Anti-SIRP alpha antibody formulations and uses thereof

Abstract

Anti-SIRPα antibody formulations, and the use of these formulations in the treatment of cancer.

Description

ANTI-SIRPa ANTIBODY FORMULATIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to United States Provisional Application No. 63 / 677,299, filed on July 30, 2024, the entire contents of which are incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates to anti-SIRPa antibodies, as well as use of these antibodies in the treatment of diseases.BACKGROUND OF THE INVENTION

[0003] Signal regulatory protein alpha (SIRPa) is membrane glycoprotein from the SIRP family. Members of the SIRP family share certain common structural motifs. These include a transmembrane segment and an N-terminal extracellular domain that contains three Ig-like loops connected by three pairs of disulfide bonds. The C-terminal intracellular domain, however, differs between SIRP family members. SIRPa has an extended intracellular domain containing four tyrosine residues that form two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), while SIRPpi contains a lysine residue in the transmembrane domain followed by a short intracellular tail lacking ITIMs serving as a receptor for DAP12. Eight SIRPa single nucleotide polymorphisms have been identified, with the most prevalent being SIRPaVl and SIRPaV2 (Takenaka et al., Nat. Immunol. 2007, 8: 1313-23).

[0004] “Eat-me” signals (i.e. “altered self’) are extracellular players specifically produced by and displayed on the surface of apoptotic cells, but not healthy cells, and are key to the initiation of phagocytosis by activating phagocytic receptors and subsequent signaling cascades. Eat-me signals require extracellular trafficking in order to be displayed on apoptotic cells. A particular category of eat-me signals is provided by membrane-anchored proteins such as phosphatidylserine (PtdSer) and calreticulin (CRT). Externalized PtdSer binds to its receptors on phagocytes to facilitate clearance of apoptotic cells (a process known as efferocytosis). Likewise, CRT is upregulated on the surface of apoptotic cells and binds to LDL-receptor-related protein 1 (LRP1) on the phagocyte thereby mediating engulfment.

[0005] SIRPa is broadly expressed on phagocytes (e.g., macrophages, granulocytes, and dendritic cells) and acts as an inhibitory receptor through its interaction with a transmembrane protein CD47. This interaction mediates a response referred to as the "don’t eat me" signal. This interaction negatively regulates effector function of innate immune cells such as host cell phagocytosis. As CD47 is often present on tumor cells, this “don’t eat me” signal is thought to contribute to the resistance of tumors to phagocyte-dependent clearance. Despite the similarities in the extracellular domains of SIRPa and SIRPpi functional differences exist among the SIRP family members. For example, SIRPpi does not bind CD47 at detectable levels and so does not mediate the "don’t eat me" signal. Instead, SIRPpi is involved in the activation of myeloid cells.

[0006] Disruption of CD47-SIRPa signalling (e.g., by antagonistic monoclonal antibodies that bind to either CD47 or SIRPa) reportedly results in enhanced phagocytosis of both solid and hematopoietic tumor cells, including increased phagocytosis of glioblastoma cells in vitro and significant anti-tumor activity in vivo.

[0007] PCT / NL2018 / 050234, published as WO2018190719, which is hereby incorporated by reference in its entirety, describes certain SIRPa antibodies referred to therein as hSIRPa.40A and hSIRPa.50A, including humanized forms thereof. In particular, an antibody known as “ADU-1805” is a humanized IgG2 variant of hSIRPa.40A. The functional characteristics of the ADU-1805 antibody are described further in Voets et al., J. Immunother. Cancer: 7: 340 (doi: 10.1186 / s40425-019-0772-0), which is also incorporated by reference in its entirety.SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide formulations of an anti-SIRPa antibody, and in certain embodiments of ADU-1805, for use in therapeutic applications. It is a further object of the invention to use such formulations for administration to individuals in need thereof for the treatment of cancer.

[0009] In a first aspect, the invention relates to an antibody formulation suitable for pharmaceutical infusion or subcutaneous injection, comprising: an anti-SIRPa antibody that inhibits signaling through the SIRPa-CD47 axis at a concentration of between about 20 mg / mL to about 100 mg / mL; a buffering component selected from the group consisting of about 20 mM L- histidine, and 20 mM sodium phosphate;a disaccharide comprising an a-glycosidic linkage at a concentration of about 8% w / v; about 0.01 wt % polysorbate 20; and a pH of about 5.7 to about 6.3; wherein the antibody formulation is free of glycine, arginine, carbonate, HEPES, citrate, and acetate.

[0010] In various embodiments, the antibody formulation comprises the anti-SIRPa antibody at a concentration of about 20 mg / mL.

[0011] Preferably, the anti-SIRPa antibody binds to a cell expressing human SIRPaVl protein with an EC 50 < 10 nM; binds to a cell expressing human SIRPaV2 protein with an EC50 < 10 nM; exhibits at least a 100-fold higher EC50 for SIRPaVl (P74A) having the sequence of SEQ ID NO: 62 as compared to the EC50 for human SIRPaVl protein; and exhibits at least a 100-fold higher EC50 for human SIRPpi protein as compared to the EC50 for human SIRPaVl protein.

[0012] In various embodiments, the anti-SIRPa antibody comprises one of the following combinations of heavy chain sequence and light chain sequence:SEQ ID NO: 80 and SEQ ID NO: 90,SEQ ID NO: 80 and SEQ ID NO: 92,SEQ ID NO: 80 and SEQ ID NO: 94,SEQ ID NO: 80 and SEQ ID NO: 96,SEQ ID NO: 80 and SEQ ID NO: 98, orSEQ ID NO: 80 and SEQ ID NO: 100.

[0013] In certain embodiments, the formulation consists of 20 mg / mL anti-SIRPa antibody, 20 mM histidine, 8% w / v sucrose, 0.01% wt % polysorbate 20 at a pH of about 6.0. Most preferably, the anti-SIRPa antibody is ADU-1805.

[0014] In another aspect, the present invention relates to single-use or multi-use vials comprising an anti-SIRPa antibody formulation as described herein; or pre-filled syringes, autoinjectors, or injector pens comprising the anti-SIRPa antibody formulation as described herein.

[0015] In yet another aspect, the present invention relates to methods of administering an anti-SIRPa antibody to an individual in need thereof comprising administering an anti- SIRPa antibody formulation as described herein.

[0016] In certain embodiments, the method comprises repeating the administration over at least 2 administration cycles. Such repeated administrations may be, for example, each week, every other week, every three weeks, every month, every other month, quarterly, etc. In preferred embodiments, adminstration of the anti-SIRPa antibody formulation is on at least an every three week (Q3W) schedule for at least 2 administration cycles, and most preferably administration is Q3W.

[0017] Administration of the anti-SIRPa antibody formulation may be by any parenteral route. In certain embodiments, the administration route is intravenous administration. In certain embodiments the administration route is subcutaneous administration.

[0018] In preferred embodiments, the dose of anti-SIRPa antibody being adminstered in a single administration is between 1 mg / kg body weight and 30 mg / kg body weight. By way of example, the administration may be 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, or 30 mg / kg of anti-SIRPa antibody. In certain embodiments, the anti-SIRPa antibody formulation may be diluted in normal saline or 5% dextrose in water prior to intravenous administration of the desired amount of anti-SIRPa antibody. For example, the anti-SIRPa antibody concentration of the undiluted anti-SIRPa antibody formulation is between about 20 mg / mL to about 100 mg / mL, and the formulation is diluted to between 1 mg / mL and 10 mg / mL prior to administration.

[0019] In various embodiments, the individual in need thereof has a histologically and / or cytologically confirmed diagnosis of cancer. By way of example, the individual may have a confirmed diagnosis of a solid tumor. Examples include adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, ductal carcinoma, angiosarcoma, osteosarcoma, fibroblastic sarcoma, rhabdomyosarcoma, blastoma, melanoma, germ cell carcinoma, etc. The solid tumor may be a tumor of thelung, bladder, colon, pancreas, thyroid, ovary, testicle, or endometrium. These lists are not meant to be limiting. In certain embodiments, the cancer is metastatic or unresectable.

[0020] In certain embodiments, the anti-SIRPa antibody formulation of the invention is administered as a combination therapy with a checkpoint inhibitor. Preferably, the checkpoint inhibitor is a PD-1 or PD-L1 checkpoint inhibitor such as pembrolizumab. By way of example only, a 200 mg dose of pembrolizumab may be administered before, after, or simultaneously with administration of the anti-SIRPa antibody formulation. In most preferred embodiments, the anti-SIRPa antibody formulation and the pembrolizumab are each administered to the individual by intravenous administration within a 12 hour period to provide the combination therapy.BRIEF DESCRIPTION OF THE FIGURES

[0021] Fig. 1 shows a clinical study design for use with the formulations described herein.

[0022] Fig. 2 shows a schedule of events for use in the clinical study design.

[0023] Fig. 3 shows a dose escalation plan for use in the clinical study design.DETAILED DESCRIPTION

[0024] In healthy individuals, active immune surveillance is preventing aberrant tumor cells to develop into cancer. Active interplay between adaptive and innate immunity results in, among others, a cytotoxic CD8 T cell response that destroys tumor cells before they can develop into a cancer. However, tumor (cells) evolve multiple mechanisms to bypass immune surveillance leading to inadequate T cell activation. Over the last decade checkpoint inhibitors (CPIs) were developed that reinvigorates anti-cancer T cell response. Despite the fact that CPIs have transformed cancer treatment, a majority of about 70% of patients with solid tumors do not respond to CPIs. Subsequent attention has focused on other mediators in the human immune system to enhance a more broad anti-tumor immune response.

[0025] One critical immune checkpoint on innate immune cells, like macrophages and dendritic cells, is the “do not eat-me” CD47-SIRPa pathway, which is co-opted by cancer cells to escape from uptake by myeloid cells and subsequent antigen-presentation to (cytotoxic) T cells. Signal-regulatory protein a (SIRPa) is expressed on dendritic cells, macrophages, monocytes and neutrophils [1] and has several ligands including the surfactant proteins (e.g., Sp-A and Sp-D) [2], and CD47 [3], CD47 serves as a “self-molecule” signal with its best-characterized functions in the homeostasis of complementer Ig-opsonized red blood cells (RBCs) and platelets. Binding of CD47 to SIRPa inhibits phagocytosis of these cells by macrophages thereby preventing their homeostatic clearance, while downregulation of CD47 on ‘old’ RBCs or platelets is making them prone to phagocytosis and clearance from circulation [4, 5], The overexpression of CD47 on numerous human cancers [6-11] indicates that tumor cells employ the CD47-SIRPa pathway to evade phagocytosis and clearance. Targeting of the SIRPa / CD47 axis using an anti-CD47 blocking antibody enhances phagocytosis of acute myeloid leukemia (AML) cells [6], In addition, inhibiting the SIRPa / CD47 axis enhances tumor growth inhibition in combination with tumor-targeting monoclonal antibody (mAb) therapies (e.g. rituximab, trastuzumab, alemtuzumab, daratumumab and cetuximab) [8, 12-14] and synergizes with other treatments including chemotherapy

[0015] , radiotherapy

[0016] , targeted therapy using small-molecule drugs

[0017] as well as immunotherapeutic agents blocking the PD-1 / PD-L1 axis [18, 19],

[0026] Several agents blocking the SIRPa-CD47 pathway have been developed thus far including anti-CD47 and anti-SIRPa antibodies, and soluble SIRPa-Fc protein, of which several are currently being evaluated in clinical trials. Of these, Hu5F9-G4, TTL 621 and ALX148 are furthest in development and have shown encouraging clinical data either alone or in combination with other agents [14, 20, 21,35], Nevertheless, the systemic use of CD47-targeting agents is hampered by the broad expression of CD47, which manifests itself by severe depletion of RBCs and platelets, leading to acute anemia and thrombocytopenia in treated patients [20, 22] as well as requiring substantial amounts of agent to block CD47 on all immune cells (i.e. the “antigen sink”). Furthermore, CD47 is also a receptor for thrombospondin- 1 (TSP1)

[0023] and blocking this interaction using anti-CD47 mAbs may have additional undesirable effects

[0024] , It is therefore anticipated that targeting of the SIRPa / CD47 axis with an anti-SIRPa blocking mAb

[0025] will display a more favorable safety profile due to the more restricted expression of SIRPa.

[0027] SIRPa, SIRPP and SIRPy belong to a family of paired receptors comprising separate genes that encode proteins with similar extracellular domains but different transmembrane or cytoplasmic regions. SIRPa and SIRPP are expressed in myeloid lineage cells, while SIRPy is expressed on T-cells, NK cells and NKT cells. SIRPa binds to CD47 thereby transmitting an inhibitory signal, while the ligand for SIRPP has been unidentified and has been reported to transduce an activating signal into the same cells[36, 37], In contrast, SIRPy binds CD47 albeit with a 10-fold weaker affinity than SIRPa

[0027] , and based on the lack of signal transduction domains intracellularly, is considered to function as a decoy receptor. Targeting of SIRPy has been suggested to inhibit T cell activation and recruitment

[0028]

[0028] The membrane distal extracellular Ig-like V-type (IgV) domain of SIRPa is highly polymorphic and thus far 10 human SIRPa alleles have been described

[0026] , which has presented researchers with a challenge to develop anti-SIRPa antibodies that can be used across human alleles and population. In fact, OSE-172 / BI 765063 was shown to target only the SIRPa VI allele

[0028] that is most frequently occurring in Europe, while less frequently occurring in other geographies.

[0029] ADU-1805

[0030] ADU-1805 is a humanized IgG2 monoclonal antibody entering clinical development for the potential use as a monotherapy or in combination with other antitumor therapies. ADU-1805 is a selective pan-allele anti-SIRPa antibody that blocks the SIRPa-CD47 innate immune checkpoint.

[0031] Summary of Nonclinical Studies with ADU-1805

[0032] ADU-1805 was identified to block the SIRPa-CD47 pathway via its binding toSIRPa, where ADU-1805 is characterized by its binding to all known human alleles of SIRPa. ADU-1805 blocks the interaction of SIRPa with CD47, does not bind the SIRPpi activating receptor and by reduced binding to SIRPy does not affect T cell activation. The binding of ADU-1805 to SIRPa enhances tumor cell uptake by macrophages and neutrophils. By targeting SIRPa and not the more broadly expressed CD47 protein, ADU-1805 does not demonstrate binding to red blood cells and platelets, not does it induce hemagglutination of RBCs or aggregation / activation of platelets. In contrast to CD47 targeting, ADU-1805 does not affect CD4 T-cell activation

[0027] , ADU-1805 does not elicit effector function in antibody-dependent cell cytotoxicity or complementdependent cell cytotoxicity assays, nor does ADU-1805 induce cytokine release when incubated in human PBMCs. Human tissue cross-reactivity using tissue panels showed staining consistent with SIRPa expression on immune cells and brain tissue.

[0033] A series of nonclinical studies in non-human primates (cynomolgus) evaluated the pharmacology, PK, and toxicology of ADU-1805. The cynomolgus monkey was selected as the relevant species for Good Laboratory Practice (GLP) evaluation of ADU-1805 based on the cross-reactivity of ADU-1805 to cynomolgus monkey SIRPa and a lack of binding to mouse and rat SIRPa. The affinity of ADU-1805 binding to cynomolgus monkey SIRPa is approximately similar to human SIRPa (~2 fold more potent affinity for cynomolgus monkey vs. human SIRPa).

[0034] In a GLP study, ADU-1805 was evaluated in Cynomolgus monkeys of both sexes at doses ranging from 3 to 30 mg / kg. ADU-1805 was administered once weekly by the intravenous route (30-minute infusion) in the cynomolgus monkey for 5 consecutive weeks. Parameters evaluated included daily mortality / morbidity checks, clinical signs including general and local observations, full clinical examination, body weight, qualitative food consumption, ophthalmological examination, urinalysis, measurements of cardiovascular and respiratory parameters. Blood samples were collected to evaluate hematology, coagulation and clinical chemistry. A toxicokinetic and anti-drug antibody (ADA) analysis was also performed. In addition, cytokine releases, receptor occupancy and effect of ADU-1805 on the immune cells was evaluated. At necropsy, organs weight, macroscopic and microscopic lesions were analyzed by a pathologist.

[0035] No mortality related to the test item occurred in animals dosed with ADU- 1805. There were no test item-related clinical signs, nor changes in body weight, food consumption, ophthalmological examination and in cardiovascular and respiratory parameters during the treatment or the recovery period. Compared to predose values and / or with the vehicle control group, there were no test item-related changes in hematology, coagulation, blood chemistry parameters and in semi -quantitative urinary parameters during the treatment period or at the end of the recovery period. No test-item related relevant changes in cytokines levels were identified for IL-1 / ?, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-cr, IFN-y or CCL2. There were no test item-related changes in organ weights, macroscopic findings, or microscopic changes at the end of the treatment period or at the end of the recovery period.

[0036] The results of receptor occupancy (RO) showed the sensitivity of animals to the test item as RO increased in all treated groups compared to the vehicle control group. Full saturation was maintained on monocytes at 30 mg / kg over the course of dosing, while receptor saturation decreased on Day 11 and Day 18 respectively in animals treated with 3 or 10 mg / kg. Similar profiles were observed in lymphocytes. The saturation was observed until Day 71 in both monocytes and lymphocytes. The data generated from immunophenotyping of leukocyte subsets from each group showed a decrease ofleukocytes cells observed after vehicle or test item injection, occurring 2 hours after injection. In most animals, the decrease in almost all leukocytes’ subsets after each injection was rapidly followed by an expansion of classical monocytes, that persisted till the next injection. No major change in leukocytes distribution could obviously be identified between the groups. No CD80 expression was detected among monocytes subsets, whatever the group tested. However, a trend of increase of CD86 expression was observed in animals treated with the test item compared to vehicle group on Day 8 up to Day 11. This trend was observed in all monocytes sub-populations. However, no dose response was observed as all treated groups behaved similarly.

[0037] The systemic exposure to ADU-1805 increased dose-proportionally in males and females on Days 1 and 8 as the dosage increased from 3 to 30 mg / kg. At the later days, the dose-proportionality was influenced by decreased exposures in individual animals because of the presence of ADA. No marked sex differences in systemic exposure were observed at any dose level.

[0038] Plasma elimination half-life of ADU-1805 was very long especially at the highest dose of 30 mg / kg. It was estimated during the last dosing session (Day 29) for two males up to 95.5 hours and 204 hours for one female with a long mean retention time (MRT) of 259.8 hours and 361.9 hours, respectively.

[0039] Analysis for the presence of ADA was performed before each dosing on Day 8, Day 15, Day 22 and Day 29 and weekly during the recovery period. ADA formation was reliably detected in animals in all groups but predominantly in the group treated with 3 mg / kg. The detection of ADA corelated with a reduced exposure to ADU-1805. Sporadic samples were screened to be positive for ADA with no impact on ADU-1805 exposure.

[0040] In conclusion, under the experimental conditions of the study, ADU-1805 administered once a week at 3, 10 and 30 mg / kg by a 30-minute intravenous infusion induced no adverse effects in male or female cynomolgus monkeys. Based on these results, the No Observed Adverse Effect Level (NOAEL) was considered to be 30 mg / kg in both genders.

[0041] Overall, nonclinical studies support use of ADU-1805 in humans at the proposed dose levels. A complete summary of nonclinical information on ADU-1805 is provided in the IB.

[0042] Rationale for the use of ADU-1805 as Cancer Immunotherapy

[0043] Therapeutic blockade of the “do not eat-me” CD47-SIRPa pathway has reported encouraging clinical signal in both hematological and solid tumors. Based on the overexpression of CD47 on numerous human cancers [6-11] these agents aim to inhibit this “do not eat-me” pathway and enhance uptake of tumor cells, as a single agent (AML) [6] or in combination with tumor-targeting monoclonal antibody (mAb) therapies (e.g. rituximab, trastuzumab and cetuximab) [8, 12-14] or with other treatments including chemotherapy [15, 35], Enhanced tumor cell uptake has been shown to also result in enhanced antigen presentation and subsequent priming of T-cells

[0039] , thereby potentially explaining the observed synergy with immunotherapeutic agents blocking the PD-l / PD- L1 axis [18, 19, 38],

[0044] ADU-1805 blocks the SIRPa-CD47 pathway via its binding to SIRPa, whereby ADU-1805 is binding to all known human alleles of SIRPa. ADU-1805 does not bind the SIRPpi activating receptor that has been implicated to enhance phagocytosis by myeloid cells

[0037] , In addition, ADU-1805 displays reduced binding to SIRPy and does not affect T cell activation

[0027] , The binding of ADU-1805 to SIRPa enhances tumor cell uptake by macrophages and neutrophils. By targeting SIRPa and not the more broadly expressed CD47 protein, ADU-1805 does not demonstrate binding to red blood cells and platelets, nor does it induce hemagglutination of RBCs or aggregation / activation of platelets.Abbreviations

[0045] Throughout the detailed description and examples of the invention the following abbreviations will be used:ADCC Antibody-dependent cellular cytotoxicityADCP Antibody-dependent cellular phagocytosisCDC Complement-dependent cytotoxicityCDR Complementarity determining region in the immunoglobulin variable regions, defined using the Kabat numbering systemCHO Chinese hamster ovaryEC50 Concentration at which 50% of the total binding signal is observedELISA Enzyme-linked immunosorbant assayFR Antibody framework region: the immunoglobulin variable regions excluding the CDR regions.HRP Horseradish peroxidaseIFN interferonIC50 concentration resulting in 50% inhibitionIgG Immunoglobulin GKabat An immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) mAb or Mab or MAb Monoclonal antibodySEB Staphylococcus Enterotoxin BTT Tetanus toxoidV region The segment of Ig chains which is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.VH Immunoglobulin heavy chain variable regionVK Immunoglobulin kappa light chain variable regionVL Immunoglobulin light chain variable regionDefinitions

[0046] So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise.

[0048] Administration" and "treatment," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.

[0049] Treat" or "treating" means to administer a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of the present invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.

[0050] “Recombinant expression” of a protein means the transcription and translation of an exogenous gene in a host organism to generate the protein, which is referred to herein as a “recombinant protein.”

[0051] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. The term antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." Preferred therapeutic antibodies are intact IgG antibodies. The term “intact IgG” as used herein is meant as a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises IgGl, IgG2, IgG3, and IgG4. In mice this class comprises IgGl, IgG2a, IgG2b, IgG3. The known Ig domains in the IgG class of antibodies are VH, Cyl, Cy2, Cy3, VL, and CL.

[0052] As used herein, a “full length antibody” is, in the case of an IgG, a bivalent molecule comprising two heavy chains and two light chains. Each heavy chain comprises a VH domain followed by a constant domain (CHI), a hinge region, and two more constant (CH2 and Cm) domains; while each light chain comprises one VL domain and one constant (CL) domain. A full length antibody in the case of an IgM is a decavalent or dodecavalent molecule comprising 5 or 6 linked immunoglobulins in which immunoglobulin each monomer has two antigen binding sites formed of a heavy and light chain.

