Construct having SIRP-alpha domain or variant thereof

Abstract

To provide a high affinity SIRP-α D1 domain or variant thereof that binds CD47 with higher affinity than wild-type SIRP-α (signal-regulatory protein α), which is useful 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.SOLUTION: The present invention provides a polypeptide, comprising: a 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.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference This application is a continuation of U.S. Provisional Patent Application No. 62 / 202,772, filed August 7, 2015. , U.S. Provisional Patent Application No. 62 / 202,775, filed August 7, 2015 U.S. Provisional Patent Application No. 62 / 202,779, filed August 7, 2016, January 8, 2016 U.S. Provisional Patent Application No. 62 / 276,801, filed December 10, 2015 U.S. Provisional Patent Application No. 62 / 265,887, filed January 8, 2016, U.S. Provisional Patent Application No. 62 / 276,796, and U.S. Provisional Patent No. filed June 6, 2016 This application claims the benefit of application Ser. No. 62 / 346,414, each of which is incorporated herein by reference. and the like, all of which are incorporated herein by reference in their entireties. Summary of the Invention [Means for solving the problem]

[0002] In certain embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or A polypeptide comprising a SIRP-α D1 variant, including a fragment thereof. In this embodiment, the wild-type SIRP-α D1 domain is a sequence selected from the group consisting of SEQ ID NOs: 1 to 10. In some embodiments, the SIRP-α D1 domain has a sequence similar to that of wild-type SIR For the P-α D1 domain, residues 6, 27, 31, 47, 53, and 1 to 9 residues selected from the group consisting of residue 54, residue 56, residue 66, and residue 92 In some embodiments, the SIRP-α D1 barrier comprises an additional amino acid mutation. The amino acid sequence is EEELQX1IQPDKSVLVAAGETATLRCTX2T SLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTV SDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSP DDVEFKSGAGTELSVRAKPS (SEQ ID NO: 49), wherein X1 is V , L, or I, X2 is A, I, V, or L, and X3 is I, F, S, or X4 is E, V, or L, X5 is K or R, and X6 is E or Q X7 is H, P, or R, X8 is L, T, S, or G, and X9 is A and X 10 is V or I, and the SIRP-α D1 variant is at least two amino acid substitutions relative to the wild-type SIRP-α D1 domain having the sequence In some embodiments, the SIRP-α D1 variant has the sequence of SEQ ID NO:7. In some embodiments, the SIRP has any one of the amino acid sequences of 8 to 85. The -α D1 variant has the amino acid sequence EEELQX1IQPDKSVLVAAGETA TLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX 7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAG TYYCX 13KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 2 18), wherein X1 is V, L, or I, and X2 is A, V, L, or I. X3 is I, S, T, or F, X4 is E, L, or V, and X5 is K or or R, X6 is E or Q, X7 is H, R, or P, and X8 is S, G , L, or T, X9 is any amino acid, and X 10 is any amino acid, X 11 is any amino acid, and X 12 is any amino acid, and X 13 is V or I and the SIRP-α D1 variant is a wild-type SIRP-α D1 variant having the sequence of SEQ ID NO: 1. At least two amino acid substitutions relative to the α D1 domain. In some embodiments, X9 is A. In some embodiments, X9 is N. In this state, X 10 In some embodiments, X9 is N and X10 is P. In some embodiments, X9 is N and X11 is any group other than S, T, or C. In some embodiments, X is an amino acid. 11 is T. In some embodiments, is X 11 is an amino acid other than T. In some embodiments, X 12 is P. In some embodiments, X is N and X 12 is any amino acid other than P. In some embodiments, the SIRP-α D1 variant has the amino acid sequence EEELQX 1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAG PGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRI GX9ITX10 ADAGTYYCX 11 KFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO: 219), wherein X1 is V, L, or I; and X2 X1 is A, V, L, or I, X2 is I, S, T, or F, and X3 is E, L, or or V, X5 is K or R, X6 is E or Q, and X7 is H, R, or is P, X8 is S, G, L, or T, X9 is N, and X 10 is a non-P X is an amino acid 11 is V or I, and the SIRP-α D1 variant is For the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 1, at least two In some embodiments, the SIRP-α D1 variant has an amino acid substitution of , amino acid sequence EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX 3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6T KRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS (SEQ ID NO: 52), wherein X1 is V, L, or I X2 is A, I, or L, X3 is I, T, S, or F, and X4 is K or R, X5 is H, P, or R, X6 is L, T, or G, and X7 is A, and the SIRP-α D1 variant is a wild-type SIRP-α D1 variant having the sequence of SEQ ID NO: 1. It has at least two amino acid substitutions relative to the RP-α D1 domain. In an embodiment, X1 is V or I, X2 is A or I, and X3 is I or F. X4 is K or R, X5 is H or P, X6 is L or T, and X 7 is A. In some embodiments, the SIRP-α D1 variant is SEQ ID NO: 1, a wild-type SIRP-α D1 domain having a sequence of at least three amino acids In some embodiments, the SIRP-α D1 variant has an amino acid substitution. For the wild-type SIRP-α D1 domain having the sequence of No. 1, at least four amino acids In some embodiments, the SIRP-α D1 variant has the sequence At least five amino acids were identified for the wild-type SIRP-α D1 domain having sequence number 1. In some embodiments, the SIRP-α D1 variant has a amino acid substitution. For the wild-type SIRP-α D1 domain having the sequence of column number 1, at least six In some embodiments, the SIRP-α D1 variant has an amino acid substitution. At least seven wild-type SIRP-α D1 domains having the sequence of SEQ ID NO: 1 In some embodiments, X1 is I. In some embodiments, X2 is I. In some embodiments, X3 is F. In some embodiments, X4 is R. In some embodiments, X5 is P. In some embodiments, X6 is T. In some embodiments, X1, X2, X3, X4, X5, and In some embodiments, each of X and X6 is not a wild-type amino acid. The D1 variant has the amino acid sequence of any one of SEQ ID NOs: 81 to 85. In some embodiments, the SIRP-α D1 variant has the amino acid sequence EEELQX1I QPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPA RELIYNQX4EG X5FPRVTTVSEX6TKRENMDFSISISX7 ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS(distribution column number 212), where X1 is V, L, or I, and X2 is V, I, or L, X3 is I, T, S, or F, X4 is K or R, and X5 is H, P or R, X6 is S, T, or G, and X7 is A, The D1 variant is a variant of the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide has at least two amino acid substitutions. is a K D Approximately 5x10 -9 In some embodiments, The polypeptide further comprises F linked to the N-terminus or C-terminus of the polypeptide. The Fc domain monomer comprises a human IgG1, IgG2, or In some embodiments, the Fc domain monomer is an IgG4 Fc region. At least one mutation has been introduced into the Fc region of native human IgG1, IgG2, or IgG4. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 135, SEQ ID NO: 13 6, or the amino acid sequence of any one of SEQ ID NO: 137. In the Fc domain monomer, (a) the following amino acid sequence is present relative to wild-type human IgG1: One of the substitutions: T366W, T366S, L368A, Y407V, T366Y, T 394W, F405W, Y349T, Y349E, Y349V, L351T, L351H , L351N, L351K, P353S, S354D, D356K, D356R, D35 6S, E357K, E357R, E357Q, S364A, T366E, L368T, L 368Y, L368E, K370E, K370D, K370Q, K392E, K392D , T394N, P395N, P396T, V397T, V397Q, L398T, D39 9K, D399R, D399N, F405T, F405H, F405R, Y407T, Y 407H, Y407I, K409E, K409D, K409T, or K409I, or or (b) (i) a mutation of N297A in the Fc region of human IgG1; For the Fc region of IgG1, mutations L234A, L235A, and G237A were added. (ii) i) L234A, L235A, G237A, and N2 for the Fc region of human IgG1 (iv) a N297A mutation in the Fc region of human IgG2; (v) a N297A mutation in the Fc region of human IgG2; (vi) A330S and P331S mutations in the Fc region of human IgG2; (vii) A330S, P331S, and N297A mutations in the Fc region of IgG1; S228P, E233P, F234V, L235A, and and delG236 mutations, or (viii) S2 against the Fc region of human IgG4 28P, E233P, F234V, L235A, delG236, and N297A mutations In some embodiments, the Fc domain The IgG monomer has (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405 W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L3 51K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368 E, K370E, K370D, K370Q, K392E, K392D, T394N, P3 95N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407 I, K409E, K409D, K409T, or K409I, and (b) the Fc The domain monomer further comprises (i) an N297A mutation relative to the Fc region of human IgG1. (ii) L234A, L235A, and G237 relative to the Fc region of human IgG1 A mutation, (iii) L234A, L235A, G2 37A and N297A mutations, (iv) N297A for the Fc region of human IgG2 (v) A330S and P331S mutations in the Fc region of human IgG2, vi) A330S, P331S, and N297A mutations in the Fc region of human IgG2 (vii) S228P, E233P, F234V in the Fc region of human IgG4 , L235A, and delG236 mutations, or (viii) the Fc region of human IgG4 In contrast, S228P, E233P, F234V, L235A, delG236, and N In some embodiments, the polypeptide comprises a mutation at position 297A. In comparison with a polypeptide having the Fc region of wild-type human IgG, In some embodiments, the Fc domain monomer is a second Fc domain monomer. The Fc domain is linked to a second polypeptide containing the Fc domain, forming an Fc domain dimer. In embodiments, the second Fc domain monomer is linked to an additional polypeptide. In some embodiments, the additional polypeptide comprises an antibody variable domain. In some embodiments, the antibody variable domain targets an antigen expressed on a cell. In some embodiments, the cell is a cancer cell. The domain targets cell surface proteins involved in immune cell regulation. In some embodiments, the additional polypeptide comprises a therapeutic protein. The therapeutic protein may be a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory In some embodiments, the additional polypeptide is an anti-inflammatory agent, or an immunomodulatory agent. In some embodiments, the polypeptide further comprises a SIRP-α D1 variant. Additionally, it comprises human serum albumin (HSA) (SEQ ID NO: 12). , the HSA contains an amino acid substitution of C34S or K573P relative to SEQ ID NO: 12. In some embodiments, the polypeptide is any one of SEQ ID NOs: 152-159. In some embodiments, the polypeptide further comprises the amino acid sequence of In some embodiments, the albumin-binding peptide comprises a DI In some embodiments, the amino acid sequence of CLPRWGCLW (SEQ ID NO: 160) is The polypeptide may further comprise a polyethylene glycol (PEG) polymer. In some embodiments, the PEG polymer is linked to a cysteine ​​substitution in the polypeptide. are.

[0003] In certain embodiments, disclosed herein is a polypeptide comprising a signal It is a variant of the signal transduction regulatory protein α (SIRP-α) D1 and has the amino acid sequence E EX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10TSL X 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FP RVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 IT X 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGT ELSVRX 31 KPS (SEQ ID NO: 47), wherein X1 is E or G and X2 is L; X3 is V, L, or I, X4 is S or F, and X5 is X6 is S or T, X7 is A or V, and X8 is I or T, X9 is H, R, or L, and X 10 is A, V, I, or L, and X1 1 is I, T, S, or F, and X 12 is A or G, and X 13 is E, V, or L and X 14 is K or R, and X 15 is E or Q, and X 16 H, P, and or R, and X 17 is D or E, and X 18 is S, L, T, or G, and X1 9 is K or R, X 20 is E or N, and X 21 is S or P, and X 22 is S or R, and X 23 is S or G, and X 24is any amino acid, and X2 5 is any amino acid, and X 26 is V or I, and X 27 is F, L, or V Ri, X 28 is D or absent, and X 29 is T or V, and X 30 F also is V and X 31 is A or G, and has any one of the sequences of SEQ ID NOs: 1 to 10. The SIRP-α D1 domain has at least two amino acid substitutions relative to the wild-type SIRP-α D1 domain. The SIRP-α D1 variant, and an Fc domain having two Fc domain monomers. It is an Fc variant containing a domain dimer, and each Fc domain monomer has its own unique (i) a human IgG1 Fc region containing the mutation N297A; (ii) a human IgG1 Fc region containing the mutation L234A; (iii) a human IgG1 Fc region containing the mutations L234A and G237A; A human IgG1 Fc region containing the mutations A, L235A, G237A, and N297A ( (iv) the Fc region of human IgG2 containing the mutations N297A, (v) A330S and P331 (vi) the Fc region of human IgG2 containing the mutations A330S, P331S, and N29 (vii) the Fc region of human IgG2 containing the mutations 7A, S228P, E233P, and F23 a human IgG4 Fc region containing the mutations 4V, L235A, and delG236, or viii) S228P, E233P, F234V, L235A, delG236, and N A polypeptide comprising the Fc variant, which is an Fc region of human IgG4 containing the 297A mutation. In some embodiments, the Fc domain monomer of the Fc domain dimer is One of them is a human Ig containing the mutations L234A, L235A, G237A, and N297A. In some embodiments, the polypeptide comprises the Fc region of SEQ ID NO: 98-1. One of the following amino acids: 04, 107-113, 116-122, or 135-137 In some embodiments, the Fc variant comprises the amino acid sequence of the Fc region of human IgG. Shows elimination or reduction of binding to Fcγ receptors compared to wild-type versions. In some embodiments, the IgG1 or IgG2 Fc variant is a human IgG1 or IgG2 Compared to the wild-type version of the Fc region of and ablation or reduction of CD64 binding to Fcγ receptors. The IgG4 Fc variant has the following characteristics compared to the wild-type version of the human IgG4 Fc region: This shows the elimination or reduction of binding of CD16a and CD32b to Fcγ receptors. In some embodiments, the IgG1 or IgG2 Fc variant is human IgG1 or Shows ablation or reduction of binding to C1q compared to wild-type versions of IgG2 Fc fusions In some embodiments, the Fc variant has K D Approximately 5x1 0 -6 Combines over M.

[0004] In certain embodiments, disclosed herein are polypeptides comprising Fc variants. The Fc variant is an Fc domain peptide having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L234A; The Fc region of human IgG1 consisting of the mutations L235A, G237A, and N297A (i i) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) S228P, E233P, F234V, L235A, delG236; and a polypeptide selected from the Fc region of human IgG4 containing the N297A mutation. In some embodiments, at least one of the Fc domain monomers has the amino acid sequence L234A, The Fc region of human IgG1 consists of the mutations L235A, G237A, and N297A. In some embodiments, at least one of the Fc domain monomers is A330S, The Fc region of human IgG2 consists of the mutations P331S and N297A. In embodiments, the Fc variants have an Fcγ In some embodiments, the Fc variant exhibits ablated or reduced binding to the Fc receptor. The Fc region of human IgG was found to be significantly higher in CD16a, CD32a, and CD32 compared to the wild-type version. b, elimination or reduction of binding of CD32c and CD64 to Fcγ receptors. In some embodiments, the Fc variant has a nucleotide sequence similar to that of a wild-type Fc fusion of human IgG. In some embodiments, the Fc domain is abolished or reduced binding to C1q. At least one of the monomers is S228P, E233P, F234V, L235A, The Fc region of human IgG4 contains the delG236 and N297A mutations. In one embodiment, the Fc variant has an Fc region that is different from the Fc region of wild-type human IgG4. In some embodiments, the Fc barrier exhibits ablated or reduced binding to γ ​​receptors. The IgG4 Fc region is expressed in a variety of CD16a and CD32b regions compared to the wild-type version. In some embodiments, the Fc The variant binds to the Fcγ receptor and D Approximately 5x10-6 Combined with M. In embodiments, the polypeptide further comprises a CD47 binding polypeptide. In embodiments, the Fc variants have a Fc region that is shorter than that of a wild-type version of the Fc region of human IgG. In some embodiments, the CD47 The binding polypeptides do not cause acute anemia in rodents and non-human primates. In some embodiments, the CD47 binding polypeptide is a compound that induces acute anemia in humans. In some embodiments, the CD47 binding polypeptide does not induce signal regulation. In some embodiments, the polypeptide is a SIRP-α polypeptide or a fragment thereof. the SIRP-α polypeptide has the amino acid sequence EEELQX1IQPDKSVLV AAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQ X5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGT YYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 51) wherein X1 is V or I and X2 is X3 is A or I, X4 is I or F, X5 is K or X6 is H or P, X7 is L or T, and X8 is any atom other than N. and X9 is V or I. In some embodiments, the SIRP-α poly The peptide is a peptide in which X1 is V or I, X2 is A or I, and X3 is I or F. X4 is E, X5 is K or R, X6 is H or P, and X7 is L or is T, including SIRP-α D1 variants in which X8 is not N and X9 is V.

[0005] In certain embodiments, disclosed herein is a polypeptide comprising a signal SIRP-α D1 variant and a non-naturally occurring high The affinity of the SIRP-α D1 domain is less than that of the naturally occurring D1 domain. the SIRP-α D1 variant that binds to human CD47 with 10-fold higher affinity than either of the SIRP-α D1 variants; and a second polypeptide comprising a second Fc domain monomer and a second Fc domain monomer; and Fc domains in which the Fc domain is removed or reduced. The polypeptides are those containing the Fc domain monomers that have vector function. In embodiments, the non-naturally occurring high affinity SIRP-α D1 domain has an amino acid sequence at residue 80. Including acid mutations.

[0006] In certain embodiments, disclosed herein is a signal regulatory protein alpha ( A polypeptide comprising a SIRP-α D1 variant, Ant, K D binds to CD47 from the first species at less than 250 nM, 1 variant, K D binds to CD47 from a second species at less than 250 nM and K against CD47 from various species D and K for CD47 from the second species. D are mutually 1 100-fold, and the first and second species are selected from the group consisting of humans, rodents, and non-human primates. In some embodiments, the SIRP-α polypeptide is selected from the group consisting of: The D1 variant binds to CD47 from at least three different species. In embodiments, the non-human primate is a cynomolgus monkey.

[0007] In certain embodiments, disclosed herein is a polypeptide comprising: )K D Signal-regulatory receptor protein alpha (SIRP-α) binds to human CD47 at less than 250 nM (a) a D1 domain, and (b) a polypeptide linked to the N-terminus or C-terminus of the SIRP-α D1 domain. It contains a fused Fc domain monomer and induces acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a human S In some embodiments, the polypeptide is a non-naturally occurring variant of IRP-α. In vivo administration of results in a decrease in hemoglobin of less than 50% during the first week after administration. In some embodiments, administration of the polypeptide in humans results in a 50% or greater decrease in leukemia-associated ... In some embodiments, the polypeptide further and at least one Fc variant, wherein the Fc variant comprises: (i) L2 The Fc region of human IgG1 consists of the mutations 34A, L235A, G237A, and N297A. (ii) the Fc region of human IgG2, which consists of the mutations A330S, P331S, and N297A region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. In embodiments, the Fc variant comprises L234A, L235A, G237A, and N29 In some embodiments, the Fc region of human IgG1 comprises a mutation at position 7A. The variant is a human IgG2 F mutant consisting of the A330S, P331S, and N297A mutations. In some embodiments, the Fc variant is S228P, E233P Human IgG4 containing the mutations F234V, L235A, delG236, and N297A The Fc region of

[0008] In certain embodiments, disclosed herein are methods for treating an individual with a disease or disorder. a method of treating a subject comprising administering to said subject a polypeptide disclosed herein. In some embodiments, the polypeptide is a wild-type signal regulator protein. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or In some embodiments, the polypeptide comprises a SIRP-α D1 variant comprising a fragment thereof. The peptide contains a signal-regulatory receptor alpha (SIRP-α) D1 variant and The SIRP-α D1 variant has the amino acid sequence EEX1X2QX3IQPDKX4VX 5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 R X 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX2 7RKGSPDX 28 X29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 4 7), wherein X1 is E or G, X2 is L, I, or V, and X3 is X4 is S or F, X5 is L or S, and X6 is S or T, X7 is A or V, X8 is I or T, and X9 is H, R, or or L and X 10 is A, V, I, or L, and X 11 is I, T, S, or F Yes, X 12 is A or G, and X 13 is E, V, or L, and X 14 is K or R and X 15 is E or Q, and X 16 is H, P, or R, and X 17 Is D or E and X 18 is S, L, T, or G, and X 19 is K or R, and X2 0 is E or N, X 21 is S or P, and X 22 is S or R, and X 23 is S or G, and X 24 is any amino acid, and X 25 is any amino acid Ri, X 26 is V or I, and X 27 is F, L, or V, and X 28 is D or not present and X 29 is T or V, and X 30 is F or V, and X 31 teeth A or G, and the SIRP-α D1 variant is any one of SEQ ID NOs: 1 to 10. For wild-type SIRP-α D1 domains with one sequence, and the polypeptide has two Fc domain monomers. and Fc variants including indimers, wherein each Fc domain monomer independently (i) the Fc region of human IgG1 containing the mutation N297A, (ii) L234A, L2 (iii) a human IgG1 Fc region containing the mutations G234A, G35A, and G237A; The Fc region of human IgG1 containing the mutations L235A, G237A, and N297A (iv ) the Fc region of human IgG2 containing the mutation N297A, (v) the mutations A330S and P331S (vi) the Fc region of human IgG2 containing the mutations A330S, P331S, and N297A; (vii) the Fc region of human IgG2 containing the mutations S228P, E233P, and F234V , L235A, and delG236 mutations in the Fc region of human IgG4, or ii) S228P, E233P, F234V, L235A, delG236, and N29 In some embodiments, the polypeptide is an Fc region of human IgG4 comprising the mutation at position 7A. The antibody comprises an Fc variant, and the Fc variant comprises two Fc domain monoclonal antibodies. and an Fc domain dimer having an Fc domain monomer, each Fc domain monomer independently having: (i ) Human IgG1 consisting of the mutations L234A, L235A, G237A, and N297A Fc region, (ii) human IgG consisting of A330S, P331S, and N297A mutations 2, or (iii) S228P, E233P, F234V, L235A, d The Fc region of human IgG4 is selected from the Fc region of human IgG4 containing the mutations e1G236 and N297A. In some embodiments, the polypeptide is signal-regulatory protein alpha (SIRP-α). D1 variant and a non-naturally occurring high-affinity SIRP-α D1 domain It binds to human CD47 with an affinity at least 10-fold higher than that of the naturally occurring D1 domain. the SIRP-α D1 variant that binds to the Fc domain monomer; and linked to a second polypeptide comprising a second Fc domain monomer to form an Fc domain. and the Fc domain model having an effector function in which the Fc domain is deleted or reduced. In some embodiments, the non-naturally occurring high affinity SIRP-α D1 domain comprises a In some embodiments, the polypeptide comprises an amino acid mutation at residue 80. comprises a signal regulatory protein alpha (SIRP-α) D1 variant, The D1 variant is K D binds to CD47 from the first species at less than 250 nM and the SIR The P-α D1 variant is K D binds to CD47 from a second species at less than 250 nM, and a K for CD47 from the first species. D and K for CD47 from the second species. D are within 100-fold of each other, and the first and second species are human, rodent, and non-human spirits. In some embodiments, the polypeptide is selected from the group consisting of (a) K D Signal-regulatory receptor protein alpha (SIRP-α) binds to human CD47 at less than 250 nM and (b) a SIRP-α D1 domain linked to the N-terminus or C-terminus of the SIRP-α D1 domain. and the polypeptide comprises an Fc domain monomer selected from rodents and non-human primates. In some embodiments, the disease or disorder is: In some embodiments, the disease or The disorder is cancer, and the cancer may be a solid tumor cancer, a blood cancer, an acute myeloid leukemia, a chronic phospholipid Myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma Lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, tracheal cancer Cancer of the bladder, liver cancer, ovarian cancer, colon and rectum cancer, stomach cancer er, 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 , virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer, renal cell carcinoma, kidney Pelvic cancer, leukemia, lymphoma, non-epithelial malignant tumors, glioma, brain cancer, and epithelial malignant tumors In some embodiments, the disease or disorder is selected from an autoimmune disease or an inflammatory disease. The autoimmune or inflammatory disease is an autoimmune or inflammatory disease, and the autoimmune or inflammatory disease is multiple sclerosis, rheumatoid arthritis, spinal Spondyloarthropathy, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune diseases, graft versus Host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjogren's syndrome, progressive Systemic sclerosis, scleroderma, acute coronary syndrome, ischemia-reperfusion, Crohn's disease, endometriosis, glomeruli Nephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis In some embodiments, the SIRP-α is selected from the group consisting of inflammatory autoimmune myositis, inflammatory bowel disease, and inflammatory autoimmune myositis. D1 variants are SEQ ID NOs: 78-85, 98-104, 107-113, 116-12 2, 135 to 137, or 152 to 159. In embodiments, the method further comprises administering at least one additional agent. In some embodiments, the at least one additional agent is an antibody, a tumor-associated antigen, or a non-antibody therapeutic agent. In some embodiments, at least two additional agents are In some embodiments, the at least two additional agents are administered two antibodies. In some embodiments, the at least two additional agents include an antibody and a tumor-associated In some embodiments, the at least one additional agent is an antibody. In some embodiments, the antibody is a human IgG1 isotype antibody. In some embodiments, the antibody is a human IgG2 isotype antibody. In some embodiments, the antibody is a human IgG4 isotype antibody. The antibodies are anti-HER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, and anti-CD38 antibody. Antibody, anti-EGFR antibody, anti-PD1 antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 Antibodies, anti-CD274 antibodies, anti-CTLA-4 antibodies, anti-CD137 antibodies, anti-4-1BB antibodies, 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 an anti-VEGFR2 antibody. In some embodiments, the antibody is selected from an anti-H ER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-PD In some embodiments, the antibody is selected from an anti-PD-L1 antibody, an anti-RANKL antibody, or an anti-PD-L1 antibody. In one embodiment, the at least one additional agent is at least one antibody, , cetuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, ME DI0680, MED16469, atezolizumab, avelumab, durvalumab, ME DI6383, RG7888, ipilimumab, tremelimumab, urelumab, PF-05 082566, enoblituzumab, vanticutumab antictumab, varlilumab, mogamalizumab ogamalizumab), SAR650984, daratumumab, trastuzumab, rastuzumab emtansine, pertuzumab, elotuzumab, Tuximab, ofatumumab, obinutuzumab, RG7155, FPA008, panitum Mab, brentuximab vedotin, MSB0010718C, belimumab, bevacizumab , denosumab, panitumumab, ramucirumab, necitumumab, nivolumab, pembrolizumab Mab, avelumab, atezolizumab, durvalumab, MEDI0680, pidilisma In some embodiments, the antibody is selected from a trans In some embodiments, the SIRP-α D1 variant is stuzumab. Numbers 78-85, 98-104, 107-113, 116-122, 135-137, or 152 to 159. In some embodiments, the antibody In some embodiments, the SIRP-α D1 variant is rituximab. Row numbers 78-85, 98-104, 107-113, 116-122, 135-137, or any one of sequences 152 to 159. In some embodiments, the antibody is cetuximab. In some embodiments, the SIRP-α D1 variant is SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137 or any one of sequences 152 to 159. In some embodiments, the antibody is daratumumab. D1 variants are SEQ ID NOs: 78-85, 98-104, 107-113, 116-12 2, 135 to 137, or 152 to 159. In embodiments, the antibody is belimumab. D1 variants are SEQ ID NOs: 78-85, 98-104, 107-113, 116-12 2, 135 to 137, or 152 to 159. In embodiments, the antibody is bevacizumab. α D1 variants are SEQ ID NOs: 78-85, 98-104, 107-113, 116- 122, 135 to 137, or 152 to 159. In some embodiments, the antibody is denosumab. α D1 variants are SEQ ID NOs: 78-85, 98-104, 107-113, 116- 122, 135 to 137, or 152 to 159. In some embodiments, the antibody is pantimumab. In embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104 , 107–113, 116–122, 135–137, or 152–159 In some embodiments, the antibody is ramucirumab. In this embodiment, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-10 4, 107-113, 116-122, 135-137, or 152-159 In some embodiments, the antibody is necitumumab. In some embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 In some embodiments, the antibody is nivolumab. In some embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 In some embodiments, the antibody is pembrolizumab. In some embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 9 8-104, 107-113, 116-122, 135-137, or 152-159 In some embodiments, the antibody is avelumab. In some embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 9 8-104, 107-113, 116-122, 135-137, or 152-159 In some embodiments, the antibody is atezolizumab. In some embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85. , 98-104, 107-113, 116-122, 135-137, or 152-1 59. In some embodiments, the antibody is durvalumab In some embodiments, the SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78 to 79. 85, 98-104, 107-113, 116-122, 135-137, or 152 In some embodiments, the antibody has the sequence of any one of MEDI0 to 159. 680. In some embodiments, the SIRP-α D1 variant is SEQ ID NO: 78-85, 98-104, 107-113, 116-122, 135-137, or In some embodiments, the antibody has any one of the sequences 152 to 159. In some embodiments, the SIRP-α D1 variant is tRNA-GAL ... Numbers 78-85, 98-104, 107-113, 116-122, 135-137, or 152 to 159. In some embodiments, the antibody In some embodiments, the SIRP-α D1 variant is BMS-93659. are SEQ ID NOs: 78 to 85, 98 to 104, 107 to 113, 116 to 122, 135 to 1 37, or any one of 152 to 159. In some embodiments, The at least one additional agent is a tumor-associated antigen, the tumor-associated antigen being a tumor-associated antigen that induces an immune response. In some embodiments, the at least one additional agent is an antibody. The antibodies target HLA / peptide or MHC / peptide complexes. In embodiments, the antibody is selected from the group consisting of NY-ESO-1 / LAGE1, SSX-2, and MAGE family members. Lee (MAGE-A3), gp100 / pmel17, Melan-A / MART-1, gp 75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature lamina Nin receptor, MOK / RAGE-1, WT-1, Her2 / neu, EphA3, SAP -1, BING-4, Ep-CAM, MUC1, PRAME, survivin, mesothelin, BRCA1 / 2 (mutation), CDK4, CML66, MART-2, p53 (mutation), Ra s (mutation), β-catenin (mutation), TGF-βRII (mutation), HPV E6, or targets HLA / peptide or MHC / peptide complexes containing E7. In embodiments, the antibody is ESK1, RL1B, Pr20, or 3.2G1. [Brief explanation of the drawings]

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of this point may be obtained by reference to the following detailed description of exemplary embodiments, in which the principles of the present invention are utilized, and the accompanying drawings, in which: Reference to the detailed description is provided. [Figure 1] FIG. 1 shows a SIRP-α construct comprising a SIRP-α D1 domain or variant thereof tethered to a first Fc domain monomer that forms an Fc domain with a second Fc domain monomer. [Figure 2] FIG. 1 shows a SIRP-α construct comprising a SIRP-α D1 domain or a variant thereof tethered to a first Fc domain monomer and an antibody variable domain tethered to a second Fc domain monomer, wherein the first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain. [Figure 3] FIG. 1 shows a SIRP-α construct comprising a SIRP-α D1 domain or a variant thereof tethered to a first Fc domain monomer and a Therapeutic protein tethered to a second Fc domain monomer, wherein the first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain. [Figure 4]Figure 4A shows a SIRP-α construct comprising a SIRP-α D1 domain or variant thereof tethered to a first Fc domain monomer having a knob mutation, which forms an Fc domain with a second Fc domain monomer having a hole mutation. Figure 4B shows a SIRP-α construct comprising a SIRP-α D1 domain or variant thereof tethered to a first Fc domain monomer having a hole mutation, which forms an Fc domain with a second Fc domain monomer having a knob mutation. [Figure 5] Figure 5A shows a SIRP-α construct comprising a SIRP-α D1 domain or variant thereof tethered to an Fc domain monomer, and Figure 5B shows a SIRP-α construct that is a homodimer of the construct shown in Figure 5A. [Figure 6] 1 shows SPR binding data for monofunctional and bifunctional SIRP-α constructs containing the SIRP-α D1 domain. [Figure 7] 1 shows phagocytosis of DLD-1-GFP-luciferase tumor cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 8] 1 shows phagocytosis of DLD-1-GFP-luciferase tumor cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 9] 1 shows phagocytosis of DLD-1-GFP-luciferase tumor cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 10] 1 shows the stability of the half-life of SIRP-α polypeptides over time. [Figure 11] 1 shows hemagglutination assay data for SIRP-α polypeptide constructs. [Figure 12] Figure 1 shows survival curves for mouse syngeneic tumor models treated with SIRP-α polypeptide constructs and anti-mPD-L1. [Figure 13]Figure 10 shows tumor volume analysis of a mouse syngeneic tumor model treated with SIRP-α polypeptide constructs in combination with anti-mPD-L1. [Figure 14] Binding of various concentrations of C1q complement to SIRP-α-Fc fusions is shown. [Figure 15] 1 shows phagocytosis of MM1R cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 16] 1 shows phagocytosis of MM1R cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 17] 1 shows phagocytosis of N87 cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 18] 1 shows the molecular weight analysis of the SIRP-α D1 variant with the P83V mutation. [Figure 19A] Figure 1 shows tumor growth of human GFP-Luc-Raji lymphoma cells in the NOD scid gamma (NSG) mouse model of cancer treated with various SIRP-α constructs with or without rituximab. [Figure 19B] 19B shows a scatter plot of tumor volumes for the tumors shown in FIG. 19A. [Figure 19C] Hemoglobin levels of treated mice shown in Figure 19A are shown. [Figure 20] Shown are red blood cell counts collected from mice treated with either the wild-type IgG1 Fc construct of SIRP-α or the IgG1 Fc variant construct of SIRP-α. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition The terms "about" or "approximately" mean within an acceptable error range for a particular value, as determined by one of ordinary skill in the art. This means that the value is measured or determined at the limit of the measurement system. For example, "about" may mean a range of values ​​that, according to the practice of the art, may be within 1 or more standard deviations. Alternatively, "about" can mean within 20%, or within 10%, of a given value. %, up to 5%, or up to 1%. Alternatively, it can refer to a range, particularly in biological systems. Or with respect to a biological process, the term refers to a value that is within 10-fold, preferably within 5-fold, more preferably within Preferably, it can mean within 2 times. When a particular value is described in a range, the term "about" refers to an acceptable error for the particular value. It shall be assumed to mean within a range.

