitution according to the EU numbering system. In some embodiments of any of the IgG2 modified Fcs, the antibody is of an IgG2 isotype with a light chain kappa constant domain that contains a C214S amino acid substitution according to the EU numbering system.

In some embodiments, any of the IgG2 modified Fcs, the Fc contains a C219S amino acid substitution according to the EU numbering system. In some embodiments of any of the IgG2 modified Fcs, the antibody is of an IgG2 isotype with a light chain kappa constant domain that contains a C214S amino acid substitution according to the EU numbering system.

In some embodiments of any of the IgG2 modified Fcs, the Fc comprises an IgG2 isotype constant domain 1 (CH1) and a heavy chain hinge region (White et al. Cancer Cell 27:138148(2015)). In certain embodiments of any of the IgG2 modified Fcs, the IgG2 isotype CH1 and the hinge region comprises the sequence of amino acids 118-230 according to EU numbering. 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 system.

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

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

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

In certain embodiments of any of the IgG1 and/or IgG2 modified Fcs, the Fc 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 according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E430G and P331S according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E430G and K322A according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E430G, A330S, and P331S according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc comprises an amino acid

- 54 044628 replacement in positions E430G, K322A, A330S and P331S according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E430G, K322A, and A330S according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E430G, K322A, and P331S according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions S267E and L328F according to EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at position C127S according to the EU numbering. In some embodiments, any of the IgG1 and/or IgG2 modified Fcs, the Fc contains an amino acid substitution at positions E345R, E430G, and S440Y according to EU numbering.

In some embodiments of any of the antibodies provided herein, the modified Fc antibody is an IgG4 modified Fc. In some embodiments, the IgG4 modified Fc contains one or more modifications. For example, in some embodiments, the IgG4 modified Fc contains one or more amino acid substitutions (eg, relative to the wild-type Fc region of the same isotype). In some embodiments of any of the IgG4 modified Fc, 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 system. In some embodiments of any of the IgG4 modified Fcs, the Fc may further comprise L235A, G237A, and E318A according to the EU numbering system. In some embodiments of any of the IgG4 modified Fcs, the Fc may further comprise S228P and L235E according to the EU numbering system. 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 system.

In some embodiments, any of the IgG4 modified Fcs, the IgG4 modified Fcs may be combined with the S228P mutation according to the EU numbering system (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 improve antibody stabilization.

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

In some embodiments, any of the IgG4 modified Fcs, the Fc comprises L235E according to the EU numbering. In certain embodiments of any of the IgG4 modified Fcs, the Fc contains 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, any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at positions E430G and P331S according to the EU numbering. In some embodiments, any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at positions E430G and K322A according to EU numbering. In some embodiments, any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at position E430 according to the EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc region contains an amino acid substitution at positions E430G and K322A according to EU numbering. In some embodiments, any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at positions S267E and L328F according to the EU numbering. In some embodiments, any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at position C127S according to the EU numbering. In some embodiments, any of the IgG4 modified Fcs, the Fc contains an amino acid substitution at positions E345R, E430G, and S440Y according to the EU numbering.

Other antibody modifications.

In some embodiments of any of the antibodies, the antibody is a derivative. The term derivative refers to a molecule that involves chemical modifications other than insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, the derivatives contain covalent modifications, including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic groups. In certain embodiments, a chemically modified antigen binding protein may have a longer half-life in the bloodstream than an antigen binding protein that is not chemically modified. In certain embodiments, the chemically modified antigen binding protein may have improved targeting efficiency to desired cells, tissues and/or organs. In some embodiments, the antigen binding protein derivative is covalently modified to include one or more water-soluble polymeric moieties, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or

- 55 044628 propylene glycol. See, for example, US patents No. 4640835, 4496689, 4301144, 4670417, 4791192 and 4179337. In certain embodiments, the anti -agent protein contains one or more polymers, including, but not limited, monometoxopypolietilen glycol, dextrin, cellulomes, subpoles of ethylenegliko. La/ propylene glycol, carboxymethylcellulose, polyvinylpyrrolidodine, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), poly(Nvinylpyrrolidodine)-polyethylene glycol, propylene glycol homopolymers , polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerin) and polyvinyl alcohol, as well as mixtures of such polymers.

In certain embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are linked at one or more specific positions, for example, at the amino terminus of the derivative. In certain embodiments, one or more water-soluble polymers are optionally attached to one or more side chains of the derivative. In certain embodiments, PEG is used to improve therapeutic efficacy for an antigen binding protein. In certain embodiments, PEG is used to improve the therapeutic efficacy of a humanized antibody. Certain such methods are discussed, for example, in US Pat. No. 6,133,426, which is incorporated herein by reference for all purposes.

Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of template peptides. These types of non-peptide compounds are called peptide mimetics or peptidomimetics. Fauchere, J. Adv. Drug Res., 15:29 (1986); and Evans et al. J. Med. Chem., 30:1229 (1987), which are incorporated herein by reference for all purposes. Such compounds are often developed using computerized molecular modeling. Peptidomimetics, which are structurally similar to therapeutically used peptides, can be used to achieve similar therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to a model polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity) such as a human antibody, but have one or more peptide bonds, optionally replaced by a bond selected from: -CH ₂ NH -, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH) _CH2- and -CH2SO-, by methods well known in the art. Systematic replacement of one or more amino acids of a consensus sequence with a D-amino acid of the same type (eg, D-lysine instead of L-lysine) can be used in certain embodiments to create more stable peptides. In addition, peptides with limited conformational freedom containing a consensus sequence or a substantially identical variant of the consensus sequence can be created 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 the addition of internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

Drug conjugation involves combining a biologically active cytotoxic (anticancer) load or drug with an antibody that specifically targets a specific tumor marker (eg, a polypeptide that is ideally found only on or in tumor cells). Antibodies track these proteins in the body and themselves attach to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal into the tumor cell, which then absorbs or internalizes the antibody along with the cytotoxin. Once the ADC is internalized, the cytotoxic drug is released and kills the cancer. Thanks to this targeting, ideally the drug has fewer side effects and a wider therapeutic window than other chemotherapy agents. Methods for conjugating antibodies are described and known in the art (see, for example, Jane de Lartigue OncLive July 5, 2012; ADC Review on antiboby-drug conjugates; and Ducry et al. Bioconjugate Chemistry 21 (1):5-13 (2010) .

Antibody framework regions.

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

- 56 044628 sequence of a human antibody of a particular subgroup of the variable region of the light or heavy chains (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol. , 151:2623 (1993)); human mature (somatically mutated) frameworks or human germline frameworks (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening KO libraries (see, for example, Vasa et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

In some embodiments, an anti-SIRPA antibody of the present 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, the anti-SIRPA antibody of the present 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, the anti-SIRPA antibody of the present 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, the anti-SIRPA antibody of the present invention comprises a heavy chain variable region comprising VH FR3, wherein VH FR3 comprises the amino acid sequence of SEQ ID No: 27. B In some embodiments, the anti-SIRPA antibody of the present 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, the anti-SIRPA antibody of the present invention comprises a heavy chain variable region comprising VH FR1 containing the amino acid sequence SEQ ID No: 25, VH FR2 containing the amino acid sequence SEQ ID No: 26, VH FR3 containing the amino acid sequence SEQ ID No: 27 and VH FR4 containing the amino acid sequence SEQ ID No: 28.

In some embodiments, the anti-SIRPA antibody of the present invention comprises a heavy chain variable region comprising VH FR1, VH FR2, VH FR3 and VH FR4 anti-SIRPA antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9 -5, 3F9-6, 3F9-7, 3F9-8, 3F9-9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F914, 3F9-15, 3F9-16, 3F9-17, 3F9 -18, 3F9-19, 3F9-20, 3F9-21, 3F9-22, 3F9-23, 3F9-24 or 3F9-25.

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

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

In some embodiments, the anti-SIRPA antibody of the present invention comprises a heavy chain variable region comprising VH FR1 containing the amino acid sequence of SEQ ID No: 25, VH FR2 containing the amino acid sequence of SEQ ID No: 26, VH FR3 containing the amino acid sequence of SEQ ID No.: 27, and VH FR4 containing the amino acid sequence SEQ ID No.: 28, and a light chain variable region containing VL FR1 containing the amino acid sequence SEQ ID No.: 29, VL FR2 containing the amino acid sequence SEQ ID No.: 30, VL FR3 comprising the amino acid sequence of SEQ ID No: 31 and VL FR4 containing the amino acid sequence of SEQ ID No: 32. In some embodiments, the anti-SIRPA antibody of the present invention comprises a heavy chain variable region comprising VH FR1, VH FR2, VH FR3 and VH FR4 antibodies 3F9-1, 3F9-2, 3F9-3, 3F94, 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 variable light chain region containing VL FR1, VL FR2, VL FR3 and VL FR4 antibodies 3F9-1, 3F9-2, 3F9-3, 3F9-4, 3F9-5, 3F9-6, 3F9-7, 3F9-8, 3F9 -9, 3F9-10, 3F9-11, 3F9-12, 3F9-13, 3F9-14, 3F9-15, 3F9-16, 3F9-17, 3F9-18, 3F9-19, 3F9-20, 3F9- 57 044628

21, 3F9-22, 3F9-23, 3F9-24 or 3F9-25.

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 contains the amino acid sequence of SEQ ID No: 50.

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 contains the amino acid sequence of SEQ ID No: 50.

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 contains the amino acid sequence of SEQ ID No: 50.

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 contains the amino acid sequence of SEQ ID No: 50.

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 contains the amino acid sequence of SEQ ID No: 50.

Nucleic acids, vectors and host cells.

The anti-SIRPA antibodies of the present invention can be produced using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. In some embodiments, isolated nucleic acids are provided having nucleotide sequences encoding any of the anti-SIRPA antibodies of the present invention. Such nucleic acids may encode an amino acid sequence containing the V _L and/or an amino acid sequence containing the V H of the anti-SIRPA antibody (eg, the light and/or heavy chains of the antibody). In some embodiments, one or more vectors (eg, expression vectors) containing such nucleic acids are provided. In some embodiments, a host cell containing such nucleic acids is also provided. In some embodiments, the host cell contains (eg, has been transduced with): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing a V _L antibody and an amino acid sequence containing a V _H antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing a V _L antibody and a second vector containing a nucleic acid that encodes an amino acid sequence containing a V _H antibody. In some embodiments, the host cell is a eukaryotic, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (eg, a Y0, NS0, Sp20 cell). The host cells of the present invention also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

Methods for producing the anti-SIRPA antibody of the present invention are provided. In some embodiments, the method includes culturing a host cell of the present invention containing a nucleic acid encoding an anti-SIRPA antibody under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

To recombinantly produce the anti-SIRPA antibody of the present invention, the nucleic acid encoding the anti-SIRPA antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional techniques (eg, using oligonucleotide probes that are capable of binding specifically to the genes encoding the heavy and light chains of the antibody).

Suitable vectors containing a nucleic acid sequence encoding the anti-SIRPA antibodies of the present invention or cell surface expressed fragments or polypeptides thereof, the polypeptides (including antibodies) described herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors, which can be constructed by standard methods or can be selected from a large number of cloning vectors, are available in the art. Although the cloning vector selected may vary according to the host cell selected for use, useful cloning vectors typically have the ability to self-replicate, may contain a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in the selection of clones containing the vector . Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and bifunctional vectors such as pSA3 and pAT28 . These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, anti-SIRPA antibodies of the present

- 58 044628 of the invention can be obtained in bacteria, in particular when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria (e.g. patents

US Nos. 5648237, 5789199 and 5840523). Once expressed, the antibody can be isolated from the bacterial cell paste in the soluble fraction, and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as hyphomycetes 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 an antibody with a partial or complete human glycosylation pattern (eg , Gerngross Nat. Biotech. 22:1409-1414 (2004); and Li et al. Nat. Biotech. 24:210215 (2006)).

Suitable host cells for expression of the glycosylated antibody can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrates include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (eg, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, colonies of mammalian cells that are adapted to be grown in suspension may be useful. Other examples of useful mammalian host cell colonies include CV1 monkey kidney cell colony transformed with SV40 (C0S-7); a colony of human embryonic kidney cells (293 or 293 cells, as described, for example, in Graham et al. J. Gen Virol. 36:59 (1977)); newborn hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, 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 (HELA) cells; canine kidney cells (MDCK); gray rat kidney cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (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 useful colony mammalian host cells include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al. Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell colonies such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell colonies suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Pharmaceutical compositions/formulations.

Provided herein are pharmaceutical compositions and/or pharmaceutical formulations containing the anti-SIRPA antibodies of the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier is preferably non-toxic to recipients at the dosages and concentrations used. The antibodies described herein may be formulated in solid, semi-solid, liquid or gaseous form. Examples of such formulations include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres and aerosols. Pharmaceutically acceptable carriers may include, depending on the desired formulation, pharmaceutically acceptable non-toxic diluent carriers, which are carriers commonly used for formulating pharmaceutical compositions for administration to animals or humans. In certain embodiments, the pharmaceutical composition may contain constituent materials to modify, maintain or maintain, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition.

In certain embodiments, pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); excipients (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); dyes, flavors and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronics, PEGs, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery means; thinners; excipients and/or pharmaceutical adjuvants. Additional examples of formulations suitable for various types of administration can be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press 22nd ed. (2013). For a brief review of drug delivery methods, see Langer, Science 249:1527-1533 (1990).

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents and solutes that render the formulation isotonic to the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents. agents, solubilizers, thickeners, stabilizers and preservatives.

Formulations may be optimized for retention and stabilization in the brain or central nervous system. When an agent is administered into the cranial compartment, it is desirable that the agent remains in the compartment and does not spread or otherwise cross the blood-brain barrier. Stabilization methods include cross-linking, multimerization or coupling to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. to achieve an increase in molecular weight.

Other strategies to increase retention include entrapping an antibody, such as the anti-SIRPA antibody of the present invention, in a biodegradable or bioerodible implant. The rate of release of the therapeutically active agent is controlled by the rate of transport through the polymer matrix and biodegradation of the implant. The implants may be particles, sheets, patches, plaques, fibers, microcapsules and the like, and may be of any size or shape compatible with the chosen insertion site. Biodegradable polymer compositions that can be used can be organic esters or ethers that, when degraded, lead to physiologically acceptable degradation products, including monomers. Anhydrides, amides, orthoesters or the like may find use, alone or in combination with other monomers. The polymers will be condensation polymers. The polymers may be cross-linked or non-cross-linked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids, either homo- or copolymers and polysaccharides. Polyesters of interest include polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof. Polysaccharides of interest include calcium alginate and functionalized celluloses, in particular carboxymethylcellulose esters, characterized by being water soluble, having a molecular weight of about 5 kDa to 500 kDa, etc. Biodegradable hydrogels can also be used in the implants of the present invention. Hydrogels are typically a copolymer material characterized by their ability to absorb liquid.

Therapeutic use.

As described herein, the anti-SIRPA antibodies of the present invention can be used to prevent, reduce the risk of, or treat diseases and disorders.

In one aspect of the invention, an agent that negatively regulates SIRPA, for example an anti-SIRPA antibody, is used as a therapeutic agent. 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. A treatment regimen is accomplished by identifying a subject, for example a human patient, suffering from (or at risk of developing) a disease or disorder associated with SIRPA expression, activity and/or signaling, such as cancer or other oncoproliferative diseases, using standard methods. In some embodiments, cells having 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.

As described in detail below, an agent that negatively regulates SIRPA, such as an anti-SIRPA antibody, can be used in combination with an additional therapeutic agent that is used to treat a disease or pathology associated with SIRPA expression, activity and/or signaling. The terms combined and jointly are used interchangeably in the present invention. The additional therapeutic agent administered in combination with the anti-SIRPA antibody may be administered before, after, or simultaneously with an agent that negatively regulates SIRPA, such as an anti-SIRPA antibody.

In one aspect of the present invention, an anti-SIRPA antibody preparation, for example comprising an anti-SIRPA antibody, which reduces cell surface expression of SIRPA but does not substantially

- 60 044628 blocks the binding of a ligand, for example CD47, to SIRPA, administered to a person. Administration of an antibody may suppress or inhibit or interfere with expression, activity and/or signaling function

SIRPA, which is mediated by ligand binding, such as CD47 binding.

In one embodiment, the disease or disorder with SIRPA expression is cancer. In some embodiments, the anti-SIRPA antibody is administered to a patient who has a cancer, such as a hematologic myeloid cell proliferative disorder, that express SIRPA. In exemplary embodiments, the anti-SIRPA antibody is administered to a patient who has cancer expressing CD47.

In certain embodiments, cancers preventable and treatable 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 cell non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, non-squamous cell NSCLC, glioma, abdominal cancer, hepatocellular carcinoma, gastrointestinal cancer, gastrointestinal or stomach cancer, including gastrointestinal cancer, gastrointestinal stromal cancer, kidney cancer (eg, clear cell carcinoma), cancer ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (eg, renal cell carcinoma (RCC)), prostate cancer (eg, hormone-resistant prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme) , cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastrointestinal cancer, germ cell tumor, childhood sarcoma, sinonasal natural killer cell carcinoma, melanoma (eg, metastatic malignant melanoma such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, cancer small intestine, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid childhood tumors, ureteral cancer, renal pelvic carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, myelodysplastic syndromes, colorectal neoplasms, solid tumors, tumor angiogenesis, spinal axis tumor, brain stem cancer, eosinophilic adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, cancers caused by environmental conditions, including those caused by asbestos, viral cancers (eg, human papillomavirus (HPV) tumors) and hematological malignancies derived from either of two blood cell colonies, i.e., myeloid cell colonies (which produce granulocytes, red blood cells, platelets, macrophages and mast cells) or colonies of lymphoid cells (which produce B, T, NK and plasma cells), such as all types of leukemias, lymphomas and myelomas, such as acute, chronic, lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular mutation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 eosinophilia variant [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), indolent lymphoma, large B-cell diffuse lymphoma, B-cell hematological malignancy such as B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B- cellular lymphoma, mucosal lymphoid tissue lymphoma (MTLL), anaplastic (eg, Ki 1+) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, lymphoblastic T-cell progenitor lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors lymphoid cells, acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL) (including patients with DLBCL who are initially refractory to treatment, refractory to treatment with Rituximab, refractory to treatment with Rituximab/CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), recurrent or treatment refractory (R/R) DLBCL and/or CAP-T unsuitable DLBCL patients), Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, B-lymphoblast progenitor cell lymphoma, cutaneous T-cell lymphoma (CTCL) ) (also called mycosis fungoides or Sézary syndrome) and lymphoplasmacytic lymphoma (LPL) with Waldenström's macroglobulinemia; myelomas such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called low-grade myeloma), isolated plasmacytoma and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid cells, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, hematopoietic lymphoid cell tumors, such as T-cell and B-cell tumors, including, but not limited to, T-cell disorders such as T-cell prolymphocytic leukemia (T-PLL), including small cell and medullary cell types; large granular lymphocyte leukemia (LGL), preferably T-cell type; a/d T-NHL semisyllabic lymphoma; peripheral/post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, colon cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these cancers. The anti-SIRPA antibody of the present invention can also be used to treat metastatic cancer.

In some embodiments, 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, melanoma, lymphoma, pancreatic cancer , prostate cancer, ovarian cancer and fibrosarcoma. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer may be an early stage cancer or an advanced stage cancer. In some embodiments, the cancer may be a primary tumor. In some embodiments, the cancer may be a second site metastatic tumor derived from any of the above cancer types.