[0053] As used herein, unless otherwise indicated, "antibody fragment" or "antigenbinding fragment" refers to antigen -binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antigenbinding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

[0054] A "Fab fragment" is comprised of one light chain and the CHI and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0055] The present invention includes anti-SIRPa antibodies which comprise an Fc region and methods of use thereof. An "Fc" region contains two heavy chain fragments comprising the CH3 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0056] A "Fab1fragment" contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the C H1 domain and also the region between the CHI and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.

[0057] A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab') 2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bondbetween the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody.

[0058] The term "single-chain Fv" or "scFv" antibody refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen-binding. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946, 778 and 5,260,203.

[0059] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0060] The present invention includes anti-SIRPa bivalent antibodies and methods of use thereof. A "bivalent antibody" comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).

[0061] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. Duobodies are described in Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23 : 1126-1136.

[0062] Typically, an antibody or antigen-binding fragment of the invention which is modified in some way retains at least 10% of its binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%,80%, 90%, 95% or 100% or more of the SIRPa binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function conserved variants" of the antibody) that do not substantially alter its biologic activity.

[0063] As used herein, a "chimeric antibody" is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Set. USA 81 : 6851-6855). Typically, the variable domains are obtained from an antibody from an experimental animal (the "parental antibody"), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., mouse) antibody.

[0064] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure). As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0065] Isolated nucleic acid molecule" or “isolated polynucleotide” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that "a nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules "comprising" specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or mayinclude operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and / or may include vector sequences.

[0066] The phrase "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0067] A nucleic acid or polynucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, but not always, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0068] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0069] As used herein, "germline sequence" refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health.Mouse germline sequences may be obtained, for example, as described in Giudicelli et al.(2005) Nucleic Acids Res. 33: D256-D261.SIRPa and associated proteins

[0070] The present invention provides formulations of antibodies or antigen-binding fragments thereof that bind human SIRPa and uses of such antibodies for the treatment of cancer.

[0071] SIRPa belongs to a class of membrane proteins known as “paired receptors” that contain several genes coding for proteins (e.g., SIRPa, SIRPpi, and SIRPy) with similar extracellular regions but different transmembrane and / or cytoplasmic regions having opposite (activating or inhibitory) signaling abilities. Like SIRPa, there are several examples of paired receptors on NK cells and some on myeloid cells, including the SIRP and CD200 receptor families (Hatherley et al., Mol Cell. 2008; 31: 266-277).

[0072] SIRPa contains an extracellular region that can be subdivided into three separate domains: the Ig-like (immunoglobulin-like) V-type (IgV), Ig-like Cl -type (IgCl), and Ig-like C2-type (IgC2) domain. The IgV domain is also known as the ligandbinding N-terminal domain of SIRPa. Like SIRPa, also the related proteins SIRPpi and SIRPy comprise an extracellular region that can be subdivided into an IgV, IgCl, and IgC2 domain. However, SIRPa, SIRPpi and SIRPy have different cytoplasmic regions. SIRPpi has a very short cytoplasmic region of only 6 amino acids and lacks signalling motifs for association with phosphatases. Instead, this protein associates with DNAX activation protein 12 (DAP 12), a dimeric adaptor protein that binds an amino acid with a basic side chain in the transmembrane region of SIRPpi and is able to transmit activating signals through its immunoreceptor tyrosine-based activation motif (IT AM). SIRPy also has a short cytoplasmic region of 4 amino acids, but it lacks a charged amino-acid side chain in the transmembrane region and therefore does not associate with DAP12. Hence, SIRPy is annotated as a non-signalling protein (Barclay, A.N. and Brown, M.H., Nat Rev Immunol. 2006; 6: 457-464).

[0073] The major ligand of SIRPa is CD47, which consists of one extracellular IgV domain, a five times transmembrane-spanning domain, and a short cytoplasmic tail. CD47 functions as a cellular ligand with binding mediated through the NH2 -terminal IgV domain of SIRPa. Evidence that CD47 contributes to recognition of self comes from the observation that splenic macrophages derived from CD47-expressing mice clear infused blood cells from CD47 / _mice (Oldenborg et al., Science. 2000; 288: 2051-2054).

[0074] In addition to CD47, two other SIRPa ligands have been reported, known as surfactant proteins A and D (Sp-A and Sp-D), both of which belong to the collectin family. Sp-D has been reported to bind to the membrane-proximal IgC2 domain of SIRPa in a calcium- and saccharide-dependent manner. It is thought that Sp-A and Sp-D help maintain an anti-inflammatory environment in the lung by stimulating SIRPa on alveolar macrophages (Gardai etal., Cell. 2003; 115: 13-23).

[0075] The amino acid sequence of eight human SIRPa variants are listed in SEQ ID NOs: 34, 36, 44, 46, 48, 50, 52, and 54; exemplary nucleic acid sequences encoding these variants are listed in SEQ ID NOs: 33, 35, 43, 45, 47, 49, 51, and 53, respectively.

[0076] For comparison, the amino acid sequence of human SIRPpi and SIRPy are listed in SEQ ID NOs: 38 and 40, respectively, and exemplary nucleic acid sequences in SEQ ID NOs: 37 and 39, respectively.

[0077] The amino acid sequence of human CD47 is listed in SEQ ID NO: 42, and an exemplary nucleic acid sequence in SEQ ID NO: 41.

[0078] Modified SIRPa polypeptides hSIRPa-VpC 1 aC2a, hSIRPa-VaC 1 pC2a, hSIRPa-VaClaC2p, and hSIRPaVl(P74A) discussed hereinafter are listed in SEQ IDNOs: 56, 58, 60, and 62; exemplary nucleic acid sequences encoding these variants are listed in SEQ ID NOs: 55, 57, 59, and 61, respectively.

[0079] Whether an antibody specifically binds to a polypeptide sequence (e.g., human SIRPa, hSIRPpi, etc.) can be determined using any assay known in the art. Examples of assays known in the art to determining binding affinity include surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

[0080] The present invention includes formulations of humanized anti-SIRPa antibodies (e.g., rat or mouse antibodies that have been humanized) and methods of use thereof. As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from both human and non-human (e.g., mouse or rat) antibodies. In general, the humanized antibody will comprise substantially of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc). For more details about humanized antibodies, see, e.g., Jones et al., Nature,321 :522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op.Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21 : 397-402 (2000).

[0081] In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

[0082] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.

[0083] Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al , National Institutes of Health, Bethesda, MD; 5thed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) JAfo / . Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0084] Antibodies and fragments that bind to the same epitope as any of the anti- SIRPa antibodies or antigen-binding fragments thereof of the present invention also form part of the present invention. In one embodiment, the invention provides an antibody or antigen binding fragment thereof that binds to the same epitope of human SIRPa as anantibody comprising one of the following combinations of heavy chain sequence / light chain sequence (or in each case an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto):SEQ ID NO: 10 / SEQ ID NO: 20 (referred to herein as hSIRPa.50A.HlLl)SEQ ID NO: 10 / SEQ ID NO: 22 (referred to herein as hSIRPa.50A.HlL2)SEQ ID NO: 10 / SEQ ID NO: 24 (referred to herein as hSIRPa.50A.HlL3)SEQ ID NO: 10 / SEQ ID NO: 26 (referred to herein as hSIRPa.50A.HlL4)SEQ ID NO: 10 / SEQ ID NO: 28 (referred to herein as hSIRPa.50A.HlL5)SEQ ID NO: 12 / SEQ ID NO: 20 (referred to herein as hSIRPa.50A.H2Ll)SEQ ID NO: 12 / SEQ ID NO: 22 (referred to herein as hSIRPa.50A.H2L2)SEQ ID NO: 12 / SEQ ID NO: 24 (referred to herein as hSIRPa.50A.H2L3)SEQ ID NO: 12 / SEQ ID NO: 26 (referred to herein as hSIRPa.50A.H2L4)SEQ ID NO: 12 / SEQ ID NO: 28 (referred to herein as hSIRPa.50A.H2L5)SEQ ID NO: 14 / SEQ ID NO: 20 (referred to herein as hSIRPa.50A.H3Ll)SEQ ID NO: 14 / SEQ ID NO: 22 (referred to herein as hSIRPa.50A.H3L2)SEQ ID NO: 14 / SEQ ID NO: 24 (referred to herein as hSIRPa.50A.H3L3)SEQ ID NO: 14 / SEQ ID NO: 26 (referred to herein as hSIRPa.50A.H3L4)SEQ ID NO: 14 / SEQ ID NO: 28 (referred to herein as hSIRPa.50A.H3L5)SEQ ID NO: 16 / SEQ ID NO: 20 (referred to herein as hSIRPa.50A.H4Ll)SEQ ID NO: 16 / SEQ ID NO: 22 (referred to herein as hSIRPa.50A.H4L2)SEQ ID NO: 16 / SEQ ID NO: 24 (referred to herein as hSIRPa.50A.H4L3)SEQ ID NO: 16 / SEQ ID NO: 26 (referred to herein as hSIRPa.50A.H4L4)SEQ ID NO: 16 / SEQ ID NO: 28 (referred to herein as hSIRPa.50A.H4L5)SEQ ID NO: 18 / SEQ ID NO: 20 (referred to herein as hSIRPa.50A.H5Ll)SEQ ID NO: 18 / SEQ ID NO: 22 (referred to herein as hSIRPa.50A.H5L2)SEQ ID NO: 18 / SEQ ID NO: 24 (referred to herein as hSIRPa.50A.H5L3)SEQ ID NO: 18 / SEQ ID NO: 26 (referred to herein as hSIRPa.50A.H5L4)SEQ ID NO: 18 / SEQ ID NO: 28 (referred to herein as hSIRPa.50A.H5L5)SEQ ID NO: 78 / SEQ ID NO: 90 (referred to herein as hSIRPa.40A.HlLl)SEQ ID NO: 78 / SEQ ID NO: 92 (referred to herein as hSIRPa.40A.HlL2)SEQ ID NO: 78 / SEQ ID NO: 94 (referred to herein as hSIRPa.40A.HlL3)SEQ ID NO: 78 / SEQ ID NO: 96 (referred to herein as hSIRPa.40A.HlL4)SEQ ID NO: 78 / SEQ ID NO: 98 (referred to herein as hSIRPa.40A.HlL5)SEQ ID NO: 78 / SEQ ID NO: 100 (referred to herein as hSIRPa.40A.HlL6)SEQ ID NO: 80 / SEQ ID NO: 90 (referred to herein as hSIRPa.40A.H2Ll)SEQ ID NO: 80 / SEQ ID NO: 92 (referred to herein as hSIRPa.40A.H2L2)SEQ ID NO: 80 / SEQ ID NO: 94 (referred to herein as hSIRPa.40A.H2L3)SEQ ID NO: 80 / SEQ ID NO: 96 (referred to herein as hSIRPa.40A.H2L4)SEQ ID NO: 80 / SEQ ID NO: 98 (referred to herein as hSIRPa.40A.H2L5)SEQ ID NO: 80 / SEQ ID NO: 100 (referred to herein as hSIRPa.40A.H2L6)SEQ ID NO: 82 / SEQ ID NO: 90 (referred to herein as hSIRPa.40A.H3Ll)SEQ ID NO: 82 / SEQ ID NO: 92 (referred to herein as hSIRPa.40A.H3L2)SEQ ID NO: 82 / SEQ ID NO: 94 (referred to herein as hSIRPa.40A.H3L3)SEQ ID NO: 82 / SEQ ID NO: 96 (referred to herein as hSIRPa.40A.H3L4)SEQ ID NO: 82 / SEQ ID NO: 98 (referred to herein as hSIRPa.40A.H3L5)SEQ ID NO: 82 / SEQ ID NO: 100 (referred to herein as hSIRPa.40A.H3L6)SEQ ID NO: 84 / SEQ ID NO: 90 (referred to herein as hSIRPa.40A.H4Ll)SEQ ID NO: 84 / SEQ ID NO: 92 (referred to herein as hSIRPa.40A.H4L2)SEQ ID NO: 84 / SEQ ID NO: 94 (referred to herein as hSIRPa.40A.H4L3)SEQ ID NO: 84 / SEQ ID NO: 96 (referred to herein as hSIRPa.40A.H4L4)SEQ ID NO: 84 / SEQ ID NO: 98 (referred to herein as hSIRPa.40A.H4L5)SEQ ID NO: 84 / SEQ ID NO: 100 (referred to herein as hSIRPa.40A.H4L6)SEQ ID NO: 86 / SEQ ID NO: 90 (referred to herein as hSIRPa.40A.H5Ll)SEQ ID NO: 86 / SEQ ID NO: 92 (referred to herein as hSIRPa.40A.H5L2)SEQ ID NO: 86 / SEQ ID NO: 94 (referred to herein as hSIRPa.40A.H5L3)SEQ ID NO: 86 / SEQ ID NO: 96 (referred to herein as hSIRPa.40A.H5L4)SEQ ID NO: 86 / SEQ ID NO: 98 (referred to herein as hSIRPa.40A.H5L5)SEQ ID NO: 86 / SEQ ID NO: 100 (referred to herein as hSIRPa.40A.H5L6)SEQ ID NO: 88 / SEQ ID NO: 90 (referred to herein as hSIRPa.40A.H6Ll)SEQ ID NO: 88 / SEQ ID NO: 92 (referred to herein as hSIRPa.40A.H6L2)SEQ ID NO: 88 / SEQ ID NO: 94 (referred to herein as hSIRPa.40A.H6L3)SEQ ID NO: 88 / SEQ ID NO: 96 (referred to herein as hSIRPa.40A.H6L4)SEQ ID NO: 88 / SEQ ID NO: 98 (referred to herein as hSIRPa.40A.H6L5)SEQ ID NO: 88 / SEQ ID NO: 100 (referred to herein as hSIRPa.40A.H6L6).

[0085] There are several methods available for mapping antibody epitopes on target antigens, including: H / D-Ex mass spectrometry, crosslinking coupled mass spectrometry, X-ray crystallography, pepscan analysis and site directed mutagenesis. For example, HDX (Hydrogen Deuterium Exchange) coupled with proteolysis and mass spectrometry can be used to determine the epitope of an antibody on a specific antigen Y. HDX-MS relies on the accurate measurement and comparison of the degree of deuterium incorporation by an antigen when incubated in D2O on its own and in presence of its antibody at various time intervals. Deuterium is exchanged with hydrogen on the amide backbone of the proteins in exposed areas whereas regions of the antigen bound to the antibody will be protected and will show less or no exchange after analysis by LC- MS / MS of proteolytic fragments., Crosslinking coupled mass spectrometry begins by binding the antibody and the antigen with a mass labeled chemical crosslinker. Next the presence of the complex is confirmed using high mass MALDI detection. Because after crosslinking chemistry the Ab / Ag complex is extremely stable, many various enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high resolution mass spectrometry and MS / MS techniques. Identification of the crosslinked peptides is determined using mass tag linked to the cross-linking reagents. After MS / MS fragmentation and data analysis, both epitope and paratope are determined in the same experiment.

[0086] The scope of the present invention also includes formulations of anti-SIRPa antibodies (e.g., humanized antibodies), comprising a variant of an immunoglobulin chain set forth herein, wherein the variant exhibits one or more of the following properties: binds human SIRPaVl protein having the sequence of SEQ ID NO: 34 with an ECso < 1 nM; and exhibits at least a 100-fold higher ECso for SIRPaVl (P74 A) having the sequence of SEQ ID NO: 62; and optionally also at least a 100-fold higher ECso for human SIRPpi protein having the sequence of SEQ ID NO: 38 (in each case wherein the reduced ECso is relative to the ECso for human SIRPaVl protein having the sequence of SEQ ID NO: 34, and in each case preferably when measured by cellular ELISA (CELISA) as described hereinafter; binds to a cell expressing human SIRPaVl protein with an ECso < 10 nM, preferably < 5 nM, more preferably < 1.5 nM, still more preferably < 1.0 nM, even more preferably < 0.5 nM, and most preferably about 0.3nM or less; binds to a cell expressing human SIRPaV2 protein with an ECso < 10 nM, preferably < 5 nM, more preferably < 1.5 nM, still more preferably < 1.0 nM, even more preferably < 0.5 nM, and most preferably about 0.3nM or less; does not appreciably bind to SIRPpi protein at an antibody concentration of 50 nM, preferably 67 nM, and more preferably 100 nM; or alternatively at a concentration that is 10-fold greater, preferably 50-fold greater, more preferably 100-fold greater, and still more preferably 200-fold greater than the antibody’s ECso for SIRPaVl or SIRPaV2; inhibits binding between human SIRPa and CD47 with an IC50 < 10.0 nM, more preferably < 5.0 nM, still more preferably < 2.5 nM, and most preferably about 1.0 nM or less; and exhibits a T20 “humanness” of at least 79, and more preferably 85%.

[0087] In other embodiments, the invention provides formulations of antibodies or antigen-binding fragment thereof that bind human SIRPa (e.g., humanized antibodies) and have VH domains and VL domains with at least 90% sequence identity with SEQ IDNOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human SIRPa (e.g., humanized antibodies) and have VH domains and VL domains with at least 95% sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human SIRPa (e.g., humanized antibodies) and have VH domains and VL domains with at least 97% sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human SIRPa (e.g., humanized antibodies) and have VH domains and VL domains with at least 98% sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human SIRPa (e.g., humanized antibodies) and have VH domains and VL domains with at least 99% sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. Preferably, in each case, the sequence differences between SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32 and the variants consist of conservative substitutions and are most preferably limited to substitutions within the framework residues.

[0088] The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Camacho, C. et al. (2009): BMC Bioinformatics 10:421; Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W ., et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., 1996) Meth. Enzymol. 266: 131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al., (1993) Comput. Chem. 17: 149-163; Hancock, J.M. et al., (1994) Comput. Appt. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found.,Washington, DC; Schwartz, R.M., et aL, "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3." M.O. Dayhoff (ed.), pp.353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., 1999) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S.F. "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York. In the present application, percent identity comparisons are preferably performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g. expect threshold: 10; word size: 6; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment).

[0089] "Conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth the following Table 1

[0090] Table 1. Exemplary Conservative Amino Acid Substitutions

[0091] Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. "Function-conservative variants," as used herein, refers to antibodies or fragments in which one or more amino acid residues have been changed without altering a desired property, such an antigen affinity and / or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table 1. Also provided are isolated polypeptides comprising the VL domains of the anti-SIRPa antibodies of the invention (e.g., SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100, 8, 20, 22, 24, 26, 28, and 32), and isolated polypeptides comprising the VH domains of the anti-SIRPa antibodies of the invention (e.g, SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 7, 10, 12, 14, 16, 18, and 30) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, and preferably conservative substitutions.

[0092] By way of example, and not limitation, the antibodies and antigen-binding fragments disclosed herein may bind human SIRPa bivalently with a KD value of 10 x 109M or lower) as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or bio-layer interferometry (OCTET)). In one embodiment, the antibodies and antigen-binding fragments disclosed herein may bind human SIRPa orbivalently with a KD value of about 5-10 x 10'9M as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET). Affinity is calculated as KD = koff / kon(koff is the dissociation rate constant, Konis the association rate constant and KD is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r / c = K(n-r): where r = moles of bound ligand / mole of receptor at equilibrium; c = free ligand concentration at equilibrium; K = equilibrium association constant; and n = number of ligand binding sites per receptor molecule. By graphical analysis, r / c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.Humanness

[0093] For purposes of this document, “humanness” is measured using the T20 score analyzer to quantify the humanness of the variable region of monoclonal antibodies as described in Gao SH, Huang K, Tu H, Adler AS. Monoclonal antibody humanness score and its applications. BMC Biotechnology . 2013: 13:55. doi: 10.1186 / 1472-6750-13-55).

[0094] A web-based tool is provided to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com. In computing a T20 score, an input VH, VK, or VL variable region protein sequence is first assigned Kabat numbering, and CDR residues are identified. The full-length sequence or the framework only sequence (with CDR residues removed) is compared to every sequence in a respective antibody database using the blastp protein-protein BLAST algorithm. The sequence identity between each pairwise comparison is isolated, and after every sequence in the database has been analyzed, the sequences are sorted from high to low based on the sequence identity to the input sequence. The percent identity of the Top 20 matched sequences is averaged to obtain the T20 score.

[0095] For each chain type (VH, VK, VL) and sequence length (full-length or framework only) in the “All Human Databases,” each antibody sequence was scored with its respective database using the T20 score analyzer. The T20 score was obtained for the top 20 matched sequences after the input sequence itself was excluded (the percent identity of sequences 2 through 21 were averaged since sequence 1 was always the input antibody itself). The T20 scores for each group were sorted from high to low. Thedecrease in score was roughly linear for most of the sequences; however the T20 scores for the bottom -15% of antibodies started decreasing sharply. Therefore, the bottom 15 percent of sequences were removed and the remaining sequences formed the T20 Cutoff Human Databases, where the T20 score cutoff indicates the lowest T20 score of a sequence in the new database.

[0096] As used herein, a “Human” antibody is one that has a T20 humanness score of at least 79%, and more preferably at least 85%.Ability of Anti-hSIRPa Antibodies to Block Binding to CD47

[0097] The anti-SIRPa antibodies or antigen binding fragments of the invention are able to block binding of human SIRPa to human CD47. The ability to block binding of human SIRPa to human CD47 can be determined using any method known in the art. In one embodiment, the ability of the antibodies to block binding of human SIRPa to human CD47 is determined using an ELISA assay.Methods of Making Antibodies and Antigen-binding Fragments Thereof

[0098] The anti-SIRPa antibodies finding use in the present formulations may be produced recombinantly (e.g., in an A. colU l expression system, a mammalian cell expression system or a lower eukaryote expression system). In this embodiment, nucleic acids encoding the antibody immunoglobulin molecules of the invention (e.g., VH or VL) may be inserted into a pET-based plasmid and expressed in the E. colUTl system. For example, the present invention includes methods for expressing an antibody or antigenbinding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E.coli such as BL21 or BL21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, in an embodiment of the invention, a bacterial host cell, such as a E. coh. includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl- b eta-D-thi ogal actopy ranosi de) .

[0099] There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567.

[0100] Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

[0101] Thus, the present invention includes methods for making an anti-SIRPa antibody or antigen-binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising introducing a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (e.g., heavy and / or light immunoglobulin chain); culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under condition favorable to such expression and, optionally, isolating the antibody or fragment or chain from the host cell and / or medium in which the host cell is grown.

[0102] Anti-SIRPa antibodies can also be synthesized by any of the methods set forth in U.S. Patent No. 6,331,415.

