CONSTRUCTS HAVING A SIRP-ALPHA DOMAIN OR VARIANT THEREOF

• US 20170107270A1
• Filed: 08/05/2016
• Published: 04/20/2017
• Est. Priority Date: 08/07/2015
• Status: Active Grant

• Current Assignee: ALX Oncology Inc.
• Original Assignee: Alexo Therapeutics, Inc.
• Inventors: DEMING, Laura, GOODMAN, Corey, SIM, Bang Janet, KAUDER, Steven Elliot, WAN, Hong, PONS, Jaume, KUO, Tracy Chia-Chien
• Granted Patent: US 10,259,859 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 38/177 Receptors; Cell surface antigens; Cell surface determinants
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/6811 the drug being a protein or peptide, e.g. transferrin or bleomycin
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 : Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 : Antibacterial agents
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/4703 : Inhibitors; Suppressors
• CPC Class Codes: C07K 14/70503 : Immunoglobulin superfamily
• CPC Class Codes: C07K 14/70596 : Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/00 : Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2818 : against CD28 or CD152
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 : multispecific
• CPC Class Codes: C07K 2317/524 : CH2 domain
• CPC Class Codes: C07K 2317/526 : CH3 domain
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/732 : Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present disclosure features signal-regulatory protein α (SIRP-α) polypeptides and constructs that are useful, e.g., to target a cell (e.g., a cancer cell or a cell of the immune system), to increase phagocytosis of the target cell, to eliminate immune cells such as regulatory T-cells, to kill cancer cells, to treat a disease (e.g., cancer) in a subject, or any combinations thereof. The SIRP-α constructs include a high affinity SIRP-α D1 domain or variant thereof that binds CD47 with higher affinity than a wild-type SIRP-α. The SIRP-α polypeptides or constructs include a SIRP-α D1 variant fused to an Fc domain monomer, a human serum albumin (HSA), an albumin-binding peptide, or a polyethylene glycol (PEG) polymer. Compositions provided herein include (i) a polypeptide including a signal-regulatory protein α (SIRP-α) D1 variant and (ii) an antibody.

Claims

1. A polypeptide, comprising:

a signal-regulatory protein α

(SIRP-α

) D1 variant comprising a SIRP-α

D1 domain, or a fragment thereof, having an amino acid mutation at residue 80 relative to a wild-type SIRP-α

D1 domain; and

at least one additional amino acid mutation relative to a wild-type SIRP-α

D1 domain at a residue selected from the group consisting of;

residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

2. The polypeptide of claim 1, wherein the wild type SIRP-α

D1 domain has a sequence according to any one of SEQ ID NOs;

1-10.

3. The polypeptide of claim 1, wherein the SIRP-α

D1 domain comprises between one and nine additional amino acid mutations relative to a wild-type SIRP-α

D1 domain at a residue selected from the group consisting of;

residue 6, residue 27, residue 31, residue 47, residue 53, residue 54 residue 56, residue 66, and residue 92.

4. The polypeptide of claim 1, wherein the SIRP-α

D1 variant comprises the amino acid sequence,

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO;

     49), wherein X₁ is V, L, or I;

X₂ is A, I, V, or L;

X₃ is I, F, S, or T;

X₄ is E, V, or L;

X₅ is K or R;

X₆ is E or Q;

X₇ is H, P, or R;

X₈ is L, T, S, or G;

X₉ is A; and

X₁₀ is V or I; and

wherein the SIRP-α

D1 variant has at least two amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

5. The polypeptide of claim 4, wherein the SIRP-α

D1 variant has an amino acid sequence according to any one of SEQ ID NOs;

78-85.

6. The polypeptide of claim 1, wherein the SIRP-α

D1 variant comprises the amino acid sequence,

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO;

     218), wherein X₁ is V, L, or I;

X₂ is A, V, L, or I;

X₃ is I, S, T, or F;

X₄ is E, L, or V;

X₅ is K or R;

X₆ is E or Q;

X₇ is H, R, or P;

X₈ is S, G, L, or T;

X₉ is any amino acid;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is any amino acid; and

X₁₃ is V or I; and

wherein the SIRP-α

D1 variant has at least two amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

