SIRP-Alpha Domain or a construct having a variant thereof

Abstract

The present invention provides signal-regulating protein a(SIRP-α) polypeptides and constructs useful for targeting cells (e.g., cancer cells or immune system cells), increasing the phagocytosis of said target cells, eliminating immune cells such as regulatory T cells, killing cancer cells, treating diseases (e.g., cancer) in a target, or any combination thereof, as well as methods for treating cancer. [Solution] A construct is provided comprising a high-affinity SIRP-αD1 domain or a variant thereof that binds to CD47 with higher affinity than wild-type SIRP-α.

Description

[Technical Field]

[0001] cross reference This application is U.S. Provisional Patent Application No. 62 / 202,772, filed on August 7, 2015. U.S. Provisional Patent Application No. 62 / 202,775, filed on August 7, 2015. U.S. Provisional Patent Application No. 62 / 202,779, filed on August 7, 2016, January 8, 2016 U.S. Provisional Patent Application No. 62 / 276,801, filed on December 10, 2015. U.S. Provisional Patent Application No. 62 / 265,887, filed on January 8, 2016, in the United States. Provisional Patent Application No. 62 / 276,796 and the U.S. Provisional Patent filed on June 6, 2016 We claim the benefits of application No. 62 / 346,414. These applications are referred to respectively. The whole of these is incorporated herein by reference. [Overview of the project] [Means for solving the problem]

[0002] In a particular embodiment, the wild-type signal regulatory protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment. Several implementations In this state, the wild-type SIRP-α D1 domain is one of the sequence numbers 1 to 10. It has columns. In some embodiments, the SIRP-α D1 domain is wild-type SIR For the P-α D1 domain, residues 6, 27, 31, 47, 53, remain 1 to 9 residues selected from the group consisting of group 54, residue 56, residue 66, and residue 92 Further amino acid mutations are included. In some embodiments, the SIRP-α D1 barrier The amino acid sequence is EEELQX1IQPDKSVLVAAGETATLRCTX2T SLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTV SDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSP Includes DDVEFKSGAGTELSVRAKPS (sequence number 49), where X1 is V X2 is A, I, V, or L, and X3 is I, F, S, or X4 is T, X5 is E, V, or L, X6 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 X 10 is V or I, and the SIRP-α D1 variant is of SEQ ID NO: 1 For the wild-type SIRP-α D1 domain having the sequence, at least two amino acids are added. It has substitution. In some embodiments, the SIRP-α D1 variant is represented by Sequence ID No. 7 It has one amino acid sequence of any of 8 to 85. In some embodiments, the SIRP -α D1 variant has the amino acid sequence EEELQX1IQPDKSVLVAAGETA TLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX 7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAG TYYCX 13KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 2 18), where X1 is V, L, or I, X2 is A, V, L, or I and X3 is I, S, T, or F, X4 is E, L, or V, X5 is K or R, X6 is E or Q, X7 is H, R, or P, X8 is S, G , L, or T, X9 is any amino acid, X 10 is any amino acid, X 11 is any amino acid, X 12 is any amino acid, X 13 is V or I and the SIRP-α D1 variant has at least two amino acid substitutions relative to the wild-type SIRP- α D1 domain having the sequence of SEQ ID NO: 1. In some embodiments , X9 is A. In some embodiments, X9 is N. In some embodiments , X 10 is I. In some embodiments, X9 is N and X10 is P . In some embodiments, X9 is N and X11 is any amino acid other than S, T, or C . In some embodiments, X 11 is T. In some embodiments , X 11 is an amino acid other than T. In some embodiments, X 12 is P. In some embodiments, X9 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 Includes VRAKPS (sequence number 219), where X1 is V, L, or I, and X2 X3 is A, V, L, or I, X4 is I, S, T, or F, and X4 is E, L, X5 is V, X6 is K or R, X7 is E or Q, and X7 is H, R, and is P, X8 is S, G, L, or T, X9 is N, X 10 Other than P It is an amino acid, X 11 is V or I, and the SIRP-α D1 variant is For a wild-type SIRP-α D1 domain having the sequence of sequence number 1, at least two It has the amino acid substitution. In some embodiments, the SIRP-α D1 variant is , amino acid sequence EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX 3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6T KRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKS Includes GAGTELSVRAKPS (sequence number 52), where 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, X7 is A, and the SIRP-α D1 variant has the sequence of sequence number 1, wild-type SI The RP-α D1 domain has at least two amino acid substitutions. In this embodiment, X1 is V or I, X2 is A or I, and X3 is I or F. Yes, X4 is K or R, X5 is H or P, X6 is L or T, X 7 is A. In some embodiments, the SIRP-α D1 variant is sequence number For the wild-type SIRP-α D1 domain having sequence 1, at least three amino acids It has acid substitution. In some embodiments, the SIRP-α D1 variant has sequence number For the wild-type SIRP-α D1 domain having sequence 1, at least four amino acids It has no acid substitution. In some embodiments, the SIRP-α D1 variant has a sequence For the wild-type SIRP-α D1 domain having sequence number 1, at least 5 A It has a amino acid substitution. In some embodiments, the SIRP-α D1 variant is distributed For a wild-type SIRP-α D1 domain having the sequence at column number 1, at least six It has amino acid substitutions. In some embodiments, the SIRP-α D1 variant is For a wild-type SIRP-α D1 domain having the sequence of sequence number 1, at least 7 It has the amino acid substitution. In some embodiments, X1 is I. In this state, X2 is I. In some embodiments, X3 is F. In this state, X4 is R. In some embodiments, X5 is P. In this state, X6 is T. In some embodiments, X1, X2, X3, X4, X5, and Each of X6 is not a wild-type amino acid. In some embodiments, the SIRP-α The D1 variant has one of the amino acid sequences from sequence numbers 81 to 85. In that embodiment, the SIRP-α D1 variant has the amino acid sequence EEELQX1I QPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPA RELIYNQX4EG X5FPRVTTVSEX6TKRENMDFSISISX7 ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS(distribution (including column number 212), where X1 is V, L, or I, and X2 is V, I, or L is X3 is I, T, S, or F, X4 is K or R, and X5 is H or P , or R, X6 is S, T, or G, X7 is A, and the SIRP-α The D1 variant is compared to the wild-type SIRP-α D1 domain which has the sequence of SEQ ID NO: 2. and has at least two amino acid substitutions. In some embodiments, the polypeptide K is used against human CD47. D Approximately 5x10 -9 Joins less than M. Several embodiments Then, the polypeptide further has an F attached to its N-terminus or C-terminus. It contains a c-domain monomer, and the Fc-domain monomer is human IgG1, IgG2, and This is the Fc region of IgG4. In some embodiments, the Fc domain monomer is a field At least one mutation in the Fc region of viable human IgG1, IgG2, or IgG4 It contains differences. In some embodiments, the polypeptide is SEQ ID NO: 135, SEQ ID NO: 13 Having one of the amino acid sequences of 6 or SEQ ID NO: 137. Several embodiments Therefore, the Fc domain monomer has the following amino acids relative to (a) 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, (b)(i) a mutation in the Fc region of human IgG1, (ii) human Mutations in the Fc region of IgG1, L234A, L235A, and G237A, (ii i) L234A, L235A, G237A, and N2 for the Fc region of human IgG1 (iv) Mutation of 97A in the Fc region of human IgG2, (v) (vi) Mutations in the Fc region of IgG2, A330S and P331S, (vi) human IgG Mutations A330S, P331S, and N297A in the Fc region of 2, (vii) S228P, E233P, F234V, L235A, and (viii) mutations in the delG236, or in the Fc region of human IgG4, S2 Mutations of 28P, E233P, F234V, L235A, delG236, and N297A This includes the Fc domain. In some embodiments, the Fc domain The monomer is (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 domain monomer further comprises (i) a mutation of N297A relative to the Fc region of human IgG1, (ii) mutations of L234A, L235A, and G237A relative to the Fc region of human IgG1, (iii) mutations of L234A, L235A, G237A, and N297A relative to the Fc region of human IgG1, (iv) a mutation of N297A relative to the Fc region of human IgG2, (v) mutations of A330S and P331S relative to the Fc region of human IgG2, (vi) mutations of A330S, P331S, and N297A relative to the Fc region of human IgG2, (vii) mutations of S228P, E233P, F234V, L235A, and delG236 relative to the Fc region of human IgG4, or (viii) mutations of S228P, E233P, F234V, L235A, delG236, and N297A relative to the Fc region of human IgG4. In some embodiments, the polypeptide shows a reduction in phagocytosis compared to a polypeptide having the Fc In one embodiment, the antibody variable domain targets an antigen expressed on the cell. In some embodiments, the cell is a cancer cell. In some embodiments, the antibody variable domain targets a cell surface protein involved in immune cell regulation. In some embodiments the additional polypeptide comprises a therapeutic protein. In some embodiments the therapeutic protein is a cytokine, interleukin, antigen, steroid, anti-inflammatory drug, or immunomodulatory drug. In some embodiments, the additional polypeptide comprises a SIRP-α D1 variant. In some embodiments, the polypeptide further comprises human serum albumin (HSA) (SEQ ID NO: 12). In some embodiments the HSA comprises an amino acid substitution of C34S or K573P relative to SEQ ID NO: 12 . In some embodiments, the polypeptide has an amino acid sequence of any one of SEQ ID NOs: 152-159 . In some embodiments, the polypeptide further comprises an albumin binding peptide. In some embodiments, the albumin binding peptide comprises the amino acid sequence of DICLPRWGCLW (SEQ ID NO: 160). In some embodiments the polypeptide further comprises a polyethylene glycol (PEG) polymer. In some embodiments, the PEG polymer is linked to the cysteine substitution of the polypeptide .

[0003] In certain embodiments, disclosed herein is a polypeptide that is a signal regulatory protein α (SIRP-α) D1 variant and has the amino acid sequence EX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX E 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 It contains KPS (sequence number 47), where X1 is E or G, and X2 is L. X3 is I or V, X4 is V, L or I, X5 is S or F, X5 is It is L or S, X6 is S or T, X7 is A or V, X8 is I or T is, X9 is H, R, or L, 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, X 15 is E or Q, and X 16 H, P, ma or is R, X 17 is D or E, X 18 is S, L, T, or G, X1 9 is K or R, X 20 is E or N, X 21 is S or P, X 22 is S or R, X 23 is S or G, X 24is any amino acid, X2 5 is any amino acid, X 26 is V or I, X 27 is F, L, or V ri, X 28 is either D or does not exist, X 29 is T or V, X 30 is F also is V, X 31 It is A or G and has one of the sequences with sequence numbers 1 to 10. The wild-type SIRP-α D1 domain has at least two amino acid substitutions. The SIRP-α D1 variant, as well as Fc having two Fc domain monomers It is an Fc variant containing a domain dimer, and each Fc domain monomer is (i) the Fc region of human IgG1 containing the N297A mutation, (ii) L234A, (iii) L234 The Fc region of human IgG1, including mutations A, L235A, G237A, and N297A, ( iv) Fc region of human IgG2 containing the N297A mutation, (v) A330S and P331 The Fc region of human IgG2 containing the S mutation, (vi) A330S, P331S, and N29 Human IgG2 Fc region containing the 7A mutation, (vii)S228P, E233P, F23 The Fc region of human IgG4 containing mutations in 4V, L235A, and delG236, or ( viii) S228P, E233P, F234V, L235A, delG236, and N A polypeptide containing the Fc variant, which is the Fc region of human IgG4 containing the 297A mutation. In some embodiments, the Fc domain dimer is the Fc domain monomer. One of them is human Ig, which includes mutations L234A, L235A, G237A, and N297A. It includes the Fc region of G1. In some embodiments, the polypeptide has an amino acid sequence of any one of SEQ ID NOs: 98 to 1 04, 107 to 113, 116 to 122, or 135 to 137. In some embodiments, the Fc variant shows removal or reduction of binding to Fcγ receptors as compared to the wild-type version of the Fc region of human IgG. In some embodiments, the Fc variant of IgG1 or IgG2 shows removal or reduction of binding to Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared to the wild-type version of the Fc region of human IgG1 or IgG2. In some embodiments, the Fc variant of IgG4 shows removal or reduction of binding to Fcγ receptors CD16a and CD32b as compared to the wild-type version of the Fc region of human IgG4. In some embodiments, the Fc variant of IgG1 or IgG2 shows removal or reduction of binding to C1q as compared to the wild-type version of the Fc fusion of human IgG1 or IgG2. In some embodiments, the Fc variant binds to Fcγ receptors at approximately 5x1 0 M or more. In certain embodiments, disclosed herein is a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, and each Fc domain monomer independently has (i) the Fc region of human IgG1 consisting of the mutations L234A, D L235A, G237A, and N297A, (ii) the Fc region of human IgG2 consisting of the mutations A330S, P331S, and N297A. -6

[0004] In certain embodiments, disclosed herein is a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, and each Fc domain monomer independently has (i) the Fc region of human IgG1 consisting of the mutations L234A, L235A, G237A, and N297A, (ii) the Fc region of human IgG2 consisting of the mutations A330S, P331S, and N297A, (ii) Or (iii) S228P, E233P, F234V, L235A, delG236, and polypeptides selected from the Fc region of human IgG4, including the N297A mutation. In some embodiments, at least one of the Fc domain monomers is L234A, The Fc region of human IgG1 consists of mutations L235A, G237A, and N297A. In some embodiments, at least one of the Fc domain monomers is A330S, This is the Fc region of human IgG2 consisting of mutations P331S and N297A. In this embodiment, the Fc variant is compared to the wild-type version of the Fc region of human IgG with Fcγ This indicates the removal or reduction of binding to the receptor. In some embodiments, the Fc Varian Compared to the wild-type version of the Fc region of human IgG, CD16a, CD32a, CD32 This indicates the removal or reduction of binding of b, CD32c, and CD64 to the Fcγ receptor. In some embodiments, the Fc variant is compared to the wild-type version of the human IgG Fc fusion. This indicates the removal or reduction of the bond to C1q. In some embodiments, the Fc dome At least one of the in monomers is S228P, E233P, F234V, L235A, This is the Fc region of human IgG4 containing mutations in delG236 and N297A. In this embodiment, the Fc variant is compared to the Fc region of wild-type human IgG4. This indicates the removal or reduction of binding to the γ receptor. In some embodiments, the Fc barrier The test compared the wild-type version of the Fc region of human IgG4 with CD16a and CD32b This indicates the removal or reduction of binding to the Fcγ receptor. In some embodiments, the Fc The variant has K for the Fcγ receptor. D Approximately 5x10-6 They are joined in a greater than M size. In some embodiments, the polypeptide further comprises a CD47-conjugated polypeptide. In this embodiment, the Fc variant is compared to the wild-type version of the Fc region of human IgG, This indicates the removal or reduction of binding to the cγ receptor. In some embodiments, the CD47 The conjugated polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the CD47-binding polypeptide induces acute anemia in humans. It does not cause a signal control. In some embodiments, the CD47-binding polypeptide controls the signal control. This is a protein α(SIRP-α) polypeptide or a fragment thereof. Several embodiments Therefore, the SIRP-α polypeptide has the amino acid sequence EEELQX1IQPDKSVLV AAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQ X5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGT YYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (Sequence ID 51) Includes a SIRP-α D1 variant, where X1 is V or I and X2 is X3 is A or I, X4 is I or F, X5 is E or V, X5 is K or R is, X6 is H or P, X7 is L or T, and X8 is any A other than N. It is a sine acid, and X9 is V or I. In some embodiments, the SIRP-α poly The peptide is such that X1 is V or I, X2 is A or I, and X3 is I or F. Furthermore, X4 is E, X5 is K or R, X6 is H or P, and X7 is L. It includes a SIRP-α D1 variant where is T, X8 is not N, and X9 is V.