[0103] Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the antibodies or fragments or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g, Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia fmlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp.,Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknow ense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium luckno ense, any Fusarium sp., Yarrow ia lipolytica, and Neurospora crassa. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, and / or the light chain or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody or fragment or chain in the host cells or secretion into the culture medium in which the host cells are grown.

[0104] Antibodies and immunoglobulin chains can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies and immunoglobulin chains of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0216846, 0256055, and 0323997 and 0338841. Thus, in an embodiment of the invention, the mammalian host cells (e.g., CHO) lack a glutamine synthetase gene and are grown in the absence of glutamine in the medium wherein, however, the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell.

[0105] The present invention includes methods for purifying an anti-SIRPa antibody or antigen-binding fragment thereof of the present invention comprising introducing a sample comprising the antibody or fragment to a purification medium (e.g., cation exchange medium, anion exchange medium, hydrophobic exchange medium, affinity purification medium (e.g., protein-A, protein-G, protein-A / G, protein-L)) and either collecting purified antibody or fragment from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bound antibody or fragment from the medium and collecting the eluate. In anembodiment of the invention, the medium is in a column to which the sample is applied. In an embodiment of the invention, the purification method is conducted following recombinant expression of the antibody or fragment in a host cell, e.g., wherein the host cell is first lysed and, optionally, the lysate is purified of insoluble materials prior to purification on a medium.

[0106] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, comprise the instant invention, independent of the glycosylation pattern the antibodies may have. Similarly, in particular embodiments, antibodies with a glycosylation pattern comprising only non- fucosylated -gl yeans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These antibodies with non-fucosylated N- glycans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

[0107] The present invention includes bispecific and bifunctional antibodies and antigen-binding fragments having a binding specificity for SIRPa and another antigen such as, for example, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, c-Met, CEA, EGFR, EpCAM, HER2, HER3, PSMA, PTHR2, mesothelin, PD-1, PD-L1, TIM3, and methods of use thereof. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J Immunol. 148: 1547- 1553. In addition, bispecific antibodies may be formed as "diabodies" (Holliger, et al., (1993) PNAS USA 90:6444- 6448) or as "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52). Included are “Duobodies,”which are bispecific antibodies with normal IgG structures (Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150).

[0108] The present invention further includes anti-SIRPa antigen-binding fragments of the anti-SIRPa antibodies disclosed herein. The antibody fragments include F(ab)2 fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine.

[0109] Immunoglobulins may be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. In some embodiments, different constant domains may be appended to humanized VL and VH regions derived from the CDRs provided herein. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3 and IgG4; IgAl and IgA2. The invention comprises antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.

[0110] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g. a human constant region, such as yl, y2, y3, or y4 human heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g. a human light chain constant region, such as lambda or kappa human light chain region or variant thereof. By way of example, and not limitation the human heavy chain constant region can be y4 and the human light chain constant region can be kappa. In an alternative embodiment, the Fc region of the antibody is y4 with a Ser228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).

[0111] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgGl subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG2 subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG4 subtype.Antibody Engineering

[0112] The anti-SIRPa antibodies finding use in the formulations of the invention may be engineered antibodies to include modifications to framework residues within thevariable domains the antibody, e.g. to improve the properties of the antibody or fragment. Typically, such framework modifications are made to decrease the immunogenicity of the antibody or fragment. This is usually accomplished by replacing non-CDR residues in the variable domains (i.e. framework residues) in a parental e.g. rodent) antibody or fragment with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g. human residues in the case of human therapeutics. Such an antibody or fragment is referred to as a "humanized" antibody or fragment. In some cases, it is desirable to increase the affinity, or alter the specificity of an engineered e.g. humanized) antibody. One approach is to mutate one or more framework residues to the corresponding germline sequence. More specifically, an antibody or fragment that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody or fragment framework sequences to the germline sequences from which the antibody or fragment is derived. Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g. humanized) antibody, e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues. (See, e.g., U.S. Patent No. 5,693,762, U.S. Patent No. 5,585,089 and U.S. Patent No. 5,530,101).

[0113] In certain embodiments, the anti-SIRPa antibodies are engineered (e.g. humanized) to include modifications in the framework and / or CDRs to improve their properties. Such engineered changes can be based on molecular modelling. A molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al:, National Institutes of Health, Bethesda, MD; 5thed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and coworkers carefully examined conformations of the loops in crystal structures of antibodies and proposed hypervariable loops. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There are variations between regions classified as “CDRs” and “hypervariable loops”. Later studies(Raghunathan et al, (2012) J. Mol Recog. 25, 3, 103-113) analyzed several antibody - antigen crystal complexes and observed that the antigen binding regions in antibodies donot necessarily conform strictly to the “CDR” residues or “hypervariable” loops. The molecular model for the variable region of the non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In practice the potential antigen binding regions based on the model differ from the conventional “CDR”s or “hypervariable” loops. Commercial scientific software such as Discovery Studio (BIO VIA, Dassault Systems)) can be used for molecular modeling. Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs. For FR4 (framework 4) in VH, VJ regions for the human germlines are compared with the corresponding non-human region. In the case of FR4 (framework 4) in VL, J-kappa and J-Lambda regions of human germline sequences are compared with the corresponding non-human region. Once suitable human frameworks are identified, the CDRs are grafted into the selected human frameworks. In some cases, certain residues in the VL-VH interface can be retained as in the non-human (parental) sequence. Molecular models can also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen. In some cases, these residues are retained as in the non-human (parental) sequence. Molecular models can also be used to identify solvent exposed amino acids that can result in unwanted effects such as glycosylation, deamidation and oxidation. Developability filters can be introduced early on in the design stage to eliminate / minimize these potential problems.

[0114] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent No. 7,125,689.

[0115] In particular embodiments, it will be desirable to change certain amino acids containing exposed side-chains to another amino acid residue in order to provide for greater chemical stability of the final antibody, so as to avoid deamidation or isomerization. The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect). Isomerization can occur at DG, DS, DA or DT sequences. In certain embodiments, the antibodies of the present disclosure do not contain deamidation or asparagine isomerism sites.

[0116] For example, an asparagine (Asn) residue may be changed to Gin or Ala to reduce the potential for formation of isoaspartate at any Asn-Gly sequences, particularly within a CDR. A similar problem may occur at a Asp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60: 1281. Isoaspartate formation may debilitate or completely abrogate binding of an antibody to its target antigen. See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In one embodiment, the asparagine is changed to glutamine (Gin). It may also be desirable to alter an amino acid adjacent to an asparagine (Asn) or glutamine (Gin) residue to reduce the likelihood of deamidation, which occurs at greater rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, any methionine residues (typically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id. Additionally, in order to prevent or minimize potential scissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Pro combinations found in a CDR to Gin-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are subsequently screened to ensure that the substitutions do not decrease the affinity or specificity of the antibody for SIRPa, or other desired biological activity to unacceptable levels.

[0117] Table 2. Exemplary stabilizing CDR variants

[0118] Another type of framework modification involves mutating one or more residues within the framework regions to prevent aggregation. The risk of an antibody to aggregate can be assessed using the spatial aggregation propensity -See, Chennamsetty, N et al (2010) J. Phys. Chem. 114, 6614-6624. The method requires the calculation of the Solvent Accessible Area (SAA) for each atom. The molecular aggregation score is then calculated as the sum of all atomic scores. For a given radius and size of molecule, this is an approximate indication of its overall tendency to aggregate. Residues with a high aggregation score are replaced by residues with a lower score (e.g. more hydrophilic amino acids).Antibody Engineering of the Fc region

[0119] The anti-SIRPa antibodies (e.g., humanized antibodies) finding use in the formulations described herein can also be engineered to include modifications within the Fc region, typically to alter one or more properties of the antibody, such as serum halflife, complement fixation, Fc receptor binding, and / or effector function (e.g., antigendependent cellular cytotoxicity). Furthermore, the antibodies disclosed herein can be chemically modified (e.g., one or more chemical moi eties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more properties of the antibody or fragment. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0120] The anti-SIRPa antibodies also include antibodies and fragments with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; W02003 / 086310; W02005 / 120571; W02006 / 0057702. Such modifications can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc regions. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

[0121] In one embodiment, the anti-SIRPa antibody of the invention is an IgG4 isotype antibody or fragment comprising a Serine to Proline mutation at a position corresponding to position 228 (S228P; EU index; SEQ ID NO: 66) in the hinge region of the heavy chain constant region. This mutation has been reported to abolish theheterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al (1993). Mol. Immunol. 30: 105-108; position 241 is based on the Kabat numbering system).

[0122] In one embodiment of the invention, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of CHI is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0123] In another embodiment, the Fc hinge region of an antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody or fragment has impaired Staphylococcyl protein A (SpA) binding relative to native Fc- hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745.

[0124] In another embodiment, the antibody or antigen-binding fragment of the invention is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.

[0125] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand and retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0126] In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such thatthe antibody has altered Clq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551.

[0127] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94 / 29351.

[0128] The anti-SIRPa antibodies of the invention, which are preferably IgG antibodies, may have altered (e.g., relative to an unmodified antibody) FcyR binding properties (examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (KD), dissociation and association rates (koff and konrespectively), binding affinity and / or avidity) and that certain alterations are more or less desirable. It is known in the art that the equilibrium dissociation constant (KD) is defined as koff / kon, and Kais the reciprocal of KD.

[0129] The affinities and binding properties of an Fc region for its ligand, may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-FcyR interactions, i.e., specific binding of an Fc region to an FcyR including but not limited to, equilibrium methods (e.g., enzyme- linked immuno absorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g. BIACORE®, Octet®, or KinExa® analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.

[0130] In certain embodiments, the anti-SIRPa antibodies of the present invention bind to one or more human FcyRs selected from the group consisting of FcyRI, FcyRIIB, FcyRIIC, FcyRIIIA-F158, and FcyRIIIA-V158 with an affinity at least 10-fold, preferably at least 30-fold, and more preferably at least 100-fold, less than equivalent protein having a wild-type human IgGl heavy chain constant domain (SEQ ID NO: 119) Fc region or a wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66) Fc region.

[0131] In various embodiments, the anti-SIRPa antibodies of the invention comprise an immunoglobulin Fc region comprising an immunoglobulin C2 region and an immunoglobulin C3 region and an immunoglobulin hinge region. By way of example, theimmunoglobulin Fc region may be an IgG Fc region, an IgE Fc region, or an IgA Fc region. In certain preferred embodiments, the protein comprises two immunoglobulin Fc regions, each immunoglobulin Fc region comprising an immunoglobulin C2 region and an immunoglobulin C3 region and an immunoglobulin hinge region, wherein the hinge region of one of the immunoglobulin Fc regions is bound to the hinge region of the other immunoglobulin Fc region to form a dimeric Fc structure. Most preferably, such a protein is a human or humanized IgG protein.

[0132] In certain embodiments, the anti-SIRPa antibodies of the invention comprise a mutated IgG4 Fc region, and preferably the protein is an IgG comprising two mutated IgG4 Fc regions to form a dimeric Fc structure. By way of example, a mutated IgG4 Fc region may comprise one of the mutations, or mutational combinations, recited in Table 3. The numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position. For those entries that include combinations of more than one mutation, each mutation in the combination is separated by a “ / ”.

[0133] Table 3:

[0134] In certain embodiments, the anti-SIRPa antibodies of the invention comprise a mutated IgGl Fc region, and preferably the protein is an IgG comprising two mutated IgGl Fc regions to form a dimeric Fc structure. By way of example, a mutated IgGl Fc region may comprise one of the mutations recited in Table 4. The numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat etal. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position.

[0135] Table 4:

[0136] In certain embodiments, a mutated IgGl Fc region may comprise one of the mutational combinations recited in Table 5. The numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position. For each of the combinations of more than one mutation, each mutation in the combination is separated by a “ / ” and deletions are indicated by a “A".

[0137] Table 5:

[0138] In certain embodiments, the anti-SIRPa antibodies of the invention comprise a wild type or mutated IgG2 Fc region, and preferably the protein is an IgG comprising two wild type or mutated IgG2 Fc regions to form a dimeric Fc structure. A mutated IgG2 Fc region may comprise one of the mutations, or mutational combinations, recited in Table6. The numbering system of the constant region referred to in this table is that of the EUindex as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position. For those entries that include combinations of more than one mutation, each mutation in the combination is separated by a

[0139] Table 6:Pharmaceutical Formulations

[0140] The invention comprises pharmaceutical formulations of an anti-human anti- SIRPa antibody. To prepare pharmaceutical or sterile compositions, the antibody, in particular an antibody or fragment thereof, is admixed with a pharmaceutically acceptable carrier or excipient, see, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). Formulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al., 2001, Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, McGraw- Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY;Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0141] Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with another agent, such as the usual anti-cancer drugs, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0142] Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration and oral administration. Administration of antibodies, used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection. In one embodiment, the antibody of the invention is administered intravenously. In another embodiment, the antibody of the invention is administered subcutaneously.

[0143] Alternatively, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into the site of action, often in a depot or sustained release formulation. Furthermore, one may administer the antibody in a targeted drug delivery system.

[0144] The formulations of the present invention can be provided in a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the antibodies or antigen-binding fragments of the invention or a pharmaceutical composition thereof. The present invention also provides an injection device comprising any of the anti-SIRPa antibodies of the invention or a pharmaceutical composition thereof. An injection device is a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., antibody orfragment or a pharmaceutical composition thereof), a needle for piecing skin and / or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the invention, an injection device that comprises an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device includes the antibody or fragment or a pharmaceutical composition thereof in a cannula or trocar / needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline; or lactated ringer solution comprising NaCl, sodium lactate, KC1, CaCh and optionally including glucose) introduced into the body of the patient through the cannula or trocar / needle. The antibody or fragment or a pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line). In an embodiment of the invention, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a patient’s body. External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a patient’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.

[0145] The formulations disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or4,596,556. Such needleless devices comprising the pharmaceutical composition are alsopart of the present invention. The formulations disclosed herein may also be administered by infusion. Examples of well-known implants and modules for administering the pharmaceutical compositions include those disclosed in: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art and those comprising the pharmaceutical compositions of the present invention are within the scope of the present invention.

[0146] Alternately, one may administer the anti-SIRPa antibody or antigen-binding fragment formulations of the invention in a local rather than systemic manner, for example, via injection directly into a tumor. Furthermore, one may administer the anti- SIRPa antibodies in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, a tumor. The liposomes will be targeted to and taken up selectively by the afflicted tissue. Such methods and liposomes are part of the present invention.

[0147] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody or antigen-binding fragment, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody or fragment to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies or fragments is available (see, e.g., Wawrzynczak 1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, el al. (2003) New Engl. J. Med. 348:601-608; Milgrom el al. (1999) New Engl. J. Med. 341 : 1966-1973; Slamon el al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al.(2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594- 1602).

[0148] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, humanized and fully human antibodies may be desirable.

[0149] Antibodies or antigen-binding fragments thereof disclosed herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 pg / kg body weight, more generally at least 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 100 pg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / mL, 10 mg / kg, 25 mg / kg, 50 mg / kg or more (see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346: 1692-1698; Liu, et al. (1999) J.Neurol. Neurosurg. Psych. 67: 451-456; Portielji, et al. (20003) Cancer Immunol. Immunother. 52: 151-144). Doses may also be provided to achieve a pre-determined target concentration of anti-SIRPa antibody in the subject’s serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 pg / mL or more. In other embodiments, An anti-SIRPa antibody of the present invention is administered, e.g., subcutaneously or intravenously, on a weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject.

[0150] As used herein, the term "effective amount" refers to an amount of an anti- SIRPa in a formulation of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of disease, for example cancer or the progression of cancer. An effective dose further refers to that amount of the anti-SIRPa antibodies sufficient to result in at least partial amelioration of symptoms, e.g., tumorshrinkage or elimination, lack of tumor growth, increased survival time. When applied to an individual active ingredient administered alone, an effective dose refers to that ingredient alone. When applied to a combination, an effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.

[0151] A preferred dose protocol is one involving the maximal dose or dose frequency that achieves a desired therapeutic effect (e.g., blocking the CD40 / SIRPa axis) while avoiding significant undesirable side effects. Dosing of the antibodies as described herein can be about every week, about every two weeks, about every three weeks, about every 4 weeks, about every 8 weeks, etc., either buy intraveneous injection, or by subcutaneous injection (e.g., into the thigh, abdomen, upper arm, etc.). The dose per injection or infusion may be about 10 to 1350 mg, e.g. about 50 mg., about 150 mg, about 300 mg, about 450 mg, about 600 mg, about 750 mg, about 1000 mg, or about 1350 mg. In certain embodiments, dosing of the anti-SIRPa antibody will be by subcutaneous injection, with a dose per dosing event (where a “dosing event” refers to one or more deliveries, such as injections, intended to provide a single administration to the individual, where the administrations are given in the same or different sites on the individual) of about 600 mg, with a dosing frequency of once every week, or once every two weeks. A preferred formulation for intraveneous dosing is an aqueous buffered solution at a concentration of about 10-100 mg / mL, or about 20 mg. Formulations for intraveneous infusion can be diluted in sterile saline (0.9%) prior to infusion, for example the desired amount of anti-SIRPa antibody can be diluted to a volume of about 250 mL, for example 15 mL of a 20 mg / mL formulation of antibody can be diluted with 235 mL of sterile saline solution prior to infusion of a 300 mg dose. The formulation for subcutaneous injection can be used without further dilution.

[0152] The therapeutically effective amount and the frequency of administration of, and the length of treatment with, an anti-SIRPa antibody disclosed herein to treat an antibody- associated condition may depend on various factors, including the nature andseverity of the condition, the potency of the antibody, the mode of administration, the age, body weight, general health, gender and diet of the subject, and the response of the subject to the treatment, and can be determined by the treating physician. The anti-SIRPa antibody can be administered once daily, once every 2 days, once every 3 days, twice weekly, once weekly, once every 2 weeks, once every 3 weeks, once monthly, once every 6 weeks, once every 2 months or once every 3 months, or as deemed appropriate by the treating physician.

[0153] The anti-SIRPa antibody can be administered over a period of at least about 1 week, 2 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer, or as deemed appropriate by the treating physician. An anti- SIRPa antibody can also be administered in an irregular manner to treat an antibody- associated condition. Early achievement of an effective target antibody concentration (a therapeutic dose level) with a loading dose followed by maintenance dosing with the antibody (frontloading) may be more effective than conventional therapy in terms of requiring a lower total antibody dose and faster time to maximum target engagement. As used herein, such an administration protocol is referred to as a “loading / maintenance administration protocol.” An effective target antibody concentration may be reached in 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, most preferably 1 week or less, including 1 day or less using a loading dose. The target serum concentration is then maintained by administration of an equal or smaller (or less frequent) maintenance dose during the remainder of the treatment regimen or until suppression of disease symptoms is achieved.

[0154] The term “frontloading” when referring to drug administration refers to the initial loading dose, followed by the maintenance dose. The initial loading dose (single or multiple) is intended to more quickly increase the serum drug concentration of an animal or human patient to an effective target serum concentration. In various embodiments, frontloading is accomplished by initial dosing delivered over 3 weeks or less so that the antibody reaches the target serum concentration. Preferably, the loading dose or series of doses is administered for 2 weeks or less, more preferably 1 week or less, e.g. 1 day or less. Most preferably, the loading dosing is a single dosing, with no maintenance dosing thereafter for at least one week, and the loading dosing is administered in 1 day or less. In order to avoid adverse immune reactions to antibody drugs, it may be preferred to deliver the loading dose of antibody is administered by intravenous injection. The presentinvention includes loading and maintenance doses of frontloading drug delivery by intravenous or subcutaneous administration.

[0155] Administration of the loading dose can be, for example, one or more dosings at a time interval of at least about 1, 2, 3, 4, 5, 6, 7 or 8 weeks apart. In some embodiments, the at least one loading dose is administered by one or more intravenous injections and then at least one maintenance dose by one or more intravenous or subcutaneous administrations. In other embodiments, the instructions can be for administering at least one loading dose by, for example, one or more intravenous or subcutaneous administrations and at least one maintenance dose by one or more intravenous or subcutaneous administrations. In certain embodiments, both the at least one loading dose as well as the at least one maintenance dose is administered subcutaneously. In other embodiments, the at least one loading dose is administered by intravenous infusion followed by at least one maintenance dose administered subcutaneously. For example, the method of treatment can comprise administering a loading dose of 150-1350 mg of the anti-SIRPa antibody by intravenous infusion or subcutaneous injection. After the loading dose (e.g. 1 week, 2 weeks, 3 weeks or 4 weeks after the loading dose), a maintenance dose of 600 mg or less of the anti-SIRPa antibody can be administered every 4 weeks or less, preferably every 3 weeks or less, more preferably every 2 weeks or less, and in embodiments every 1 week or less, by subcutaneous injection. The choice of loading and maintenance dosages and intervals can be made according to the ability of the animal or human patient to tolerate administration of the antibody to the body and according to a desired serum level of SIRPA to achieve.

[0156] A loading dose of a drug can be larger (e.g., about 1.5, 2, 3, 4 or 5 times larger) than a subsequent maintenance dose. The one or more therapeutically effective maintenance doses can be any therapeutically effective amount described herein. The loading dose can be about 2 or 3 times larger than the maintenance dose. The anti-SIRPa antibody can be administered in two (or more) loading doses prior to the maintenance dose. A first loading dose of the antibody or fragment thereof can be administered on day 1, a second loading dose can be administered, e.g., about 1 or 2 weeks later, and a maintenance dose can be administered, e.g., once weekly or once every 2 weeks thereafter for the duration of treatment. The first loading dose can be about 3 or 4 times larger than the maintenance dose, and the second loading dose can be about 2, 3, 4, 5, or more times larger than the maintenance dose.

[0157] As used herein, “inhibit” or “treat” or “treatment” includes a postponement of development of the symptoms associated with disease and / or a reduction in the severity of such symptoms that will or are expected to develop with said disease. The terms further include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject with a disease.

[0158] The antibody of the present invention for therapeutic purposes is administered in a therapeutically effective amount. As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an anti-SIRPa antibody or fragment thereof, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the disease or condition to be treated. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of therapeutic will decrease the symptoms typically by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%.

[0159] Methods for co-administration or treatment with a second therapeutic agent are well known in the art, see, e.g., Hardman, et al. (eds.), 2001, Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.), 2001, Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA.

[0160] The pharmaceutical composition of the invention may also contain other agents, including but not limited to a cytotoxic, chemotherapeutic, cytostatic, anti- angiogenic or antimetabolite agents, a tumor targeted agent, an immune stimulating or immune modulating agent or an antibody conjugated to a cytotoxic, cytostatic, orotherwise toxic agent. The pharmaceutical composition can also be employed with other therapeutic modalities such as surgery, chemotherapy and radiation.Therapeutic Uses of Anti-SIRPa antibodies

[0161] Further provided are methods for treating subjects, including human subjects, in need of treatment with the isolated antibodies or antigen-binding fragments thereof disclosed herein. In one embodiment of the invention, such subject suffers from an infection or an infectious disease.