[0011] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a," "an," and " "the" is intended to include the plural as well, unless the context clearly indicates otherwise. Furthermore, the terms "including," "includes," "having," "has," "accompanying," or variations thereof To the extent used in the description or claims, such terms shall mean "comprising" The term "includes" is intended to be as inclusive as the term "includes."

[0012] As used herein, the term "antibody" refers to an intact antibody, an antibody fragment (which may possess a desired biological activity, e.g., For example, monoclonal antibodies, polyclonal antibodies, etc., may be used, provided that they exhibit epitope binding. antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-like proteins Refers to quality.

[0013] As used herein, the term "antibody variable domain" refers to the complementarity determining regions (CDRs, e.g., For example, CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3. DR H3) and the light chain of the antibody containing the amino acid sequence of the framework region (FR) Refers to the heavy chain portion.

[0014] As used herein, the term "linker" refers to a linker that connects two elements, e.g., a protein domain. In some embodiments, a linker is a covalent bond or a spacer. The term "spacer" refers to a spacer between two polypeptides or polypeptide domains. To give space or flexibility (or both), two polypeptides Moieties occurring between peptide or polypeptide domains (e.g., polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids) In some embodiments, the amino acid spacer is part of the primary sequence of the polypeptide. (e.g., polypeptides or polypeptides spaced apart through the polypeptide backbone) connected to the domain).

[0015] As used herein, the term "therapeutically effective amount" refers to a therapeutically effective amount for treating a disease, e.g., cancer, e.g., solid tumors. and (iii) a compound sufficient and effective to achieve the desired therapeutic effect in the treatment of patients with tumors or hematologic cancers. and a pharmaceutical composition comprising the polypeptide or polypeptide described herein, which is effective in treating a variety of conditions, such as a heart attack, stroke ... pulmonary circulation, or heart failure. , refers to the amount of a polypeptide having the SIRP-α D1 domain or a variant thereof In some embodiments, a therapeutically effective amount of the polypeptide avoids adverse side effects.

[0016] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition containing an active ingredient and an excipient or diluent (e.g., or both excipients and diluents) so that the active ingredient can be administered in a suitable manner. In some embodiments, the pharmaceutical formulations disclosed herein refer to a pharmaceutical or pharmaceutical formulation that The pharmaceutical composition comprises pharmaceutically acceptable ingredients that are compatible with the polypeptide. In form, the pharmaceutical composition may be in tablet or capsule form for oral administration, or in the form of e.g. , in aqueous form for intravenous or subcutaneous administration by injection.

[0017] As used herein, the terms "subject," "individual," and "patient" are used interchangeably. , refers to vertebrates, for example, mammals. Mammals include mice, monkeys, humans, livestock, and sports In vivo derived and Also encompassed are tissues, cells, and their progeny of biological entities cultured in vitro. None of the terms require the guidance of a medical professional.

[0018] As used herein, the term "affinity" or "binding affinity" refers to the binding affinity between two molecules. Generally, binding affinity refers to the strength of the binding between a molecule and its binding partner, e.g., indicates the strength of the total non-covalent interactions between the SIRP-α D1 variant and CD47. Unless otherwise indicated, binding affinity refers to a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is usually expressed as a dissociation constant (K D )Also is the association constant (K A ) Two molecules that have low binding affinity for each other are Generally binds slowly, tends to dissociate easily, and has a high K D High relative to each other Two molecules that have affinity generally bind more easily and tend to remain bound longer. Small K D In some embodiments, the K D is a known It is measured using methods and techniques, such as surface plasmon resonance (SPR). D is k off / k on It can be calculated as the ratio of

[0019] As used herein, the term "smaller than K" D " is the K D For values, numbers Small value of K D As used herein, the term "more" refers to a higher binding affinity and a higher affinity for the target molecule. Bigger K D " is the K D For values ​​of K, numerically large D Value and decreasing bond Refers to affinity.

[0020] As used herein, the term "acute anemia" refers to anemia occurring during the first 5 days after administration of a compound or treatment. This refers to a 30% reduction in red blood cell mass or hemoglobin between 2000 and 2005.

[0021] I. Signal Regulatory Receptor Protein-α (SIRP-α) D1 Domain and Its Variants In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0022] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the polypeptide selected from the Fc region of human IgG4 containing the mutation N297A. It is Do.

[0023] Signal-regulating protein α ("SIRP-α" or "SIRP-alpha") is a It is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed in the membranes of marrow cells SIRP-α interacts with CD47, a protein that is widely expressed on many cell types in the body. The interaction of SIRP-α with CD47 inhibits the production of proteins that would otherwise be recognized by the immune system. High expression of CD47 on tumor cells prevents phagocytosis of "self" cells that may be ingested. It acts as a negative prognostic factor for survival in chronic leukemia and some solid tumor cancers. is recognized as being obtainable.

[0024] Native SIRP-α contains three highly homologous immunoglobulin (Ig)-like extracellular domains. The SIRP-α D1 domain ("D1 domain") comprises the D1, D2, and D3 domains. The term "in" refers to the membrane-distal, or extracellular, domain of SIRP-α, and is related to CD47. As used herein, the term "SIRP-α polypeptide" refers to a polypeptide that mediates the binding of SIRP-α. "SEQ ID NO: 1" means any SIRP-α polypeptide or fragment thereof capable of binding to CD47. There are at least 10 variants of wild-type human SIRP-α. Table 1 shows: The D1 domains of the 10 naturally occurring wild-type human SIRP-α D1 domain variants The amino acid sequences of SIRP- In some embodiments, the SIRP-α polypeptide comprises a SIRP-α D1 domain. The RP-α polypeptide may comprise a wild-type D1 domain, such as those shown in SEQ ID NOs: 1-10. In some embodiments, the SIRP-α polypeptide comprises a wild-type human SIRP-α polypeptide. -α D2 or D3 domain (or both D2 and D3 domains) (Table 3) . [Table 1-1] [Table 1-2] [Table 1-3]

[0025] As used herein, the term "high affinity SIRP-α D1 variant," "high affinity "SIRP-α variant" or "SIRP-α D1 variant" refers to a variant of SIRP-α D1 domain, or the CD47-binding portion of the SIRP-α polypeptide, High affinity SIRP-α refers to a polypeptide that has a higher affinity for wild-type SIRP-α. The α D1 variant has at least one amino acid substitution relative to wild-type SIRP-α: It may contain deletions or insertions (or a combination thereof).

[0026] In some embodiments, the high affinity SIRP-α D1 variants disclosed herein comprises a SIRP-α D1 domain or a variant thereof. The high affinity SIRP-α D1 variants are the wild-type D1 variants shown in SEQ ID NOs: 1 to 10. Each of the amino acid sequences contains one or more amino acid substitutions, insertions, additions, or deletions relative to the main sequence. Exemplary amino acids in SIRP-α D1 domain variants (SEQ ID NOS: 13-22) In some embodiments, the SIRP-α D1 domain polypeptide or Alternatively, high affinity SIRP-α D1 variants may comprise fragments of the D1 domain. In embodiments, the fragment of the SIRP-α polypeptide or the high affinity SIRP-α barrier Fragments of the nucleotides may be less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, or about 30 amino acids, approximately 40 amino acids in length, approximately 50 amino acids in length, approximately 60 amino acids in length, approximately 70 amino acids, approximately 80 amino acids in length, approximately 90 amino acids in length, approximately 100 amino acids in length, or comprises an amino acid sequence greater than about 100 amino acids in length. -A fragment of the α D1 domain retains the ability to bind to CD47.

[0027] In some embodiments, the polynucleotides of the present disclosure, including high affinity SIRP-α D1 variants, The peptide binds to CD47 with higher binding affinity than the wild-type human SIRP-α D1 domain. In some embodiments, the high affinity SIRP-α D1 variant is a naturally occurring At least 1-fold (e.g., at least 1.5-fold, 2-fold, Binds to human CD47 with affinity of 2.5x, 3x, 3.5x, 4x, 5x, or greater than 5x In some embodiments, the high affinity SIRP-α D1 variant is a naturally occurring at least 1-fold (e.g., at least 10-fold, 100-fold, It binds to human CD47 with an affinity of 1000-fold or more.

[0028] As used herein, the term "optimized affinity" or "optimized binding affinity" refers to a high affinity between the polypeptides disclosed herein, including SIRP-α D1 variants, and CD47 For example, in some embodiments, the polypeptide The peptide binds primarily or with higher affinity to CD47 on cancer cells and not on non-cancer cells. It does not substantially bind or binds with lower affinity to CD47 on cells. In some embodiments, the binding affinity between the polypeptide and CD47 is such that the interaction is clinically Variants that do not cause clinically significant toxicity or bind with maximal affinity In some embodiments, the compounds described herein are optimized to reduce toxicity compared to the compounds described herein. High-affinity SIR to achieve optimized binding affinity between the provided polypeptide and CD47 The polypeptide containing the P-α D1 variant has the highest achievable binding to CD47. In some embodiments, the present invention is developed to have a lower binding affinity than that of the The high-affinity SIRP-α variants disclosed herein are expressed in rodents, non-human primates (NHPs), and , and cross-reacts with human CD47.

[0029] As used herein, the term "immunogenic" means to have an effect on the host as if it were a foreign antigen. It refers to the property of a protein (e.g., a therapeutic protein) to elicit an immune response in a subject. The immunogenicity of proteins can be assessed in vitro using a variety of different methods, including in vitro T cell proliferation assays. It can be assayed in vitro.

[0030] As used herein, the term "minimally immunogenic" refers to a protein that is minimally immunogenic, e.g., by amino acid substitution. , the immunogenicity is lower than that of the protein before the amino acid substitutions are introduced (e.g., the unmodified protein) ( (e.g., at least 10%, 25%, 50%, or 100% lower) In some embodiments, the immunogenicity of a protein (e.g., a therapeutic protein) is In some embodiments, proteins (e.g., therapeutic proteins) are modified to have minimal immunogenicity. and provokes little or no immune response from the host, even if it is a foreign antigen. .

[0031] In some embodiments, the high affinity SIRP-α D1 variant is a minimally immunized In some embodiments, the SIRP-α polypeptide of the present disclosure administered to a subject has The peptides are designed to have amino acid changes that increase the affinity of the SIRP-α D1 variant. and a SIRP-α polypeptide having the same amino acid sequence as that of the SIRP-α polypeptide in the subject's biological sample. In some embodiments, variants of the polypeptides disclosed herein have an anti- Reduces the risk of side effects compared to CD47 antibodies or wild-type SIRP-α. In some embodiments, variants of the polypeptides disclosed herein are anti-CD47 antibodies or or wild-type SIRP-α. Variants of the polypeptides disclosed herein may be expressed in rodents or non-human primates (NH P) Does not cause acute anemia in the study.

[0032] Table 2 shows the high affinity SIRP-α D1 variants for each D1 domain sequence. In some embodiments, the high affinity SIRP-α D1 antibody The variant may have one or more of the substitutions shown in Table 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 1 In some embodiments, the nucleotide sequence includes 0, 11, 12, 13, 14, or more. High-affinity SIRP-α D1 variants have up to 14 affinity domains relative to the wild-type D1 domain. In some embodiments, the high affinity SIRP-α D1 variant comprises an amino acid substitution. , comprising up to 10 amino acid substitutions relative to the wild-type D1 domain. High-affinity SIRP-α D1 variants have up to seven D1 domains relative to the wild-type D1 domain. In some embodiments, the high affinity SIRP-α D1 of the present disclosure comprises an amino acid substitution of The variant should have a sequence similar to that of the wild-type D1 domain, with a sequence similar to that of the wild-type D1 domain being at least 90% (e.g., at least The amino acid sequence identity of the nucleotides is 92%, 95%, 97%, or greater than 97%.

[0033] In some embodiments, the high affinity SIRP-α D1 variant comprises two or more wild-type SIRP-α D1 variants. a portion of a wild-type D1 domain or a variant thereof (e.g., one wild-type D1 domain or a portion of the wild-type D1 domain or a variant thereof and another portion of the wild-type D1 domain or a variant thereof In some embodiments, the chimeric high affinity SIRP-α D1 variant is High-affinity SIRP-α D1 variants are characterized by the wild-type D1 domain or its variants. At least two moieties (e.g., three, four, five, or more moieties), Each of the D1 domains is derived from a different wild-type D1 domain. The compatible SIRP-α D1 variants further include one or more of the amino acid substitutions shown in Table 2. nothing. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0034] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVE X 14 KSGAGTELSVRAKPS (SEQ ID NO: 13), where X1 is L, I, or is V, X2 is V, L, or I, X3 is A or V, and X4 is A, I, or L, X5 is I, T, S, or F, and X6 is E, V, or L; X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I , X 13is F, L, or V, and X 14 SIRP- having a sequence that is F or V α D1 variants, which variants are wild-type SIRP having the sequence of SEQ ID NO: 1. -has at least one amino acid substitution relative to the α D1 domain.

[0035] In some embodiments, the polypeptide is EEGX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVE X 14 KSGAGTELSVRAKPS (SEQ ID NO: 16), where X1 is L, I, or is V, X2 is V, L, or I, X3 is A or V, and X4 is A, I, or L, X5 is I, T, S, or F, and X6 is E, V, or L; X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I , X 13 is F, L, or V, and X 14 SIRP- having a sequence that is F or V α D1 variant, the variant having the sequence of SEQ ID NO: 4. -has at least one amino acid substitution relative to the α D1 domain.

[0036] In some embodiments, the polypeptide is EEEX1QX2IQPDKFVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVE X 14 KSGAGTELSVRAKPS (SEQ ID NO: 17), where X1 is L, I, or is V, X2 is V, L, or I, X3 is A or V, and X4 is A, I, or L, X5 is I, T, S, or F, and X6 is E, V, or L; X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I , X 13 is F, L, or V, and X 14 SIRP- having a sequence that is F or V α D1 variant, the variant having the sequence of SEQ ID NO: 5. -has at least one amino acid substitution relative to the α D1 domain.

[0037] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFPIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVE X 14 KSGAGTELSVRAKPS (SEQ ID NO: 18), wherein X1 is L, I, or is V, X2 is V, L, or I, X3 is A or V, and X4 is A, I, or L, X5 is I, T, S, or F, and X6 is E, V, or L; X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I , X 13 is F, L, or V, and X 14 SIRP- having a sequence that is F or V α D1 variant, the variant having the sequence of SEQ ID NO: 6. -has at least one amino acid substitution relative to the α D1 domain.

[0038] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRISNITPADAGTYYCX 12 KX 13 RKGSPDDVE X 14 KSGAGTELSVRAKPS (SEQ ID NO: 21), where X1 is L, I, or is V, X2 is V, L, or I, X3 is A or V, and X4 is A, I, or L, X5 is I, T, S, or F, and X6 is E, V, or L; X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I , X 13 is F, L, or V, and X 14 SIRP- having a sequence that is F or V α D1 variant, which variant is a wild-type SIRP having the sequence of SEQ ID NO: 9. -has at least one amino acid substitution relative to the α D1 domain.

[0039] In any of the above embodiments, the polypeptide is selected from the group consisting of SEQ ID NOs: 13, 16-18, and 21, wherein X1 is L, I, or V. In any of the above embodiments, X2 is V, L, or or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I, or L. and X5 is I, T, S, or F. In any of the above embodiments, X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. and X9 is H, P, or R. In any of the above embodiments, X 10 is L, T or G. In any of the above embodiments, X 11 is K or R. In any of the embodiments, X 12 is V or I. In any of the above embodiments, X 13 is F, L, or V. In any of the above embodiments, X 14 is F or In some embodiments, the polypeptide of this aspect of the disclosure is SEQ ID NO: 1, 4 For wild-type SIRP-α D1 domains with any one of the sequences ~6 and 9 , containing six or fewer amino acid substitutions.

[0040] In some embodiments, the polypeptide is any of SEQ ID NOs: 1, 4-6, and 9. At least 10-fold higher binding than wild-type SIRP-α D1 domain with one sequence In some embodiments, the polypeptide binds to CD47 with affinity. SIRP-α D1 domain having any one of the sequences 4 to 6 and 9. In some embodiments, the polypeptide binds to CD47 with at least 100-fold greater binding affinity. The polypeptide is a wild-type SIR having any one of the sequences of SEQ ID NOs: 1, 4 to 6, and 9. It binds to CD47 with at least 1000-fold higher binding affinity than the P-α D1 domain. In some embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is , KD less than 1x10-8M, less than 5x10-9M, less than 1x10-9M, 5x10-1 binds to CD47 at less than 0 M, less than 1x10-10 M, or less than 1x10-11 M. In some embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is , KD approx. 500nM~100nM, approx. 100nM~50nM, approx. 50nM~10nM, approx. 10nM~5nM, approx. 5nM~1nM, approx. 1nM~500pM, approx. 500pM~100p M, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0041] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14KSGAGTELSVRAKPS (SEQ ID NO: 14), wherein X1 is L, I, or X2 is V, L, or I, X3 is A or V, and X4 is V, I, or or L, X5 is I, T, S, or F, X6 is E, V, or L, and X X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X1 0 is S, T, or G, and X 11 is K or R, and X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α with a sequence that is F or V D1 variant, which variant comprises a wild-type SIRP-D1 variant having the sequence of SEQ ID NO: 2. It has at least one amino acid substitution relative to the α D1 domain.

[0042] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 15), wherein X1 is L, I, or X2 is V, L, or I, X3 is A or V, and X4 is V, I, or or L, X5 is I, T, S, or F, X6 is E, V, or L, and X X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X1 0 is S, T, or G, and X 11 is K or R, and X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α with a sequence that is F or V D1 variant, which variant comprises a wild-type SIRP-D1 variant having the sequence of SEQ ID NO: 3. It has at least one amino acid substitution relative to the α D1 domain.

[0043] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRGKPS (SEQ ID NO: 19), wherein X1 is L, I, or X2 is V, L, or I, X3 is A or V, and X4 is V, I, or or L, X5 is I, T, S, or F, X6 is E, V, or L, and X X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X1 0 is S, T, or G, and X 11 is K or R, and X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α with a sequence that is F or V D1 variant, which variant comprises a wild-type SIRP-D1 variant having the sequence of SEQ ID NO: 7. It has at least one amino acid substitution relative to the α D1 domain.

[0044] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 22), wherein X1 is L, I, or X2 is V, L, or I, X3 is A or V, and X4 is V, I, or or L, X5 is I, T, S, or F, X6 is E, V, or L, and X X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X1 0 is S, T, or G, and X 11 is K or R, and X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α with a sequence that is F or V D1 variant, the variant having the sequence of SEQ ID NO: 10. -has at least one amino acid substitution relative to the α D1 domain;

[0045] In any of the above embodiments of this aspect of the disclosure, the polypeptide comprises SEQ ID NO: Any one of Nos. 14, 15, 19, and 22, wherein X1 is L, I, or V. In any of the above embodiments, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. and X4 is V, I, or L. In any of the above embodiments, X5 is I, T, In any of the above embodiments, X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P, or R. In any of the above embodiments, X 10 is S, T, or G. In any of the above embodiments, X 11 is K or R. In any of the above embodiments In X 12 is V or I. In any of the above embodiments, X 13 is F, L, or V. In any of the above embodiments, X 14 is F or V. In some embodiments, the polypeptides of this aspect of the disclosure are selected from the group consisting of SEQ ID NOs: 2, 3, 7, and 1. 6 or less for a wild-type SIRP-α D1 domain having any one of the sequences of Contains amino acid substitutions of

[0046] In some embodiments, the polypeptide is any of SEQ ID NOs: 2, 3, 7, and 10. At least 10-fold higher binding than wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the polypeptide binds to CD47 with a specific affinity. , 3, 7, and 10. In some embodiments, the antibody binds to CD47 with at least 100-fold greater binding affinity than the antibody. The polypeptide may be a wild-type polypeptide having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. Binds to CD47 with at least 1000-fold higher binding affinity than the SIRP-α D1 domain In some embodiments, the SIRP-α D1 variant polypeptide or The fragment is K D 1x10 -8Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x1 0 -10 Under M, 1x10 -10 Less than M or 1x10 -11 Binds to CD47 at less than M In some embodiments, the SIRP-α D1 variant polypeptide or The fragment is K D Approx. 500nM~100nM, Approx. 100nM~50nM, Approx. 50nM~10n M, approx. 10nM~5nM, approx. 5nM~1nM, approx. 1nM~500pM, approx. 500pM~1 Binds to CD47 at 00pM, approximately 100pM to 50pM, or approximately 50pM to 10pM .

[0047] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 20), wherein X1 is L, I, or X2 is V, L, or I, X3 is A or V, and X4 is A, I, or or L, X5 is I, T, S, or F, X6 is E, V, or L, and X X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X1 0 is S, T, or G, and X 11 is K or R, and X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α with a sequence that is F or V D1 variant, which variant comprises a wild-type SIRP-D1 variant having the sequence of SEQ ID NO: 8. It has at least one amino acid substitution relative to the α D1 domain.

[0048] In some embodiments, the polypeptide is SEQ ID NO: 20, wherein X1 is L, I, or V. In any of the above embodiments of this aspect of the disclosure, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I, or L. In any of the above embodiments, X5 is I, T, S, or F. , X6 is E, V, or L. In any of the above embodiments, X7 is K or R In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X is H, P, or R. 10 is S, T, or G. In any of the above embodiments, X 11 is K or R In any of the above embodiments, X 12 is V or I. In either case, X 13 is F, L, or V. In any of the above embodiments, , X 14 is F or V. In some embodiments, the polypeptide of this aspect of the disclosure is a sequence of 6 or less amino acids relative to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 8. Contains amino acid substitutions.

[0049] In some embodiments, the polypeptide is a wild-type SIR having the sequence of SEQ ID NO:8. It binds to CD47 with at least 10-fold higher binding affinity than the P-α D1 domain. In some embodiments, the polypeptide is a wild-type SIRP-α having the sequence of SEQ ID NO:8. It binds to CD47 with at least 100-fold higher binding affinity than the D1 domain. In one embodiment, the polypeptide is a wild-type SIRP-α D having the sequence of SEQ ID NO:8. It binds to CD47 with at least 1000-fold higher binding affinity than the 1 domain. In embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is D 1 x10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 It binds to CD47 at less than M. In embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is D about 5 00nM~100nM, approx. 100nM~50nM, approx. 50nM~10nM, approx. 10nM~ 5nM, approx. 5nM~1nM, approx. 1nM~500pM, approx. 500pM~100pM, approx. 10 Binds to CD47 at 0 pM to 50 pM, or approximately 50 pM to 10 pM.

[0050] In some embodiments, the polypeptide is EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TS LX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 F PRVTTVSX 17 X 18 TX19 RX 20 NMDFX 21 IX 22 IX 23 NITP ADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELS VRX 29 KPS (SEQ ID NO: 23), wherein X1 is E or G and X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, and X5 is L or S, X6 is S or T, X7 is A or V, and X8 is I or T and X9 is H or R, and X 10 is A, V, I, or L, and X 11 is I, T, S, or F and X 12 is A or G, and X 13 is E, V, or L , X 14 is K or R, and X 15 is E or Q, and X 16 is H, P, or R Yes, X 17 is D or E, and X 18 is S, L, T, or G, and X 19 Ha K or R, and X 20 is E or D, and X 21 is S or P, and X 22 is S also is R and X 23 is S or G, and X 24 is V or I, and X 25 is F, L, V and X 26 is D or absent, and X 27 is T or V, and X 28 teeth F or V, X 29 SIRP-α D1 variants with a sequence of A or G The variant includes a wild-type SI having any one of the sequences of SEQ ID NOs: 1 to 10. It has at least one amino acid substitution in the RP-α D1 domain.

[0051] In any of the above embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the above embodiments, X3 is V, L, or I. In any of the above embodiments, X4 is S or F. In any of the above embodiments, X5 is L or or S. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is A or V. In any of the above embodiments, X 8 is I or T. In any of the above embodiments, X9 is H or R. In any of the above embodiments, X 10 is A, V, I, or L. In either case, X 11 is I, T, S, or F. In any of the above embodiments, X 12 is A or G. In any of the above embodiments, X 13 is E, V, or L. In any of the above embodiments, X 14 is K or R. In any of the embodiments, X 15 is E or Q. In any of the above embodiments Te, X 16 is H, P, or R. In any of the above embodiments, X 17 Is D or E. In any of the above embodiments, X 18 is S, L, T, or G In any of the above embodiments, X 19 is K or R. In either case, X20 is E or D. In any of the above embodiments, X 21 is S or P. In any of the above embodiments, X 22 is S or R. In any of the embodiments, X 23 is S or G. In any of the above embodiments Te, X 24 is V or I. In any of the above embodiments, X 25 is F, L, V In any of the above embodiments, X 26 is D or does not exist. In any of the above embodiments, X 27 is T or V. In any of the above embodiments, Hey, X 28 is F or V. In any of the above embodiments, X 29 A also is G. In some embodiments, the polypeptide of this aspect of the disclosure is 6 or more for a wild-type SIRP-α D1 domain having any one of the sequences of 10 Contains the following amino acid substitutions:

[0052] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NOs: 1-10. with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain 47. In some embodiments, the polypeptide binds to any of SEQ ID NOs: 1-10. at least 100-fold higher than the wild-type SIRP-α D1 domain with either sequence In some embodiments, the polypeptide binds to CD47 with binding affinity. A wild-type SIRP-α D1 domain having any one of the sequences of Nos. 1 to 10 Both SIRs bind to CD47 with 1000-fold higher binding affinity. The P-α D1 variant polypeptide or a fragment thereof may be D 1x10 -8 Less than M, 5 x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M , or 1x10 -11 In some embodiments, the SIR binds to CD47 at less than M. The P-α D1 variant polypeptide or a fragment thereof may be D Approximately 500nM~100nM , about 100nM~50nM, about 50nM~10nM, about 10nM~5nM, about 5nM~1 nM, approximately 1nM to 500pM, approximately 500pM to 100pM, approximately 100pM to 50pM, or It binds to CD47 at approximately 50 pM to 10 pM.

[0053] In some embodiments, the polynucleotides of the present disclosure, including high affinity SIRP-α D1 variants, The peptide further comprises a D2 having the sequence of SEQ ID NO: 24 of wild-type SIRP-α as shown in Table 3. domain, a D3 domain having the sequence of SEQ ID NO: 25, or a D4 domain having the sequence of SEQ ID NO: 24 and a D3 domain having the sequence of SEQ ID NO: 25. In some embodiments, the high affinity SIRP-α D1 variant further comprises a fragment of the D2 domain. or variants, or fragments or variants of the D3 domain. In embodiments, the high affinity SIRP-α D1 variant further comprises a fragment of the D2 domain. or variants, and fragments or variants of the D3 domain. In this state, the high-affinity SIRP-α D1 variant is linked to the D2 or D3 domain via a linker. In some embodiments, the high affinity SIRP-α D1 variant is tethered to a domain. is connected to the D2 and D3 domains via a linker. [Table 3]

[0054] In some embodiments, the polynucleotides of the present disclosure, including high affinity SIRP-α D1 variants, The peptide improves the pharmacokinetic properties of the polypeptide, e.g., prolongs serum half-life. For this purpose, an Fc domain monomer, human serum albumin (HSA) or its variants , serum-binding proteins or peptides, or organic molecules, e.g., polymers (e.g., In some embodiments, the high affinity SIRP-α D1 The variants are attached to Fc domain monomers that are unable to dimerize. In some embodiments, Fc domain monomers, HSA proteins, and serum binding proteins are also included. or peptides, and organic molecules such as PEG, may increase the serum half-life of the polypeptides described herein. In some embodiments, the high affinity SIRP-α D1 barrier The polypeptide of the present disclosure, including the compound, includes any of the sequences of SEQ ID NOs: 26 to 36 shown in Table 4. Not at all. [Table 4-1] [Table 4-2]

[0055] In some embodiments, the polypeptides and polypeptide constructs described herein include: It is used in in vitro binding assays, such as immunoassays. For example, in some embodiments Thus, the polypeptides and polypeptide constructs described herein may be used in liquid phase or or bound to a solid support. In some embodiments, the polymers used in immunoassays The polypeptides may be detectably labeled in a variety of ways.

[0056] In some embodiments, the polypeptides and polypeptide constructs described herein include: They are bound to various carriers and used to detect the presence of specific antigen-expressing cells. Examples include glass, polystyrene, polypropylene, polyethylene, dextran, and nanofibers. Iron, amylase, natural and modified cellulose, polyacrylamide, agarose, The carrier may be soluble or insoluble in nature.