[0005] In a particular embodiment, the following is disclosed herein: a polypeptide, which is sig It is a non-spontaneously occurring high-level regulatory protein α(SIRP-α)D1 variant. It is an affinity SIRP-α D1 domain, and has less affinity than naturally occurring D1 domains. The SIRP-α D1 variant, which binds to human CD47 with 10 times higher affinity, and It is an Fc domain monomer, and also a second monomer containing a second Fc domain monomer. F is linked to a ptide to form an Fc domain, and the Fc domain is removed or reduced. This polypeptide contains the Fc domain monomer having an enzyme function. In the application form, the non-naturally occurring high-affinity SIRP-α D1 domain has an amino acid at residue 80. Includes acid mutations.

[0006] In a particular embodiment, the signal regulatory protein α( A polypeptide comprising the SIRP-α)D1 variant, wherein the SIRP-α D1 variant Ant, K D Binds to CD47 derived from the first species at a M of less than 250 nM, and the SIRP-α D One variant is K D It binds to CD47 derived from the second species at a M of less than 250 nM, and the first K for CD47 of species origin D And, K for CD47 derived from the second species D each The ratio is within 00, and the first and second species are derived from humans, rodents, and non-human primates. The polypeptide is selected from the group. In some embodiments, the SIRP-α The D1 variant binds to CD47 from at least three different species. In terms of morphology, the non-human primate in question is the crab-eating macaque.

[0007] In a particular embodiment, the following is disclosed herein: a polypeptide, (a )K D Signal regulatory protein α (SIRP-) binds to human CD47 at a M of less than 250 nM. α) D1 domain, and (b) connected to the N-terminus or C-terminus of the SIRP-α D1 domain It contains a linked Fc domain monomer and causes acute anemia in rodents and non-human primates. It is a polypeptide that does not cause seroconjugation. In some embodiments, the polypeptide is human S It is a non-spontaneously occurring variant of IRP-α. In some embodiments, the polypeptide In vivo administration results in a hemoglobin reduction of less than 50% during the first week after administration. In some embodiments, administration of the polypeptide to humans resulted in a 50% reduction in the first week after administration. This results in a decrease in hemoglobin levels. In some embodiments, the polypeptide further (i)L2 The Fc region of human IgG1 consisting of mutations 34A, L235A, G237A, and N297A. (ii)Fc of human IgG2 consisting of mutations in region, A330S, P331S and N297A Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A. In the embodiment, the Fc variant is L234A, L235A, G237A, and N29 This is the Fc region of human IgG1 consisting of the 7A mutation. In some embodiments, the Fc region Rianto is a human IgG2 F2 variant consisting of mutations A330S, P331S, and N297A. This is the c region. In some embodiments, the Fc variant is S228P, E233P Human IgG4 including mutations F234V, L235A, delG236, and N297A This is the Fc region.

[0008] In certain embodiments, the following is disclosed herein: an individual having a disease or disorder A method for treating a subject, comprising administering a polypeptide disclosed herein to the subject. This is the method. In some embodiments, the polypeptide is a wild-type signal-regulating protein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or Includes a SIRP-α D1 variant containing a fragment. In some embodiments, the polyp Chido contains the signal regulatory protein α(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) and wherein X1 is E or G, X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, X5 is L or S, X6 is S or T, X7 is A or V, X8 is I or T, X9 is H, R, or L, X 10 is A, V, I, or L, X 11 is I, T, S, or F and X 12 is A or G, X 13 is E, V, or L, X 14 is K or R, X 15 is E or Q, X 16 is H, P, or R, X 17 is D[[ID=J]] or E, X 18 is S, L, T, or G, X 19 is K or R, X2 0 is E or N, X 21 is S or P, X 22 is S or R, X 23 is S or G, X 24 is any amino acid, X 25 is any amino acid and X 26 is V or I, X 27 is F, L, or V, X 28 is D or does not exist, X 29 is T or V, X 30 is F or V, X 31 is A or G, and the SIRP-α D1 variant has at least two amino acids relative to the wild-type SIRP-α D1 domain having any one of the sequences of SEQ ID NOs: 1 to 10 The polypeptide has acid substitution and the polypeptide has two Fc domain monomers. It contains an Fc variant including an indimer, where each Fc domain monomer is independently (i) the Fc region of human IgG1 containing the N297A mutation, (ii) L234A, L2 (iii)L234A, the Fc region of human IgG1 including mutations 35A and G237A, The Fc region of human IgG1, including mutations in L235A, G237A, and N297A, (iv ) The Fc region of human IgG2 containing the N297A mutation, (v) A330S and P331S The Fc region of human IgG2 containing mutations, (vi)A330S, P331S, and N297A The Fc region of human IgG2 containing the following mutations: (vii)S228P, E233P, F234V The Fc region of human IgG4, including mutations in L235A and delG236, or (vi ii) S228P, E233P, F234V, L235A, delG236, and N29 This is the Fc region of human IgG4 containing the 7A mutation. In some embodiments, the polyp The drug contains an Fc variant, and the Fc variant has two Fc domain mono It contains an Fc domain dimer having a monomer, and each Fc domain monomer is independently (i ) Human IgG1 consisting of mutations L234A, L235A, G237A, and N297A Human IgG consisting of mutations in the Fc region, (ii)A330S, P331S, and N297A Fc region of 2, or (iii) S228P, E233P, F234V, L235A, d The mutations are selected from the Fc region of human IgG4, including the elG236 and N297A mutations. In some embodiments, the polypeptide is a signal-regulating protein α (SIRP-α) It is a D1 variant and a non-naturally occurring high-affinity SIRP-α D1 domain. Furthermore, it has at least 10 times higher affinity for human CD47 than the affinity of naturally occurring D1 domains. The SIRP-α D1 variant and Fc domain monomer to be bound, It is linked to a second polypeptide containing a second Fc domain monomer to form an Fc domain. and the Fc domain has an effector function in which the Fc domain is removed or reduced. Contains nomer. In some embodiments, the non-spontaneously occurring high affinity SIRP-α D1 The main molecule contains an amino acid mutation at residue 80. In some embodiments, the polypeptide This includes the signal regulatory protein α(SIRP-α)D1 variant, and the SIRP-α The D1 variant is K D The SIR binds to CD47 derived from the first species at a M of less than 250 nM. The P-α D1 variant is K D It binds to CD47 from the second species at a M of less than 250 nM, Furthermore, K for CD47 derived from the first species D And, K for CD47 derived from the second species D They are no more than 100 times each other, and the first and second species are humans, rodents, and non-human spirits. Selected from the group consisting of long species. In some embodiments, the polypeptide is (a)K D Signal regulatory protein α (SIRP-α) that binds to human CD47 at a M of less than 250 nM. (b) a D1 domain, and (b) a domain attached to the N-terminus or C-terminus of the SIRP-α D1 domain The polypeptide contains a modified Fc domain monomer, and the polypeptide is found in rodents and non-human primates. It does not cause acute anemia in the same class. In some embodiments, the disease or disorder is Cancer, autoimmune disease, or inflammatory disease. In some embodiments, the disease or The disorder is cancer, and the cancer is a solid tumor cancer, hematological cancer, acute myeloid leukemia, chronic phosphorus cancer. Passive-type leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin's Pancreas, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, trachea Cancers of the bronchial tuberculosis, liver cancer, ovarian cancer, colorectal cancer, stomach cancer ER, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer Head and neck cancer, oral and pharyngeal 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 malignancies, gliomas, brain cancer, and epithelial malignancies Selected from. In some embodiments, the disease or disorder is an autoimmune disease or inflammation. It is a symptomatic disease, and such autoimmune or inflammatory diseases include multiple sclerosis, rheumatoid arthritis, spinal cord disease. Spondyloarthritis, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune diseases, graft pairs Host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjögren's syndrome, progressive Systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn's disease, endometriosis, glomeruli Nephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis , and inflammatory autoimmune myositis are selected. In some embodiments, the SIRP-α The D1 variants are sequence numbers 78-85, 98-104, 107-113, and 116-12. 2, has one of the sequences 135-137 or 152-159. In embodiments, the method further includes administering at least one additional agent. In some embodiments, the at least one further agent is an antibody, a tumor-associated antigen, or non-antibody therapeutic agents. In some embodiments, at least two further agents It is administered. In some embodiments, the at least two further agents are two antibodies This includes, in some embodiments, the at least two further agents are antibodies and tumor-related. Contains linked antigens. In some embodiments, the at least one further agent is an antibody. In some embodiments, the antibody is a human IgG1 isotype antibody. In that embodiment, the antibody is a human IgG2 isotype antibody. Several embodiments In this state, the antibody is a human IgG4 isotype antibody. In some embodiments, The antibodies include 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 selected from anti-VEGFR2 antibodies. In some embodiments, the antibody is anti-H ER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-PD Selected from -1 antibody, anti-RANKL antibody, or anti-PD-L1 antibody. Several implementations In form, the at least one further agent is at least one antibody, and the antibody is Cetuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, ME DI0680, MED16469, atezolizumab, avelumab, durvalumab, ME DI6383, RG7888, Ipilimumab, Tremelimumab, Urelumab, PF-05 082566, enoblituzumab, vanchikutsumab (v antictumab, varlilumab, mogamalizumab (m ogamalizumab), SAR650984, daratumumab, trastuzumab, to Lastuzumab emtansine, pertuzumab, elotuzumab, li Tuximab, ofatumumab, obinutuzumab, RG7155, FPA008, panitum Mab, Brentuximab Vedotin, MSB0010718C, Belimumab, Bevacizumab Denosumab, panitumumab, ramucirumab, nesitumumab, nivolumab, pembroliz Mab, avelumab, atezolizumab, durvalumab, MEDI0680, pidilizumab Selected from B or BMS-93659. In some embodiments, the antibody is tra It is sutuzumab. In some embodiments, the SIRP-α D1 variant is sequence Numbers 78-85, 98-104, 107-113, 116-122, 135-137, It has one of the sequences 152 to 159. In some embodiments, the antibody It is rituximab. In some embodiments, the SIRP-α D1 variant is distributed Row numbers 78-85, 98-104, 107-113, 116-122, 135-137, or having one of the sequences 152 to 159. In some embodiments, the antibody is cetuximab. In some embodiments, the SIRP-α D1 variant is Sequence numbers 78-85, 98-104, 107-113, 116-122, 135-137 , or has one of the sequences 152-159. In the application form, the antibody is daratumumab. In some embodiments, the SIRP-α The D1 variants are sequence numbers 78-85, 98-104, 107-113, and 116-12. 2, has one of the sequences 135-137 or 152-159. In some embodiments, the antibody is belimumab. In some embodiments, the SIRP-α The D1 variants are sequence numbers 78-85, 98-104, 107-113, and 116-12. 2, has one of the sequences 135-137 or 152-159. In some embodiments, the antibody is bevacizumab. In some embodiments, the SIRP- The α D1 variant is sequence numbers 78-85, 98-104, 107-113, 116- It has one of the following sequences: 122, 135-137, or 152-159. How many? In one embodiment, the antibody is denosumab. In some embodiments, the SIRP- The α D1 variant is sequence numbers 78-85, 98-104, 107-113, 116- It has one of the following sequences: 122, 135-137, or 152-159. How many? In one embodiment, the antibody is pantimumab. In this embodiment, the SIRP-α D1 variant is sequence numbers 78-85, 98-104 107-113, 116-122, 135-137, or 152-159 It has one sequence. In some embodiments, the antibody is ramucirumab. In this embodiment, the SIRP-α D1 variant is sequence numbers 78-85, 98-10 4. Any of the following: 107-113, 116-122, 135-137, or 152-159 It has one sequence. In some embodiments, the antibody is nesitumumab. In that embodiment, the SIRP-α D1 variant is sequence numbers 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 It has one of the sequences. In some embodiments, the antibody is nivolumab. In that embodiment, the SIRP-α D1 variant is sequence numbers 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 It has one of the sequences. In some embodiments, the antibody is pembrolizumab. In some embodiments, the SIRP-α D1 variant is represented by sequence numbers 78-85, 9 8-104, 107-113, 116-122, 135-137, or 152-159 It has one of the following sequences. In some embodiments, the antibody is avelumab. In some embodiments, the SIRP-α D1 variant is represented by sequence numbers 78-85, 9 8-104, 107-113, 116-122, 135-137, or 152-159 It has one of the sequences. In some embodiments, the antibody is atezolizumab. In some embodiments, the SIRP-α D1 variant is represented by sequence numbers 78-85. , 98-104, 107-113, 116-122, 135-137, or 152-1 It has any one of the sequences 59. In some embodiments, the antibody is durvalumab In some embodiments, the SIRP-α D1 variant is represented by sequence number 78~ 85, 98-104, 107-113, 116-122, 135-137, or 152 It has one of the sequences ~159. In some embodiments, the antibody is MEDI0 It is 680. In some embodiments, the SIRP-α D1 variant is sequence number 78-85, 98-104, 107-113, 116-122, 135-137, or It has one of the sequences 152 to 159. In some embodiments, the antibody is Pide It is ilizumab. In some embodiments, the SIRP-α D1 variant is sequence Numbers 78-85, 98-104, 107-113, 116-122, 135-137, It has one of the sequences 152 to 159. In some embodiments, the antibody This is BMS-93659. In some embodiments, the SIRP-α D1 variant This includes sequence numbers 78-85, 98-104, 107-113, 116-122, 135-1 It has one of the following arrays: 37 or 152-159. In some embodiments, The at least one further agent is a tumor-associated antigen, which triggers an immune response. It induces. In some embodiments, the at least one further agent is an antibody. The antibody targets HLA / peptide or MHC / peptide complexes. In the embodiment, the antibody is 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 laminaria Nin receptor, MOK / RAGE-1, WT-1, Her2 / neu, EphA3, SAP -1, BING-4, Ep-CAM, MUC1, PRAME, Survivalbin, Mesothelin, BRCA1 / 2 (mutant), CDK4, CML66, MART-2, p53 (mutant), Ra s (mutation), β-catenin (mutation), TGF-βRII (mutation), HPV E6, or It targets HLA / peptide or MHC / peptide complexes containing E7. In the embodiment, the antibody is ESK1, RL1B, Pr20, or 3.2G1. [Brief explanation of the drawing]