[0162] In another embodiment of the invention, such subject suffers from cancer. In one embodiment the cancer is , e.g., osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm’s cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer, breast cancer or gastric cancer. In an embodiment of the invention, the cancer is metastatic cancer, e.g., of the varieties described above.

[0163] Cancers that may be treated by the antibodies or antigen-binding fragments, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the aboveidentified conditions.

[0164] In one embedment, cancers that may be treated by the antibodies or antigenbinding fragments thereof disclosed herein, compositions and methods of the invention include, but are not limited to: breast cancer, gastric cancer, esophageal cancer,gastroesophageal junction carcinoma, colorectal cancer, head and neck cancer, non-small cell lung cancer, osteosarcoma, neuroblastoma, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, lung cancer, squamous cell carcinoma, melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, kidney cancer, renal cell carcinoma, thyroid cancer, glioblastoma multiforme, fallopian tube cancer, peritoneal cancer, angiosarcoma, hepatocellular carcinoma, choriocarcinoma, soft tissue sarcoma, chronic lymphocytic leukemia, chronic myelocytic leukemia, non-Hodgkin's lymphoma, B-cell non-hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, myelodysplastic syndrome, acute myelocytic leukemia, T-cell lymphoma, natural killer cell lymphoma, extranodal marginal zone B-cell lymphoma, acute lymphocytic leukemia, multiple myeloma.

[0165] In one embodiment, the antibodies or antigen-binding fragments thereof disclosed herein may be used for the treatment of infections and infectious diseases. As used herein, the term “infection” refers to any state in at least one cell of an organism (i.e., a subject) is infected by an infectious agent (e.g., a subject has an intracellular pathogen infection, e.g., a chronic intracellular pathogen infection). As used herein, the term “infectious agent” refers to a foreign biological entity (i.e. a pathogen) that induces CD47 expression (e.g., increased CD47 expression) in at least one cell of the infected organism. For example, infectious agents include, but are not limited to bacteria, viruses, protozoans, and fungi.

[0166] Intracellular pathogens are of particular interest. Infectious diseases are disorders caused by infectious agents. Some infectious agents cause no recognizable symptoms or disease under certain conditions, but have the potential to cause symptoms or disease under changed conditions. The subject methods can be used in the treatment of chronic pathogen infections, for example including but not limited to viral infections, e.g. retrovirus, lentivirus, hepadna virus, herpes viruses, pox viruses, human papilloma viruses, etc.; intracellular bacterial infections, e.g. Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, Helicobacter pylori etc.; and intracellular protozoan pathogens, e.g. Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmania sp., etc.

[0167] In an embodiment, the invention provides methods for treating subjects using an anti-SIRPa antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a viral infection. In one embodiment, the viral infection is aninfection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV- II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus or arboviral encephalitis virus.

[0168] In an embodiment, the invention provides methods for treating subjects using an anti-SIRPa antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a bacterial infection. In one embodiment, the bacterial infection is infection with a bacteria selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

[0169] In an embodiment, the invention provides methods for treating subjects using an anti-SIRPa antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a fungal infection. In one embodiment, the fungal infection is an infection with a fungus selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.). Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.). Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

[0170] In an embodiment, the invention provides methods for treating subjects using an anti-SIRPa antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a parasitic infection. In one embodiment, the parasitic infection is infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Nippostrongylus brasiliensis.

[0171] A “subject” may be a mammal such as a human, dog, cat, horse, cow, mouse, rat, monkey (e.g., cynomolgous monkey, e.g., Macaca fascicularis) or rabbit. In preferred embodiments of the invention, the subject is a human subject.

[0172] The term “in association with” indicates that the components administered in a method of the present invention (e.g., an anti-SIRPa antibody (e.g., humanized antibody) or antigen-binding fragment thereof along with an anti-cancer agent can be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at several intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route.

[0173] In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used alone, or in association with other, further therapeutic agents and / or therapeutic procedures, for treating or preventing any disease such as cancer, e.g., as discussed herein, in a subject in need of such treatment or prevention. Compositions, e.g., pharmaceutical compositions comprising a pharmaceutically acceptable carrier, comprising such antibodies and fragments in association with further therapeutic agents are also part of the present invention.

[0174] Therefore, the present invention provides a method of treating cancer in a human subject, comprising administering to the subject an effective amount of the antibody or antigen binding fragment disclosed herein, optionally in association with a further therapeutic agent or therapeutic procedure. The present invention also provides a method of treating an infection or infectious disease in a human subject, comprising administering to the subject an effective amount of the antibody or antigen binding fragment disclosed herein, optionally in association with a further therapeutic agent or therapeutic procedure. The present invention also provides a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragment disclosed herein. In one embodiment, the method is used for: the treatment of cancer; the treatment of an infection or infectious disease; or as a vaccine adjuvant.

[0175] In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used alone, or in association with tumor vaccines.Examples of tumor vaccines include but are not limited to vaccines for Human Papillomavirus (HPV) infection caused cancer such as Gardasil®, Gardisil9® and Cervarix®; vaccines that prevent hepatitis B virus caused liver cancer such as Engerix-B® and Recombivax HB®; oncolytic virus therapy that triggers immune response such as Imlygic®; DNA vaccines such as Synchotrope MA2M plasmid DNA vaccine and ZYC101; mammaglobin-a DNA vaccine (see Clinical Cancer Res. 2014 20(23):5964-75); vector based vaccines such as PSA-TRICOM (prostvac), PANVAC-VF, Listeria monocytogenes-based vaccines (see, e.g., Therapeutic Advances in Vaccines, 2014, 2(5) 137-148), Listeria-based vaccines (Listeria expressing one or more cancer vaccines such as Listeria-mesothelin (e.g., CRS-207), ADXS-HPV, Axalimogene Filolisbac, Listeria- HER2 / Neu, Listeria-EGFRvIII), Adeno-CEA; allogeneic vaccines such as GV AX, BLP- 25 (anti-Ankara-mucin 1), Belagenpumatucel-L, TG4010, CIMAvax epidermal growth factor vaccine, NY-ESO, GM.CD40L-CCL21; autologous vaccines such as:Adeno- CD40L, BCG, INGN-225, Dendritic cell vaccines such as Provenge®(Sipuleucel-T), rF- CEA-MUC1 -TRICOM (panvac-DC); antigen vaccines such as MUC-1 (stimuvax), NY- ESO-1, GP-100, MAGE-A3 (melanoma antigen encoding gene A3), INGN-225 (see Pharmacology & Therapeutics 153 (2015) 1-9).

[0176] Eat-me signals could be elevated by cytotoxic therapies like radiotherapy or chemotherapeutic agents including, but not limited to anthracyclines (doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone), oxaliplatin, bortezomib, cyclophosphamide, bleomycin, vorinostat, paclitaxel, 5-fluorouracil, cytarabine, prednisolone, docetaxel, mitomycin C, topotecan / camptothecin, etoposide, zoledronic acid, methotrexate, ibrutinib, aflibercept, bevacizumab, toremifene, vinblastine, vincristine, idelalisib, mercaptopurine, thalidomide, sorafenib. Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used in association with chemotherapeutic agents, in association with radiation therapy, etc. In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used alone, or in association with targeted therapies. Examples of targeted therapies include: hormone therapies, signal transduction inhibitors (e.g., EGFR inhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva)); CD20 inhibitors (e.g., rituximab (Rituxan) and ofatumumab (Arzerra)); CD38 inhibitors (e.g., daratumumab (DARZALEX)); CD52 inhibitors (e.g., alemtuzumab (Campath)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (such as imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib(Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators (e.g., decitabine (Dacogen) and Vorinostat (Zolinza)), apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)), immunomodulatory imide drugs (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast), monoclonal antibodies attached to toxins (e.g., brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla)).

[0177] The antibodies or antigen-binding fragments thereof disclosed herein may preferably find use in association with targeted therapies in which antibodies are employed to mediate ADCC / ADCP. Functional bioassays are available to analyze the mode of action of an antibody drug and to distinguish ADCP as a mode of action from ADCC. By way of example, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay typically utilizes normal human peripheral blood mononuclear cells (PBMCs) or effector cells isolated thereof. Assay variation can be reduced by using selective donor pools with defined Fey receptor Ila (FcyRIIa / CD32a), Illa (FcyRIIIa / CD16a) or Illb (FcyRIIIb / CD16b) gene copy number variation (CNV) or genotypes such as FcyRIIIa-158 V / V versus V / F or F / F, FcyRIIIa-131 H / H versus H / R or R / R, and the FcyRIIIb-NAl and -NA2 polymorphic variants. Alternatively, effector cells such as PBMCs, PBMC-derived natural killer (NK) cells, granulocytes, monocytes, monocyte-derived macrophages, or dendritic cells (DCs) can be replaced with a FcyRIIIa-expressing cell line (for example, engineered NK92). Killing of the target cells can be assessed by measuring the release of specific probes from pre-labelled target cells, using51chromium (Cr51) or fluorescent dyes such as calcein-acetoxymethyl (calcein-AM), carboxyfluorescein succinimidyl ester (CFSE), 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), europium (Eu) or propidium iodide (PI), or by measuring the release of cytosolic enzymes such as lactate dehydrogenase (LDH) or the release of nucleoside triphosphate (ATP).

[0178] In contrast, antibody-dependent cellular phagocytosis (ADCP) may be assessed by measuring the destruction of target cells via granulocyte, monocyte, dendritic cell, or macrophage-mediated phagocytosis. ADCP assays use PBMC-derived cells or myeloid cell lines such as HL-60, THP-1, and U937 cells differentiated into macrophages or granulocytes. Stimuli that are commonly used to induce macrophage differentiation in monocytic cell lines include phorbol-12-myristate-13-acetate (PMA), 1,25- dihydroxyvitamin D3 (VD3), and retinoic acid (RA). RA is also known to induceterminal granulocytic differentiation of for example HL-60 cells. Phagocytosis of the target cells can be assessed by monitoring effector cells for the internalization of specific probes from target cells pre-labelled with fluorescent dyes such as cell proliferation dye eFluor450, CFSE, and pH-sensitive dyes including pHrodo and CypHer5E. Phagocytosis is measured by an increase in fluorescently labelled effector cells using flow cytometry or fluorescence microscopy. “Reporter gene” assays are also available to assess ADCP. In order to measure ADCP function in a reporter gene assay, target cells are first incubated with a titration of an antibody of interest. Once the antibody is bound to its cognate target on the target cell surface, engineered Jurkat effector cells are added. If ADCP pathway activation ensues, the Jurkat cells produce a luciferase product by expression of the reporter gene NFAT-RE-luc2. Luciferase activity is then measured following a 4-24 hour induction period, after addition of the luciferase assay reagent. The dose-dependent response in the microtiter plate-based assay can be used to quantify the relative biological activity of the therapeutic antibody compared to the dose-response curve of a suitable reference item.

[0179] In particular embodiments, the anti-SIRPa antibodies or antigen-binding fragments thereof of the invention may be used in combination with an anti-cancer therapeutic agent or immunomodulatory drug such as an immunomodulatory receptor inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to the receptor.

[0180] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with one or more of: an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecules (SLAM proteins), an activating NK cell receptor, a Toll like receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, ICAM-1, LFA-1 (CD1 la / CD18), 4- IBB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB- A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), SLAM7, BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83; or an inhibitor of CD47, PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, CEACAM (e.g., CEACAM-1, -3 and / or -5), VISTA, BTLA, TIGIT, LAIR1, IDO, TDO, CD 160 and / or TGFR beta.

[0181] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with one or more cyclic dinculeotides or other STING pathway agonists. STING (stimulator of interferon genes, also known as TMEM173, MITA, ERIS, and MPYS) is a transmembrane protein localized to the ER that undergoes a conformational change in response to direct binding of cyclic dinucleotides (CDNs), resulting in a downstream signaling cascade involving TBK1 activation, IRF-3 phosphorylation, and production of IFN-P and other cytokines. The STING pathway in tumor-resident host antigen presenting c3ellss is involved in the induction of a spontaneous CD8+ T cell response against tumor-derived antigens. Activation of this pathway and the subsequent production of IFN-P also reportedly contributes to the anti-tumor effect of radiation. STING agoinists and their uses are described in, for example, US20060040887, US20080286296, US20120041057, US20140205653, WO2014179335, WO 2014179760, US20150056224, WO 2015185565, WO 2016096174, WO 2016145102, WO 2017011444, WO 2017027645, WO 2017027646, WO 2017123657, WO 2017123669, WO 2017175147, WO 2017175156, WO 2018045204, WO 2018009648, WO 2018006652, WO 2018013887, WO 2018013908, US20180002369, US20180092937, and US20180093964.

[0182] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with one or more of anti-CD47 antibody, anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, anti-PDLl antibody, anti-TIGIT antibody, anti -APRIL antibody, anti-CTLA4 antibody, anti-CSl antibody (e.g., elotuzumab), anti -KIR2DL 1 / 2 / 3 antibody (e.g., lirilumab), anti-CD137 antibody (e.g., urelumab), anti-GITR antibody (e.g., TRX518), anti-PD-Ll antibody (e.g., BMS- 936559, MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILTl antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody, anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody, anti-ILT8 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS, anti-KIR2DLl antibody, anti-KIR2DL2 / 3 antibody, anti-KIR2DL4 antibody, anti-KIR2DL5A antibody, anti-KIR2DL5B antibody, anti-KIR3DLl antibody, anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E antibody, anti-4-lBB antibody (e.g., PF-05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 or PEGylated IL-10, or any small organic molecule inhibitor of such targets.

[0183] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD20 antibody (e.g., rituximab, ofatumumab, ocrelizumab, obinutuzumab, ocaratuzumab, ublituximab, veltuzumab, ibritumomab tiuxetan, tositumomab, BVX-20, SCT-400 or PROD 1921).

[0184] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD38 antibody (e.g., daratumumab, isatuximab or MOR202).

[0185] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-EGFR antibody (e.g., cetuximab, CetuGEX, panitumumab, nimotuzumab, depatuxizumab or AFM-21).

[0186] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-HER2 antibody (e.g., trastuzumab, TrasGEX, pertuzumab, margetuximab or ADCT-502).

[0187] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-HER3 antibody (e.g., lumretuzumab, patritumab or LJM716).

[0188] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD19 antibody (e.g., inebilizumab, blinatumomab, DLB4, MDX-1342, MEDI-551, MOR208 or 4-G7SDIE).

[0189] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD52 antibody (e.g., alemtuzumab).

[0190] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-EpCAM antibody (e.g., adecatumumab, catumaxomab, edrecolomab or ING-1).

[0191] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-SLAMF7 antibody (e.g., elotuzumab or ABBV-838).

[0192] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab).

[0193] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-PD-Ll antibody (e.g., BMS-936559, MSB0010718C or MPDL3280A).

[0194] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CTLA4 antibody (e.g., ipilimumab or tremelimumab).

[0195] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD137 antibody (e.g., urelumab).

[0196] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-GITR antibody (e.g., TRX518 or FPA154).

[0197] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-OX40 antibody (e.g., MEDI6469, MOXR0916 or INC AGNI 949).

[0198] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD40 antibody (e.g., lucatumumab, dacetuzmumab, APX005M, ChiLob7 / 4, CP-870,893 or JNJ-64457107)In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CSl antibody.

[0199] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti -KIR2DL 1 / 2 / 3 antibody.

[0200] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD137 (e.g., urelumab) antibody.

[0201] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-GITR (e.g., TRX518) antibody.

[0202] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-PD-L2 antibody.

[0203] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITLl antibody.

[0204] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL2 antibody.

[0205] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL3 antibody.

[0206] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL4 antibody.

[0207] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL5 antibody.

[0208] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL6 antibody.

[0209] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL7 antibody.

[0210] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ITL8 antibody.

[0211] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-CD40 antibody.

[0212] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-OX40 antibody.

[0213] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR2DLl antibody.

[0214] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR2DL2 / 3 antibody.

[0215] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR2DL4 antibody.

[0216] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR2DL5A antibody.

[0217] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR2DL5B antibody.

[0218] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR3DLl antibody.

[0219] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR3DL2 antibody.

[0220] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-KIR3DL3 antibody.

[0221] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-NKG2A antibody.

[0222] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-NKG2C antibody.

[0223] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-ICOS antibody.

[0224] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-4-lBB antibody.

[0225] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-IL-10 antibody.

[0226] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with an anti-TSLP antibody.

[0227] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with IL- 10 or PEGylated IL- 10.

[0228] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with one or more of an inhibitor (e.g., a small organic molecule or an antibody or antigen -binding fragment thereof) such as: an MTOR (mammalian target of rapamycin) inhibitor, a cytotoxic agent, a platinum agent,an EGFR inhibitor, a VEGF inhibitor, a microtubule stabilizer, a taxane, a CD20 inhibitor, a CD52 inhibitor, a CD30 inhibitor, a RANK (Receptor activator of nuclear factor kappa-B) inhibitor, a RANKL (Receptor activator of nuclear factor kappa-B ligand) inhibitor, an ERK inhibitor, a MAP Kinase inhibitor, an AKT inhibitor, a MEK inhibitor, a PI3K inhibitor, a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a Bcl2 inhibitor, a CD22 inhibitor, a CD79b inhibitor, an ErbB2 inhibitor, or a farnesyl protein transferase inhibitor.

[0229] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with any one or more of: 13-cis- retinoic acid, 3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone, 4- hydroxytamoxifen, 5-deooxyuridine, 5'-deoxy-5-fluorouridine, 5-fluorouracil, 6- mecaptopurine, 7-hydroxy staurosporine, A-443654, abirateroneacetate, abraxane, ABT- 578, acolbifene, ADS-100380, ALT-110, altretamine, amifostine, aminoglutethimide, amrubicin, Amsacrine, anagrelide, anastrozole, angiostatin, AP -23573, ARQ-197, arzoxifene, AS-252424, AS-605240, asparaginase, AT-9263, atrasentan, axitinib, AZDI 152, Bacillus Calmette -Guerin (BCG) vaccine, batabulin, BC-210, besodutox, bevacizumab, bicalutamide, Biol 11, BIO140, bleomycin, BMS-214662, BMS-247550, BMS-275291, BMS-310705, bortezomib, buserelin, busulfan, calcitriol, camptothecin, canertinib, capecitabine, carboplatin, carmustine, CC8490, Cediranib, CG-1521, CG-781, chlamydocin, chlorambucil, chlorotoxin, cilengitide, cimitidine, cisplatin, cladribine, clodronate, COL-3, CP-724714, cyclophosphamide, cyproterone, cyproteroneacetate, cytarabine, cytosinearabinoside, dacarbazine, dacinostat, dactinomycin, dalotuzumab, danusertib, dasatanib, daunorubicin, decatanib, deguelin, denileukin, deoxycoformycin, depsipeptide, diarylpropionitrile, diethylstilbestrol, diftitox, docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin, yttrium-90 labeled-edotreotide, edotreotide, EKB- 569, EMD121974, endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B, ERA- 923, Erbitux, erlotinib, estradiol, estramustine, etoposide, everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol, floxuridine, fludarabine, fludrocortisone, fluoxymesterone, flutamide, FOLFOX regimen, Fulvestrant, galeterone, gefitinib, gemcitabine, gimatecan, goserelin, goserelin acetate, gossypol, GSK461364, GSK690693, HMR-3339, hydroxyprogesteronecaproate, hydroxyurea, IC87114, idarubicin, idoxyfene, ifosfamide, IM862, imatinib, IMC-1C11, INCB24360, INO1001, interferon, interleukin- 12, ipilimumab, irinotecan, JNJ-16241199, ketoconazole, KRX- 0402, thalidomide, lenalidomide, pomalidomide, apremilast,lapatinib, lasofoxifene,letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, liposome entrapped paclitaxel, lomustine, lonafamib, lucanthone, LY292223, LY292696, LY293646, LY293684, LY294002, LY317615, marimastat, mechlorethamine, medroxyprogesteroneacetate, megestrolacetate, melphalan, mercaptopurine, mesna, methotrexate, mithramycin, mitomycin, mitotane, mitoxantrone, tozasertib, MLN8054, neovastat, Neratinib , neuradiab, nilotinib, nilutimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide, ofatumumab, oregovomab, orteronel, oxaliplatin, paclitaxel, palbociclib, pamidronate, panitumumab, pazopanib, PD0325901, PD 184352, PEG- interferon, pemetrexed, pentostatin, perifosine, phenylalaninemustard, PI-103, pictilisib, PIK-75, pipendoxifene, PKI-166, plicamycin, porfimer, prednisone, procarbazine, progestins, PX-866, R-763, raloxifene, raltitrexed, razoxin, ridaforolimus, rituximab, romidepsin, RTA744, rubitecan, scriptaid, Sdxl02, seliciclib, selumetinib, semaxanib, SF1126, sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668, streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib, synthetic estrogen, talampanel, talimogene laherparepvec, tamoxifen, temozolomide, temsirolimus, teniposide, tesmilifene, testosterone, tetrandrine, TGX-221, thalidomide, thioguanine, thiotepa, tremelimumab, tipifamib, tivozanib, TKI-258, TLK286, topotecan, toremifene citrate, trabectedin, trastuzumab, tretinoin, trichostatin A, triciribinephosphate monohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproic acid, valrubicin, vandetanib, vatalanib, VEGF trap, vinblastine, vincristine, vindesine, vinorelbine, vitaxin, vitespan, vorinostat, VX-745, wortmannin, Xr311, zanolimumab, ZK186619, ZK- 304709, ZM336372, ZSTK474.