In some embodiments, the cancer is selected from glioblastoma multiforme; clear cell renal carcinoma; adrenocortical carcinoma; urothelial carcinoma of the bladder; diffuse large B-cell lymphoma; lung adenocarcinoma; pancreatic adenocarcinomas, renal cell cancer, non -watering lymphoma, acute lymphoblastic leukemia (OLL), acute myeloidal leukemia (OML), chronic lymphocytic leukemia (HLL), chronic myeloidal leukemia, multiple myeloma, invasive carcinoma of the feud a cervical carcinoma, the uterine endocervical carcinoma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal carcinoma, squamous cell carcinoma of the head and neck, chromophobe kidney cancer, papillary kidney cancer, low-grade glioma, hepatocellular carcinoma, squamous cell carcinoma of the lung, mesothelioma, serous cystadenocarcinoma ovarian, pancreatic adenocarcinoma, pheochromocytomas and paragangliomas, prostate adenocarcinoma, rectal adenocarcinoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumors, thyroid carcinoma, thymoma, endometrial carcinoma of the uterine body, uterine carcinosarcoma and uveal melanoma.

In some embodiments, the present invention provides methods for preventing, reducing the risk of, or treating an individual suffering from cancer, where the individual is refractory to immune checkpoint inhibitor therapy, by administering to the individual a therapeutically effective amount of an anti-SIRPA antibody of the present invention.

In some embodiments, the present invention provides methods for preventing, reducing the risk of, or treating an individual suffering from cancer by administering to the individual a therapeutically effective amount of an anti-SIRPA antibody of the present invention.

In some embodiments, an anti-SIRPA antibody of the present invention may be co-administered with a therapeutic agent that acts as an immune checkpoint inhibitor. In some embodiments, the method further includes administering to the individual at least one antibody that specifically binds to an inhibitory immune checkpoint molecule and/or other standard or investigational anticancer therapy. In some embodiments, the inhibitory immune checkpoint molecule is selected from PD1, PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39 and CD73. In exemplary embodiments, the therapeutic agent is an antibody to an immune checkpoint inhibitor selected from D1, PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7- H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39 or CD73. In some embodiments, at least one antibody that specifically binds to an inhibitory immune checkpoint molecule is administered in combination with an anti-SIRPA antibody of the present invention. In some embodiments, a combination of an antibody directed against immune checkpoint inhibitors is co-administered with an anti-SIRPA antibody of the present invention.

In some embodiments, an anti-SIRPA antibody of the present invention may be co-administered with at least one agonistic antibody that specifically binds to a stimulatory immune checkpoint protein, e.g., an anti-CD40 antibody agonist, an anti-OX40 antibody agonist, an -ICOS antibodies, agonist anti-CD28 antibodies,

- 62 044628 agonistic anti-TREM1 antibody, agonistic anti-TREM2 antibody, agonist anti-CD137/4-1BB antibody, agonist anti-CD27 antibody, agonist anti-glucocorticoid TNFR-related protein GITR antibody, agonist anti-CD30 antibody, agonist anti-BTLA antibody, agonist anti-HVEM antibody, anti-CD2 antibody agonist, anti-CD5 antibody agonist, and any combination thereof.

In some embodiments, the method further includes administering to the individual at least one antibody that specifically binds to an inhibitory immune checkpoint molecule and/or other standard or investigational anticancer therapy. In some embodiments, at least one antibody that specifically binds to an inhibitory immune checkpoint molecule is administered in combination with an anti-SIRPA antibody of the present invention. In some embodiments, the at least one antibody that specifically binds to an inhibitory immune checkpoint molecule is selected from an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody, an -B7-H3 antibodies, anti-B7-H4 antibodies and anti-HVEM antibodies, anti-B and T lymphocyte attenuator antibodies (BTLA), anti-killer cell inhibitory receptor (KIR) antibodies, anti-GAL9 antibodies, anti -T1M3 antibodies, anti-A2AR antibodies, anti-LAG-3 antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies and any combination thereof. In some embodiments, the standard or investigational anticancer therapy is one or more therapies selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab (Herceptin®), adoptive cell transfer (ACT), chimeric antigen receptor T- cell transfer (CAP-T), vaccine therapy and cytokine therapy. In some embodiments, the method further includes administering to the individual at least one antibody that specifically binds to the inhibitory cytokine. In some embodiments, at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with an anti-SIRPA antibody of the present invention. In some embodiments, the at least one antibody that specifically binds to the inhibitory cytokine is selected from an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-[L-2 antibody, and any combination thereof.

In some embodiments, the method further includes administering to the individual at least one agonistic antibody that specifically binds to a stimulatory immune checkpoint protein. In some embodiments, at least one agonist antibody that specifically binds to a stimulatory immune checkpoint protein is administered in combination with an anti-SIRPA antibody of the present invention. In some embodiments, the at least one agonist antibody that specifically binds to a stimulatory immune checkpoint protein is selected from an anti-CD40 antibody agonist, an anti-OX40 antibody agonist, an anti-ICOS antibody agonist, an anti-CD28 antibody agonist, an anti-CD137/4-1BB antibody, anti-CD27 antibody agonist, antiglucocorticoid-induced TNFR-related protein GITR antibody agonist, and any combination thereof.

In some embodiments, the anti-SIRPA antibody of the present invention is administered in combination with radiation therapy and/or a chemotherapeutic agent. Chemotherapeutic agents include, for example, the following groups: antimetabolites/anticancer agents such as pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogues, folate antagonists and related inhibitors (methotrexate, pemetrexed, mercaptopurine, thioguanine, pentostatin and 2-chlorodeosciadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, eribuline and navelbine; epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea , plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylene thiophosphoramide and etoposide (VP 16)); DNA methyltransferase inhibitors (azacytidine); antibiotics such as dactinomycin (Actinomycin D), daunorubicin, doxorubicin (Adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase, which systematically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes ( dacarbazine (DTIC)); antiproliferative/antimitotic antimetabolites such as folic acid analogues (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues

- 63 044628 (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migration agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP470, genistein, pomalidomide) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors such as zivaflibercept; fibroblast growth factor (FGF) inhibitors); inhibitors of apoptosis protein (IAP) antagonists (birinapant); histodine deacetylase (HDAC) inhibitors (vorinostat, romidepsin, chidamide, panobinostat, mocetinostat, abexinostat, belinostat, etinostat, resminostat, givinostat, quisinostat, SB939); proteasome inhibitors (ixazomib); angiotensin receptor blocker; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, panitumumab, pertuzumab, cetuximab, adalimumab, golimumab, infliximab, rituximab, ocrelizumab, ofatumumab, obinutuzumab, alemtuzumab, abciximab, atlizumab, daclizumab, donezumab, efalizumab, elotuzumab, rovelizumab, ruplizumab, ustekinumab, visilizumab, gemtuzumab, ozogamicin, brentuximab, vedotin); chimeric antigen receptors; cell cycle inhibitors (flavopiridol, roscovitine, bryostatin-1) and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (Adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone , prednisone and prednisolone); PARP inhibitors (niraparib, olaparib); focal adhesion kinase (FAK) inhibitors (defactinib (VS-6063), VS-4718, VS-6062, GSK2256098); growth factor signal transduction kinase inhibitors (cediranib, galuniserib, rociletinib, vandetanib, afatanib, EGF816, AZD4547); c-Met inhibitors (capmatinib, INC280); ALK inhibitors (ceritinib, crizotinib); inducers of mitochondrial dysfunction, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin and caspase activators; and chromatin breakers. In some embodiments, the chemotherapeutic agent is a B-Raf inhibitor, a MEK inhibitor, a VEGF inhibitor, a VEGFR inhibitor, a tyrosine kinase inhibitor, an anti-mitotic agent, or any combination thereof.

In some embodiments, the anti-SIRPA antibody of the present invention is administered in combination with adoptive cell transfer (ACT) therapy, chimeric antigen receptor T-cell transfer (CAP-T) therapy, vaccine therapy, and/or cytokine therapy.

In some embodiments, an anti-SIRPA antibody of the present invention is administered in combination with at least one antibody that specifically binds to an inhibitory cytokine, e.g., an inhibitory cytokine such as an anti-CCL2 antibody, an anti-CSF-1 antibody, or an anti-SIRPA antibody. -IL-2 antibody.

In some embodiments, the anti-SIRPA antibody of the present invention is administered in combination with at least one stimulatory cytokine. In some embodiments, which may be combined with any of the embodiments herein, the at least one stimulatory cytokine is selected from IFN-a4, IFN-β, ΓΕ-1β, TNF-α, IL-6, IL-8, CRP , family members IL-20, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33 , MCP-1, MIP-1-beta and any combination thereof.

In some embodiments, an agent that negatively regulates SIRPA, such as an anti-SIRPA antibody, is administered to a patient who has a neurological disorder or is administered to reduce the risk of, delay the onset of, or prevent a neurological disorder. In some embodiments, the neurological disorder is dementia, including dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mild cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, tauopathy, or multiple sclerosis.

In some embodiments, an agent that negatively regulates SIRPA, such as an anti-SIRPA antibody, is administered to a patient who has Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, or multiple sclerosis. In some embodiments, the agent is administered to a patient who has Creutzfeldt-Jakob disease, normal pressure hydrocephalus, Nasu-Hakol disease, stroke, infection, traumatic brain injury, progressive supranuclear palsy, boxer's dementia (chronic traumatic encephalopathy), parkinsonism-related chromosome 17, Lytic-Bodiga disease (Parkinson-dementia complex in Guam), dementia with a predominance of neurofibrillary tangles, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervoorden-Spatz disease, lipofuscinosis, Pick's disease, core ticobasal degeneration , a disease characterized by the appearance of argyrophilic granules, frontotemporal lobar degeneration, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy or cortical degeneration of the subcortical ganglia.

- 64 044628

Dementia.

Dementia is a non-specific syndrome (ie, a set of signs and symptoms) that represents a severe loss of overall cognitive ability in a previously normal person, beyond what would be expected from normal aging. Dementia can be static, resulting from a unique global brain injury. Alternatively, dementia can be progressive, resulting in long-term decline due to damage or disease in the body. Although dementia is much more common in older people, it can also occur before the age of 65. Cognitive areas affected by dementia include, but are not limited to, memory, attention span, language, and problem-solving ability. Typically, symptoms must be present for at least six months before a person is diagnosed with dementia.

Typical forms of dementia include, but are not limited to, frontotemporal dementia, Alzheimer's disease, vascular dementia, semantic dementia and dementia with Lewy bodies.

In some embodiments, administration of an anti-SIRPA antibody of the present invention can prevent, reduce the risk of, and/or cure dementia. In some embodiments, an anti-SIRPA antibody may modulate one or more SIRPA activities in an individual having dementia.

Frontotemporal dementia.

Frontotemporal dementia (FTD) is a condition that occurs when the frontal lobe of the brain progressively deteriorates. Over time, degeneration may spread to the temporal lobe. Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for 20% of cases of presenile dementia. Clinical features of FTD include memory deficits, behavioral abnormalities, personality changes, and language impairment (Cruts, M. & Van Broeckhoven, C, Trends Genet. 24:186-194 (2008); Neary, D., et al., Neurology 51: 1546-1554 (1998); Ratnavalli, E., Brayne, C., Dawson, K. & Hodges, J. R., Neurology 58:1615-1621 (2002)).

A significant proportion of cases of FTD are inherited in an autosomal dominant pattern, but even within the same family, symptoms can span the spectrum from FTD with behavioral disturbances to primary progressive aphasia to corticobasal ganglion degeneration. FTD, like most neurodegenerative diseases, can be characterized by the abnormal presence of certain protein aggregates in the affected brain. Historically, the first descriptions of FTD recognized the presence of intraneuronal accumulations of hyperphosphorylated tau protein in neurofibrillary tangles or Pick bodies. The causative role of microtubule-associated protein tau has been supported by the identification of mutations in the gene encoding tau protein in several families (Hutton, M., et al., Nature 393:702-705 (1998). However, in most brains, FTD lesions do not show accumulation of hyperphosphorylated tau protein, but do demonstrate immunoreactivity for ubiquitin (Ub) and TAR DNA binding protein (TDP43) (Neumann, M., et al., Arch. Neural. 64:1388-1394 (2007) Most cases of FTD with Ub inclusions (FTD-U) have been shown to have mutations in the progranulin gene.

In some embodiments, administration of an anti-SIPRA antibody of the present invention can prevent, reduce the risk of, and/or treat FTD. In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having FTD.

Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia. There is no cure for the disease and it worsens as it progresses and ultimately leads to death. Most often, asthma is diagnosed in people over 65 years of age. However, the less common early-onset Alzheimer's disease can occur much earlier. Common symptoms of Alzheimer's disease include behavioral symptoms such as difficulty remembering recent events; cognitive symptoms, confusion, irritability and aggression, mood swings, speech problems and long-term memory loss. As the disease progresses, body functions are lost, eventually leading to death. Alzheimer's disease develops over an unknown and variable period of time before becoming fully evident, and can progress without diagnosis for many years.

In some embodiments, administration of an anti-SIPRA antibody of the present invention may prevent, reduce the risk of, and/or treat Alzheimer's disease. In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having Alzheimer's disease.

Parkinson's disease.

Parkinson's disease, which may be called idiopathic or primary parkinsonism, hypokinetic rigid syndrome (HRS), or shaking palsy, is a neurodegenerative disease of the brain that affects the control of the motor system. The progressive death of brain cells that produce dopamine leads to the main symptoms of Parkinson's disease. Parkinson's disease is most often diagnosed in people over 50 years of age. Parkinson's disease is idiopathic (has no known cause) in most people. However, genetic factors also play a role in the disease.

Symptoms of Parkinson's disease include, but are not limited to, tremors of the hands, arms, legs, jaws and face, stiffness in the muscles of the limbs and trunk, slowness of movement (bradykinesia), postural instability, difficulty walking, neuropsychiatric problems, changes in speech or behavior, depression, anxiety, pain, psychosis, dementia, hallucinations and sleep problems.

In some embodiments, administration of an anti-SIPRA antibody of the present invention may prevent, reduce the risk of, and/or treat Parkinson's disease. In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having Parkinson's disease.

Amyotrophic lateral sclerosis (ALS).

In the context of this document, amyotrophic lateral sclerosis (ALS), or motor neuron disease, or Lou-Gehrig's disease, is used interchangeably and refers to a disabling disease of various etiologies, characterized by rapidly progressive weakness, muscle atrophy and fasciculations, muscle spasticity, difficulty speaking (dysarthria) , difficulty swallowing (dysphagia) and difficulty breathing (shortness of breath).

Progranulin has been shown to play a role in ABS (Schymick, JC et al., (2007) J[0343] Neurol Neurosurg Psychiatry.;78:754-6) and protects against damage caused by ABS-causing proteins such as TDP- 43 (Laird, AS et al., (2010). PLoS ONE 5: e13368). It has also been demonstrated that proNGF causes p75-mediated death of oligodendrocytes and corticospinal neurons after spinal cord injury (Beatty et al., Neuron (2002), 36, pp. 375-386; Giehl et al, Proc. Natl. Acad. Sci USA (2004), 101, pp 6226-30).

In some embodiments, administration of an anti-SIPRA antibody of the present invention may prevent, reduce the risk of, and/or treat ABS. In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having amyotrophic lateral sclerosis.

Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an anautosomal dominant mutation in the Huntingtin gene (HTT). Propagation of the cytokine-adenine-guanine (CAG) triplet repeat in the Huntingtin gene results in the production of a mutant form of the Huntingtin protein (Htt) encoded by this gene. This mutant huntingtin protein (mHtt) is toxic and promotes neuronal death. Symptoms of Huntington's disease most often appear between the ages of 35 and 44, although they can appear at any age.

Symptoms of Huntington's disease include, but are not limited to, motor control problems, jerky movements, random movements (chorea), abnormal eye movements, balance problems, seizures, difficulty chewing, difficulty swallowing, cognitive problems, speech impairment, memory deficits, difficulty thinking, insomnia , fatigue, dementia, personality changes, depression, anxiety and compulsive behavior.

In some embodiments, administration of an anti-SIRPA antibody of the present invention can prevent, reduce the risk of, and/or cure Huntington's disease (HD). In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having Huntington's disease.

Tauopathy.

Taupathic diseases, or tauopathies, are a class of neurodegenerative diseases caused by the aggregation of microtubule-associated protein tau in the brain. Alzheimer's disease (AD) is the best known taupathic disease and involves the accumulation of tau protein in neurons in the form of insoluble neurofibrillary tangles (NFTs). Other taupathic diseases and disorders include progressive supranuclear palsy, boxer's dementia (chronic traumatic encephalopathy), frontotemporal dementia and parkinsonism associated with chromosome 17, Lytic-Bodiga disease (Guam Parkinson-dementia complex), dementia with glomerular predominance, gangioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervoorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic grain disease (AGD), Huntington's disease and frontotemporal lobe degeneration.

In some embodiments, administration of an anti-SIPRA antibody of the present invention may prevent, reduce the risk of, and/or treat tauopathy. In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having tauopathy.

Multiple sclerosis.

Multiple sclerosis (MS) is also called multiple sclerosis or multiple encephalomyelitis. MS is an inflammatory disease in which the fatty myelin sheaths around axons in the brain and spinal cord are damaged, leading to demyelination and scarring, as well as a wide range of signs and symptoms. PC affects the ability of nerve cells in the brain and spinal cord to communicate effectively with each other. Nerve cells communicate by sending electrical signals called action potentials along long fibers called axons, which are contained in an insulating substance called myelin. In MS, the body's own immune system attacks and damages myelin. When myelin is lost, axons can no longer conduct signals effectively. The onset of multiple sclerosis usually occurs in young people and is more common in women.

Symptoms of PC include, but are not limited to, changes in sensation such as loss of sensation or tingling; piercing pain or numbness such as hypoesthesia and paresthesia; muscle weakness; clonus; muscle spasms; difficulty in movement; difficulties with coordination and balance, such as ataxia; speech problems such as dysarthria, or swallowing problems such as dysphagia; vision problems such as nystagmus, optic neuritis including phosphene and diplopia; fatigue; acute or chronic pain; and problems with the bladder and intestines; cognitive impairment of varying degrees; emotional symptoms of depression or unstable mood; Uchthoff's sign, which is an exacerbation of existing symptoms due to exposure to higher than normal ambient temperatures; and Lhermitte's sign, which is an electrical sensation that runs down the back when the neck is flexed.

In some embodiments, administration of an anti-SIPRA antibody of the present invention can prevent, reduce the risk of, and/or treat multiple sclerosis. In some embodiments, administration of an anti-SIPRA antibody may modulate one or more SIPRA activities in an individual having multiple sclerosis.

In some embodiments, the subject or individual is a mammal. Mammals include, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs and horses), primates (eg, humans and non-human primates such as monkeys), rabbits and rodents (eg, mice and rats). In some embodiments, the subject or individual is a human.

The antibody provided herein (and any additional therapeutic agent) can be administered by any suitable routes, including parenteral, intrapulmonary, intranasal, intralesional, intracerobospinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusions include intramuscular, intravenous bolus or continuous infusion over a period of time, intra-arterial, intra-articular, intraperitoneal or subcutaneous administration. In some embodiments, the administration is intravenous. In some embodiments, administration is subcutaneous. Dosing may be accomplished by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing regimens are discussed herein, including, but not limited to, single or multiple dosing at different times, bolus dosing, and pulse infusion.

The antibodies presented herein may be formulated, dosed and administered in accordance with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to the medical practitioner. The antibody is optionally, but optionally, formulated with one or more agents that are currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. They are generally used at the same dosages and routes of administration as described herein, or from 1 to 99% of the dosages described herein, or at any dosage and route of administration that are empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether administered antibody for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the judgment of the treating physician. The antibody is usually administered to the patient at one time or over several courses of treatment.

Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of antibody may be an initial candidate dose to administer to the patient, e.g., in one or more divided administrations or continuous infusion. One typical daily dose may range from about 1 mcg/kg to 100 mg/kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, treatment is usually extended until desired suppression of the resulting painful symptoms is achieved. One standard

- 67 044628 the dose of the antibody will be from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5, 2.0, 4.0 or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered periodically, for example, every week or every three weeks (eg, so that the patient receives from about two to about twenty or, for example, about six doses of the antibody). In certain embodiments, the dosing frequency is three times daily, twice daily, once daily, once every other day, once weekly, once every two weeks, once every four weeks, once every five weeks. , once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks or once a month, once every two months, once every three months or longer. A higher initial dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of this therapy is easily monitored by conventional methods and tests.