[0057] A variety of different labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, , fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. A variety of techniques are available for attaching labels to the polypeptides disclosed herein.

[0058] In some embodiments, the polypeptides are conjugated to low molecular weight haptens. The phenant is then specifically detected by a second reaction. For example, in some embodiments In this case, the hapten biotin is used in conjunction with avidin or The amines dinitrophenol, pyridoxal, or fluorescein have been shown to have anti-inflammatory effects on certain anti-inflammatory drugs. anti-dinitrophenol antibodies (e.g., anti-dinitrophenol antibodies, anti-pyridoxal antibodies, and and anti-fluorescein antibodies).

[0059] II. High-affinity SIRP-α D1 domain with altered glycosylation In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0060] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the polypeptide selected from the Fc region of human IgG4 containing the mutation N297A. It is Do.

[0061] In some embodiments, the polypeptides in the compositions disclosed herein are glycosylated. Includes high-affinity SIRP-α D1 variants with reduced or minimal sylation. The D1 domains of each of the 10 wild-type human SIRP-α proteins (SEQ ID NOs: 1 to 3 in Table 1) were 10) has a single potential N-linked glycosylation site at amino acid N80 of the sequence N80ITP. The SIRP-α D1 domain in Chinese hamster ovary (CHO) cells Expression of the 16 kDa (non-glycosylated) major band was observed and was eliminated by Endo Hf. Endo Hf is a phospholipid-binding protein that binds to endoglycosyltransferase (endoglycosyltransferase), resulting in a minor band of higher molecular weight. It is a recombinant protein fusion of maltose-binding protein and maltose-binding protein. Hf is a N-terminal oligosaccharide within the high-mannose chitobiose core and within some hybrid oligosaccharides. This means that the proline at amino acid position 83 is involved in the cleavage of glycoproteins. This can reduce the efficiency of glycosylation and lead to proteins with different degrees of glycosylation and therefore heterogeneity. For pharmaceutical development, heterogeneity is a challenge in process development. Therefore, the homogeneity of high-affinity SIPR-α D1 variants is important. To explore the possibility of generating glycosylated forms, in some embodiments, SIPR- Amino acid N80 of the αD1 variant is mutated to Ala. to produce a non-glycosylated high-affinity SIRP-α D1 variant, thus enabling the development of high-affinity SIRP-α D1. The amino acid N80 of the IRP-α D1 variant may be any natural or unnatural amino acid. In some embodiments, the amino acid substitutions are any amino acid substitutions, such as N80A and N80Q. In this state, the high-affinity SIRP-α D1 variant contains the N80A mutation and at least one additional mutations (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional mutations). In some embodiments, the additional mutations are In some embodiments, the additional mutation is in the D1 domain, which is in the binding site for CD47. The hydrophobic core of the

[0062] In some embodiments, the polypeptide in the compositions disclosed herein is a wild-type polypeptide. High-affinity SIRP-α with increased glycosylation compared to the SIRP-α D1 domain Another option to increase the homogeneity of the final product is to use a nucleotide sequence at amino acid N80, which contains the D1 variant. High-affinity SIRP-1 with enhanced glycosylation efficiency and increased glycosylation compared to wild-type In some embodiments, the sequence NITP83 is used to generate α D1 variants. The amino acid P83 of the ribosomal protein affects the degree of glycosylation at amino acid N80. In embodiments, changing P83 to any amino acid can improve the efficiency of glycosylation at N80. In some embodiments, the amino acid sequence of the high affinity SIRP-α D1 variant is P83 can be substituted with any amino acid, including natural and unnatural amino acids, e.g., P83 In some embodiments, the polynucleotides of the present disclosure are P83A, P83I, and P83L. The peptides can be produced, for example, by genetic manipulation of the cell line (e.g., genetically engineered yeast or mammalian cells). by altering cell culture conditions, such as the addition of prokaryotic or mammalian hosts or kifunensine. By using a host that does not naturally glycosylate, such as a host of bacteria (such as E. coli), the cells The proteins expressed in the cells are optimized not to glycosylate the proteins expressed by the cells. can be.

[0063] Table 5 shows the high affinity SIRP-α D1 variants for each D1 domain variant sequence. In some embodiments, the high affinity SIRP- α D1 variants may have one or more of the substitutions shown in Table 5 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more). In this form, the SIRP-α D1 variant is not or minimally glycosylated. In some embodiments, the SIRP-α D1 variant is glycosylated by It is fully glycosylated or nearly fully glycosylated. In this state, the high-affinity SIRP-α D1 variants have up to 100% affinity to the wild-type D1 domain. In some embodiments, the high affinity SIRP-α D1 variant comprises 14 amino acid substitutions. The ant contains up to 10 amino acid substitutions relative to the wild-type D1 domain. In embodiments, the high affinity SIRP-α D1 variant has, relative to the wild-type D1 domain: In some embodiments, the high affinity SIRP- The α D1 variants should have at least 90% (e.g., , at least 92%, 95%, 97%, or more than 97% amino acid sequence identity .

[0064] In some embodiments, the high affinity SIRP-α D1 variant comprises two or more wild-type SIRP-α D1 variants. a portion of a wild-type D1 domain or a variant thereof (e.g., one wild-type D1 domain or a portion of the wild-type D1 domain or a variant thereof and another portion of the wild-type D1 domain or a variant thereof In some embodiments, the chimeric high affinity SIRP-α D1 variant is High-affinity SIRP-α D1 variants are characterized by the wild-type D1 domain or its variants. At least two moieties (e.g., three, four, five, or more moieties), Each of the D1 domains is derived from a different wild-type D1 domain. The compatible SIRP-α D1 variants further include one or more of the amino acid substitutions shown in Table 5. nothing. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0065] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X1 is L, X2 is V, L, or I; X3 is A or V; and X4 is X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Yes, X 10 is L, T, or G, and X 11 is K or R, and X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y Ri, X 13are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y and X 14 is V or I, and X 15 is F, L, or V , X 16 The variant comprises a SIRP-α D1 variant having a sequence that is F or V, The ant is at least 100% SIRP-α D1 domain with respect to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 1. Both have one amino acid substitution.

[0066] In some embodiments, the polypeptide is EEGX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 40), wherein X1 is L, X2 is V, L, or I; X3 is A or V; and X4 is X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Yes, X 10 is L, T, or G, and X 11 is K or R, and X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y Ri, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y and X 14is V or I, and X 15 is F, L, or V , X 16 The variant comprises a SIRP-α D1 variant having a sequence that is F or V, The ant is at least 100% SIRP-α D1 domain with the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 4. Both have one amino acid substitution.

[0067] In some embodiments, the polypeptide is EEEX1QX2IQPDKFVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 41), wherein X1 is L, X2 is V, L, or I; X3 is A or V; and X4 is X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Yes, X 10 is L, T, or G, and X 11 is K or R, and X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y Ri, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y and X 14 is V or I, and X 15 is F, L, or V , X 16The variant comprises a SIRP-α D1 variant having a sequence that is F or V, The antibody was found to bind to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:5 at least Both have one amino acid substitution.

[0068] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFPIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 42), wherein X1 is L, X2 is V, L, or I; X3 is A or V; and X4 is X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Yes, X 10 is L, T, or G, and X 11 is K or R, and X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y Ri, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y and X 14 is V or I, and X 15 is F, L, or V , X 16 The variant comprises a SIRP-α D1 variant having a sequence that is F or V, The antibody was found to bind to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 6. Both have one amino acid substitution.

[0069] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX1 1RNNMDFSIRISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 45), wherein X1 is L, X2 is V, L, or I; X3 is A or V; and X4 is X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Yes, X 10 is L, T, or G, and X 11 is K or R, and X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y Ri, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y and X 14 is V or I, and X 15 is F, L, or V , X 16 The variant comprises a SIRP-α D1 variant having a sequence that is F or V, The antibody was at least 50 ... Both have one amino acid substitution.

[0070] In any of the above embodiments of this aspect of the disclosure, the polypeptide is SEQ ID NO: 37, 40 to 42, and 45, wherein X1 is L, I, or V. In any of the above embodiments, X2 is V , L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I, or L. In any of the above embodiments, X5 is I, T, S, or F. X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, In any of the above embodiments, X is H, P, or R. 10 is L, T, or G. In any of the above embodiments, X 11 is K or R In any of the above embodiments, X 12 are N, A, C, D, E, F, G, H, and I , K, L, M, P, Q, R, S, T, V, W, or Y. Any of the above embodiments In X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S , T, V, W, or Y. In any of the above embodiments, X 14 is V or I In any of the above embodiments, X 15 are F, L, and V. In either case, X 16 is F or V.

[0071] In some embodiments, the polypeptides provided herein are selected from the group consisting of SEQ ID NOs: 1, 4-6, and 9, for a wild-type SIRP-α D1 domain having any one of the sequences 10 In some embodiments, the polypeptides provided herein contain no more than one amino acid substitution. The wild-type SIRP-α having any one of the sequences set forth in SEQ ID NOs: 1, 4 to 6, and 9 is Contains up to seven amino acid substitutions relative to the D1 domain.

[0072] In some embodiments, the polypeptide is any of SEQ ID NOs: 1, 4-6, and 9. At least 10-fold higher binding than wild-type SIRP-α D1 domain with one sequence In some embodiments, the polypeptide binds to CD47 with affinity. SIRP-α D1 domain having any one of the sequences 4 to 6 and 9. In some embodiments, the polypeptide binds to CD47 with at least 100-fold greater binding affinity. The polypeptide is a wild-type SIR having any one of the sequences of SEQ ID NOs: 1, 4 to 6, and 9. It binds to CD47 with at least 1000-fold higher binding affinity than the P-α D1 domain. In some embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is , K. D 1x10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -1 0 Under M, 1x10 -10 Less than M or 1x10 -11 Binds to CD47 at less than M In some embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is , K. D Approx. 500nM~100nM, Approx. 100nM~50nM, Approx. 50nM~10nM, Approx. 10nM~5nM, approx. 5nM~1nM, approx. 1nM~500pM, approx. 500pM~100p M, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0073] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 38), where X1 is L, I , or V, X2 is V, L, or I, X3 is A or V, and X4 is V , I, or L, X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Ri, X 10 is S, T, or G, and X 11 is K or R, and X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y Ri, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T , V, W, or Y, and X 14 is V or I, and X 15 is F, L, or V Ri, X 16 The variant comprises a SIRP-α D1 variant having a sequence that is F or V, The variant was at least 100% identical to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:2. It has at least one amino acid substitution.

[0074] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 39), where X1 is L, I , or V, X2 is V, L, or I, X3 is A or V, and X4 is V , I, or L, X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Ri, X 10 is S, T, or G, and X 11 is K or R, and X 12 is N, A, C , D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y , X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V , W, or Y, and X 14 is V or I, and X 15 is F, L, or V, X 16 comprises a SIRP-α D1 variant having a sequence that is F or V, The compound has at least one sequence similar to that of the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:3. It also has one amino acid substitution.

[0075] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DTEX 16 KSGAGTELSVRGKPS (SEQ ID NO: 43), where X1 is L, I , or V, X2 is V, L, or I, X3 is A or V, and X4 is V , I, or L, X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Ri, X 10 is S, T, or G, and X 11 is K or R, and X 12 is N, A, C , D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y , X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V , W, or Y, and X 14 is V or I, and X 15 is F, L, or V, X 16 comprises a SIRP-α D1 variant having a sequence that is F or V, The compound has at least one nucleotide sequence similar to that of the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:7. Both have one amino acid substitution.

[0076] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 46), where X1 is L, I , or V, X2 is V, L, or I, X3 is A or V, and X4 is V , I, or L, X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Ri, X 10 is S, T, or G, and X 11 is K or R, and X 12 is N, A, C , D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y , X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V , W, or Y, and X 14 is V or I, and X 15 is F, L, or V, X 16 comprises a SIRP-α D1 variant having a sequence that is F or V, The compound showed at least a 5'-fold increase in the activity of the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 10. It has at least one amino acid substitution.

[0077] In any of the above embodiments of this aspect of the disclosure, the polypeptide is SEQ ID NO: 38, 39, 43, and 46, wherein X1 is L, I, or V. In any of the above embodiments, X2 is V , L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is V, I, or L. In any of the above embodiments, X5 is I, T, S, or F. X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, In any of the above embodiments, X is H, P, or R. 10 is S, T, or G. In any of the above embodiments, X 11 is K or R In any of the above embodiments, X 12 are N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. Hey, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the above embodiments, X 14 is V or I In any of the above embodiments, X 15 is F, L, or V. In either state, X 16 is F or V.

[0078] In some embodiments, the polypeptide is any of SEQ ID NOs: 2, 3, 7, and 10. A single sequence with 10 or fewer amino acid substitutions relative to the wild-type SIRP-α D1 domain. In some embodiments, the polypeptide comprises a SIRP-α D1 variant having a substitution. The wild-type SIRP-α having any one of the sequences set forth in SEQ ID NOs: 2, 3, 7, and 10. SIRP-α D1 variants with seven or fewer amino acid substitutions in the D1 domain Includes.

[0079] In some embodiments, the polypeptide is any of SEQ ID NOs: 2, 3, 7, and 10. At least 10-fold higher binding than wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the polypeptide binds to CD47 with a specific affinity. , 3, 7, and 10. In some embodiments, the antibody binds to CD47 with at least 100-fold greater binding affinity than the antibody. The polypeptide may be a wild-type polypeptide having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. Binds to CD47 with at least 1000-fold higher binding affinity than the SIRP-α D1 domain In some embodiments, the SIRP-α D1 variant polypeptide or The fragment is K D 1x10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x1 0 -10 Under M, 1x10 -10 Less than M or 1x10 -11 Binds to CD47 at less than M In some embodiments, the SIRP-α D1 variant polypeptide or The fragment is K D Approx. 500nM~100nM, Approx. 100nM~50nM, Approx. 50nM~10n M, approx. 10nM~5nM, approx. 5nM~1nM, approx. 1nM~500pM, approx. 500pM~1 Binds to CD47 at 00pM, approximately 100pM to 50pM, or approximately 50pM to 10pM .

[0080] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 44), wherein X1 is L, I , or V, X2 is V, L, or I, X3 is A or V, and X4 is A , I, or L, X5 is I, T, S, or F, and X6 is E, V, or L X7 is K or R, X8 is E or Q, and X9 is H, P, or R. Ri, X 10 is S, T, or G, and X 11 is K or R, and X 12 is N, A, C , D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y , X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V , W, or Y, and X 14 is V or I, and X 15 is F, L, or V, X 16 comprises a SIRP-α D1 variant having a sequence that is F or V, The compound has at least one nucleotide sequence similar to that of the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:8. Both have one amino acid substitution.

[0081] In some embodiments, the polypeptide is SEQ ID NO:44, wherein X1 is L, I, or V. In any of the above embodiments of this aspect of the disclosure, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I, or L. In any of the above embodiments, X5 is I, T, S, or F. , X6 is E, V, or L. In any of the above embodiments, X7 is K or R In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X is H, P, or R. 10 is S, T, or G. In any of the above embodiments, X 11 is K or R In any of the above embodiments, X 12 are N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In either case, X 13 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the above embodiments, X 14 is V or In any of the above embodiments, X 15 is F, L, or V. In any of the embodiments, X 16 is F or V.

[0082] In some embodiments, the polypeptide is a wild-type SIRP having the sequence of SEQ ID NO:8. -SIRP-α D1 variants with 10 or fewer amino acid substitutions in the α D1 domain In some embodiments, the polypeptide comprises a polypeptide having the sequence of SEQ ID NO:8. SIRP-α with seven or fewer amino acid substitutions relative to the wild-type SIRP-α D1 domain Includes the D1 variant.

[0083] In some embodiments, the polypeptide is a wild-type SIR having the sequence of SEQ ID NO:8. It binds to CD47 with at least 10-fold higher binding affinity than the P-α D1 domain. In some embodiments, the polypeptide is a wild-type SIRP-α having the sequence of SEQ ID NO:8. It binds to CD47 with at least 100-fold higher binding affinity than the D1 domain. In one embodiment, the polypeptide is a wild-type SIRP-α D having the sequence of SEQ ID NO:8. It binds to CD47 with at least 1000-fold higher binding affinity than the 1 domain. In embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is D 1 x10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 It binds to CD47 at less than M. In embodiments, the SIRP-α D1 variant polypeptide or fragment thereof is D about 5 00nM~100nM, approx. 100nM~50nM, approx. 50nM~10nM, approx. 10nM~ 5nM, approx. 5nM~1nM, approx. 1nM~500pM, approx. 500pM~100pM, approx. 10 Binds to CD47 at 0 pM to 50 pM, or approximately 50 pM to 10 pM.

[0084] In another aspect, the present disclosure provides: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TS LX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X15 GX 16 F PRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 I TX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAG TELSVRX 31 KPS (SEQ ID NO: 47), wherein X1 is E or G and X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, and X 5 is L or S, X6 is S or T, X7 is A or V, and X8 is I or or T, X9 is H, R, or L, and X 10 is A, V, I, or L, X 11 is I, T, S, or F, and X 12 is A or G, and X 13 E, V, and or L and X 14 is K or R, and X 15 is E or Q, and X 16 is H, P , or R and X 17 is D or E, and X 18 is S, L, T, or G, X 19 is K or R, and X 20 is E or N, and X 21 is S or P, and X 22 is S or R, and X 23 is S or G, and X 24 is any amino acid, X 25 is any amino acid, and X 26is V or I, and X 27 are F, L, and V , X 28 is D or absent, and X 29 is T or V, and X 30 is F or V and X 31 includes SIRP-α D1 variants having a sequence that is A or G. and the variant is a wild-type SIRP-α having any one of the sequences set forth in SEQ ID NOs: 1 to 10. A polypeptide having at least one amino acid substitution relative to the D1 domain. do.

[0085] In some embodiments, the polypeptide is SEQ ID NO: 47, wherein X1 is E or In any of the above embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the above embodiments, X3 is V, L, or I. In any of the above embodiments, X4 is S or F. In any of the above embodiments, In any of the above embodiments, X5 is L or S. In any of the above embodiments, X6 is S or In any of the above embodiments, X7 is A or V. In any of the above embodiments, X8 is I or T. In any of the above embodiments, X9 is H, R, or L. In any of the above embodiments, X 10 is A, V, I, or is L. In any of the above embodiments, X 11 is I, T, S, or F. In any of the above embodiments, X 12 is A or G. In any of the above embodiments In X 13 is E, V, or L. In any of the above embodiments, X 14 is K or R. In any of the above embodiments, X 15 is E or Q. In any of the above embodiments, X 16 is H, P, or R. In either case, X 17 is D or E. In any of the above embodiments, X 18 teeth S, L, T, or G. In any of the above embodiments, X 19 is K or R In any of the above embodiments, X 20 is E or N. In either case, X 21 is S or P. In any of the above embodiments, X 22 is S or R. In any of the above embodiments, X 23 is S or G. In any of the above embodiments, X 24 is N, A, C, D, E, F, G, H, I, K, L , M, P, Q, R, S, T, V, W, or Y. , X 25 are P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V , W, or Y. In any of the above embodiments, X 26 is V or I. In any of the above embodiments, X 27 is F, L, or V. In any of the above embodiments In X 28 is D or absent. In any of the above embodiments, X 29 is T or V. In any of the above embodiments, X 30 is F or V In any of the above embodiments, X 31 is A or G.

[0086] In some embodiments, the polypeptide of this aspect of the disclosure is any of SEQ ID NOs: 1-10. For wild-type SIRP-α D1 domains with either one of the sequences, In some embodiments, the polypeptide of this aspect of the disclosure comprises a nucleotide sequence similar to SEQ ID NO: 7 for a wild-type SIRP-α D1 domain having any one of the sequences 1 to 10 Contains up to 10 amino acid substitutions.

[0087] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NOs: 1-10. with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain 47. In some embodiments, the polypeptide binds to any of SEQ ID NOs: 1-10. at least 100-fold higher than the wild-type SIRP-α D1 domain with either sequence In some embodiments, the polypeptide binds to CD47 with binding affinity. A wild-type SIRP-α D1 domain having any one of the sequences of Nos. 1 to 10 Both SIRs bind to CD47 with 1000-fold higher binding affinity. The P-α D1 variant polypeptide or a fragment thereof may be D 1x10 -8 Less than M, 5 x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M , or 1x10 -11 In some embodiments, the SIR binds to CD47 at less than M. The P-α D1 variant polypeptide or a fragment thereof may be D Approximately 500nM~100nM , about 100nM~50nM, about 50nM~10nM, about 10nM~5nM, about 5nM~1 nM, approximately 1nM to 500pM, approximately 500pM to 100pM, approximately 100pM to 50pM, or It binds to CD47 at approximately 50 pM to 10 pM.

[0088] In some embodiments, the polypeptide is

[0089] EEELQX1IQPDKSVX2VAAGEX3AX4LX5CTX6TSLX7P VGPIQWFRGAGPX8RX9LIYNQX 10 X 11 GX 12 FPRVTTVS X 13 X 14 TKRX 15 NMDFSIX 16 IX 17 X 18 ITPADAGTYYCX 19 KFRKGX 20 X 21 X 22 DX 23 EFKSGAGTELSVRAKPS(array No. 48), wherein X1 is V or I, X2 is L or S, and X3 is T or is S, X4 is T or I, X5 is R or H, and X6 is A, V, or X7 is I, R, Y, K or F, X8 is G or A, and X9 is E or or V, and X 10 is K or R, and X 11 is E, D or Q, and X 12 is H or P and X 13 is D or E, and X 14 is S, L or T, and X 15 teeth N or E, X 16 is R or S, and X 17 is G or S, and X 18 is N Or A and X 19 is V or I, and X 20is S, I or M, and X 21 teeth P or absent, X 22 is D or P, and X 23 is V or T The present invention also includes a SIRP-α D1 variant having a sequence similar to that of the SIRP-α D1 variant, or a fragment thereof.

[0090] In another aspect, the present disclosure provides: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNM DFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAG TELSVRAKPS (SEQ ID NO: 49), wherein X1 is V, L, or I; and X2 X1 is A, I, V, or L, X2 is I, F, S, or T, and X3 is E, V, or or L, X5 is K or R, X6 is E or Q, and X7 is H, P, or is R, X8 is L, T, S, or G, X9 is A, and X 10 is V or I and a SIRP-α D1 variant having a sequence which is SEQ ID NO: 1. a wild-type SIRP-α D1 domain having any one of the sequences: Polypeptides with one amino acid substitution are characterized.

[0091] In some embodiments, the polypeptide is SEQ ID NO:49, wherein X1 is V, L or or I. In any of the above embodiments of this aspect of the disclosure, X2 is In any of the above embodiments, X3 is I, F, S, or or T. In any of the above embodiments, X4 is E, V, or L. In any of the above embodiments, X5 is K or R. and X6 is E or Q. In any of the above embodiments, X7 is H, P, or In any of the above embodiments, X8 is L, T, S, or G. In any of the above embodiments, X is A. 10 is V or I.

[0092] In some embodiments, the polypeptide comprises each of X1, X2, X3, X4, X5, X6 , X7, X8, X9, and X 10 For SEQ ID NO: 49, where the wild-type amino acid is not at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%) %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and It has a high-affinity SIRP-α D1 domain with 100% sequence identity to the SIRP-α D1 domain.

[0093] In some embodiments, the polypeptide of this aspect of the disclosure is any one of SEQ ID NO: 1. The wild-type SIRP-α D1 domain has one sequence, but the D1 domain has 10 or fewer amino acid substitutions. In some embodiments, the polypeptide of this aspect of the disclosure comprises any of SEQ ID NO: 1. A wild-type SIRP-α D1 domain having one of the following sequences is Contains acid substitutions.

[0094] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NO:1. SIRP-α D1 domain binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to any one of the sequences set forth in SEQ ID NO:1. with at least 100-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to CD47. at least 1000-fold higher than the wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the SIRP-α D1 barrier binds to CD47 with a binding affinity. The polypeptide or fragment thereof is D 1x10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 - 11 In some embodiments, the SIRP-α D1 barrier binds to CD47 at less than M. The polypeptide or fragment thereof is D Approximately 500nM~100nM, Approximately 100nM~5 0nM, approximately 50nM~10nM, approximately 10nM~5nM, approximately 5nM~1nM, approximately 1nM~5 00pM, about 500pM to 100pM, about 100pM to 50pM, or about 50pM to 1 Binds to CD47 at 0 pM.

[0095] In another aspect, the present disclosure provides: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQ WFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENM DFSISISX9ITPADAGTYYCX 10 KFRKGSPDTEFKSGAGT ELSVRAKPS (SEQ ID NO: 50), wherein X1 is V or I and X2 is V or I. or I, X3 is I or F, X4 is E or V, and X5 is K or R. X6 is E or Q, X7 is H or P, X8 is S or T, and X 9 is N or A, X 10 SIRP-α D1 variants with sequences that are V or I ant, wherein the variant comprises a wild-type SIR having the sequence of any one of SEQ ID NOs: 2. A polypeptide having at least one amino acid substitution relative to the P-α D1 domain is identified. It is a sign.

[0096] In some embodiments, the polypeptide is SEQ ID NO: 50, wherein X1 is V or In any of the above embodiments of this aspect of the disclosure, X2 has a sequence that is V or I. or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is E or V. In any of the above embodiments, X 5 is K or R. In any of the above embodiments, X6 is E or Q. In any of the above embodiments, X7 is H or P. In any of the above embodiments, X8 is S or R. In any of the above embodiments, X9 is N or A. In any of the above embodiments, X 10 is V or I.

[0097] In some embodiments, the polypeptide comprises each of X1, X2, X3, X4, X5, X6 , X7, X8, X9, and X 10 For SEQ ID NO: 50, where the wild-type amino acid is not at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%) %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and It has a high-affinity SIRP-α D1 domain with 100% sequence identity to the SIRP-α D1 domain.

[0098] In some embodiments, the polypeptide of this aspect of the disclosure is any one of SEQ ID NOs: 2 The wild-type SIRP-α D1 domain has one sequence, but the D1 domain has 10 or fewer amino acid substitutions. In some embodiments, the polypeptide of this aspect of the disclosure comprises any of SEQ ID NO:2. A wild-type SIRP-α D1 domain having one of the following sequences is Contains acid substitutions.

[0099] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NOs:2. SIRP-α D1 domain binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to any one of the sequences of SEQ ID NO:2. with at least 100-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to CD47. at least 1000-fold higher than the wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the SIRP-α D1 barrier binds to CD47 with a binding affinity. The polypeptide or fragment thereof is D 1x10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 - 11 In some embodiments, the SIRP-α D1 barrier binds to CD47 at less than M. The polypeptide or fragment thereof is D Approximately 500nM~100nM, Approximately 100nM~5 0nM, approximately 50nM~10nM, approximately 10nM~5nM, approximately 5nM~1nM, approximately 1nM~5 00pM, about 500pM to 100pM, about 100pM to 50pM, or about 50pM to 1 Binds to CD47 at 0 pM.

[0100] In another aspect, the present disclosure provides: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMD FSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTE LSVRAKPS (SEQ ID NO: 51), wherein X1 is V or I and X2 is A or X3 is I or F, X4 is E or V, and X5 is K or R X6 is H or P, X7 is L or T, and X8 is any amino acid other than N. and X9 is V or I, the variant is a wild-type SIRP-α D1 domain having any one of the sequences of SEQ ID NO: 1. The present invention features polypeptides having at least one amino acid substitution relative to the amino acid sequence of the present invention.

[0101] In some embodiments, the polypeptide is SEQ ID NO: 51, wherein X1 is V or In any of the above embodiments of this aspect of the disclosure, X2 has a sequence that is A or I. or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is E or V. In any of the above embodiments, X 5 is K or R. In any of the above embodiments, X6 is H or P. In any of the above embodiments, X7 is L or T. In any of the above embodiments, X8 is N or A. In any of the above embodiments, X9 is V or I. In some embodiments, X4 is not V.

[0102] In some embodiments, the polypeptide is SEQ ID NO: 51, wherein X8 is A. In any of the above embodiments of this aspect of the disclosure, X is A and X 1 is V or I. In any of the above embodiments of this aspect of the disclosure, X8 is A. and X2 is A or I. In any of the above embodiments, X8 is A and X X3 is I or F. In any of the above embodiments, X8 is A and X4 is E or or V. In some embodiments, X4 is not V. In any of the above embodiments, In any of the above embodiments, X8 is A and X5 is K or R. X8 is A and X6 is H or P. In any of the above embodiments, X8 is A and X7 is A or V. In any of the above embodiments, X8 is A; X9 is V or I.

[0103] In some embodiments, the polypeptide is SEQ ID NO: 51, wherein X8 is A. In any of the above embodiments of this aspect of the disclosure, X is A and X In any of the above embodiments of this aspect of the disclosure, X is A and X 2 is I. In any of the above embodiments, X8 is A and X3 is F. In any of the above embodiments, X8 is A and X4 is V. In any of the above embodiments, X8 is A and X5 is R. X8 is A and X6 is P. In any of the above embodiments, X8 is A and X 7 is T. In any of the above embodiments, X8 is A and X9 is I.

[0104] In some embodiments, the polypeptide comprises each of X1, X2, X3, X4, X5, X6 51, wherein X7, X8, and X9 are not wild-type amino acids; 5% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%) %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 It has a high-affinity SIRP-α D1 domain with 100% sequence identity to the SIRP-α D1 domain.

[0105] In some embodiments, the polypeptide of this aspect of the disclosure is any one of SEQ ID NO: 1. The wild-type SIRP-α D1 domain has one sequence, but the D1 domain has 10 or fewer amino acid substitutions. In some embodiments, the polypeptide of this aspect of the disclosure comprises any of SEQ ID NO: 1. A wild-type SIRP-α D1 domain having one of the following sequences is Contains acid substitutions.

[0106] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NO:1. SIRP-α D1 domain binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to any one of the sequences set forth in SEQ ID NO:1. with at least 100-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to CD47. at least 1000-fold higher than the wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the SIRP-α D1 barrier binds to CD47 with a binding affinity. The polypeptide or fragment thereof is D 1x10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 - 11 In some embodiments, the SIRP-α D1 barrier binds to CD47 at less than M. The polypeptide or fragment thereof is D Approximately 500nM~100nM, Approximately 100nM~5 0nM, approximately 50nM~10nM, approximately 10nM~5nM, approximately 5nM~1nM, approximately 1nM~5 00pM, about 500pM to 100pM, about 100pM to 50pM, or about 50pM to 1 Binds to CD47 at 0 pM.

[0107] In another aspect, the present disclosure provides: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDF SIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO: 52), wherein X1 is V, L, or I and X2 is A, I , or L, X3 is I, T, S, or F, X4 is K or R, and X5 X is H, P, or R, X6 is L, T, or G, and X7 is N or A. and a SIRP-α D1 variant having the sequence of SEQ ID NO: 1. having at least one amino acid substitution relative to the wild-type SIRP-α D1 domain The present invention is characterized by a polypeptide comprising:

[0108] In some embodiments, the polypeptide has the sequence of SEQ ID NO: 52, wherein X1 is V, L or or I. In any of the above embodiments of this aspect of the disclosure, X2 is A, I, or L. In any of the above embodiments, X3 is I, T, S, or In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is is L, T, or G. In any of the above embodiments, X7 is N or A.