[0230] Non-limiting examples of suitable anti-cancer agents to be used in combination with an anti-SIRPa antibody or antigen-binding fragment thereof of the invention include cytostatic agents, immune modulating imide drugs, cytotoxic agents, targeted therapeutic agents (small molecules, biologies, siRNA and microRNA) against cancer and neoplastic diseases,1) anti-metabolites (such as methotrexate, 5-fluorouracil, gemcitabine, fludarabine, capecitabine);2) alkylating agents, such as temozolomide, cyclophosphamide,3) DNA interactive and DNA damaging agents, such as cisplatin, oxaliplatin, doxorubicin,4) Ionizing irradiation, such as radiation therapy,) topoisomerase II inhibitors, such as etoposide, doxorubicin, ) topoisomerase I inhibitors, such as irinotecan, topotecan, ) tubulin interacting agents, such as paclitaxel, docetaxel, Abraxane, epothilones,) kinesin spindle protein inhibitors, ) spindle checkpoint inhibitors, 0) Poly(ADP -ribose) polymerase (PARP) inhibitors, such as olaparib, MK-4827 and veliparib 1) Matrix metalloprotease (MMP) inhibitors 2) Protease inhibitors, such as cathepsin D and cathepsin K inhibitors3)Proteosome or ubiquitination inhibitors, such as bortezomib, 4) Activator of mutant p53 to restore its wild-type p53 activity 5) Adenoviral -p53 6) Bel -2 inhibitors, such as ABT-263 7) Heat shock protein (HSP) modulators, such as geldanamycin and 17-AAG8) Histone deacetylase (HD AC) inhibitors, such as vorinostat (SAHA), 9) sex hormone modulating agents, a. anti-estrogens, such as tamoxifen, fulvestrant, b. selective estrogen receptor modulators (SERM), such as raloxifene, c. anti-androgens, such as bicalutamide, flutamide d. LHRH agonists, such as leuprolide, e. 5a-reductase inhibitors, such as finasteride, f. Cytochrome P450 C17 lyase (CYP450cl7, also called 17aC); g. aromatase inhibitors, such as letrozole, anastrozole, exemestane,0) EGFR kinase inhibitors, such as geftinib, erlotinib, laptinib 1) dual erbBl and erbB2 inhibitors, such as Lapatinib 2) multi -targeted kinases (serine / threonine and / or tyrosine kinase) inhibitors, a. ABL kinase inhibitors, imatinib and nilotinib, dasatinib b. VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK and ERK inhibitors, such as sunitinib, sorafenib, Vandetanib, pazopanib, PLX-4032, Axitinib, PTK787, GSK-1120212 c. Polo-like kinase inhibitors d. Aurora kinase inhibitors e. JAK inhibitor f. c-MET kinase inhibitorsg. Cyclin-dependent kinase inhibitors, such as CDK1 and CDK2 inhibitor Dinaciclib SCH 727965 (see Parry et al, Molecular Cancer Therapeutics 9 (8): 2344-53 (2010)) and CDK4 / 6 inhibitors, such as Riboci clib, Palbociclib, Abemaciclib, and Trilaciclib. h. PI3K and mTOR inhibitors, such as GDC-0941, BEZ-235, BKM-120 and AZD-8055 i. Rapamycin and its analogs, such as Temsirolimus, everolimus, and deforolimus ) and other anti-cancer (also know as anti -neoplastic) agents include but are not limited to ara-C, adriamycin, cytoxan, Carboplatin, Uracil mustard, Clormethine, Ifosfsmide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, Vinblastine, Vincristine, Vindesine, Vinorelbine, Navelbine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, teniposide, cytarabine, pemetrexed, Idarubicin, Mithramycin, Deoxy coformycin, Mitomycin-C, L-Asparaginase, Teniposide, Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Flutamide Medroxyprogesteroneacetate, Toremifene, goserelin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, Drolloxafine, Hexamethylmelamine, Bexxar, Zevalin, Trisenox, Profimer, Thiotepa, Altretamine, Doxil, Ontak, Depocyt, Aranesp, Neupogen, Neulasta, Kepivance.)Farnesyl protein transferase inhibitors, such as, SARASAR™(4-[2-[4-[(l lR)- 3,10-dibromo-8-chloro-6, 1 l-dihydro-5H-benzo[5,6]cyclohepta[l,2-b]pyridin-l 1- yl-]-l-piperidinyl]-2-oxoethyl]-piperidinecarboxamide, tipifamib ) interferons, such as Intron A, Peg-Intron, ) anti-erbBl antibodies, such as cetuximab, panitumumab, ) anti-erbB2 antibodies, such as trastuzumab, ) anti-CD52 antibodies, such as Alemtuzumab, ) anti-CD20 antibodies, such as Rituximab ) anti-CD33 antibodies, such as Gemtuzumab ozogamicin31) anti-VEGF antibodies, such as Avastin,32) TRIAL ligands, such as Lexatumumab, mapatumumab, and AMG-65533) anti-CTLA-4 antibodies, such as ipilimumab34) antibodies against CTA1, CEA, CD5, CD19, CD22, CD30, CD44, CD44V6, CD55, CD56, EpCAM, FAP, MHCII, HGF, IL-6, MUC1, PSMA, TAL6, TAG- 72, TRAILR, VEGFR, IGF-2, FGF,35) anti-IGF-lR antibodies, such as dalotuzumab (MK-0646) and robatumumab (SCH 717454).

[0231] “Estrogen receptor modulators” refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-l- oxopropoxy-4-methyl-2-[4-[2-(l-piperidinyl)ethoxy]phenyl]-2H-l-benzopyran-3-yl]- phenyl-2,2-dimethylpropanoate, 4,4’-dihydroxybenzophenone-2,4-dinitrophenyl- hydrazone, and SH646.

[0232] “Androgen receptor modulators” refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

[0233] “Retinoid receptor modulators” refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis- retinoic acid, oc-difluoromethylomithine, ILX23-7553, trans-N-(4’ -hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.

[0234] “Cytotoxic / cytostatic agents” refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell’s functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors / microtubule- stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal / anti-hormonal therapeutic agents, haematopoietic growth factors,monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

[0235] Examples of cytotoxic / cytostatic agents include, but are not limited to, platinum coordinator compounds, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cisaminedi chi oro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-l,6-diamine)-mu-[diamine- platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3 ’-deamino-3’ -morpholino- 13 -deoxo- 10- hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-dem ethoxy-3 - deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00 / 50032).

[0236] An example of a hypoxia activatable compound is tirapazamine.

[0237] Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).

[0238] Examples of microtubule inhibitors / microtubule-stabilising agents include taxanes in general. Specific compounds include paclitaxel (Taxol®), vindesine sulfate, 3’,4’-didehydro-4’-deoxy-8’-norvincaleukoblastine, docetaxol (Taxotere®), rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS 184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L- prolyl-L-proline-t-butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS 188797.

[0239] Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3’,4’-O-exo-benzylidene-chartreusin, 9- methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, 1-amino- 9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH,12H-benzo[de]pyrano[3’,4’:b,7]- indolizino[l,2b]quinoline-10,13(9H,15H)dione, lurtotecan, 7-[2-(N- isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942,etoposide phosphate, teniposide, sobuzoxane, 2’-dimethylamino-2’-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole- 1 -carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N- methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9- hexohydrofuro(3’,4’ :6,7)naphtho(2,3-d)-l,3-dioxol-6-one, 2, 3-(m ethylenedi oxy)-5- methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2- aminoethyl)amino]benzo[g]isoguinoline-5, 10-dione, 5-(3-aminopropylamino)-7,10- dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,l-de]acridin-6-one, N-[l- [2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]- 3-hydroxy-7H-indeno[2,l-c] quinolin-7-one, and dimesna.

[0240] Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in Publications W003 / 039460, W003 / 050064, W003 / 050122, WO03 / 049527, WO03 / 049679, WO03 / 049678, WO04 / 039774, WO03 / 079973, W003 / 099211, W003 / 105855, W003 / 106417, W004 / 037171, W004 / 058148, W004 / 058700, WO04 / 126699, W005 / 018638, W005 / 019206, WO05 / 019205, WO05 / 018547, W005 / 017190, US2005 / 0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL.

[0241] Examples of “histone deacetylase inhibitors” include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T. A. et al. J. Med. Chem. 46(24): 5097-5116 (2003).

[0242] “ Inhibitors of kinases involved in mitotic progression” include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-Rl. An example of an “aurora kinase inhibitor” is VX-680.

[0243] “Antiproliferative agents” includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2’ -deoxy -2’ -methylidenecytidine, 2’ -fluoromethylene-2’ -deoxy cytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N’-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2- [2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno- heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8- tetrahydro-3H-pyrimidino[5,4-b][l,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, l l-acetyl-8-(carbamoyloxymethyl)-4-formyl- 6-methoxy-14-oxa-l,l l-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2’-cyano-2’-deoxy-N4- palmitoyl-l-B-D-arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

[0244] Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.

[0245] “Prenyl -protein transferase inhibitor” refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including famesyl- protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl -protein transferase type-II (GGPTase-II, also called Rab GGPTase).

[0246] Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96 / 30343, WO 97 / 18813, WO 97 / 21701, WO 97 / 23478, WO 97 / 38665, WO 98 / 28980, WO 98 / 29119, WO 95 / 32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94 / 19357, WO 95 / 08542, WO 95 / 11917, WO 95 / 12612, WO 95 / 12572, WO 95 / 10514, U.S. Patent No. 5,661,152, WO 95 / 10515, WO 95 / 10516, WO 95 / 24612, WO 95 / 34535, WO 95 / 25086, WO 96 / 05529, WO 96 / 06138, WO 96 / 06193, WO 96 / 16443, WO 96 / 21701, WO 96 / 21456, WO 96 / 22278, WO 96 / 24611, WO 96 / 24612, WO 96 / 05168, WO 96 / 05169, WO 96 / 00736, U.S. Patent No. 5,571,792, WO 96 / 17861, WO 96 / 33159, WO 96 / 34850, WO 96 / 34851, WO 96 / 30017, WO 96 / 30018, WO 96 / 30362, WO 96 / 30363, WO 96 / 31111, WO 96 / 31477, WO 96 / 31478, WO 96 / 31501, WO 97 / 00252, WO 97 / 03047, WO 97 / 03050, WO 97 / 04785, WO 97 / 02920, WO 97 / 17070, WO 97 / 23478, WO 97 / 26246, WO 97 / 30053, WO 97 / 44350, WO 98 / 02436, and U.S. Patent No. 5,532,359. For anexample of the role of a prenyl-protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).

[0247] “Angiogenesis inhibitors” refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2), inhibitors of epidermal- derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-a, interleukin- 12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy -genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Leters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Inti. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6- O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin- 1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105: 141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00 / 44777; and WO 00 / 61186).

[0248] Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2- butenyl)oxiranyl]-l-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5- amino-l-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-lH-l,2,3-triazole-4- carb oxami de, CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2- pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(l,3-naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

[0249] Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem.La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80: 10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res . 101 :329-354 (2001)). TAFIa inhibitors have been described in U.S. Ser. Nos. 60 / 310,927 (filed August 8, 2001) and 60 / 349,925 (filed January 18, 2002).

[0250] “Agents that interfere with cell cycle checkpoints” refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHK1 1 and CHK12 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS- 387032.

[0251] “Agents that interfere with receptor tyrosine kinases (RTKs)” refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume- Jensen and Hunter, Nature, 411 :355-365, 2001.

[0252] “ Inhibitors of cell proliferation and survival signalling pathway” refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine / threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02 / 083064, WO 02 / 083139, WO 02 / 083140, US 2004-0116432, WO 02 / 083138, US 2004-0102360, WO 03 / 086404, WO 03 / 086279, WO 03 / 086394, WO 03 / 084473, WO 03 / 086403, WO 2004 / 041162, WO 2004 / 096131, WO 2004 / 096129, WO 2004 / 096135, WO 2004 / 096130, WO 2005 / 100356, WO 2005 / 100344, US 2005 / 029941, US 2005 / 44294, US 2005 / 43361, 60 / 734188, 60 / 652737, 60 / 670469), inhibitors of Raf kinase (for example PLX-4032 ), inhibitors of MEK (for example Arry-162, RO-4987655 and GSK-1120212), inhibitors of mTOR (for example AZD-8055, BEZ-235 and everolimus), and inhibitors of PI3K (for example GDC-0941, BKM-120).

[0253] As used above, “integrin blockers” refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the a. Psintegrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiologicalligand to the avP5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the avP3 integrin and the avP5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the avPe, avPs, aiPi, (X2P1, asPi, aePi, and aeP4 integrins. The term also refers to antagonists of any combination of avPs, avPs, avPs, aiPi, (X2P1, asPi, aePi, and aeP4 integrins.

[0254] Some specific examples of tyrosine kinase inhibitors include N- (trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5- yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro- 4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3- ethynylphenyl)-6,7-bis(2 -methoxy ethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro- 10-(hydroxymethyl)- 10-hydroxy-9-methyl-9, 12-epoxy- 1H- diindolo[l,2,3-fg:3’,2’,r-kl]pyrrolo[3,4-i][l,6]benzodiazocin-l-one, SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3- d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7- dimethoxyquinazoline, 4-(4’-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-l-phthalazinamine, and EMD121974.

[0255] Combinations of the instantly claimed antibodies or antigen binding fragments with PPAR-y (i.e., PPAR-gamma) agonists and PPAR-6 (i.e., PPAR-delta) agonists may be useful in the treatment of certain malignancies. PPAR-y and PPAR-6 are the nuclear peroxisome proliferator-activated receptors y and 6. The expression of PPAR-y on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31 : 909-913; J. Biol. Chem. 1999; 274: 9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41 : 2309-2317). More recently, PPAR-y agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice.(Arch. Ophthamol. 2001; 119: 709-717). Examples of PPAR-y agonists and PPAR- y / a agonists include, but are not limited to, Lynparza®, Rucaparib®, Talazoparib®, niraparib, Veliparib®, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7- dipropyl-3-trifluoromethyl-l,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy )propoxy)-2-ethylchromane-2-carboxylic acid.

[0256] The antibody or antigen binding fragment of the instant invention may also be useful for treating or preventing breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane.

[0257] The antibody or antigen binding fragment of the instant invention may also be useful for treating cancer in combination with the following chemotherapeutic agents: abarelix (Plenaxis depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®);Alemtuzumab (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bendamustine hydrochloride (Treanda®); bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); brefeldin A; busulfan intravenous (Busulfex®); busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC- Dome®); dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection (Fragmin®); daratumumab (DARZALEX®); Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); degarelix (Firmagon®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®); dexrazoxane hydrochloride (Totect®); didemnin B; 17-DMAG; docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®); dromostanolone propionate (Dromostanolone ®); dromostanolone propionate (Masterone Injection®);eculizumab injection (Soliris®); Elliott's B Solution (Elliott's B Solution®); eltrombopag (Promacta®); epirubicin (Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); ethinyl estradiol; etoposide phosphate (Etopophos®); etoposide, VP- 16 (Vepesid®); everolimus tablets (Afinitor®); exemestane (Aromasin®); ferumoxytol (Feraheme Injection®); Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin implant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (Intron A®); iobenguane I 123 injection (AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinib tablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®); mitramycin; nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®); nilotinib (Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (K epivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanib tablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor (Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); pralatrexate injection (Folotyn®); procarbazine (Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®); raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin(Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-26 (Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiopurine; thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab / I-131 tositumomab (Bexxar®); trans-retinoic acid; Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin; and zoledronate (Zorn eta®).

[0258] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is in association with one or more antiemetics including, but not limited to: casopitant (GlaxoSmithKline), Netupitant (MGLHelsinn) and other NK-1 receptor antagonists, palonosetron (sold as Al oxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, NJ), diphenhydramine (sold as Benadryl® by Pfizer; New York, NY), hydroxyzine (sold as Atarax® by Pfizer; New York, NY), metoclopramide (sold as Reglan® by AH Robins Co,; Richmond, VA), lorazepam (sold as Ativan® by Wyeth; Madison, NJ), alprazolam (sold as Xanax® by Pfizer; New York, NY), haloperidol (sold as Haldol® by Ortho-McNeil; Raritan, NJ), droperidol (Inapsine®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, GA), dexamethasone (sold as Decadron® by Merck and Co.; Rahway, NJ), methylprednisolone (sold as Medrol® by Pfizer; New York, NY), prochlorperazine (sold as Compazine® by Glaxosmithkline; Research Triangle Park, NC), granisetron (sold as Kytril® by Hoffmann-La Roche Inc.; Nutley, NJ), ondansetron ( sold as Zofiran® by Glaxosmithkline; Research Triangle Park, NC), dolasetron (sold as Anzemet® by Sanofi- Aventis; New York, NY), tropisetron (sold as Navoban® by Novartis; East Hanover, NJ).

[0259] Other side effects of cancer treatment include red and white blood cell deficiency. Accordingly, in an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof is in association with an agent which treats or prevents such a deficiency, such as, e.g., filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa or darbepoetin alfa.

[0260] In an embodiment of the invention, an anti-SIRPa antibody or antigen-binding fragment thereof of the invention is administered in association with anti-cancer radiation therapy. For example, in an embodiment of the invention, the radiation therapy is external beam therapy (EBT): a method for delivering a beam of high-energy X-rays to the location of the tumor. The beam is generated outside the patient (e.g., by a linear accelerator) and is targeted at the tumor site. These X-rays can destroy the cancer cells and careful treatment planning allows the surrounding normal tissues to be spared. No radioactive sources are placed inside the patient's body. In an embodiment of the invention, the radiation therapy is proton beam therapy: a type of conformal therapy that bombards the diseased tissue with protons instead of X-rays. In an embodiment of the invention, the radiation therapy is conformal external beam radiation therapy: a procedure that uses advanced technology to tailor the radiation therapy to an individual's body structures. In an embodiment of the invention, the radiation therapy is brachytherapy: the temporary placement of radioactive materials within the body, usually employed to give an extra dose — or boost — of radiation to an area.

[0261] In an embodiment of the invention, a surgical procedure is administered in association with an anti-SIRPa antibody or antigen-binding fragment thereof is surgical tumorectomy.Kits

[0262] Further provided are kits comprising one or more components that include, but are not limited to, an anti-SIRPa antibody formulation as described herein in association with one or more additional components. In one embodiment, the kit includes an anti- SIRPa antibody formulation of the invention in one container (e.g., in a sterile glass or plastic vial) and a second therapeutic agent and a pharmaceutical composition thereof in another container (e.g., in a sterile glass or plastic vial).

[0263] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above.

[0264] The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding acombination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and patent information.

[0265] The kit can also comprise a second therapeutic, for example one or more of: anti-CD47 antibody, anti-APRIL antibody, anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, anti-PDLl antibody, anti-TIGIT antibody, anti-CTLA4 antibody, anti- CS1 antibody (e.g., elotuzumab), anti -KIR2DL 1 / 2 / 3 antibody (e.g., lirilumab), anti- CD137 antibody (e.g., urelumab), anti-GITR antibody (e.g., TRX518), anti-PD-Ll antibody (e.g., BMS-936559, MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILTl antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody, anti- ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody, anti-ILT8 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS, anti-KIR2DLl antibody, anti-KIR2DL2 / 3 antibody, anti-KIR2DL4 antibody, anti-KIR2DL5 A antibody, anti-KIR2DL5B antibody, anti-KIR3DLl antibody, anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E antibody, anti-4-lBB antibody (e.g., PF- 05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 or PEGylated IL-10, or any small organic molecule inhibitor of such targets; an antibody or antigen binding fragment thereof binds to an antigen selected from the group consisting of AMHR2, AXL, BCMA, CA IX, CD4, CD 16, CD 19, CD20, CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b, folate receptor alpha, fucosyl-GMl, HER2, HER3, IL1RAP, kappa myeloma antigen, MS4A1, prolactin receptor, TA-MUC1, and PSMA; Rituximab, ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, Dinituximab, Trastuzumab, Cetuximab, Rituximab-RLI, C.60C3- RLI, Hul4.18-IL2, KM2812, AFM13, and (CD20)2xCD16, erlotinib (Tarceva), daratumumab, alemtuzumab, pertuzumab, brentuximab, elotuzumab, ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab, carotuximab, epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21, AFM22, LY-3022855, SNDX-6352, AFM-13, BL 836826, BMS-986012, BVX-20, mogamulizumab, ChiLob-7 / 4, leukotuximab, isatuximab, DS-8895, FPA144, GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213, PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX- 1097, MOR202, MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomabtiuxetan, ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine (Kadcyla); radiotherapy or chemotherapeutic agents including, but not limited to Anthracyclines (Doxorubicin, Epirubicin, Daunorubicin, Idarubicin, Mitoxantrone), Oxaliplatin, Bortezomib, Cyclophosphamide, Bleomycin, Vorinostat, Paclitaxel, 5-Fluorouracil, Cytarabine, Prednisolone, Docetaxel, Mitomycin C, Topotecan / Camptothecin, Etoposide, Zoledronic acid, Methotrexate, Ibrutinib, Aflibercept, Bevacizumab, Toremifene, Vinblastine, Vincristine, Idelalisib, Mercaptopurine, Thalidomide, Sorafenib; a cyclic dinculeotide or other STING pathway agonist; etc.PREFERRED EMBODIMENTS

[0266] Embodiment 1. An anti-SIRPa antibody formulation suitable for parenteral administration, comprising: an anti-SIRPa antibody that inhibits signaling through the SIRPa-CD47 axis at a concentration of between about 20 mg / mL to about 100 mg / mL; a buffering component selected from the group consisting of about 20 mM L- histidine, and 20 mM sodium phosphate; a disaccharide comprising an a-glycosidic linkage at a concentration of about 8% w / v; about 0.01 wt % polysorbate 20; and a pH of about 5.7 to about 6.3; wherein is free of glycine, arginine, carbonate, HEPES, citrate, and acetate.

[0267] Embodiment 2. An antibody formulation according to embodiment 1, wherein the anti-SIRPa antibody is at a concentration of about 20 mg / mL.

[0268] Embodiment 3. An anti-SIRPa antibody formulation according to embodiment 1 or 2, wherein the anti-SIRPa antibody binds to a cell expressing human SIRPaVl protein with an EC 50 < 10 nM; binds to a cell expressing human SIRPaV2 protein with an EC50 < 10 nM; exhibits at least a 100-fold higher EC50 for SIRPaVl (P74A) having the sequence of SEQ ID NO: 62 as compared to the EC50 for human SIRPaVl protein; andexhibits at least a 100-fold higher EC50 for human SIRPpi protein as compared to the EC50 for human SIRPaVl protein.

[0269] Embodiment 4. An anti-SIRPa antibody formulation according to one of embodiments 1-3, wherein the anti-SIRPa antibody comprises one of the following combinations of heavy chain sequence and light chain sequence:SEQ ID NO: 80 and SEQ ID NO: 90,SEQ ID NO: 80 and SEQ ID NO: 92,SEQ ID NO: 80 and SEQ ID NO: 94,SEQ ID NO: 80 and SEQ ID NO: 96,SEQ ID NO: 80 and SEQ ID NO: 98, orSEQ ID NO: 80 and SEQ ID NO: 100.

[0270] Embodiment 5. An anti-SIRPa antibody formulation according to embodiment 4, wherein the anti-SIRPa antibody formulation consists of 20 mg / mL anti- SIRPa antibody, 20 mM histidine, 8% w / v sucrose, 0.01% wt % polysorbate 20 at a pH of about 6.0

[0271] Embodiment 6. An anti-SIRPa antibody formulation according to one of embodiments 1-5, wherein the anti-SIRPa antibody is ADU-1805.