Diagnostic application.

In some embodiments, any of the antibodies, any of the anti-SIRPA antibodies provided herein, are useful for detecting the presence of SIRPA in a sample or individual. The term detection in the context of this document covers quantitative or qualitative detection. This document provides methods for using the antibodies of the present invention for diagnostic purposes, such as the detection of SIRPA in an individual or in tissue samples obtained from an individual. In some embodiments, the individual is a human.

The detection method may include quantifying an antibody bound to an antigen. Detection of antibodies in biological samples can occur by any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, SCAF assay, immunoprecipitation, or micropositron emission tomography. In certain embodiments, the antibody is radiolabeled, for example, with ¹⁸ F, and is subsequently detected using micropositron emission tomography analysis. Antibody binding can also be quantified in a patient using noninvasive techniques such as positron emission tomography (PET), X-ray computed tomography, single photon emission computed tomography (SPECT), computed tomography (CT), and computed axial tomography (CAT).

Finished goods.

Provided herein are finished products (eg, a kit) containing the anti-SIRPA antibody described herein. The finished product may include one or more containers containing the antibody described herein. Containers can be any suitable packaging, including, but not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags) and the like. Containers may be unit doses, bulk units (eg, multi-dose units), or subunit units.

In some embodiments, the kits may further include a second agent. In some embodiments, the second agent is a pharmaceutically acceptable buffer or diluent, including, but not limited to, bacteriostatic water for injection (BWI), phosphate buffered saline, Ringer's solution, and dextrose solution. In some embodiments, the second agent is a pharmaceutically active agent as described above.

In some embodiments of any of the finished products, the finished products further include instructions for use in accordance with the methods of the present description. The instructions typically include information on dosage, dosage schedule, and route of administration for the intended treatment. In some embodiments, these instructions include a description of administering an isolated anti-SIRPA antibody of the present invention (e.g., the anti-SIRPA antibody described herein) for the prevention, risk reduction, or treatment of an individual having a disease, disorder, or injury selected from dementia, frontal -temporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakola disease, stroke, acute injury, chronic injury, lupus, acute and chronic colitis, rheumatoid arthritis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, lupus of the central nervous system, Behçet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear paralysis, corticobasal ganglion degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, seizures, spinal cord injury, age-related macular degeneration, glaucoma, retinal pigmentary degeneration, retinal degeneration, respiratory tract infections, sepsis, eye infections, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteopetrosis disease, Paget's disease bone cancers including 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,

- 68 044628 pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, polycythemia vera, essential thrombocytosis, primary or idiopathic myelofibrosis, primary or idiopathic myelosclerosis, tumors of myeloid origin, tumors expressing SIRPA, thyroid cancer, infections, herpes central nervous system, parasitic infections, Trypanosome infections, Cruzi infections, Pseudomonas aeruginosa infections, Leishmania donovani infections, group B infections Streptococcus infections, Campylobacter jejuni infections, Neisseria meningiditis infections, HIV type I and Haemophilus influenzae infections, according to any methods of this invention. In some embodiments, the instructions include instructions for use of the anti-SIRPA antibody and a second agent (eg, a second pharmaceutically active agent).

The present invention will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the present invention. All citations throughout the invention are incorporated herein by reference.

Examples

The following examples are provided for illustrative purposes only and not as limitations. Those skilled in the art will readily recognize many parameters that can be changed or modified to obtain substantially similar results.

Example 1. Preparation of anti-SIRPA antibodies.

The amino acid sequence of the human SIRPA protein is set forth below in SEQ ID No.: 1. Human SIRPA contains a signal peptide located at amino acid residues 1-30 of SEQ ID No.: 1. Human SIRPA contains an extracellular immunoglobulin-like variable type (IgV) domain located at amino acid residues 32-137 from SEQ ID No: 1; additional constant-type extracellular immunoglobulin-like domain (IgC) sequences located at amino acid residues 148-247 and 254-348 from SEQ ID No: 1; transmembrane domain located at amino acid residues 374-394 of SEQ ID No: 1; and an intracellular domain located at amino acid residues 395-504 of SEQ ID No: 1.

Amino acid sequence of human SIRPAv1 (SEQ ID No: 1):

10 20 thirty 40 50 MEPAGPAPG R LGPLLCLLLLA ASCAWSGVAG EEELQVIQPD KSVLVAAGE T 60 70 80 90 100 ATLRCTATSL IPVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN BY 120 130 140 150 NMDFSIRIGN ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPSA 160 170 180 190 200 PVVSGPAAR TPQHTVSFTC ESHGFSPRDI TLKWFKNGNE LSDFQTNVDP A 210 220 230 240 250 VGESVSYSIH STAKVVLTRE DVHSQVICEV AHVTLQGDPL RGTANLSETI 260 270 280 290 300 RVPPTLEVTQ QPVRAENQVN VTCQVRKFYP QRLQLTWLEN GNVSRTETAS 310 320 330 340 350 TVTENKDGT Y NWMSWLLVN V SAHRDDVKLT CQVEHDGQPA VSKSHDLKVS 360 370 380 390 400 AHPKEQGSN AAENTGSNER NIYIVVGVVC TLLVALLMAA LYLVRIRQKK T 410 420 430 440 450 AQGSTSSTRL HEPEKNAREI TQDTNDITYA DLNLPKGKKP APQAAEPNN N 460 470 480 490 500 TEYASIQTSP QPASEDTLTY ADLDMVHLN R TPKQPAPKPE PSFSEYASVQ

VPRK

Crystal structure analysis of SIRPA-CD47 complexes separates the ligand binding site from the variable loops that bind the β-sheet strands in the IgV domain of SIRPA. CD47-binding

- 69 044628 interface consists of amino acid residues S59-P65, L96-F104 and K123-D130 SIRPA.

Numerous SIRPA polymorphisms have been identified in humans. Since most sequence variations lie outside the CD47 ligand binding site, most SIRPA variants examined are likely to bind CD47 with similar affinity. Another SIRP family member, SIRPB1, shares high sequence homology with SIRPA but does not bind CD47. A single A57M substitution is sufficient to rearrange the S59-P65 ligand binding interface to prevent SIRPB1 binding to CD47. Moreover, the SIRPA-CD47 interaction is highly species specific. Human SIRPA cannot bind mouse or rat or bovine CD47 (of the species tested), and human CD47 only recognizes a distinct allelic variant of mouse SIRPA expressed by the mouse NOD strain.

Protein BLAST analysis of SIRPA sequences from multiple species showed a significant degree of identity with human SIRPA (Fig. 1). The high sequence homology within the SIRP family poses a significant challenge in generating SIRPA-specific antibodies while maintaining cross-reactivity with non-human SIRPA antigens.

Example 2 Characterization of Humanized Anti-SIRPA Antibody.

The murine SIRPA antibody m3F9 is discussed herein and contains a heavy chain variable region containing the amino acid sequence of SEQ ID No: 2 and a light chain variable region containing the amino acid sequence of SEQ ID No: 5.

In table 4 and 5 below list the hybridoma-derived heavy chain variable region and light chain variable region amino acid sequences of the mouse anti-SIRPA antibody, m3F9, as well as the corresponding humanized sequences (denoted h3F9). The first humanized antibody sequence for the heavy chain variable domain of the anti-SIRPA antibody 3F9 (h3F9-H1) is an OKO replacement with no change in the human framework sequence.

The next humanized variable region heavy chain sequence (h3F9-H2) has altered framework residues at certain CCR boundaries. (See FIG. 2A.) In FIG. 2A lists potential humanized sequences of the heavy chain variable domain of the anti-SIRPA antibody 3F9. The humanized sequences are based on the IGHV3-23*01 acceptor framework region and the IGHJ4*01 connecting region.

The first humanized sequence for the light chain variable domain of anti-SIRPA antibody 3F9 (h3F9-L1) is an OCR replacement of the light chain variable domain without changes to the human framework sequence. Subsequent humanized light chain variable region sequences alter the amino acid residues of the framework region (h3F9-L2 and h3F9-L3). (See FIG. 2B.) In FIG. 2B lists potential humanized light chain variable domain sequences of the anti-SIRPA antibody 3F9.

The humanized sequence is based on the IGKV3-11*01 acceptor framework region and the IGKJ2*01 connecting region. OKO sequences are highlighted in bold. OKO definitions are AbM from the website www.bioinf.org.uk/abs/. b indicates masked side chain; p means partially masked; i denotes the side chain at the interface between the VH and VL domains. Sequences that differ between human and mouse germline sequences are indicated with an asterisk (*). Potential additional mutations in the framework regions are noted below the sequence. Potential changes to the CCO sequences are noted below each CCO sequence. They can prevent deamidation of asparagine (N). All six different humanized versions of the anti-SIRPA antibody 3F9 (based on the following combinations of heavy chain and light chain variable regions: h3F9-H1/L1; h3F9-H1/L2; h3F9-H1/L3; h3F9-H2/L1; h3F9H2/L2 ; and h3F9-H2/L3) were grafted onto a human IgG4 backbone recombinantly produced in CHOK1 cells and further characterized as described below.

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Table 4

Antibody Heavy chain variable region sequences SEQ ID#: m3F9 VH EVKLVESGGGLVKPGGSLKLSCAASGFFSSYAMSWVRQTPE KRLEWVATISDYGGSYTYYPDSVKGRFTISRDNAKYTLYLQM SSLRS3flTKLYYXAPPPYDDYYGGFAYWGQGTLVTVSA 2 h3F9-Hl EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVSTISDYGGSYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKPPYDDYYGGFAYWGQGTLVTVSS 3 h3F9-H2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVATISDYGGSYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYXAPPPYDDYYGGFAYWGQGTLVTVSS 4

Table 5

Antibody Light chain variable region sequences SEQ ID#: m3F9 VL DIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQ KPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEED AATYYCQHNRELPCTFGGGTKLEIK 5 h3F9-Ll DIQMTQSPSSLSSASVGDRVTHTKRASKSVSSSGYSYMHWYQQ KPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQHNRELPCTFGQGTKLEIK 6 h3F9-L2 DIQLTQSPSSSLSASVGDRVTHTKRASKSVSSSGYSYMHWYQQ KPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSVQPED 7 FATYYCQHNRELPCTFGQGTKLEIK h3F9-L3 DIQLTQSPSSSLSVSVGDRATHTKRASKSVSSSGYSYMHWYQQ KPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSVQPED FATYYCQHNRELPCTFGQGTKLEIK 8

Affinity measurements for anti-SIRPA antibodies are performed as follows. Biolayer interferometry (BSI) data are collected on a Pall ForteBio Octet RED96 instrument. Data analysis is carried out using ForteBio Data Analysis Software, version 9.0. Standard kinetic buffer (BSF, 0.1% ABS, 0.02% Tween-20, pH 7.2) was used for analysis and to prepare reagents. After sensory equilibration in buffer, each of the humanized 3F9 anti-SIRPA antibodies (2.5 μg/ml, loading time 300 s) was captured on an Anti-Human IgG Fc Capture Dip and Read Biosensors (Pall ForteBio, Menlo Park, CA). Various concentrations (0–200 nM) of histidine-tagged human SIRPA (Sino Biological, Beijing, China) are then bound to the captured surface of anti-SIRPA (association time 300 s, dissociation time 600 s). The resulting BSI signal is obtained as the difference with the response from the reference (2.5 μg/ml IgG+O nM SIRPA) sensor. A null ligand control (0 μg/mL IgG+200 nM SIRPA) showed no measurable non-specific binding of SIRPA to the probe tip surface. Affinity measurements for the parent m3F9 antibody are performed using a similar method, except for the antibody captured on Anti-Mouse IgG Fc biosensors. Multi-concentration kinetic analysis is performed using a 1:1 interaction model to obtain association and dissociation rate constants (ka and kd, respectively) for each antibody. Binding affinity constants (BA) are calculated from the kd/ka ratio.

Anti-mouse IgG Fc or anti-human IgG Fc antibodies captured by mouse anti-SIRPA antibody 3F9 and humanized anti-SIRPA antibody 3F9 are immobilized on the biosensors, respectively. Increasing concentrations of histidine-tagged huSIRPA isoform 1 (10–500 nM) were infused through captured anti-SIRPA antibody 3F9 to record traces. The best-fitting traces from the resulting sensorgrams are analyzed to calculate association rates, dissociation rates, and affinity values. The three variants of the humanized anti-SIRPA antibody 3F9 are mAb14,70,1 (variable heavy chain sequence from SEQ ID No: 3 and variable light chain sequence from SEQ ID No: 6), mAb14,70,2 (variable heavy chain sequence from SEQ ID No.: 3 and variable light chain sequence from SEQ ID No.: 7) and mAb 14,70.4 (variable heavy chain sequence from SEQ ID No.: 4 and variable light chain sequence from SEQ ID No.: 6). The measured binding kinetics of humanized anti-SIRPA 3F9 antibodies are summarized in

- 71 044628 table. 6 below, in the form of highlighted values of ka, kd and CD. Of the six h3F9 variants obtained, 3 anti-SIRPA antibodies (mAb14.70.1: H1/L1; mAb14.70.2: H1/L2; mAb14.70.4: H2/L1) show similar affinity to the soluble SIRPA antigen as parent m3F9 anti-SIRPA antibody (variable heavy chain sequence from SEQ ID No: 2 and variable light chain sequence from SEQ ID No: 5).

Table 6

Clone 3F9 antibody Cop (M- Is- 1) Koff (s-1) KD (nM) Original m3F9 IgGl 5.08E+04 1.00E-03 18 mAb 14.70.1 1D0E+05 2.00E-03 19 mAb 14.70.2 1D0E+05 2.20E-03 20 mAb 14.70.4 8.60E+04 9.40E-04 eleven

In addition to soluble antigen binding, cell-based affinity measurements were also performed to establish the apparent affinity of the h3F9 anti-SIRPA antibody relative to the m3F9 anti-SIRPA antibody for the membrane-bound antigen SIRPA v1. EC50 values determine relative affinity by adding increasing concentrations of anti-SIRPA 3F9 antibody to BWZ cells overexpressing human SIRPA. Receptor-bound antibodies are determined by staining cells with anti-mouse IgG Fc APC or anti-human IgG PE secondary antibody. Serial dilutions of monoclonal antibodies are added to 10 ⁵ BWZ cells overexpressing huSIRPA (BWZ-huSIRPA) and allowed to reach binding equilibrium at 4°C. After the addition of fluorescently labeled secondary antibody and rapid washing steps, mean fluorescence intensity (AFI) values as a function of the titrated antibody concentration are recorded through the AFFI assay. Curves were fitted using nonlinear regression analysis using Graphpad Prism 6. Cell-based titration experiments with h3F9 anti-SIRPA antibodies mAb14.70.1, mAb14.70.2 and mAb14.70.4 gave EC50 values of 1.065 nM, 0.9682 nM and 1.607 nM, respectively. The humanized 3F9 anti-SIRPA antibody produces lower EC50 values than the m3F9 anti-SIRPA antibody (EC50=2.1 nM), indicating improved binding affinity of the humanized antibody relative to that determined for the murine 3F9 anti-SIRPA antibody.

A competition assay was also performed to calculate IC50 values of anti-SIRPA antibodies, where increasing concentrations of humanized and mouse 3F9 anti-SIRPA antibodies were added to BWZ-huSIRPAv1 cells to displace the fluorophore-labeled reference antibody. IC50 values provide another comparison of relative affinities. In a competition assay, increasing concentrations of anti-SIRPA 3F9 antibodies are added to cells expressing huSIRPA to displace the fluorophore-labeled reference antibody. The following IC50 values are determined: m3F9 =11.54 nM; mAb14.70.1=3.303 nM; mAb 14.70.2=2.185 nM; mAb 14.70.4=4.606 nM. These results demonstrate that the humanized anti-SIRPA antibodies of the present invention were more effective competitors than the reference m3F9 anti-SIRPA antibody in binding to SIRPA.

The ability of h3F9 anti-SIRPA antibodies to reduce cell surface expression of SIRPA was assessed in primary human macrophages (huMacs). Human monocytes are isolated from the peripheral blood of healthy donors and differentiated into macrophages in vitro by adding human M-CSF to the culture medium. Following differentiation, huMacs were harvested and seeded into 96-well tissue culture plates with increasing concentrations of isotype control or soluble anti-SIRPA antibodies and incubated overnight at 37°C. Cells were analyzed by flow cytometry to determine surface expression of SIRPA using DyLight650 conjugated anti-human SIRPA antibody (clone SA56), which belongs to a distinct epitope group of anti-SIRPA antibody 3F9. As shown in FIG. 3, h3F9 variants of anti-SIRPA antibodies (mAb14.70.1 (70.1), mAb14.70.2 (70.2) and mAb 14.70.4 (70.4)) negatively regulate SIRPA receptor expression levels in macrophages in a dose-dependent manner. However, the parent m3F9 anti-SIRPA antibody exhibits greater maximum receptor downregulation than the humanized antibodies despite similar antigen-binding properties discussed above. These results confirm that non-receptor binding properties of the anti-SIRPA antibody determine the functional activity of the anti-SIRPA antibody 3F9 on macrophages.

Example 3. Anti-SIRPA antibodies requiring Fc gamma receptors (FcyRs) for functional activity.

The principal difference between human and mouse 3F9 anti-SIRPA antibodies is the ability to attract Fc gamma receptors (FcyRs). Humanized anti-SIRPA antibodies are grafted onto a human IgG4 Fc backbone, which is known to have poor FcyR binding properties. Hybridoma m3F9 anti-SIRPA antibody, despite being a murine IgG1 antibody, still retains binding activity against certain human FcyRs. To verify that FcyRs modulate the functional activity of anti-SIRPA antibodies, the m3F9 anti-SIRPA antibody was treated with EndoSto, enzymatically removing the Fc glycan at Asn297, a key determinant mediating the IgG-FcyR interaction. Primary human macrophages from 2 healthy donors were treated overnight with increasing concentrations of glycosylated or deglycosylated m3F9 anti-SIRPA antibody. The cells are then stained with DyLight650-conjugated anti-SIRPA reference antibody (SA56-DyL650), which binds to a specific epitope group and then analyzed for surface expression of SIRPA by the SCAF assay.

As shown in FIG. 4A, both glycoforms of the m3F9 anti-SIRPA antibody significantly negatively regulated cell surface expression of SIRPA. However, in both donors, the deglycosylated m3F9 variant of the anti-SIRPA antibody exhibited reduced maximum activity compared to that observed for the glycosylated form of the antibody. Specifically, the m3F9 glycosylated anti-SIRPA antibody negatively regulates SIRPA expression by as much as 90 and 85% in donor (Dnr) 413 and donor 414, respectively; while m3F9 deglycosylated anti-SIRPA antibody achieved only 70 and 75% of SIRPA receptor down-regulation in macrophages from the same donor, respectively. This finding suggests that anti-SIRPA antibodies, particularly the m3F9 antibody, require FcyR engagement for enhanced negative regulation of cell surface SIRPA expression. The results in Fig. 4A are presented as the percentage of reference antibody binding obtained by dividing the UPC value of samples treated with anti-SIRPA antibodies by the UPC value of samples treated with isotype control.