[0109] In some embodiments, the polypeptide is SEQ ID NO: 52, wherein X1 is V or In any of the above embodiments of this aspect of the disclosure, X2 has a sequence that is A or I. or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X 5 is H or P. In any of the above embodiments, X6 is L or T. In any of the above embodiments, X7 is N or A.

[0110] In some embodiments, the polypeptide is SEQ ID NO: 52, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A and X 1 is V or I. In any of the above embodiments of this aspect of the disclosure, X7 is A. and X2 is A or I. In any of the above embodiments, X7 is A and X X3 is I or F. In any of the above embodiments, X7 is A and X4 is K or or R. In any of the above embodiments, X7 is A and X5 is H or P. In any of the above embodiments, X7 is A and X6 is L or T.

[0111] In some embodiments, the polypeptide is SEQ ID NO: 52, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A and X In any of the above embodiments of this aspect of the disclosure, X7 is A and X 2 is I. In any of the above embodiments, X7 is A and X3 is F. In any of the above embodiments, X7 is A and X4 is R. In any of the above embodiments, X7 is A and X5 is P. 7 is A and X6 is T.

[0112] In some embodiments, the polypeptide comprises each of X1, X2, X3, X4, X5, X6 and SEQ ID NO: 52, in which X7 is not the wild-type amino acid. Uniformity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It has a high-affinity SIRP-α D1 domain with phospholipid activity.

[0113] In some embodiments, the polypeptide of this aspect of the disclosure is any one of SEQ ID NO: 1. The wild-type SIRP-α D1 domain has one sequence, but the D1 domain has 10 or fewer amino acid substitutions. In some embodiments, the polypeptide of this aspect of the disclosure comprises any of SEQ ID NO: 1. A wild-type SIRP-α D1 domain having one of the following sequences is Contains acid substitutions.

[0114] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NO:1. SIRP-α D1 domain binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to any one of the sequences set forth in SEQ ID NO:1. with at least 100-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to CD47. at least 1000-fold higher than the wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the fragment binds to CD47 with a binding affinity of 10 amino acids. less than about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, and about 40 amino acids in length 50 amino acids in length, 60 amino acids in length, 70 amino acids in length, 80 amino acids in length 100 amino acids in length, 90 amino acids in length, 100 amino acids in length, or more than 100 amino acids in length The fragments contain SIRP polypeptides. The fragments retain the ability to bind to CD47. Preferably, the fragments contain SIRP polypeptides. -α D1 variant polypeptides and fragments thereof, wherein the SIRP-α polypeptide is a CD4 7. For example, in some embodiments, The SIRP-α D1 variant polypeptide or a fragment thereof may be D 1x10 -8 M not yet Full, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 In some embodiments, the ATP binds to CD47 with a binding affinity of less than M. The SIRP-α D1 variant polypeptide or a fragment thereof may be D Approximately 500nM~10 0nM, approx. 100nM~50nM, approx. 50nM~10nM, approx. 10nM~5nM, approx. 5n M~1nM, approx. 1nM~500pM, approx. 500pM~100pM, approx. 100pM~50p M, or approximately 50 pM to 10 pM, binds to CD47.

[0115] In another aspect, the present disclosure provides: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQ WFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDF SISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSV RAKPS (SEQ ID NO: 212), wherein X1 is V, L, or I and X2 is V, I , or L, X3 is I, T, S, or F, X4 is K or R, and X5 X is H, P, or R, X6 is S, T, or G, and X7 is A. and a SIRP-α D1 variant comprising any one of SEQ ID NOs: 2. at least one amino acid substitution relative to the wild-type SIRP-α D1 domain having the sequence The present invention is characterized by a polypeptide having the following structure:

[0116] In some embodiments, the polypeptide is SEQ ID NO: 212, wherein X1 is V, L or I. In any of the above embodiments of this aspect of the disclosure, X2 is V, I, or L. In any of the above embodiments, X3 is I, T, S, or is F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is S, T, or G. In any of the above embodiments, X7 is A.

[0117] In some embodiments, the polypeptide is SEQ ID NO: 212, wherein X1 is V or In any of the above embodiments of this aspect of the disclosure, X2 has a sequence where X2 is V or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is K or R. X5 is H or P. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is A.

[0118] In some embodiments, the polypeptide is SEQ ID NO: 212, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A, X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X7 is A and X2 is V or I. In any of the above embodiments, X7 is A and X X3 is I or F. In any of the above embodiments, X7 is A and X4 is K or or R. In any of the above embodiments, X7 is A and X5 is H or P. In any of the above embodiments, X7 is A and X6 is S or T.

[0119] In some embodiments, the polypeptide is SEQ ID NO: 212, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A, X1 is I. In any of the above embodiments of this aspect of the disclosure, X7 is A; X2 is I. In any of the above embodiments, X7 is A and X3 is F. In any of the above embodiments, X7 is A and X4 is R. In any of the above embodiments, X7 is A and X5 is P. X7 is A and X6 is T.

[0120] In some embodiments, the polypeptide comprises each of X1, X2, X3, X4, X5, X6 and at least 85% of the sequence of SEQ ID NO: 212, where X7 is not the wild-type amino acid. Identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It has a high-affinity SIRP-α D1 domain with a single domain identity.

[0121] In some embodiments, the polypeptide of this aspect of the disclosure is any one of SEQ ID NOs: 2 The wild-type SIRP-α D1 domain has one sequence, but the D1 domain has 10 or fewer amino acid substitutions. In some embodiments, the polypeptide of this aspect of the disclosure comprises any of SEQ ID NO:2. A wild-type SIRP-α D1 domain having one of the following sequences is Contains acid substitutions.

[0122] In some embodiments, the polypeptide has the sequence of any one of SEQ ID NOs:2. SIRP-α D1 domain binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to any one of the sequences of SEQ ID NO:2. with at least 100-fold higher binding affinity than the wild-type SIRP-α D1 domain In some embodiments, the polypeptide binds to CD47. at least 1000-fold higher than the wild-type SIRP-α D1 domain with one of the sequences In some embodiments, the fragment binds to CD47 with a binding affinity of 10 amino acids. less than about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, and about 40 amino acids in length 50 amino acids in length, 60 amino acids in length, 70 amino acids in length, 80 amino acids in length 100 amino acids in length, 90 amino acids in length, 100 amino acids in length, or more than 100 amino acids in length The fragments contain SIRP polypeptides. The fragments retain the ability to bind to CD47. Preferably, the fragments contain SIRP polypeptides. -α D1 variant polypeptides and fragments thereof, wherein the SIRP-α polypeptide is a CD4 7. For example, in some embodiments, The SIRP-α D1 variant polypeptide or a fragment thereof may be D 1x10 -8 M not yet Full, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 In some embodiments, the ATP binds to CD47 with a binding affinity of less than M. The SIRP-α D1 variant polypeptide or a fragment thereof may be D Approximately 500nM~10 0nM, approx. 100nM~50nM, approx. 50nM~10nM, approx. 10nM~5nM, approx. 5n M~1nM, approx. 1nM~500pM, approx. 500pM~100pM, approx. 100pM~50p M, or approximately 50 pM to 10 pM, binds to CD47.

[0123] In some embodiments, disclosed herein is EEELQX1IQPDKS VLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LI YNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 KFRKGSPDDVEFKSGAGTELSVRA KPS (SEQ ID NO: 218), wherein X1 is V, L, or I and X2 is A, V, L , or I, X3 is I, S, T, or F, and X4 is E, L, or V , X5 is K or R, X6 is E or Q, and X7 is H, R, or P; X8 is S, G, L, or T, X9 is any amino acid, and X 10 is any mesh X 11 is any amino acid, and X 12 is any amino acid, and X 13 teeth V or I, The D1 variant is a variant of the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 1. A polypeptide having at least two amino acid substitutions.

[0124] In some embodiments, the polypeptide is SEQ ID NO: 212, wherein X9 is A. In any of the above embodiments of this aspect of the disclosure, X9 is N. In any of the above embodiments of this aspect of the disclosure, X 10 is I. This aspect of the disclosure In any of the above embodiments, X9 is N and X10 is P. In any of the above embodiments, X9 is N and X11 is S, T, or C or a combination thereof. In any of the above embodiments of this aspect of the disclosure, X 11 is T. In any of the above embodiments of this aspect of the disclosure, X 11 is any value other than T In any of the above embodiments of this aspect of the disclosure, X 12 is P In any of the above embodiments of this aspect of the disclosure, X is N and X 12 P and above Any amino acid other than the above.

[0125] In some embodiments, disclosed herein are EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNM DFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSG AGTELSVRAKPS (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, and X4 is E, L or V, X5 is K or R, X6 is E or Q, and X7 is H, R , or P, X8 is S, G, L, or T, X9 is N, and X 10 P and above Any amino acid other than X 11 SIRP-α D1 having a sequence that is V or I and a variant thereof, wherein the SIRP-α D1 variant is a wild-type SIRP-α D1 variant having the sequence of SEQ ID NO: 1. Polypeptides with at least two amino acid substitutions in the type SIRP-α D1 domain It's Chido.

[0126] In another embodiment of the present disclosure, SIRP-α D1 having the amino acid sequence of SEQ ID NO: 48 Compositions comprising the variant polypeptides, or fragments thereof, are disclosed. In this case, the SIRP-α D1 variant polypeptide or a fragment thereof is a SIRP-α polypeptide. It binds to CD47 with a higher affinity than the affinity of the polypeptide that binds to CD47. In some embodiments, the SIRP-α D1 variant polypeptide is D 1x1 0 -8 Less than M or 1x10 -9 Under M, 1x10 -10 Less than M or 1x10 -1 1 In some embodiments, the SIRP-α D1 barrier binds to CD47 with less than M. The ant polypeptide is conjugated or fused to a second polypeptide. In this embodiment, the second polypeptide may be, without limitation, an Fc polypeptide, an Fc variant, or the like. , HSA polypeptide, albumin peptide, PEG polymer, or fragments thereof. can be done.

[0127] Without limiting the above, in some embodiments, SIRP-α D1 variant polypeptides The polypeptide is selected from any one of SEQ ID NOs: 53 to 87 and 213 shown in Table 6. . [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0128] In some embodiments, the polypeptide has a sequence similar to that of any of the variants shown in Table 6. At least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0129] In some embodiments, the polypeptide is SEQ ID NO: 80, 81, or 85 in Table 6. at least 85% sequence identity (e.g., at least 86%, 87%, 88% ,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% , 99%, or 100% sequence identity) Includes.

[0130] III. Fc Domain Variants and Fusion Constructs In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0131] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0132] Antibodies that target cell surface antigens involve the engagement of Fc receptors (FcRs) on immune cells. It can induce immune stimulation and effector functions that contribute to immune responses. IgG (gamma receptor) , IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors) There are multiple Fc receptors specific for particular antibody classes, including: Fc receptors on the cell surface Binding of the Fc region to the body facilitates phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, also known as ADCP), removal of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cytotoxicity). ADCC), as well as the release of inflammatory mediators and placental passage They can elicit many biological responses, including the regulation of immune responses, immune responses, and the regulation of immunoglobulin production. Furthermore, binding of the C1 component of complement to antibodies can activate the complement system. Complement activation can be important for the lysis of cellular pathogens. Activation can also stimulate inflammatory responses, which are involved in autoimmune hypersensitivity and other immune disorders. Variant Fc antibodies that have reduced or eliminated binding to certain Fc receptors may also be used. The c region targets, activates, and mediates ligand function without damaging or destroying local cells or tissues. to develop therapeutic antibodies and Fc fusion polypeptide constructs that act by neutralizing It is useful for

[0133] In some embodiments, the SIRP-α D1 polypeptide construct is an effector function. Non-covalently linked to Fc domain monomers forming Fc domains with ablated or reduced activity Contains naturally occurring high-affinity SIRP-α D1 variants.

[0134] In some embodiments, an Fc domain monomer refers to a second and third antibody constant domain. In some embodiments, the term "polypeptide chain" refers to a polypeptide chain comprising a CH2 and a CH3. In some embodiments, the Fc domain monomer also comprises a hinge domain. The c domain monomers can be used to identify any immune system, including IgG, IgE, IgM, IgA, and IgD. Furthermore, in some embodiments, the antibody is of the immunoglobulin antibody isotype. The main monomer can be any IgG subtype (e.g., IgG1, IgG2, IgG2a , IgG2b, IgG2c, IgG3, and IgG4). In this state, the Fc domain monomers contain as many as 10 changes from the wild-type Fc domain monomer sequence. (e.g., 1 to 10, 1 to 8, 1 to 6, or 1 to 4 amino acid substitutions, additions, insertions, or deletions) or a combination thereof), which alters the interaction between the Fc domain and the Fc receptor. To transform.

[0135] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. In a wild-type Fc domain, two Fc domain monomers are connected to two CH3 antigens. Interactions between the Fc domains and the cytoplasmic constant domains of two dimerized Fc domain monomers Dimerization occurs via one or more disulfide bonds between the didomains. In some embodiments, the Fc domain may be modified to lack effector function, e.g., a "dead Fc" domain. In some embodiments, each Fc domain in the Fc domain is mutated to a "Fc domain". The monomer contains an amino acid substitution in the CH2 antibody constant domain, and the Fc domain and F c receptors, such as Fcγ receptors (FcγR), Fcα receptors (FcαR), or Fc ε (FcεR) interaction or binding.

[0136] In some embodiments, high affinity SIRP-α D1 variants (e.g., Tables 2, 5 , and any of the variants shown in 6) is an immunoglobulin Fc domain monomer or is fused to a fragment of an Fc domain monomer. The Fc domain monomer or fragment of an Fc domain monomer may be amplified by another Fc domain monomer. In some embodiments, the immunoglobulin may be a soluble form of an Fc domain. The Fc domain monomer or fragment of the Fc domain monomer of a globulin can be expressed by another Fc domain. In some embodiments, the Fc domain cannot be formed with the guanine monomer. the Fc domain monomer or fragment of the Fc domain is fused to a polypeptide of the disclosure, In some embodiments, the polypeptides of the present disclosure are The Fc domain monomer or fragment of the Fc domain monomer fused to the second Fc dimerize with domain monomers to form Fc domains that bind to Fc receptors; or Or alternatively, the Fc domain monomer binds to an Fc receptor. The present invention relates to an Fc domain fused to a polypeptide to extend the serum half-life of the polypeptide. Neither the main nor fragments of the Fc domain induce any immune system-related responses.

[0137] In some embodiments, the SIRP-α polypeptide or construct provided herein The present invention relates to a SIRP-α D1 domain or its derivatives tethered to a first Fc domain monomer. and a second Fc domain monomer, and a second Fc domain monomer is linked to form an Fc domain (e.g., a heterodimeric Fc The Fc domain is a protein found at the C-terminus of immunoglobulins. The Fc domain is a dimer formed by interactions between the CH3 antibody constant domains. The wild-type Fc domain contains two Fc domain monomers that bind to Fc receptors, e.g. For example, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIII b, and forms a minimal structure that binds to FcγRIV.

[0138] The Fc domain is not directly involved in binding an antibody to its antigen, but is involved in antibody-dependent cell proliferation. These antibodies may be involved in various effector functions, such as the involvement of the antibody in cytotoxicity. In some embodiments, the Fc domain of a SIRP-α polypeptide or construct of the disclosure comprises: Decreased effector function, e.g., decreased antibody-dependent cell-mediated cytotoxicity (ADCC), Decreased complement-dependent cytolysis (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof, resulting in an amino acid substitution, addition or insertion, deletion, or In some embodiments, the SIRP-α polypeptide of the present disclosure is The peptide or construct may have reduced binding to human Fc receptors (e.g., minimal binding or no binding) and reduced binding to the complement protein C1q (e.g., minimal binding In some embodiments, the SIRP-α of the present disclosure is characterized by: The constructs include human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, Fc γRIIIB, or any combination thereof, and reduced binding to C1q (e.g., Characterized by antibody-dependent effector functions, e.g., minimal or no binding. For example, altering or reducing ADCC, CDC, ADCP, or any combination thereof. Therefore, in some embodiments, the Fc domain of the SIRP-α constructs of the present disclosure is IgG class, and one or more amino acid substitutions are E233, L234, L235, G2 36, G237, D265, D270, N297, E318, K320, K322, A3 27, A330, P331, or P329 (numbering is based on the EU numbering in Kabat) Sequences of Proteins of Immuno logical interest,5th Ed. Public Health S ervice,National Institutes of Health,Bet hesda, MD. (1991)).

[0139] In some embodiments, a polypeptide construct comprising a non-native Fc region as described herein compared to polypeptide constructs containing the native Fc region, CD16a, CD32a, C Decreased binding of CD32b, CD32c, and CD64 to at least one of the Fcγ receptors In some cases, the polypeptide constructs described herein exhibit a reduction or elimination of , CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors. This shows a reduction or elimination of binding to the

[0140] CDC is a process in which the complement cascade is activated by the complement component C1q, which binds to the Fc of antibodies. In some embodiments, the non-native Fc region described herein is a cytotoxic form of A polypeptide construct comprising a wild-type Fc region has a significantly increased Fc-dependent cleavage rate compared to a polypeptide construct comprising a wild-type Fc region. At least 5%, 10%, 15%, 20%, 30%, 40%, 50% in C1q binding , 60%, 70%, 80%, 90% or more reduction. Thus, the polypeptide constructs comprising the non-native Fc region described herein may be constructed by incorporating a wild-type Fc region. In some embodiments, the polypeptide constructs exhibit reduced CDC compared to polypeptide constructs comprising the polypeptides described herein. The polypeptide constructs comprising a non-native Fc region described herein are polypeptides comprising a wild-type Fc region. At least 5%, 10%, 15%, 20%, 30%, 40% compared to the peptide construct , 50%, 60%, 70%, 80%, 90% or greater reduction in CDC. In some cases, polypeptide constructs comprising the non-naturally occurring Fc variants described herein The constructs exhibit negligible CDC compared to polypeptide constructs containing wild-type Fc regions. show.

[0141] In some embodiments, the Fc variants herein have a minimum of 200 nucleotides compared to the wild-type sequence. In some embodiments, the glycosylated protein is minimally glycosylated or has reduced glycosylation. Glycosylation can be achieved by mutation of N297A or by changing N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by altering the motif. N-Xaa1-Xaa2-Xaa3, where N=asparagine and Xaa1=P(Pro Xaa2 = T (threonine), S (serine) or C (cysteine). steine), and disruption of motifs where Xaa3 = any amino acid other than P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300 indicated according to Kabat et al., 1991. In some embodiments, any one or more of N, Xaa1, Xaa2, or Xaa3 Mutations to result in deglycosylation of the Fc variant.

[0142] In some embodiments, variants of antibody IgG constant regions (e.g., Fc variants) are used. ) have a reduced ability to specifically bind to Fcγ receptors or to induce phagocytosis. In some embodiments, variants of antibody IgG constant regions (e.g., For example, Fc variants have a reduced ability to specifically bind to Fcγ receptors, and For example, in some embodiments, the Fc domain The Fc domain is mutated to lack effector function, characteristic of a "dead" Fc domain. For example, in some embodiments, the Fc domain is a cytoplasmic domain that binds to an Fcγ receptor. It contains specific amino acid substitutions known to minimize interactions with In embodiments, the Fc domain monomer is derived from an IgG1 antibody and contains the amino acid substitution L234 A, L235A, G237A, and N297A (Kabat et al., 1991 In some embodiments, the numbering system includes one or more of the following: One or more additional mutations are included in such IgG1 Fc variants. Non-limiting examples of such additional mutations for the Fc variant of 1 include E318A and In some instances, the Fc variants of human IgG1 include wild-type Compared with the human IgG1 sequence, up to 12, 11, 10, 9, 8, 7, 6, 5, and 10 total sequences were identified. In some embodiments, the nucleotide sequence has one or more mutations. Further deletions are included in such IgG1 Fc variants. For example, in some embodiments In this embodiment, the C-terminal lysine of the IgG1 heavy chain constant region of Fc shown in SEQ ID NO: 88 in Table 7 is For example, the polypeptide is deleted when it is produced in bacterial or mammalian cells. In some instances, human IgG1 Fc variants are Compared with the native human IgG1 sequence, a total of 12, 11, 10, 9, 8, 7, 6, 5, In some embodiments, there are four or fewer deletions of IgG1. The Fc variant is any of SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137. It has one sequence.

[0143] In some embodiments, the Fc domain monomer is derived from an IgG2 or IgG4 antibody. and the amino acid substitutions A330S, P331S, or both A330S and P331S The amino acid positions are defined according to Kabat, et al. (1991). The Kabat numbering of amino acid residues is based on the "standard" Kabat numbering for a given antibody. The sequence number can be determined by aligning the homologous regions of the antibody sequence with the t-number sequence. In some embodiments, the Fc variant comprises A330S, P331S, and N297A. Amino acid substitutions (EU numbering system according to Kabat et al. (1991) In some embodiments, the Fc sequence of human IgG2 includes one or more of the following: In the present study, one or more additional mutations are included in the Fc variants of such IgG2. Non-limiting examples of such additional mutations to the G2 Fc variant include V234A , G237A, P238S, V309L, and H268A (Kabat et al. ( Some examples include the EU numbering system (as designated by the EU numbering system in 1991). In total, the Fc variants of human IgG2 have the highest number of Fc variants compared to the wild-type human IgG2 sequence. 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer mutations In some embodiments, one or more additional deletions are made to such IgG2 Fc variants. For example, in some embodiments, the Fc fragment shown in SEQ ID NO: 89 in Table 7 is included. The C-terminal lysine of the IgG2 heavy chain constant region is deleted, and the polypeptide is In some instances, the homogeneity of the polypeptide is enhanced when produced in cellular or mammalian cells. The Fc variants of human IgG2 have a total of up to 1000 nucleotides compared to the wild-type human IgG2 sequence. 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions do.

[0144] When the Fc variant is an IgG4 Fc variant, in some embodiments: Such Fc variants are characterized by the S228P mutation (Kabat et al. (1991) In some instances, the Fc variant of human IgG4 includes a wild-type Compared with the human IgG4 sequence, a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, Possessing 3, 2, or 1 mutation.

[0145] In some embodiments, the Fc variant comprises the mutation L234A in the Fc region of IgG1. , L235A, G237A, or N297A, or at least one of It contains at least one of the following Fc region mutations: A330S, P331S, or N297A. In some embodiments, the Fc variant comprises the mutation L234A in the Fc region of IgG1, At least two of L235A, G237A, or N297A, or F of IgG2 Contains at least two of the c region mutations A330S, P331S, or N297A. In some embodiments, the Fc variant comprises the mutations L234A, L234B, L234C, L234D, L234E, L234F, L234G, L234H, L234H, L234I ... 235A, G237A, or N297A, or IgG The Fc region of the antibody consists of mutations A330S, P331S, and N297A. In one embodiment, the Fc variant comprises the mutations L234A, L235A, G237A, and N29 It consists of 7A.

[0146] In some embodiments, the Fc variant has a sequence similar to that of a wild-type human IgG Fc region. In some embodiments, the Fc barrier The compound exhibits ablation of binding to the Fc receptor of interest compared to the Fc region of wild-type human IgG. In some embodiments, the Fc variant has a Fc region that is greater than or equal to that of wild-type human IgG. In some embodiments, the Fc variants exhibit reduced phagocytosis compared to wild-type human Fc variants. The Fc region of human IgG shows a reduction in phagocytosis.

[0147] SEQ ID NO: 88 and SEQ ID NO: 89 are the amino acid sequences of the IgG1 and IgG2 heavy chain constant regions of the Fc. In some embodiments, the Fc variants are SEQ ID NOs: 90-99 shown in Table 7. Any variant of 95. [Table 7-1] [Table 7-2]

[0148] Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, is a method for the treatment of certain cells. Fc receptors present on cytotoxic cells (e.g., natural killer (NK) cells and neutrophils) Secreted Ig bound to FcRs allows these cytotoxic effector cells to target antigen-bearing cells. Cytotoxicity that specifically binds to and subsequently kills target cells Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, is a Molecules that bind to Fc receptors (FcR) present on certain phagocytes (e.g., macrophages) These phagocytic effector cells specifically bind to antigen-bearing target cells, and the secreted Ig It refers to a cytotoxic form of the substance that is then able to engulf and digest the target cell. Ligand-specific high-affinity IgG antibodies against the surface of target cells inhibit the cytotoxic or phagocytic cell proliferation. Cells can be stimulated and used for such killing. In other words, the polypeptide constructs comprising the Fc variants described herein may be constructed in a manner similar to that described above, but containing a wild-type Fc region. Some experiments show reduced ADCC or ADCP compared to polypeptide constructs containing In embodiments, the polypeptide constructs comprising the Fc variants described herein comprise a wild-type Fc at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater ADCC or a reduction in ADCP. In some embodiments, the Fc variants described herein The polypeptide construct comprising the Fc region has a higher Fc activity than the polypeptide construct comprising the wild-type Fc region. Shows removal of ADCC or ADCP.

[0149] Complement-dependent cytotoxicity, also referred to herein as CDC, is the process by which the complement cascade binds to antibodies. It refers to the form of cytotoxicity activated by the complement component C1q, which binds to c. In embodiments, the polypeptide constructs comprising the Fc variants described herein comprise a wild-type Fc at least 5%, 10%, 15%, 20% compared to a polypeptide construct containing the c region , 30%, 40%, 50%, 60%, 70%, 80%, 90% or more C1q In some cases, the antibodies containing the Fc variants described herein show reduced binding. The polypeptide constructs exhibited reduced CDC compared to polypeptide constructs containing wild-type Fc regions. In some embodiments, polypeptides comprising the Fc variants described herein The polypeptide construct has at least 5%, 1%, or more Fc regions than the wild-type Fc region-containing polypeptide construct. 0%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or In some cases, the Fc balance described herein exhibits a greater reduction in CDC. The polypeptide constructs containing the variants were compared to polypeptide constructs containing wild-type Fc regions. and exhibits negligible CDC.

[0150] As used herein, an Fc variant refers to a variant that has a Fcγ For example, in some embodiments, Fc receptor binding is reduced. As shown in the Examples, the riants bind to the Fcγ receptor expressed by the Fc region of wild-type human IgG. In some instances, the Fc barrier exhibits lower binding to Fcγ than to the receptor. The markers showed a 10%, 20%, 30%, 40%, and 50% reduction in binding to Fcγ receptors. , 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the binding is 100% (complete elimination of effector function). The reduction may be any one or more Fcγ receptors, e.g., CD16a, CD32a, CD32b , CD32c, or CD64.

[0151] In some examples, the Fc variants disclosed herein have the same Fc as that of wild-type human IgG. Such Fc variants exhibit reduced phagocytosis compared to the wild-type human Ig c region. The Fc region of G exhibits a decrease in phagocytosis, and the decrease in phagocytosis activity is, for example, 10%. , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% , 97%, 98%, 99%, or 100%. shows ablation of phagocytosis compared to its wild-type human IgG Fc region.

[0152] In some embodiments, the Fc variants disclosed herein comprise one or more fusion proteins. In some cases, the fusion partner is a therapeutic moiety. In some cases, the fusion partner may be used to target, purify, or otherwise enhance the expression of the expressed protein. In some embodiments, the fusion The binding partner also influences the degree of Fc receptor binding or the reduction of phagocytosis. As described herein, in some embodiments, the Fc variant is linked to a fusion partner. When linked, it forms the polypeptide construct shown below.

[0153] In some embodiments, the fusion partner binds the Fc variant via a linker sequence. In some embodiments, the linker sequence is generally a small number of amino acids. , e.g., containing less than 10 amino acids, although longer linkers are also utilized. In some cases, the linker is less than 10, 9, 8, 7, 6, or 5 amino acids. In some cases, the linker has a length of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 amino acids Optionally, in some embodiments, the cleavable phosphorus Car is used.

[0154] In some embodiments, the fusion partner comprises an Fc variant protein and any associated targeting or signaling that directs the associated fusion partner to a desired cellular location or extracellular medium; In some embodiments, certain signal sequences are expressed in the growth medium or Targeting proteins secreted either into the periplasmic space, located between the inner and outer membranes of cells In some embodiments, the fusion partner allows for purification or screening. Such fusion partners include sequences encoding peptides or proteins that , immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+ Polyhistidine tag (His tag) for use with 2 affinity columns For example, His6 and His10) or other tags, GST fusions, MBP fusions, St The rep-tag, the BSP biotinylation target sequence for the bacterial enzyme BirA, and the antibody-targeted Examples of such epitope tags include those that can be used for the detection of genomic DNA (e.g., c-myc tag, flag tag, etc.). Not limited.

[0155] In some embodiments, such tags are useful for purification, screening, or both. For example, in some embodiments, the Fc variants utilize a His tag, which was purified by immobilization on a Ni+2 affinity column, and then after purification, The His tag is used to immobilize antibodies to Ni+2-coated plates for ELISA and other binding assays. Said fusion partners are carried out as described elsewhere herein. This allows for the use of the selection methods described herein to screen for Fc variants. do.

[0156] A variety of fusion partners are available that allow for different selection methods. by fusing members of the variant library to gene III proteins In some embodiments, the fusion partner may be Alternatively, in some embodiments, the fusion protein may be labeled. The promoter binds to a specific sequence in the expression vector and binds to the fusion partner and associated Fc antigen. The reagents are covalently or non-covalently linked to the nucleic acids that encode them. To do so.

[0157] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety can be, for example, The Fc variant of the present disclosure is a peptide, protein, antibody, siRNA, or small molecule. Non-limiting examples of therapeutic antibodies conjugated to the antibody include antibodies that recognize CD47. Therapeutic peptides conjugated to the Fc variants of the present disclosure include, but are not limited to: Typical examples include CD47-binding polypeptides, including SIRP-α polypeptides. In such examples, the CD47 binding polypeptide may be any of the CD47 binding polypeptides of the present disclosure, but is not limited to these. Examples of CD47-binding polypeptides include anti-C CD47 antibody or fragment thereof, and a ligand of CD47, such as SIRP-α or Further examples of CD47-binding polypeptides include, but are not limited to, fragments thereof. Examples include, but are not limited to, naturally occurring forms of SIRP-α and mutants thereof. Not determined.

[0158] In some embodiments, disclosed herein are polypeptides comprising Fc variants. peptide, wherein the Fc variant is an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L234A, L235 (ii) the Fc region of human IgG1 consisting of the mutations A, G237A, and N297A; the Fc region of human IgG2 consisting of the mutations 30S, P331S and N297A, or ii) S228P, E233P, F234V, L235A, delG236, and N29 The polypeptide is selected from the Fc region of human IgG4 containing the mutation of 7A. In some embodiments, the Fc domain monomers are identical (ie, homodimers). In some embodiments, the Fc domain monomers are different (i.e., heterodimers). In some embodiments, at least one of the Fc domain monomers of the Fc domain dimer The other is human I, which consists of the mutations L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain of the Fc domain dimer is the Fc region of IgG1. At least one of the amino acid monomers contains the A330S, P331S, and N297A mutations. In some embodiments, the Fc variant is a human IgG2 Fc region. The Fc region of IgG exhibits ablation or reduction of binding to Fcγ receptors compared to the wild-type version. In some embodiments, the Fc variants have a similar structure to the wild-type version of the Fc region of human IgG. In comparison, CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors In some embodiments, the Fc variant exhibits ablated or reduced binding to the receptor. Eliminates or reduces binding to C1q compared to wild-type versions of Fc fusions of human IgG In some embodiments, at least one of the Fc domain monomers of the Fc domain dimer At least one of S228P, E233P, F234V, L235A, delG236, and and N297A mutations. The Fc variants have enhanced binding to Fcγ receptors compared to the Fc region of wild-type human IgG4. In some embodiments, the Fc variant exhibits elimination or reduction of the human I Compared to the wild-type version of the Fc region of IgG4, In some embodiments, the Fc variant exhibits ablated or reduced binding to Fc It binds to the gamma receptor with a KD of approximately 5x10-6M or greater.