7. The polypeptide of claim 6, wherein X₉ is A.

8. The polypeptide of claim 6, wherein X₉ is N.

9. The polypeptide of claim 6, wherein X₁₀ is I.

10. The polypeptide of claim 6, wherein X₉ is N and X₁₀ is P.

11. The polypeptide of claim 6, wherein X₉ is N and X₁₁ is any amino acid other than S, T, or C.

12. The polypeptide of claim 6, wherein X₁₁ is T.

13. The polypeptide of claim 6, wherein X₁₁ is an amino acid other than T.

14. The polypeptide of claim 6, wherein X₁₂ is P.

15. The polypeptide of claim 6, wherein X₉ is N and X₁₂ is any amino acid other than P.

16. The polypeptide of claim 1, wherein the SIRP-α

D1 variant comprises the amino acid sequence,

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO;

     219), wherein X₁ is V, L, or I;

X₂ is A, V, L, or I;

X₃ is I, S, T, or F;

X₄ is E, L, or V;

X₅ is K or R;

X₆ is E or Q;

X₇ is H, R, or P;

X₈ is S, G, L, or T;

X₉ is N;

X₁₀ is any amino acid other than P; and

X₁₁ is V or I; and

wherein the SIRP-α

D1 variant has at least two amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

17. The polypeptide of claim 1, wherein the SIRP-α

D1 variant comprises the amino acid sequence,

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO;

     52), wherein X₁ is V, L, or I;

X₂ is A, I, or L;

X₃ is I, T, S, or F;

X₄ is K or R;

X₅ is H, P, or R;

X₆ is L, T, or G; and

X₇ is A; and

wherein the SIRP-α

D1 variant has at least two amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

18. The polypeptide of claim 17, wherein X₁ is V or I, X₂ is A or I, X₃ is I or F, X₄ is K or R, X₅ is H or P, X₆ is L or T, and X₇ is A.

19. The polypeptide of claim 17, wherein the SIRP-α

D1 variant has at least three amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

20. The polypeptide of claim 17, wherein the SIRP-α

D1 variant has at least four amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

21. The polypeptide of claim 17, wherein the SIRP-α

D1 variant has at least five amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

22. The polypeptide of claim 17, wherein the SIRP-α

D1 variant has at least six amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

23. The polypeptide of claim 17, wherein the SIRP-α

D1 variant has at least seven amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to SEQ ID NO;

1.

24. The polypeptide of claim 17, wherein X₁ is I.

25. The polypeptide of claim 17, wherein X₂ is I.

26. The polypeptide of claim 17, wherein X₃ is F.

27. The polypeptide of claim 17, wherein X₄ is R.

28. The polypeptide of claim 17, wherein X₅ is P.

29. The polypeptide of claim 17, wherein X₆ is T.

30. The polypeptide of claim 17, wherein each of X₁, X₂, X₃, X₄, X₅, and X₆ is not a wild-type amino acid.

31. The polypeptide of claim 17, wherein the SIRP-α

D1 variant has an amino acid sequence according to any one of SEQ ID NOs;

81-85.

32. The polypeptide of claim 1, wherein the SIRP-α

D1 variant comprises the amino acid sequence,

EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO;

     212), wherein X₁ is V, L, or I;

X₂ is V, I, or L;

X₃ is I, T, S, or F;

X₄ is K or R;

X₅ is H, P, or R;

X₆ is S, T, or G; and

X₇ is A; and

wherein the SIRP-α

D1 variant has at least two amino acid substitutions relative to a wild-type SIRP-α

D1 domain having the sequence of SEQ ID NO;

2.

33. The polypeptide of claim 1, wherein the polypeptide binds to human CD47 with a K_D less than about 5×

10<sup>−

9</sup> M.

34. The polypeptide of claim 1, further comprising an Fc domain monomer linked to the N-terminus or the C-terminus of the polypeptide, wherein the Fc domain monomer is a human IgG1, IgG2, or IgG4 Fc region.

35. The polypeptide of claim 34, wherein the Fc domain monomer comprises at least one mutation relative to a wild-type human IgG1, IgG2, or IgG4 Fc region.

36. The polypeptide of claim 35, wherein the polypeptide has the amino acid sequence of any one of SEQ ID NO:

135, SEQ ID NO;

136, or SEQ ID NO;

137.