In addition to the antibody-mediated negative regulation of the receptor described above, the role of FcyRs is assessed in the 3F9 anti-SIRPA antibody-mediated improvement in tumor cell phagocytosis. The tumor cell phagocytosis assay is developed based on pHrodo fluorescence. Primary human macrophages are treated overnight with isotype control antibody (ICA) or the indicated anti-SIRPA 3F9 antibody variants. Raji cells labeled with pHrodo dye are added to macrophages and incubated at 37°C for 2 hours. Red Avidin (Invitrogen) is a streptavidin molecule conjugated to pHrodo Red, a fluorogenic marker that acquires fluorescence in acidic environments such as the phagosome. For targeted tumor cell labeling, 500 nM Red Avidin was mixed with 15 nM biotinylated Lens Culinaris Agglutinin (LCA; Vector Labs). Red Avidin-LCA complexes are then mixed at a 1:1 volume ratio with 400,000 Raji cells in serum-free RPMI medium on ice. The sugar binding properties of LCA bind Red Avidin to carbohydrate structures on the surface of the tumor cell. After quick wash steps, Red Avidin-LCA-labeled Raji cells are mixed with monocyte-derived human macrophages in serum-free RPMI medium and incubated at 37°C for 2 hours. Macrophages are then collected and stained on ice with anti-CD14 APC in SCAF buffer containing FcyR-blocking antibodies. Phagocytotic activity is measured by counting the percentage of CD14/pHrodo double positive macrophages. As a control, untagged Raji cells were mixed with macrophages to establish background fluorescence levels. The results in Fig. 4B are presented as fold increases in the proportion of the pHrodo/CD14 double positive macrophage population.

As shown in FIG. 4B, macrophages treated overnight with glycosylated m3F9 anti-SIRPA antibody exhibit a ~2-fold increase in tumor cell phagocytosis compared to that observed for isotype control antibody-treated macrophages. Macrophages treated with m3F9 deglycosylated anti-SIRPA antibody exhibited a ~1.5-fold increase in tumor cell phagocytosis compared to isotype control antibody-treated macrophages. This ~1.5-fold increase in tumor cell phagocytosis represents a statistically significant ~50% reduction in activity relative to that observed for the m3F9 glycosylated anti-SIRPA antibody. Moreover, macrophages treated with h3F9 IgG4 (mAb14.70.1 or mAb14.70.2) did not demonstrate any ability to improve tumor cell phagocytosis above basal levels.

Previous studies establish a correlation between increased phagocytic activity and CD14 down-regulation when 3F9 macrophages are stimulated with anti-SIRPA antibody. To determine whether this correlation also depends, at least in part, on FcyRs, primary human macrophages are treated overnight with either glycosylated m3F9 or h3F9 variant IgG4 anti-SIRPA antibodies (mAb14.70.1, mAb14.70.2 and mAb14,70,4) and analyzed for tumor cell phagocytosis and CD14 expression. As shown in FIG. 5A, only macrophages treated with the m3F9 anti-SIRPA antibody showed improved phagocytosis of Raji cells relative to that observed for macrophages treated with the isotype control antibody. Results are presented as fold increases in the proportion of the pHrodo/CD14 double positive macrophage population. Humanized anti-SIRPA IgG4 antibodies failed to improve phagocytosis of Raji cells. Also, only stimulation of m3F9 with anti-SIRPA antibody negatively regulates CD14 expression on macrophages, while h3F9 variants of IgG4 anti-SIRPA antibody do not alter CD14 expression (Fig. 5B). In addition to supporting a correlation between the level of CD14 expression and phagocytic activity in macrophages, these results confirm that the functional activity of the 3F9 anti-SIRPA antibody is dependent, at least in part, on FcyRs.

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Example 4: Negative regulation of CD32 by anti-SIRPA antibodies.

In addition to the target antigen of interest, cells of the myeloid lineage also express a variety of Fc receptors capable of binding to the Fc domain of a therapeutic antibody. Fc gamma receptors (FcyRs) constitute the best characterized and most effective class of receptors for mediating Fc-dependent effector functions. FcyRs are composed of both ITAM-associated activating receptors (CD64/FcyRI, CD32A/FcyRIIA, and CD16A/FcyRIIIA) and ITAM-bearing inhibitory receptors (CD32B/FcyRIIB), and coexpression of activating/inhibitory receptors on the same cell sets the threshold for cellular activation. In general, ligation of activating FcyRs by immune complexes initiates several signaling cascades that lead to cellular activation and subsequent induction of effector functions. These activities vary among myeloid cell types, but may include antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and positive regulation of several proinflammatory cytokines and chemokines, etc. Conversely, ligation of the inhibitory receptor, FcyRIIB, by immune complexes interferes with the immunostimulatory activation signals of FcyRs, maintaining tissue homeostasis. For example, several studies have found that genetic knockout of FcyRIIB causes enhanced proinflammatory macrophage activity in mouse models of immune complex-mediated inflammation. Because FcyRIIB is the only FcyR with an inhibitory effect, it plays a central role in regulating FcyR-mediated inflammation in myeloid cells. In the context of the tumor microenvironment, FcyRIIB expression levels may determine the polarization state of tumor-associated macrophages and regulate macrophage effector function in vivo.

To determine which FcyR promotes the in vitro activity of 3F9 anti-SIRPA antibodies, monocyte-derived macrophages are collected from two healthy donors, treated overnight with either an isotype control antibody or the anti-SIRPA antibody m3F9, and then assessed for surface expression levels of FcyRIIIA (CD16 ), FcyRI (CD64) and FcyRIIA/B (CD32A/B). As shown in FIG. 6A, treatment of m3F9 with anti-SIRPA antibody substantially reduced surface expression of CD32A/B relative to that observed in isotype control antibody-treated macrophages. Because the identifying antibody used to measure surface levels of FcyRII (clone FUN-2; Biolegend) does not distinguish between the activating receptor FcyRIIA and the inhibitory receptor FcyRIIB, this assay is repeated with receptor-specific antibodies. As described previously, monocyte-derived macrophages from two healthy donors were treated overnight with either an isotype control antibody or the indicated 3F9 anti-SIRPA antibody variant. In fig. 6B shows that the anti-SIRPA antibody m3F9 significantly down-regulated FcyRIIA in macrophages by ∼70-85% relative to that observed in isotype control antibody-treated cells. This effect is Fc domain dependent, as both the h3F9 IgG4 variant (mAb14.70.2) and the deglycosylated form of the mouse antibody neutralize the down-regulation of FcyRIIA.

When assessing the surface expression of FcyRIIB, treatment with the anti-SIRPA antibody m3F9 reduced expression of the inhibitory receptor to essentially undetectable levels relative to those observed in isotype control antibody-treated macrophages (Fig. 6B). In contrast, no significant down-regulation of FcyRI/CD64 or FcyRIIIA/CD16A was observed in anti-SIRPA antibody 3F9-treated macrophages compared with isotype control-treated cells, indicating that anti-SIRPA 3F9 antibody treatment does not alter CD16 and CD64 expression on primary human macrophages (data not shown).

To confirm that CD32A/B is required for the functional activity of the anti-SIRPA antibody m3F9, selective FcyR blockade experiments were performed to evaluate the role of individual FcyRs in antibody-mediated SIRPA down-regulation and tumor cell phagocytosis. In blocking experiments, primary human macrophages from two healthy donors were preincubated with anti-CD16 or anti-CD32A/B blocking antibodies for 15 minutes on ice. Cells are then incubated overnight with either isotype control (MOPC21) or m3F9 anti-SIRPA antibodies. SIRPA expression was determined with DyLight650-conjugated anti-SIRPA reference antibody (SA56-DyL650). The results are presented as the percentage of binding of the reference antibody obtained by dividing the UPC value of samples treated with anti-SIRPA antibodies by the UPC value of samples treated with isotype control. As shown in FIG. 7A, CD16 blockade does not abolish the down-regulation of SIRPA following treatment with the anti-SIRPA antibody m3F9 relative to that observed in macrophages not treated with FcyR-blocking antibodies. In contrast, m3F9-mediated down-regulation of SIRPA was significantly impaired in macrophages preincubated with anti-CD32A/B blocking antibodies.

To ensure that CD32A/B blockade also delayed phagocytosis, primary human macrophages were preincubated with anti-CD32A/B blocking antibody and treated overnight with isotype control antibody or anti-SIRPA antibody m3F9. Raji cells labeled with dye

- 74 044628 pHrodo body, mixed with macrophages and incubated at 37°C for 2 hours. Results are presented as fold increase in the percentage of the pHrodo/CD14 double positive macrophage population. As shown in FIG. 7B, m3F9-treated macrophages from both donors improved phagocytosis of Raji cells ~2-fold relative to that shown in isotype control antibody-treated macrophages. However, CD32A/B blockade partially or completely blocks m3F9-mediated improvement in tumor cell phagocytosis. Taken together, these results establish that CD32A/B engagement is functionally required for the activity of the anti-SIRPA antibody m3F9.

Example 5: Affinity maturation of h3F9 anti-SIRPA antibody.

Affinity maturation of the humanized h3F9 anti-SIRPA antibody is carried out. Briefly, certain amino acid residues in the heavy and light chain are selectively mutagenized, and mutants that improve binding are selected through additional rounds of screening. This process simultaneously improves specificity, interspecies cross-reactivity, and expression profiles, allowing fine-tuning of properties involved in the desired mechanism of action, bioassay performance, and preclinical modeling. Characterization includes Forte Bio and MSD affinity measurements, cellular binding and several manifestation assays. After the first round of affinity maturation, antibodies with improved affinity also exhibit improved polyspecific reactivity (SMR), which is used to determine nonspecific binding of the antibody. Thus, a second round of affinity maturation is performed to improve the affinity without increasing the PSR.

Heavy chain and light chain variable domain sequences.

Using standard methods, the amino acid sequences of the light chain variable domains and heavy chain variable domains of the affinity mature antibodies described herein are determined. The antibody light chain HVR sequences are presented in Table. 7-8. The antibody light chain HVR sequences are presented in Table. 7. The HVR sequences of the heavy chain of antibodies are presented in table. 8. The sequences of the framework regions of the light chain and heavy chain (HC) of antibodies are presented in table. 9. The sequences of the variable region of the heavy chain and the variable region of the light chain of antibodies are presented below in table. 10.

Table 7

Light chain HVR sequences of affinity mature anti-SIRPA antibodies

SEQ ID#: mAb ID (3F9-#) HVR-L1 RASKSVSS GGYSYMH 9 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24 and 25 HVR-L2 LASNLES 10 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24 and 25 HVR-L3 QHNRELPS t eleven 1, Yui 18 QHNRELPC t 12 2 QHNRELPIT 13 3, I and 19 QHNRELPP t 14 4, 12 and 20 QHNRELPT T 15 5, 13 and 21 QHNRELPV t 16 6, 14 and 22 QHNRELPA t 17 7, 15 and 23 QHNRELPG t 18 8, 16 and 24 QHNRELPW t 19 9, 17 and 25

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Table 8

Heavy chain HVR sequences of affinity mature anti-SIRPA antibodies

SEQ ID No.: mAb ID (3F9-#) HVR-H1 GFTFSSYAM S 20 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24 and 25 HVR-H2 TISEYGGSYT YYAESVKG 21 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24 and 25 HVR-H3 PPYDDYYGG FAY 22 1,2,3,4, 5, 6, 7, 8 and 9 PPYDDYYGG FRY 23 10, I, 12, 13, 14, 15, 16 and 17 PPYDDYYGG FQY 24 18, 19, 20,21,22, 23, 24 and 25

Table 9

Heavy and light chain framework sequences of affinity mature anti-SIRPA antibodies

FR VH SEQ ID No.: VL SEQ ID No.: FR 1 EVQLLESGGGLVQPGG SLRLSCAAS 25 DIQLTQSPSSLSSASV GDRVTTITC 29 FR2 WVRQAPGKGLEWVA 26 WYQQKPGKAPKLL IY thirty FR3 RFTISRDNSKNTLYLQM NSLRAEDTAVYYXAP 27 GVPSRFSGSGSGTD FTLTISSVQPEDFAT YYC 31 FR4 WGQGTLVTVSS 28 FGQGTKLEIK 32

Table 10

Heavy chain variable region SEQ ID No.: mAb ID (3F9-#) EVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVATISEYG GSYTYYAESVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYXAPPPYDDYYGGF AYWGQGTLVTVSS 33 1,2,3,4, 5, 6, 7, 8 and 9 EVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVATISEYG GSYTYYAESVKGRFTISRDNSKNTLYLQ 34 10, 11, 12, 13, 14, 15, 16 and 17

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MNSLRAEDTAVYYXAPPPYDDYYGGFR YWGQGTLVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVATISEYG GSYTYYAESVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYXAPPPYDDYYGGF QYWGQGTLVTVSS 35 18, 19, 20,21,22, 23, 24 and 25 Light chain variable region SEQ ID No.: mAb ID (3F9-#) DIQLTQSPSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFS GS GS GTDFTLTIS S VQPE DFATYYCQHNRELPSTFGQGTKLEIK 36 1, 10 and 18 DIQLTQSPSSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPCTFGQGTKLEIK 37 2 DIQLTQSPSSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPITFGQGTKLEIK 38 3, I and 19 DIQLTQSPSSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPPTFGQGTKLEIK 39 4, 12 and 20 DIQLTQSPSSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPTTFGQGTKLEIK 40 5, 13 and 21 DIQLTQSPSSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPVTFGQGTKLEIK 41 6, 14 and 22 DIQLTQSPSSLSSASVGDRVTITCRASKSV 42 7, 15 and 23 SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPATFGQGTKLEIK DIQLTQSPSSLSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFS GS GS GTDFTLTIS S VQPE DFATYYCQHNRELPGTFGQGTKLEIK 43 8, 16 and 24 DIQLTQSPSSLSSASVGDRVTITCRASKSV SSGGYSYMHWYQQKPGKAPKLLIYLAS NLESGVPSRFSGSGSGTDFTLTISSVQPE DFATYYCQHNRELPWTFGQGTKLEIK 44 9, 17 and 25

Example 6: Characterization of affinity mature anti-SIRPA antibodies.

Initial characterization of the affinity mature anti-SIRPA antibodies described here involves performing acquired antigen affinity measurements using biolayer interferometry (BSI) on a Pall ForteBio Octet RED96 instrument. Anti-mouse IgG Fc or anti-human IgG Fc antibodies captured by mouse anti-SIRPA 3F9 antibody and humanized anti-SIRPA 3F9 antibody are immobilized on the biosensors, respectively. Single-shot kinetics were performed with 25 nM huSIRPA or 50 nM huSIRPB1 flowing over captured 3F9 antibody for trace recording. Ana

- 77 044628 data lysis is carried out using ForteBio Data Analysis Software, version 9.0. Standard kinetic buffer (BSF, 0.1% ABS, 0.02% Tween-20, pH 7.2) was used for analysis and to prepare reagents. After equilibration of the sensor in buffer, anti-SIRPA antibody h3F9 (2 μg/ml, loading time 300 s) or affinity-mature variants of anti-SIRPA antibody h3F9 are captured on Anti-Human IgG Fc Capture Dip and Read Biosensors (Pall ForteBio, Menlo Park, CA). 50 nM histidine-tagged human SIRPA v1 or human SIRPB1 (Novoprotein, Summit, NJ, USA) is then bound to the anti-SIRPA antibody-captured surface coated (association time 200 s, dissociation time 900 s). The resulting BSI signal is obtained as the difference in responses from the reference (2 μg/ml 3F9+0 nM SIRPA) sensor. The null-ligand control (0 μg/ml 3F9 + 200 nM SIRPA) showed no measurable non-specific binding of SIRPA to the probe tip surface. Single curve kinetic analysis is performed using a 1:1 interaction model to obtain association and dissociation rate constants (ka and kd, respectively) for each antibody. Affinity constants (AC) are calculated from the ratio kd/ka. Soluble huSIRPA and huSIRPB1 binding sensorgrams are obtained for 25 anti-SIRPA progeny antibodies and one parent antibody, and the best-matching traces are used to calculate association rates, dissociation rates, and affinity values. Affinity measurements (ka, kd, and KD) obtained from curve-fitting analysis of the sensorgrams obtained in these experiments are summarized in Table 1. 11. All -mature anti-SIRPA antibodies having no measurable cross-reactivity with huSIRPB1 (data not shown) and six affinity-mature anti-SIRPA antibodies (3F9-18, 3F9-12, 3F9-23, 3F9-14, 3F9- 22 and 3F9-20) demonstrate a ~10-fold increase in affinity for soluble huSIRPA relative to that observed for the parent anti-SIRPA antibody h3F9.

Table 11

Binding kinetics of affinity-mature h3F9 antibodies

Ab ID (3F9-#) KD(Μ) kon(Ms)-l koff(s-1) 18 4D9E-10 Ε37Ε+05 5.74Ε-05 12 4.29Ε-10 Ε39Ε+05 5.96Ε-05 23 5.39Ε-10 Ε69Ε+05 9D1E-05 14 6D8E-10 Ε65Ε+05 Ε02Ε-04 22 6.46Ε-10 1.74Ε+05 1DZE-04 20 8.50Ε-10 Ε50Ε+05 1D8E-04 16 1D7E-09 1D9E+05 Ε51Ε-04 21 Ε03Ε-09 1D8E+05 Ε52Ε-04 24 Ε07Ε-09 1D4E+05 Ε55Ε-04 25 Ε06Ε-09 Ε53Ε+05 Ε62Ε-04 7 Ε81Ε-09 9.92Ε+04 Ε80Ε-04 19 1D9E-09 1D1E+05 Ε81Ε-04 eleven 1.43Ε-09 1.34Ε+05 1D2E-04 6 Ε65Ε-09 1D6E+05 1D2E-04 13 1.38Ε-09 1.46Ε+05 2.01Ε-04 10 Ε51Ε-09 1.34Ε+05 2.01Ε-04 4 Ε79Ε-09 1DZE+05 2.03Ε-04 1 2.09Ε-09 Ε06Ε+05 2D2E-04 2 2.06Ε-09 Ε08Ε+05 2D2E-04 17 1J0E-09 1D0E+05 2D8E-04 5 2.95Ε-09 9.36Ε+04 2.76Ε-04 8 3.60Ε-09 7.66Ε+04 2.76Ε-04 9 ZD8E-09 8.94Ε+04 3.02Ε-04 15 6.28Ε-09 5.76Ε+04 3.62Ε-04 3 4.43Ε-09 9.39Ε+04 4D6E-04 h3F9 6.48Ε-09 Ε84Ε+05 1D9E-03

Cellular affinity measurements are also performed to verify the apparent affinity of the affinity mature 3F9 anti-SIRPA antibodies for the cell surface expressed antigen. Serial dilutions of each anti-SIRPA monoclonal antibody were added to 105 BWZ-huSIRPA cells and allowed to reach binding equilibrium at 4°C. After the addition of a fluorescently labeled secondary antibody and rapid washing steps, the UPC values as a function of the titrated

- 78 044628 antibody concentrations are recorded via SCAF analysis. EC50 values (listed below) determine the relative affinity by adding increasing concentrations of 3F9 antibody variants to BWZ cells overexpressing human SIRPA. Receptor-bound antibodies are determined by staining cells with an anti-human IgG PE secondary antibody. Curves were fitted using nonlinear regression analysis using Graphpad Prism 6. Cellular titration experiments with mouse 3F9 and humanized affinity mature anti-SIRPA antibodies showed improved EC50 values of humanized affinity mature anti-SIRPA antibodies; two anti-SIRPA antibodies show significant improvement in EC50 values (mAb3F9-14=0.42 nM and mAb 3F9-22=0.49 nM) compared to those obtained for the anti-SIRPA 3F9 mIgG1 antibody (0.72 nM) . See table. 12 below.

Table 12

3F9-14 3F9-17 3F9-18 3F9-20 3F9-21 3F9-22 3F9-25 3F9 mlgGl EC50 (nM) 0.42 0.61 0.51 0.49 0.64 0.49 0.96 0.72

Example 7. Cross-reactivity of affinity mature anti-SIRPA antibodies with SIRPA antigenic variants.

The CD47-binding domain of human SIRPA is highly polymorphic, which may make it difficult to develop CD47-blocking anti-SIRPA antibodies capable of recognizing all SIRPA allelic variants expressed in human populations. In fact, the two most common alleles of human SIRPA (v1 and v2) are also the most divergent in sequence, with 13 residues differing in the IgV domain. Because competition studies with CD47 showed that the anti-SIRPA antibody m3F9 binds a region different from the CD47 ligand binding site, binding assays were performed to test cross-reactivity between human SIRPA v1 and v2, as well as cross-species reactivity to identify appropriate animal models for toxicology studies .