[0159] In some embodiments, the Fc variant further comprises a CD47 binding polypeptide. In some embodiments, the Fc variant comprises a wild-type version of the Fc region of human IgG. In some embodiments, the antibody exhibits ablated or reduced binding to Fcγ receptors compared to The CD47-binding polypeptide induces acute anemia in rodents and non-human primates. In some embodiments, the CD47 binding polypeptide does not induce the following in humans: Does not cause acute anemia.

[0160] In some embodiments, the CD47 binding polypeptide is signal-regulatory protein alpha. In some embodiments, the SIRP-α polypeptide is a SIRP-α polypeptide or a fragment thereof. The RP-α polypeptide has the amino acid sequence EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMD FSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTE LSVRAKPS (SEQ ID NO: 51), wherein X1 is V or I and X2 is A or X3 is I or F, X4 is E or V, and X5 is K or R X6 is H or P, X7 is L or T, and X8 is any amino acid other than N. and X9 is V or I. In some embodiments, the SIRP-α polypeptide has a structure in which X1 is V or I and X2 is A. or I, X3 is I or F, X4 is E, and X5 is K or R; S where X6 is H or P, X7 is L or T, X8 is not N, and X9 is V Contains IRP-α D1 variant.

[0161] In some embodiments, disclosed herein are polypeptides, Naturally occurring high-affinity SIRP-α D1 domain, with the affinity of the naturally occurring D1 domain SIRP-α D1 variant binds to human CD47 with at least 10-fold higher affinity than and a second polypeptide comprising a second Fc domain monomer linked to the Fc domain. and an Fc domain monomer that forms an Fc domain, wherein the Fc domain is removed or reduced. The polypeptides are those containing the Fc domain monomers that have vector function. In embodiments, the non-naturally occurring high affinity SIRP-α D1 domain has an amino acid sequence at residue 80. Including acid mutations.

[0162] In some embodiments, disclosed herein are SIRP-α D1 variants. wherein the SIRP-α D1 variant has a KD of less than 250 nM and and the SIRP-α D1 variant binds to CD47 from the same species, and the KD is less than 250 nM. binds to CD47 from a second species at a KD for CD47 from the first species; the KD for CD47 from the second species are within 100-fold of each other, and the first and second species The species is selected from the group consisting of humans, rodents, and non-human primates. In one embodiment, the SIRP-α D1 variant is expressed in CD4+ cells from at least three different species. 7. In some embodiments, the non-human primate is a cynomolgus monkey.

[0163] In some embodiments, disclosed herein are compounds that: (a) have a KD of less than 250 nM; (b) a SIRP-α D1 domain that binds to human CD47, and (b) the SIRP-α D1 A polypeptide containing an Fc domain monomer linked to the N- or C-terminus of the domain. In this case, the polypeptide induces acute anemia in rodents and non-human primates. In some embodiments, the polypeptide does not induce a non-naturally occurring mutation in human SIRP-α. In some embodiments, the in vivo administration of the polypeptide comprises: In some embodiments, the administration results in a decrease in hemoglobin of less than 50% during the first week after administration. The study found that administration of the polypeptide in humans resulted in a hemoglobin reduction of less than 50% during the first week after administration. In some embodiments, the polypeptide further comprises at least one and the Fc variant comprises (i) L234A, L235A , G237A, and N297A mutations in the Fc region of human IgG1; (ii) A33 an Fc region of human IgG2 consisting of the mutations 0S, P331S and N297A, or (ii i) S228P, E233P, F234V, L235A, delG236, and N297 The Fc domain monomer is selected from the Fc region of human IgG4 containing the A mutation. In some embodiments, the Fc domain monomer comprises the amino acid sequence L234A, L235A, G237 and N297A mutations. In this case, the Fc domain monomer consists of the A330S, P331S, and N297A mutations. This is the Fc region of human IgG2.

[0164] SIRP-α constructs of the present disclosure can be prepared by conventional genetic or chemical methods, e.g., chemical conjugation The Fc domain monomer is then linked to its C-terminus by a linker at the N-terminus of the Fc domain monomer. In some embodiments, the SIRP-α domain comprises a phosphoprotein or a variant thereof. A carrier (e.g., a spacer) is inserted between the polypeptide and the Fc domain monomer. In some embodiments, a polyclonal antibody of the present disclosure comprising a high affinity SIRP-α D1 variant is The peptide is fused to an Fc domain monomer that is incapable of forming a dimer. In some embodiments, the polypeptides of the present disclosure form dimers, e.g., heterodimers. It is fused to an Fc domain monomer that can form with another Fc domain monomer. In some embodiments, the polypeptides of the invention are fused to an Fc domain monomer, The fusion protein forms a homodimer. In some embodiments, the poly(A)- The peptide is fused to a first Fc domain monomer and is coupled to a different protein or peptide ( For example, an Fc domain (e.g., an antibody variable region) is fused to a second Fc domain monomer. In this embodiment, the SIRP-α D1 domain or a variant thereof is a first Fc domain monoclonal antibody. tethered to a marker and capable of delivering therapeutic proteins (e.g., cytokines, interleukins, antigens, a steroid, anti-inflammatory drug, or immunomodulatory drug) is tethered to the second Fc domain monomer. In some embodiments, the first and second Fc domain monomers are heterodimers. Form.

[0165] Without limiting the above, in some embodiments, SIRP-α D1 variant polypeptides The polypeptide (e.g., any of the variants shown in Tables 2, 5, and 6) is an Fc polypeptide. The antibody may be fused to an Fc domain monomer or an Fc variant polypeptide, for example, an Fc domain monomer. SIRP-α D1 variant polypeptide and fused Fc variant polypeptide Examples of polypeptides containing the above include those of SEQ ID NOs: 96 to 137, 214, and 21 shown in Table 8. 6 are included, but are not limited to. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12] [Table 8-13] [Table 8-14]

[0166] In some embodiments, the polypeptide has a sequence similar to that of any of the variants shown in Table 8. At least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0167] In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NOS: 98-104, 107-109, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 120-121, At least 85% sequence identity to 113, 116-122, or 135-137 Sex (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity ) containing a high-affinity SIRP-α D1 domain.

[0168] In some embodiments, the polypeptide comprises (a) signal-regulatory protein alpha (SI and a SIRP-α D1 variant, wherein the SIRP-α D1 variant is an amino acid sequence of Acid sequence EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TS LX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 F PRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 I TX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAG TELSVRX 31 KPS (SEQ ID NO: 47), wherein X1 is E or G; X2 is L, I, or V, X3 is V, L, or I, and X4 is S or F. X5 is L or S, X6 is S or T, X7 is A or V, and X8 is I or T, X9 is H, R, or L, and X 10 is A, V, I, or L Yes, X 11 is I, T, S, or F, and X 12 is A or G, and X 13 is E, V, or L, and X 14 is K or R, and X 15 is E or Q, and X 16 teeth H, P, or R and X 17 is D or E, and X 18 is S, L, T, or G Yes, X 19 is K or R, and X 20 is E or N, and X 21 is S or P Ri, X 22 is S or R, and X 23 is S or G, and X 24 is any amino acid Yes, X 25 is any amino acid, and X 26 is V or I, and X 27 F, L, and or V, and X 28 is D or absent, and X 29 is T or V, X3 0 is F or V, X 31 is A or G, and the SIRP-α D1 variant is a wild-type SIRP-α D1 domain having any one of the sequences of SEQ ID NOs: 1 to 10; and (b) the polypeptide has at least two amino acid substitutions relative to the and an Fc variant comprising an Fc domain dimer having an Fc domain monomer, Each Fc domain monomer independently consists of (i) a human IgG1 containing the N297A mutation; (ii) a human IgG containing the mutations L234A, L235A, and G237A (iii) the Fc region of IgG1, and the mutations L234A, L235A, G237A, and N297A (iv) an Fc region of human IgG1 containing the N297A mutation; (v) the Fc region of human IgG2 containing the A330S and P331S mutations; (vi) A a human IgG2 Fc region containing the mutations 330S, P331S, and N297A; ) containing the mutations S228P, E233P, F234V, L235A, and delG236 the Fc region of human IgG4, or (viii) S228P, E233P, F234V, L The Fc region of human IgG4 contains the mutations 235A, delG236, and N297A. .

[0169] In some embodiments, the polypeptide comprises an SI polypeptide comprising the amino acid sequence of SEQ ID NO:47. RP-α D1 variant, an Fc domain dimer with two Fc domain monomers wherein the Fc domain monomer of the Fc domain dimer comprises One of the mers is a human genomic DNA fragment containing the mutations L234A, L235A, G237A, and N297A. Contains the Fc region of IgG1.

[0170] Dimerization of Fc domain monomers

[0171] In some embodiments, a SIRP-α D1 variant polypeptide (e.g., Table 2) , 5, and 6) is attached to the N-terminus or In some embodiments, the first Fc domain monomer is fused at the C-terminus or the C-terminus. In some embodiments, the Fc domain is unable to form a dimer. One Fc domain monomer binds to a second Fc domain monomer, In some embodiments, the first and second Fc domain monomers form a dimer. - an amino acid substitution that promotes heterodimerization between the first and second domain monomers Includes.

[0172] In some embodiments, each of the two Fc domain monomers of the Fc domain is In some embodiments, the amino acid substitutions promote heterodimerization of the two monomers. The SIRP-α construct may comprise, for example, a SIRP-α fusion domain monomer fused to a first Fc domain monomer. a first subunit comprising an αD1 variant polypeptide and a second Fc domain molecule; (e.g., SIRP-α D1 variant polypeptides or any other polypeptides. In some embodiments, the construct is formed from a second subunit (which does not include any peptides). The product contains a single SIRP-α D1 variant polypeptide linked to an Fc domain. In some embodiments, the construct is linked to an Fc domain (e.g., single arm). and having two SIRP-α D1 variant polypeptides (e.g., double-arm). In some embodiments, K D SIRP-α D1 variant with approximately 500 nM In some embodiments, K D Approximately 50n SIRP-α D1 variants with M are particularly useful in double-armed constructs. In some embodiments, K D SIRP-α D1 variants with an activity of approximately 5 nM were In some embodiments, K is useful for double-armed constructs and single-armed constructs. D A SIRP-α D1 variant with approximately 500 pM was identified in both the double-arm construct and the single-arm construct. In some embodiments, K D SI of approximately 100 pM RP-α D1 variants are useful in double-arm and single-arm constructs. In some embodiments, K D SIRP-α D1 variants with approximately 50 pM In some embodiments, the present invention is useful for double-armed and single-armed constructs. , K. D A SIRP-α D1 variant with a β-glucan nucleotide sequence of approximately 10 pM was obtained using a double-armed construct and Useful for single arm constructs.

[0173] In some embodiments, heterodimerization of Fc domain monomers involves the formation of two Fc domains. The main monomer contains different but compatible substitutions, e.g., "knob-and-hole" residue pairs and charged The knob-hole interaction is facilitated by introducing a pair of residues. whereas knob-knob and hole-hole interactions are favored due to steric clashes and The lack of favorable interactions prevents homodimer formation. This refers to the space that is created when an amino acid is replaced with a different amino acid with a smaller side chain volume. A knob is a structure in which the original amino acid of a protein is replaced with a different amino acid with a larger side chain volume. For example, in some embodiments, the substituted amino acid is , located in the CH3 antibody constant domain of the Fc domain monomer, and two Fc domain monomers In some embodiments, the knob and hole amino acids are involved in the dimerization of two Fc domains. One acts to promote or prefer heterodimerization of the main monomer. The hole in the CH3 antibody constant domain is shaped to accommodate the knob of another CH3 antibody constant domain. In some embodiments, the hole in one CH3 antibody constant domain is formed by It is designed to better accommodate the original amino acids of the antibody constant domain. In one embodiment, the knob of one CH3 antibody constant domain is attached to the knob of another CH3 antibody constant domain. The amino acid sequence is formed to provide further interactions with the amino acid of the target molecule.

[0174] In some embodiments, the cavity is formed by amino acids with large side chains, such as tyrosine and tryptophan. Substitute amino acids with amino acids with small side chains, such as alanine, valine, or threonine. For example, the Y407V mutation in the CH3 antibody constant domain Similarly, in some embodiments, the knobs allow amino acids with small side chains to be linked to amino acids with large side chains. For example, the T3 of the CH3 antibody constant domain can be substituted with an amino acid having a side chain. In some embodiments, one Fc domain monomer is generated by the 66W mutation. One Fc domain monomer contains a knob mutation, T366W, and the other Fc domain monomer contains a hole mutation, In some embodiments, the high affinity The polypeptides of the present disclosure, including SIRP-α D1 variants, contain a knob mutation, T36 It is fused to an Fc domain monomer containing 6W, which inhibits unwanted knob-knob homodimer formation. Examples of knob-hole amino acid pairs include, but are not limited to, those listed in Table 9. Examples of Fc variants and SIRP-α-Fc fusions are shown in Table 10. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0175] In addition to the knobs and holes approach, in some embodiments, dimers of Fc domain monomers are also used. Electrostatic steering is also used to control the electrostatic force. Peptides, protein domains, and proteins are used to control the formation of subsequent protein molecules. This refers to the use of favorable electrostatic interactions between oppositely charged amino acids in proteins. uses electrostatic steering to control the dimerization of Fc domain monomers, thus One or more amino acid residues that form the interface between CH3 and CH4 are introduced into the The positively or negatively charged amino acids are electrostatically favored or unfavorable depending on the specific charged amino acid. In some embodiments, the positively charged amino acid at the interface is substituted with an amino acid residue: For example, lysine, arginine, or histidine may be used as a negatively charged amino acid, e.g., In some embodiments, the negative side of the interface is substituted with a disulfide or glutamate. The charged amino acid is substituted with a positively charged amino acid. Charged amino acids are introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, the interacting CH3 antibody constant domains of two Fc domain monomers The introduction of charged amino acids into the main chain results in electrostatic stearic acid formation due to interactions between charged amino acids. Selective formation of heterodimers of Fc domain monomers as controlled by the ATPase effect Examples of electrostatic steering amino acid pairs include, but are not limited to, Table 11. I can't. [Table 11]

[0176] Other methods used to control heterodimerization of Fc domain monomers include can be used in connection with the construction of bispecific antibodies.

[0177] In some embodiments, a first Fc domain monomer and a second Fc domain monomer - each contains one or more of the following amino acid substitutions: relative to the sequence of human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405 W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L3 51K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368 E, K370E, K370D, K370Q, K392E, K392D, T394N, P3 95N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407 I, K409E, K409D, K409T, and K409I.

[0178] In some embodiments, the Fc domain monomer: (a) is a IgG1 analogue to a wild-type human IgG1 and one of the following amino acid substitutions: T366W, T366S, L368A, Y407V, T 366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T , L351H, L351N, L351K, P353S, S354D, D356K, D35 6R, D356S, E357K, E357R, E357Q, S364A, T366E, L 368T, L368Y, L368E, K370E, K370D, K370Q, K392E , K392D, T394N, P395N, P396T, V397T, V397Q, L39 8T, D399K, D399R, D399N, F405T, F405H, F405R, Y 407T, Y407H, Y407I, K409E, K409D, K409T, or K 409I, or (b)(i) a N297A mutation relative to the Fc region of human IgG1; (ii) L234A, L235A, and G237A in the Fc region of human IgG1 (iii) mutations L234A, L235A, G237 relative to the Fc region of human IgG1; (iv) the N297A mutation in the Fc region of human IgG2 (v) A330S and P331S mutations in the Fc region of human IgG2; (vi) ) A330S, P331S, and N297A mutations relative to the Fc region of human IgG2; (vii) S228P, E233P, F234V, L for the Fc region of human IgG4 235A and delG236 mutations, or (viii) in the Fc region of human IgG4 In contrast, S228P, E233P, F234V, L235A, delG236, and N2 In some embodiments, the Fc domain monomer comprises: (a) a wild-type One of the following amino acid substitutions relative to human IgG1: T366W, T366S, L368 A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y3 49V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364 A, T366E, L368T, L368Y, L368E, K370E, K370D, K3 70Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405 H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K4 and (b) further comprising (i) a human IgG1 Fc region, (ii) the mutations N297A and N234A for the Fc region of human IgG1; (iii) mutations L23 (iv) the Fc region of human IgG2; (v) the N297A mutation in the Fc region of human IgG2; and (vi) the A330S and and P331S mutations, and (vi) A330S, P331 for the Fc region of human IgG2. S and N297A mutations, (vii) S228P relative to the Fc region of human IgG4, E233P, F234V, L235A, and delG236 mutations, or (viii) S228P, E233P, F234V, L235A, Contains the delG236 and N297A mutations.

[0179] In some embodiments, the first and second Fc domain monomers contain different amino acids In some embodiments, the first Fc domain monomer comprises a substitution of T366W. In some embodiments, the second Fc domain monomer comprises T366S, L36 In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 8A, and SEQ ID NO: 8Y407V. In some embodiments, the second Fc domain monomer comprises D399K. Includes 409D.

[0180] IV. Serum Albumin In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0181] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0182] Fusion to serum albumin can improve the pharmacokinetics of protein drugs, and several In some embodiments, the present invention provides a method for the treatment of SIRP-α D1 comprising administering to a subject person skilled in the art .... The polypeptide shown is linked to serum albumin.

[0183] Serum albumin is a globular protein abundant in mammalian blood. It is produced in the liver and can constitute about half of the serum proteins. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other Transporting proteins throughout the body, neutralizing pH, and properly distributing fluids between blood vessels and body tissues In a preferred embodiment, serum albumin is a human serum albumin. In some embodiments, HSA is a polypeptide of the present disclosure. In some embodiments, the C-terminus of the polypeptide is tethered to extend the serum half-life of the polypeptide. In some embodiments, the N-terminus of HSA is tethered to the C-terminus of a polypeptide of the present disclosure. In this embodiment, HSA is attached to the C-terminus of the polypeptide either directly or via a linker. In some embodiments, the HSA is tethered to a or attached to the N-terminus of the polypeptide.

[0184] In some embodiments, human serum albumin is selected from the group consisting of the UniProt identifiers shown in Table 12. No. P02768, containing the amino acid sequence 25 to 609 (SEQ ID NO: 12). In this embodiment, high affinity SIRP-α D1 variants (e.g., those listed in Tables 2, 5, and 6) are used. HSA tethered to any SIRP-α D1 variant shown was identified by UniProt It contains amino acids 25 to 609 of the sequence of ID No. P02768 (SEQ ID NO: 12). In embodiments, the HSA comprises a C34S or K573P substitution relative to SEQ ID NO: 12. In some embodiments, the HSA has the amino acid sequence C34S and K57 relative to SEQ ID NO: 12. Contains 3P substitutions. [Table 12]

[0185] In some embodiments, serum albumin is genetically fused to a polypeptide of the present disclosure. or linked to the polypeptide by chemical means, e.g., chemical bonding. In some embodiments, a spacer is inserted between the polypeptide and the HSA. Some examples of pacers are described in detail elsewhere herein. In some embodiments, the spacer is A or AAAL. The fusion of HSA in the amide not only results in long-term retention of the polypeptide, but also in a half-life. The period will also be extended.

[0186] Polypeptides containing SIRP-α D1 variant polypeptides and fused HSA Examples include, but are not limited to, SEQ ID NOs: 150 to 159 shown in Table 13. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6]

[0187] In some embodiments, the polypeptide is a variant of any of the variants shown in Table 13. , at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or 100% sequence identity).

[0188] In some embodiments, the polypeptide is selected from SEQ ID NOs: 154, 155, and 159, at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, High-affinity SIRP-α D1 domains with 98%, 99%, or 100% sequence identity Including the main.

[0189] V. Albumin-binding peptides In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0190] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0191] Binding to serum proteins can improve the pharmacokinetics of protein pharmaceuticals, particularly In some embodiments, the polypeptides described herein are serum protein binding peptides. The gene is fused to a gene or protein.

[0192] As used herein, the term "albumin-binding peptide" refers to a peptide that binds to serum albumin protein. An amino acid sequence of about 12 to 16 amino acids that has affinity for and functions as a binding agent to a substance. In some embodiments, the albumin binding peptide is a human, mouse, or is of rat origin.

[0193] In some embodiments, high affinity SIRP-α D1 variants (e.g., Tables 2, 5 The polypeptides of the present disclosure, including the polypeptides shown in (and any variant shown in 6), have binding properties to serum albumin. The polypeptide is fused to an albumin-binding peptide that exhibits binding activity to the albumin-binding peptide, thereby extending the half-life of the polypeptide. Various albumin-binding peptides that can be used in the methods and compositions described herein are In some embodiments, the albumin-binding peptide is a DI CLPRWGCLW (SEQ ID NO: 160). The amino acid-binding peptide may be genetically fused to the polypeptide of the present disclosure or chemically fused to the polypeptide. It is attached to the polypeptide by methods such as chemical coupling.

[0194] In some embodiments, a linker (e.g., a spacer) may be provided between the polypeptide and the amino acid sequence. The fusion protein is inserted between the phosphodiesterase-binding peptides, providing additional structural and spatial flexibility to the fusion protein. Specific linkers (e.g., spacers) and their amino acid sequences are In some embodiments, the albumin binding peptide is It is fused to the N-terminus or C-terminus of a polypeptide of the present disclosure. The N-terminus of the conjugated peptide is fused directly to the C-terminus of the polypeptide of the disclosure via a peptide bond. In another example, the C-terminus of the albumin-binding peptide is linked to the C-terminus of the present invention via a peptide bond. In some embodiments, the albumin binding peptide is fused directly to the N-terminus of the polypeptide. The fusion of a peptide to a polypeptide of the present disclosure can enhance the long-term retention of the polypeptide in its serum albumin. This is mediated through binding to phospholipids.

[0195] VI. Polyethylene Glycol (PEG) Polymers In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0196] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0197] In some embodiments, the high affinity SIRP-α D1 domain (e.g., Tables 2, 5, and any variant shown in 6) can be used with a polymer (e.g., polyethylene In some embodiments, the polymer is fused to a protein. Binding to the agent "masks" the protein agent from the host's immune system. In some embodiments, certain polymers, e.g., hydrophilic polymers, may be hydrophobic polymers. For example, in some embodiments, such polymers provide water solubility to proteins and drugs. Examples include PEG, polysialic acid chains, and PAS chain molecules. In this embodiment, a polymer such as PEG is covalently attached to a cysteine ​​substitution or addition in the polypeptide. In some embodiments, the cysteine ​​substitutions in the polypeptide are selected from those listed in Tables 2, 5, and 6. For any one of the sequences shown in 45C, G45C, G79C, S79C, or A84C. The addition of cysteine ​​residues to the polypeptide can be achieved by peptide synthesis, genetic engineering, molecular cloning, etc. In some embodiments, the gene is introduced using a gene encoding the gene encoding the polypeptide, such as a polypeptide encoding the polypeptide, ... The polymer, e.g., PEG, is attached to a cysteine ​​residue using a cysteine-maleimide bond. In some embodiments, polymers such as PEG are attached to the A high-affinity SIRP-α D1 variant-containing polypeptide was synthesized using chemical methods. Covalent attachment may occur at either the terminus or C-terminus, or at an internal position.

[0198] VII. Bispecific constructs In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0199] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0200] In some embodiments, high affinity SIRP-α D1 variants (e.g., Tables 2, 5 , and any variant shown in 6) comprise a bispecific construct. A bispecific construct refers to a construct that has two target interaction domains. In embodiments, the bispecific construct comprises an Fc domain and two target interacting domains, all of which (1) SIRP-α D1 domain or a variant thereof (e.g., Tables 2, 5, and and any variant shown in 6) and (2) an antibody variable domain. In the present specification, the bispecific construct comprises a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide has the formula ALB, where A is SI RP-α D1 domain or a variant thereof, wherein L is a linker, and B is a first In some embodiments, the second polypeptide comprises an Fc domain monomer. '-L'-B', where A' comprises an antibody variable domain and L' is a linker. and B' comprises a second Fc domain monomer. In some embodiments, the first and The orientation of the two polypeptides is B-L-A and B'-L'-A', respectively. In some embodiments, the first and second Fc domain monomers are combined to form the bispecific construct. In some embodiments, the bispecific construct forms the Fc domain of any immunoglobulin. immunoglobulin antibody isotypes (e.g., IgG, IgE, IgM, IgA , and IgD). The SIRP-α D1 domain variants are similar to wild-type human SIRP-α and one or more amino acid substitutions relative to the wild-type D1 domain (e.g., For example, any of the SIRP-α D1 variants shown in Tables 2, 5, and 6. In this state, the SIRP-α D1 variants have a larger domain size than the wild-type human SIRP-α D1 domain. In some embodiments, the bispecific construct binds to CD47 with high binding affinity. The antibody variable domain in the antibody targets a cellular antigen (e.g., a cellular antigen of a cancer cell).

[0201] An antibody variable domain comprises complementarity determining regions (CDRs, e.g., CDR L1, CDR L2 , CDR L3, CDR H1, CDR H2, and CDR H3) and framework The term refers to the light and heavy chain portions of an antibody that contain the amino acid sequences of the antibody variable region (FR). The main purpose of the antibody is to confer the ability to bind to a specific antigen. In some embodiments, the antibody variable domain molecules used Interleukin-16 variable domain molecules include, but are not limited to, single chain Fvs.

[0202] In some embodiments, the antibody variable domains in the bispecific construct are Targeting proteins (e.g., cellular antigens on cancer cells or immune cells). , are expressed at higher levels in cancer cells than in non-cancerous cells. For example, cancer antigens include Proteins that are preferentially expressed by cancer cells (e.g., that are more abundant than non-cancer cells) is expressed at higher levels in cancer cells), and in some instances, it In some embodiments, the protein, e.g., high affinity SIRP-α, is expressed only in the antibody variable domains, together with their variants, to generate the Fc domain. Proteins expressed by cancer cells that can be targeted include 5T4, AGS-16 , ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD 33, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, C D98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4 , DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2 , fibronectin, FR-alpha, GCC, GD2, glypican-3, GPNMB, HER-2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LI V-1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4, Not ch 2, Notch 1, PD-L1, PD-L2, PDGFR-a, PS, PSMA , SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK In some embodiments, the CSF-1R may be CSF-1R, but is not limited to CSF-1R. In the bispecific construct, the antibody variable domains are engineered to bind to a human protein. Not operated.

[0203] In some embodiments, the first and second Fc domains of the bispecific construct Each of the Fc domain monomers is a heterodimer of the first and second Fc domain monomers. Heterodimer of Fc domain monomers containing one or more amino acid substitutions that promote ligation. Methods for promoting cleavage are described in more detail herein, for example, knob and hole methods. and electrostatic steering methods.

[0204] In some embodiments, the Fc domain of the bispecific construct is a "dead Fc domain." These proteins are mutated to lack one or more effector functions that are characteristic of the target protein. In an embodiment, the Fc domain of the bispecific construct is derived from an IgG1 antibody and is represented by SEQ ID NO: 161 (Table 14), which contains the amino acid substitutions L14A, L15A, and G17A. This reduces the interaction or binding between the Fc domain and the Fcγ receptor. In embodiments, the Fc domain monomer is derived from an IgG1 antibody and contains the amino acid substitution L234 A, L235A, G237A, and N297A (Kabat et al., 1991 In some embodiments, the numbering system includes one or more of the following: The Fc variants described herein may be minimally glycosylated or glycosylated. In some embodiments, deglycosylation is achieved by a mutation at N297A or or N297 to any amino acid other than N (Kabat et al. (1991) This is achieved by mutating the nucleotide sequence into a sequence (as indicated by the EU numbering system). In some embodiments, the bispecific construct is capable of binding to a target cell type that is expressed by different cell types. It is designed to have selective binding to a protein (e.g., a receptor such as an Fc receptor). The hinge, constant domains (e.g., CH2 and CH3 constant domains) of an antibody, or Amino acid substitutions in the hinge and constant domains allow for expression in different cell types (e.g., regulatory T specific receptors (e.g., Fc receptors) expressed on T cells (e.g., T cell lysates and effector T cells) Studies have shown that amines can effectively alter the binding affinity of antibodies. IgG with acid substitutions A111S and P112S (relative to SEQ ID NO: 162 in Table 14) 2 exhibits significantly enhanced binding to FcγRIIIa 131 H compared to wild-type IgG2. In some embodiments, the Fc variants herein exhibit reduced glycosylation. In some embodiments, the protein is deglycosylated or has reduced glycosylation. is a mutation of N297A, or N297 can be replaced with any amino acid other than N (Kabat, (1991) and the EU numbering system) In some embodiments, the bispecific construct is an IgG2 or In some embodiments, the antibody comprises an Fc domain of the IgG4 subclass. The bispecific construct containing the Fc domain of the ras was amplified against SEQ ID NO: 162 (Table 14). In some embodiments, the Fc variant comprises the amino acid substitutions A111S and P112S. The mutant has the amino acid substitutions A330S, P331S, and N297A (Kabat, et al. (1991) and the EU numbering system It contains the Fc sequence of human IgG2. [Table 14]

[0205] A SIRP-α D1 domain connected via a linker to a first Fc domain monomer. or a variant thereof and a second Fc domain monomer, An example of a SIRP-α construct in which domain monomers combine to form an Fc domain is shown in Figure 1. In some embodiments, the protein bound to the second Fc monomer In some embodiments, the SIRP-α construct is free of any protein or antibody variable domain. The SIRP-α D1 domain is connected to the first Fc domain monomer via a linker. or variants thereof, and an antibody linked to a second Fc domain monomer via a linker. and a first and second Fc domain monomer are combined to form an Fc domain. (as shown in Figure 2). In some embodiments, the SIRP-α construct SIRP-α D1 domain or a SIRP-α D2 domain tethered to the first Fc domain monomer via a carboxyl group and variants thereof, and therapeutics tethered to a second Fc domain monomer via a linker. Proteins (e.g., cytokines, interleukins, antigens, steroids, anti-inflammatory drugs) or an immunomodulatory agent), wherein the first and second Fc domain monomers are linked to form an Fc domain In some embodiments, the SIRP-α structure described above forms a cytoplasmic domain (as shown in FIG. 3). The two Fc domains of the Fc domains of the construct (e.g., the SIRP-α construct shown in Figures 1-3) Each of the dimer monomers contains an amino acid substitution that promotes heterodimerization of the two monomers. Different methods (e.g., nodimerization) can be used to promote heterodimerization of two Fc domain monomers. The amino acid substitutions in the Fc domain (e.g., the hole-and-hole method, the electrostatic steering method) and the Fc domain are described herein. For example, Figure 4A shows a mutant ribosomal protein containing a knob mutation, e.g., T366W. SIR tethered to Fc domain monomers restricts knob-knob homodimer formation SIRP-α constructs with the P-α D1 domain or its variants are shown in Figure 4B. The Fc domain model contains hole mutations, e.g., T366S, L358A, and Y407V. SIRP with SIRP-α D1 domain or variant thereof tethered to a SIRP-α D1 domain In some embodiments, a similar Fc domain heterodimerization method is used. The method applies to the Fc domain in the constructs shown in Figures 2 and 3. In this state, the SIRP-α construct comprises SIRP-α D1 tethered to an Fc domain monomer. domain or its variants (as shown in Figure 5A). In this embodiment, the fusion protein forms a homodimer (as shown in Figure 5B).