[0009] The novel features of the present invention are specifically described in the appended claims. Features and benefits of the present invention A better understanding of the points can be found in the exemplary embodiments utilizing the principles of the present invention and the following in the accompanying drawings. This can be obtained by referring to the detailed explanation. [Figure 1] This figure shows a SIRP-α construct containing a SIRP-α D1 domain or a variant thereof, linked to a first Fc domain monomer that forms an Fc domain together with a second Fc domain monomer. [Figure 2] This figure shows a SIRP-α construct comprising a SIRP-α D1 domain or a variant thereof linked to a first Fc domain monomer and an antibody variable domain linked to a second Fc domain monomer, wherein the first Fc domain monomer and the second Fc domain monomer bind to form an Fc domain. [Figure 3] This figure shows a SIRP-α construct comprising a SIRP-α D1 domain or a variant thereof linked to a first Fc domain monomer and a therapeutic protein linked to a second Fc domain monomer, wherein the first Fc domain monomer and the second Fc domain monomer bind to form an Fc domain. [Figure 4]Figure 4A shows a SIRP-α construct containing a SIRP-α D1 domain or a variant thereof, linked to a first Fc domain monomer having a knob mutation, which forms an Fc domain together with a second Fc domain monomer having a hole mutation. Figure 4B shows a SIRP-α construct containing a SIRP-α D1 domain or a variant thereof, linked to a first Fc domain monomer having a hole mutation, which forms an Fc domain together with a second Fc domain monomer having a knob mutation. [Figure 5] Figure 5A shows a SIRP-α construct containing a SIRP-α D1 domain or a variant thereof linked to an Fc domain monomer. Figure 5B shows a SIRP-α construct that is a homodimer of the construct shown in Figure 5A. [Figure 6] SPR binding data for monofunctional and bifunctional SIRP-α constructs containing the SIRP-α D1 domain are shown. [Figure 7] This study demonstrates the phagocytic activity of human monocyte-derived macrophages on DLD-1-GFP-luciferase tumor cells in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 8] This study demonstrates the phagocytic activity of human monocyte-derived macrophages on DLD-1-GFP-luciferase tumor cells in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 9] This study demonstrates the phagocytic activity of human monocyte-derived macrophages on DLD-1-GFP-luciferase tumor cells in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 10] This demonstrates the stability of the SIRP-α polypeptide's half-life over a certain period of time. [Figure 11] The hemagglutination assay data for the SIRP-α polypeptide construct are shown. [Figure 12] The survival curves of mouse syngeneic tumor models treated with SIRP-α polypeptide constructs and anti-mPD-L1 are shown. [Figure 13]This shows tumor volume analysis of a mouse syngeneic tumor model treated with a SIRP-α polypeptide construct in combination with anti-mPD-L1. [Figure 14] This shows the binding of C1q complement to SIRP-α-Fc fusion products at various concentrations. [Figure 15] This study demonstrates the phagocytic activity of MM1R cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 16] This study demonstrates the phagocytic activity of MM1R cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 17] This study demonstrates the phagocytic activity of N87 cells by human monocyte-derived macrophages in the presence of various concentrations of SIRP-α polypeptide constructs. [Figure 18] The molecular weight analysis of the SIRP-α D1 variant with the P83V mutation is shown. [Figure 19A] This shows tumor growth of human GFP-Luc-Raji lymphoma cells in a NOD scid gamma (NSG) mouse model of cancer treated with various SIRP-α constructs, with or without rituximab. [Figure 19B] Figure 19A shows a scatter plot of the tumor volume of the tumor. [Figure 19C] Figure 19A shows the hemoglobin levels of the treated mice. [Figure 20] This shows the number of red blood cells collected from mice treated with either the SIRP-α wild-type IgG1 Fc construct or the SIRP-α IgG1 Fc variant construct. [Modes for carrying out the invention]

[0010] definition The terms "about" or "approximately" refer to a range of tolerances for a particular value determined by those skilled in the art. This means that the method of measuring or determining a value, i.e., the limits of the measurement system, are It depends partially. For example, "about" means that for the implementation of the technology, one or more standards are applied. It can mean within the difference. Alternatively, "approximately" can mean up to 20% of a given value, up to 10% It can mean a range of %, up to 5%, or up to 1%. Alternatively, especially in biological systems Or, in relation to biological processes, the term means within 10 times the value, preferably within 5 times, more preferably "Mashiku" can mean up to twice the original amount. Unless otherwise stated, this application and the claims. When a specific value is given within a range, the term "approximately" means the acceptable error for that specific value. It is assumed that this means "within the range".

[0011] The terms used herein are for illustrative purposes only and are not limited to any specific context. This is not intended to be the case. The singular forms "a", "an", and " Unless the context clearly indicates otherwise, "the" is intended to include plurals as well. Furthermore, the terms "include" and "includes" are also included. "having," "has," "accompany," or variations thereof are detailed. To the extent that such terms are used in detailed descriptions or claims, such terms are "comprising It is intended to be inclusive, similar to the term "g (including)".

[0012] As used herein, the term "antibody" refers to an intact antibody, an antibody fragment (with desired biological activity (e.g.) For example, monoclonal antibodies and polyclonal antibodies (under the condition that they exhibit epitope binding). Body, monospecific antibody, multispecific antibody (e.g., bispecific antibody), and antibody-like protein It refers to quality.

[0013] As used herein, the term "antibody variable domain" refers to a complementarity-determining region (CDR, for example) For example, CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and C The light chain of the antibody containing the amino acid sequence of DR H3) and the framework region (FR) This refers to the heavy chain portion.

[0014] As used herein, the term "linker" refers to two elements, for example, protein domains. It refers to the bond between two elements. In some embodiments, the linker is a covalent bond or a spacer. There is a spacer. The term "spacer" refers to a space between two polypeptides or polypeptide domains. To give it space or flexibility (or both space and flexibility), two polyp The moiety that forms between the 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. (For example, a polypeptide or polypeptide spaced apart via the polypeptide skeleton) (Connects to the domain).

[0015] As used herein, the term “therapeutic dose” refers to a disease, e.g., cancer, e.g., solid tumor. In the treatment of patients with ulcers or blood cancers, sufficient and effective in achieving the desired therapeutic effect. The efficacy of the polypeptide or the pharmaceutical composition comprising the polypeptide described herein, for example This refers to the amount of polypeptides having the SIRP-α D1 domain or its variants. In some embodiments, a therapeutically effective amount of polypeptide is used to avoid adverse side effects.

[0016] As used herein, the term "pharmaceutical composition" refers to the active ingredient and excipients or diluents. (or both excipients and diluents) so that the active ingredient is administered in an appropriate manner. This refers to a pharmaceutical prescription or formulation prescription. In some embodiments, the medical The pharmaceutical composition contains pharmaceutically acceptable components compatible with the polypeptide. Several implementations In terms of form, the pharmaceutical composition may be in the form of tablets or capsules for oral administration, or, for example, It is an aqueous form for intravenous or subcutaneous administration by injection.

[0017] As used herein, the terms “subject,” “individual,” and “patient” are to be used interchangeably. This refers to vertebrates, such as mammals. Examples of mammals include mice, monkeys, humans, livestock, and sports. This includes, but is not limited to, animals and pets. It is also obtained in vivo. This also includes tissues, cells, and their offspring of biological entities cultured in vitro. None of these terms require guidance from a medical professional.

[0018] As used herein, the terms "affinity" or "binding affinity" refer to the mutual binding between two molecules. This refers to the strength of the effect. Generally, binding affinity refers to the relationship between a molecule and its binding partner, for example, high affinity. This refers to the total strength of the non-covalent interaction between the sex SIRP-α D1 variant and CD47. Unless otherwise specified, binding affinity is opposed to the 1:1 interaction between members of a binding pair. This refers to the intrinsic binding affinity. The binding affinity between two molecules is usually expressed by the dissociation constant (K). D )Also The association constant (K A This is shown by: Two molecules that have low binding affinity to each other, Generally, they tend to bond slowly and dissociate easily, and have high K D This indicates that they are high relative to each other. Two molecules with affinity generally tend to bind easily and remain bound for longer periods. small K D This shows that in some embodiments, the K of two interacting molecules D is known Methods and techniques, such as surface plasmon resonance (SPR), are used for measurement. D is, k off / k on It can be calculated as a ratio of .

[0019] The term "~less than K" as used in this specification. D " is the K listed D For a value, number A small K in terms of value D This refers to the value and increasing binding affinity. As used herein, the term "~yo" Large K D " is the K listed D A numerically large K relative to the value D Value and decreasing combination It refers to affinity.

[0020] As used herein, the term “acute anemia” refers to the first five days after administration or treatment of the compound. This refers to a 30% decrease in red blood cell count or hemoglobin.

[0021] I. Signal regulatory protein α(SIRP-α)D1 domain and its variants In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0022] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 The polypeptide selected from the Fc region of human IgG4, including mutations 36 and N297A It is Do.

[0023] Signal regulatory protein α ("SIRP-α" or "SIRP-alpha") is involved in bone It is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed in the membranes of myelin cells. SIRP-α interacts with CD47, a protein widely expressed in many cell types in the body. They interact. The interaction between SIRP-α and CD47 is otherwise not recognized by the immune system. It prevents the phagocytosis of "self" cells that can be engulfed. High expression of CD47 in tumor cells is associated with acute bone marrow In sexually transmitted leukemia and some solid tumor cancers, it acts as a negative prognostic factor for survival. It is permitted to obtain it.

[0024] Natural SIRP-α consists of three highly homologous immunoglobulin (Ig)-like extracellular domains. , that is, including D1, D2, and D3. The SIRP-α D1 domain ("D1 domain") The term "in" refers to the distal membrane portion of SIRP-α, i.e., the extracellular domain, and is opposed to CD47. It mediates the binding of SIRP-α. The term "SIRP-α polypeptide" as used herein "D" refers to any SIRP-α polypeptide or its cleavage capable of binding to CD47. It refers to a single variant. Wild-type human SIRP-α has at least 10 variants. Table 1 shows: The D1 domain of the 10 naturally occurring wild-type human SIRP-α D1 domain variants The amino acid sequences of yin are shown (SEQ ID NOs: 1-10). In some embodiments, SIRP- The α polypeptide contains the SIRP-α D1 domain. In some embodiments, SI RP-α polypeptides have a wild-type D1 domain, for example, as shown in SEQ ID NOs: 1-10. This includes the following. In some embodiments, the SIRP-α polypeptide is wild-type human SIRP. -Includes the D2 or D3 domain of α (or both D2 and D3 domains) (Table 3) . [Table 1-1] [Table 1-2] [Table 1-3]

[0025] The terms used herein are "high affinity SIRP-α D1 variant" and "high affinity SIRP-α D1 variant". "SIRP-α variant" or "SIRP-α D1 variant" is SIRP-α The D1 domain, or the CD47 binding portion of the SIRP-α polypeptide, contains CD47 This refers to polypeptides with higher affinity for wild-type SIRP-α. The α D1 variant has at least one amino acid substitution compared to wild-type SIRP-α. Includes deletions or insertions (or a combination thereof).

[0026] In some embodiments, the high affinity SIRP-α D1 variant disclosed herein This includes the SIRP-α D1 domain or a variant thereof. In some embodiments, The high-affinity SIRP-α D1 variant is the wild-type D1 variant shown in SEQ ID NOs: 1-10. The main molecule contains one or more amino acid substitutions, insertions, additions, or deletions. Table 2 shows each Exemplary amino acids in SIRP-α D1 domain variants (SEQ ID NOs: 13-22) Acid substitution is shown. In some embodiments, the SIRP-α D1 domain polypeptide Alternatively, high-affinity SIRP-α D1 variants contain fragments of the D1 domain. In this embodiment, a fragment of the SIRP-α polypeptide or a high-affinity SIRP-α barrier is used. The fragments of the nucleotide are less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, and about 30 amino acids, length approximately 40 amino acids, length approximately 50 amino acids, length approximately 60 amino acids, length approximately 70 amino acids, length approximately 80 amino acids, length approximately 90 amino acids, length approximately 100 amino acids, and It contains an amino acid sequence of more than approximately 100 amino acids in length. In some embodiments, the SIRP -α The D1 domain fragment retains its ability to bind to CD47.

[0027] In some embodiments, the poly(P) of the Disclosure includes a high affinity SIRP-α D1 variant. The peptide binds to CD47 with a higher binding affinity than the wild-type human SIRP-α D1 domain. In some embodiments, the high affinity SIRP-α D1 variant is naturally At least 1x the affinity of the existing D1 domain (for example, at least 1.5x, 2x, It binds to human CD47 with affinity (2.5 times, 3 times, 3.5 times, 4 times, 5 times, or more than 5 times). In some embodiments, the high-affinity SIRP-α D1 variant is found in nature. The affinity of the existing D1 domain is at least 1 (for example, at least 10 times, 100 times, It binds to human CD47 with an affinity of 1000 times or more.

[0028] As used herein, the terms “optimization affinity” or “optimization coupling affinity” mean high affinity Polypeptides disclosed herein, including the sexual SIRP-α D1 variant, and CD47 This refers to the optimized bond interaction strength of the polyp Butido primarily binds to CD47 on cancer cells, or with higher affinity, and to non-cancer cells. It either does not bind to CD47 in cells at all, or binds with a lower affinity. In some embodiments, the binding affinity between the polypeptide and CD47 is such that the interaction is appropriate. Variants that do not cause floor-significant toxicity or bind with maximum affinity. It is optimized to reduce toxicity compared to [another method]. In some embodiments, as specified herein To achieve optimal binding affinity between the polypeptide to be provided and CD47, high affinity SIR The polypeptide containing the P-α D1 variant is maximally achievable for CD47. It is developed to have a lower binding affinity than the present invention. In some embodiments, The high-affinity SIRP-α variant disclosed in the detailed document is used in rodents and non-human primates (NHPs). It also cross-reacts with human CD47.

[0029] As used herein, the term "immunogenicity" refers to a state in which a host reacts to an antigen as if it were an exogenous antigen. This refers to the properties of proteins that trigger an immune response (e.g., therapeutic proteins). The immunogenicity of proteins can be determined by various methods, such as in vitro T cell proliferation assays. It can be used for assays with Toro.

[0030] As used herein, the term "minimal immunogenicity" refers to, for example, the absence of amino acid substitutions. , the immunogenicity is lower than that of the protein before the amino acid substitution was introduced (e.g., the unmodified protein). For example, modified to be at least 10%, 25%, 50%, or 100% lower. This refers to the immunogenicity of a protein (e.g., a therapeutic protein). In some embodiments, this refers to the immunogenicity of a protein. Proteins (e.g., therapeutic proteins) are modified to have minimal immunogenicity. Therefore, even if it is an exogenous antigen, it does not trigger any or very little immune response from the host. .

[0031] In some embodiments, the high-affinity SIRP-α D1 variant provides minimal immunity. It has a toxic effect. In some embodiments, the SIRP-α-polypeptide of the Disclosure is administered to a subject. Butido eliminates the amino acid changes that increase the affinity of the SIRP-α D1 variant. Furthermore, the same amino acid sequence as that of the SIRP-α polypeptide in the target biological sample is obtained. It has. In some embodiments, the polypeptide variants disclosed herein are anti It reduces the risk of side effects compared to CD47 antibodies or wild-type SIRP-α. In that embodiment, the polypeptide variant disclosed herein is an anti-CD47 antibody Alternatively, it reduces the risk of anemia compared to wild-type SIRP-α. In some embodiments, The polypeptide variants disclosed herein are rodents or non-human primates (NH P) In the trial, it did not cause acute anemia.

[0032] Table 2 shows the high affinity SIRP-α D1 variants for each D1 domain sequence. It exhibits specific amino acid substitutions. In some embodiments, high affinity SIRP-α D1 A riant is one or more of the substitutions shown in Table 2 (for example, 2, 3, 4, 5, 6, 7, 8, 9, 1 Including 0, 11, 12, 13, 14, or more. In some embodiments, The high-affinity SIRP-α D1 variant has up to 14 atypical features compared to the wild-type D1 domain. Includes mino acid substitution. In some embodiments, the high affinity SIRP-α D1 variant is , including up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, The high-affinity SIRP-α D1 variant has up to 7 properties relative to the wild-type D1 domain. This includes amino acid substitutions. In some embodiments, the high affinity SIRP-α D1 of the present disclosure The variant is at least 90% (for example, less) of the wild-type D1 domain sequence. They all have amino acid sequence identity of 92%, 95%, 97%, or more than 97%.

[0033] In some embodiments, the high affinity SIRP-α D1 variant is found in two or more wild-type variants. A type D1 domain or a portion of its variant (for example, one wild-type D1 domain or (including the variant portion and another wild-type D1 domain or its variant portion) It is a chimeric high-affinity SIRP-α D1 variant. In some embodiments, it is a chimeric The high affinity SIRP-α D1 variant is a wild-type D1 domain or a variant thereof. It includes at least two parts (for example, three, four, five, or more parts), and the part Each of these is derived from a different wild-type D1 domain. In some embodiments, the chimeric high parentage is used. The Japanese SIRP-α D1 variant further contains 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 (Sequence ID 13), where X1 is L, I, and X2 is V, X3 is V, L, or I, X4 is A or V, X4 is A, I or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is L, T, or G, X 11 is K or R, X 12 is V or I , X 13is F, L, or V, and X 14 SIRP- has a sequence that is F or V The α D1 variant is included, and the variant has the sequence of SEQ ID NO: 1, which is a wild-type SIRP. - The D1 domain has at least one amino acid substitution.