In the ELISA assay, 96-well Immulon HBX plates were coated with His- or Fc-tagged SIRPA of the species as indicated at 1 μg/ml in PBS overnight at 4°C. The plates are blocked in 5% (wt/vol) bovine serum albumin in PBS for 1 hour, after which they are washed 3 times with PBS/0.05% Tween 20 (BGF/T).

Anti-SIRPA antibodies are added to the plate in serial dilution and incubated at room temperature for 2 hours, after which unbound antibody is removed by washing with PBS/T. Horseradish peroxidase-conjugated anti-mouse IgG or anti-human kappa light chain (Jackson Immunoresearch) is then added to the wells and incubated for 30 minutes. Unbound antibody is removed by washing with PBS/T, after which bound antibody is detected using tetramethylbenzidine (TMB). The reaction is stopped with 2N H2SO4, after which the plates are read at A450.

The parent mouse 3F9 anti-SIRPA antibody binds both v1 and v2 versions of human SIRPA-Fc with similar EC50 values (0.695 and 0.789 nM, respectively). A partial decrease in apparent affinity is observed with His-tagged human antigen, suggesting greater avid binding with Fc-tagged SIRPA. Additionally, the parent mouse 3F9 anti-SIRPA antibody shows weak cross-reactivity with Fc- and His-tagged cynomolgus SIRPA (EC50=1.09 nM). No detectable binding was observed with rat SIRPA.

Following affinity maturation of the parent antibody as described above, two progeny anti-SIRPA antibodies, mAb3F9-14 and mAb3F9-22, are matched to the same panel of SIRPA antigens to assess cross-reactivity. Regarding the anti-SIRPA antibody m3F9, both humanized and affinity mature anti-SIRPA antibodies retain binding activity against human SIRPA v1 and v2, but with significantly lower EC50 values, confirming the increased affinity observed in biosensor experiments and cell binding assays. described above. Despite increased affinity for human SIRPA v1 and v2, anti-SIRPA antibody 3F9-14 shows weak cross-reactivity with cynomolgus SIRPA; The binding curves give EC50 values (EC50=0.879 nM) similar to those observed for the anti-SIRPA antibody m3F9. In contrast, anti-SIRPA antibody 3F9-22 exhibits binding activity to cynomolgus SIRPA equivalent to both human SIRPA variants (EC50=0.107 nM). Neither 3F9-14 nor 3F9-22 antibody binds to rat SIRPA (data not shown). The EC50s calculated from the binding curves are listed below and establish that all anti-SIRPA antibodies bind both allelic variants of human SIRPA. Only the anti-SIRPA antibody hu3F9-22 binds cynomolgus SIRPA with similar binding affinity to human SIRPA. In table 13 below shows the EC50 values of anti-SIRPA antibodies with different SIRPA proteins (nM).

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Table 13

ELISA huSIPRA v.l CAA71403.1 huSIRPA v.2 NP542970.1 Cynomolgus macaque SIRPA XPO15313153.1 3F9 mlgGl 0.789 0.695 1,089 3F9-14 0.093 0.080 0.879 3F9-22 0.108 0.082 0.107

Similar ELISA experiments were performed to determine the EC50 values for the anti-SIRPA antibody 9C2 (see below for amino acid sequences of the anti-SIRPA antibody 9C2) and 3F9 to a panel of different SIRPA and SIRPB proteins. In table 14 below shows the EC50 values of these anti-SIRPA antibodies for different SIRP proteins (nM).

Table 14

SIRPA v2 SIRPB 1- Fc iso. 1 SIRPB 1- Fc iso. 3 SIRPB 1- His iso. 3 SIRPA cynomolgus macaque SIRPB 1 Javanese toque 0.3578 n.s. 0.3822 0.4251 7,439 n.s. 0.3528 H.C. 0.3188 0.4353 0.6582 n.s.

Accession numbers: CAA71403.1 (SIRPA v2); 000241.5 (SIRPB1 iso. 1); Q5TFQ8,1 (SIRPB1 image 3);

NP001271679.1 (cynomolgus SIRPA); XP005568598 (cynomolgus SIRPB1); n.s. = no binding.

Given the high sequence similarity between SIRPA proteins of several species, as shown in FIG. 1, anti-SIRPA antibody progeny clones were also tested for cross-reactivity against additional SIRPA antigens from different mammalian species. Fc-labeled marmoset SIRPA (Accession No. JAB51896) or canine SIRPA (Accession No. XP005634938) or rabbit SIRPA (Accession No. G1U0I5) were applied to the plates and incubated with increasing concentrations of anti-SIRPA antibodies hu3F9-14 and hu3F9-22. Both anti-SIRPA mAb3F9-14 and mAb 3F9-22 recognize marmoset and dog SIRPA, although with lower affinity than observed with human SIRPA. For example, the EC50 values for 3F9-14 binding of monkey and dog SIRPA are 1.2 nM and 0.64 nM, respectively. Also, the EC50 values for 3F9-22 binding of monkey and dog SIRPA are 4.3 nM and 0.83 nM, respectively. Neither the anti-SIRPA antibody clone 3F9-14 nor 3F9-22 binds rabbit SIRPA.

Example 8 Affinity mature anti-SIRPA antibodies negatively regulate SIRPA and CD32A/B.

Previous experiments have established that the anti-SIRPA antibody m3F9 noncompetitively antagonizes CD47 binding to SIRPA through decreased surface expression of SIRPA, resulting in an increased ability to stimulate tumor cell engulfment by macrophages compared to CD47-blocking anti-SIRPA antibodies. Affinity-mature anti-SIRPA antibodies are assessed to determine whether they retain similar functional properties to the murine antibody.

Since the anti-SIRPA antibody m3F9 (on the mouse IgG1 backbone) negatively regulates SIRPA and CD32A/B in an Fc-dependent manner, chimeric antibodies are produced in which the Fab sequence of the original anti-SIRPA antibody m3F9 is hybridized with the Fc domain of wild-type human IgG1 (3F9 huIgG1 ) or human IgG1 Fc carrying the S267E/L328F mutation (3F9-SELF) in the heavy chain. Fc SELF mutations enhance binding to human FcyR2B/CD32B >100-fold relative to wild-type human IgG1 Fc. Human monocytes are isolated from the peripheral blood of healthy donors and differentiated in vitro into macrophages with human M-CSF. After differentiation, huMacs are harvested and seeded into 96-well tissue culture plates with increasing concentrations of isotype control or soluble anti-SIRPA antibody and incubated overnight at 37°C. Cells were analyzed by flow cytometry to determine the surface expression of SIRPA and C32A/B using DyLight650-conjugated anti-human SIRPA antibody (clone SA56) and FITC-labeled anti-CD32A/B (FUN-2). Consistent with previous observations, the anti-SIRPA antibody 3F9 mIgG1 exhibits robust dose-dependent down-regulation of both SIRPA and CD32A/B. However, the chimeric anti-SIRPA 3F9 huIgG1 antibody showed reduced efficiency of receptor downregulation compared with the mouse antibody (data not shown). These results provide further evidence that the Fc domain of the antibody plays an important role in activity. Construction of SELF mutations in the Fc domain of chimeric 3F9 (3F9-SELF) restores the ability to negatively regulate SIRPA and CD32A/B to levels similar to those observed with the anti-SIRPA antibody 3F9 mIgG1. This result confirms that CD32A/B serves as an important co-receptor for m3F9 activity on macrophages.

Earlier experiments showed that humanized versions of the anti-SIRPA antibody 3F9 grafted onto a human IgG4 backbone lose functional activity. Affinity-mature varians

- 80 044628 you anti-SIRPA antibody 3F9 is produced on the human IgG1 backbone and evaluated for its ability to negatively regulate SIRPA and CD32A/B compared to the parent anti-SIRPA antibody m3F9 and the chimeric anti-SIRPA antibody 3F9 huIgG1. The expression of SIRPA and CD32A/B was determined with anti-SIRPA DyLight650 (clone SA56) and anti-CD32A/B FITC (clone FUN2). Affinity-mature antibodies 3F9-14, 3F9-18, 3F9-20, and 3F9-22 negatively regulate SIRPA to levels similar to those observed for the parent anti-SIRPA m3F9 antibody (data not shown). However, the same affinity-mature anti-SIRPA antibodies only minimally improved CD32A/B down-regulation compared with that observed with the chimeric anti-SIRPA 3F9 huIgG1 antibody. These results indicate that improving antibody-antigen affinity compensates for Fc-dependent negative regulation at the target receptor (ie, SIRPA), but not Fc-mediated interactions with FcyRs.

To determine whether Fc engineering further enhances the negative regulation of SIRPA by affinity-mature anti-SIRPA antibodies, SELF point mutations were introduced into the Fc domain of the anti-SIRPA 3F9-22 huIgG1 antibody. Primary human macrophages from 4 healthy donors were treated overnight with increasing concentrations of humanized anti-SIRPA 3F9-22 antibody carrying wild-type human IgG1 Fc or human IgG1 S267E/L328F Fc (SELF). SIRPA expression was determined with anti-SIRPA DyLight650 (clone SA56). Blood donors are genotyped for the CD32A-R/H131 polymorphism by qPCR. In fig. 8A and 8B show typical results of these experiments compared to the activity of both Fc variants. Among individuals homozygous for the CD32A-H131 allele (donors 516 and 517 were verified by qPCR), both Fc variant anti-SIRPA 3F9-22 antibodies negatively regulated SIRPA to levels similar to those observed in human macrophages (Fig. 8B). This result indicates that improved binding to FcyR2B does not enhance antibody-mediated down-regulation of SIRPA.

Among individuals heterozygous for both the CD32A-H131 and -R131 alleles, the anti-SIRPA 3F9-22 hIgG1 Fc variant showed greater maximum down-regulation of SIRPA than that observed for the SELF Fc variant (Fig. 8A). Rather than enhancing negative regulation of SIRPA, SELF Fc may limit this function. It should be noted that SELF mutations increase Fc affinity for CD32B, as well as the CD32A-R131 allele. Given that human macrophages express higher levels of CD32A than CD32B, macrophages from individuals expressing CD32A-R131 may sequester the anti-SIRPA antibody 3F9-22 from SIRPA or CD32B to limit maximum down-regulation activity.

In table 15 below is a summary of the results of antibody-mediated down-regulation of SIRPA and CD32 on a colony of human macrophages U937. In these studies, the anti-SIRPA antibodies of the present invention are compared with the previously described anti-SIRPA antibody KWAR23 (disclosed in International Patent Application Publication No. WO 2015/138600). As shown in table. 15, the anti-SIRPA antibodies of the present invention negatively regulate or reduce the cell surface expression of SIRPA in U937 cells, with a maximum downregulation percentage of 68-76%. In comparison, the anti-SIRPA antibody KWAR23 is only able to down-regulate cell surface SIRPA expression by 9%.

In table 15 also shows that the anti-SIRPA antibodies of the present invention effectively negatively regulate or reduce the cell surface expression of CD32 in U937 cells. As shown in the table, the anti-SIRPA antibodies of the present invention negatively regulate or reduce the cell surface expression of CD32 in these cells with a maximum downregulation percentage of 49-73%.

Table 15

SIRPA CD32

Antibody IC50 (nM) Max % OR IC50 (nM) Max % OR 3F9 huIgGl 0.804 68% 0.9807 49% 3F9 mlgGI 0.105 75% 0.005609 73% 3F9-14 1D7 69% 2,028 50% 3F9-18 2.19 71% 4,861 52% 3F9-20 0.152 75% 0.2706 58% 3F9-22 0.187 76% 0.4305 60% KWAR23 15.52 9% 0.07118 63%

Example 9 Affinity mature anti-SIRPA antibodies destroy SIRPA in macrophages.

Measurement of antibody-mediated down-regulation of the receptor by SCAF confirms that targeted binding of the anti-SIRPA antibody reduces cell surface expression. To determine whether internalized receptors are degraded and subsequently recycled to the cell surface, human macrophages are analyzed for total protein levels

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SIRPA by Western blotting as follows. Primary human monocyte-derived macrophages are treated overnight with 5 μg/ml of either isotype control or the Fc variant anti-SIRPA 3F9-22 A330S/P331S (ASPS). Cells were collected, washed with cold PBS and lysed with RIPA buffer supplemented with HALT protease/phosphatase inhibitors on ice for 15 minutes. The lysed cells are centrifuged to pellet the membrane fraction, and the supernatant fraction, including the total cell lysate, is collected. As shown in FIG. 9A, increasing amounts of the cell lysate fraction from macrophages treated with isotype control antibody or anti-SIRPA antibody 3F9-22 were loaded onto SDS-PAGE and immunoblotted for SIRPA protein with an antigen-specific antibody. In fig. 9A, 5 μg, 10 μg, and 20 μg of whole cell lysate were loaded onto SDS-PAGE and immunoblotted with anti-SIRPA cytoplasmic domain (aSIRPAct). A band migrating with the predicted molecular weight of SIRPA is detected in macrophages treated with isotype control but is not observed in macrophages treated with the anti-SIRPA 3F9-22 antibody. In a similar experiment, cell lysates of macrophages treated with isotype control or 3F9-22 ASPS antibody were loaded onto SDS-PAGE and immunoblotted for SIRPA and SHP-2, a tyrosine phosphatase that can bind to SIRPA and other ITIM receptors. In fig. 9B, 20 μg of whole cell lysate was loaded onto SDS-PAGE and probed with a-SIRPAct and anti-SHP2. Blots demonstrate that SIRPA protein levels are reduced by mAb3F9-22 treatment. As shown in FIG. 9B, stimulation with anti-SIRPA antibody 3F9-22 results in disruption of SIRPA but not SHP2.

Example 10 Affinity-mature anti-SIRPA antibodies stimulate phagocytosis of tumor cells in macrophages.

Previous experiments confirmed that affinity mature anti-SIRPA antibodies retain the ability to negatively regulate SIRPA and CD32A/B, resulting in the destruction of SIRPA in human macrophages. The affinity mature antibodies of the present invention are tested for their ability to induce phagocytosis of tumor cells. Primary human macrophages are treated overnight with isotype control or 3F9-22 human IgG1 or 3F9-22 SELF to assess Fc-dependent effects on antibody function. Raji cells labeled with pHrodo are mixed with macrophages and incubated for 2 hours at 37°C. Phagocytic activity is measured by counting the percentage of CD14/pHrodo double positive macrophages. Results are presented as fold increase in the percentage of the pHrodo/CD14 double positive macrophage population. Additionally, CD14 expression levels following phagocytosis are shown. As shown in FIG. 10A, both Fc variants of the anti-SIRPA antibody 3F9-22 enhance phagocytosis of Raji cells compared to macrophages treated with isotype control. Although the 3F9-22 SELF antibody appears to induce greater phagocytosis than the 3F9-22 huIgG1 antibody, this difference does not reach statistical significance. Additionally, both Fc variants of anti-SIRPA 3F9-22 suppressed CD14 expression to similar levels, indicating a similar challenge to phagocytic activity and macrophage activation status.

A hallmark of antagonism of the SIRPA-CD47 signaling axis is increased antibody-dependent phagocytosis of opsonized tumor cells. In fig. 10B, phagocytosis of Raji cells with or without the addition of the anti-CD20 opsonizing huIgG1 was assessed on macrophages treated with the anti-SIRPA antibody 3F9-22. In isotype control-treated macrophages, Raji cell opsonizing anti-CD20 huIgG1 enhanced tumor cell phagocytosis by ~50% compared to non-opsonized Raji cells. Results are presented as percentage of the pHrodo/CD14 double positive macrophage population. As previously shown, macrophages stimulated with either the anti-SIRPA antibody 3F9-22 SELF or 3F9-22 ASPS increased phagocytosis of non-opsonized Raji cells by ∼30–40% compared to isotype control-treated macrophages. Similarly, macrophages treated with the anti-SIRPA antibody 3F9-22 increased phagocytosis by ∼30% compared to macrophages treated with a control mixed with opsonized anti-CD20 Raji cells. Addition of opsonized anti-CD20 Raji cells to macrophages treated with the anti-SIRPA antibody 3F9-22 showed an additive effect on phagocytosis with a ~2-fold increase compared to that observed for control-treated macrophages mixed with non-opsonized Raji cells. Although both Fc variants of the 3F9-22 antibody induce phagocytosis of tumor cells, the SELF Fc variant demonstrates statistically significantly greater phagocytic activity than the ASPS Fc variant. These results confirmed that the affinity mature anti-SIRPA antibodies of the present invention enhance phagocytosis of tumor cells in macrophages by antagonizing the SIRPACD47 pathway.

In table 16 below is a summary of the IC50 values determined for antibody-mediated down-regulation of SIRPA and CD32 in primary human macrophages. Additionally, in table. 16 shows the fold increase in phagocytosis associated with various anti-SIRPA antibodies of the present invention in primary human macrophages. In these studies, the anti-SIRPA antibodies of the present invention are compared with the previously described anti-SIRPA antibody HEFLB (disclosed in International Patent Application Publication No: WO 2017/178653). As shown in Table 16, anti- 82 044628

The SIRPA affinity mature antibodies of the present invention have lower IC50 values compared to those observed for the anti-SIRPA 3F9 huIgG1 antibody. Additionally, anti-SIRPA antibodies 3F914, 3F9-18, 3F9-20 and 3F9-22 showed lower IC50 values than anti-SIRPA antibody HEFLB.

Likewise, the anti-SIRPA antibodies of the present invention showed better phagocytic activity compared to the anti-SIRPA antibody HEFLB.

Table 16

SIRPA CD32 Phagocytosis* Antibody IC50 (nM) Max % OP IC50 (nM) Max % OP 3F9 huIgGl 0.1722 90% 0.679 67% 1.77 3F9 mlgGl 0.00905 89 0.0623 82% 1.51 3F9-14 0.0372 89% 0.135 62% 1.56 3F9-18 0.0174 90% 0.0915 65% 1.52 3F9-20 0.0283 90% 0.114 66% 1.62 3F9-22 0.0542 91% 0.133 66% 1.68 OSE HEFLB 0.0959 13% H.O. H.O. 1.05

But. = unspecified, * multiple increase to baseline activity.

Example 11 Affinity-mature anti-SIRPA antibodies stimulate phagocytosis of tumor cells in M1 and M2 macrophages.

Macrophages undergo profound phenotypic transformation in response to microenvironmental signals, acquiring various pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes. The anti-SIRPA antibodies of the present invention are evaluated for their ability to induce phagocytosis of tumor cells in homeostatic (M2-like) macrophages and inflammatory (M1-like) macrophages. Monocytes are isolated from the blood of healthy volunteers using density gradient centrifugation over Ficoll. M1-like and M2-like macrophages are prepared by culturing monocytes in the presence of GM-CSF (800 U/ml; Peprotech) or M-CSF (25 ng/ml; Peprotech) for 6 days. Polarized macrophages are treated overnight with isotype control antibody or Fc variant anti-SIRPA antibody 3F9-22. Fluorescently labeled Raji cells with or without the opsonizing anti-CD20 huIgG1 are mixed with macrophages for 2 hours at 37°C. Alternatively, Raji cells are opsonized with either anti-CD47 IgG4 alone (clone hu5F9) or anti-CD47 and anti-CD20 huIgG1 and added to untreated macrophages.

Phagocytic activity is measured by counting the percentage of CD14/pHrodo double positive macrophages.

As shown in FIG. 11A, both Fc variants of 3F9-22 antibody-treated M2-like macrophages enhance phagocytosis of tumor cells compared to control-treated macrophages. Consistent with previous observations, the SELF Fc variant antibody showed a statistically significant increase in phagocytosis of non-opsonized and opsonized Raji cells compared to the 3F9-22 huIgG1 antibody. In addition, M2-like macrophages treated with the anti-SIRPA antibody 3F9-22 SELF enhanced phagocytosis of non-opsonized and opsonized Raji cells to the same extent as anti-CD47 IgG4-treated Raji cells.