[0272] Embodiment?. A single-use or multi-use vial comprising the anti-SIRPa antibody formulation of one of embodiments 1-6.

[0273] Embodiment 8. A pre-filled syringe, autoinjector, or injector pen comprising the anti-SIRPa antibody formulation of one of embodiments 1-6.

[0274] Embodiment 9. A method of administering an anti-SIRPa antibody to an individual in need thereof comprising administering the anti-SIRPa formulation of one of embodiments 1-6 by a subcutaneous or intravenous administration route into the individual.

[0275] Embodiment 10. A method according to embodiment 9, wherein the method comprises repeating the administration on at least an every three week (Q3W) schedule for at least 2 administration cycles.

[0276] Embodiment 11. A method according to embodiment 9 or 10, wherein the administration route is intravenous administration.

[0277] Embodiment 12. A method according to one of embodiments 9-11, wherein the administered dose of anti-SIRPa antibody is 1 mg / kg body weight, 2 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, or 30 mg / kg body weight.

[0278] Embodiment 13. A method according to one of embodiments 9-12, wherein the anti-SIRPa antibody formulation is diluted in normal saline or 5% dextrose in water prior to intravenous administration.

[0279] Embodiment 14. A method according to embodiment 13, wherein the anti- SIRPa antibody concentration of the diluted anti-SIRPa antibody formulation is between 1 mg / mL and 10 mg / mL.

[0280] Embodiment 15. A method according to one of embodiments 9-14, wherein the individual in need thereof has a histologically and / or cytologically confirmed diagnosis of cancer.

[0281] Embodiment 16. A method according to embodiment 15, wherein the cancer is a solid tumor.

[0282] Embodiment 17. A method according to embodiment 16, wherein the solid tumor is adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, ductal carcinoma, angiosarcoma, osteosarcoma, fibroblastic sarcoma, rhabdomyosarcoma, blastoma, melanoma, or germ cell carcinoma.

[0283] Embodiment 18. A method according to one of embodiments 9-17, wherein the anti-SIRPa antibody formulation is administered as a combination therapy with a checkpoint inhibitor.

[0284] Embodiment 19. A method according to embodiment 18, wherein the checkpoint inhibitor is a PD-1 or PD-L1 checkpoint inhibitor.

[0285] Embodiment 20. A method according to embodiment 19, wherein the checkpoint inhibitor is pembrolizumab.

[0286] Embodiment 21. A method according to embodiment 20, wherein the administered dose of pembrolizumab is 200 mg.

[0287] Embodiment 22. A method according to embodiment 17, wherein the anti- SIRPa antibody formulation and the pembrolizumab are each administered to theindividual by intravenous administration within a 12 hour period to provide the combination therapy.GENERAL METHODS

[0288] Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2ndEdition, 2001 3rdEdition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0289] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. ( .QQ ) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0290] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane ( \ iUL) Antibodies A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160: 1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272: 10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0291] An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1 :837-839; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol. Today 21 :371- 377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0292] Single chain antibodies and diabodies are described (see, e.g., Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al. (2001 ) . / . Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol. Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods 231 : 177-189; and U.S. Pat. No. 4,946,778).Bifunctional antibodies are provided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al. (2001) Protein Engineering 14:815-823; Segal, et al. (2001 ) . / . Immunol. Methods 248: 1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al. (1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229: 1202-1207; Traunecker, et al. (1991) EMBO J. 10:3655- 3659; and U.S. Pat. Nos. 5,932,448, 5,532,210, and 6,129,914).

[0293] Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998) J. Immunol. 161 :3493; Kita et o / . (1999) J. Immunol. 162:6901; Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276: 12999; Propst et al. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).Purification of antigen is not necessary for the generation of antibodies. Animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals, and the splenocytes can fused with a myeloma cell line to produce a hybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al.(2000) Immunity 13:233-242; Preston et al. , supra, Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0294] Antibodies can be conjugated, e.g., to small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes, and include antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g., colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

[0295] Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nded:, Wiley -Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0296] Standard methods of histology of the immune system are described (see, e.g., Muller-Harmelink (ed.)Human Thymus: Histopathology and Pathology, SpringerVerlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0297] Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (see, e.g., GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16: 741- 742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).EXAMPLES

[0298] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

[0299] Example 1 : ADU-1805 Formulation Development

[0300] ADU-1805 is a recombinant, humanized, IgG2 mAb that blocks the interaction of SIRPa-CD47 interaction by binding to SIRPa. The biological activity of ADU-1805 is measured by the ability to bind to recombinant human SIRPa and is quantified using an enzyme-linked immunosorbent assay (ELISA).

[0301] The ADU-1805 DP formulation was selected based on a DS formulation development program including a pre-formulation screening study. During the formulation development study, formulations with different combinations of excipients and surfactants in different pH / buffer systems were screened and evaluated. In the preformulation screening, 20 mg / mL ADU-1805 was formulated with a range of buffers (Sodium Acetate, Citrate-Phosphate, Histidine, Sodium Phosphate), salt strengths (135 mM NaCl, 0 mM NaCl), stabilizers (Sucrose, Sorbitol, Glycine, Arginine, Glutamate) and pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5). Before and after shake stress and freeze / thaw stress conditions several physical analyses (automated visual appearance, sub-visible particles, Differential Scanning Calorimetry (DSC) and Dynamic Light Scattering (DLS)) were executed to select the best conditions to progress. In the next stage of formulation development, ten ADU-1805 formulations prepared in sodium phosphate buffer and 0.01 % Polysorbate 20 over the range of pH 6.0 to 7.0, containing different concentrations (3 % or 5 %) of either Sorbitol, Trehalose or Sucrose and either Glycine or Arginine (50 mM or 75 mM) and one formulation containing 20 mM Histidine, 8 % Sucrose (w / v) and 0.01 % Polysorbate 20 were selected to be evaluated in a stability study over 4 weeks (see Table 7). The samples were subjected to a buffer exchange, up-concentrated to the protein-target concentration of 20 mg / mL and 0.2 pm filtered. The 11 batches were then stored in aliquots at - 70 °C, 5 °C, and 40 °C to study their respective stability by determination of appearance, purity, aggregation level and quality assessments.Additionally, a freeze / thaw study was conducted including three freeze / thawing cycles.

[0302] Table 7: Screening formulation study

[0303] After 4 weeks, the top four formulations (1, 2, 3, and 5 in Table 7) were selected based on appropriate protein stability indicating properties (evaluated by visual appearance, sub-visible particles, dynamic light scattering, differential scanning calorimetry, pH, Osmolality, GC-MALS and purity / impurity profile by SEC and icIEF (charged variants)) and investigated further for a total of 13 weeks in a stability study (-70 °C, 5 °C, and 40 °C condition (8 weeks)).

[0304] Superior protein stability was observed in formulation 1 (consisting of 20 mM Histidine, 8 % Sucrose (w / v), 0.01 % Polysorbate 20 at pH 6.0), which was used as final formulation for GLP and GMP material. In order to provide conditions that support the stability of the ADU-1805 DP, the pH is targeted at 6.0, with a specification range of 5.7 to 6.3. The pH is buffered by the 20 mM L-histidine solution. In order to assess the biological activity at appropriate dose levels, ADU-1805 DP is formulated at a concentration of 20 mg / mL. The desired concentration is achieved at the DS stage.

[0305] ADU-1805 is manufactured, packaged, and labeled according to GoodManufacturing Practice (GMP) regulations.

[0306] Example 2: Use of ADU-1805 in adults with solid malignant tumors

[0307] This open-label, multicenter, multi-arm dose-escalation evaluates the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of ADU-1805 as monotherapy andin combination with pembrolizumab (anti-PD-1 antibody). The primary objective of the study is to determine the RP2D of ADU-1805 monotherapy and ADU-1805 plus pembrolizumab administered by IV infusion Q3W.

[0308] The study starts with the ADU-1805 monotherapy dose escalation arm. The ADU-1805 monotherapy dose escalation design is a recently proposed i3+3 design

[0046] until the ADU-1805 monotherapy recommended phase II dose (RP2D) is defined. The RP2D is represented by the dose that is tolerated while not exceeding the maximum tolerated dose (MTD). The RP2D is selected on the basis of a cumulative review of available safety and tolerability data, and may also include PK, target engagement level, PD, and other available data.

[0309] The ADU-1805 plus pembrolizumab dose escalation arm starts after clearance of the ADU-1805 monotherapy dose level achieving maximum target engagement (e.g > 90% target engagement). The starting dose for ADU-1805 plus pembrolizumab is at least one dose level lower compared to the highest dose level declared safe, during DLT assessment, in the ADU-1805 monotherapy dose escalation phase. The starting dose is selected by SRC based upon safety, PK and target engagement data collected in monotherapy arm. The pembrolizumab dose remains fixed to 200 mg every 3 weeks, while subjects are assigned to an ADU-1805 dose level. An i3+3 design is used; all terms as described above for ADU-1805 monotherapy dose escalation are applicable for the ADU-1805 plus pembrolizumab dose escalation arm. The ADU-1805 plus pembrolizumab dose escalation arm occurs independently of ADU-1805 monotherapy dose escalation arm but any ADU-1805 dose level studied in combination would have to be deemed safe as ADU-1805 monotherapy.

[0310] Overall, up to 90 subjects receive treatment with ADU-1805 at approximately 15-20 sites globally. Safety expansion phase is defined in a future substantial protocol amendment after the ADU-1805 monotherapy and / or ADU-1805 plus pembrolizumab RP2D is determined and will begin after having received the required approvals. Subject safety is monitored throughout the study by a Safety Review Committee (SRC).

[0311] A study design is depicted in Fig. 1. Objectives and endpoints are described in Table 8.

[0312] Table 8:Characterize the pharmacodynamics (PD) of ADU-1805 monotherapy and ADU-1805 plus pembrolizumab by assessing changes from baseline in selected blood and tissue biomarkers (immune monitoring)Explore preliminary clinical activity of ADU-1805 monotherapy and ADU-1805 plus pembrolizumab by occurrence of overall response, duration of response, disease control, duration of disease control and progression-free survival per the Response Evaluation Criteria in Solid Tumors (RECIST) vl.l and modified RECIST 1.1 for immune-based therapeutics (iRECIST); assess overall survivalInvestigate the mechanism of action of ADU-1805 monotherapy and ADU-1805 plus pembrolizumab using tumor tissue and peripheral blood-based biomarkers.

[0313] Enrollment Plan and Dose Escalation Rules

[0314] The ADU-1805 monotherapy dose escalation arm uses the recently proposed i3+3 design as shown in Fig. 3. Dose escalation proceeds at the proposed dose levels following the dose escalation rules. Cohorts of 3-9 evaluable subjects are treated with ADU-1805 until the RP2D or maximum tolerated dose (MTD) can be determined.

[0315] Subjects are considered evaluable for dose-escalation assessment if the DLT evaluation period (Cycle 1 Day 1 through Cycle 2 Day 1; 21 days) is completed, or the subject experiences a DLT-defining event. If the subject is not administered the full dose, or could not complete scheduled assessments (due to reasons other than toxicities) in the DLT evaluation period (e.g. disease progression, missed appointments, non-compliance, subject withdrawal), the subject is considered non-evaluable for DLT and will be replaced with a new subject.

[0316] In the i3+3 dose escalation part, the first subject in the first cohort is observed for one week after the Cycle 1 Day 1 dose, prior to the second subject receiving their first dose. All subsequent subjects enrolled into the cohort and subsequent cohorts has their Cycle 1 Day 1 dose staggered by at least 24 hours. Evaluation of at least 3 evaluable subjects is required prior to determining the dose for the next cohort.

[0317] To proactively ensure at least 3 subjects are considered to be evaluable, a 4thsubject may be enrolled and treated. If 4 subjects are included, the escalation rules are modified accordingly per i3+3.

[0318] No dose escalation decisions is made until all subjects enrolled at a given dose level have completed the DLT evaluation period. If a subject experiences a DLT, the cohort may expand to 6 evaluable subjects. Up to 9 subjects may be treated at a given dose level. This cap is selected using the anticipated rates of DLTs at each dose level, which suggest that a decision to escalate or de-escalate is made with 6 subjects or less subjects in a cohort. However, discretion may be used to continue enrollment at that same dose level. Dose Escalation Phase.

[0319] ADU-1805 monotherapy dose escalation arm: A minimum of 3 subjects per dose level and up to 45 subjects are enrolled. ADU-1805 plus pembrolizumab dose escalation arm: A minimum of 3 subjects per dose level and up to 45 subjects are enrolled. To better understand the safety, tolerability, PK, and PD of ADU-1805, additional subjects may be enrolled in previously cleared cohorts (for up to a total of 9 subjects per cohort), or additional cohorts of up to 9 subjects may be enrolled atintermediate dose levels; before or while proceeding with further dose escalation, or to confirm the RP2D.

[0320] Pembrolizumab characteristics

[0321] Pembrolizumab is a sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow solution that requires dilution for intravenous infusion. Each vial contains 100 mg of pembrolizumab in 4 mL of solution. Each 1 mL of solution contains 25 mg of pembrolizumab and is formulated in: L-histidine (1.55 mg), polysorbate 80 (0.2 mg), sucrose (70 mg), and Water for Injection. Additional information on physical and chemical properties of pembrolizumab may be found in the SmPC

[0047] or national label.

[0322] ADU-1805 and pembrolizumab Infusion

[0323] ADU-1805 is diluted in 0.9% NaCl saline as described in the PharmacyManual to achieve the assigned dose level prior to administration. ADU-1805 is administered by IV infusion over approximately 2 hours for the initial dose, reducing to 90 minutes for subsequent doses provided there are no tolerability issues. ADU-1805 may be administered using central lines or other venous access devices including an in-line 0.2 mm filter. The start and stop times for each infusion is recorded.

[0324] Subjects in the ADU-1805 plus pembrolizumab arms will receive pembrolizumab (200 mg) by IV infusion over 30 (-5 / +10) minutes followed by ADU- 1805 by IV infusion. For cycle 1, ADU-1805 infusion begins approximately 90 minutes after completion of the pembrolizumab infusion. Any IRR during these 90 mins should not constitute a DLT. For Cycle 2+, the ADU-1805 infusion begins approximately 30 minutes after completion of the pembrolizumab infusion

[0325] Use of pre-medications (e.g. anti-pyretic or anti-emetic) for infusions is based on Investigator discretion, institutional guidelines, and the subject’s tolerance of prior infusions. Section 9.4.5 provides guidance for subsequent premedication recommendations following an ADU-1805 or pembrolizumab-related infusion reaction.

[0326] Dose Escalation Rules and Definitions

[0327] Dose escalation decisions are primarily based on available safety and tolerability data. Dose escalation decisions and identification of the RP2D (or MTD) is made by the SRC. The following definitions and dose escalation rules are followed:Maximum -tolerated Dose (MTD) is defined as an acceptable level of toxicity as evidenced by a DLT rate of 30% and an indifference interval of 0.25 to 0.35. The MTD is the dose with DLT rate closest and lower than 35%. This is determined at the end of the trial.Recommended Phase 2 Dose (RP2D) is represented by the dose that is tolerated while not exceeding the maximum tolerated dose (MTD). The RP2D is selected on the basis of a cumulative review of available safety and tolerability data, and may also include PK, target engagement level, PD, and other available data.If DLTs are observed, a traditional step-up step-down ruleset (i3+3) is triggered; escalation is stopped if the MTD appears to have been reached. Subjects who are enrolled into the cohort exceeding the MTD will continue treatment at a dose reduced to the next lowest dose level.Dose escalations is based on the dose levels that have been selected to represent 2 to 10-fold increases. Dose escalations will account for observed safety in the preceding cohort and will allow for appropriate dose escalation to potentially therapeutically effective dose-levels.Escalation is stopped if ADU-1805 exposure has not increased and / or is not expected to increase to a clinically relevant extent.

[0328] Dose Limiting Toxicity

[0329] During dose-escalation, the DLT evaluation period is defined as the first 21 days of treatment (Cycle 1 Day 1 through Cycle 2 Day 1). The occurrence of any of the toxicities presented in Table 9 is considered a DLT unless clearly and incontrovertibly due to disease progression or extraneous causes.

[0330] Table 9: Criteria for Dose-limiting Toxicities

[0331] DLTs should be assessed and reported. Toxi cities that occur outside the DLT evaluation period are recorded and considered in decisions regarding the RP2D. Emergent safety data will continue to be evaluated throughout the study on an ongoing basis by the SRC.

[0332] ADU-1805 plus Pembrolizumab Dose Escalation

[0333] The ADU-1805 plus pembrolizumab dose escalation arm starts after clearance of the ADU-1805 monotherapy dose level achieving maximum target engagement (e.g > 90% target engagement). The starting dose is selected by SRC based upon safety, PK and target engagement data collected in monotherapy arm. The starting dose for ADU-1805 plus pembrolizumab is at least one dose level lower compared to the highest dose level declared safe, during DLT assessment, in the ADU-1805 monotherapy dose escalation phase. The pembrolizumab dose will remain fixed to 200 mg every 3 weeks, while subjects are assigned to an ADU-1805 dose level. The i3+3 design will also be used; The Dose Escalation Rules and Definitions and Dose Limiting Toxicity terms, reporting and assessment as described above for ADU-1805 monotherapy dose escalation are applicable for the ADU-1805 plus pembrolizumab dose escalation arm. For ADU-1805 pluspembrolizumab dose escalation phase late toxicities identified at least 90 days post last study dose are considered in the determination of the maximal tolerated dose / RP2D and dose schedule. The ADU-1805 plus pembrolizumab dose escalation arm will occur independently of ADU-1805 monotherapy dose escalation arm but any ADU-1805 dose level studied in combination would have to be deemed safe as ADU-1805 monotherapy.

[0334] Enrollment Plan and Dose Escalation Rules

[0335] Enrollment in ADU-1805 plus pembrolizumab dose-escalation cohorts follow the i3+3 dose escalation plan as specified in Table 10. Dose escalation proceeds at the proposed dose levels following the dose escalation rules.

[0336] Table 10: ADU-1805 plus pembrolizumab Dose-Escalation Enrollment Plan

[0337] During dose escalation, the first subject in the first cohort is observed for one week after the Cycle 1 Day 1 dose, prior to the second subject receiving their first dose. All subsequent subjects enrolled into the cohort and subsequent cohorts has their Cycle 1 Day 1 dose staggered by at least 24 hours. Cohorts of 3-9 evaluable subjects are treatedwith ADU-1805 plus pembrolizumab (200 mg) until the RP2D or maximum tolerated dose (MTD) appears to have been achieved.

[0338] Subjects are considered evaluable for dose-escalation assessment if both scheduled doses (ADU-1805 and pembrolizumab) are administered and the DLT evaluation period (Cycle 1 Day 1 through Cycle 2 Day 1; 21 days) is completed, or the subject experiences a DLT-defining event. If the subject is not administered both full doses, or could not complete scheduled assessments (due to reasons other than toxicities) in the DLT evaluation period (e.g. disease progression, missed appointments, non- compliance, subject withdrawal), the subject is considered non-evaluable for DLT and is replaced with a new subject.

[0339] Evaluation of a cohort of at least 3 evaluable subjects is required prior to determining the dose for the next cohort. To proactively ensure at least 3 subjects are considered evaluable, a 4thsubject may be enrolled and treated. No dose escalation decisions is made until all subjects enrolled at a given dose level have completed the DLT evaluation period. If one subject experiences a DLT, the cohort may expand to 6 evaluable subjects. Up to 9 subjects may be treated at a given dose level. This cap is selected using the anticipated rates of DLTs at each dose level, which suggest that a decision to escalate or de-escalate is made with 6 subjects or less subjects in a cohort. However, discretion may be used to continue enrollment and that same dose level.

[0340] Safety expansion

[0341] Safety expansion arm is defined in a future substantial protocol amendment after the ADU-1805 monotherapy and / or ADU-1805 plus pembrolizumab RP2D has been determined.

[0342] Study Duration

[0343] Subjects receive study drug(s) until study completion (after maximum of 35 cycles), the subject meets criteria for treatment discontinuation (Section 8.3.2), consent is withdrawn or Sponsor termination of the study, whichever occurs first.

[0344] Following the End of Treatment Visit, subjects will complete 4 follow-up visits for safety monitoring, disease evaluations (as applicable), and survival assessment. The end of the subject participation is defined as the date when a subject has completed the final protocol-specified safety assessment and / or discontinued study participation (withdrawal of consent or lost to follow-up), whichever occurs first.

[0345] End of Study

[0346] The end of the study is defined as the date when all subjects have completed the final protocol-specified safety assessment (i.e. Follow Up 4) and / or discontinued study participation (withdrawal of consent or lost to follow-up), whichever occurs first. Following the in-clinic portion of study; additional biomarker analyses may take place prior to Clinical Study Report submission.

[0347] The study may be terminated at any time for any reason. Should the study be terminated, subjects is contacted to complete the End of Treatment (EOT) visit and protocol -defined safety follow-up procedures.

[0348] Stopping Rules

[0349] Subjects are monitored throughout the study for DLTs; provisions are in place for dose escalation, dose modification, and oversight by the SRC. If the SRC considers the safety profile, exposure, and / or biological activity of ADU-1805 inadequate, enrollment may be stopped or the study may be terminated at any time.

[0350] The following additional safety rules apply outside the 21 -day DLT evaluation period to further mitigate potential risk to subjects:Grade 5 toxicity (death) in any subject within 21 days of receipt of study drug(s) unless clearly related to an alternative cause other than study drug.DLT rates indicating that the MTD dose may have been exceeded.

[0351] Should these events occur the SRC evaluates available data within 72 hours of notification of the event(s) and recommends whether to stop the study, suspend dosing and / or enrollment, or determine whether additional dose adjustments are warranted.

[0352] Eligibility

[0353] Eligible subjects receive study drug(s) every 3 weeks (Q3W) intravenously until disease progression, development of unacceptable toxicity, withdrawal of consent, study completion (after maximum of 35 cycles) or termination of the study, whichever occurs first. Following the End of Treatment Visit, subjects complete 4 follow-up visits for safety monitoring, disease evaluations (as applicable), and survival assessment. The end of the subject participation is defined as the date when a subject has completed the final protocol-specified safety assessment and / or discontinued study participation (withdrawal of consent or lost to follow-up), whichever occurs first.