37. The polypeptide of claim 35, wherein the Fc domain monomer comprises:

(a) one of the following amino acid substitutions relative to wild type human IgG1;

T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I;

or

(b)(i) a N297A mutation relative to a human IgG1 Fc region;

(ii) a L234A, L235A, and G237A mutation relative to a human IgG1 Fc region;

(iii) a L234A, L235A, G237A, and N297A mutation relative to a human IgG1 Fc region;

(iv) a N297A mutation relative to a human IgG2 Fc region;

(v) a A330S and P331S mutation relative to a human IgG2 Fc region;

(vi) a A330S, P331S, and N297A mutation relative to a human IgG2 Fc region;

(vii) a S228P, E233P, F234V, L235A, and delG236 mutation relative to a human IgG4 Fc region;

or (viii) a S228P, E233P, F234V, L235A, delG236, and N297A mutation relative to a human IgG4 Fc region.

38. The polypeptide of claim 35, wherein the polypeptide exhibits a reduction of phagocytosis in a phagocytosis assay compared to a polypeptide with a wild-type human IgG Fc region.

39. The polypeptide of claim 35, wherein the Fc domain monomer is linked to a second polypeptide comprising a second Fc domain monomer to form an Fc domain dimer.

40. The polypeptide of claim 39, wherein the second Fc domain monomer is linked to an additional polypeptide.

41. The polypeptide of claim 40, wherein the additional polypeptide comprises an antibody variable domain.

42. The polypeptide of claim 41, wherein the antibody variable domain targets an antigen expressed on a cell.

43. The polypeptide of claim 42, wherein the cell is a cancer cell.

44. The polypeptide of claim 43, wherein the antibody variable domain targets a cell surface protein involved in immune cell regulation.

45. The polypeptide of claim 40, wherein the additional polypeptide comprises a therapeutic protein.

46. The polypeptide of claim 45, wherein the therapeutic protein is a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulatory agent.

47. The polypeptide of claim 45, wherein the additional polypeptide comprises a SIRP-α

D1 variant.

48. The polypeptide of claim 1, further comprising a human serum albumin (HSA) (SEQ ID NO:

12).

49. The polypeptide of claim 48, wherein the HSA comprises a C34S or K573P amino acid substitution relative to SEQ ID NO:

12.

50. The polypeptide of claim 48, wherein the polypeptide has an amino acid sequence according to any one of SEQ ID NOs:

152-159.

51. The polypeptide of claim 1, further comprising an albumin-binding peptide.

52. The polypeptide of claim 51, wherein the albumin-binding peptide comprises the amino acid sequence DICLPRWGCLW (SEQ ID NO:

160).

53. The polypeptide of claim 1, further comprising a polyethylene glycol (PEG) polymer.

54. The polypeptide of claim 53, wherein the PEG polymer is joined to a cysteine substitution in the polypeptide.

94. A method of treating an individual having a disease or disorder, the method comprising administering to the individual the polypeptide of claim 1.

95. The method of claim 94, wherein the disease or disorder is a cancer, an autoimmune disease, or an inflammatory disease.

96. The method of claim 94, wherein the disease or disorder is a cancer, and the cancer is selected from solid tumor cancer, hematological cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, bronchus cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, Merkel cell carcinoma, virally induced cancer, neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cell cancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, brain tumor, and carcinoma.

97. The method of claim 94, wherein the disease or disorder is an autoimmune disease or an inflammatory disease, and the autoimmune disease or the inflammatory disease is selected from multiple sclerosis, rheumatoid arthritis, a spondyloarthropathy, systemic lupus erythematosus, an antibody-mediated inflammatory or autoimmune disease, graft versus host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjogren'"'"'s syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn'"'"'s Disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis.

98. The method of claim 94, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

99. The method of claim 98, further comprising administration of at least one additional agent.

100. The method of claim 99, wherein the at least one additional agent is an antibody, tumor associated antigen, or a non-antibody therapeutic.

101. The method of claim 100, wherein at least two additional agents are administered.

102. The method of claim 101, wherein the at least two additional agents comprise two antibodies.

103. The method of claim 101, wherein the at least two additional agents comprise an antibody and a tumor associated antigen.