M1-like macrophages differentiated from monocytes cultured in the presence of GM-CSF have properties distinct from M2-like macrophages derived from MCSF. For example, GM-CSF-derived M1-like macrophages reduce the expression of CD14, CD32A/B, and SIRPA. Additionally, M1-like macrophages appeared to induce phagocytosis of non-opsonized Raji cells at a higher rate than M2-like macrophages (Fig. 11B) obtained from the same healthy donor. When stimulated with any of the Fc variants of the anti-SIRPA antibody 3F9-22, M1-pod6nys macrophages increase phagocytosis of non-opsonized Raji cells by -50% compared to control-treated macrophages. Opsonization of Raji cells with anti-CD20 huIgG1 did not show an additive effect on phagocytosis by M1-like macrophages treated with 3F9-22 antibody, which differs from observations made with M2-like macrophages. Similarly, Raji cells opsonized with anti-CD47 IgG4 with or without anti-CD20 huIgG1 also failed to increase phagocytosis above basal levels when added to M1-like macrophages (Fig. 11B). Because GM-CSF-derived macrophages negatively regulate FcgR expression, these cells may have a lower potential to mediate antibody-dependent phagocytosis. These results also suggest that anti-SIRPA antibody 3F9-22 acts through antagonism with SIRPA to induce phagocytosis in both M1 and M2 macrophages, whereas anti-CD47 antibodies act primarily as opsonizing agents that require engagement of FcgR on M2- like macrophages to induce phagocytosis.

Example 12 Combination therapies improve the antitumor activity of affinity mature anti-SIRPA antibodies.

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A small subset of cancer cells, called tumor-initiating cells (TICs) or cancer stem cells (CSCs), form a source of self-sustaining cancer cells that have the ability to self-renew and maintain tumor volume. Data suggest that human solid tumor cells and CSCs upregulate CD47 expression to evade immune surveillance mechanisms allowing tumor cell proliferation and metastasis. Tumorsphere culture models, in which cancer cells grow as three-dimensional spheroid cell clumps, are widely used to analyze the self-renewal capacity of CSCs and to better simulate in vivo cell growth conditions. Tumorsphere formation is based on culturing cancer cells on ultra-low attachment plates in a serum-free medium supplemented with growth factors such as epidermal growth factor and basic fibroblast growth factor. Co-culture of Tumorsphere with primary human macrophages allows anti-SIRPA antibodies to be assessed for tumor cell viability either as a single agent or in combination with other antitumor therapies.

The MDA-MB-231 cell colony is a well-characterized colony of human triple-negative breast cancer cells that has previously been shown to form a Tumorsphere. To measure tumor cell viability in co-culture assays, MDA-MB-231 cells were transduced with lentivirus to constitutively express luciferase and GFP (MB231-Luc). To form Tumorsphere, 10,000 MB231-Luc cells per well were seeded in 96-well plates in StemXVivo Serum-Free Tumorsphere Media (R&D Systems) supplemented with heparin and hydrocortisone. Tumorsphere media is also supplemented with MCSF to support macrophage viability. MB231-Luc is cultured for 2-3 days either without or in the presence of 50,000 macrophages/well. Tumor cell viability was quantified by measuring luciferase activity with OneGlo reagent (Promega) added to each well and incubating the sample for 3 min at room temperature on a plate shaker. The luminescence signal is recorded using a BioTek Synergy™ microplate reader using GEN5™ 2.04 software.

MDA-MB-231 breast cancer cells constitutively expressing luciferase were cultured either alone or in the presence of human macrophages in serum-free Tumorsphere medium supplemented with MCSF. Cells are treated for 2 days at 37°C with anti-EGFR (2 μg/ml), anti-PDL1 (2 μg/ml) or paclitaxel (0.5 μM), with or without anti-SIRPA antibody 3F9-22 . For comparison, tumor cells alone or in the presence of macrophages are treated with anti-CD47 IgG4. Tumor cell viability is quantified by measuring luciferase activity using OneGlo substrate reagent. As shown in FIG. 12A, MB231-Luc cells proliferate in culture with or without macrophages based on luminescence values. Anti-SIRPA antibody 3F9-22 inhibited tumor cell viability only when MB231-Luc cells formed Tumorsphere in the presence of macrophages. This result demonstrates that macrophages retain tumoricidal potential under culture conditions optimized for tumor viability. Under similar conditions, anti-CD47 IgG4 did not significantly inhibit the viability of MB231-Luc.

After testing the effectiveness of the single agent anti-SIRPA antibody 3F9-22 in this Tumorsphere viability assay, additional studies were conducted to show the effect of the combination treatment. Since the viability of tumor cells depends on the EGF present in the medium, the addition of an anti-EGFR blocking antibody showed strong inhibition of tumor growth when only MB231-Luc cells were cultured. The antitumor activity of anti-EGFR antibody is not enhanced in the presence of untreated macrophages. However, macrophages treated with the anti-SIRPA antibody 3F9-22 potentiated the antitumor activity of the anti-EGFR antibody to a statistically significant extent. Under similar culture conditions, the anti-PDL1 antibody showed an unexpected phenotype by inducing sustained apoptosis of tumor cells, regardless of the presence of macrophages. MB231-Luc cell viability was also reduced when exposed to 500 nM paclitaxel, a microtubule-stabilizing chemotherapeutic agent commonly used to treat a variety of cancers. Combining paclitaxel with macrophages treated with the anti-SIRPA antibody 3F9-22 also showed a statistically significant reduction in tumor cell viability. These results demonstrated that the anti-SIRPA antibodies of the present invention enhance the activity of antitumor therapies that function through different mechanisms of action other than opsonization of tumor cells.

MDA-MB-231 breast cancer cells constitutively expressing luciferase were cultured either alone or in the presence of human macrophages in serum-free Tumorsphere medium supplemented with MCSF or MCSF+IL-4+IL-10. Where indicated, cells were treated with either anti-SIRPA antibody 3F9-22 or anti-CD47 IgG4 for 3 days at 37°C. Tumor cell viability is determined by measuring luminescence values after adding OneGlo reagent containing luciferase substrate. Occasionally, monocyte-derived macrophages from healthy volunteers co-cultured with MB231-Luc cells inhibit tumor cell viability without

- 84 044628 treatment (Fig. 12B). To ensure that macrophages cocultured with MB231-Luc cells acquired a more tumor-like, immunosuppressive phenotype, IL-4 and IL-10 were added to Tumorsphere medium. As shown in FIG. 12B, MB231-Luc cells cultured alone proliferated equally well in media supplemented with MCSF or MCSF plus IL-4 and IL-10. However, in media containing only MCSF, macrophages from donor 570 reduced tumor cell viability to a statistically significant extent. This inhibition was reversed by the addition of IL-4 and IL-10, demonstrating that different culture strategies polarize macrophages to modulate tumor cell growth. Importantly, treatment of macrophages with the anti-SIRPA antibody 3F9-22 significantly reduced tumor cell viability under both growth conditions.

Example 13 Affinity mature anti-SIRPA antibodies improve T cell proliferation.

T cells form an immunological synapse with antigen presenting cells (APCs) to initiate T cell activation and proliferation. CD47 expression on T cells may counteract activation by transmitting an inhibitory signal to APCs via SIRPA. However, T cells also express SIRPG, another member of the SIRP family, capable of binding CD47 expressed on APCs. The SIRPG (T cell) - CD47 (APC) interaction has been shown to stabilize the immunological synapse and promote T cell activation and proliferation. Although anti-CD47 antibodies block the inhibitory signal delivered through SIRPA, anti-CD47 antibodies also block the stimulatory effect of SIRPG binding and thus may have a detrimental effect on enhancing effective antitumor T cell responses. Therefore, antagonistic anti-SIRPA antibodies circumvent this potential drawback by inhibiting SIRPA signaling without disrupting critical interactions at the immunological synapse. To test this hypothesis, anti-SIRPA antibodies were assessed by one-way and two-way MLR assays.

The principle of the MLR assay is that APCs derived from a single donor, usually DCs, present peptides on MHC molecules to T cells isolated from a single donor. A small fraction of T cells will express TCRs capable of recognizing the MHC:peptide complex and proliferating upon costimulation. In such a single-factor MLR, cells from one donor participate in antigen presentation, and cells from the other donor respond by proliferation. In two-factor MLR, both donors contribute APCs and T cells to the reaction; thus, two separate populations of T cells respond by proliferation.

The effect of anti-SIRPA antibodies on T cell proliferation is initially assessed using a two-factor MLR assay. PBMCs were isolated from healthy donors and cocultured for 3 days in the presence of increasing concentrations of Fc variants 3F9-22 or isotype control. T cell proliferation is assessed by quantifying the presence of metabolically active cells as an indicator of cell number. Metabolic activity is quantified with CellTiter Glo reagent (Promega), which generates a luminescent signal proportional to the amount of ATP present. As shown in FIG. 13A, PBMCs treated with the anti-SIRPA antibody 3F9-22 showed a ~1.5-fold increase in luminescence compared to isotype-treated PBMCs, indicating increased T cell proliferation. In contrast to the phagocytosis assays described previously, the Fc variant of the 3F9-22 huIgG1 antibody tends to stimulate T cell proliferation more effectively than the SELF Fc variant.

To determine whether the increased luminescence observed with the anti-SIRPA antibody 3F9-22 is specific for the anti-SIRPA antibodies, the two-way MLR assay was repeated with the anti-CD47 antibody. PBMCs are isolated from two healthy donors and 100,000 cells from each donor are mixed with increasing concentrations of test antibody or isotype control antibody. Cell proliferation was measured with CellTiter Glo reagent after 3 days of coculture. As shown in FIG. 13B, anti-SIRPA antibodies 3F9-14 and 3F9-22 huIgG1 and Fc variants of ASPS increase the luminescence signal by ~70% compared to that observed for isotype control-treated PBMCs. Anti-SIRPA antibodies to SELF Fc (3F9-14 SELF and 3F9-22 SELF) do not increase the luminescence signal compared to control under the same conditions, having a different Fc-dependent effect on antibody function. In contrast to the 3F9-22 huIgG1 antibody, anti-CD47 treatment showed a slight increase in luminescence, ~10%. Taken together, the results of the two-factor MLR experiments suggest that antagonism of SIRPA by the anti-SIRPA antibody 3F9-22 huIgG1 promotes T cell proliferation.

Because quantification of metabolic activity by two-factor MLR is an indirect measurement of T cell proliferation, more traditional one-way MLR assays were performed to confirm the stimulatory effect observed with the anti-SIRPA antibody 3F9-22 huIgG1. Primary human dendritic cells (DCs) are obtained from monocytes from healthy donors and cocultured with allogeneic T cells labeled with CFSE dye in a 1:5 ratio. Dendritic cells are treated with either the Fc variant anti-SIRPA antibody 3F9-22 or anti-CD47 (hu5F9) or isotype control antibody and incubated at 37°C for 5 days. T cell proliferation is measured by anti-CD3 APC staining of T cells and gating to a low CFSE population. In fig. 14A shows that DCs from different donors have been processed

- 85 044628 huIgGl or ASPS antibody 3F9-22 increased T cell proliferation by ~50% compared to DCs treated with either isotype or anti-CD47. This increase in T cell proliferation is proportional to the increase in luminescence observed for PBMCs treated with 3F9-22 huIgG1 antibody in two-factor MLR. In fig. 14B shows a typical SCAF plot for the data presented in FIG. 14A. Thus, the results of one- and two-factor MLRs suggest that antagonism of SIRPA with the anti-SIRPA antibody 3F9-22 huIgG1 without blocking the SIRPG-CD47 interaction may promote T cell proliferation.

Example 14 Affinity mature anti-SIRPA antibodies improve pro-inflammatory cytokine release.

To further differentiate the activity of Fc variants of the anti-SIRPA antibody 3F9-22, primary human macrophages from two healthy volunteers were stimulated overnight with a low dose of LPS in combination with the anti-SIRPA antibody 3F9-22 or an isotype control antibody. In fig. 15A, primary human macrophages from 2 healthy donors were stimulated overnight at 37°C with 0.5 ng/ml LPS in combination with either the Fc variant anti-SIRPA antibody 3F9-22 or an isotype control antibody. Supernatant fractions are collected and TNFa levels are analyzed by ELISA. In fig. 15B, primary human macrophages are stimulated overnight at 37°C with 0.5 ng/ml LPS in combination with either increasing concentrations of Fc variant anti-SIRP A antibody 3F9-22 or isotype control antibody. The supernatant fraction is collected and analyzed by ELISA for TNFa release. As shown in FIG. 15A and 15B, macrophages costimulated with the 3F9-22 ASPS antibody showed a significant increase in TNFa secretion compared to cells costimulated with either the isotype control antibody or the 3F922 SELF antibody. The Fc variant of the 3F9-22 SELF antibody exhibits minimal co-stimulatory activity. Titration of anti-SIRPA antibodies showed a similar pattern, with the 3F9-22 huIgG1 antibody and ASPS variants clearly increasing TNFa release in a dose-dependent manner from LPS-primed macrophages, whereas the 3F9-22 SELF antibody showed a reduced TNFa response with high variability. These results indicated that non-CD47 blocking anti-SIRPA antibodies antagonize SIRPA in an Fc-dependent manner, relieving the inhibition of ITIM signaling.

Example 15 Epitope mapping of anti-SIRPA antibody binding sites.

Epitope mapping of anti-SIRPA antibodies is performed using an alanine scanning library generated by fractional mutagenesis of the human SIRPA cDNA sequence. The SIRPA expression construct encoding the C-terminal V5 epitope tag was subjected to high-throughput alanine scanning mutagenesis (described in Davidson and Doranz, 2014 Immunology 143, 13-20) to generate a complete mutation library. Each of the residues representing the extracellular domain of SIRPA (amino acids 31-374 SEQ ID NO: 1) was mutated, most to alanine, while the alanine codons were mutated to serine.

SIRPA mutant library clones plated in a 384-well microplate were individually transfected into HEK-293T cells and allowed to be expressed for 22 hours. Antibodies are digested to produce Fab, after which cells are incubated with Fab diluted in 10% normal goat serum (NSS) (Sigma-Aldrich, St. Louis, MO). Prior to library screening, primary Fab concentrations are determined using an independent immunofluorescence titration curve against cells expressing wild type SIRPA to ensure that signals are within the linear region of detection. Fab was detected using 7.5 μg/ml AlexaFluor488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Westgrove, PA) in 10% NSC. Cells are washed twice with PBS and resuspended in Cellstripper (Cellgro, Manassas, VA) with 0.1% ABS (Sigma-Aldrich, St. Louis, MO). In some cases, higher stringency conditions are used, including increased pH, increased temperature and increased dissociation time. Average cellular fluorescence was determined using an Intellicyt high throughput flow cytometer (HTFC, Intellicyt, Albuquerque, NM). Fab reactivity against each mutant clone is calculated relative to the reactivity of wild-type SIRPA protein by subtracting the signal from mock-transfected controls and normalizing to the signal of transfected wild-type SIRPA controls.

Mutated residues in the library clones were identified as critical for a Fab binding epitope if it did not support the reactivity of the tested Fab but supported the reactivity of the commercially available reference antibody, MAB4546 (R&D Systems) or additional anti-SIRPA Fab. This counter-screening strategy helps exclude SIRPA mutants that were locally misfolded or that had a defective expression.

Anti-SIRPA antibody 9C2 is described in international patent application No. PCT/US2017/65366.

mAL9S2. Heavy chain variable domain sequence

EFQLQQSGAELVKPGASVKISCKASGYSLTGYNMNWVKQSRGKSLEWIGNINPH

YGSSTYNQNFKDKATLTVDKSSSAAYMQFNSLTSEDSAVYYXAPEGYDGVFDYWGQG

TTLTVSS (SEQ Ш No: 45)

- 86 044628 tAL9S2. Light chain variable domain sequence

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYVTSNLAS

GVPTRFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIK (SEQ ID No.:

46)

In table 17 below presents the average binding reactivity values and ranges for all residues identified as critical in the screens. Primary critical residues were defined as residues in which mutations were negative for test antibody binding (<30% WT binding) but positive for control antibody (>80% WT). In fig. 16 depicts models of the crystal structure of SIRPA (PDB ID 2WNG; Hatherley et al., 2009, J Biol Chem, 284:26613-9) highlighting the critical residues for binding of the anti-SIRPA antibodies 3F9 and 9C2 as shaded spheres.

As indicated in table. 18, critical SIRPA residues involved in binding of the anti-SIRPA antibody 3F9 include amino acid residues R282, Q284 and G337 of the human SIRPA v1 sequence shown above. The critical SIRPA residues involved in binding by the 9C2 antibody (as described in International Patent Application No. PCT/US2017/65366) include amino acid residues Q281, R282, Q284, L285, W287, R295, E297, V302 and W315 of the human SIRPA v1 sequence. described above. These residues are located in the membrane-proximal Ig domain of SIRPA, referred to in the SIRPA scientific literature as the D3 domain, which corresponds to amino acids 254-348 of the human SIRPA v1 sequence. Multiple published reports demonstrate that the D3 domain of human SIRPA binds to structure recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively).

Table 17

Critical residues for binding 3F9 and 9C2

Binding reactivity (% DT) Mutation 3F9 Fab 9С2 Fab Q281A 151.1 (14) 14.1 (Π) R282A 3.8 (2) 4.7 (2) Q284A 3.3 (1) 6.5 (5) L285A 107.0(12) 17.9 (1) W287A 124.8 (12) 2.4 (2) R295A 120.9 (10) 1.8 (2) E297A 116.9(14) 5.3 (0) V302A 92.3 (29) 4.9 (0) W315A 117.3 (10) 19.0 (6) G337A 17.9 (9) 36.3 (68)

Table 18

Residues involved in anti-SIRPA antibody binding

Antibody Critical remnants of SIRPA 3F9 R282, Q284, G337 9S2 Q281, R282, Q284, L285, W287, R295, E297, V302, W315

Example 16 Anti-SIRPa antibodies improve the antitumor activity of immune checkpoint inhibitors in a synergistic tumor model.

Therapeutic antibodies targeting the SIRPA-CD47 pathway could rely on xenograft tumor models for studies to confirm the mechanism of action, in which immunodeficient NOD or NOG mice support the growth of implanted human cancer cells. Because the allelic variant of mouse SIRPA expressed by NOD mice binds human CD47, this interaction allows engraftment of human cells into a mouse host. However, these mice may also be exaggerating the role of this pathway because NOD SIRPA binds human CD47 with greater affinity than human SIRPA. Moreover, Prkdc ^SCID and IL2Ry ^Nu11 mutations in immunodeficient mouse strains eliminate T cells, B cells, and NK cells, thus making myeloid cells and the innate immune system the only effector cells available to inhibit tumor growth. Tumor xenograft models do not allow the investigation of therapies that target innate immune cells to prime or enhance the adaptive antitumor immune response. Thus, to evaluate the human anti-SIRPA antibodies of the present invention in the context of immunocompetent animal hosts, human SIRPA and human CD47 genes were introduced into a C57BL6/J mouse strain.

Briefly, bacterial artificial chromosomes (BAR) carrying human SIRPA or human CD47-encoding nucleic acids with flanking sequences for

- 87 044628 inclusions of relevant human gene regulatory elements were identified using the UPCK Genome Browser and CloneDB from NCBI. One IIR clone (BACRP11-636L228) was identified, which was predicted to contain coding sequences for the human SIRPA gene. Another IIR clone (BACRP11-671F8) was identified that was predicted to contain coding sequences for the human CD47 gene. Mice carrying the IIR clones of interest were then generated by injecting purified IIR DNA into C57BL6/J mouse zygotes using standard pronuclear injection techniques. The zygotes were returned to the female mice, and the resulting mouse pups were genotyped for the desired transgene. Founder animals containing the transgene are then crossed with non-transgenic animals, and the progeny are screened for transgene expression by PCR using standard techniques, and additionally by SCAF staining of the animals' peripheral blood cells. Transgenic IHR mice carrying each individual gene of interest are crossed to produce humanized mice expressing both human SIRPA and human CD47.