[0206] The Fc variants herein linked to a fusion partner are preferably native or wild-type. at least 1.5 times higher than a similar polypeptide construct comprising a native (non-mutated) antibody Fc region. four Fcγ receptors, CD16a, CD32a, CD32b, CD32c, and CD64 In some cases, the Fc binding domains described herein exhibit reduced or eliminated binding to the Fc domains. The nucleotides or fusion partners are CD16a, CD32a, CD32b, CD32c, and reduced or eliminated binding of CD64 to Fcγ receptors.

[0207] In some embodiments, the Fc variants of the present disclosure linked to a fusion partner are The complement component is increased compared to a similar polypeptide construct containing a natural or wild-type (non-mutated) Fc region. In some cases, the Fc barrier exhibits reduced binding to C1q and CDC. The constructs exhibit at least 5%, 10% or more of the Fc region-specific ... , 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more In some cases, the Fc variant exhibits reduced C1q binding beyond that of natural Reduced CDC compared to polypeptide constructs containing a native or wild-type (non-mutated) Fc region. In some embodiments, the Fc variant comprises a polypeptide comprising a wild-type Fc region. Compared to the chid construct, at least 5%, 10%, 15%, 20%, 30%, 40%, 5 Demonstrating a reduction in CDC of 0%, 60%, 70%, 80%, 90% or more.

[0208] VIII. Linker In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0209] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0210] In the present disclosure, a linker is a polypeptide or protein domain or related In some embodiments, the term "protein" is used to indicate a bond or connection between non-protein moieties. In the example, the linker is an Fc domain monomer, an albumin-binding peptide, or HSA, and the high-affinity SIRP-α D1 variant. In embodiments, the linker connects the two polypeptides together in series. The C-terminus of the SIRP-α D1 variant and the N-terminus of the Fc domain monomer, The HSA is then attached to a amine-binding peptide.

[0211] In some embodiments, the linker is a simple covalent bond, e.g., a peptide bond, a synthetic Polymers, such as polyethylene glycol (PEG) polymers, or polymers formed from chemical reactions When the linker is a peptide bond, the linker is any type of bond formed, for example, a chemical bond. In some embodiments, the C-terminal carboxylic acid group of one protein domain is substituted with another It reacts with the N-terminal amino group of the protein domain in a condensation reaction to form a peptide bond. In some embodiments, peptide bonds can be synthesized by synthetic means, such as by conventional organic chemistry reactions. or by natural production from the host cell, in which case both proteins in tandem (e.g. For example, the DNA sequences of the Fc domain monomer and the high-affinity SIRP-α D1 variant The nucleic acid molecule encoding the required molecular machinery of the host cell (e.g., DNA polymerase and directly transcribed and bound to adjacent polypeptides encoding both proteins by the ribosome Can be translated.

[0212] When the linker is a synthetic polymer (e.g., a PEG polymer), in some embodiments The polymer is attached to each end of the two proteins so as to react with the terminal amino acids at the connecting ends of the two proteins. is functionalized with reactive chemical functional groups.

[0213] When the linker (other than the peptide bond described above) comprises a chemical reaction, some embodiments In this case, chemical functional groups (e.g., amines, carboxylic acids, esters, azides, or other functional groups) ) are synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. In some embodiments, the two functional groups are then reacted by synthetic chemistry to form a chemical bond. The cleavage site forms a bond, thus connecting the two proteins.

[0214] Spacer In some embodiments of the present disclosure, an Fc domain monomer, an albumin binding peptide The linker between the peptide, or HSA, and the polypeptide of the present disclosure is about 1 to 200 amino acids. Suitable peptide spacers include flexible amino acids. and peptide linkers containing residues such as glycine and serine. Examples of G are shown in Table 15. In some embodiments, the spacer is a motif, e.g., G S, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164 ), or SGGG (SEQ ID NO: 165) motifs. In embodiments, the spacer is a GS motif, e.g., GS, GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGS GSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170) In some embodiments, the spacer comprises 2 to 12 amino acids including the GGS motif. , for example, GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 1 72), and 3 to 12 amino acids including GGSGGSGGSGGS (SEQ ID NO: 173) In some embodiments, the spacer comprises the motif GGSG (SEQ ID NO: 164), For example, GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or G It contains 4 to 12 amino acids including GSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer comprises a GGGGS (SEQ ID NO: 163) motif, e.g., GG GGSGGGGSGGGGS (SEQ ID NO: 176). The sequence contains amino acids other than glycine and serine, for example, AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180) ), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182) Sequence number 182), AEAAAKEAAAKA (sequence number 183), KESGSVSSEQ LAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GEN LYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGS AGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 19 0), AAANSSIDLISVPVDSR (SEQ ID NO: 191), or GGSGGGS Contains EGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 192) nothing.

[0215] In some embodiments, the spacer comprises a motif, e.g., EAAAK (SEQ ID NO: 193 ) in some embodiments. In some embodiments, the spacer comprises a motif, e.g. For example, multiple or repeated motifs of proline-rich sequences, e.g., where X is any amino acid (e.g., (XP)n, where n is 1 to 5, and PAPAP (sequence Number 194). [Table 15-1] [Table 15-2]

[0216] In some embodiments, the peptide spacer and amino acid length used may be Depending on the two proteins and the desired degree of flexibility in the final protein fusion polypeptide, In some embodiments, the length of the spacer is adjusted to accommodate proper protein folding. In some embodiments, the H The spacer, such as the spacer between SA and a polypeptide disclosed herein, may be A or AAAL. (SEQ ID NO: 178).

[0217] IX. Vectors, Host Cells, and Protein Production In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0218] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0219] In some embodiments, the polypeptides of the present disclosure are produced from host cells. The term "cells" refers to the polypeptides and fusion polypeptides described herein that are synthesized from their corresponding nucleic acids. It refers to a medium containing cellular components, e.g., organelles, necessary for expression of a gene. In some embodiments, the nucleic acid may be transformed, transfected, electroporated, phosphorylated, or Introduction into host cells by calcium precipitation, direct microinjection, infection, etc. In some embodiments, the selection of the nucleic acid vector is based on the In some embodiments, the host cell is a prokaryote (e.g., a bacterium). ) or eukaryotic (e.g., mammalian) origin.

[0220] In some embodiments, the polypeptide, e.g., a SIRP-α D1 variant (e.g., any of the variants shown in Tables 2, 5, and 6) and a fusion partner, e.g., F The polypeptide construct comprising the c variant, HSA, and albumin-binding peptide is a nucleic acid Preferably, the polypeptide construct (e.g., Fc variants, linkers, and fusion proteins) A host cell transformed with an expression vector containing a nucleic acid encoding a target protein (a target protein) is culturing under conditions suitable to induce or cause expression of the polypeptide construct; In some embodiments, the conditions suitable for expression are produced by a selected expression vector. In some embodiments, the vectors vary depending on the vector and host cell. A wide variety of suitable host cells may be used, including, but not limited to, yeast and yeast. For example, various cell lines that find use in this disclosure are those listed in the American Type Culture Collection. Available from the ATCC® Cell Line Collection In some embodiments, the Fc variants of the disclosure are obtained by genetically engineering the cell line. Alternatively, by changing cell culture conditions such as the addition of kifunensine, or by using prokaryotes (Ec By using a host that does not naturally glycosylate, such as oli, The expressed protein is expressed in cells that are optimized to not glycosylate, and several In some cases, modification of the glycosylation sequence of the Fc is not necessary.

[0221] Nucleic Acid Vector Constructs and Host Cells Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be obtained by a variety of methods. These methods include oligonucleotide-mediated (or site-specific) These include, but are not limited to, (heterologous) mutagenesis and PCR mutagenesis. In some embodiments, nucleic acid molecules encoding polypeptides of the disclosure can be prepared using standard techniques, e.g., Alternatively, the wild-type SIRP-α D1 domain may be obtained using gene synthesis. Nucleic acid molecules encoding the genes can be cloned using standard techniques, e.g., QuikChange™ clones. Mutagenesis is used to modify the sequence to include specific amino acid substitutions. Thus, the nucleic acid molecules are synthesized using a nucleotide synthesizer or PCR technology.

[0222] In some embodiments, a polypeptide construct, such as a SIRP-α D1 variant, a variant (e.g., any variant shown in Tables 2, 5, and 6) and a fusion partner, e.g., For example, a polypeptide construct comprising an Fc variant, HSA, and an albumin-binding peptide. The encoding nucleic acid is incorporated into an expression vector to express the protein. A variety of expression vectors are available for protein expression. They can include replicating, extrachromosomal vectors, or vectors that integrate into the host genome. It may also contain various components or elements. For example, in some embodiments The vector components include transcriptional and translational regulatory sequences, such as promoter sequences, ribonucleotide sequences, and the like. thrombin-binding site, signal sequence, transcription start and stop sequences, translation start and stop sequences, 3' and and 5' untranslated region (UTR), as well as enhancer or activator sequences, replication origin a selectable marker gene, and a nucleic acid sequence encoding a polypeptide of interest, and a transcription factor In some embodiments, the expression vector includes, but is not limited to, a termination sequence. may contain control or regulatory sequences, selectable markers, any fusion partners, additional elements, or any combination thereof. "Linked to" means that the nucleic acid is placed into a functional relationship with another nucleic acid sequence. Generally, these expression vectors contain a nucleic acid sequence operably linked to a nucleic acid encoding an Fc variant. The nucleic acid sequence includes the transcriptional and translational regulatory nucleic acids linked to the nucleic acid sequence, and is typically used to express the protein. Suitable for host cells. Antibiotic resistance genes, fluorescent protein genes, etc. Selection genes or markers include, but are not limited to, those that allow host cells containing the expression vector to be isolated, e.g., Can be used for selection by antibiotics or fluorescent expression. Child is available.

[0223] In some embodiments, the components or elements of the vector are such that the expression vector is compatible with the host cell type. Expression vectors that find use in the present disclosure are optimized to be compatible with mammalian cells. These include those that allow protein expression in bacteria, insect cells, yeast, and in vitro systems. These include, but are not limited to:

[0224] In some embodiments, mammalian cells are host cells for producing the polypeptides of the present disclosure. Examples of mammalian cell types include human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO) cells, He La, COS, PC3, Vero, MC3T3, NS0, SP2 / 0, VERY, BHK , MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, N S0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7 Some examples include, but are not limited to, O3O and HsS78Bst cells. In embodiments, E. coli cells are used as host cells to produce the polypeptides of the present disclosure. Examples of E. coli strains that can be used include E. coli 294 (ATCC ) 31,446), E. coli λ 1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025), and E coli RV308 (ATCC (registered trademark) 31,608), Not limited.

[0225] Different host cells may have different post-translational processing and modifications (e.g., glycosylation) of protein products. In some embodiments, the expressed polypeptide has a characteristic and specific mechanism of action (protein synthesis). An appropriate cell line or host system is selected to ensure correct modification and processing of the peptide. When the vector is introduced into a host cell for protein production, the host cell Modifications are made depending on the induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence. It is cultivated on standard nutrient medium decorated with

[0226] In some embodiments, a polypeptide construct, such as a SIRP-α D1 variant, a variant (e.g., any variant shown in Tables 2, 5, and 6) and a fusion partner, e.g., For example, a polypeptide construct comprising an Fc variant, HSA, and an albumin-binding peptide The expression construct is expressed in the mammalian cell line using a virus such as a retrovirus or an adenovirus. In some embodiments, the vector is expressed in a mammalian expression system, including a system introduced into a cell. Human, mouse, rat, hamster, or primate cells are utilized. Suitable cells include: Also included are known research cells, such as Jurkat T cells, NIH3T3, CHO, COS, and Alternatively, in some embodiments, In some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art. , Escherichia coli (E.coli), Bacillus subti lis, Streptococcus cremoris, and Streptococcus In some cases, Fc variants are included. The polypeptide constructs can be expressed in insect cells such as, but not limited to, Sf9 and Sf21 cells. , or Saccharomyces, Pichia, Kluyveromyces, H Microorganisms derived from the genera ansenula and Yarrowia, including, but not limited to, enzymes In some cases, the polypeptide construct containing the Fc variant is produced in the host cell. The product may be expressed in vitro using a cell-free translation system, such as a prokaryotic (e.g., E. coli) In vitro translation from both ribosomal and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells Systems are available that, in some embodiments, allow for the expression level and function of proteins of interest. For example, in vitro translation, as will be understood by those skilled in the art, may be used. is required for some display technologies, e.g., ribosome display. In some embodiments, the Fc variants are synthesized using liquid phase peptide synthesis and solid phase peptide synthesis. They may be produced by chemical synthesis methods, such as, but not limited to, synthesis; In the case of in vitro transcription using a glycosylation system, the Fc is converted to a glycosylated Fc having no natural glycosylation sites. Even if present, it is not glycosylated, thus resulting in equivalent inactivation of the Fc.

[0227] In some embodiments, the polypeptide construct comprises the same amino acids as naturally occurring amino acids. unnatural amino acids, amino acid analogs, amino acid mimetics, or their equivalents that function in a Naturally encoded amino acids include any combination of the 20 common amino acids ( Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamine Amino acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine threonine, tryptophan, tyrosine, and valine ) as well as pyrrolysine and selenocysteine. Amino acid analogs refer to naturally occurring amino acids. It has the same basic chemical structure as the amino acids that are produced, i.e., a carbon bonded to a hydrogen atom, a carboxyl group, and an amino acid. and compounds having R groups, such as homoserine, norleucine, methionine sulfoxide, In some embodiments, such analogs are , modified R groups (e.g., norleucine) or modified peptide backbones, but They usually have the same basic chemical structure as a naturally occurring amino acid.

[0228] Protein production, recovery, and purification In some embodiments, host cells used to produce the polypeptides of the present disclosure The cells are grown in a medium suitable for culturing the selected host cells. Examples of these include minimal essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), E xpi293™ expression medium, DMEM supplemented with fetal bovine serum (FBS), and R Examples of media suitable for bacterial host cells include PMI-1640. Necessary nutrient supplements, such as Luria Broth (LB) containing ampicillin, are included. In some embodiments, the host cells are grown at a suitable temperature, for example, from about 20°C to about 39°C, e.g. , about 25°C to about 37°C, preferably 37°C, and a CO2 level, for example, about 5% to 10% In some embodiments, the pH of the medium is about pH 6, depending primarily on the host organism. pH 7.0 to 7.8, e.g., pH 7.0. When the promoter is activated, protein expression can be induced under conditions appropriate for activation of the promoter. .

[0229] In some embodiments, protein recovery is achieved by, for example, osmotic shock, sonication, or It involves the destruction of host cells by lysis or lysis. Once the cells are disrupted, the cell debris can be removed by centrifugation or The protein can then be further purified. In embodiments, the polypeptides of the disclosure can be purified by various protein purification methods, e.g., by cloning. chromatography (e.g., ion exchange chromatography, affinity chromatography) (feedback and size exclusion column chromatography), centrifugation, differential solubility, or tank The protein may be purified by any other standard technique for protein purification. For example, in some embodiments In this case, the protein is purified by affinity column, e.g., Protein A column (e.g., POROS Protein A chromatography) and a chromatography column (e.g., P OROS HS-50 cation exchange chromatography) and isolated and purified by filtration, ultrafiltration, desalting, and dialysis procedures. In embodiments, the polypeptide is linked to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a nickel-functionalized agarose affinity column. A hexahistidine peptide (His6) capable of binding to HIV with micromolar affinity was Alternatively, an epitope derived from the influenza hemagglutinin protein can be used. A hemagglutinin "HA" tag corresponding to the HA group can be used.

[0230] In some embodiments, a polypeptide of the disclosure, e.g., a SIRP-α D1 barrier nucleotides (e.g., any variant shown in Tables 2, 5, and 6) and fusion partners, e.g., For example, polypeptide constructs comprising Fc variants, HSA, and albumin-binding peptides. The present disclosure provides a method for the production of a gene by cells of a subject (e.g., a human), for example, in the context of gene therapy. A viral vector (e.g., a retroviral vector) containing a nucleic acid molecule encoding a polypeptide. vectors, adenovirus vectors, poxvirus vectors (e.g., mutant vaccinia virus vectors), Vaccinia virus vectors such as MVA (Mugenol Ankara), adeno-associated virus vectors, vectors, such as alphavirus vectors, Once inside the subject's cells (e.g., transformation, transfection), the vector electroporation, calcium phosphate precipitation, direct microinjection, Such vectors, such as those described herein, can be used to express the polypeptides disclosed herein. In some cases, the polypeptide is secreted from the cell. If treatment of the disease or disorder is the desired outcome, no further action is necessary. In this embodiment, when recovery of the protein is desired, blood is drawn from the subject and collected by a variety of methods. The protein is purified from the blood.

[0231] X. Pharmaceutical Compositions and Drug Products In some embodiments, disclosed herein is a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the main, as well as the wild-type signal regulatory protein α ( Residues 6, 27, 31, 47, and 5 for the SIRP-α D1 domain 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0232] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0233] The present disclosure provides polypeptides described herein, e.g., high affinity SIRP-α D1 antibodies. In some embodiments, the present invention features a pharmaceutical composition comprising a polypeptide having an agonist. The pharmaceutical compositions of the present disclosure include the polypeptides of the present disclosure as therapeutic proteins. In certain embodiments, the pharmaceutical compositions of the present disclosure comprising the polypeptides described herein are useful in therapy. In some cases, other agents or compositions (e.g., therapeutic agents, biologics, small molecules, or any of these) may be used. In some embodiments, one or more additional therapeutic agents are used in combination with Therapeutic agents, e.g., small molecules, chemical compounds, or biological compounds, e.g., siR Polynucleotides, including but not limited to NAs, short polypeptides, and therapeutic antibodies The nucleotides and polypeptides are optionally incorporated into pharmaceutical compositions of the polypeptides described herein. In some embodiments, the formulation of the polypeptide constructs described herein is For storage, the polypeptide constructs described herein having the desired purity can be administered to any pharmaceutical in the form of a lyophilized formulation or aqueous solution together with a commercially acceptable carrier, excipient, or stabilizer. In some embodiments, the pharmaceutical compositions of the present disclosure are prepared by mixing Nucleic acid molecules (DNA or RNA, e.g., mRNA) encoding the disclosed polypeptides or a vector containing such a nucleic acid molecule.

[0234] Acceptable carriers, excipients, or stabilizers in pharmaceutical compositions are preferably In some embodiments, the compound is non-toxic to the recipient at the dosage and concentration. Carriers, excipients, and stabilizers that may be used include buffers, e.g., phosphate, citrate, HEPE, S, TAE, and other organic acids, antioxidants such as ascorbic acid and methionine, preservatives (e.g., hexamethonium chloride, octadecyldimethylbenzylammonium chloride, salt Benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol parabens, such as methyl or propyl paraben, catechol, resorcinol, ol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (e.g. polypeptides, proteins, e.g., less than about 10 residues, e.g., human serum albumin, latins, dextrans, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, lysines, amino acids such as glycine, glutamine, histidine, and lysine, monosaccharides, disaccharides Sugars and other carbohydrates, such as glucose, mannose, sucrose, and sorbitol chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose; sugars, or sorbitol, sweeteners and other flavorings, bulking agents, e.g., microcrystalline cellulose Sugars, lactose, corn and other starches, binders, additives, colorants, salt forms Counterions, e.g., sodium, metal complexes (e.g., Zn-protein complexes), non-ionic Polycarboxylic surfactants, such as TWEEN™, PLURONICS™, and polyols. polyethylene glycol (PEG), or any combination thereof.

[0235] In some embodiments, pharmaceutical compositions comprising the polypeptides described herein are water-soluble. and is intended to include, for example, both acid and base addition salts. The term "pharmaceutically acceptable acid addition salt" refers to a biologically acceptable salt of the free base. Inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, Acids such as nitric acid, phosphoric acid, etc., and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvate, etc. Acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid Aromatic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid The term "pharmaceutically acceptable base addition salt" refers to a salt formed with carboxylic acid, carboxylic acid, or the like. Sodium, potassium, lithium, ammonium, calcium, magnesium, iron Examples of suitable inorganic bases include those derived from inorganic bases such as zinc, copper, manganese, and aluminum salts. Particularly preferred are ammonium, potassium, sodium, calcium, and magnesium. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary and secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and to basic ion exchange resins, e.g., isopropylamine, trimethylamine, diethylamine, amine, triethylamine, tripropylamine, and ethanolamine salts. Formulations to be used for in vivo administration are preferably sterile, e.g., by filtration through sterile membranes. Or it can be achieved in other ways.

[0236] In some embodiments, the pharmaceutical compositions of the present disclosure are administered parenterally in the form of an injectable formulation. In some embodiments, the pharmaceutical composition for injection is a sterile solution or any pharmaceutically acceptable Pharmaceutically acceptable vehicles include sterile water, Physiological saline, and cell culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-modified Eagle's Medium (α-MEM), and F-12 medium). A variety of formulation methods are available.

[0237] In some embodiments, the polypeptides described herein are formulated as immunoliposomes. Liposomes are a variety of lipid, liposome-containing molecules that are useful for delivering therapeutic agents to mammals. Liposomes containing antibodies or Fc fusions are small vesicles containing phospholipids or surfactants. can be prepared by a variety of methods known in the art. In general, the components of liposomes are arranged in a bilayer structure similar to the lipid arrangement of biological membranes. In some embodiments, the liposomes contain phosphatidylcholine, cholesterol, and PE. A lipid composition containing G-derivatized phosphatidylethanolamine (PEG-PE) was used. In some embodiments, liposomes are prepared by reverse phase evaporation. The liposomes are extruded through filters of a predetermined pore size to obtain In some embodiments, the chemotherapeutic agent or other therapeutically active agent is optionally delivered in a liposome. Included within.

[0238] In some embodiments, the polypeptide constructs and other therapeutically effective The drugs that can be used are those that are prepared by coacervation techniques, interfacial polymerization (e.g., hydroxymethylcellulose, etc.). or gelatin microcapsules or poly(methyl methacrylate) microcapsules. capsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres), emulsions, microemulsions, nanoparticles, and nanocapsules), and macroemulsions Microcapsules prepared by methods including, but not limited to, It is enclosed.

[0239] In some embodiments, sustained release formulations are prepared. Suitable examples of sustained release formulations include: Semipermeable matrices of solid hydrophobic polymers are included, and these matrices are Examples of sustained release matrices include: Examples include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) ) or poly(vinyl alcohol)), polylactide, L-glutamic acid and L-glutamine Copolymer of gamma ethyl phosphate, non-degradable ethylene-vinyl acetate, LUPRON DEP OT (trademark) (an injectable mixture of lactic acid-glycolic acid copolymer and leuprolide acetate) Degradable lactic acid-glycolic acid copolymers such as poly(D-(-)-3-hydroxybenzoates) and poly(D-(-)-3-hydroxybenzoates) in a matrix of hydroxybutyric acid and poly-DL-lactide-co-glycolide (PLG). ProL, a microsphere-based delivery system consisting of incorporated desired bioactive molecules ease® (available from Alkermes). Some sustained release formulations allow for release of molecules over several months, e.g., 1-6 months, while others The formulations release the pharmaceutical compositions of the present disclosure for a shorter period of time, for example, several days to several weeks.

[0240] In some embodiments, the concentration of a polypeptide described herein in a pharmaceutical formulation is about 0. In some cases, the amount of the polymers described herein may vary from 0.1 to 100% by weight. The concentration of the peptide ranges from 0.003 to 1.0 molar. In some cases, the formulation The concentration of the polypeptide in the formulation is about 5 mg / mL to about 50 mg / mL (e.g., about 10 mg / mL to approximately 40 mg / mL or approximately 20 mg / mL to approximately 30 mg / mL) In some embodiments, the polypeptides described herein are administered to treat a patient. A therapeutically effective dose is administered. The term "therapeutically effective dose" refers to a dose that produces the effect for which it is administered. The exact dose will depend on the purpose of the treatment. In some embodiments, the dose is , 0.01 to 100 mg / kg body weight or more, e.g., 0.1, 1, 5, 10, 1 ranges from 5, 20, 25, 30, 35, 40, 45 or 50 mg / kg body weight. In embodiments, degradation of polypeptide constructs, systemic versus local delivery, and new proteases are The enzyme synthesis rate, as well as age, weight, general condition, sex, diet, administration time, drug interactions, and It needs to be adjusted according to the severity of the condition.

[0241] In some embodiments, the compositions and formulations described herein are administered to a subject in need thereof. In some embodiments, such administration occurs in vivo. In some embodiments, such administration is performed ex vivo. Administration of pharmaceutical compositions containing the polypeptides described herein can be by oral, subcutaneous, intravenous, intranasal, Intraotically, transdermally, topically (e.g., gels, ointments, lotions) , creams, etc.), intraperitoneal, intramuscular, intrapulmonary (e.g., commercially available from Aradigm Inhalable technology AERx®, or Inhale Therapeutics Inhance™, a commercially available pulmonary delivery system from Pharma, Inc., vaginal, parenteral, rectal, or The procedure is performed in a variety of ways, including but not limited to intraocularly. The pharmaceutical composition is then formulated appropriately depending on the method of administration.

[0242] In some embodiments, the pharmaceutical composition for gene therapy is in an acceptable diluent. or a sustained release matrix in which the gene delivery vehicle is embedded. In this state, vectors used as in vivo gene transfer vehicles include retroviral vectors. vectors, adenovirus vectors, poxvirus vectors (e.g., mutant vaccinia vaccinia virus vectors such as Virus Ankara), adeno-associated virus vectors, and and alphavirus vectors.

[0243] XI. Route, Dosage, and Administration In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0244] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0245] In some embodiments, pharmaceutical compositions comprising a polypeptide of the present disclosure as a therapeutic protein are provided. The composition may be administered, for example, intravenously, parenterally, subcutaneously, intramuscularly, intraarterially, intrathecally, or intravenously. In some embodiments, the pharmaceutical composition is formulated for intravenous or intraperitoneal administration. Formulated for or intended for oral, nasal, spray, aerosol, rectal, or vaginal administration For injection preparations, a variety of effective pharmaceutical carriers are available.

[0246] In some embodiments, nucleic acid molecules encoding polypeptides of the present disclosure or Pharmaceutical compositions containing vectors containing acid molecules are administered by gene transfer. Gene transfer methods are available. In some embodiments, in vivo gene transfer and expression The vectors used include retroviral vectors, adenoviral vectors, and pop vaccinia virus vectors (e.g., vaccinia vectors such as mutant vaccinia virus Ankara (MVA)) niavirus vectors), adeno-associated virus vectors, and alphavirus vectors In some embodiments, the polypeptides of the present disclosure include, but are not limited to: The mRNA molecule encoding the gene is administered directly to the subject.

[0247] The dosage of the pharmaceutical composition of the present disclosure will depend on the route of administration, the disease being treated, and the physical characteristics of the subject. For example, it depends on factors including age, weight, and general condition. The amount of polypeptide of the present disclosure included in the administration is sufficient to effectively treat the disease without inducing significant toxicity. In some embodiments, the pharmaceutical compositions of the present disclosure are The product contains a polypeptide of the present disclosure at 0.01 to 500 mg / kg (e.g., 0.01, 0.1 ,0.2,0.3,0.4,0.5,1,2,3,4,5,10,15,20,25,3 0, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 40 0, 450, or 500 mg / kg), and in more specific embodiments, from about 0.1 to about 50 mg / kg, and in more specific embodiments, doses ranging from about 1 to about 30 mg / kg. In some embodiments, the dosage may be adjusted as directed by a physician depending on the extent of the subject's disease and different parameters. It is adapted by

[0248] In some embodiments, the toxicity of the therapeutic agents and polypeptides described herein is comparable to standard by various pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (50 of the group % lethal dose) or LD 100 (a dose that is lethal to 100% of the population) In some embodiments, these cell culture assays Data from animal studies have been used to formulate a dose range that is non-toxic for human use. The doses of proteins described herein are preferably used to minimize toxicity. In some embodiments, the therapeutic dose is within a range of circulating concentrations that include some or no effective dose. The amount may vary within this range depending upon the dosage form employed and the route of administration utilized. In this embodiment, the exact formulation, route of administration, and dosage will be determined individually taking into account the patient's condition. Selected by the doctor.

[0249] In some embodiments, the pharmaceutical composition is administered to treat a disease or condition in a manner compatible with the dosage formulation. It is administered in an amount that is therapeutically effective to bring about an improvement or remediation of the symptoms of the disorder. In some embodiments, the pharmaceutical compositions are available in various dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug-release capsules) Generally, therapeutic proteins are administered at a dose of 0.1 to 100 mg / kg, for example, 1 to 50 mg / kg. In some embodiments, a pharmaceutical composition comprising a polypeptide of the present disclosure is administered For those who need it, for example, daily, weekly, monthly, semi-annually, once a year or more ( Doses may be administered in single or multiple doses (e.g., 1 to 10 or more doses), or as medically necessary. can be given in any of multiple dose regimens. In some embodiments, administration The timing between treatments may decrease as the condition improves or as the patient's health condition improves. increases as the condition worsens.

[0250] XII. Treatment method In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0251] Also disclosed herein in some embodiments are Fc variants. polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0252] Further disclosed herein, in some embodiments, is a wild-type SIRP-α Amino acid mutation at residue 80 relative to the D1 domain and wild-type signal regulatory protein For the SIRP-α D1 domain, residues 6, 27, 31, and 47 a residue selected from the group consisting of residue 53, residue 54, residue 56, residue 66, and residue 92 a SIRP-α D1 domain having at least one additional amino acid mutation in The method includes administering a polypeptide containing a SIRP-α D1 variant, including a fragment thereof. This is a treatment method that

[0253] In some embodiments, the present disclosure provides a method for treating a disease associated with SIRP-α or CD47 activity. or disorders, such as cancer and immune disorders (e.g., autoimmune and inflammatory diseases) The present invention provides pharmaceutical compositions and methods for treating patients with several In some embodiments, the polypeptides described herein are administered to a subject to induce the delivery of target cells (e.g., In some embodiments, the antibody is administered in a manner that increases phagocytosis of the antibody (e.g., cancer cells). The polypeptide is administered to a subject in a manner that kills cancer cells in the subject. In some embodiments, the polypeptide is administered to a subject to deplete regulatory T cells in the subject. In some embodiments, the polypeptides described herein are administered to a subject in a manner that is administered to a subject in a manner that increases hematopoietic stem cell engraftment in the subject, In some embodiments, the method comprises modulating the interaction between SIRP-α and CD47. The polypeptides described herein can be administered to a subject to alter the subject's immune response (e.g., In some embodiments, the method further comprises: In some embodiments, disclosed herein are methods for treating a disease. The combination of a polypeptide (eg, a SIRP-α D1 variant) and a second therapeutic agent. In some embodiments, the combination comprises a polypeptide (e.g., SIRP-α D1 variant). ant) and a second therapeutic agent, wherein the second therapeutic agent is an antibody. , the combination comprising an amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain; and residues 6, 27, 31, and 32 relative to the wild-type SIRP-α D1 domain. residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation at a residue The present invention also includes SIRP-α D1 variants containing the SIRP-α D1 antibody or fragments thereof, as well as antibodies. In some embodiments, the combination comprises SEQ ID NOs: 78-85, 98-104, 107-113, having any one of the sequences 116 to 122, 135 to 137, or 152 to 159 This includes polypeptides, as well as antibodies.