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

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

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

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

[0039] In any of the above embodiments, the polypeptide is sequence numbers 13, 16-18, and A high affinity SI having one of the 21 sequences where X1 is L, I, or V. Includes RP-α D1 variant. In any of the above embodiments, X2 is V, L, or is I. In any of the above embodiments, X3 is A or V. The above embodiment In any of the above embodiments, X4 is A, I, or L. X5 is I, T, S, or F. In any of the above embodiments, X6 is E, It is V or L. In any of the above embodiments, X7 is K or R. In any of the embodiments, X8 is E or Q. X9 is H, P, or R. In any of the above embodiments, X 10 L, T , or G. In any of the above embodiments, X 11 is either K or R. In any of the embodiments, X 12 is V or I. And, X 13 are F, L, and V. In any of the above embodiments, X 14 is F or V is the polypeptide of this embodiment of the present disclosure, SEQ ID NOs: 1, 4 For wild-type SIRP-α D1 domains having one of sequences ~6 and 9 , containing six or fewer amino acid substitutions.

[0040] In some embodiments, the polypeptide is one of SEQ ID NOs: 1, 4-6, and 9. At least 10 times higher binding parentage than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 in a saturating manner. In some embodiments, the polypeptide is sequence number 1, Fewer than the wild-type SIRP-α D1 domain which has one of sequences 4-6 and 9. At the very least, it binds to CD47 with a binding affinity 100 times higher. In some embodiments, the port The lipeptide is a wild-type SIR having one of the sequences of SEQ ID NOs: 1, 4-6, and 9. It binds to CD47 with at least 1000 times higher binding affinity than the P-α D1 domain. In some embodiments, the SIRP-α D1 variant polypeptide or a fragment thereof is KD less than 1x10⁻⁸M, less than 5x10⁻⁹M, less than 1x10⁻⁹M, 5x10⁻¹ Combine to CD47 if the size is less than 0M, less than 1x10⁻¹⁰M, or less than 1x10⁻¹¹M. In some embodiments, the SIRP-α D1 variant polypeptide or a 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 It binds to CD47 at M, approximately 100 pM to 50 pM, or approximately 50 pM to 10 pM.

[0041] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14KSGAGTELSVRAKPS (Sequence ID 14), where X1 is L, I, or X2 is V, X3 is A or V, X4 is V, I, t is L, X5 is I, T, S, or F, X6 is E, V, or L, X 7 is K or R, X8 is E or Q, X9 is H, P or R, X1 0 is S, T, or G, and X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α has a sequence that is F or V Includes a D1 variant, the variant having the sequence of SEQ ID NO: 2, wild-type SIRP- It has at least one amino acid substitution in the α D1 domain.

[0042] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (Sequence ID 15), where X1 is L, I, or X2 is V, X3 is A or V, X4 is V, I, t is L, X5 is I, T, S, or F, X6 is E, V, or L, X 7 is K or R, X8 is E or Q, X9 is H, P or R, X1 0 is S, T, or G, and X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α has a sequence that is F or V The D1 variant is included, and the variant has the sequence of SEQ ID NO: 3, which is wild-type SIRP- It has at least one amino acid substitution in the α D1 domain.

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

[0044] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (Sequence ID 22), where X1 is L, I, or X2 is V, X3 is A or V, X4 is V, I, t is L, X5 is I, T, S, or F, X6 is E, V, or L, X 7 is K or R, X8 is E or Q, X9 is H, P or R, X1 0 is S, T, or G, and X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α has a sequence that is F or V Includes the D1 variant, which is a wild-type SIRP having the sequence of SEQ ID NO: 10. The -α D1 domain has at least one amino acid substitution.

[0045] In any of the embodiments described above in this aspect of the present disclosure, the polypeptide is sequence number One of numbers 14, 15, 19, and 22, where X1 is L, I, or V. It has an array. In any of the above embodiments, X2 is V, L, or I. In any of the embodiments, X3 is A or V. X4 is V, I, or L. In any of the above embodiments, X5 is I, T, It is 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 this, 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 It is S, T, or G. In any of the above embodiments, X 11 is K or R. 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 It is F or V. In some embodiments, the polypeptides of this embodiment of the Disclosure are as follows: SEQ ID NOs: 2, 3, 7, and 1 For wild-type SIRP-α D1 domains containing any one of the sequences 0, there are 6 or fewer. Includes the amino acid substitution.

[0046] In some embodiments, the polypeptide is any of SEQ ID NOs: 2, 3, 7, and 10. Binding is at least 10 times higher than that of wild-type SIRP-α D1 domains with only one sequence. It binds to CD47 by affinity. In some embodiments, the polypeptide is sequence number 2. A wild-type SIRP-α D1 domain having one of sequences 3, 7, and 10 It binds to CD47 with at least 100 times higher binding affinity. In some embodiments, The polypeptide is wild-type having one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. It binds to CD47 with at least 1000 times 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 -8Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x1 0 -10 Less than M, 1x10 -10 Less than M, or 1x10 -11 Binds to CD47 if 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 It binds to CD47 at 00 pM, approximately 100 pM to 50 pM, or approximately 50 pM to 10 pM. .

[0047] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (Sequence ID 20), where X1 is L, I, or V is X2 is V, L, or I, X3 is A or V, X4 is A, I, t is L, X5 is I, T, S, or F, X6 is E, V, or L, X 7 is K or R, X8 is E or Q, X9 is H, P or R, X1 0 is S, T, or G, and X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, and X 14 SIRP-α has a sequence that is F or V The D1 variant is included, and the variant has the sequence of SEQ ID NO: 8, which is wild-type SIRP- The α D1 domain has at least one amino acid substitution.

[0048] In some embodiments, the polypeptide is sequence number 20, where X1 is L, I, or has an array that is V. In any of the embodiments of this disclosure described herein, 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, X9 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 embodiment of the present disclosure This refers to a wild-type SIRP-α D1 domain having the sequence of sequence number 8, with 6 or fewer Includes 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 times higher binding affinity than the P-α D1 domain. In one embodiment, the polypeptide is wild-type SIRP-α having the sequence of SEQ ID NO: 8. It binds to CD47 with at least 100 times higher binding affinity than the D1 domain. In this embodiment, the polypeptide is wild-type SIRP-α D having the sequence of SEQ ID NO: 8. It binds to CD47 with a binding affinity at least 1000 times higher than a single domain. In the embodiment, the SIRP-α D1 variant polypeptide or a fragment thereof is K D 1 x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 -11 It binds to CD47 with a minimum of M. In the embodiment, the SIRP-α D1 variant polypeptide or a fragment thereof is K 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 It 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 (Sequence ID 23), where X1 is E or G, and X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, X5 is L or S, X6 is S or T, X7 is A or V, X8 is I or T And X9 is H or R, X 10 is A, V, I, or L, and X 11 is I, It is 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, X 15 is E or Q, and X 16 is H, P, or R Yes, X 17 is D or E, X 18 is S, L, T, or G, X 19 haKma or is R, X 20 is E or D, X 21 is S or P, X 22 is S also is R, and X 23 is S or G, X 24 is V or I, X 25 is F, L, V and X 26 is either D or does not exist, X 27 is T or V, X 28 teeth F or V, X 29 This is a SIRP-α D1 varian having a sequence that is A or G. The variant includes a wild-type SI having one sequence from sequence numbers 1 to 10. The RP-α D1 domain has at least one amino acid substitution.

[0051] In any of the embodiments described herein, 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 is S. In any of the above embodiments, X6 is S or T. The above embodiment In any of the above embodiments, X7 is A or V. 8 is I or T. In any of the above embodiments, X9 is H or R. In any of the embodiments described, X 10 is A, V, I, or L. In either case, X 11 is I, T, S, or F. And, 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 application methods, X 15 is E or Q. In any of the above embodiments, X 16 is H, P, or R. In any of the above embodiments, X 17 D or is E. In any of the above embodiments, X 18 It is S, L, T, or G. In any of the above embodiments, X 19 is K or R. Any of the above embodiments In 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 application methods, X 23 is S or G. In any of the above embodiments, X 24 is V or I. In any of the above embodiments, X 25 F, L, V In any of the above embodiments, X 26 It is either D or does not exist. In any of the embodiments described, X 27 is T or V. X 28 is F or V. In any of the above embodiments, X 29 A also is G. In some embodiments, the polypeptides of this embodiment of the present disclosure are SEQ ID NOs: 1 to For a wild-type SIRP-α D1 domain having one of the 10 sequences, 6 or more Includes the following amino acid substitutions.

[0052] In some embodiments, the polypeptide is one of the sequences of sequence numbers 1 to 10. CD has a binding affinity at least 10 times higher than that of the wild-type SIRP-α D1 domain. It binds to 47. In some embodiments, the polypeptide is one of the sequence numbers 1 to 10. At least 100 times higher than wild-type SIRP-α D1 domains with any one of the sequences. It binds to CD47 by binding affinity. In some embodiments, the polypeptide is sequence number Fewer than the wild-type SIRP-α D1 domain having one of sequences 1-10 Both bind to CD47 with a binding affinity 1000 times higher. In some embodiments, SIR P-α D1 variant polypeptide or a fragment thereof is K D 1x10 -8 Less than M, 5 x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M , or 1x10 -11 It binds to CD47 less than M. In some embodiments, SIR P-α D1 variant polypeptide or a fragment thereof is K D Approximately 500nM~100nM , about 100nM~50nM, about 50nM~10nM, about 10nM~5nM, about 5nM~1 nM, approximately 1nM~500pM, approximately 500pM~100pM, approximately 100pM~50pM, It binds to CD47 at a concentration of approximately 50 pM to 10 pM.

[0053] In some embodiments, the poly(P) of the Disclosure includes a high affinity SIRP-α D1 variant. The peptide further has the sequence of wild-type SIRP-α sequence number 24 shown in Table 3. A domain, a D3 domain having the sequence of sequence number 25, or having the sequence of sequence number 24 It includes the D2 domain and the D3 domain having the sequence of sequence number 25. Morphologically, the high-affinity SIRP-α D1 variant also contains fragments of the D2 domain. This includes a variant or a fragment or variant of the D3 domain. In the application form, the high-affinity SIRP-α D1 variant further comprises fragments of the D2 domain. or variants, and fragments or variants of the D3 domain. Several implementations In this state, the high-affinity SIRP-α D1 variant is transmitted via the linker to D2 or D3. Connects to the main. In some embodiments, a high-affinity SIRP-α D1 variant It is connected to the D2 and D3 domains via a linker. [Table 3]

[0054] In some embodiments, the poly(P) of the Disclosure includes a high affinity SIRP-α D1 variant. The peptide improves the pharmacokinetic properties of the polypeptide, for example, by extending its serum half-life. Therefore, Fc domain monomer, human serum albumin (HSA) or its variant , serum-binding proteins or peptides, or organic molecules, for example, polymers (e.g., It is bonded to a PEG polymer. In some embodiments, high affinity SIRP-α D1 The variant binds to an Fc domain monomer that cannot be dimerized. In some embodiments, the Fc domain monomer, HSA protein, and serum-binding protein are also included. Alternatively, peptides and organic molecules such as PEG may be used to reduce the serum half-life of polypeptides as described herein. It works to extend the period. In some embodiments, a high affinity SIRP-α D1 barrier The polypeptides of this disclosure, including the nucleotide, contain any of the sequences 26-36 shown in Table 4. No. [Table 4-1] [Table 4-2]

[0055] In some embodiments, the polypeptides and polypeptide constructs described herein are It is used in vitro for binding assays such as immunoassays. For example, in several embodiments. Therefore, the polypeptides and polypeptide constructs described herein are used in a liquid phase, Alternatively, it is bound to a solid support. In some embodiments, it is used in immunoassays. Lipeptides can be labeled in a way that makes them detectable by various methods.

[0056] In some embodiments, the polypeptides and polypeptide constructs described herein are It is bound to various carriers and used to detect the presence of cells expressing specific antigens. Examples include glass, polystyrene, polypropylene, polyethylene, dextran, and Iron, amylase, natural and modified cellulose, polyacrylamide, agarose, Magnetite is another example. The properties of the carrier may be soluble or insoluble.

[0057] Various different labeling methods and techniques are known. Examples of labeling include enzymes and radioactive isotopes. Examples include fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Various techniques are available for attaching labels to the polypeptides disclosed in this document.

[0058] In some embodiments, the polypeptide is bound to a low molecular weight hapten. Butene is then specifically detected by a second reaction. For example, in several embodiments So, is biotin, the hapten in question, used together with avidin, or is the hapten used together with avidin? Dinitrophenol, pyridoxal, or fluorescein are known to be effective against certain anti-inflammatory drugs. Buten antibodies (for example, anti-dinitrophenol antibody, anti-pyridoxal antibody, and It is detected by (and anti-fluorescein antibodies).

[0059] II. High-affinity SIRP-α D1 domain with altered glycosylation In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0060] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 The polypeptide selected from the Fc region of human IgG4, including mutations 36 and N297A It is Do.

[0061] In some embodiments, the polypeptide in the compositions disclosed herein is glyco 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. 1-1 in Table 1) 10) is amino acid N80 of sequence N80ITP, with a single latent N-linked glycosylation site. Includes the position. SIRP-α D1 dormancy in Chinese hamster ovary (CHO) cells. In expression is removed by the major band at 16kDa (non-glycosylated) and Endo Hf. The result is a band with a lower molecular weight than expected. Endo Hf is endoglycerin It is a recombinant protein fusion of sidase H and maltose-binding protein. Hf is found in high-mannose chitobiose cores and in some hybrid oligosaccharides. It is cleaved from the bound glycoprotein. This means that the proline at amino acid position 83 is cleaved from the glyco This can reduce the efficiency of sylation, resulting in proteins with different degrees of glycosylation, and consequently, heterogeneity. It means bringing quality. For pharmaceutical development, heterogeneity is a challenge in process development. This can cause problems. Therefore, the uniform non- To investigate the possibility of generating glycosylated forms, in some embodiments, SIPR- The amino acid N80 of the α D1 variant is mutated to Ala. In some embodiments To produce a non-glycosylated high-affinity SIRP-α D1 variant, high-affinity S The amino acid N80 of the IRP-α D1 variant includes any natural and non-natural amino acids. It is substituted with any amino acid, for example, N80A and N80Q. Several implementations In this state, the high-affinity SIRP-α D1 variant is characterized by the N80A mutation and at least one other mutation. Further mutations (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) (or further mutations beyond that). In some embodiments, the further mutations include , located at the CD47 binding site. In some embodiments, the further mutation is D1 domain It is located in the hydrophobic core.

[0062] In some embodiments, the polypeptides in the compositions disclosed herein are wild-type High-affinity SIRP-α with increased glycosylation compared to the SIRP-α D1 domain. Contains the D1 variant. Another option to increase the homogeneity of the final product is to use a glycerin at amino acid N80. High affinity SIRP- The goal is to generate an α D1 variant. In some embodiments, the sequence NITP83 The amino acid P83 affects the degree of glycosylation at amino acid N80. In the application method, changing P83 to any amino acid improves the efficiency of glycosylation at N80. To enhance the amino acids of the high affinity SIRP-α D1 variant. In some embodiments, P83 can be substituted with any amino acid, including natural and non-natural amino acids, for example, P83 These are V, P83A, P83I, and P83L. In some embodiments, the poly of the present disclosure Peptides are used, for example, in genetically modified cell lines (e.g., genetically modified yeast or pyocytes). (Mammalian host) or by changing cell culture conditions such as adding kifunensin, or prokaryotic By using a host organism that does not naturally undergo glycosylation, such as E. coli, the details can be improved. Proteins expressed by cells are optimized to prevent glycosylation. It can be done.