[0354] General Inclusion CriteriaIndividuals eligible to participate in this study must meet the following criteria:Male or female aged >18 years at the time consent is obtainedHistologically and / or cytologically confirmed diagnosis of metastatic or unresectable solid tumors that are refractory to standard therapy or for which no standard therapy exists. Subjects who are considered intolerant of or ineligible for standard therapy(ies), may also be eligible.Measurable disease according to RECIST (vl.l) [NOT required during Dose Escalation]Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1Adequate organ and marrow function at Screening, as defined by the following laboratory parameters (Table 11)

[0355] Table 11 :a If receiving anticoagulant therapy, values should be within therapeutic range of intended use.Provide written informed consent prior to any study-related procedures and is willing and able to comply with all study procedures.Women of childbearing potential (WOCBP) and fertile males with WOCBP partners must use highly effective contraception per (CTFG 2020)] throughout the study and for 4 months following the last dose of study drug.Affiliated with, or the beneficiary of, a social security system (France only)

[0356] General Exclusion Criteria

[0357] Individuals who meet any of the following exclusion criteria will not be eligible to participate in the study:Patients that suffer from melanoma, brain tumors, glioblastoma, sarcoma and pancreatic ductal adenocarcinoma (PDAC) are to be excluded.Active untreated brain metastases. Subjects with prior brain metastases treated and who are clinically stable and do not require corticosteroid treatment between 2 weeks prior to the first screening visit of SRP-22C102 study and first dosing of ADU-1805 are allowed.Prior biological agents, including monoclonal antibodies and immunotherapies, within 28 days prior to the first dose of ADU-1805.Prior chemotherapy, targeted small molecule therapy, hormonal therapy, or radiation therapy within 21 days prior to the first dose of ADU-1805 and within 42 days for nitrosoureas and mitomycin C. Ongoing bisphosphonate therapy is allowed for supportive / palliative care.Prior treatment with anti- SIRPa or anti-CD47-directed therapy.Participated in any other study in which receipt of an investigational new drug or investigational device occurred within 28 days of first dose of ADU-1805 (with the exception of COVID-19 vaccines approved by emergency use authorization)Active infection requiring systemic therapy. Prior oral or IV antibiotics, antifungals, or antiviral medications must be discontinued at least 14 days before the first dose of ADU-1805.Impaired cardiac function or clinically significant cardiac disease, including any of the following:Left ventricular ejection fraction (LVEF) < 50% as measured by an echocardiogram (ECHO) or multigated acquisition (MUGA) scan at Screening.Clinically significant cardiac arrhythmia.Uncontrolled hypertension.Acute myocardial infarction or unstable angina pectoris <6 months prior to the first dose of ADU-1805.QT interval corrected for heart rate using Fridericia’s formula (QTcF) >470 msec at Screening.Marked limitation of physical activity due to symptoms, or unable to carry on any physical activity without discomfort (i.e. New York Heart Association Functional Class III-IV).Current (unresolved) Grade >2 toxicity (except alopecia, ototoxicity, and peripheral neuropathy which are excluded only if Grade >3) related to prior anti-cancer therapy.History of, or current, drug-induced Grade 3 or 4 immune-related adverse reaction, including interstitial lung disease, pneumonitis, endocrinopathy, colitis, rash, nephritis, hepatitis / transaminitis, immune-related neuropathies, and encephalitis. Individuals with history of drug-induced endocrinopathy who are adequately treated with hormone replacement therapy should not be excluded.Prior severe hypersensitivity (Grade >3) to other monoclonal antibodies or ADU- 1805 excipients.If WOCBP, is pregnant, planning to become pregnant while enrolled in this study or within 4 months after the last dose of study drug, or is breastfeeding.Had major surgery (e.g. requiring general anesthesia) within 4 weeks before the planned first dose of ADU-1805, or will not have fully recovered from surgery, or has surgery planned during the time the subject is expected to participate in the study orwithin 4 weeks after the last dose of study drug. Subjects with minor planned surgical procedures to be conducted under local anesthesia within 1 week before the planned first dose of ADU-1805 may participate.Received a vaccine containing live virus within 28 days prior to the first dose of study drug. Seasonal flu vaccines that do not contain live virus are permitted. Please note that mRNA-based vaccinations are allowed if received >14 days prior to 1st dose of ADU-1805.Received a diagnosis (or tests positive at Screening) for hepatitis B or hepatitis C for which there is no clear evidence of natural immunity, immunity subsequent to vaccination, or successful eradication of the virus following antiviral therapy (individuals who are hepatitis C antibody positive or positive by anti-hepatitis B core antibody may be enrolled if negative viral load confirmed at Screening).History of human immunodeficiency virus (HIV), or tests positive for HIV, or evidence of active tuberculosis (history and / or radiology findings) at Screening. Please note that subjects who test positive for HIV at screening and have CD4+ T cell (CD4+) counts > 350 cells / uL and are not taking active or require HIV therapy are eligible for study enrollment in line with July 2020 FDA Guidance for Industry: https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / cancer- clinical-trial-eligibility-criteria-patients-hiv-hepatitis-b-virus-or-hepatitis-c-virus

[0048] ,Active other malignancy requiring treatment with the exception of any of the following:Adequately treated basal cell carcinoma.Squamous cell carcinoma of the skin, or in situ cervical cancer.Low-risk prostate cancer (i.e. Gleason score < 7 and prostate specific antigen < 10 ng / mL); orAny other cancer from which the individual has been disease-free for > 3 years.Active, known or suspected autoimmune disease or a documented history of autoimmune disease, except vitiligo or resolved childhood asthma / atopy. Individuals with vitiligo, type I diabetes, residual hypothyroidism only requiring hormone replacement, psoriasis not requiring systemic treatment or conditions not expected torecur, history of Hashimoto’s Thyroiditis on stable dose of thyroid hormone replacement therapy should not be excluded.Systemic chronic steroid therapy (>10 mg / day prednisone or equivalent) or any immunosuppressive therapy within 14 days of the first dose of study drug.Intercurrent illness that is either life-threatening or of clinical significance such that it might limit compliance with study requirements, or in the Investigator’s assessment would place the subject at an unacceptable risk for study participation.Has had an allogenic tissue / solid organ transplant.

[0358] Dosage Forms and Route of Administration:

[0359] ADU-1805 is supplied as a sterile solution intended for IV administration.ADU-1805 is diluted in 0.9% NaCl saline to the assigned dose level and administered once every 3 weeks (Q3W) by IV infusion over approximately 2 hours for the initial dose, reducing to 90 minutes for subsequent doses provided there are no tolerability issues. The subject’s weight from prior visit / cycle may be used for ADU-1805 dose calculations.

[0360] Pembrolizumab is a sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow solution that requires dilution for intravenous infusion. Each vial contains 100 mg of pembrolizumab in 4 mL of solution. Each 1 mL of solution contains 25 mg of pembrolizumab and is formulated in: L-histidine (1.55 mg), polysorbate 80 (0.2 mg), sucrose (70 mg), and Water for Injection. Subjects in the ADU- 1805 plus pembrolizumab arms will receive pembrolizumab (200 mg) by IV infusion over 30 (-5 / +10) minutes followed by ADU-1805 by IV infusion. For cycle 1, ADU-1805 infusion begins approximately 90 minutes after completion of the pembrolizumab infusion. For Cycle 2+, the ADU-1805 infusion begins approximately 30 minutes after completion of the pembrolizumab infusion.

[0361] Efficacy Analyses

[0362] Tumor Imaging and Response Assessments

[0363] Radiographic tumor evaluation includes baseline computed tomography (CT) scans with contrast of the chest, abdomen, and pelvis. Magnetic resonance imaging (MRI) is conducted if there are known or suspected CNS metastases. A bone scan is conducted for known or suspected bone metastases.

[0364] If CT scan is contraindicated (e.g. allergy to contrast dye), MRI is performed. Tumor imaging is performed using the same assessment technique throughout the study. Imaging will not be performed at EOT if conducted within previous 30 days. If a subject enters the Follow-up Period without confirmed disease progression, continue imaging throughout the Follow-up Period until documented disease progression, the start of new anti-cancer therapy, or withdrawal of consent.

[0365] For each scan, tumor measurements and assessment should be obtained using RECIST vl. l

[0032] ; response assessment is determined by the local Investigator, lesions used for paired biopsies should not be selected as target lesions for response assessment

[0033] , The investigator / local radiology review will also use iRECIST to assess tumor response and progression, and make treatment decisions.

[0366] Clinical response to study drug(s) is determined by the investigator’s assessment. Response related clinical activity endpoints is defined and analyzed according to RECIST vl. l

[0032] (primary evaluation) and iRECIST

[0033] (secondary evaluation).

[0367] Exploratory Efficacy Variables and Analyses

[0368] The following exploratory efficacy endpoints are derived and summarized descriptively for ADU-1805 monotherapy and ADU-1805 plus pembrolizumab RP2DObjective Response Rate (ORR) is defined as the proportion of subjects with a best overall response of partial response (PR) or complete response (CR) according to RECIST 1.1.Duration of response (DOR) is defined as the time from the first tumor assessment that supports the subject’s objective disease response to the time of disease progression or death due to any cause.Disease control rate (DCR) is defined as subjects with CR, PR, or stable disease (SD) per RECIST 1.1 criteria.Duration of disease control (DODC) is defined as the time from the first tumor assessment that supports the subject’s disease control (CR, PR, SD) to the time of disease progression or death due to any cause.Progression-free survival (PFS) is defined as the time from first dose of study drug(s) to first documentation of disease progression or death due to any cause.For DOR, DODC, and PFS, data is censored for subjects who do not experience progressive disease and are alive at the time of last evaluable tumor assessment.Overall Survival (OS) defined as the time from first dose of study drug(s) until date of death due to any cause. Subjects without documentation of death at the time of analysis is censored as of the date the subject is last known to be alive.PK parameters is assessed by non-compartmental analysis. The following PK parameters is calculated: maximum concentration (Cmax), time of maximum concentration (Tmax) and the area under the curve (AUCtlast). If data permit, also the terminal half-life (t 1 / 2) and thus AUCinf, AUMC (area under the first moment), CL, Vss and Vdz is determined.Analysis of PK, PD data, and PK-PD modeling and simulation may be performed in support of dose escalation analysis and in support of RP2D dose selection. These analyses is described outside the context of this protocol and SAP and may be described in a separate report.

[0369] Survival and Subsequent Anti-Cancer Therapy Follow-Up

[0370] During the Follow-up Period, vital status (survival) and subsequent cancer- related therapy are assessed until completion of the Follow-up Period, death of the subject, or withdrawal of consent.

[0371] For subjects who withdraw from the study prior to completion of the Follow-up Period, reasonable efforts are made to collect survival outcome. Sites will attempt to obtain vital status data from public records or other external sources where possible if a subject withdraws consent from study (i.e. refuse follow-up for vital status) or is documented as lost to follow up. All deaths must be reported on the eCRF.

[0372] Safety Assessments

[0373] Safety of study drug(s) is assessed by collection of data on ECOG performance status, vital signs, weight, physical examination, ECG parameters, TEAEs, concomitant medications, and routine clinical laboratory assessments. Clinically significant changes from pre-treatment values in safety assessments are reported as AEs. Safety assessments described below is conducted according to the Schedule of Events.

[0374] Eastern Cooperative Oncology Group Scale of Performance Status

[0375] The ECOG Scale of Performance Status is recognized as a standard tool to measure disease impact on daily living activities

[0034] , The ECOG scale is used by site personnel to determine eligibility and characterize a subject’s level of functioning (self- care, daily activity, and basic physical ability) as indicated.

[0376] Vital Signs

[0377] Vital signs, including blood pressure, pulse rate, respiratory rate, and temperature are obtained at each indicated visit (prior to dosing, as applicable). On dosing days, collect prior to dosing (- 10 min) and at least every 30 minutes (± 10 min) during and after the infusion until the subject has completed the required post-infusion monitoring or is considered clinically stable. Additional measurements should be obtained if clinically indicated.

[0378] Comprehensive Physical Examination

[0379] Complete physical examinations are conducted at Screening and EOT. Comprehensive physical examinations must be performed by a medically qualified individual such as a licensed Physician, Physician's Assistant, or an advanced Registered Nurse Practitioner, as local law permits.

[0380] The comprehensive physical examination includes the following organ or body system assessments: skin; head, eyes, ears, nose, and throat; thyroid; lungs; cardiovascular system; abdomen (liver and spleen); extremities; lymph nodes; and a brief neurological examination.

[0381] Symptom-directed Physical Examination

[0382] Symptom-directed physical examinations may be conducted at all other visits as indicated (up to 3 days prior to dosing). The Investigator or medically qualified designee will perform a symptom-directed evaluation as clinically indicated. The targeted physical examination includes assessment(s) of the body systems or organs, as indicated by subject symptoms, AEs, or other findings.

[0383] Weight (kilograms) is obtained at each indicated visit (prior to dosing, if applicable).

[0384] Routine 12-lead ECG is performed in triplicate after the subject has rested in a recumbent or semi-recumbent position for >5 minutes. Where indicated on dosing days, ECGs is performed pre-dose. If blood sampling or vital sign measurement is scheduledfor the same time point as an ECG recording, the procedures should be performed in the following order: ECG, vital signs, blood draw.

[0385] On dosing days additional timepoints may be assessed as clinically indicated. Additional ECGs may also be performed throughout the study if clinically indicated.

[0386] ECG parameters to be evaluated include heart rate, PR interval, QT interval, QRS duration, and QTcF (Fridericia's correction). The ECG is interpreted by the Investigator as normal, not clinically significant abnormal, or clinically significant abnormal.

[0387] Echocardiogram (ECHO) or multigated acquisition (MUGA) scan

[0388] A MUGA scan (using technetium-99m-pertechnetate) or an echocardiogram to assess left ventricular ejection fraction (LVEF) is performed at screening and clinically indicated while on study treatment. MUGA or echocardiogram scans should be performed locally in accordance with the institution’s standard practice. MUGA scans are the preferred modality; however, whichever modality is used for an individual subject at screening should be repeated for subsequent LVEF assessments for that subject as clinically indicated. LVEFs as assessed by the institution is entered onto the eCRF.

[0389] Safety Analyses

[0390] Adverse Events (Aes) are coded according to the most current version of the Medical Dictionary for Regulatory Activities (MedDRA) and assessed for severity using NCI-CTCAE v. 5.0. TEAEs is summarized by system organ class and preferred term and presented in decreasing order of frequency. Changes from baseline in ECOG, vital signs, electrocardiogram parameters, and safety laboratory parameters are examined and presented in shift tables. The incidence of potentially clinically significant (PCS) changes in laboratory parameters is summarized abnormal laboratory parameters is listed.

[0391] After signing informed consent, and prior to the first study drug administration, any medical occurrence considered related to screening procedures (e.g. tumor biopsy, venipuncture) is captured as an AE; all other medical events is captured in the subject’s medical history.

[0392] Safety reporting periods for this study are defined in Table 12. The determination, evaluation, reporting, and follow-up of AEs is performed.

[0393] Table 12: Safety Reporting Periods

[0394] Prior and Concomitant Medications

[0395] Medications used within 28 days prior to the first dose of study drug(s) are recorded as prior medications.

[0396] Any concomitant medication administered from Cycle 1 Day 1 until the EOT visit is recorded in the eCRF (including pre-medications and all over the counter medications, herbal remedies and dietary supplements). The generic name, dosage, duration, and reason for the concomitant medication should be included. Changes in the use of concomitant medications is captured at each study visit.

[0397] Following the EOT visit, concomitant medications will only be collected if associated with the management of an ongoing AE, SAE, or AESI.

[0398] Clinical Laboratory Evaluation for Safety

[0399] Routine hematology, serum chemistry, coagulation, urinalysis, and endocrine function testing is performed throughout the study as a safety measure. The Medical Monitor may, depending on study criteria, be consulted before enrollment about a potential subject with abnormal laboratory values that are not considered clinically significant by the Investigator.

[0400] The clinical significance of laboratory parameter findings is determined by the Investigator throughout the study. More frequent clinical laboratory tests may be performed if indicated by the overall clinical condition of the subject or by abnormalities that warrant more frequent monitoring.

[0401] Additional safety laboratory assessments include immunogenicity (ADA; neutralization assays).

[0402] Laboratory Assessments

[0403] Laboratory assessments on blood, urine, and tumor tissue samples are used to characterize the study population, assess safety and efficacy, and characterize PK and PD throughout the study per the Schedule of Events. Collect Screening blood samples after requisite tests for eligibility have been completed. Samples indicated on dosing days must be pre-dose and may be obtained up to 3 days before Day 1 of each dosing cycle, unless otherwise indicated.

[0404] Blood and urine samples are obtained at screening to confirm eligibility for each subject. These initial laboratory assessments are conducted at the institution’s local laboratory:Virology / Serology: Tubercolosis, HIV antibody, Hepatitis B surface antigen, antihepatitis B core antibody, Hepatitis C antibody, Hepatitis B and C viral load (if indicated)Urinalysis (dipstick): bilirubin, blood, glucose, ketones, leukocytes, nitrite, pH, protein, and specific gravityIf dipstick result is abnormal, microscopy is used to measure sediment (red blood cells, white blood cells, epithelial cells, crystals, casts, bacteria)Additional urinalysis may be performed throughout the study if clinically indicated

[0405] Safety Laboratory Assessments

[0406] Routine hematology, serum chemistry, coagulation, urinalysis, and endocrine function testing is conducted to assess eligibility and as a measure of safety per protocol requirements. All clinical laboratory evaluations are performed by the institution’s local laboratory. Laboratory assessments for continued dosing must be confirmed prior to dosing; testing may be completed up to 3 days prior to study drug administration for each cycle. Fasting is not required.

[0407] The following parameters is evaluated:Hematology: complete blood count (hematocrit, hemoglobin, platelets, red blood cells, white blood cells with differential), and CD4 lymphocyte count [absolute count and percentage]Serum chemistry: albumin, alkaline phosphatase, ALT, AST, amylase, bicarbonate, calcium, chloride, lactate dehydrogenase, lipase, sodium, potassium, creatinine, glucose, magnesium, phosphate (inorganic phosphorus), bilirubin (total, direct), blood urea nitrogen, uric acid, total proteinEndocrine function: thyroid stimulating hormone (TSH), free tri-iodothyronine (FT3), free thyroxine (FT4), adrenocorticotropic hormone (ACTH)Coagulation panel: prothrombin time (PT), activated partial thromboplastin time (aPTT), and International normalized ratio of prothrombin time (INR)

[0408] The effects of ADU-1805 on a fetus in utero or on the composition of sperm are unknown. Therefore, WOCBP and fertile males must consent to use highly effective contraception [per (CTFG 2020) while receiving study drug and for 4 months after the last dose of ADU-1805.A WOCBP is defined as any female who has experienced menarche and who has not undergone surgical sterilization (hysterectomy or bilateral oophorectomy) and is not postmenopausal. Menopause is defined clinically as 12 months of amenorrhea in a woman over age 45 years in the absence of other biological or physiological causes.For study eligibility, WOCBP must have a negative serum pregnancy test (HCG) at Screening and within 24 hours prior to first dose of study drug. A urine pregnancy test may be performed at all other indicated visits. In case of delayed menstrual period (>1 month), confirm absence of pregnancy prior to next ADU-1805 dose or next study visit, whichever occurs first. If a urine pregnancy test is positive, the results should beconfirmed with a serum pregnancy test. Pregnancy of a subject or partner must be reported and followed.

[0409] Immunogenicity (Anti-drug Antibodies)

[0410] Samples for the detection of ADA are collected and processed by sites as outlined in the Laboratory Manual. ADA to ADU-1805 is assessed by a central laboratory using a validated method. Immunogenicity testing utilizes a 3 -tiered approach; if ADA are detected, samples are also be used to assess and further establish assays for specificity confirmation (i.e. titer) and neutralizing antibodies. Serum samples collected for ADA may also be evaluated for ADU-1805 serum concentration to enable interpretation of the antibody data.

[0411] Drug Concentration Measurements (Pharmacokinetics)

[0412] Blood samples for analysis of ADU-1805 serum concentrations are obtained to characterize the PK profile of ADU-1805 following a single dose (Cycle 1 Day 1), and repeat-dosing (Cycle 2 and sparse sampling during Cycles 3-4) as indicated in Table .

[0413] Table 13: Sampling Schedule for Drug Concentration Measurements1Post-dose is defined as time from completion of ADU-1805 infusion.

[0414] Scheduled time points are relative to the start of ADU-1805 administration. For planned time points up to 48 hours post-infusion, blood samples must be taken from a peripheral vein contralateral to the arm / location into which ADU-1805 is administered. The time and date of each sample must be recorded; samples is collected and processed by sites as outlined in the Laboratory Manual. All analyses is conducted by the Sponsor or designee.

[0415] Research Laboratory Assessments

[0416] Biological samples are collected from all enrolled subjects (where not prohibited by local regulations or policies) for biomarker assessments to characterize the mechanism of action of ADU-1805. The results may help to inform dose selection and monitor baseline and on-treatment immune responses and cancer progression. The Sponsor may retain samples and their derivatives (e.g. DNA, RNA, and protein) for possible future research beyond the end of the study. Samples are collected and stored according to applicable FDA / EMA guidances (FDA 2013, 2018; EMA 2012) and will not carry personal health information. Samples are destroyed 10 years after study completion (or term defined per local policies), or if the subject withdraws consent during the study and also requests sample destruction.

[0417] Evaluations that may be performed in this study include but are not limited to:DNA sequencing of SIRPa allelesTarget engagement by ADU-1805Detection of soluble shed SIRPaDetection of CD80 and CD86 on myeloid cellsGene expression changes in tumor microenvironment and periphery (such as peripheral blood mononuclear cells)Tumor microenvironment assessment of markers associated with immune infiltrate.

[0418] ADU-1805 has been shown to bind to all human alleles of SIRPa in preclinical studies. Blood is collected to allow to study the SIRPa allele frequency in patients treated in this study. ADU-1805 via binding to SIRPa is blocking the SIRPa- CD47 interaction. To guide dose-escalation and RP2D selection, blood is collected that may be used for determining target engagement by ADU-1805. At distinct timepoints, blood is collected from dosed patients and using (isolated) blood cells, binding of fluorescently-labeled ADU-1805 is determined to study the amount of SIRPa receptor that is occupied by the administered ADU-1805 to the patients. Analyses may include detection of binding of a second anti- SIRPa antibody that does not compete with the binding of ADU-1805 to detect SIRPa membrane expression. Similarly, in serum of treated patients the amount of soluble shed SIRPa protein may be studied as soluble shed SIRPa is stabilized by binding to ADU-1805

[0039] , Based on preclinical studies that demonstrated SIRPa blockade is enhancing CD80 and CD86 expression on SIRPa expressing cells, using (isolated) blood cells from dosed patients the upregulation of these co-stimulatory molecules may be studied. To understand the impact of blocking SIRPa by ADU-1805, alone or in combination with pembrolizumab the change of tumor microenvironment may be studied using immunohistochemical approaches or RNA expression analyses of (paired) tumor biopsies.

[0419] Blood (serum, whole blood, and plasma) and tumor tissue (biopsy) samples are collected and processed by sites as outlined in the Laboratory Manual. All analyses are conducted by the Sponsor or designee.