[0254] In some embodiments, the methods include administering the polypeptide to a patient suffering from a disease for which administration of the polypeptide is a treatment option. Non-limiting examples of the above include antibodies or protein fragments. For example, in some embodiments, the antibody or protein fragment is and administered in combination with the Fc variant polypeptides disclosed herein. In embodiments, the polypeptide constructs disclosed herein improve the phagocytosis of other agents It is used for this purpose.

[0255] The treatment method comprises administering to a subject having a disease (e.g., cancer) (i) SIRP-α D1 and optionally (ii) administering an antibody to the subject. In some embodiments, prior to treatment of a disease (e.g., cancer) in a subject, the subject is SIRP- The amino acid sequence(s) of SIRP-α may be, for example, one of two alleles encoding the SIRP-α gene. In this treatment method, the S in a biological sample derived from the subject is determined. The amino acid sequence(s) of the IRP-α polypeptide are first determined. A therapeutically effective amount of a polypeptide of the present disclosure is administered. In some embodiments, The high-affinity SIRP-α D1 variant binds to CD47 of the SIRP-α polypeptide. SIR in the subject's biological sample, except for the introduction of amino acid changes that increase affinity for The P-α polypeptide has the same amino acid sequence as that of the P-α polypeptide. The SIRP-α D1 variant is preferably administered to the subject after administration of the polypeptide. It has minimal immunogenicity in

[0256] In some embodiments, the antibody is administered in addition to the polypeptides disclosed herein. In some embodiments, the antibody is co-administered with the polypeptide. In embodiments, the antibody may be administered simultaneously, e.g., to a pharmaceutical having both the polypeptide and the antibody. Alternatively, the antibody may be administered in a pharmaceutical composition before or after administration of the polypeptide. In some embodiments, the polypeptide and the antibody are administered to simultaneously (e.g., 1 week from each other, 6, 5, 4, 3, 2, 1 day, 12, 6, 3, 2, 1 Within a certain time period, or substantially simultaneously), and then the antibody is administered alone. In embodiments, the antibody is administered first, and then the polypeptide and the antibody are administered substantially simultaneously. (i.e., within 1 week, 6, 5, 4, 3, 2, 1 day, 12, 6, 3, 2, 1 hour of each other) or substantially simultaneously).

[0257] Co-administered or provided in a composition or provided by the methods disclosed herein Antibodies target cells such as cancer cells and immune system cells such as T cells (e.g., regulatory T cells). An antibody refers to an antibody of any immunoglobulin antibody isotype, e.g., IgG, It may be of IgE, IgM, IgA, or IgD. In some embodiments, the The antibody is a human IgG1 isotype antibody. In some embodiments, the antibody is a human IgG1 isotype antibody. In some embodiments, the antibody is a human IgG4 isotype antibody. It is an isotype antibody.

[0258] The term "antibody" is used herein in the broadest sense to refer to any antibody, so long as it exhibits the desired activity. Monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) The present invention encompasses various antibody structures, including but not limited to antibody fragments, antibody fragments, and antibody-like proteins. "Antibody fragment" includes a portion of an intact antibody, preferably a fragment of an intact antibody. The antibody fragments include the antigen-binding region or the variable region. Examples of antibody fragments include Fab, Fab', F (ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multi-antibodies Monoclonal antibodies are antibodies obtained from a population of substantially homogeneous antibodies. For example, individual antibodies in the population may be present in trace amounts. They have the same primary sequence except for naturally occurring mutations. Monoclonal antibodies are highly specific. It is specific and can be directed against a single antigenic site (e.g., an epitope of a cancer antigen). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different epitopes, Each monoclonal antibody is generally directed against a single epitope on an antigen. The modifier "monoclonal" refers to the characteristic of an antibody being obtained from a population of substantially homogeneous antibodies. and is not to be construed as requiring production of antibodies by any particular method. In some embodiments, the antibodies in the compositions of the present disclosure are capable of inhibiting antibody-dependent cell proliferation. It induces phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC). Non-limiting examples of diseases that may be treated using the methods include cancers, such as blood cancers, e.g., leukemia (e.g., acute myeloid leukemia), immune disorders (e.g., impaired or reduced immune function in a subject), to enhance an immune response in a subject or to limit an overactive immune response in a subject), and Infections include:

[0259] In some embodiments, the methods disclosed herein comprise administering to a subject a polypeptide as described herein. (e.g., SIRP-a D1 variants), and administering antibodies targeting cancer antigens. In some embodiments, the targeting is by an antibody or antibody-like protein. Cancer antigens are expressed in the form of complexes with human leukocyte antigen (HLA) class I molecules on the surface of tumor cells. Exposed peptides derived from tumor-associated antigens (TAA) (also known as MHC / peptide complexes) Such cancer antigens, for example, antibodies or antibody-like proteins in the compositions of the present disclosure, Peptides in complex with HLA molecules exposed on the surface of cancer cells are targeted by proteins Non-limiting examples include: NY-ESO-1 / LAGE1, SSX-2, MAGE family Lee (MAGE-A3), gp100 / pmel17, Melan-A / MART-1, gp 75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature lamina Nin receptor, MOK / RAGE-1, WT-1, Her2 / neu, EphA3, SAP -1, BING-4, Ep-CAM, MUC1, PRAME, survivin, mesothelin, BRCA1 / 2 (mutation), CDK4, CML66, MART-2, p53 (mutation), Ra s (mutation), β-catenin (mutation), TGF-βRII (mutation), HPV E6, E7 Examples of such antibodies include ESK1 (WT-1), RL1B (Her2-E 75), Pr20 (PRAME), and 3.2G1 (hCGβ).

[0260] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide containing the variant, and an antibody targeting the cancer antigen. In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. an amino acid mutation at residue 80 relative to the main, as well as the wild-type SIRP-α D1 domain Residues 6, 27, 31, 47, 53, 54, 56, and At least one additional amino acid residue selected from the group consisting of group 66 and residue 92 SIRP-α D1 domain containing an acid mutation, or a fragment thereof Polypeptides containing NY-ESO-1 / LAGE1, SSX-2, and the like , MAGE family (MAGE-A3), gp100 / pmel17, Melan-A / M ART-1, gp75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG- 72, immature laminin receptor, MOK / RAGE-1, WT-1, Her2 / neu, E phA3, SAP-1, BING-4, Ep-CAM, MUC1, PRAME, Survivi , mesothelin, BRCA1 / 2 (mutation), CDK4, CML66, MART-2, p5 3 (mutation), Ras (mutation), β-catenin (mutation), TGF-βRII (mutation), HP In some embodiments, the present invention provides a method for treating a patient with a HIV-1-associated ... The method disclosed herein involves adding an amino acid sequence at residue 80 relative to the wild-type SIRP-α D1 domain. and mutations at residues 6, 27, and 8 relative to the wild-type SIRP-α D1 domain. From the group consisting of residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 SIRP-α D having at least one additional amino acid mutation at a residue selected from a polypeptide comprising a SIRP-α D1 variant comprising one or more of the SIRP-α D1 domains, or a fragment thereof; and ESK1 (WT-1), RL1B (Her2-E75), Pr20 (PRAME ), and 3.2G1 (hCGβ).

[0261] In some embodiments, the methods disclosed herein comprise the steps of: Sequence of 04, 107-113, 116-122, 135-137, or 152-159 The method includes administering a polypeptide having the formula (I) and an antibody targeting a cancer antigen. In embodiments, the methods disclosed herein involve the use of SEQ ID NOs: 78-85, 98-104, 107 One of the following allocations: ~113, 116~122, 135~137, or 152~159 polypeptides having the sequence, and NY-ESO-1 / LAGE1, SSX-2, MAGE fragments 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, S AP-1, BING-4, Ep-CAM, MUC1, PRAME, survivin, mesothelial BRCA1 / 2 (mutation), CDK4, CML66, MART-2, p53 (mutation), Ras (mutation), β-catenin (mutation), TGF-βRII (mutation), HPV E6, E In some embodiments, the method comprises administering an antibody that targets 7. The method for cloning is as follows: A polypeptide having any one of the sequences of 5 to 137, or 152 to 159, and ESK1 (WT-1), RL1B (Her2-E75), Pr20 (PRAME), and and 3.2G1 (hCGβ).

[0262] In some embodiments, the antibody is directed to a protein expressed by, for example, a cancer cell. Some proteins target cancer cells by binding to them more than non-cancer cells. For example, cancer antigens are expressed at high levels by cancer cells. It is a protein that is differentially expressed (e.g., it is expressed in cancer cells more than in non-cancerous cells). In some cases, it is expressed only in cancer cells. proteins, for example, proteins expressed by cancer cells targeted by the antibodies in the compositions of the present disclosure. Non-limiting examples of proteins that may be used include: 4-1BB, 5T4, AGS-16, ALK1 , ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CCR4, CD123, CD19, CD20, CD22, CD27, EpCAM, CD30, CD3 2b, CD33, CD37, CD38, CD40, CD52, CD70, CD74, CD 79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CTLA-4, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA 3, ETBR, FGFR2, fibronectin, FR-alpha, Frizzled receptor Body, GCC, GD2, glypican-3, GPNMB, HER-2, HER3, HLA-D R, ICAM-1, IGF-1R, IL-3R, LAG-3, LIV-1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4, Notch 2, Notch 1, OX40, PD-1, PD-L1, PD-L2, PDGFR-α, PS, PSMA , SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK -1, CSF-1 R, or any combination thereof. In this regard, the polypeptides described herein may be used in combination with checkpoint inhibitors, e.g., CTLA- 4 antibody inhibitors (e.g., ipilimumab, tremelimumab), PD-1 antibody inhibitors (e.g., For example, nivolumab, pidilizumab, MK3475 (also known as pembrolizumab), and BMS93 6559, and MPDL3280A), and antibody inhibitors of LAG-3 (e.g., BM It is administered in combination with S986016).

[0263] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of Targeting variant-containing polypeptides and proteins expressed by cancer cells In some embodiments, the methods disclosed herein comprise administering an antibody to , an amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain, as well as wild-type For the type SIRP-α D1 domain, residues 6, 27, 31, 47, and residue 53, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain with at least one additional amino acid mutation, or and a polypeptide comprising a SIRP-α D1 variant comprising a fragment of 4-1BB, 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CCR4, CD123, CD19, CD20, CD22, CD27 , EpCAM, CD30, CD32b, CD33, CD37, CD38, CD40, CD 52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN 18.2, CLDN6, CS1, CTLA-4, CXCR4, DLL-4, EGFR, E GP-1, ENPP3, EphA3, ETBR, FGFR2, fibronectin, FR- alpha, Frizzled receptor, GCC, GD2, glypican-3, GPNMB, H ER-2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV -1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4, Notc h2, Notch 1, OX40, PD-1, PD-L1, PD-L2, PDGFR- α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, Antibodies targeting CD27L, DKK-1, CSF-1R, or any combination thereof In some embodiments, the methods disclosed herein include administering to the body a wild-type The SIRP-α D1 domain contains an amino acid mutation at residue 80, as well as wild-type SIR For the P-α D1 domain, residues 6, 27, 31, 47, 53, and at least one residue selected from the group consisting of residue 54, residue 56, residue 66, and residue 92; The SIRP-α D1 domain, or a fragment thereof, having one additional amino acid mutation. and a polypeptide comprising a SIRP-α D1 variant containing the SIRP-α D1 variant, as well as an antibody inhibitor of CTLA-4. anti-PD-1 antibody inhibitors (e.g., ipilimumab, tremelimumab), and Lumab, pidilizumab, MK3475, also known as pembrolizumab, BMS936559, and MPDL3280A), or antibody inhibitors of LAG-3 (e.g., BMS98601 6).

[0264] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 The target polypeptides are those having one of the sequences, and proteins expressed by cancer cells. In some embodiments, the methods disclosed herein include administering an antibody targeting the are SEQ ID NOs: 78 to 85, 98 to 104, 107 to 113, 116 to 122, 135 to 1 37, or 152 to 159, and a polypeptide having any one of the sequences of 4-1B B, 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fm s, c-Met, CA6, CCR4, CD123, CD19, CD20, CD22, CD 27, EpCAM, CD30, CD32b, CD33, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CL DN18.2, CLDN6, CS1, CTLA-4, CXCR4, DLL-4, EGFR , EGP-1, ENPP3, EphA3, ETBR, FGFR2, fibronectin, F R-alpha, Frizzled receptor, GCC, GD2, glypican-3, GPNMB , HER-2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, L AG-3, LIV-1, mesothelin, MUC16, MUC1, NaPi2b, Necti n-4, Notch 2, Notch 1, OX40, PD-1, PD-L1, PD-L 2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEG FR, CD25, CD27L, DKK-1, CSF-1R, or any combination thereof In some embodiments, the method comprises administering an antibody that targets the target gene. The method for cloning is as follows: A polypeptide having any one of the sequences of 5 to 137, or 152 to 159, and CTLA-4 antibody inhibitors (e.g., ipilimumab, tremelimumab), PD-1 inhibitors inhibitors (e.g., nivolumab, pidilizumab, MK3475, also known as pembrolizumab) , BMS936559, and MPDL3280A), or antibody inhibitors of LAG-3 (e.g. This includes administering an antibody that is an antibody against a cancer-causing agent (e.g., BMS986016).

[0265] In some embodiments, the methods disclosed herein comprise administering to a subject a polypeptide as described herein. (e.g., SIRP-α D1 variants), and immuno-oncology antibodies. In some embodiments, the antibody used in the compositions of the present disclosure includes cetuximab. , necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469, atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, ipilimumab, tremelimumab, urelumab, PF-05082566, Enoblituzumab, vantictum mab), varlilumab, mogamalizumab zumab), SAR650984, daratumumab, trastuzumab, trastuzumab Mutansine, pertuzumab, elotuzumab, rituximab, Fatumumab, obinutuzumab, RG7155, FPA008, panitumumab, Brentz Ximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, Panitumumab, ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab ib, atezolizumab, durvalumab, MEDI0680, pidilizumab, or BM S-93659, anti-HER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, anti- CD38 antibody, anti-EGFR antibody, anti-PD1 antibody, anti-RANKL antibody, anti-OX40 antibody, anti- PD-1 antibody, anti-PD-L1 antibody, anti-CD274 antibody, anti-CTLA-4 antibody, anti-CD13 7 antibody, anti-4-1BB antibody, anti-B7-H3 antibody, anti-FZD7 antibody, anti-CD27 antibody, anti-C CR4 antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CSF antibody, anti-CD30 antibody, anti-B Antibodies include, but are not limited to, AFF antibodies, anti-VEGF antibodies, or anti-VEGFR2 antibodies. In some embodiments, the methods disclosed herein do not involve the use of wild-type SIRP-α D. The amino acid mutation at residue 80 for the D1 domain, as well as the wild-type SIRP-α D1 domain For Yin, residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56 , residue 66, and residue 92. SIRP-α containing the SIRP-α D1 domain or a fragment thereof with an amino acid mutation Polypeptides containing the D1 variant, as well as anti-HER2 antibodies, anti-CD20 antibodies, anti-C D19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-EGFR antibody, anti-PD1 antibody, anti-RAN KL antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD274 antibody, anti- CTLA-4 antibody, anti-CD137 antibody, anti-4-1BB antibody, anti-B7-H3 antibody, anti-FZD 7 antibody, anti-CD27 antibody, anti-CCR4 antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CS F antibody, anti-CD30 antibody, anti-BAFF antibody, anti-VEGF antibody, or anti-VEGFR2 antibody In some embodiments, the methods disclosed herein comprise administering an antibody which is , an amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain, as well as wild-type For the type SIRP-α D1 domain, residues 6, 27, 31, 47, and residue 53, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain with at least one additional amino acid mutation, or and a polypeptide comprising a SIRP-α D1 variant comprising a fragment of cetuximab. , necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469, atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, ipilimumab, tremelimumab, urelumab, PF-05082566, Enoblituzumab, vantictum mab), varlilumab, mogamalizumab zumab), SAR650984, daratumumab, trastuzumab, trastuzumab Mutansine, pertuzumab, elotuzumab, rituximab, Fatumumab, obinutuzumab, RG7155, FPA008, panitumumab, Brentz Ximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, Panitumumab, ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab ib, atezolizumab, durvalumab, MEDI0680, pidilizumab, or BM This involves administering an antibody that is S-93659.

[0266] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide comprising the variant, and an antibody that is trastuzumab. nothing.

[0267] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, and an antibody that is rituximab. .

[0268] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, and an antibody that is cetuximab. .

[0269] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, and an antibody that is daratumumab. .

[0270] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide comprising the variant, and an antibody that is belimumab.

[0271] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, and an antibody that is bevacizumab. .

[0272] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide comprising the variant, and an antibody that is denosumab.

[0273] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and polypeptides containing the variants, as well as pantimumab. The method includes administering an antibody.

[0274] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, and an antibody that is ramucirumab. .

[0275] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, and an antibody that is necitumumab. .

[0276] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide comprising the variant, and an antibody that is nivolumab.

[0277] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide containing the variant, and an antibody that is pembrolizumab. include.

[0278] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide comprising the variant, as well as an antibody that is avelumab.

[0279] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide comprising the variant, and an antibody that is atezolizumab. nothing.

[0280] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide containing the variant, and an antibody that is durvalumab. nothing.

[0281] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of and administering a polypeptide comprising the variant, as well as an antibody that is MEDI0680. include.

[0282] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of The method includes administering a polypeptide containing the variant, and an antibody that is pidilizumab. nothing.

[0283] In some embodiments, the methods disclosed herein involve administering to a subject a wild-type SIRP-α D1 domain. The amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, and 66, and at least one additional amino acid at a residue selected from the group consisting of residue 92 SIRP-α D1 containing a SIRP-α D1 domain or a fragment thereof having a mutation of administering a polypeptide comprising the variant, as well as an antibody that is BMS-93659 Includes.

[0284] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 a polypeptide having any one of the sequences, as well as an anti-HER2 antibody, an anti-CD20 antibody, an anti-C D19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-EGFR antibody, anti-PD1 antibody, anti-RAN KL antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD274 antibody, anti- CTLA-4 antibody, anti-CD137 antibody, anti-4-1BB antibody, anti-B7-H3 antibody, anti-FZD 7 antibody, anti-CD27 antibody, anti-CCR4 antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CS F antibody, anti-CD30 antibody, anti-BAFF antibody, anti-VEGF antibody, or anti-VEGFR2 antibody In some embodiments, the methods disclosed herein comprise administering an antibody which is , SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-13 7, or 152-159, as well as cetuximab, necits Mumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED1 6469, atezolizumab, avelumab, durvalumab, MEDI6383, RG78 88, ipilimumab, tremelimumab, urelumab, PF-05082566, Enobili Enoblituzumab, vantictumab , varlilumab, mogamalizumab b), SAR650984, daratumumab, trastuzumab, trastuzumab emtansine pertuzumab, elotuzumab, rituximab, ofatum Mab, obinutuzumab, RG7155, FPA008, panitumumab, brentuximab Vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, panitum Mab, ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab, ate zolizumab, durvalumab, MEDI0680, pidilizumab, or BMS-93 659.

[0285] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and a polypeptide having one of the sequences, and an antibody, which is the antibody trastuzumab. This includes:

[0286] In some embodiments, the methods disclosed herein involve the use of SEQ ID NO: 7, SEQ ID NOs: 78-8 5, 98-104, 107-113, 116-122, 135-137, or 152- 159, and an antibody that is rituximab. This includes administering.

[0287] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 administering a polypeptide having one of the sequences, and an antibody that is cetuximab Includes:

[0288] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 administering a polypeptide having one of the sequences of daratumumab and an antibody that is daratumumab. Includes:

[0289] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences of belimumab to a subject. include.

[0290] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 administering a polypeptide having one of the sequences, and an antibody that is bevacizumab Includes:

[0291] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, and an antibody that is denosumab. include.

[0292] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 a polypeptide having one of the sequences, and pantimumab The method comprises administering an antibody which is

[0293] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 administering a polypeptide having one of the sequences, and an antibody that is ramucirumab Includes:

[0294] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 administering a polypeptide having one of the sequences, and an antibody that is necitumumab. Includes:

[0295] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 The present invention includes a polypeptide having any one of the sequences, as well as an antibody that is nivolumab.

[0296] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, as well as an antibody that is pembrolizumab. This includes:

[0297] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, as well as an antibody that is avelumab. include.

[0298] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, as well as an antibody that is atezolizumab. Includes:

[0299] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, as well as an antibody that is durvalumab. Includes:

[0300] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, as well as an antibody that is MEDI0680. This includes:

[0301] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 The present invention includes a polypeptide having any one of the sequences, as well as an antibody that is pidilizumab.

[0302] In some embodiments, the methods disclosed herein comprise the steps of: 04, 107-113, 116-122, 135-137, or 152-159 and administering a polypeptide having one of the sequences, as well as an antibody that is BMS-93659. This includes:

[0303] In some embodiments, the polypeptides disclosed herein inhibit the anti-tumor activity of rituximab. In some embodiments, the polypeptides disclosed herein enhance Raji activity. -Enhances the antitumor activity of rituximab in NSG xenograft models. In some embodiments, the polypeptides disclosed herein inhibit rituximab in non-human primates (NHPs). Enhances mab-mediated B cell depletion.

[0304] In some embodiments, the polypeptides and pharmaceutical compositions of the present disclosure are useful in treating various cancers. Cancers suitable for treatment according to the present disclosure include solid tumor cancers, hematological cancers, acute bone marrow cancers, and rheumatoid arthritis. Myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, non-Hodgkin's Lymphoma, Hodgkin's lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, intrauterine cancer Membrane cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer (st gallbladder cancer, gastrointestinal stromal cancer Tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin Cancer, Merkel cell carcinoma, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney Cancer, renal cell carcinoma, renal pelvic cancer, leukemia, lymphoma, non-epithelial malignant tumor, glioma, brain cancer In some embodiments, tumors include, but are not limited to, pulmonary tumors, pulmonary fibrosis ... Cancerous conditions suitable for treatment in accordance with the present disclosure include metastatic cancer. Thus, the cancers suitable for treatment according to the present disclosure are solid tumors or hematological cancers.

[0305] In some embodiments, the antibody binds to a protein expressed by a cell of the immune system. By doing so, cells of the immune system such as T cells, e.g., regulatory T cells, are targeted. In some embodiments, the methods disclosed herein involve the use of a polypeptide described herein (e.g., By administering antibodies that target cells of the immune system (e.g., SIRP-α D1 variant), Examples of proteins expressed by cells of the immune system include 41BB, CD4 0, CD40L, CD163, CD206, CTLA4, PD1, TIM-3, BTLA , VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27, H VEM, CCR4, CD25, CD103, KIrg1, Nrp1, CD278, Gpr 83, including but not limited to TIGIT, CD154, CD160, and PD1H In some embodiments, the antibody is designed to inhibit T cells relative to other cells of the immune system. For proteins (e.g., receptors) expressed by cells (e.g., regulatory T cells) In some embodiments, the compositions of the present disclosure are designed to have selective binding to The antibodies in the present invention comprise an Fc domain of the IgG1, IgG2, or IgG4 subclass.

[0306] In some embodiments, the methods of the present disclosure involve altering an immune response in a subject. The method includes administering to the subject a polypeptide comprising a high affinity SIRP-α D1 variant. administering peptides and antibodies to the subject, thereby altering the immune response in the subject. In some embodiments, altering the immune response includes suppressing the immune response. Includes:

[0307] In some embodiments, the polypeptides and pharmaceutical compositions of the present disclosure are used to treat immune disorders. Autoimmune and inflammatory diseases suitable for treatment according to the present disclosure are Diseases include multiple sclerosis, rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, and Mediated inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerosis atherosclerosis, Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia Hemolytic reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma These include pulmonary emphysema, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis. Not limited to these.

[0308] In some embodiments, delivery of a polypeptide to a cell is achieved by administering to the cell a polypeptide of the composition described herein. This involves contacting the cell with one or more

[0309] The effective dose for such treatment options will depend on the method of administration, the target site, the physiological condition of the patient, and the patient's Whether human or animal, other medications administered, and whether treatment is prophylactic or therapeutic In some embodiments, the patient is a human. However, non-human mammals, such as companion animals such as dogs, cats, and horses, rabbits, and mice, Experimental animals such as mice, rats, etc. may also be treated. In some embodiments, the therapeutic dose is The dose is titrated to optimize safety and effectiveness.

[0310] In some embodiments, the therapeutic dose is about 0.0001 to 100 mg / kg of host body weight. g, more usually ranging from 0.01 to 30 mg / kg. The dose is 1 mg / kg body weight or 30 mg / kg body weight, or 1 to 30 mg / kg body weight. In some embodiments, an exemplary treatment regimen is once weekly or It involves administration once every two weeks, once a month, or once every three to six months. In some embodiments, the therapeutic agents and polypeptide constructs described herein are administered multiple times. In some embodiments, the administration interval is weekly, monthly, or yearly. The interval may also be determined by measuring the patient's blood level of the therapeutic entity. Alternatively, the therapeutic agent or polypeptide constructs described herein may be administered indefinitely. is administered as a sustained release formulation, which allows for less frequent administration. In embodiments, the dosage and frequency will vary depending on the half-life of the polypeptide in the patient.

[0311] In prophylactic applications, in some embodiments, a relatively low dose may be administered in a relatively infrequent manner. In some embodiments, the patient receives a 20 mg dose of the 20 mg 20 mg 100 mg 20 ...200 mg 300 mg 400 mg 500 mg 600 mg 700 mg 800 mg 900 mg 100 mg 1100 mg 1200 mg 1300 mg 1400 mg 1500 In other therapeutic applications, the progression of the disease is reduced or halted. preferably until the patient shows partial or complete remission of the symptoms of the disease. , relatively high doses at relatively short intervals are required. The patient is then administered a prophylactic regimen.

[0312] As used herein, the terms "treatment," "treating," and the like refer to any treatment for the purpose of obtaining a benefit. In some embodiments, the effect is is prophylactic in that it completely or partially prevents a disease or its symptoms. In some embodiments, the effect is a partial or complete cure of the disease or symptoms of the disease. It is therapeutic in that it has an impact on

[0313] XIII. Kit In some embodiments, disclosed herein are wild-type signal regulatory proteins. The amino acid mutation at residue 80 in the D1 domain of SIRP-α, as well as the wild-type For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3, residue 54, residue 56, residue 66, and residue 92. a SIRP-α D1 domain having at least one additional amino acid mutation, or The polypeptide comprises a SIRP-α D1 variant, including a fragment thereof.

[0314] In some embodiments, also disclosed herein are Fc variants. A polypeptide, wherein the Fc variant comprises an Fc domain having two Fc domain monomers. and each Fc domain monomer independently comprises: (i) L23 The Fc region of human IgG1 consisting of the mutations 4A, L235A, G237A, and N297A (ii) Fc of human IgG2 consisting of A330S, P331S, and N297A mutations region, or (iii) S228P, E233P, F234V, L235A, delG2 36, and the Fc region of human IgG4 containing the mutation N297A. It's Chido.

[0315] Also provided are kits containing the polypeptides described herein and instructions for their use. Optionally, the kit may further comprise at least one additional reagent. As a non-limiting example, a chemotherapeutic agent or an anti-tumor antibody may be administered in combination with at least one additional agent. In some embodiments, the kit may serve as a means for identifying the contents of the kit. The term label includes any label provided on the kit indicating the intended use. or any part supplied with or otherwise accompanying the kit. This includes any written or recorded matter.

[0316] In some embodiments, the kit comprises: (i) a high affinity SIRP-α D1 variant; and optionally (ii) an antibody, and (iii) a subject with a disease. and instructions for administering (i) and (ii) (if provided). In one embodiment, the kit includes: (i) a polypeptide comprising a high-affinity SIRP-α D1 variant; and (ii) administering (i) to a subject having a disease using an antibody, e.g., an antibody provided in the kit. In some embodiments, the kit comprises: (i) an antibody; and (ii) administering (i) to a subject having the disease with a high-affinity SIRP-α D The present invention also includes instructions for administering the polypeptide containing the 1 variant together with the 1 variant.

[0317] In some embodiments, the kits are used to treat cancers, e.g., solid tumor cancers, hematological cancers, acute myeloid Leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer Cancer of the lung, bronchus, liver, ovaries, colon and rectum, stomach ach 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, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer , renal cell carcinoma, renal pelvic cancer, leukemia, lymphoma, non-epithelial malignant tumor, glioma, brain cancer, and any combination thereof. In some embodiments, the kit is for administering to a subject with a solid tumor cancer or a hematological cancer. It is used to treat

[0318] In some embodiments, the kit is used to treat a subject having an immune disorder. In some embodiments, the immune disease is an autoimmune disease or an inflammatory disease; For example, multiple sclerosis, rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, antibody-mediated Inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerotic Atherosclerosis, Sjögren'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, inflammatory autoimmune myositis, or any of these It is a combination. [Example]

[0319] Example 1 - SIRP-α D1 variant polypeptides Production of Polypeptides of the Disclosure The polypeptides of the present disclosure, including high-affinity SIRP-α D1 variants, are capable of binding to conventional molecular cleavage sites. It is produced using cloning and protein expression technology. The possible amino acid substitutions in SIRP-α D1 variants relative to the amino acid sequence are shown in Table 2 and Table 3. Nucleic acid molecules encoding the polypeptides of the present disclosure can be synthesized using well-known molecular biology techniques. Cloning into vectors optimized for expression in bacterial or mammalian cells using methods After induction of protein expression, the cells are harvested and the expressed polypeptide is isolated by affinity chromatography. The purified polypeptide is purified from the cell culture supernatant using column chromatography. The peptides were then analyzed by SDS-PAGE and then stained with Coomassie blue to confirm the expected size. Check for the presence of protein bands.

[0320] Purified polypeptides can be purified using techniques available in the art, e.g., phage display, enzyme Mother presentation, surface plasmon resonance (SPR), scintillation proximity assay, ELISA, ORIGEN Immunoassay (IGEN), fluorescence quenching, fluorescence transfer, or any suitable bioassay The desired polypeptides are screened for binding to CD47 using an immunoassay. The peptide binds to CD47, for example, human CD47, with higher affinity than wild-type SIRP-α. Combine.