[0063] Table 5 shows the high affinity SIRP-α D1 variants for each D1 domain variant sequence. This shows specific amino acid substitutions in the t. In some embodiments, high affinity SIRP- The α D1 variant is one or more of the substitutions shown in Table 5 (e.g., 2, 3, 4, 5, 6, 7, Including 8, 9, 10, 11, 12, 13, 14 or more. Several implementations In terms of form, the SIRP-α D1 variant is either not glycosylated or minimally glycosylated. It is glycosylated. In some embodiments, the SIRP-α D1 variant is Completely glycosylated or almost completely glycosylated. Several implementations In this state, the high-affinity SIRP-α D1 variant has the maximum affinity for the wild-type D1 domain. It contains 14 amino acid substitutions. In some embodiments, it is a high affinity SIRP-α D1 variant. Ant contains up to 10 amino acid substitutions compared to the wild-type D1 domain. In terms of application, the high-affinity SIRP-α D1 variant, compared to the wild-type D1 domain, Includes up to 7 amino acid substitutions. In some embodiments, the high affinity SIRP- The α D1 variant is at least 90% (for example) of the wild-type D1 domain sequence. Having amino acid sequence identity of at least 92%, 95%, 97%, or more than 97% .

[0064] In some embodiments, the high affinity SIRP-α D1 variant is found in two or more wild-type variants. A type D1 domain or a portion of its variant (for example, one wild-type D1 domain or (including the variant portion and another wild-type D1 domain or its variant portion) It is a chimeric high-affinity SIRP-α D1 variant. In some embodiments, it is a chimeric The high affinity SIRP-α D1 variant is a wild-type D1 domain or a variant thereof. It includes at least two parts (for example, three, four, five, or more parts), and the part Each of these is derived from a different wild-type D1 domain. In some embodiments, the chimeric high parentage is used. The Japanese SIRP-α D1 variant further contains 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 (sequence number 37), where X1 is L. X2 is I or V, X3 is V, L, or I, X4 is A or V, X4 is X5 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. Yes, X 10 is L, T, or G, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V , X 16 The variant includes a SIRP-α D1 variant having an array that is F or V, and the variant Ant has less than the wild-type SIRP-α D1 domain with sequence number 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 (Sequence ID 40), where X1 is L. X2 is I or V, X3 is V, L, or I, X4 is A or V, X4 is X5 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. Yes, X 10 is L, T, or G, X 11 is K or R, 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, X 14is V or I, X 15 is F, L, or V , X 16 The variant includes a SIRP-α D1 variant having an array that is F or V, and the variant Ant has less than the wild-type SIRP-α D1 domain with sequence number 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 (Sequence ID 41), where X1 is L. X2 is I or V, X3 is V, L, or I, X4 is A or V, X4 is X5 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. Yes, X 10 is L, T, or G, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V , X 16The variant includes a SIRP-α D1 variant having an array that is F or V, and the variant Ant has less than the wild-type SIRP-α D1 domain with sequence number 5. 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 (sequence number 42), where X1 is L. X2 is I or V, X3 is V, L, or I, X4 is A or V, X4 is X5 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. Yes, X 10 is L, T, or G, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V , X 16 The variant includes a SIRP-α D1 variant having an array that is F or V, and the variant Ant has less than the wild-type SIRP-α D1 domain with sequence number 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 (sequence number 45), where X1 is L. X2 is I or V, X3 is V, L, or I, X4 is A or V, X4 is X5 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. Yes, X 10 is L, T, or G, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V , X 16 The variant includes a SIRP-α D1 variant having an array that is F or V, and the variant Ant has less of the wild-type SIRP-α D1 domain with sequence number 9. Both have one amino acid substitution.

[0070] In any of the embodiments of this disclosure described herein, the polypeptide is SEQ ID NO: 37, The sequence has one of 40-42 and 45, where X1 is L, I, or V. Includes the SIRP-α D1 variant. In any of the above embodiments, X2 is V It is L, or I. In any of the above embodiments, X3 is A or V. In any of the embodiments described above, X4 is A, I, or L. In the above embodiment, X5 is I, T, S, or F. 6 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 the above embodiment, X9 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 The letters are N, A, C, D, E, F, G, H, 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 These are F, L, and V. In either case, X 16 It is either F or V.

[0071] In some embodiments, the polypeptides provided herein are SEQ ID NOs: 1, 4-6. For a wild-type SIRP-α D1 domain having one of the sequences of and 9, 10 Includes one or fewer amino acid substitutions. In some embodiments, the polypeptide provided herein D is a wild-type SIRP-α having one of the sequences 1, 4-6, and 9. The D1 domain contains seven or fewer amino acid substitutions.

[0072] In some embodiments, the polypeptide is one of SEQ ID NOs: 1, 4-6, and 9. At least 10 times higher binding parentage than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 in a saturating manner. In some embodiments, the polypeptide is sequence number 1, Fewer than the wild-type SIRP-α D1 domain which has one of sequences 4-6 and 9. At the very least, it binds to CD47 with a binding affinity 100 times higher. In some embodiments, the port The lipeptide is a wild-type SIR having one of the sequences of SEQ ID NOs: 1, 4-6, and 9. It binds to CD47 with at least 1000 times higher binding affinity than the P-α D1 domain. In some embodiments, the SIRP-α D1 variant polypeptide or a fragment thereof is , K D 1x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -1 0 Less than M, 1x10 -10 Less than M, or 1x10 -11 It binds to CD47 if it is less than M. In some embodiments, the SIRP-α D1 variant polypeptide or a 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 It binds to CD47 at M, approximately 100 pM to 50 pM, or approximately 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 (Sequence ID 38), where X1 is L, I X2 is V, L, or I, X3 is A or V, and X4 is V X5 is I, T, S, or F, and X6 is E, V, or L. Yes, 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, X 11 is K or R, X 12 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, X 14 is V or I, X 15 is F, L, or V ri, X 16 It comprises a SIRP-α D1 variant having an array that is F or V, and the The rianto has less of the wild-type SIRP-α D1 domain with 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 (Sequence ID 39), where X1 is L, I X2 is V, L, or I, X3 is A or V, and X4 is V X5 is I, T, S, or F, and X6 is E, V, or L. Yes, 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, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V, X 16 The barrier comprises a SIRP-α D1 variant having an array that is F or V, and the barrier The nt is at least compared to the wild-type SIRP-α D1 domain having the sequence of Sequence 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 (Sequence ID 43), where X1 is L, I X2 is V, L, or I, X3 is A or V, and X4 is V X5 is I, T, S, or F, and X6 is E, V, or L. Yes, 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, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V, X 16 The barrier comprises a SIRP-α D1 variant having an array that is F or V, and the barrier The nt, compared to the wild-type SIRP-α D1 domain having the sequence of sequence number 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 (sequence number 46), where X1 is L, I X2 is V, L, or I, X3 is A or V, and X4 is V X5 is I, T, S, or F, and X6 is E, V, or L. Yes, 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, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V, X 16 The barrier comprises a SIRP-α D1 variant having an array that is F or V, and the barrier The nt is less than the wild-type SIRP-α D1 domain having the sequence of sequence number 10. It has at least one amino acid substitution.

[0077] In any of the embodiments described herein, the polypeptide is SEQ ID NO: 38, One of 39, 43, and 46 has an array where X1 is L, I, or V. Includes the SIRP-α D1 variant. In any of the above embodiments, X2 is V It is L, or I. In any of the above embodiments, X3 is A or V. In any of the embodiments described above, X4 is V, I, or L. In the above embodiment, X5 is I, T, S, or F. 6 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 the above embodiment, X9 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 N, A, C, D, E, F, G, H, I, The elements are 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, It is T, V, W, or Y. In any of the above embodiments, X 14 is V or I Yes, in any of the above embodiments, X 15 is F, L, or V. (The above implementation form) In any of the states, X 16 It is either F or V.

[0078] In some embodiments, the polypeptide is one of SEQ ID NOs: 2, 3, 7, and 10. For a wild-type SIRP-α D1 domain with one sequence, there are 10 or fewer amino acids. Includes a SIRP-α D1 variant having a substitution. In some embodiments, polypeptide D is a wild-type SIRP-α having one of the sequences 2, 3, 7, and 10. SIRP-α D1 variant having 7 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. Binding is at least 10 times higher than that of wild-type SIRP-α D1 domains with only one sequence. It binds to CD47 by affinity. In some embodiments, the polypeptide is sequence number 2. A wild-type SIRP-α D1 domain having one of sequences 3, 7, and 10 It binds to CD47 with at least 100 times higher binding affinity. In some embodiments, The polypeptide is wild-type having one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. It binds to CD47 with at least 1000 times 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 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x1 0 -10 Less than M, 1x10 -10 Less than M, or 1x10 -11 Binds to CD47 if 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 It binds to CD47 at 00 pM, approximately 100 pM to 50 pM, or approximately 50 pM to 10 pM. .

[0080] In some embodiments, the polypeptide is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGP IQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX1 1RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSP DTEX 16 KSGAGTELSVRAKPS (Sequence ID 44), where X1 is L, I X2 is V, L, or I, X3 is A or V, and X4 is A X5 is I, T, S, or F, and X6 is E, V, or L. Yes, 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, X 11 is K or R, 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, X 14 is V or I, X 15 is F, L, or V, X 16 The barrier comprises a SIRP-α D1 variant having an array that is F or V, and the barrier The nt is less than the wild-type SIRP-α D1 domain having the sequence of sequence number 8. Both have one amino acid substitution.

[0081] In some embodiments, the polypeptide is sequence number 44, where X1 is L, I, or has an array that is V. In any of the embodiments of this disclosure described herein, 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, X9 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 N, A, C, D, E, F, G, H, 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, It is S, T, V, W, or Y. In any of the above embodiments, X 14 is V or I is in any of the above embodiments, X 15 is F, L, or V. In any of the application methods, X 16 It is either 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 variant having 10 or fewer amino acid substitutions in the α D1 domain Contains ant. In some embodiments, the polypeptide has the sequence of sequence number 8. SIRP-α with 7 or fewer amino acid substitutions in the bio-type SIRP-α D1 domain Includes 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 times higher binding affinity than the P-α D1 domain. In one embodiment, the polypeptide is wild-type SIRP-α having the sequence of SEQ ID NO: 8. It binds to CD47 with at least 100 times higher binding affinity than the D1 domain. In this embodiment, the polypeptide is wild-type SIRP-α D having the sequence of SEQ ID NO: 8. It binds to CD47 with a binding affinity at least 1000 times higher than a single domain. In the embodiment, the SIRP-α D1 variant polypeptide or a fragment thereof is K D 1 x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 -11 It binds to CD47 with a minimum of M. In the embodiment, the SIRP-α D1 variant polypeptide or a fragment thereof is K 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 It binds to CD47 at 0 pM to 50 pM, or approximately 50 pM to 10 pM.

[0084] In another embodiment, this disclosure is, 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 (sequence number 47), where X1 is E or G, and X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, X 5 is L or S, X6 is S or T, X7 is A or V, X8 is I t is T, X9 is H, R, or L, X 10 is A, V, I, or L, X 11 is I, T, S, or F, X 12 is A or G, and X 13 is E, V, ma ta is L, X 14 is K or R, X 15 is E or Q, and X 16 H, P , or R, X 17 is D or E, X 18 is S, L, T, or G, X 19 is K or R, X 20 is E or N, X 21 is S or P, X 22 is S or R, X 23 is S or G, X 24 is any amino acid, X 25 is any amino acid, X 26is V or I, X 27 These are F, L, and V. , X 28 is either D or does not exist, X 29 is T or V, X 30 is F or V and X 31 It contains a SIRP-α D1 variant having a sequence that is A or G. The variant has a sequence of any one of sequence numbers 1 to 10, which is wild-type SIRP-α. The polypeptide is characterized by having at least one amino acid substitution in the D1 domain. do.

[0085] In some embodiments, the polypeptide is sequence number 47, where X1 is E or It has an array that is G. In any of the above embodiments of this disclosure, X2 is L, It is 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 this embodiment, X5 is L or S. In any of the above embodiments, X6 is S or T is. In any of the above embodiments, X7 is A or V. In either case, X8 is I or T. In any of the above embodiments, X9 is It is H, R, or L. In any of the above embodiments, X 10 is A, V, I, also is L. In any of the above embodiments, X 11 It is I, T, S, or F. In any of the above embodiments, X 12 is A or G. 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 It is E or Q. In any of the embodiments described, X 16 is H, P, or R. In any of the following, X 17 is D or E. In any of the above embodiments, X 18 teeth It is S, L, T, or G. In any of the above embodiments, X 19 is K or R Yes, 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 It is S or G. In any of the embodiments described, X 24 is N, A, C, D, E, F, G, H, I, K, L , M, P, Q, R, S, T, V, W, or Y. In any of the above embodiments , 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 It is either V or I. In any of the above embodiments, X 27 These are F, L, and V. Any of the above embodiments In X 28 is either D or does not exist. 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 It is either A or G.

[0086] In some embodiments, the polypeptides of this embodiment of the present disclosure are among SEQ ID NOs: 1 to 10 For wild-type SIRP-α D1 domains containing any one of the sequences, 10 or fewer ami Includes no acid substitution. In some embodiments, the polypeptide of this embodiment of the present disclosure is SEQ ID NO For a wild-type SIRP-α D1 domain having one of the sequences 1 to 10, 7 Contains one or fewer amino acid substitutions.

[0087] In some embodiments, the polypeptide is one of the sequences of sequence numbers 1 to 10. CD has a binding affinity at least 10 times higher than that of the wild-type SIRP-α D1 domain. It binds to 47. In some embodiments, the polypeptide is one of the sequence numbers 1 to 10. At least 100 times higher than wild-type SIRP-α D1 domains with any one of the sequences. It binds to CD47 by binding affinity. In some embodiments, the polypeptide is sequence number Fewer than the wild-type SIRP-α D1 domain having one of sequences 1-10 Both bind to CD47 with a binding affinity 1000 times higher. In some embodiments, SIR P-α D1 variant polypeptide or a fragment thereof is K D 1x10 -8 Less than M, 5 x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M , or 1x10 -11 It binds to CD47 less than M. In some embodiments, SIR P-α D1 variant polypeptide or a fragment thereof is K D Approximately 500nM~100nM , about 100nM~50nM, about 50nM~10nM, about 10nM~5nM, about 5nM~1 nM, approximately 1nM~500pM, approximately 500pM~100pM, approximately 100pM~50pM, It binds to CD47 at a concentration of 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) Number 48) where X1 is V or I, X2 is L or S, and X3 is T or X4 is S, X5 is T or I, X6 is R or H, X6 is A, V, or X7 is I, X7 is I, R, Y, K or F, X8 is G or A, and X9 is E. ta is V, X 10 is K or R, X 11 is E, D, or Q, and X 12 is H or P, X 13 is D or E, X 14 is S, L or T, X 15 teeth N or E, X 16 is R or S, X 17 is G or S, X 18 is N Or A, X 19 is V or I, X 20is S, I or M, X 21 teeth P is either not present or X 22 is D or P, X 23 The distribution is V or T. Includes a SIRP-α D1 variant having a column, or a fragment thereof.

[0090] In another embodiment, this disclosure is, EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNM DFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAG TELSVRAKPS (Sequence ID 49), where X1 is V, L, or I, and X2 X3 is A, I, V, or L, X4 is I, F, S, or T, and X4 is E, V, X5 is L, X6 is K or R, X7 is E or Q, and X7 is H, P, and is R, X8 is L, T, S, or G, X9 is A, X 10 is V or I The SIRP-α D1 variant has the sequence, and the variant is sequence number 1 For a wild-type SIRP-α D1 domain having any one of the following sequences, at least It is characterized by a polypeptide having one amino acid substitution.

[0091] In some embodiments, the polypeptide is sequence number 49, where X1 is V, L is X2 has an array that is I. In any of the above embodiments of this disclosure, X2 is It is A, I, V, or L. In any of the above embodiments, X3 is I, F, S, or or is T. In any of the above embodiments, X4 is E, V, or L. In any of the embodiments, X5 is K or R. X6 is E or Q. In any of the above embodiments, X7 is H, P, or R is R. In any of the above embodiments, X8 is L, T, S, or G. In any of the above embodiments, X9 is A. In any of the above embodiments, X 10 It is either V or I.