[0420] Based on emerging data, site-specific collection feasibility, or for other operational reasons, the timing for sample collection may be adjusted, or certain samples may not be collected and / or analyzed.

[0421] Additional analytical assessments and methodologies may be used at the Sponsor’s discretion, and may include: additional phenotyping and cellular function analysis in the periphery, and gene expression changes in the tumor or periphery

[0422] Tumor Biopsies

[0423] Tumor biopsies are encouraged when feasible. If patients opt in, paired tumor biopsies are collected at pre-treatment / baseline (Screening) and on-treatment (Cycle 2 between Days 8 and 15), as applicable.

[0424] A biopsy is obtained from appropriate tumor site(s) using standard techniques to yield adequate tissue for analysis. The Investigator should select a representative lesion amenable to paired biopsies and preferably not a target lesion. The tissue sample should be adequate to enable immunohistochemistry analysis as described in the Laboratory Manual. Excision biopsies are preferred (if accessible), core needle biopsies are acceptable; fine needle aspirations will not be acceptable.

[0425] An optional post-treatment biopsy may also be collected from all subjects at the time of disease progression, provided the lesion for biopsy is accessible and tissue collection is clinically feasible. If additional biopsies or relevant samples (e.g. pleural fluid) are collected for routine care during the course of study, a sample should be retained if possible for Sponsor research evaluation.

[0426] Pharmacodynamic Markers and Indicators of Biological Activity

[0427] Blood samples to assess PD markers and indicators of biological activity are collected at time points indicated in Table 14:1Post-dose is defined as time from completion of ADU-1805 infusion.

[0428] Appropriateness of Measures

[0429] The safety parameters to be evaluated in this study include standard assessments such as recording of medical history, AEs and SAEs, physical examination, vital signs, ECGs, serum chemistry and hematology, urinalysis, concomitant medications, and other routine clinical and laboratory procedures. The DLT criteria for dose-escalation decisions are based on available ADU-1805 nonclinical data combined general practices in the development of immunotherapeutic agents. Since the safety profile of ADU-1805 has not been characterized, additional safety precautions (i.e. dosing eligibility, prespecified AESI, and extended safety-reporting period) have been included to monitor for serious risks which have been associated with other immunotherapies.

[0430] PK sampling times are driven by typical profiles observed for monoclonal antibodies delivered by IV infusion and available nonclinical data. As may occur with any therapeutic monoclonal antibody, ADU-1805 may elicit an immune response resulting in development of ADA. Subjects will therefore be monitored for the development of ADA in parallel with select PK time points to assess potential impacts on exposure, PD effects, and neutralization of activity.

[0431] A schedule of events is shown in Fig. 2. Footnotes for the figure are as follows:1 Cycle length is 21 days in dose escalation unless otherwise advised by the SRC. If a dose during Cycle 2 or beyond is delayed, shift scheduled assessments accordingly.2 EOT Visit occurs 28 days (+7 days) after the last dose of study drug or prior to commencing new anti -cancer therapy, whatever comes first.3 Follow-up: Visits may be conducted at site or via phone for vital status assessment, any subsequent cancer-related therapies, and safety follow-up, as indicated. Timing of visits should be adjusted to align with a monthly visit schedule based on date of last dose to complete the 5 month (150-day) safety reporting period. .For the combination cohort the follow up visit that is at least 90 days after the last dose of study treatment is in person to monitor for long term toxicities due to immunotherapy.4 Medical History, Height: includes demographic data as allowed by local regulations (year of birth, age, gender, ethnicity, and race). Medical history includes all active conditions and any condition considered to be clinically significant by theInvestigator. Record details of solid tumor: date of diagnosis, primary tumor histology, mutation status, prior surgery(ies), radiation therapy, chemo- and biological therapies, and stage of cancer. Obtain standing height.5 Tumor imaging: See Section

[0354] , Screening tumor assessments using CT of the chest, abdomen, and pelvis and other areas of known disease or newly suspected disease should be performed within 28 days prior to C1D1. Scans of the abdomen, pelvis, and other areas of the body may be done with MRI instead of CT, but evaluation of the chest should be done with CT. CT scans should be performed with oral and iodinated IV contrast and MRI scans with IV gadolinium chelate unless there is a medical contraindication to contrast. If iodinated IV contrast is contraindicated, chest CT should be done without IV contrast. Objective responses must be confirmed at least 4 weeks later (eg, generally at the next tumor assessment time point). Suspected PD must also be confirmed at least 4 weeks after the imaging that initially indicated progression. Perform tumor imaging at EOT if imaging not conducted within previous 30 days. If a subject enters the Follow-up Period without confirmed disease progression, perform imaging at Follow-up Visits 2 and 4, or until documented disease progression, the start of new anti -cancer therapy, or withdrawal of consent.6 Vital Signs: Blood pressure, pulse, respiratory rate, and temperature. On dosing days, collect prior to dosing (-10 min) and at least every 30 minutes (± 10 min) during and after the infusion until subject considered clinically stable; perform additional monitoring as clinically indicated.7 Physical Examination: Complete physical examinations at Screening and EOT; all other indicated visits are symptom-directed physical examinations. Assessment may be done up to 3 days prior to visit.8 Electrocardiogram (ECG): Perform routine 12-lead ECG in triplicate with subject in recumbent or semi-recumbent position after 5 minutes rest at Screening Visit, then pre-dose on Day 1 of Cycles 1 and 2, and EOT. On dosing days additional timepoints may be assessed as clinically indicated. Additional ECGs may be performed throughout the study if clinically indicated. If blood sampling or vital sign measurement is scheduled for the same time point as an ECG recording, the procedures should be performed in the following order: ECG, vital signs, blood draw.9 Adverse events: Collect from date of informed consent according to safety-reporting periods.10 Virology, Hematology, Chemistry, Coagulation, Endocrine function, Urinalysis, Pregnancy Tests (WOCBP only): Collect samples as outlined in the Laboratory Manual; testing performed at local laboratory unless otherwise indicated. Collect Screening blood samples after requisite tests for eligibility have been completed. Samples indicated on dosing days must be pre-dose and may be obtained up to 3 days before Day 1 of each dosing cycle, unless otherwise indicated.11 Immunogenicity, PK, PD Samples: Collect samples; process and ship to central laboratory for analysis per Laboratory Manual. During the Follow-up Period, only collect if subject completes visit at site.12 Immunogenicity: blood for ADA, specificity and neutralizing antibodies (as applicable); on dosing days, collect prior to dosing.13 ADU-1805 PK Samples: For planned time points up to 48 hours post infusion, blood samples for PK assessment must be taken from a peripheral vein contralateral to the arm / location into which ADU-1805 is administered. On dosing days, collect blood samples within 60 min prior to start of infusion and 30 min (± 10 min) following completion of the infusion. Additional samples on Cycle 1 Day 1 and Cycle 3 Day 1 required at 2 hours (± 15 min), and one sample between 4 and 8 hours following completion of the infusion. Record start time of infusion, completion time, and sample collection time.14 Tumor biopsy is encouraged if feasible. If patients opt in, On-treatment biopsy may be conducted between Days 8 - 15 of Cycle 2; collect an additional biopsy at disease progression if feasible. Collect samples.15 ADU-1805: Administer premedication as indicated. Administer ADU-1805 by IV infusion over approximately 2 hours for the initial dose, reducing to 90 minutes for subsequent doses provided there are no tolerability issues. Observe for at least 4 hours after the first infusion (or following any infusion-related AE) and release once considered clinically stable. For subsequent dosing, monitor at least 1 hour after infusion and release only when considered clinically stable.16 Echocardiogram (ECHO) or multigated acquisition (MUGA) to be performed at screening and when clinically indicated during ADU-1805 treatment.17 Subjects may continue to receive study treatment until disease progression, development of unacceptable toxicity, withdrawal of consent, study completion (after maximum of 35 cycles in treatment and extension phase combined) or Sponsor termination of the study.18 Pembrolizumab infusion: Administer pre-medi cation as indicated: Subjects in the ADU-1805 plus pembrolizumab dose arms will receive pembrolizumab (200 mg) by IV infusion over 30 (-5 / +10) minutes followed by ADU-1805 by IV infusion over 90 (± 15) minutes. For cycle 1, ADU-1805 infusion begins approximately 90 minutes after completion of the pembrolizumab infusion. For Cycle 2+, the ADU-1805 infusion begins approximately 30 minutes after completion of the pembrolizumab infusion.19 SIRPa genotype: Blood sample to determine the genotype of the subject at a single nucleotide polymorphism (SNP) associated with SIRPa expression. Collect sample.20 Assessments to be performed Cycle 6 and every 3 cycles after (Cycle 9, 12, 15 etc.).

[0432] Abbreviations used:ADA anti-drug antibodies humanized IgG2 selective pan-allele anti- SIRPa antibodyADU-1805 that blocks the SIRPa-CD47 innate immune checkpointAE adverse eventAESI adverse event of special interestALT alanine aminotransferase aPTT activated partial thromboplastin timeAST aspartate aminotransferaseAUC area under the curveAUMC area under the first momentCFR Code of Federal RegulationsCmax maximum concentrationCR complete responseCRA clinical research associateCT computed tomographyCI checkpoint inhibitorsCTCAE Common Terminology Criteria For Adverse EventsDCR disease control rateDLT dose-limiting toxicityDMC data monitoring committeeDOR duration of responseEAS evaluable analysis setEC ethics committeeECG el ectrocardi ogramECHO echocardiogramECOG Eastern Cooperative Oncology Group eCRF electronic case report formEDC electronic data captureEOT end of TreatmentEU European UnionFDA United States Food and Drug AdministrationFT3 free triiodothyronineFT4 free thyroxineGCP Good Clinical PracticeGLP Good Laboratory PracticeGMP Good Manufacturing PracticeHIV human immunodeficiency virusHNSTD highest non-severely toxic doseIB investigator’s brochureICF informed consent formICH International Council on HarmonisationINR International normalized ratio of prothrombin time irAE immune-related adverse eventIRB institutional review boardIRR infusion-related reaction iRECIST modified RECIST1.1 for immune-based therapeuticsIV intravenous(ly)LVEF left ventricular ejection fraction mAb monoclonal antibodyMedDRA Medical Dictionary for Regulatory ActivitiesMRI magnetic resonance imagingMRSD maximal recommended starting doseMRT mean retention timeMTD maximum tolerated doseMUGA multigated acquisitionNOAEL no observed adverse effect levelNCI National Cancer InstituteNSCLC non-small cell lung cancerORR objective response rateOS overall survivalPD pharmacodynamic(s)PDX patient-derived xenograftPFS progression-free survivalPK pharmacokinetic(s)PR partial responsePT prothrombin timeQTcF QT interval corrected (Fridericia’s)QxW once every x weeksRBCs red blood cellsRECIST response evaluation criteria in solid tumorsRP2D recommended Phase 2 doseSAE serious adverse eventSAP statistical analysis planSIRPa Signal Regulatory Protein-alphaSRC safety review committeeTl / 2 terminal half-lifeTEAE treatment-emergent adverse eventTmaxtime of maximum concentrationTTL target toxicity levelULN upper limit of normalWOCBP women of child-bearing potential

[0433] Example 3 : Animal study results and selection of dose

[0434] Consideration of the proposed starting dose for the first-in-human (FIH) study in cancer patients has taken into account the CHMP guideline on identifying and mitigating risk for such studies (EMEA / CHMP / SWP / 28367 / 07, issued 19 July 2007)

[0040] , the ICH S9 guideline on Nonclinical Evaluation for Anticancer Pharmaceuticals (ICH, 2009)

[0041] , White Paper: Strategies and Recommendations for Using a Data- Driven and Risk-Based Approach in the Selection of First-in-Human Starting Dose: An International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Assessment

[0042] , the 29-day repeated dose GLP Study (ERBC 20210125TCYPB), the cytokine release study (Prolmmune 35456) and the Translational model development ADU-1805 for FIH dose estimation (Certara report SAIR-PMX-ADU1805-3381).

[0435] Based on the guidance in the White Paper, a non-MABEL approach for setting the First-in-Human dose is chosen. This is based on the Mechanism of Action of ADU- 1805 (blocking interaction with CD47), the safety of the compound observed in the 29- day repeated dose GLP study, the lack of cytokine release in human in vitro assays as well as in vivo in cynomolgus monkeys and the presence of clinical safety data with comparable compounds. Each of these aspects is discussed below.

[0436] Taken together, despite ADU-1805 having a high level of safety, a starting dose of ADU-1805 for the FIH clinical study of 1.0 mg / kg is proposed.

[0437] In the pivotal GLP intravenous repeated dose toxicity study in cynomolgus monkeys, no adverse effects are observed up to the highest dose of 30 mg / kg. The highest non-severely toxic dose (HNSTD) was, therefore, determined to be 30 mg / kg. Using a more conservative approach and a scaling factor for translation of the monkey dose to a human equivalent dose, the HNSTD correlates with a human equivalent dose of 9.4 mg / kg. Using one-sixth of the HNSTD, as specified in the ICH S9 guideline, this human equivalent dose would support a starting dose in humans of 1.6 mg / kg. For a patient with a body weight of 70 kg this corresponds to a flat dose of 109 mg.

[0438] Table 15: MRSD calculation from the HNSTD determined in the pivotal GLP IV toxicity study in cynomolgus monkeys.MRSD = Maximal Recommended Starting Dose.Predicted human doses are expressed as flat dose, based on a patient with a body weight of 70 kg.

[0439] Based on modelling principles, the monkey PK data are used to develop a PK model. The model is extrapolated to human properties by allometrically extrapolating clearance and volume using body weight exponents of 0.85 and 1, respectively. At the MRSD in humans, the Cmax is estimated to be 32 pg / mL and the AUCo-3wk to be 884.1 h-pg / mL. (Table 10).

[0440] ADU-1805 was shown to induce no biologically relevant increases in cytokine levels in cynomolgus monkeys (GLP 29-day study) (ERBC 20210125TCYPB). In addition, in a human whole blood assay, ADU-1805 appeared to induce low / negligible levels of inflammatory cytokines and the levels of cytokine profile are comparable to Erbitux®, at concentrations up to 100 pg / mL (Study Prolmmune 34456).

[0441] Recently, clinical data are presented for 2 other SIRPa blocking mAbs dosed in human alone (BI 765063) and in combination with rituximab (CC-95251) and showed that the tolerability of these mAbs is acceptable at the high dose levels of 36 mg / kg Q3W and 20 mg / kg Q1W, respectively [43, 44],

[0442] With BI 765063 alone, in 1 of 50 patients a Grade 3 toxicity (2 instances of IRR in this patient) is observed, and other treatment-related adverse events of lower grade are IRRs (48%), fatigue (14%), headache (10%), diarrhea (8.0%) and arthralgia (8%), all not leading to treatment discontinuation. The Maximum Tolerated Dose is not reached. CC-95251 in combination with rituximab induced more pronounced treatment related effects including neutropenia (> grade 3 in 8 of 17 patients), infection, fatigue, thrombocytopenia (all > grade 3 in 3 of 17 patients) and AST elevations (> grade 3 in 4 of 17 patients). It should be noted that the SIRPa blocker CC-95251 has a reduced complement interaction related to a K322A mutation, but does retain Fey receptor interaction. However, Fey receptor interaction is strongly reduced for ADU-1805 making it more comparable to BI 765063.

[0443] The proposed starting dose of ADU-1805 for the FIH clinical study is 1.0 mg / kg. This dose level is expected to be safe and in the lower end of the potential therapeutically active dose range, based on the following considerations:Allometric scaling of PK data in monkeys is performed, including simulation of human exposure over the range of proposed dosages in the FIH study [Certara report SAIR-PMX-ADU1805-3381]. Safety margin projections based on potential systemic exposure in the FIH study are shown in Table 11. Predicted human exposures are derived after the first dose over three weeks. Safety margins are calculated relative to the observed exposures at the no observed adverse event level (NOAEL) (30 mg / kg) in the GLP repeated dose monkey toxicity study. Safety margins based on potential human exposures are high at the proposed starting dose of 1 mg / kg, at 55.8- and 137- fold based on Cmax and AUC, respectively. Positive safety margins, based on potential systemic exposure, are present up to the highest anticipated dose of 30 mg / kg (1.9 and 4.6-fold based on Cmax and AUC, respectively).In addition, total receptor occupancy at each proposed human dose is predicted. The theoretical receptor occupancy of ADU-1805 is assessed using a commonly used algorithm for calculating drug-target occupancy (based on the Michaelis-Menten equilibrium).where RO is the receptor occupancy, Ab is the concentration of antibody at a specific dose and KD is the equilibrium dissociation constant (11 nM). The RO predictions used for the proposed First-in-Human dose has been based on the values of Cav.ss. In addition, in the predictions the high Clearance value of 0.209 L / h is used (target- mediated drug disposition), which is more likely to be relevant for the First-in-Human dose.

[0444] These predictions showed that at the proposed starting dose of 1 mg / kg, the average receptor occupancy over the first three weeks after dosing would be 39.9%. Peak receptor occupancy at the Cmax following the 1 mg / kg may theoretically be higher (92.4%). Maximal average receptor occupancy (greater than 95%) over the first three weeks of dosing is achieved once the human dose is escalated to 30 mg / kg.

[0445] Table 16: Safety margin and receptor occupancy estimates at proposed dosages in the FIH study with ADU-1805.Q3W, every 3 weeks1: Assuming 70 kg patient body weight2: Average receptor occupancy over the first three weeks after dosing.3 : Parameters calculated over first three weeks after first treatment.4: Safety margins based on Day 29 exposures at 30 mg / kg NOAEL in monkeys - Cmax of 1,115 pg / mL and AUCAII of 76,087 h- pg / mL (male and female monkeys combined). (ERBC, Baugy, France; study report 20210125TCYPB).

[0446] Based on these data, a FIH starting dose of 1.0 mg / kg is proposed. This dose is expected to be safe and is assumed to be in at the lower end of the pharmacologically active dose range. Positive safety margins based on potential systemic exposure, are present up to the highest anticipated dose of 30 mg / kg.

[0447] Example 4: Sequences

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[0419] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GenelD entries), patentapplication, or patent, each of which is clearly identified in compliance with 37 C.F.R.§ 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. To the extent that the references provide a definition for a claimed term that conflicts with the definitions provided in the instant specification, the definitions provided in the instant specification shall be used to interpret the claimed invention.

[0420] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein iscome apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0421] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein iscome apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.We claim:1. An anti-SIRPa antibody formulation suitable for parenteral administration, comprising: an anti-SIRPa antibody that inhibits signaling through the SIRPa-CD47 axis at a concentration of between about 20 mg / mL to about 100 mg / mL; a buffering component selected from the group consisting of about 20 mM L- histidine, and 20 mM sodium phosphate; a disaccharide comprising an a-glycosidic linkage at a concentration of about 8% w / v; about 0.01 wt % polysorbate 20; and a pH of about 5.7 to about 6.3; wherein is free of glycine, arginine, carbonate, HEPES, citrate, and acetate.2. An antibody formulation according to claim 1, wherein the anti-SIRPa antibody is at a concentration of about 20 mg / mL.3. An anti-SIRPa antibody formulation according to claim 1 or 2, wherein the anti- SIRPa antibody binds to a cell expressing human SIRPaVl protein with an EC 50 < 10 nM; binds to a cell expressing human SIRPaV2 protein with an EC50 < 10 nM; exhibits at least a 100-fold higher EC50 for SIRPaVl (P74A) having the sequence of SEQ ID NO: 62 as compared to the EC50 for human SIRPaVl protein; and exhibits at least a 100-fold higher EC50 for human SIRPpi protein as compared to the EC50 for human SIRPaVl protein.4. An anti-SIRPa antibody formulation according to one of claims 1-3, wherein the anti-SIRPa antibody comprises one of the following combinations of heavy chain sequence and light chain sequence:SEQ ID NO: 80 and SEQ ID NO: 90,SEQ ID NO: 80 and SEQ ID NO: 92,

Claims

1. SEQ ID NO: 80 and SEQ ID NO: 94,SEQ ID NO: 80 and SEQ ID NO: 96,SEQ ID NO: 80 and SEQ ID NO: 98, orSEQ ID NO: 80 and SEQ ID NO: 100.

5. An anti-SIRPa antibody formulation according to claim 4, wherein the anti-SIRPa antibody formulation consists of 20 mg / mL anti-SIRPa antibody, 20 mM histidine, 8% w / v sucrose, 0.01% wt % polysorbate 20 at a pH of about 6.

06. An anti-SIRPa antibody formulation according to one of claims 1-5, wherein the anti-SIRPa antibody is ADU-1805.

7. A single-use or multi-use vial comprising the anti-SIRPa antibody formulation of one of claims 1-6.

8. A pre-filled syringe, autoinjector, or injector pen comprising the anti-SIRPa antibody formulation of one of claims 1-6.

9. A method of administering an anti-SIRPa antibody to an individual in need thereof comprising administering the anti-SIRPa formulation of one of claims 1-6 by a subcutaneous or intravenous administration route into the individual.

10. A method according to claim 9, wherein the method comprises repeating the administration on at least an every three week (Q3W) schedule for at least 2 administration cycles.

11. A method according to claim 9 or 10, wherein the administration route is intravenous administration.

12. A method according to one of claims 9-11, wherein the administered dose of anti- SIRPa antibody is 1 mg / kg body weight, 2 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, or 30 mg / kg body weight.

13. A method according to one of claims 9-12, wherein the anti-SIRPa antibody formulation is diluted in normal saline or 5% dextrose in water prior to intravenous administration.

14. A method according to claim 13, wherein the anti-SIRPa antibody concentration of the diluted anti-SIRPa antibody formulation is between 1 mg / mL and 10 mg / mL.

15. A method according to one of claims 9-14, wherein the individual in need thereof has a histologically and / or cytologically confirmed diagnosis of cancer.

16. A method according to claim 15, wherein the cancer is a solid tumor.

17. A method according to claim 16, wherein the solid tumor is adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, ductal carcinoma, angiosarcoma, osteosarcoma, fibroblastic sarcoma, rhabdomyosarcoma, blastoma, melanoma, or germ cell carcinoma.

18. A method according to one of claims 9-17, wherein the anti-SIRPa antibody formulation is administered as a combination therapy with a checkpoint inhibitor.

19. A method according to claim 18, wherein the checkpoint inhibitor is a PD-1 or PD-L1 checkpoint inhibitor.

20. A method according to claim 19, wherein the checkpoint inhibitor is pembrolizumab.

21. A method according to claim 20, wherein the administered dose of pembrolizumab is 200 mg.

22. A method according to claim 17, wherein the anti-SIRPa antibody formulation and the pembrolizumab are each administered to the individual by intravenous administration within a 12 hour period to provide the combination therapy.