[0321] Binding affinity of SIRP-α D1 variant polypeptides In a series of experiments, wild-type SIRP-α D1 domain and high-affinity SIRP-α D1 vectors were The derivative polypeptides are produced using conventional molecular cloning and protein expression techniques. Binding to human CD47 was measured using SPR as follows. , human CD47 (R and D Systems, Cat. No. 4670-CD or Monomeric extracellular domains (ECDs) of wild-type SIRP-α and SIRP- Binding to the α D1 variant polypeptide variants was measured using a Biacore T100 instrument. (GE Healthcare) or Proteon XPR36 (Bio-Rad, Hercules, CA) supplemented with 0.01% Tween-20. Phosphate-buffered saline (PBS, pH 7.4) (PBST) was used as the running buffer. 200-1000 RU of the ligand was dissolved in 10 mM sodium acetate buffer (pH 4.5). On the Biacore chip CM4 sensor or Proteon GLC chip, Immobilization was performed by standard amine coupling according to the manufacturer's recommendations. RP-α D1 variant polypeptide), for example, at least 0.1 Values ​​ranging from 10 to 10xKD were injected at a flow rate of 100 μL / min for 2 min, followed by 10 min. After each analyte injection, a 2:1 Pierce IgG elution buffer (Life Technologies, Cat. No. 21004) The surface was regenerated using a mixture of 4M NaCl. Complete regeneration of the surface was confirmed by measuring the baseline Analysis was confirmed by injecting the same analyte at the beginning and end of the experiment. All sensorgrams were double-referenced using a reference surface and buffer injection, with a 1:1 The analyte was primarily monomeric, Fc-free CD47. The ligands on the chip were either ECD or SIRP-α. The binding data are shown in Table 16. All SPR assays were performed at 25 The temperature was ℃. [Table 16-1] [Table 16-2]

[0322] Possession of a glutamic acid or aspartic acid residue at position 54 is essential for mouse CD47 It has also been found to improve binding of SIRP-α D1 variant polypeptides to As a non-limiting example, the SIRP-α D1 variant polypeptides identified in Table 17 below are The peptide exhibits high affinity binding to mouse CD47. 1. The binding affinity of the variant polypeptide to human CD47 may be determined by measuring the binding affinity of the variant polypeptide to human CD47. The mouse CD47 protein was used instead of 47, and as previously described, SPR was used to detect the The binding affinity of the antibody to mouse CD47 was compared with that of the antibody to mouse CD47. The results are shown in Table 18. [Table 17-1] [Table 17-2] [Table 18-1] [Table 18-2]

[0323] Partial glycosylation of certain SIRP-α D1 variant polypeptides The N80A mutation, which can minimize or eliminate the phenotype, includes SIRP-α D1 variant polypeptides, providing the functional benefits of increased homogeneity associated with the polypeptides. It has also been found that SIRP-α variant polypeptides are expressed in E. coli. When expressed, N80 is due to the lack of a glycosylation system in E. coli compared to mammalian systems. Table 19 shows the glycosylation of SIRP-α D1 produced in E. coli. This indicates that effective binding between the ribosomal polypeptide and human CD47 can still occur. Thus, deglycosylation may be a key factor in determining whether SIRP-α D1 variants are still expressed by CD4 7. The N80A mutation does not affect the binding affinity of 7. In addition, deglycosylation can be achieved by mutating N80 to any amino acid other than N. or the motif N-Xaa1-Xaa2, where N=asparagine and Xaa1= Any amino acid except P (proline), Xaa2 = T (threonine), S (serine) or is C (cysteine) and is a residue of the SIRP-α D1 variant polypeptide This can be achieved by disrupting the motif 80-82. Mutation of p83 to valine. Increased glycosylation at N80 can be achieved by increasing the N80 residue or by mutating it to a residue other than P. A homogeneously glycosylated SIRP-α D1 variant polypeptide capable of causing oxidative stress It is possible to produce polypeptides.

[0324] The amino acid P83 can also affect the degree of glycosylation. The efficiency of glycosylation at N80 can be increased by changing the amino acid to The SIRP-α D1 variant with a valine (V) at position 83 (SEQ ID NO: 213) was found to be a SIRP-α D1 variant with a valine (V) at position 83. The protein was expressed in K293FS mammalian cells. The size of the expressed protein was determined by comparing the wild-type amino acid sequence with that of the wild-type amino acid sequence. SIRP-α D1 variants (sequences) having an acid residue (e.g., proline, P) at position 83 The molecular weights of the expressed proteins were analyzed by protein gel (Figure 1). 8) shows that the variant with the P83V mutation (SEQ ID NO: 213, lane 2) has a P83V mutation at position 83 It has a higher molecular weight (e.g., approximately 22 kDa) compared to the unmutated variant (lane 1). As shown in Figure 18, when residue 83 was mutated to Val, The SIRP-α variant polypeptides expressed in the cytoplasmic host are primarily of higher molecular weight (approximately 22 kDa), which indicates that the efficiency of glycosylation at N80 can be increased. are. [Table 19-1] [Table 19-2]

[0325] Example 2 - Generation of single-arm and bispecific SIRP-α polypeptides (i) a SIRP-α-Fc fusion protein and (ii) CD47 and an antigen, e.g., EG Fc domain fused to a polypeptide such as an antigen binding domain to bind both the Fc domain and the Fc domain. The ability of constructs containing heterodimers of ATP monomers to react with ATP in SPR was demonstrated as described previously in this example. The Fc fusion proteins for forming heterodimers are shown in Table 20. Three monofunctional (eg, single target binding) SIRP-α-Fc fusions were tested. These fusion proteins are designated A, B, and C in Figure 6A. The α-Fc fusion ("A") was a homodimer of SEQ ID NO: 136. A monofunctional SIRP-α-Fc fusion is (i) a SIRP-α-Fc fusion and (ii) a fusion thereof. It was a heterodimer of Fc domain monomers with no additional polypeptide fused to it. These were generated using the knob and hole mutation engineering method shown in Figures 4A and 4B. The monofunctional SIRP-α-Fc fusion ("B") is SEQ ID NO: 139 (Fc variant) and and SEQ ID NO: 142 (SIRP-α-Fc fusion). The monofunctional SIRP-α-Fc fusion (“C”) comprises SEQ ID NO: 139 (Fc variant) and Formed from the heterodimerization of SEQ ID NO: 138 (SIRP-α-Fc fusion). The SIRP-α-Fc fusion ("D") (e.g., binding to two targets) is represented by SEQ ID NO: 1 27 (SIRP-α-Fc fusion) and SEQ ID NO: 144 (enantiomer linked to Fc variant) SEQ ID NO: 220 is the antigen-binding domain of E. coli. Represents the light chain of the Rubitux antibody. [Table 20-1] [Table 20-2] [Table 20-3]

[0326] Briefly, CD47 was attached to the Proteon GLC chip by amine chemistry as described above. In the first injection, the analytes (e.g., A, B, C, D, and Elbeta) were immobilized on a plate. Tax) was injected at 100 nM for 60 seconds at 30 μL / min in PBST to target the CD47 surface. The binding of 100 nM EGFR-ECD (HEK2 The extracellular domain of the epidermal growth factor receptor (EGFR) produced in 93 cells...

Claims

1. The wild-type signal transduction regulator protein alpha (SIRP-α) D1 domain has an affinitor at residue 80. Amino acid mutations at residues 6, 27, and 30 relative to the wild-type SIRP-α D1 domain. , residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 SIRP- having at least one additional amino acid mutation at a residue selected from the group consisting of: Polypeptides containing SIRP-α D1 variants containing the α D1 domain, or fragments thereof Chid.

2. The wild-type SIRP-α D1 domain has any one of the sequences of SEQ ID NOs: 1 to 10. The polypeptide of claim 1 .

3. The SIRP-α D1 domain has a residue Group 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, The polypeptide according to claim 1.

4. The SIRP-α D1 variant has the amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRX ４ LIYNQX ５ X ６ GX ７ FPRVTTVSDX ８ TKRNNM DFSIRIGX ９ ITPADAGTYYCE １０ KFRKGSPDDVEFKSGAG TELSVRAKPS (SEQ ID NO:49), wherein １ is V, L, or I; , X ２ is A, I, V, or L; X ３ is I, F, S, or T, and X ４ Is E, V or L, and X ５ is K or R, and X ６ is E or Q, and X ７ H, P or R, and X ８ is L, T, S, or G; X ９ is A and X １０ Is V or I, wherein the SIRP-α D1 variant is a wild-type having the sequence of SEQ ID NO:

1. At least two amino acid substitutions relative to the SIRP-α D1 domain.

2. The polypeptide described in 1.

5. The SIRP-α D1 variant has an amino acid sequence similar to any one of SEQ ID NOs: 78-85.

5. The polypeptide of claim 4 having the sequence:

6. The SIRP-α D1 variant has the amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRX ４ LIYNQX ５ X ６ GX ７ FPRVTTVSDX ８ TKRNNM DFSIRIGX ９ X １０ X １１ X １２ ADAGTYYCX １３ KFRKGSPDDVE FKSGAGTELSVRAKPS (SEQ ID NO: 218), wherein １ is V, L, or I, X ２ is A, V, L, or I; X ３ is I, S, T, or F R,X ４ is E, L, or V; X ５ is K or R, and X ６ is E or Q , X ７ is H, R, or P; X ８ is S, G, L, or T; X ９ is any a amino acid, X １０ is any amino acid, and X １１ is any amino acid, and X １２ is any amino acid, and X １３ is V or I, and the SIRP-α D1 variant The antibody has at least one nucleotide sequence corresponding to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:

1. The polypeptide of claim 1 having two amino acid substitutions.

7. X ９ The polypeptide of claim 6, wherein is A.

8. X ９ The polypeptide of claim 6, wherein is N.

9. X １０ The polypeptide of claim 6, wherein is I.

10. X ９ is N and X １０ The polypeptide of claim 6, wherein is P.

11. X ９ is N and X １１ is any amino acid other than S, T, or C. The polypeptide described.

12. X １１ The polypeptide of claim 6, wherein is T.

13. X １１ is an amino acid other than T.

14. X １２ The polypeptide of claim 6, wherein is P.

15. X ９ is N and X １２ is any amino acid other than P. Chid.

16. The SIRP-α D1 variant has the amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRX ４ LIYNQX ５ X ６ GX ７ FPRVTTVSDX ８ TKRNNM DFSIRIGX ９ ITX １０ ADAGTYYCX １１ KFRKGSPDDVEFKSG AGTELSVRAKPS (SEQ ID NO: 219), wherein １ is V, L, or I And X ２ is A, V, L, or I; X ３ is I, S, T, or F, and X ４ is E, L, or V; X ５ is K or R, and X ６ is E or Q, and X ７ teeth H, R, or P; X ８ is S, G, L, or T; X ９ is N, and X １０ is any amino acid other than P, and X １１ is V or I, and the SIRP-α D1 The variant has a low affinity to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO:

1. The polypeptide of claim 1 having at least two amino acid substitutions.

17. The SIRP-α D1 variant has the amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRELIYNQX ４ EGX ５ FPRVTTVSDX ６ TKRNNMDF SIRIGX ７ ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO:52), wherein X １ is V, L, or I; X ２ teeth A, I, or L; X ３ is I, T, S, or F; X ４ is K or R , X ５ is H, P, or R; X ６ is L, T, or G; X ７ is A, and before The SIRP-α D1 variant has a wild-type SIRP-α D1 having the sequence of SEQ ID NO:

1. The polypeptide of claim 1 having at least two amino acid substitutions per domain. Chid.

18. X １ is V or I, and X ２ is A or I, and X ３ is I or F, and X ４ but K or R, X ５ is H or P, and X ６ is L or T, and X ７ is A 18. The polypeptide of claim 17.

19. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of SEQ ID NO:

1.

18. The method of claim 17, having at least three amino acid substitutions relative to the D1 domain. Polypeptides.

20. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of SEQ ID NO:

1. The antibody of claim 17 having at least four amino acid substitutions relative to the D1 domain. Polypeptides.

21. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of SEQ ID NO:

1.

18. The method of claim 17, having at least five amino acid substitutions relative to the D1 domain. Polypeptides.

22. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of SEQ ID NO:

1.

18. The method of claim 17, having at least six amino acid substitutions relative to the D1 domain. Polypeptides.

23. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of SEQ ID NO:

1.

18. The method of claim 17, having at least seven amino acid substitutions relative to the D1 domain. Polypeptides.

24. X １ The polypeptide of claim 17, wherein is I.

25. X ２ The polypeptide of claim 17, wherein is I.

26. X ３ The polypeptide of claim 17, wherein is F.

27. X ４ The polypeptide of claim 17, wherein is R.

28. X ５ The polypeptide of claim 17, wherein is P.

29. X ６ The polypeptide of claim 17, wherein is T.

30. X １ , X ２ , X ３ , X ４ , X ５ , and X ６ each of which is not a wild-type amino acid.

8. The polypeptide according to claim 7.

31. The SIRP-α D1 variant has the amino acid sequence of any one of SEQ ID NOs: 81 to 85.

18. The polypeptide of claim 17 having the sequence:

32. The SIRP-α D1 variant has the amino acid sequence EEELQX １ IQPDKSVS VAAGESAILHCTX ２ TSLX ３ PVGPIQWFRGAGPARELIYNQ X ４ EG X ５ FPRVTTVSEX ６ TKRENMDFSISISX ７ ITPADAG TYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 212) Wherein X １ is V, L, or I; X ２ is V, I, or L; X ３ is I, T, S, or F; X ４ is K or R, and X ５ is H, P, or R R,X ６ is S, T, or G, and X ７ is A, and the SIRP-α D1 variant is at least one of the following is a sequence of SEQ ID NO:2 for a wild-type SIRP-α D1 domain: The polypeptide of claim 1 having at least two amino acid substitutions.

33. For human CD47, Ｄ Approximately 5x10 －９ The polysaccharide of claim 1, wherein the polysaccharide is linked by less than M. peptide.

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

35. The Fc domain monomer is a wild-type human IgG1, IgG2, or IgG4 Fc domain monomer.

35. The polypeptide of claim 34, comprising at least one mutation in the region.

36. Any one of the amino acid sequences of SEQ ID NO:135, SEQ ID NO:136, or SEQ ID NO:137 36. The polypeptide of claim 35 having a sequence.

37. The Fc domain monomer (a) has the following amino acid substitutions relative to wild-type human IgG1: One of the following: T366W, T366S, L368A, Y407V, T366Y, T394W , F405W, Y349T, Y349E, Y349V, L351T, L351H, L35 1N, L351K, P353S, S354D, D356K, D356R, D356S, E 357K, E357R, E357Q, S364A, T366E, L368T, L368Y , L368E, K370E, K370D, K370Q, K392E, K392D, T39 4N, P395N, P396T, V397T, V397Q, L398T, D399K, D 399R, D399N, F405T, F405H, F405R, Y407T, Y407H , Y407I, K409E, K409D, K409T, or K409I, or (b) (i) a N297A mutation in the Fc region of human IgG1; (iii) the mutations L234A, L235A, and G237A in the Fc region of For the Fc region of human IgG1, L234A, L235A, G237A, and N297A (iv) a N297A mutation in the Fc region of human IgG2; (v) a N297A mutation in the Fc region of human IgG2; (vi) A330S and P331S mutations in the Fc region of human IgG2; (vii) human Ig For the Fc region of G4, S228P, E233P, F234V, L235A, and de or (viii) a mutation at S228P in the Fc region of human IgG4. , containing the mutations E233P, F234V, L235A, delG236, and N297A.

36. The polypeptide of claim 35.

38. In a phagocytosis assay, the polypeptides were phagocytosed in comparison with the polypeptides of the Fc region of wild-type human IgG.

36. The polypeptide of claim 35, which exhibits reduced activity.

39. a second polypeptide comprising a second Fc domain monomer; 36. The polypeptide of claim 35, wherein the polypeptide is linked to and forms an Fc domain dimer.

40. 39. The method of claim 38, wherein the second Fc domain monomer is linked to an additional polypeptide. The polypeptide described in

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

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

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

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

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

46. The therapeutic protein may be a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory 46. ​​The polypeptide of claim 45, which is an immunosuppressant, an immunomodulator, or an immunomodulator.

47. The method according to claim 45, wherein the additional polypeptide comprises a SIRP-α D1 variant. The polypeptide described above.

48. The polypeptide of claim 1 further comprises human serum albumin (HSA) (SEQ ID NO: 12). Lipeptide.

49. The HSA comprises an amino acid substitution of C34S or K573P with respect to SEQ ID NO:

12.

49. The polypeptide of claim 48.

50. The method according to claim 48, having an amino acid sequence according to any one of SEQ ID NOs: 152 to 159. Polypeptides.

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

52. The albumin binding peptide has the amino acid sequence of DICLPRWGCLW (SEQ ID NO: 160).

52. The polypeptide of claim 51, comprising a amino acid sequence.

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

54. The PEG polymer is linked to a cysteine ​​substitution of the polypeptide.

54. The polypeptide described in 53.

55. A polypeptide comprising: (a) a signal regulatory protein alpha (SIRP-α) D1 barrier polypeptide; The amino acid sequence EEX １ X ２ QX ３ IQPDKX ４ VX ５ VAAGEX ６ X ７ X ８ LX ９ CTX １０ TS LX １１ PVGPIQWFRGAGPX １２ RX １３ LIYNQX １４ X １５ GX １６ F PRVTTVSX １７ X １８ TX １９ RX ２０ NMDFX ２１ IX ２２ IX ２３ X ２４ I TX ２５ ADAGTYYCX ２６ KX ２７ RKGSPDX ２８ X ２９ EX ３０ KSGAG TELSVRX ３１ KPS (SEQ ID NO: 47), wherein X １ is E or G , X ２ is L, I, or V; X ３ is V, L, or I; X ４ is S or F And X ５ is L or S, and X ６ is S or T, and X ７ is A or V Yes, X ８ is I or T, and X ９ is H, R, or L; X １０ A, V, I or L, １１ is I, T, S, or F; X １２ is A or G , X １３ is E, V, or L; X １４ is K or R, and X １５ is E, or Q and X １６ is H, P, or R; X １７ is D or E, and X １８ Is S , L, T, or G; X １９ is K or R, and X ２０ is E or N , X ２１ is S or P, and X ２２ is S or R, and X ２３ is S or G Yes, X ２４ is any amino acid, and X ２５ is any amino acid, and X ２６ V, ma or I, X ２７ is F, L, or V; X ２８ is D or absent , X ２９ is T or V, and X is ３０ is F or V, and X ３１ is A or G and a wild-type SIRP-α D1 domain having any one of the sequences of SEQ ID NOs: 1 to 10. The SIRP-α D1 variant has at least two amino acid substitutions relative to the and (b) an Fc domain dimer having two Fc domain monomers. variant, and each Fc domain monomer independently has (i) N297A (ii) a human IgG1 Fc region comprising the mutations L234A, L235A, and G237A; (iii) a human IgG1 Fc region comprising the following mutations: L234A, L235A, G237A; and (iv) a human IgG1 Fc region comprising the N297A mutation; (v) human IgG2 Fc region containing the A330S and P331S mutations; (vi) a human IgG Fc region comprising the mutations A330S, P331S, and N297A. (vii) the Fc region of S228P, E233P, F234V, L235A, and de or (viii) an Fc region of human IgG4 comprising the mutation at S228P, E2 Human I containing the mutations 33P, F234V, L235A, delG236, and N297A The polypeptide comprising the Fc variant which is an Fc region of gG4.

56. One of the Fc domain monomers of the Fc domain dimer is L234A, L23 The Fc region of human IgG1 comprising the mutations 5A, G237A, and N297A.

55. A polypeptide according to claim 55.

57. SEQ ID NO: 98-104, 107-113, 116-122, or 135-137 56. A polypeptide according to claim 55 comprising any one of the amino acid sequences.

58. The Fc variant has a specific binding affinity to Fcγ receptors compared to a wild-type version of the Fc region of human IgG.

56. The polypeptide of claim 55, which exhibits eliminated or reduced binding to the

59. The IgG1 or IgG2 Fc variant is a human IgG1 or IgG2 Fc variant. Compared to the wild-type versions of the regions, CD16a, CD32a, CD32b, CD32c, and C 56. The polypeptide of claim 55, which exhibits ablated or reduced binding of D64 to Fcγ receptors. Petite.

60. the IgG4 Fc variant is compared to a wild-type version of the human IgG4 Fc region; , which exhibits ablation or reduction in binding of CD16a and CD32b to Fcγ receptors. Item 56. The polypeptide according to item 55.

61. The IgG1 or IgG2 Fc variant is a human IgG1 or IgG2 Fc variant.

56. The method of claim 55, which exhibits ablated or reduced binding to C1q compared to a wild-type version of the fusion. The polypeptide described.

62. The Fc variant binds to an Fcγ receptor and Ｄ Approximately 5x10 －６ Binding at M or more 56. The polypeptide of claim 55.

63. A polypeptide comprising an Fc variant, said Fc variant comprising two Fc domains. and an Fc domain dimer having an Fc domain monomer, each Fc domain monomer being independently (i) human I consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human Fc region consisting of the A330S, P331S and N297A mutations; or (iii) S228P, E233P, F234V, L235 A human IgG4 Fc region comprising the mutations delG236, delG236, and N297A. The polypeptide.

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

65. At least one of the Fc domain monomers is selected from the group consisting of L234A, L235A, and G237A. and N297A mutation. Lipeptide.

66. At least one of the Fc domain monomers is selected from the group consisting of A330S, P331S, and N29 64. The polypeptide of claim 63, which is a human IgG2 Fc region consisting of the 7A mutation.

67. The Fc variant has an Fcγ receptor agonist as compared to a wild-type version of the Fc region of the human IgG.

64. The polypeptide of claim 63, which exhibits eliminated or reduced binding to the body.

68. The Fc variant has a specific function to inhibit CD16a, C, Elimination of binding of CD32a, CD32b, CD32c, and CD64 to Fcγ receptors 68. The polypeptide of claim 67, which exhibits a decrease or a decrease in

69. The Fc variant has a higher affinity for C1q compared to a wild-type version of the Fc fusion of human IgG.

64. The polypeptide of claim 63, which exhibits elimination or reduction of binding to the polypeptide.

70. At least one of the Fc domain monomers is S228P, E233P, F234V , L235A, delG236, and N297A mutations in the Fc region of human IgG4.

64. The polypeptide of claim 63.

71. The Fc variant has a higher affinity for Fcγ receptors than a wild-type human IgG4 Fc region.

71. The polypeptide of claim 70, which exhibits elimination or reduction of binding to the polypeptide.

72. The Fc variant has a CD16 72. The method of claim 71, wherein the Fcγ receptor binding of CD32a and CD32b is eliminated or reduced. The polypeptide described above.

73. The Fc variant binds to an Fcγ receptor and Ｄ Approximately 5x10 －６ Binding at M or more 64. The polypeptide of claim 63.

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

75. The Fc variant has a specific binding affinity to Fcγ receptors compared to a wild-type version of the Fc region of human IgG.

75. The polypeptide of claim 74, which exhibits elimination or reduction of binding to the polypeptide.

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

77. 8. The CD47 binding polypeptide of claim 7, wherein the CD47 binding polypeptide does not cause acute anemia in humans.

5. The polypeptide according to claim 4.

78. The CD47 binding polypeptide is a signal regulatory protein alpha (SIRP-α) polypeptide.

75. The polypeptide of claim 74, which is a peptide or a fragment thereof.

79. The SIRP-α polypeptide has the amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRX ４ LIYNQX ５ EGX ６ FPRVTTVSDX ７ TKRNNMD FSIRIGX ８ ITPADAGTYYCE ９ KFRKGSPDDVEFKSGAGTE LSVRAKPS (SEQ ID NO:51), wherein １ is V or I, X ２ A or I, X ３ is I or F, and X ４ is E or V, and X ５ is K or R And X ６ is H or P, and X ７ is L or T, and X ８ is any ami other than N X is an acid. ９ 78. The SIRP-α D1 variant of claim 78, wherein The polypeptide described in

80. The SIRP-α polypeptide is １ is V or I, and X ２ is A or I , X ３ is I or F, and X ４ is E and X ５ is K or R, and X ６ is H or P and X ７ is L or T, and X ８ is not N, but X ９ SIRP-α where V 80. The polypeptide of claim 79, comprising a D1 variant.

81. A polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant. and a non-naturally occurring high affinity SIRP-α D1 domain, and At least 10-fold higher affinity than the naturally occurring SIRP-α D1 domain that binds to the parent The SIRP-α D1 variants that bind to human CD47 with affinity, and the Fc domain and a second polypeptide comprising a second Fc domain monomer. and ligating the Fc domain to form an Fc domain, the Fc domain being an effector domain with a deletion or reduction. - function and ablated or reduced C1q binding. The polypeptide.

82. The non-naturally occurring high affinity SIRP-α D1 domain has an amino acid mutation at residue 80.

82. The polypeptide of claim 81 , comprising:

83. A polypeptide containing a signal transduction regulator protein alpha (SIRP-α) D1 variant. The SIRP-α D1 variant is Ｄ CD4 from the first species at less than 250 nM 7, and the SIRP-α D1 variant binds to K Ｄ Less than 250 nM from the second species and said K for CD47 from said first species. Ｄ And the second The K against CD47 from the species Ｄ are within 100 times of each other, and the first species and The second species is selected from the group consisting of humans, rodents, and non-human primates. Petite.

84. The SIRP-α D1 variant binds to CD47 from at least three different species. The polypeptide of claim 83 .

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

86. (a) K Ｄ Signal Regulatory Protein Alpha (SI) binds to human CD47 at less than 250 nM (b) the N-terminus or C-terminus of the SIRP-α D1 domain; A polypeptide comprising a terminally linked Fc domain monomer, The polypeptide which does not cause acute anemia in human primates.

87. 87. The method of claim 86, which is a non-naturally occurring variant of the human SIRP-α D1 domain. Polypeptides.

88. In vivo administration of the polypeptide reduces hemoglobin by less than 50% during the first week after administration.

87. The polypeptide of claim 86, which results in a reduction.

89. Administration of the polypeptide in humans reduces hemoglobin levels by less than 50% during the first week after administration.

87. The polypeptide of claim 86, which results in a reduction.

90. Further, the present invention relates to an antibody that comprises at least one Fc variant, the Fc variant comprising: i) a human IgG1 consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG Fc region consisting of the mutations A330S, P331S and N297A; or (iii) the Fc region of S228P, E233P, F234V, L235A, d and a human IgG4 Fc region comprising the mutations eIG236, eIG236, and N297A. The polypeptide described in claim 83.

91. The Fc variant comprises the mutations L234A, L235A, G237A, and N297A. The polypeptide of claim 90, which is an Fc region of human IgG1 consisting of:

92. The Fc variant comprises a human Fc variant consisting of the A330S, P331S, and N297A mutations.

91. The polypeptide of claim 90, which is an IgG2 Fc region.

93. The Fc variant is S228P, E233P, F234V, L235A, delG 91. The Fc region of claim 90, which is a human IgG4 Fc region comprising the mutations at positions 236, 237, and N297A. Polypeptides.

94. A method for treating an individual having a disease or disorder, comprising administering to said individual a compound according to any one of claims 1 to 93. The method comprises administering to the individual the polypeptide described above.

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

96. The disease or disorder is cancer, and the cancer is solid tumor cancer, blood cancer, acute myeloid cancer, Myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's leukemia lymphoma, Hodgkin's lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer Cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer (sto mach cancer, gastric cancer), gallbladder cancer, gastrointestinal stromal tumor thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer , Merkel cell carcinoma, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer renal cell carcinoma, renal pelvic cancer, leukemia, lymphoma, non-epithelial malignant tumor, glioma, brain cancer, and epithelial carcinomas.

97. The disease or disorder is an autoimmune disease or an inflammatory disease, The inflammatory disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, spondyloarthropathy, and systemic lupus erythematosus. antibody-mediated inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, diabetes, psoriasis, Atherosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary artery disease syndrome, ischemia-reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis selected from fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis.

95. The method of claim 94, wherein

98. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 95. The method of claim 94, having the following structure:

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

100. The at least one additional agent is an antibody, a tumor-associated antigen, or a non-antibody therapeutic agent.

100. The method of claim 99,

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

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

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

104. 101. 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 antibody is an anti-HER2 antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CS1 antibody, an anti-C D38 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-CC R4 antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CSF antibody, anti-CD30 antibody, anti-BA FF antibody, anti-VEGF antibody, or anti-VEGFR2 antibody. The method described.

109. The antibody is an anti-HER2 antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CS1 antibody, an anti-C D38 antibody, anti-PD-1 antibody, anti-RANKL antibody, or anti-PD-L1 antibody. The method of claim 108.

110. The at least one additional agent is at least one antibody, the antibody being Tuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI 0680, MED16469, atezolizumab, avelumab, durvalumab, MEDI 6383, RG7888, ipilimumab, tremelimumab, urelumab, PF-0508 2566, enoblituzumab, vanticutumab tictumab, varlilumab, mogamalizumab amalizumab), SAR650984, daratumumab, trastuzumab, tras Tubulomab emtansine, pertuzumab, elotuzumab, Rituximab Cimab, ofatumumab, obinutuzumab, RG7155, FPA008, panitumumab , brentuximab vedotin, MSB0010718C, belimumab, bevacizumab, de Nosumab, 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 SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 112. The method of claim 111, comprising:

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

114. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 114. The method of claim 113, comprising:

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

116. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 116. The method of claim 115, comprising:

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

118. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 118. The method of claim 117, comprising:

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

120. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 120. The method of claim 119, comprising:

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

122. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 122. The method of claim 121, comprising:

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

124. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 124. The method of claim 123, comprising:

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

126. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 126. The method of claim 125, comprising:

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

128. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 128. The method of claim 127, comprising:

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

130. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 130. The method of claim 129, comprising:

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

132. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 132. The method of claim 131, comprising:

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

134. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 134. The method of claim 133, comprising:

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

136. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 136. The method of claim 135, comprising:

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

138. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 138. The method of claim 137, comprising:

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

140. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 140. The method of claim 139, comprising:

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

142. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 142. The method of claim 141, comprising:

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

144. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 144. The method of claim 143, comprising:

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

146. The SIRP-α D1 variant is selected from the group consisting of SEQ ID NOs: 78-85, 98-104, 107- Any one of the sequences of 113, 116-122, 135-137, or 152-159 146. The method of claim 145, comprising:

147. The at least one further agent is a tumor-associated antigen, and the tumor-associated antigen is an immune response. The method of claim 110, further comprising inducing a reaction.

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

149. The antibody is selected from the group consisting of NY-ESO-1 / LAG-1, SSX-2, MAGE family (MA GE-A3), gp100 / pmel17, Melan-A / MART-1, gp75 / TR P1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor , MOK / RAGE-1, WT-1, Her2 / neu, EphA3, SAP-1, BI NG-4, Ep-CAM, MUC1, PRAME, survivin, mesothelin, BRCA1 / 2 (mutation), CDK4, CML66, MART-2, p53 (mutation), Ras (mutation) , β-catenin (mutated), TGF-βRII (mutated), HPV E6, or E7.

149. The method of claim 148, which targets HLA / peptide or MHC / peptide complexes. method.

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

151. The polypeptide of claim 1 for use in the treatment of cancer.

152. The polypeptide of claim 1 for use in the treatment of an autoimmune disease.

153. The polypeptide of claim 1 for the treatment of an inflammatory disease.

154. 13. Use of the polypeptide of claim 1 for the manufacture of a medicament for the treatment of cancer.

155. 13. Use of a polypeptide according to claim 1 for the manufacture of a medicament for the treatment of an autoimmune disease.

156. 13. Use of a polypeptide according to claim 1 for the manufacture of a medicament for the treatment of an inflammatory disease.

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