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

[0093] In some embodiments, the polypeptides of this embodiment of the present disclosure are any one of SEQ ID NO 1. For the wild-type SIRP-α D1 domain which has two sequences, 10 or fewer amino acids Includes substitution. In some embodiments, the polypeptide of this embodiment of the present disclosure is the same as in SEQ ID NO: 1. For wild-type SIRP-α D1 domains having one or less sequences, 7 or fewer ami Includes no acid substitution.

[0094] In some embodiments, the polypeptide has one of the sequences of SEQ ID NO: 1. It binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRP-α D1 domain. To bind. In some embodiments, the polypeptide is one of the components of SEQ ID NO: 1. With binding affinity at least 100 times higher than that of the wild-type SIRP-α D1 domain with columns It binds to CD47. In some embodiments, the polypeptide is one of the elements of SEQ ID NO: 1 or at least 1000 times higher than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 by binding affinity. In some embodiments, the SIRP-α D1 barrier The polymer polypeptide or its fragments are K D 1x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 - 11 It binds to CD47 at M less than M. In some embodiments, the SIRP-α D1 barrier The polymer polypeptide or its fragments are K D Approximately 500nM~100nM, Approximately 100nM~5 0nM, approximately 50nM~10nM, approximately 10nM~5nM, approximately 5nM~1nM, approximately 1nM~5 00 pM, approximately 500 pM to 100 pM, approximately 100 pM to 50 pM, or approximately 50 pM to 1 It binds to CD47 at 0 pM.

[0095] In another embodiment, this disclosure is, EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQ WFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENM DFSISISX9ITPADAGTYYCX 10 KFRKGSPDTEFKSGAGT ELSVRAKPS, (Sequence ID 50), where X1 is V or I, and X2 is V X3 is I, X4 is I or F, X5 is E or V, and X5 is K or R. Yes, X6 is E or Q, X7 is H or P, X8 is S or T, X 9 is N or A, X 10 SIRP-α D1 variant having a sequence that is V or I An ant is included, and the variant has one of the sequences of SEQ ID NO: 2, wild-type SIR A polypeptide having at least one amino acid substitution in the P-α D1 domain is specifically To use as a sign.

[0096] In some embodiments, the polypeptide is sequence number 50, where X1 is V or It has an array that is I. In any of the above embodiments of this disclosure, X2 is V or is I. In any of the above embodiments, X3 is I or F. The above embodiment In any of the above embodiments, X4 is E or V. 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 the above embodiment, X8 is S or R. In any of the above embodiments, X9 is N or A. Yes, in any of the above embodiments, X 10 It is either V or I.

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

[0098] In some embodiments, the polypeptides of this embodiment of the Disclosure are any one of the 1s in SEQ ID NO: 2 For the wild-type SIRP-α D1 domain which has two sequences, 10 or fewer amino acids Includes substitution. In some embodiments, the polypeptide of this embodiment of the present disclosure is the same as in SEQ ID NO: 2. For wild-type SIRP-α D1 domains having one or less sequences, 7 or fewer ami Includes no acid substitution.

[0099] In some embodiments, the polypeptide has one of the sequences of SEQ ID NO: 2. It binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRP-α D1 domain. To bind. In some embodiments, the polypeptide is one of the components of SEQ ID NO: 2. With binding affinity at least 100 times higher than that of the wild-type SIRP-α D1 domain with columns It binds to CD47. In some embodiments, the polypeptide is one of the numbers in SEQ ID NO: 2 or at least 1000 times higher than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 by binding affinity. In some embodiments, the SIRP-α D1 barrier The polymer polypeptide or its fragments are K D 1x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 - 11 It binds to CD47 at M less than M. In some embodiments, the SIRP-α D1 barrier The polymer polypeptide or its fragments are K D Approximately 500nM~100nM, Approximately 100nM~5 0nM, approximately 50nM~10nM, approximately 10nM~5nM, approximately 5nM~1nM, approximately 1nM~5 00 pM, approximately 500 pM to 100 pM, approximately 100 pM to 50 pM, or approximately 50 pM to 1 It binds to CD47 at 0 pM.

[0100] In another embodiment, this disclosure is, EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMD FSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTE LSVRAKPS (sequence number 51), where X1 is V or I, and X2 is A or X3 is I, X4 is I or F, X5 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. Yes, X9 includes a SIRP-α D1 variant having a sequence that is V or I, and the The riant has one of the sequences in sequence number 1, wild-type SIRP-α D1 domain The polypeptide is characterized by having at least one amino acid substitution in the original molecule.

[0101] In some embodiments, the polypeptide is sequence number 51, where X1 is V or It has an array that is I. In any of the above embodiments of this disclosure, X2 is A or is I. In any of the above embodiments, X3 is I or F. The above embodiment In any of the above embodiments, X4 is E or V. 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 the above embodiment, X8 is N or A. In any of the above embodiments, X9 is V or I. Yes, in some embodiments, X4 is not V.

[0102] In some embodiments, the polypeptide is sequence number 51, where X8 is A. It has an array. In any of the embodiments of this disclosure described above, X8 is A and X 1 is V or I. In any of the embodiments of this disclosure described above, X8 is A Yes, and X2 is A or I. In any of the above embodiments, X8 is A and X 3 is I or F. In any of the above embodiments, X8 is A and X4 is E. X4 is V. In some embodiments, X4 is not V. In this example, X8 is A, and X5 is K or R. In any of the above embodiments, X8 is A, and X6 is H or P. In any of the above embodiments, X8 is A X7 is A or V. In any of the above embodiments, X8 is A, X9 is either V or I.

[0103] In some embodiments, the polypeptide is sequence number 51, where X8 is A. It has an array. In any of the embodiments of this disclosure described above, X8 is A and X 1 is I. In any of the embodiments of this disclosure described above, X8 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. 8 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 is X1, X2, X3, X4, X5, X6 For SEQ ID NO: 51, where X7, X8, and X9 are not wild-type amino acids, at least 8 5% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%) %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 It possesses a high-affinity SIRP-α D1 domain with % sequence identity.

[0105] In some embodiments, the polypeptides of this embodiment of the present disclosure are any one of SEQ ID NO 1. For the wild-type SIRP-α D1 domain which has two sequences, 10 or fewer amino acids Includes substitution. In some embodiments, the polypeptide of this embodiment of the present disclosure is the same as in SEQ ID NO: 1. For wild-type SIRP-α D1 domains having one or less sequences, 7 or fewer ami Includes no acid substitution.

[0106] In some embodiments, the polypeptide has one of the sequences of SEQ ID NO: 1. It binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRP-α D1 domain. To bind. In some embodiments, the polypeptide is one of the components of SEQ ID NO: 1. With binding affinity at least 100 times higher than that of the wild-type SIRP-α D1 domain with columns It binds to CD47. In some embodiments, the polypeptide is one of the elements of SEQ ID NO: 1 or at least 1000 times higher than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 by binding affinity. In some embodiments, the SIRP-α D1 barrier The polymer polypeptide or its fragments are K D 1x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 - 11 It binds to CD47 at M less than M. In some embodiments, the SIRP-α D1 barrier The polymer polypeptide or its fragments are K D Approximately 500nM~100nM, Approximately 100nM~5 0nM, approximately 50nM~10nM, approximately 10nM~5nM, approximately 5nM~1nM, approximately 1nM~5 00 pM, approximately 500 pM to 100 pM, approximately 100 pM to 50 pM, or approximately 50 pM to 1 It binds to CD47 at 0 pM.

[0107] In another embodiment, this disclosure is, EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDF SIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELS VRAKPS (sequence number 52), where X1 is V, L, or I, and X2 is A or I. , or L, X3 is I, T, S, or F, X4 is K or R, X5 The arrangement is H, P, or R, X6 is L, T, or G, and X7 is N or A. Includes a SIRP-α D1 variant having a column, wherein the sequence of sequence number 1 It has at least one amino acid substitution in the wild-type SIRP-α D1 domain. It is characterized by a polypeptide.

[0108] In some embodiments, the polypeptide is sequence number 52, where X1 is V, L is X2 has an array that is I. In any of the above embodiments of this disclosure, X2 is It is A, I, or L. In any of the above embodiments, X3 is I, T, S, or F is F. In any of the above embodiments, X4 is K or R. In either case, X5 is H or P. In any of the above embodiments, X6 is It is L, T, or G. In any of the above embodiments, X7 is N or A.

[0109] In some embodiments, the polypeptide is sequence number 52, where X1 is V or It has an array that is I. In any of the above embodiments of this disclosure, X2 is A or is I. In any of the above embodiments, X3 is I or F. The above embodiment In any of the above embodiments, X4 is K or R. 5 is H or P. In any of the above embodiments, X6 is L or T. In any of the embodiments described, X7 is N or A.

[0110] In some embodiments, the polypeptide is sequence number 52, where X7 is A. It has an array. In any of the embodiments of this disclosure described herein, X7 is A and X 1 is V or I. In any of the embodiments of this disclosure described above, X7 is A. Yes, and X2 is A or I. In any of the above embodiments, X7 is A and X 3 is I or F. In any of the above embodiments, X7 is A and X4 is K. or is R. In any of the above embodiments, X7 is A and X5 is H or P. Yes. In any of the above embodiments, X7 is A, and X6 is L or T.

[0111] In some embodiments, the polypeptide is sequence number 52, where X7 is A. It has an array. In any of the embodiments of this disclosure described herein, X7 is A and X 1 is I. In any of the embodiments of this disclosure described herein, 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 embodiments described above, 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 is X1, X2, X3, X4, X5, X6 , and for SEQ ID NO: 52, where X7 is not a wild-type amino acid, at least 85% of the sequence is identical. Unisexual (for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%) 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical It possesses a high-affinity SIRP-α D1 domain with (sexual properties).

[0113] In some embodiments, the polypeptides of this embodiment of the present disclosure are any one of SEQ ID NO 1. For the wild-type SIRP-α D1 domain which has two sequences, 10 or fewer amino acids Includes substitution. In some embodiments, the polypeptide of this embodiment of the present disclosure is the same as in SEQ ID NO: 1. For wild-type SIRP-α D1 domains having one or less sequences, 7 or fewer ami Includes no acid substitution.

[0114] In some embodiments, the polypeptide has one of the sequences of SEQ ID NO: 1. It binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRP-α D1 domain. To bind. In some embodiments, the polypeptide is one of the components of SEQ ID NO: 1. With binding affinity at least 100 times higher than that of the wild-type SIRP-α D1 domain with columns It binds to CD47. In some embodiments, the polypeptide is one of the elements of SEQ ID NO: 1 or at least 1000 times higher than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 by binding affinity. In some embodiments, the fragment is 10 amino acids long. Less than, approximately 10 amino acids in length, approximately 20 amino acids in length, approximately 30 amino acids in length, approximately 40 amino acids in length No acid, approximately 50 amino acids in length, approximately 60 amino acids in length, approximately 70 amino acids in length, approximately 80 amino acids in length No acids, approximately 90 amino acids in length, approximately 100 amino acids in length, or more than approximately 100 amino acids in length Contains lipeptides. The fragments retain their ability to bind to CD47. Preferably, SIRP. -α D1 variant polypeptide and its fragments are SIRP-α polypeptides with CD4 It binds to CD47 with a higher affinity than it binds to 7. For example, in some embodiments, SIRP-α D1 variant polypeptide or its fragments are K D 1x10 -8 M not yet Full, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 -11 It binds to CD47 at less than M. In some embodiments, SIRP-α D1 variant polypeptide or its fragments are K 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 It binds to CD47 at M, or approximately 50 pM to 10 pM.

[0115] In another embodiment, this disclosure is, EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQ WFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDF SISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSV RAKPS (Sequence ID 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, X5 The sequence has H, P, or R, X6 is S, T, or G, and X7 is A. It includes a SIRP-α D1 variant, and the variant is one of the following in Sequence ID No. 2 At least one amino acid substitution in the wild-type SIRP-α D1 domain that has the sequence It is characterized by a polypeptide having [a specific characteristic].

[0116] In some embodiments, the polypeptide is sequence number 212, where X1 is V, L or has an array that is I. In any of the embodiments of this disclosure described above, X2 is V, I, or L. In any of the above embodiments, X3 is I, T, S, and is F. In any of the above embodiments, X4 is K or R. Above embodiments 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 sequence number 212, where X1 is V and X has an array that is I. In any of the embodiments of this disclosure described above, X2 is V or I. In any of the above embodiments, X3 is I or F. In any of the 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 or T. In any of the above embodiments, X7 is A.

[0118] In some embodiments, the polypeptide is sequence number 212, where X7 is A. It has an array. In any of the embodiments of this aspect of the present disclosure, X7 is A, X1 is V or I. In any of the embodiments described herein, X7 is A And X2 is V or I. In any of the above embodiments, X7 is A and X 3 is I or F. In any of the above embodiments, X7 is A and X4 is K. or is R. In any of the above embodiments, X7 is A and X5 is H or P. Yes. In any of the above embodiments, X7 is A, and X6 is S or T.

[0119] In some embodiments, the polypeptide is sequence number 212, where X7 is A. It has an array. In any of the embodiments of this aspect of the present disclosure, X7 is A, X1 is I. In any of the embodiments of this disclosure described herein, 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 either of the above embodiments, X7 is A and X5 is P. X7 is A, and X6 is T.

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

[0121] In some embodiments, the polypeptides of this embodiment of the Disclosure are any one of the 1s in SEQ ID NO: 2 For the wild-type SIRP-α D1 domain which has two sequences, 10 or fewer amino acids Includes substitution. In some embodiments, the polypeptide of this embodiment of the present disclosure is the same as in SEQ ID NO: 2. For wild-type SIRP-α D1 domains having one or less sequences, 7 or fewer ami Includes no acid substitution.

[0122] In some embodiments, the polypeptide has one of the sequences of SEQ ID NO: 2. It binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRP-α D1 domain. To bind. In some embodiments, the polypeptide is one of the components of SEQ ID NO: 2. With binding affinity at least 100 times higher than that of the wild-type SIRP-α D1 domain with columns It binds to CD47. In some embodiments, the polypeptide is one of the numbers in SEQ ID NO: 2 or at least 1000 times higher than wild-type SIRP-α D1 domains with a single sequence. It binds to CD47 by binding affinity. In some embodiments, the fragment is 10 amino acids long. Less than, approximately 10 amino acids in length, approximately 20 amino acids in length, approximately 30 amino acids in length, approximately 40 amino acids in length No acid, approximately 50 amino acids in length, approximately 60 amino acids in length, approximately 70 amino acids in length, approximately 80 amino acids in length No acids, approximately 90 amino acids in length, approximately 100 amino acids in length, or more than approximately 100 amino acids in length Contains lipeptides. The fragments retain their ability to bind to CD47. Preferably, SIRP. -α D1 variant polypeptide and its fragments are SIRP-α polypeptides with CD4 It binds to CD47 with a higher affinity than it binds to 7. For example, in some embodiments, SIRP-α D1 variant polypeptide or its fragments are K D 1x10 -8 M not yet Full, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, or 1x10 -11 It binds to CD47 at less than M. In some embodiments, SIRP-α D1 variant polypeptide or its fragments are K 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 It binds to CD47 at M, or approximately 50 pM to 10 pM.

[0123] In some embodiments, the following is disclosed herein: EEELQX1IQPDKS VLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LI YNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 KFRKGSPDDVEFKSGAGTELSVRA KPS (Sequence ID 218), where 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, and X9 is any amino acid, X 10 is any mesh It is an acid, X 11 is any amino acid, X 12 is any amino acid, X 13 teeth The SIRP-α D1 variant comprises a sequence that is V or I, and the SIRP-α The D1 variant is compared to the wild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 1. It is a polypeptide having at least two amino acid substitutions.