In addition to generating mice to express human SIRPA and human CD47 as described above, syngeneic colonies of tumor cells were also engineered to replace mouse CD47 with human CD47. Briefly, CRISPR-Cas9 technology was used to introduce Cas9/guide RNA (gRNA) complexes into target MC38 cells. Cas9/HRNA-mediated insertion/deletion formation in the target region (exon 2 of mouse CD47) results in a frameshift and/or premature arrest, thereby knocking out expression of the endogenous mouse CD47 gene. After transfection with gRNA and Cas9, clones obtained from a single cell are screened by sequencing, and homozygous clones containing the desired mutation are expanded. As shown in FIG. 17A (top panel), SCAF analysis of parental MC38 cells and a colony of mouse CD47 knockout cells (MC38-mCD47KO) stained with anti-mouse CD47 antibody confirms loss of mouse CD47 expression in these engineered cells. MC38-mCD47KO cells are then transduced with lentivirus containing the human CD47 gene insert. MC38-mCD47KOhuCD47+ cells were selected with puromycin and expanded. In fig. 17A (bottom panel) shows a SCAF histogram confirming the expression of human CD47 in a selected population of cells.

The antitumor effect of the anti-SIRPA antibodies of the present invention was assessed in vivo using a huSIRPA/huCD47 BACtg mouse colon carcinoma model engrafted with MC38-mCD47KO-huCD47+ cells in combination with a T cell immune checkpoint inhibitor. These mice are injected subcutaneously with 500,000 MC38 cells into the right flank. Antibody therapy begins when mice are randomly assigned to groups approximately ten days after cell transplantation, or when tumors have reached an average volume of 80 mm ³ . Mice receive two intraperitoneal (IP) injections per week of anti-SIRPA antibody 3F9 mIgG1 or anti-SIRPA antibody 3F9 mIgG2A at a dose of 10 mg/kg in combination with a suboptimal dose of anti-mouse PDL1 antibody (clone 10F.9G2, Bioxcell) for 3 weeks ) at a dose of 5 mg/kg. Animals are sacrificed when tumors reach a volume of ~2000 mm ³ .

In fig. 17B shows the average tumor growth curves of a huSIRPa/huCD47 IIR transgenic mouse subcutaneously implanted with MC38 cells engineered to lose murine CD47 expression and overexpress human CD47 (MC38-mCD47KO/huCD47+). Mice are treated with isotype control antibody, anti-mouse PDL1 antibody, anti-SIRPA 3F9 mIgG1 antibody plus anti-mouse PDL1 antibody, or anti-SIRPA 3F9 mIgG2A antibody plus anti-mouse PDL1 antibody as indicated. Treatment begins when the tumors average 80 mm ³ . As shown in FIG. 17B, treatment with anti-PDL1 antibody inhibited tumor growth by 20% relative to animals treated with isotype control antibody, which did not reach statistical significance. However, combination therapy with anti-PDL1 antibody with either anti-SIRPA antibody 3F9 mIgG1 or anti-SIRPA antibody 3F9 mIgG2A further inhibited tumor growth in these animals by 40 and 47%, respectively. In fig. 17C shows line graphs of the tumor volume of each animal in the treatment groups. With the exception of a few poor responders, tumor volumes did not exceed 1000 mm in most animals in the combination therapy groups, while tumor volumes in a few animals in the isotype control or anti-PDLl antibody monotherapy groups reached a limit of 2000 mm ³ .

These results demonstrate that the anti-SIRPA 3F9 antibodies of the present invention are effective in enhancing the antitumor activity of immune checkpoint inhibitors in an immunocompetent animal model encoding the human SIRPA-CD47 pathway. These results indicated that the anti-SIRPA antibodies of the present invention are useful for enhancing the antitumor activity of immune checkpoint inhibitors. Accordingly, the anti-SIRPA antibodies of the present invention are effective in the treatment of cancer when used in combination with immune checkpoint inhibitor therapy.

Example 17 Anti-SIRPa antibodies negatively regulate SIRPA expression on tumor-infiltrating myeloid cells in murine syngeneic tumors.

The previous example demonstrated the antitumor activity of the anti-SIRPA antibody of the present invention in transgenic mice with human SIRPA/CD47 IHR. To check that

- 88 044628 antitumor activity correlates with antibody-mediated negative regulation of SIRPA on tumor macrophages, tumor myeloid cells are characterized after drug administration.

Human SIRPA/huCD47 IIR transgenic mice are implanted subcutaneously in the right flank with 500,000 MC38-mCD47KO/huCD47+ cells. Tumors in mice are grown to an average volume of ~400 mm ³ before the introduction of antibodies. Mice receive two IP injections of the anti-SIRPA antibody 3F9-22 mIgG2A three days apart at a dose of 3, 10, or 30 mg/kg. Control mice receive 2 IP injections of mIgG2A isotype at a dose of 10 mg/kg. Tumor and spleen tissue was collected from mice 1 day, 4 days, and 8 days after the second dose of antibody. Spleens are reduced to a single cell suspension by mechanical dissociation through a 70 μm cell sieve. The cells are then washed with PBS, and the red blood cells are lysed with ACK lysis buffer. Cells are then resuspended in SCAF buffer consisting of PBS/2% FBS and prepared for staining. Similarly, tumor tissues are processed into single cell suspensions by enzymatic and mechanical dissociation using a MACS™ gentle dissociator (MiltenyiBiotec). The tissue homogenate was filtered through a 70 μm cell sieve and resuspended in SCAF buffer consisting of PBS/2% FBS for staining. Cells are stained using the following panel.

Table 19

Fluorochrome Marker Clone AmCyan (BV510) Alive/dead RESu7 mCD45 30-F11 Pacific Blue (BV421) mCDllb Ml/70 PerCp/Cy5.5 mF4/80 BM8 ARS/Su7 mLy6G 1A8 AF488 (FITC) mLy6G HK1.4 ARS hSIRPa SE5A5

In fig. 18A shows the relative change in SIRPA expression over time on tumor-infiltrating myeloid cells (monocytes and macrophages). Tumor-associated macrophages were defined as CD45+ CD11b+ F4/80+ Ly6C-Ly6G- cells, whereas tumor-infiltrating monocytes were defined as CD45+ CD11b+ F4/80- Ly6C+ Ly6G- cells. Compared with animals treated with isotype control antibody, animals treated with anti-SIRPA antibody showed nearly 80% reduction in cell surface SIRPA expression 1 day after antibody administration on both tumor monocytes and tumor macrophages. SIRPA expression returned to baseline levels after 4 days or 8 days in mice treated with 3 mg/kg or 10 mg/kg anti-SIRPA antibody 3F9-22 mIgG2A, respectively. SIRPA expression level stabilized to about 50% below baseline in mice treated with 30 mg/kg anti-SIRPA antibody 3F9-22 mIgG2A.

The splenic compartment was also immunoprofiled to determine if any correlation with the tumor microenvironment could be determined. Splenic monocytes were more resistant to antibody-mediated downregulation of the receptor compared to tumor monocytes (Fig. 18B). Splenic monocytes were defined as CD45+ CD11b+ F4/80- Ly6C+ Ly6G- cells. Splenic red pulp macrophages were defined as CD45+ CD11b- F4/80+ Ly6C- cells. Splenic monocytes exhibit maximal down-regulation of SIRPA by ∼40% one day after antibody administration and rapidly regain receptor expression at a later time. Splenic red pulp macrophages, on the other hand, follow the same pattern of SIRPA receptor expression observed for tumor macrophages.

Taken together, these results demonstrate that the anti-SIRPA antibodies of the present invention are effective in reducing (down-regulating) SIRPA levels on macrophages, in particular tumor-associated macrophages, and reducing or down-regulating SIRPA levels on macrophages, correlating with the antitumor activity of anti-SIRPA antibodies. SIRPA of the antibodies of the present invention.

Example 18 Anti-SIRPA antibodies negatively regulate SIRPA expression on tumor-infiltrating myeloid cells in human immune system (HIS) mice.

Since the Fc domain of anti-SIRPA antibodies plays a role in negative regulation of the receptor and antibody function, mice with reconstituted human immune systems are used to compare the activity of various Fc variants with the anti-SIRPA antibodies of the present invention. HIS mouse models require the inoculation of umbilical cord blood-derived CD34+ human hematopoietic stem cells (hHSCs) into immunodeficient mice (i.e., NOG strain) after pre-adapting them with sublethal irradiation. HSCs consistently develop large cell colonies, especially lymphocyte populations, in the peripheral blood, bone marrow, thymus, and spleen (as well as other tissues). NOG mice engineered to express human GM-CSF and human IL-3

- 89 044628 (NOG-EXL; Taconic), provide the additional benefit of supporting greater myeloid cell differentiation compared to other mouse strains. HIS mice provide the opportunity to study human tumor-immune system interactions in vivo in mice implanted with colonies of human cancer cells. Additionally, HIS mice help evaluate the mechanisms of action of therapeutic antibody candidates in an in vivo setting.

Mice are inoculated with human melanoma A3 75 cells subcutaneously in the flanking region at a cell density of 3 x 10 ⁶ cells per mouse with 50% Matrigel (Cat, 354234; Corning). Tumors were grown to an average volume of 400 mm before mice received 2 intraperitoneal injections of 3F9-22 huIgG1 P331S (PS), 3F9-22 huIgG1 N325S/L328F (NSLF), or huIgG1 isotype control at a dose of 10 mg/kg, three days apart. Blood, spleen, and tumor tissues were collected 1 day after the second antibody injection. Spleens are processed into a single cell suspension by mechanical dissociation through a 70 μm cell sieve. Cells are washed with PBS, and red blood cells are lysed with ACK lysis buffer. Cells are then resuspended in SCAF buffer consisting of PBS/2% FBS and prepared for staining. Similarly, tumor tissues are processed into single cell suspensions by enzymatic and mechanical dissociation using a MACS™ soft dissociator (MiltenyiBiotec). The tissue homogenate was filtered through a 70 μm cell sieve and resuspended in SCAF buffer consisting of PBS/2% FBS for staining. Peripheral blood cells are treated with ACK lysis buffer to lyse red blood cells and resuspended in SCAF buffer consisting of PBS/2% FBS. Cells are stained using the following panel.

Table 20

Fluorochrome Marker Clone PERCPCY5 CD3 SK7 PERCPCY5 CD19 HIBI 9 FITC CD14 M5E2 RE-Su7 CD 11b ICRF44 A1exa700 human CD45 2D1 AshSuap Alive/dead AIIK/DyL650 SIRPA interior BV421 CD16 3G8 BUV395 HLA-DR L243 PE-CF594 CD 163 GHI/61 RE CD86 IT2,2 ARS-Su7 Mouse CD45 30-F11

NOG-EXL mice are engrafted with human CD34+ hematopoietic stem cells (HSCs) obtained from umbilical cord blood from two different donors (donors A and B). All mice are implanted subcutaneously with 3 million A375 cells, a colony of human melanoma cells. Tumors are grown to an average volume of ~400 mm ³ before antibody administration. Mice received two IP injections of anti-SIRPA antibody three days apart at a dose of 10 mg/kg. Control mice are given 2 IP injections of huIgG1 isotype at a dose of 10 mg/kg. Tumor tissue was collected from mice 1 day after the second dose of antibody.

In fig. 19 shows the study design and results of antibody-mediated down-regulation of SIRPA in humanized NOG-EXL mice bearing A375 tumors in these studies. In fig. 19A shows that 8 mice were engrafted with CD34+ HSC from donor A, and 29 mice were engrafted with CD34+ HSC from donor B. Briefly, 40 mice engrafted with CD34+ HSC from 2 different donors were purchased from Taconic, NY. 10 weeks after inoculation, mice are tested for reconstitution of human leukocytes by flow cytometry before transport. The majority of mice (36/40) contained >50% huCD45+ cells in their peripheral blood.

Tumor-associated macrophages in NOG-EXL mice were defined as CD45+ CD11b+ CD14+ CD16+ CD3-CD19- cells. In fig. 19B shows the expression level of SIRPA on human tumor macrophages, plotted as either SPC values (left panel) or normalized values (right panel). Anti-SIRPA antibody treatment results in significant down-regulation of SIRPA in human tumor macrophages from both donors compared with isotype controls. Normalization of UPC values for each donor shows a ~70% reduction in SIRPA expression, which approximately corresponds to the degree of downregulation observed in monocyte-derived macrophages in vitro and in tumor macrophages from IHR transgenic mice. In these experiments, no significant difference in activity was observed between the Fc variant of the anti-SIRPA 3F9-22 PS and the Fc variant of the NSLF anti-SIRPA antibody.

In addition to SIRPA expression, tumor macrophages from these experiments were also profiled to examine the expression of M1 markers. Tumor macrophages were identified as huCD45+

- 90 044628 huCD11b+ CD14+ huCD16+ cells. As shown in FIG. 20A, the anti-SIRPA antibodies of the present invention positively regulate the expression of HLA-DR. Normalization of UPC values for each donor demonstrates that the Fc variant of the anti-SIRPA antibody 3F9-22 PS increases HLA-DR expression ~3-fold in both donors compared to that observed with isotype control antibody treatment. In contrast, the Fc variant of the anti-SIRPA antibody 3F9-22 NSLF increased HLA-DR expression ~1.5-2-fold compared to that observed with isotype control antibody treatment. Only the increase measured on donor B macrophages reached statistical significance with the Fc variant of the anti-SIRPA antibody 3F9-22 NSLF. Similarly, the anti-SIRPA antibodies of the present invention increase the expression of CD86 on tumor macrophages only from donor B (Fig. 20B). Although both the Fc variant PS and NSLF increased CD86 expression by ~40% compared to the isotype control antibody group, only the Fc variant PS reached statistical significance for increasing CD86 expression. These results demonstrate that both Fc variant PS and NSLF anti-SIRPA antibodies 3F9-22 suppress SIRPA and increase M1 markers on human tumor macrophages; however, in these experiments the PS variant appears to be partially more effective than the NSLF variant.

Antibody-mediated down-regulation of SIRPA was also assessed in peripheral blood monocytes and spleen monocytes to compare the activity of the anti-SIRPA antibodies of the present invention in tumor and peripheral immune system compartments. Human monocyte populations were defined as CD45+ CD11b+ CD14+ CD16- CD3-CD19- cells. Mice receive two IP injections of anti-SIRPA antibodies three days apart at a dose of 10 mg/kg. Control mice receive 2 IP injections of huIgG1 control antibody at a dose of 10 mg/kg. Spleens and blood were collected from mice 1 day after the second dose of antibody. As shown in FIG. 21A, Fc variant of anti-SIRPA antibody 3F9-22 NSLF showed higher down-regulation of SIRPA compared to that observed with Fc variant of anti-SIRPA antibody 3F9-22 PS in spleen monocytes. However, in peripheral blood monocytes, both Fc variants exhibit similar activity (Fig. 21B). The anti-SIRPA antibodies of the present invention show greater receptor downregulation in monocytes obtained from donor B than from donor A, which may correlate with the higher baseline level of SIRPA expression on monocytes from donor B than from donor A.

Taken together, these results demonstrate that the anti-SIRPA antibodies of the present invention are effective in negatively regulating SIRPA levels in both peripheral blood and spleen monocytes.

Example 19 Anti-SIRPA antibodies engineered to selectively recruit FcRs retain antibody-mediated down-regulation activity.

To further evaluate the role of FcR in the activity of the anti-SIRPA antibody of the present invention, the variable domain of the anti-SIRPA antibody 3F9-22 was expressed with different Fc domains with different FcR binding profiles. FcR affinity measurements for Fc variants of anti-SIRPA antibodies are obtained using biolayer interferometry (BSI) on a Pall ForteBio Octet RED96 instrument. Immobilized anti-human antibody Fab-CH1 captures humanized Fc variants of the anti-SIRPA antibody 3F9-22 on biosensors (10 μg/ml, loading time 300 s). Single-cycle kinetics were performed with 100 nM soluble human FcR flowing through the captured antibody to record traces (association time 300 s, dissociation time 300 s). Data analysis is carried out using ForteBio Data Analysis Software, version 9.0. Standard kinetic buffer (BSF, 0.1% ABS, 0.02% Tween-20, pH 7.2) was used for analysis and for reagent preparation.

Relative affinity measurements obtained from curve fitting analysis of the sensograms obtained in these experiments are summarized below in Table 1. 19. Fc variants exhibit predicted FcR binding patterns. For example, Fc mutations in LALAPS abrogate binding to all FcRs except the high affinity receptor, FcgR1. Fc mutations N325S/L328F (NSLF) specifically abrogate binding to human FcgR3A. Also, the IgG2 and IgG4 isotypes cannot bind FcgR3A, and only IgG4 retains binding to FcgR1.

Table 19

Fc option FcgRl FcgR2A H131 FcgR2A R131 FcgR2B FcgR3A F158 FcgR3A V158 IgGl LALAPS + - - - - - IgGl ++ + ++ ++ - - NSLF IgG2 - + + - - - IgG 4SP ++ + ++ ++ - -

Fc variants of the anti-SIRPA antibody 3F9-22 were then assessed for the ability to negatively regulate SIRPA expression or levels in human monocyte-derived macrophages. As described above, human monocytes are isolated from the peripheral blood of healthy donors and differentiated into macrophages in vitro by adding human M-CSF to the culture medium. After differentiation, human macrophages are collected and seeded into 96-well tissue culture plates with increasing concentrations of control antibody or soluble anti-SIRPA antibody and incubated overnight at 37°C. Cells were analyzed by flow cytometry for cell surface expression of SIRPA using a DyLight650-conjugated anti-human SIRPA antibody belonging to a separate epitope group from the anti-SIRPA antibody 3F922.

As shown in FIG. 22 and summarized in table. 20, most Fc variants of the anti-SIRPA antibody 3F9-22 maximally negatively regulate cell surface SIRPA levels on macrophages with similar IC50 values. However, the anti-SIRPA antibody 3F9-22 LALAPS only partially suppressed cell surface levels of SIRPA, suggesting that FcR recruitment is involved in potentiating this anti-SIRPA antibody activity. In addition, since Fc variants that do not bind FcgR3A exhibit similar negative regulation of the receptor as the anti-SIRPA antibody 3F9-22PS, which binds all FcRs, it is suggested that FcgR3A involvement is dispensable for antibody-mediated receptor internalization.

Table 20

Fc option IC50 (nM) Max%OP 3F9-22 PS 0.04701 73.825 3F9-22 LALAPS 0.04468 41,575 3F9-22 NSLF 0.045125 74.61 3F9-22 IgG2 0.02185 67,955 3F9-22 IgG4 0.032415 70,455

Example 20 Anti-SIRPA antibodies engineered to selectively recruit FcRs improve phagocytosis of tumor cells by macrophages.

In addition to the antibody-mediated negative regulation of the receptor as described above, the role of FcR in the anti-SIRPA antibody-mediated enhancement of tumor cell phagocytosis with various Fc variants of the anti-SIRPA antibody 3F9-22 was assessed. Additionally, the phagocytic activity of other antibodies targeting this pathway is assessed in parallel.

Primary human macrophages are treated overnight with control antibody or the indicated test antibody. Tumor cells are labeled with CFSE dye and added to macrophages at 37°C overnight. The next day, macrophages are collected and stained on ice with anti-CD14 APC in SCAF buffer containing FcR-blocking antibodies. Phagocytic activity is measured by counting the percentage of CD14/CFSE double positive macrophages.