104. The method of claim 100, wherein the at least one additional agent is an antibody.

105. The method of claim 104, wherein the antibody is a human IgG1 isotype antibody.

106. The method of claim 104, wherein the antibody is a human IgG2 isotype antibody.

107. The method of claim 104, wherein the antibody is a human IgG4 isotype antibody.

108. The method of claim 104, wherein the antibody is selected from an anti-HER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-EGFR antibody, anti-PD1 antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-RANKL antibody, anti-CD274 antibody, anti-CTLA-4 antibody, anti-CD137 antibody, anti-4-1BB antibody, anti-B7-H3 antibody, anti-FZD7 antibody, anti-CD27 antibody, anti-CCR4 antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CSF antibody, anti-CD30 antibody, anti-BAFF antibody, anti-VEGF antibody, or anti-VEGFR2 antibody.

109. The method of claim 108, wherein the antibody is selected from an anti-HER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-PD-1 antibody, anti-RANKL antibody, or anti-PD-L1 antibody.

110. The method of claim 104, wherein the at least one additional agent is at least one antibody and the antibody is selected from cetuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469, atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, ipilimumab, tremelimumab, urelumab, PF-05082566, enoblituzumab, vantictumab, varlilumab, mogamalizumab, SAR650984, daratumumab, trastuzumab, trastuzumab emtansine, pertuzumab, elotuzumab, rituximab, ofatumumab, obinutuzumab, RG7155, FPA008, panitumumab, brentuximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, panitumumab, ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab, atezolizumab, durvalumab, MEDI0680, pidilizumab, or BMS-93659.

111. The method of claim 110, wherein the antibody is trastuzumab.

112. The method of claim 111, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

113. The method of claim 110, wherein the antibody is rituximab.

114. The method of claim 113, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

115. The method of claim 110, wherein the antibody is cetuximab.

116. The method of claim 115, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

117. The method of claim 110, wherein the antibody is daratumumab.

118. The method of claim 117, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

119. The method of claim 110, wherein the antibody is belimumab.

120. The method of claim 119, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

121. The method of claim 110, wherein the antibody is bevacizumab.

122. The method of claim 121, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

123. The method of claim 110, wherein the antibody is denosumab.

124. The method of claim 123, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

125. The method of claim 110, wherein the antibody is pantimumab.

126. The method of claim 125, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

127. The method of claim 110, wherein the antibody is ramucirumab.

128. The method of claim 127, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

129. The method of claim 110, wherein the antibody is necitumumab.

130. The method of claim 129, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

131. The method of claim 110, wherein the antibody is nivolumab.

132. The method of claim 131, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

133. The method of claim 110, wherein the antibody is pembrolizumab.

134. The method of claim 133, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

135. The method of claim 110, wherein the antibody is avelumab.

136. The method of claim 135, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

137. The method of claim 110, wherein the antibody is atezolizumab.

138. The method of claim 137, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

139. The method of claim 110, wherein the antibody is durvalumab.

140. The method of claim 139, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

141. The method of claim 110, wherein the antibody is MEDI0680.

142. The method of claim 141, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

143. The method of claim 110, wherein the antibody is pidilizumab.

144. The method of claim 143, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

145. The method of claim 110, wherein the antibody is BMS-93659.

146. The method of claim 145, wherein the SIRP-α

D1 variant has a sequence according to any one of SEQ ID NOs;

78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.

147. The method of claim 110, wherein the at least one additional agent is a tumor associated antigen and the tumor associated antigen elicits an immune response.

148. The method of claim 110, wherein the at least one additional agent is an antibody and the antibody targets a HLA/peptide or MHC/peptide complex.

149. The method of claim 148, wherein the antibody targets a HLA/peptide or MHC/peptide complex comprising NY-ESO-1/LAGE1, SSX-2, MAGE family (MAGE-A3), gp100/pmel17, Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, Immature laminin receptor, MOK/RAGE-1, WT-1, Her2/neu, EphA3, SAP-1, BING-4, Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1/2 (mutated), CDK4, CML66, MART-2, p53 (mutated), Ras (mutated), β

-catenin (mutated), TGF-β

RII (mutated), HPV E6, or E7.

150. The method of claim 149, wherein the antibody is ESK1, RL1B, Pr20, or 3.2G1.

157. An isolated nucleic acid encoding the polypeptide of claim 1.

158. A vector comprising the nucleic acid of claim 157.

159. A host cell comprising the nucleic acid of claim 157.

160. A method of producing a polypeptide comprising culturing the host cell of claim 159 under appropriate conditions to cause expression of the polypeptide.

161. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

55. A polypeptide, comprising:

(a) a signal-regulatory protein α

(SIRP-α

) D1 variant, wherein the SIRP-α

D1 variant comprises the amino acid sequence,

EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄TX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO;

     47), wherein X₁ is E, or G;

X₂ is L, I, or V;

X₃ is V, L, or I;

X₄ is S, or F;

X₅ is L, or S;

X₆ is S, or T;

X₇ is A, or V;

X₈ is I, or T;

X₉ is H, R, or L;

X₁₀ is A, V, I, or L;

X₁₁ is I, T, S, or F;

X₁₂ is A, or G;

X₁₃ is E, V, or L;

X₁₄ is K, or R;

X₁₅ is E, or Q;

X₁₆ is H, P, or R;

X₁₇ is D, or E;

X₁₈ is S, L, T, or G;

X₁₉ is K, or R;

X₂₀ is E, or N;

X₂₁ is S, or P;

X₂₂ is S, or R;

X₂₃ is S, or G;

X₂₄ is any amino acid;

X₂₅ is any amino acid;

X₂₆ is V, or I;

X₂₇ is F, L, or V;

X₂₈ is D or absent;

X₂₉ is T, or V;

X₃₀ is F, or V; and

X₃₁ is A, or G; and

wherein the SIRP-α

D1 variant has at least two amino acid substitutions relative to a wild-type SIRP-α

D1 domain having a sequence according to any one of SEQ ID NOs;

1 to 10; and

(b) an Fc variant comprising an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer independently is (i) a human IgG1 Fc region comprising a N297A mutation;

(ii) a human IgG1 Fc region comprising L234A, L235A, and G237A mutations;

(iii) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations;

(iv) a human IgG2 Fc region comprising a N297A mutation;

(v) a human IgG2 Fc region comprising A330S and P331S mutations;

(vi) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations;

(vii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations;

or (viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations.

56. The polypeptide of claim 55, wherein one of the Fc domain monomers in the Fc domain dimer comprises a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

57. The polypeptide of claim 55, wherein the polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs:

98-104, 107-113, 116-122, or 135-137.

58. The polypeptide of claim 55, wherein the Fc variant exhibits ablated or reduced binding to an Fcγ

receptor compared to a wild-type version of a human IgG Fc region.

59. The polypeptide of claim 55, wherein the IgG1 or IgG2 Fc variant exhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ

receptors compared to a wild-type version of a human IgG1 or IgG2 Fc region.

60. The polypeptide of claim 55, wherein the IgG4 Fc variant exhibits ablated or reduced binding to CD16a and CD32b Fcγ

receptors compared to a wild-type version of the human IgG4 Fc region.

61. The polypeptide of claim 55, wherein the IgG1 or IgG2 Fc variant exhibits ablated or reduced binding to C1q compared to a wild-type version of a human IgG1 or IgG2 Fc fusion.

62. The polypeptide of claim 55, wherein the Fc variant binds to an Fcγ

receptor with a K_D greater than about 5×

10<sup>−

6</sup> M.

63. A polypeptide, comprising:

an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer independently is selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A;

(ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A;

or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

64. The polypeptide of claim 63, wherein the two Fc domain monomers are identical.

65. The polypeptide of claim 63, wherein at least one of the Fc domain monomers is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A.

66. The polypeptide of claim 63, wherein at least one of the Fc domain monomers is a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A.

67. The polypeptide of claim 63, wherein the Fc variant exhibits ablated or reduced binding to an Fcγ

receptor compared to the wild-type version of the human IgG Fc region.

68. The polypeptide of claim 67, wherein the Fc variant exhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ

receptors compared to the wild-type version of the human IgG Fc region.

69. The polypeptide of claim 63, wherein the Fc variant exhibits ablated or reduced binding to C1q compared to the wild-type version of the human IgG Fc fusion.

70. The polypeptide of claim 63, wherein at least one of the Fc domain monomers is a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

71. The polypeptide of claim 70, wherein the Fc variant exhibits ablated or reduced binding to a Fcγ

receptor compared to the wild-type human IgG4 Fc region.