[0124] In some embodiments, the polypeptide is sequence number 212, where X9 is A. It has an array. In any of the embodiments of this disclosure described above, X9 is N. In any of the embodiments described herein, X 10 is I. This aspect of the present disclosure In any of the above embodiments, X9 is N and X10 is P. The essence of the present disclosure In any of the above embodiments, X9 is N, and X11 is S, T, or C. It is any other amino acid. In any of the embodiments of this disclosure described above, X 11 is T. In any of the above embodiments of this disclosure, X 11 is any other than T It is an amino acid. In any of the embodiments of this disclosure described herein, X 12 It is P In any of the embodiments described herein, X9 is N and X 12 is P or It is any amino acid outside of this group.

[0125] In some embodiments, the following are disclosed herein: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNM DFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSG AGTELSVRAKPS (Sequence ID 219), where X1 is V, L, or I. X2 is A, V, L, or I, X3 is I, S, T, or F, and X4 is E. X5 is L or V, X6 is K or R, X7 is E or Q, and X7 is H or R , or P, X8 is S, G, L, or T, X9 is N, X 10 is P or Any amino acid outside of X 11 SIRP-α D1 has a sequence that is V or I. The variant includes a wild-type variant having the sequence of SEQ ID NO: 1. Polypeptide having at least two amino acid substitutions in the type SIRP-α D1 domain It's Chido.

[0126] In another aspect of this disclosure, SIRP-α D1 having the amino acid sequence of SEQ ID NO: 48 A composition comprising a variant polypeptide or a fragment thereof is disclosed. Several embodiments Therefore, the SIRP-α D1 variant polypeptide or its fragment is SIRP-α It binds to CD47 with a higher affinity compared to the affinity of the lipeptide to CD47. In some embodiments, the SIRP-α D1 variant polypeptide is K D 1x1 0 -8 Less than M or 1x10 -9 Less than M, 1x10 -10 Less than M, or 1x10 -1 1 It binds to CD47 at less than M. In some embodiments, the above SIRP-α D1 variant An antinucleotide is bound to or fused with a second polypeptide. Several implementations In this state, the second polypeptide can be any Fc polypeptide, Fc variant, etc., without limitation. Examples include HSA polypeptides, albumin peptides, PEG polymers, or the above fragments. It is possible.

[0127] Without limiting the above, in some embodiments, SIRP-α D1 variant The lipeptide is selected from one of the sequence numbers 53-87 and 213 shown in Table 6. . [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0128] In some embodiments, the polypeptide is used for any variant 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%, It contains a high-affinity SIRP-α D1 domain (or 100% sequence identity).

[0129] In some embodiments, the polypeptide is sequence number 80, 81, or 85 in Table 6. For this, at least 85% sequence identity (e.g., at least 86%, 87%, 88%) 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% High affinity SIRP-α D1 domain with 99% or 100% sequence identity Includes.

[0130] III. Fc Domain Variants and Fusion Structures In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0131] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0132] Antibodies that target cell surface antigens are involved in the engagement of Fc receptors (FcRs) in immune cells. It can induce immune stimulation and effector functions. IgG (gamma receptor) IgE (eta receptor), IgA (alpha receptor), and IgM (mu receptor) There are multiple Fc receptors specific to certain antibody classes, including the one mentioned. Fc receptors on the cell surface. The binding of the Fc region to the body is related to the phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis), and (ADCP), removal of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell lysis). Cell-mediated cytotoxicity (ADCC), as well as the release of inflammatory mediators, and placental cross-section. This can trigger many biological responses, including the regulation of immunoglobulin production. Furthermore, the binding of the complement C1 component to an antibody can activate the complement system. Complement activation can be important for the lysis of cellular pathogens. However, complement activation Sexualization can also stimulate inflammatory responses and is involved in autoimmune hypersensitivity and other immune disorders. In some cases, a variant F has reduced or eliminated binding ability to a specific Fc receptor. The c region targets and activates ligand function without damaging or destroying local cells or tissues, This involves developing therapeutic antibodies and Fc fusion polypeptide constructs that act by neutralization. It is useful for [something].

[0133] In some embodiments, the SIRP-α D1 polypeptide construct is used in effectors. Non- Contains the naturally occurring high-affinity SIRP-α D1 variant.

[0134] In some embodiments, the Fc domain monomer is the second and third antibody constant domain. This refers to a polypeptide chain containing (e.g., CH2 and CH3). In some embodiments, this refers to a polypeptide chain containing nucleotides (e.g., CH2 and CH3). The Fc domain monomer also includes a hinge domain. In some embodiments, the F The c-domain monomer is any immunoglobulin, including IgG, IgE, IgM, IgA, and IgD. It is an epidemic globulin antibody isotype. Furthermore, in some embodiments, Fc The main monomer is any IgG subtype (e.g., IgG1, IgG2, IgG2a) These are IgG2b, IgG2c, IgG3, and IgG4. Several implementations In this state, the Fc domain monomer undergoes as many as 10 changes from the wild-type Fc domain monomer sequence. (For example, substitution, addition, or deletion of 1-10, 1-8, 1-6, or 1-4 amino acids.) This includes the loss of (or a combination thereof) and this alters the interaction between the Fc domain and the Fc receptor. To transform.

[0135] As used herein, the term "Fc domain" refers to the two Fc domain monomers. This refers to the mer. In the wild-type Fc domain, the two Fc domain monomers are two CH3 antagonists. Interactions between constant domains in the body, and the hindrance of the two dimerized Fc domain monomers Dimerization occurs through the formation of one or more disulfide bonds between didomains. In this embodiment, the Fc domain lacks effector functionality, for example, "dead F The c domain is mutated. In some embodiments, each Fc domain in the Fc domain The in monomer contains amino acid substitutions in the constant domain of the CH2 antibody, and the Fc domain and F c receptors, for example, Fcγ receptor (FcγR), Fcα receptor (FcαR), or Fc It reduces the interaction or binding between ε(FcεR).

[0136] In some embodiments, a high-affinity SIRP-α D1 variant (e.g., Tables 2 and 5) is used. , and any of the variants shown in 6) are immunoglobulin Fc domain monomers or It is fused to a fragment of the Fc domain monomer. In some embodiments, immunoglobulin An Fc domain monomer or fragment of an Fc domain monomer is a different Fc domain monomer. It is possible to form an Fc domain together with immunoglobulins. In some embodiments, immunoglobulins Roblin's Fc domain monomer or fragment of Fc domain monomer is another Fc domain It cannot form an Fc domain with the monomer. In some embodiments, An Fc domain monomer or fragment of an Fc domain is fused to the polypeptide of this disclosure. The serum half-life of the polypeptide is extended. In some embodiments, the polypeptide of the present disclosure The Fc domain monomer or fragment of the Fc domain monomer fused to the second Fc It becomes a domain monomer and dimer, forming an Fc domain that binds to the Fc receptor, Alternatively, the Fc domain monomer binds to the Fc receptor. In some embodiments, This involves fused Fc-type compounds to the polypeptide to extend its serum half-life. Neither the main Fc domain nor any fragment of it induces any immune system-related response.

[0137] In some embodiments, the SIRP-α polypeptide or construct provided herein This is the SIRP-α D1 domain or its b linked to the first Fc domain monomer. The antibody variable domain comprises a riant and a second Fc domain monomer, the first And the second Fc domain monomer binds to the Fc domain (e.g., heterodimer Fc It forms a domain. An Fc domain is a protein found at the C-terminus of immunoglobulins. This is the qualitative structure. The Fc domain dimers through interactions between constant domains of the CH3 antibody. It contains two Fc domain monomers that are converted. The wild-type Fc domain is an Fc receptor, for example For example, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIII b forms a minimal structure that binds to FcγRIV.

[0138] The Fc domain is not directly involved in the binding of the antibody to its antigen, but rather in antibody-dependent mechanisms. It may be involved in various effector functions, such as the involvement of the antibody in cytotoxicity. In that embodiment, the Fc domain of the SIRP-α polypeptide or construct of the present disclosure is Reduced effector function, for example, decreased antibody-dependent cell-mediated cytotoxicity (ADCC), Decreased complement-dependent cell lysis (CDC), decreased antibody-dependent cell-mediated phagocytosis (ADCP), Or any combination thereof, resulting in amino acid substitutions, additions or insertions, deletions, or This includes any combination thereof. In some embodiments, the SIRP-α polypeptide of the Disclosure The cytoplasm or construct reduces binding to the human Fc receptor (e.g., minimal binding or Absence of binding and reduced binding to complement protein C1q (e.g., minimal binding) (or the absence of bonding) is a characteristic feature. In some embodiments, the SIRP-α of the present disclosure The constructs are human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, Fc γRIIIB, or any combination thereof, and a reduction in binding to C1q (e.g., Characterized by minimal or no binding. Antibody-dependent effector function, for example If so, change or decrease ADCC, CDC, ADCP, or any combination thereof. Therefore, in some embodiments, the Fc domain of the SIRP-α construct of this disclosure is IgG It is a class that has one or more amino acid substitutions, E233, L234, L235, G2 36, G237, D265, D270, N297, E318, K320, K322, A3 27, A330, P331, or P329 (numbering is Kabat EU) Follow the index (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, polypeptide constructs comprising the non-natural Fc region described herein Compared to polypeptide constructs containing the natural Fc region, CD16a, CD32a, C Reduced binding of D32b, CD32c, and CD64 to at least one of the Fcγ receptors. It indicates a small amount or removal. In some cases, the polypeptide constructs described herein are , against Fcγ receptors of CD16a, CD32a, CD32b, CD32c, and CD64 This indicates a decrease or removal of the bond.

[0140] CDC refers to the complement cascade, which is activated by the complement component C1q that binds to the Fc of antibodies. This refers to a cytotoxic form. In some embodiments, the non-natural Fc region described herein Polypeptide constructs containing the region are compared to polypeptide constructs containing the wild-type Fc region. At least 5%, 10%, 15%, 20%, 30%, 40%, and 50% of the C1q bond , showing a decrease of 60%, 70%, 80%, 90% or more. Some embodiments Therefore, polypeptide constructs containing non-natural Fc regions as described herein use the wild-type Fc region Compared to polypeptide constructs containing this, a decrease in CDC is observed. In some embodiments, this The polypeptide constructs containing the non-natural Fc region described in the specification are polypoly containing the wild-type Fc region. Compared to the peptide construct, at least 5%, 10%, 15%, 20%, 30%, 40% This indicates a decrease in CDC of 50%, 60%, 70%, 80%, 90%, or more. In some cases, polypeptide construction containing the non-natural Fc variant described herein. The substance has a negligible CDC compared to polypeptide constructs containing the wild-type Fc region. show.

[0141] In some embodiments, the Fc variants herein are minimal compared to the wild-type sequence. It is either fully glycosylated or its glycosylation is reduced. In some embodiments, it is deglycosylated. Glycosylation occurs when N297A is mutated, or when N297 is replaced with any amino acid other than N. This is achieved by causing a difference. In some embodiments, deglycosylation is performed by the motif The equation is N-Xaa1-Xaa2-Xaa3, where N = asparagine and Xaa1 = P(pro). Any amino acid other than phosphorus, Xaa2=T (threonine), S (serine), or C (syl Destruction of the motif, which is any amino acid other than stain and Xaa3=P (proline). This is achieved by the N-Xaa1-Xaa2-Xaa3 motif in one embodiment. This refers to residues 297-300, as shown according to Kabat et al., 1991. In some embodiments, one or more of N, Xaa1, Xaa2, or Xaa3 Mutations in this variant result in the deglycosylation of the Fc variant.

[0142] In some embodiments, variants of the antibody IgG constant region (e.g., Fc variants) are used. ) has a reduced ability to specifically bind to Fcγ receptors, or induces phagocytosis. The ability to do so is reduced. In some embodiments, variants in the constant region of the antibody IgG (e.g.) For example, the Fc variant has a reduced ability to specifically bind to the Fcγ receptor, and , the ability to induce phagocytosis is reduced. For example, in some embodiments, Fc dome The "dead" Fc domain is mutated to lack effector functionality. For example, in some embodiments, the Fc domain is located between the Fc domain and the Fcγ receptor. Includes certain amino acid substitutions that have been shown to minimize interactions. In the application form, the Fc domain monomer is derived from the IgG1 antibody, and the amino acid substitution L234 A, L235A, G237A, and N297A (Kabat et al., 1991) Therefore, it includes one or more of the following (as shown in the EU numbering system). In some embodiments, This is included in the Fc variant of IgG1, which undergoes one or more further mutations. Human IgG Non-limiting examples of such further mutations for the Fc variant include E318A and K322A is one example. In some cases, the Fc variant of human IgG1 is wild. Compared to the human IgG1 sequence, there are a total of up to 12, 11, 10, 9, 8, 7, 6, and 5 differences. It has four or fewer mutations. In some embodiments, it has one or more Further deletions are found in the Fc variant of IgG1. For example, several implementations In this state, the lysine at the C-terminus of the IgG1 heavy chain constant region of Fc, as shown in sequence number 88 of Table 7, Deleted, for example, when the polypeptide is produced in bacterial or mammalian cells, the polypeptide To improve the homogeneity of lipeptides. In some cases, the Fc variant of human IgG1 is used in the field Compared to the live human IgG1 sequence, the total difference was up to 12, 11, 10, 9, 8, 7, 6, 5. Or there are four or fewer deletions. In some embodiments, IgG1 The Fc variant is one of the following: SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137 It has one array.

[0143] In some embodiments, the Fc domain monomer is derived from an IgG2 or IgG4 antibody. This involves amino acid substitutions A330S, P331S, or both A330S and P331S. Includes. The aforementioned amino acid positions are defined according to Kabat, et al. (1991). The Kabat numbering of amino acid residues is applied to the "standard" Kabat for a given antibody. This can be determined by alignment of the homologous region of the antibody sequence with the t-number sequence. In some embodiments, the Fc variant is A330S, P331S, and N297A Amino acid substitution (according to Kabat et al. (1991) for EU numbering system) Includes a human IgG2 Fc sequence containing one or more of the following (indicated by Tem). Several embodiments Therefore, it is included in the Fc variant of IgG2 that involves one or more further mutations. Human Ig A non-limiting example of such further mutations for the G2 Fc variant is V234A , G237A, P238S, V309L, and H268A (Kabat et al.) Examples include those shown in the EU numbering system (1991). So, the Fc variant of human IgG2, compared to the wild-type human IgG2 sequence, is the most... It has 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer major mutations. In some embodiments, one or more further deletions of IgG2 result in the Fc variant. It is included in the set. For example, in some embodiments, Fc shown in sequence number 89 of Table 7 The lysine at the C-terminus of the IgG double-chain constant region is deleted, for example, the polypeptide can be used in bacterial cells. To enhance the homogeneity of the polypeptide when produced in cells or mammalian cells. In some examples, The Fc variant of human IgG2 is the largest in total compared to the wild-type human IgG2 sequence. There are 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer missing characters. ru.

[0144] If the Fc variant is an Fc variant of IgG4, in some embodiments, Such Fc variants are mutations in S228P (Kabat et al. (1991) (as shown by) includes. In some cases, the human IgG4 Fc variant is wild-type Compared to human IgG4 sequences, the total range is up to 12, 11, 10, 9, 8, 7, 6, 5, 4. It has 3, 2, or 1 mutation.

[0145] In some embodiments, the Fc variant is the L234A mutation in the Fc region of IgG1. , at least one of L235A, G237A, or N297A, or IgG2 Contains at least one of the following mutations in the Fc region: A330S, P331S, or N297A. In some embodiments, the Fc variant is the L234A mutation in the Fc region of IgG1. At least two of L235A, G237A, or N297A, or F of IgG2 It contains at least two mutations in the c region: A330S, P331S, or N297A. In some embodiments, the Fc variant is a mutation L234A in the Fc region of IgG1, L Contains at least three of 235A, G237A, or N297A, or IgG The mutations in the Fc region of 2 consist of A330S, P331S, and N297A. Several implementations Morphologically, the Fc variant is mutation L234A, L235A, G237A, and N29 It consists of 7A.