The results of these experiments are shown in Fig. 23 and are presented as the fold increase in the percentage of the CD14/CFSE double positive macrophage population. As shown in FIG. 23 and summarized below in table. 21, both anti-SIRPA antibody 3F9-22PS and anti-SIRPA antibody 3F9-22NSLF variants significantly enhanced phagocytosis of A375 melanoma cells compared with that observed in macrophages treated with control antibody. The observed increase in phagocytosis appeared to be comparable to the anti-CD47 antibody tested in these studies (aCD47), which primarily functions through the opsonization of CD47-expressing tumor cells for ADCP. In contrast, two different CD47-blocking anti-SIRPA antibodies KWAR23 (see WO 2015/138600) and 18D5 (see WO 2017/178653) did not show any enhancement of tumor cell phagocytosis. These results indicate that both Fc variants of PS and NSLF anti-SIRPA antibodies 3F9-22 retain the properties of enhancing tumor cell phagocytosis in the absence of an antitumor antigen opsonizing antibody. Since the anti-SIRPA antibodies of the present invention are non-blocking with respect to the SIRPA/CD47 interaction, these results also indicate that the non-blocking anti-SIRPA antibodies of the present invention were effective in increasing or enhancing phagocytosis of tumor cells compared to that observed for the non-blocking anti-SIRPA antibody. SIRPA antibodies KWAR23 and 18D5.

Table 21

Antibody EC50 (nM) Max* 3F9-22PS 0.1144575 2.06925 3F9-22NSLF 0.457625 1,774 KWAR23 0.3631 1.126 18D5 2,148 1.06446667 aCD47 0.777775 2.6235

*Multiples increase in phagocytosis to baseline activity.

Example 21 Variants of the anti-SIRPA antibody 3F9-22 do not cause changes in red blood cell and platelet counts.

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Reports show that administration of the aHmu-CD47 antibody 5F9 to non-human primates results in anemia (Liu et al, 2015, PLOS ONE, DOI: 10.1371/journal.pone.0137345) and that administration of the anti-SIRPAFc TTI621 to humans results in a dose-dependent reduction platelets (Ansell et al., 2016, Blood 128: 1812). To test whether the anti-SIRPA antibodies of the present invention have any effect on red blood cell count or platelet count in vivo, the following studies are performed. Female cynomolgus monkeys are divided into five groups and given control antibody, anti-SIRPA antibody 3F9-22 PS, anti-SIRPA antibody 3F9-22 IgG4, anti-SIRPA antibody 3F9-22 IgG2, or anti-SIRPA antibody 3F9-22 NSLF, as indicated in table. 22 below. Animals are administered antibodies by intravenous infusion through the saphenous vein.

Table 22

Group Quantity females Path Target dose concentration (mg/ml) Target dose volume (ml/kg) Test sample dosage ovani Target dose level (mg/kg) 1 3 IgG IV 10 2.5 4 2 3 3F9-22 PS IV 10 2.5 4 3 3 3F9-22 IgG4 IV 10 2.5 4 4 3 3F9-22 IgG2 IV 10 2.5 4 5 3 3F9-22 NSLF IV 10 2.5 4

Blood is collected from animals via the femoral vein for determination of red blood cell (RBC) and platelet counts once during the pre-dose phase and then approximately 72, 168, 240, 408, 576, 672 and 744 hours after antibody administration.

As shown in FIG. 24, red blood cell and platelet counts were not altered by any of the anti-SIRPA antibodies 3F9-22 PS, 3F9-22 IgG4, 3F9-22 IgG2, and 3F9-22 NSLF during these experiments.

Some additional sequences of the anti-SIRPA antibody of the present invention are shown below. The underlined amino acids indicate specific differences in the amino acid sequences of the following anti-SIRPA antibodies.

3F9-22-IgGl DT Heavy Chain (SEQ ID No: 47)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPPYDDYYGGFQY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-22 huIgGl PS Heavy Chain (SEQ ID No: 48)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPPYDDYYGGFQY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

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GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKG QPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-22 SELF Heavy Chain (SEQ ID No: 49)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPPYDDYYGGFQY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-22 hlgGl NSLF Heavy Chain (SEQ ID No: 53)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYXAPPPYDDYYGGFQY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-22 hlgGl LALAPS Heavy Chain (SEQ ID No: 54)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYXAPPPYDDYYGGFQY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-22 huIgGl Light chain (SEQ GO No: 50)

DIQLTQSPS S LS AS VGDRVTITCRAS KS VS S GG YS YMHWYQQKPGKAPKLLIYLA SNLESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQHNRELPVTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

3F9-14 huIgGl DT Heavy Chain (SEQ ID No: 51)

EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPPYDDYYGGFRY

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WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-14 PS Heavy Chain (SEQ GO No: 57)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYXAPPPYDDYYGGFRY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-14 SELF Heavy Chain (SEQ ID No: 52)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPPYDDYYGGFRY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-14 NSLF Heavy Chain (SEQ ID No: 55)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYXAPPPYDDYYGGFRY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-14 LALAPS Heavy Chain (SEQ ID No: 56)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISEY GGSYTYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYXAPPPYDDYYGGFRY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3F9-14 huIgGl Light Chain (SEQ ID No: 50)

DIQLTQSPSSLSASVGDRVTITCRASKSVSSGGYSYMHWYQQKPGKAPKLLIYLA SNLESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQHNRELPVTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

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Claims (60)

CLAIM 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 containing the amino acid sequence SEQ ID No: 21; HVR-H3 containing an amino acid sequence selected from SEQ ID Nos: 22, 23 and 24; and the light chain variable region comprises: HVRL1 containing the amino acid sequence of SEQ ID NO: 9; HVR-L2 containing the amino acid sequence SEQ ID NO: 10; and HVR-L3 containing an amino acid sequence selected from SEQ ID Nos: 11, 12, 13, 14, 15, 16, 17, 18 and 19. 2. The antibody according to claim 1, where the variable region of the heavy chain contains HVR-H1 containing the amino acid sequence SEQ ID No: 20; HVR-H2 containing the amino acid sequence SEQ ID No: 21; and HVR-H3 containing the amino acid sequence SEQ ID No: 23; and wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; HVR-L2 containing the amino acid sequence SEQ ID NO: 10; and HVR-L3 containing the amino acid sequence SEQ ID No: 16. 3. The antibody according to claim 1, where the light chain variable region contains HVR-H1 containing the amino acid sequence SEQ ID No: 20; HVR-H2 containing the amino acid sequence SEQ ID No: 21; HVR-H3 containing the amino acid sequence SEQ ID No: 24; and wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; HVRL2 containing the amino acid sequence SEQ ID NO: 10; and HVR-L3 containing the amino acid sequence SEQ ID No: 16. 4. The antibody according to claim 1, 2 or 3, wherein the heavy chain variable region contains one, two, three or four framework regions selected from VH FR1 containing the amino acid sequence SEQ ID No: 25, VH FR2 containing the amino acid sequence SEQ ID No.: 26, VH FR3 containing the amino acid sequence SEQ ID No.: 27 and VH FR4 containing the amino acid sequence SEQ ID No.: 28; and/or wherein the light chain variable region comprises one, two, three or four framework regions selected from VL FR1 containing the amino acid sequence SEQ ID No: 29, VL FR2 containing the amino acid sequence SEQ ID No: 30, VL FR3 containing the sequence amino acids SEQ ID No: 31 and VL FR4 containing the amino acid sequence SEQ ID No: 32. 5. An antibody according to claim 1, 2 or 3, wherein the heavy chain variable region contains 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; and/or, wherein the light chain variable region contains an amino acid sequence that is at least 90% or at least 95% or at least 99% identical to the amino acid sequence selected from SEQ ID Nos: 36, 37 , 38, 39, 40, 41, 42, 43 and 44. 6. An isolated antibody that binds to human SIRPA, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID Nos: 33, 34 and 35, and wherein the variable region light chain contains an amino acid sequence selected from SEQ ID Nos: 36, 37, 38, 39, 40, 41, 42, 43 and 44. 7. The antibody of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID No: 34, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID No: 41. 8. The antibody of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID No: 35, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID No: 41. 9. An antibody according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody. 10. An antibody according to any one of claims 1 to 9, wherein the antibody is a humanized antibody. 11. Antibody according to any one of claims 1 to 10, where the antibody is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment. 12. An antibody according to any one of claims 1 to 11, wherein the antibody is a multivalent antibody. 13. Antibody according to any one of claims 1 to 12, where the antibody is one of the IgG class, IgM class or IgA class. 14. The antibody of claim 13, wherein the antibody is one of the IgG class, and is an IgG1, IgG2, IgG3 or IgG4 isotype. 15. An antibody according to any one of claims 1 to 14, wherein the antibody binds to an inhibitory Fc receptor. 16. The antibody of claim 15, wherein the Fc inhibitory receptor is Fc receptor gamma inhibitory IIB (FcgRIIB). 17. The antibody of claim 16, wherein the antibody reduces cellular levels of FcgRIIB. 18. Antibody according to any one of claims 1 to 17, wherein the anti-SIRPA antibody is human or mouse - 96 044628 IgG1 isotype and contains 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 of them, where the numbering of residues is carried out according to EU numbering or contains an amino acid deletion in the Fc region at a position corresponding to glycine 236. 19. The antibody according to any one of claims 1 to 18, where the antibody contains 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, where the numbering of amino acid residues is carried out according to the EU or Cabot numbering. 20. An isolated antibody that binds human SIRPA, wherein the antibody comprises a heavy chain and a light chain, wherein (a) the heavy chain contains the amino acid sequence of SEQ ID No: 47 and the light chain contains the amino acid sequence of SEQ ID No: 50; (b) the heavy chain contains the amino acid sequence of SEQ ID No: 48 and the light chain contains the amino acid sequence of SEQ ID No: 50; (c) the heavy chain contains the amino acid sequence of SEQ ID No: 49 and the light chain contains the amino acid sequence of SEQ ID No: 50; (d) the heavy chain contains the amino acid sequence of SEQ ID No: 53 and the light chain contains the amino acid sequence of SEQ ID No: 50; (f) the heavy chain contains the amino acid sequence of SEQ ID No: 54 and the light chain contains the amino acid sequence of SEQ ID No: 50; (f) the heavy chain contains the amino acid sequence of SEQ ID No: 51 and the light chain contains the amino acid sequence of SEQ ID No: 50; (g) the heavy chain contains the amino acid sequence of SEQ ID No: 52 and the light chain contains the amino acid sequence of SEQ ID No: 50; (h) the heavy chain contains the amino acid sequence of SEQ ID No: 55 and the light chain contains the amino acid sequence of SEQ ID No: 50; (i) the heavy chain contains the amino acid sequence of SEQ ID No: 56 and the light chain contains the amino acid sequence of SEQ ID No: 50; or (j) the heavy chain contains the amino acid sequence of SEQ ID No: 57 and the light chain contains the amino acid sequence of SEQ ID No: 50. 21. The antibody according to any one of claims 1 to 20, wherein the anti-SIRPA antibody reduces surface cellular levels of SIRPA, reduces intracellular levels of SIRPA, reduces total cellular levels of SIRPA, or any combination thereof. 22. The antibody of any one of claims 1 to 21, wherein the anti-SIRPA antibody causes SIRPA degradation, causes SIRPA cleavage, causes SIRPA internalization, causes SIRPA shedding, negatively regulates SIRPA expression, or any combination thereof. 23. The antibody according to any one of claims 1 to 22, wherein the antibody reduces cell surface levels of SIRPA in vitro. 24. The antibody according to any one of claims 1 to 23, wherein the antibody reduces cell surface levels of SIRPA in vivo. 25. The antibody according to any one of claims 1 to 24, wherein the antibody negatively regulates the expression of SIRPA in human monocytes. 26. The antibody according to any one of claims 1 to 25, wherein the antibody negatively regulates the expression of SIRPA in human macrophages. 27. The antibody of claim 26, wherein the antibody negatively regulates SIRPA expression in human macrophages by about 70-95%. 28. The antibody according to any one of claims 1 to 27, where the antibody has an affinity (AK) for human SIRPA v1 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. 29. The antibody according to any one of claims 1 to 28, wherein the antibody has an affinity (Af) for human SIRPA v1 from about 0.1 to 2 nM. 30. The antibody according to any one of claims 1 to 25, where the antibody has an affinity for human SIRPA v1 with a CD that is at least 2-fold, at least 3-fold, at least 4-fold, according to 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 for human SIRPA anti-SIRPA antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID No.: 2 and a light chain variable region containing the amino acid sequence of SEQ ID No.: 5. 31. The antibody according to any one of claims 1 to 30, wherein the antibody reduces cell surface levels of SIRPA v1 in vitro with a half-maximal effective concentration (EC50) of about 0.05 to 2 nM as determined by flow cytometry. 32. The antibody according to any one of claims 1 to 31, 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, from about 0.05 to 0. 10 nM for human SIRPA v2, and/or about 0.05 to 1 nM for cynomolgus SIRPA by flow cytometry. 33. The antibody according to any one of claims 1 to 32, wherein the antibody binds human SIRPA, human - 97 044628 SIRPA vl, human SIRPA v2, cynomolgus SIRPA, marmoset SIRPA and human SIRPe3. 34. The antibody according to any one of claims 1 to 33, wherein the antibody binds to the D3 domain of human SIRPA v1 SEQ ID No: 1. 35. The antibody according to any one of claims 1 to 34, where the antibody binds to amino acid residues R282, Q284 and G337 human SIRPA v1 SEQ ID No: 1. 36. The antibody according to any one of claims 1 to 35, 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, negatively regulates CD14 expression in macrophages, and/or any combination of them. 37. The antibody according to any one of claims 1 to 36, wherein the anti-SIRPA antibody (a) improves T cell proliferation, (b) improves T cell proliferation without blocking the interaction of SIRPy and CD47, and/or (c) stimulates ROS production in monocytes and/or increases IL-8 expression in monocytes. 38. The antibody according to any one of claims 1 to 37, wherein the anti-SIRPA antibody inhibits tumor growth in vivo, reduces the number of CD14+ myeloid cells in the peripheral blood and/or increases the number of CD14+ myeloid cells in the tumor. 39. The antibody of any one of claims 1 to 38, wherein the antibody binds human SIRPA but does not substantially block CD47 binding to SIRPA. 40. An anti-SIRPA antibody according to any one of claims 1 to 39, wherein the antibody recognizes first and second antigens, wherein the first antigen is SIRPA and the second antigen is selected from: (a) an antigen that promotes transport across the blood-brain barrier; (b) an antigen that promotes transport across the blood-brain barrier selected from transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2 ) and diphtheria toxin receptor; (c) a disease-causing agent selected from disease-causing peptides or disease-causing nucleic acid proteins, wherein the disease-causing nucleic acids are antisense GGCCCC (G2C4) RNA repeat expansions, the disease-causing proteins are selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques , amyloid precursor protein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, peroxide 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, medina, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, non-ATG repeat-associated (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptides , glycine-arginine repeat (GR) peptides, proline-alanine (PA) repeat peptides, ubiquitin and proline-arginine (PR) repeat peptides; and (d) ligands and/or proteins expressed on immune cells, where the ligands and/or proteins are selected from PD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4 , PD-L2, PD1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39 , CD73 and phosphatidylserine; and a protein, lipid, polysaccharide or glycolipid expressed on one or more tumor cells. 41. 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 according to one of claims 1 to 40. 42. The method according to claim 41, where the cancer is selected from sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, small cell lung cancer , melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer and fibrosarcoma, glioblastoma multiforme; renal clear cell carcinoma; adrenocortical carcinoma; urothelial carcinoma of the bladder, diffuse large cell lymphoma, lung adenocarcinoma; pancreatic adenocarcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, slow-growing B-cell lymphoma, aggressive B-cell lymphoma, T-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, myelodysplastic syndromes, myeloproliferative neoplasms, invasive breast carcinoma, squamous cell carcinoma of the cervix, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, squamous cell carcinoma of the neck, diffuse large B-cell lymphoma , esophageal carcinoma , squamous cell carcinoma of the head and neck, chromophobe kidney cancer, papillary kidney cancer, low-grade glioma, hepatocellular carcinoma, squamous cell carcinoma of the lung, mesothelioma, serous cystadenocarcinoma of the ovary, pancreatic adenocarcinoma, pheochromo- - 98 044628 cytomas and paragangliomas, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumors, thyroid carcinoma, thymoma, endometrial carcinoma of the uterine body, uterine carcinosarcoma and uveal melanoma. 43. The method according to claim 41 or 42, where the method further comprises administering a therapeutic agent that inhibits or causes agonism of PD1, PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, CTLA4, PD-L2 , B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39 or CD73. 44. The method of any one of claims 41 to 43, further comprising administering to the individual at least one antibody that specifically binds to an inhibitory immune checkpoint molecule and/or one or more standard or experimental therapies. 45. The method of claim 44, wherein the method includes administering at least one antibody that specifically binds to an inhibitory immune checkpoint molecule. 46. The method of claim 44 or 45, wherein the at least one antibody that specifically binds to an inhibitory immune checkpoint molecule is selected from anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, anti- PD-1 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies and anti-HVEM antibodies, anti-B and T lymphocyte attenuator antibodies (BTLA), anti-killer inhibitory receptor (KIR) antibodies, anti-GAL9 antibodies , anti-TIM-1 antibodies, anti-TIM3 antibodies, anti-TIM-4 antibodies, anti-A2AR antibodies, anti-CD39 antibodies, anti-CD73 antibodies, anti-LAG3 antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-CD30 antibody, anti-TNFa antibody, anti-CD33 antibody, anti-Siglec-5 antibody, anti-Siglec-7 antibody, anti-Siglec-9 antibody, anti-Siglec-11 antibody, antagonistic anti-TREM1 antibody, antagonistic anti-TREM2 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-CD2 antibodies, anti-CD5 antibodies and any combination thereof. 47. The method of claim 44, wherein one or more standard or experimental therapies is selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer (ACT) therapy, T-cell chimeric antigen receptor therapy transfer (CHAR-T), vaccine therapy and cytokine therapy. 48. The method according to any one of claims 41-47, further comprising administering to the individual at least one antibody that specifically binds to the inhibitory cytokine. 49. The method of claim 48, wherein the at least one antibody that specifically binds to the inhibitory cytokine is selected from an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof. 50. The method of any one of claims 41 to 49, further comprising administering to the individual at least one agonistic antibody that specifically binds to an immune checkpoint stimulatory protein. 51. The method of claim 50, wherein the at least one agonist antibody that specifically binds to a stimulatory immune checkpoint protein is selected from an anti-CD40 antibody agonist, an anti-OX40 antibody agonist, an anti-ICOS antibody agonist, an anti- -CD28 antibody, agonist anti-TREM1 antibody, agonist anti-TREM2 antibody, agonist anti-CD137/41BB antibody, agonist anti-CD27 antibody, agonist anti-glucocorticoid-induced TNFR-related protein GITR antibody, agonist anti-CD30 antibody, agonist anti- BTLA antibody, anti-HVEM antibody agonist, anti-CD2 antibody agonist, anti-CD5 antibody agonist, and any combination thereof. 52. The method according to any one of claims 41-51, further comprising administering to the individual at least one stimulatory cytokine. 53. The method of claim 52, wherein the at least one stimulatory cytokine is selected from IFN-a4, IFNβ, IL-Τβ, TNF-α, IL-6, IL-8, CRP, a member of the IL-20 family, LIF , IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1 -beta and any combination thereof. 54. An isolated nucleic acid containing a nucleotide sequence encoding an antibody according to any one of claims 1 to 40. 55. A vector containing the nucleic acid according to claim 54. 56. An isolated host cell containing the nucleic acid of claim 54 or the vector of claim 55. 57. An isolated host cell that expresses the antibody of any one of claims 1 to 40. 58. A method for producing an antibody that binds to human SIRPA, comprising culturing a cell according to claim 56 or 57. 59. The method of claim 58, further comprising restoring the antibody produced by the cell. 60. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 40 and a pharmaceutically acceptable carrier. -