73. The polypeptide of claim 63, wherein the Fc variant binds to an Fcγ

receptor with a K_D greater than about 5×

10<sup>−

6</sup> M.

74. The polypeptide of claim 63, further comprising a CD47 binding polypeptide.

75. The polypeptide of claim 74, wherein the Fc variant exhibits ablated or reduced binding to an Fcγ

receptor compared to a wild-type version of a human IgG Fc region.

76. The polypeptide of claim 74, wherein the CD47 binding polypeptide does not cause acute anemia in rodents and non-human primates.

77. The polypeptide of claim 74, wherein the CD47 binding polypeptide does not cause acute anemia in humans.

78. The polypeptide of claim 74, wherein the CD47 binding polypeptide is a signal-regulatory protein α

(SIRP-α

) polypeptide or a fragment thereof.

79. The polypeptide of claim 78, wherein the SIRP-α

polypeptide comprises a SIRP-α

D1 variant comprising the amino acid sequence,

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO;

     221), wherein X₁ is V or I;

X₂ is A or I;

X₃ is I or F;

X₄ is E or V;

X₅ is K or R;

X₆ is H or P;

X₇ is L or T;

X₈ is any amino acid other than N; and

X₉ is V or I.

80. The polypeptide of claim 79, wherein the SIRP-α

polypeptide comprises a SIRP-α

D1 variant wherein X₁ is V or I;

X₂ is A or I;

X₃ is I or F;

X₄ is E;

X₅ is K or R;

X₆ is H or P;

X₇ is L or T;

X₈ is not N; and

X₉ is V.

72. The polypeptide of 71, wherein the Fc variant exhibits ablated or reduced binding to CD16a and CD32b Fcγ

receptors compared to the wild-type version of its human IgG4 Fc region.

81. A polypeptide, comprising:

a signal-regulatory protein α

(SIRP-α

) D1 variant, wherein the SIRP-α

D1 variant is a non-naturally occurring high affinity SIRP-α

D1 domain, wherein the SIRP-α

D1 variant binds to human CD47 with an affinity that is at least 10-fold greater than the affinity of a naturally occurring SIRP-α

D1 domain binding to human CD47; and

an Fc domain monomer, wherein the Fc domain monomer is linked to a second polypeptide comprising a second Fc domain monomer to form an Fc domain, wherein the Fc domain has ablated or reduced effector function and ablated or reduced C1q binding.

82. The polypeptide of claim 81, wherein the non-naturally occurring high affinity SIRP-α

D1 domain comprises an amino acid mutation at residue 80.

83. A polypeptide, comprising a signal-regulatory protein α

(SIRP-α

) D1 variant, wherein the SIRP-α

D1 variant binds CD47 from a first species with a K_D less than 250 nM; and

wherein the SIRP-α

D1 variant binds CD47 from a second species with a K_D less than 250 nM; and

the K_D for CD47 from the first species and the K_D for CD47 from the second species are within 100 fold of each other, wherein the first species and the second species are selected from the group consisting of;

human, rodent, and non-human primate.

84. The polypeptide of claim 83, wherein the SIRP-α

D1 variant binds CD47 from at least 3 different species.

85. The polypeptide of claim 83, wherein the non-human primate is cynomolgus monkey.

90. The polypeptide of claim 83, further comprising at least one Fc variant, wherein the Fc variant is selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A;

(ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A;

or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

91. The polypeptide of claim 90, wherein the Fc variant is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A.

92. The polypeptide of claim 90, wherein the Fc variant is a human IgG2 Fc region consisting of mutations A330S, P331S and N297A.

93. The polypeptide of claim 90, wherein the Fc variant is a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

86. A polypeptide, comprising:

(a) a signal-regulatory protein α

(SIRP-α

) D1 domain that binds human CD47 with a K_D less than 250 nM; and

(b) an Fc domain monomer linked to the N-terminus or the C-terminus of the SIRP-α

D1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates.

87. The polypeptide of claim 86, wherein the polypeptide is a non-naturally occurring variant of a human SIRP-α

D1 domain.

88. The polypeptide of claim 86, wherein administration of the polypeptide in vivo results in hemoglobin reduction by less than 50% during the first week after administration.

89. The polypeptide of claim 86, wherein administration of the polypeptide in humans results in hemoglobin reduction by less than 50% during the first week after administration.

151-156. -156. (canceled)