[0146] In some embodiments, the Fc variant is compared to the Fc region of wild-type human IgG. This shows a decrease in binding to the target Fc receptor. In some embodiments, the Fc barrier The test removes the binding of the target Fc receptor compared to the Fc region of wild-type human IgG. In some embodiments, the Fc variant is compared to the Fc region of wild-type human IgG. In comparison, it shows reduced phagocytosis. In some embodiments, the Fc variant is wild-type Compared to the Fc region of IgG, it shows removal of phagocytic activity.

[0147] Sequence IDs 88 and 89 are amino acids in the constant regions of the IgG1 and IgG2 double chains of Fc. The acid sequence is shown. In some embodiments, the Fc variant is sequence number 90~ shown in Table 7. It is any variant of 95. [Table 7-1] [Table 7-2]

[0148] Antibody-dependent cell-mediated cytotoxicity, also known as ADCC in this specification, refers to a specific type of cell-mediated cytotoxicity. Fc receptors present on toxic cells (e.g., natural killer (NK) cells and neutrophils) Secreted Ig bound to (FcR) allows these cytotoxic effector cells to carry antigens. Cell damage that specifically binds to target cells and subsequently allows for the killing of those target cells. This refers to the sexual morphology. Antibody-dependent cell-mediated phagocytosis, also known as ADCP in this specification, is a certain The amount that binds to the Fc receptor (FcR) present in specific phagocytic cells (e.g., macrophages) The released Ig then allows these phagocytic effector cells to specifically bind to antigen-carrying target cells. Subsequently, it refers to a cytotoxic form that enables the target cells to be taken up and digested. Ligand-specific high-affinity IgG antibodies against the surface of target cells are used to target cytotoxic cells or phagocytic cells. Cells can be stimulated and used for such killing. In some embodiments The polypeptide constructs containing the Fc variants described herein include a wild-type Fc region. Compared to polypeptide constructs, it shows a decrease in ADCC or ADCP. In application, the polypeptide construct containing the Fc variant described herein is wild-type Fc Compared to polypeptide constructs containing the region, at least 5%, 10%, 15%, 20%, ADCC of 30%, 40%, 50%, 60%, 70%, 80%, 90% or more or shows a decrease in ADCP. In some embodiments, the Fc Varian described herein. Polypeptide constructs containing the wild-type Fc region are superior to polypeptide constructs containing the wild-type Fc region. Indicates the removal of ADCC or ADCP.

[0149] Complement-dependent cytotoxicity, also known as CDC in this specification, is the cytotoxicity of the complement cascade by antibodies. This refers to a cytotoxic form activated by the complement component C1q, which binds to c. In the embodiments, the polypeptide construct comprising the Fc variant described herein is wild-type F Compared to polypeptide constructs containing the c region, at least 5%, 10%, 15%, and 20% 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of C1q It shows a decrease in bonding. In some cases, the Fc variant described herein contains Lipeptide constructs show a reduction in CDC compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, polypeptides containing the Fc variant described herein The wild-type construct is at least 5% more concentrated than the polypeptide construct containing the wild-type Fc region. 0%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and This shows a decrease in CDC that exceeds that. In some cases, the FcB described herein Polypeptide constructs containing riantos are compared to polypeptide constructs containing wild-type Fc regions. And it shows a negligible level of CDC.

[0150] In this specification, the Fc variant is defined as Fcγ compared to the Fc region of wild-type human IgG. This includes those that show a decrease in binding to the receptor. For example, in some embodiments, Fc-ba As shown in the examples, the rianto is an Fcγ receptor represented by the Fc region of wild-type human IgG. It exhibits lower binding to Fcγ than to the substance. In some cases, the Fc barrier The result was a decrease in binding to the Fcγ receptor of 10%, 20%, 30%, 40%, and 50%. , 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (complete removal of effector function). In some embodiments, the combination The decrease is due to any one or more Fcγ receptors, e.g., CD16a, CD32a, CD32b This applies to CD32c or CD64.

[0151] In some cases, the Fc variant disclosed herein is the F of wild-type human IgG. Compared to the c region, it shows reduced phagocytosis. Such Fc variants are associated with wild-type human Ig Compared to the Fc region of G, it shows a decrease in phagocytosis, and this decrease in phagocytic activity is, for example, 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% These are 97%, 98%, 99%, or 100%. In some examples, the Fc variant... This shows the removal of phagocytic activity compared to the Fc region of wild-type human IgG.

[0152] In some embodiments, the Fc variant disclosed herein is one or more fusion parts It is bonded to the toner. In some cases, the fusion partner is the treatment portion. In some cases, the fusion partner targets and purifies the expressed protein. Selected to enable screening, presentation, etc. In some embodiments, the melt The binding partner also affects the degree of binding to the Fc receptor or the degree of phagocytosis reduction. As described in the specification, in some embodiments, the Fc variant is a fusion partner When combined, it forms the polypeptide construct shown below.

[0153] In some embodiments, the fusion partner is the Fc variant via the linker array. The sequence is linked. In some embodiments, the linker sequence is generally a small number of amino acids. For example, longer linkers containing fewer than 10 amino acids are also used. In that case, the linker contains 10, 9, 8, 7, 6, or fewer than 5 amino acids. , or having a shorter length. In some cases, the linker is at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 amino acids , or having a longer length. Optionally, in some embodiments, a cleavable phosphorus. A car is used.

[0154] In some embodiments, the fusion partner is an Fc variant protein and any associated Targeting or signaling the linked fusion partner to a desired cellular location or extracellular medium It is a signal sequence. In some embodiments, a particular signal sequence is used in the growth medium, or The target is a protein secreted into either the inner or outer membrane of a cell, or into the pericellular lumen. In some embodiments, the fusion partner enables purification or screening. It is a sequence that codes for a peptide or protein. Such a fusion partner is , immobilized metal affinity chromatography (IMAC) system (e.g., Ni+ Polyhistidine tags (His tags) for use with 2 affinity columns. For example, His6 and His10) or other tags, GST fusions, MBP fusions, St rep-tag, BSP biotinylation target sequence of bacterial enzyme BirA, and antibody targeted Examples of epitope tags (for example, c-myc tag, flag tag, etc.) Not limited.

[0155] In some embodiments, such tags are used in purification, screening, or both. It is for use. For example, in some embodiments, the Fc variant uses the His tag, and It is purified by immobilizing it on a Ni+2 affinity column, and then, after purification, the same Antibodies are immobilized on Ni+2 coated plates using His tags, and then ELISA and other binding tests are performed. The process is carried out as described elsewhere in this specification. In some embodiments, the fusion partner - Enables the use of the selection method for screening Fc variants described herein. ru.

[0156] Various fusion partners are available, enabling a variety of selection methods. For example, FcB By fusing members of the riant library to the gene III protein Phage presentation methods can be employed. In some embodiments, the fusion partner is , so that the Fc variant is labeled. Alternatively, in some embodiments, the fusion The partner binds to a specific sequence of the expression vector, and the fusion partner and associated Fc vector The riant is covalently or noncovalently linked to the nucleic acid it encodes. I'll do that.

[0157] In some embodiments, if the fusion partner is a therapeutic portion, the therapeutic portion is, for example, This includes peptides, proteins, antibodies, siRNAs, or small molecules. The FcVarian of this disclosure. A non-exclusive example of a therapeutic antibody that binds to a specific host is an antibody that recognizes CD47. However, this does not limit the therapeutic peptides that can be bound to the Fc variants of this disclosure. Typical examples include CD47-binding polypeptides, including SIRP-α polypeptides. However, this is not limited to these examples. In such examples, the CD47-conjugated polypeptide is the It is bound to or fused to the Fc variant. An example of a CD47-binding polypeptide is anti-C A D47 antibody or a fragment thereof, and a CD47 ligand, such as SIRP-α or Examples of CD47-conjugated polypeptides include, but are not limited to, fragments. Examples include naturally occurring forms of SIRP-α and its mutants, but are not limited to these. It is not determined.

[0158] In some embodiments, the following is disclosed herein: polyp including Fc variant A peptide in which the Fc variant has two Fc domain monomers It contains an indimer, and each Fc domain monomer is independently (i) L234A, L235 (ii) A3 The Fc region of human IgG2 consisting of mutations 30S, P331S and N297A, or (i ii) S228P, E233P, F234V, L235A, delG236, and N29 The polypeptide is selected from the Fc region of human IgG4 containing the 7A mutation. In that embodiment, the Fc domain monomers are identical (i.e., homodimers). In some embodiments, the Fc domain monomer is different (i.e., heterodimer) ). In some embodiments, at least one of the Fc domain monomers of the Fc domain dimer Another is Human I, which consists of mutations L234A, L235A, G237A, and N297A. This is the Fc region of gG1. In some embodiments, the Fc domain of the Fc domain dimer At least one of the monomers is derived from mutations of A330S, P331S, and N297A. This is the Fc region of human IgG2. In some embodiments, the Fc variant is human Compared to the wild-type version of the IgG Fc region, it shows removal or reduction of binding to the Fcγ receptor. In some embodiments, the Fc variant is the wild-type version of the Fc region of human IgG. In comparison, the Fcγ receptors of CD16a, CD32a, CD32b, CD32c, and CD64 This indicates the removal or reduction of binding to the substance. In some embodiments, the Fc variant This involves the removal or reduction of binding to C1q compared to the wild-type version of human IgG Fc fusion. This shows that in some embodiments, the Fc domain dimer has a small amount of Fc domain monomer. At least one of them is S228P, E233P, F234V, L235A, delG236, and This is the Fc region of human IgG4 containing the N297A mutation. In some embodiments, The Fc variant, compared to the Fc region of wild-type human IgG4, binds to the Fcγ receptor. It indicates the removal or reduction of the compound. In some embodiments, the Fc variant is its human I Compared to the wild-type version of the Fc region of gG4, the CD16a and CD32b Fcγ receptors It indicates the removal or reduction of the bond. In some embodiments, the Fc variant is Fc It binds to the γ receptor with a KD of over approximately 5 x 10⁻⁶ M.

[0159] In some embodiments, the Fc variant further comprises a CD47-conjugated polypeptide. Includes. In some embodiments, the Fc variant is the wild-type version of the Fc region of human IgG. Compared to this, it shows removal or reduction of binding to the Fcγ receptor. In some embodiments, The CD47-binding polypeptide causes acute anemia in rodents and non-human primates. It does not cause. In some embodiments, the CD47-binding polypeptide is used in humans. It does not cause acute anemia.

[0160] In some embodiments, the CD47-binding polypeptide is a signal regulatory protein α (SIRP-α) polypeptide or a fragment thereof. In some embodiments, the SI RP-α polypeptide has an amino acid sequence EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMD FSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTE LSVRAKPS (sequence number 51), where X1 is V or I, and X2 is A or X3 is I, X4 is I or F, X5 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. Yes, X9 contains a SIRP-α D1 variant that includes a sequence that is V or I. How many In one embodiment, the SIRP-α polypeptide is such that X1 is V or I and X2 is A or I, X3 is I or F, X4 is E, 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. Includes IRP-α D1 variant.

[0161] In some embodiments, the polypeptide disclosed herein is non-auto It is a naturally occurring high-affinity SIRP-α D1 domain, and the affinity of the naturally occurring D1 domain SIRP-α D1 varian binds to human CD47 with at least 10 times higher affinity. The Fc domain monomer is linked to a second polypeptide containing a second Fc domain monomer. An Fc domain monomer that forms a , and in which the Fc domain is removed or reduced This polypeptide contains the Fc domain monomer having an enzyme function. In the application form, the non-naturally occurring high-affinity SIRP-α D1 domain has an amino acid at residue 80. Includes acid mutations.

[0162] In some embodiments, the SIRP-α D1 varian is disclosed herein. In this case, the SIRP-α D1 variant is less than 250nM in KD. The SIRP-α D1 variant binds to CD47 derived from this species, and has a KD of less than 250 nM. It binds to CD47 derived from the second species, and the KD for CD47 derived from the first species, The KD ratios for CD47 derived from the second species are no more than 100 times each, and the first species and the second The species are selected from a group consisting of humans, rodents, and non-human primates. Several implementations Morphologically, the SIRP-α D1 variant is derived from CD4 of at least three different species. It binds to 7. In some embodiments, the non-human primate is a cynomolgus macaque.

[0163] In some embodiments, the following are disclosed herein: (a) KD less than 250 nM (b) the SIRP-α D1 domain that binds to human CD47, and (b) the SIRP-α D1 A polypeptide comprising an Fc domain monomer ligated to the N-terminus or C-terminus of the domain. In this case, the polypeptide causes acute anemia in rodents and non-human primates. It does not cause. In some embodiments, the polypeptide is an unnatural human SIRP-α. It is a variant of the development. In some embodiments, in vivo administration of the polypeptide is This results in a reduction of less than 50% in hemoglobin during the first week after administration. In some embodiments, In humans, administration of the polypeptide resulted in a reduction of hemoglobin to less than 50% during the first week after administration. This results in a reduction. In some embodiments, the polypeptide further comprises at least one The Fc variant includes (i) L234A, L235A (ii) A33 The Fc region of human IgG2 consisting of mutations 0S, P331S and N297A, or (ii i) S228P, E233P, F234V, L235A, delG236, and N297 It contains an Fc domain monomer selected from the Fc region of human IgG4 containing mutation A. In some embodiments, the Fc domain monomer is L234A, L235A, G237 This is the Fc region of human IgG1 consisting of mutations A and N297A. Several embodiments Therefore, the Fc domain monomer is derived from mutations in A330S, P331S, and N297A. This is the Fc region of human IgG2.

[0164] The SIRP-α constructs of this disclosure can be obtained by conventional genetic or chemical methods, for example, chemical bonding. Using this, the C-terminus of the Fc domain monomer, which is linked to the N-terminus by the linker, It includes a SIRP-α domain or a variant thereof. In some embodiments, phosphorus A car (for example, a spacer) is inserted between the polypeptide and the Fc domain monomer. In some embodiments, the poly(P) of the present disclosure includes a high-affinity SIRP-α D1 variant. The peptide is fused to an Fc domain monomer that cannot form a dimer. In some embodiments, the polypeptides of the Disclosure are used to form dimers, for example, heterodimers. It can be fused to another Fc domain monomer to form a new Fc domain monomer. In some embodiments, the polypeptide of the present invention is fused to an Fc domain monomer, This fusion protein forms a homodimer. In some embodiments, the polydimer of the present disclosure A peptide is fused to the first Fc domain monomer, resulting in a different protein or peptide ( For example, the antibody variable region is fused to the second Fc domain monomer. Several implementations In this state, the SIRP-α D1 domain or its variant is the first Fc domain mono. It is linked to therapeutic proteins (e.g., cytokines, interleukins, antigens, Steroids, anti-inflammatory drugs, or immunomodulators are linked to a second Fc domain monomer. In some embodiments, the first and second Fc domain monomers are heterodimers. It forms.

[0165] Without limiting the above, in some embodiments, SIRP-α D1 variant Lipeptides (e.g., any of the variants shown in Tables 2, 5, and 6) are Fc polypeptides It is fused to a cytoplasmic or Fc variant polypeptide, for example, an Fc domain monomer. SIRP-α D1 variant polypeptide and fused Fc variant polypeptide Examples of polypeptides containing this include SEQ ID NOs. 96-137, 214, and 21 shown in Table 8. Six examples exist, but this list is not limited to these. [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 is used for any variant 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%, It contains a high-affinity SIRP-α D1 domain (or 100% sequence identity).

[0167] In some embodiments, the polypeptide is sequence numbers 98-104, 107- in Table 8. For 113, 116-122, or 135-137, at least 85% sequence identity is observed. Sex (for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 Sequence identity of 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It contains a high-affinity SIRP-α D1 domain that has the following properties.

[0168] In some embodiments, the polypeptide is (a) signal regulatory protein α(SI It contains the RP-α)D1 variant, and the SIRP-α D1 variant is amino 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 Includes KPS (sequence number 47), where 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, X8 is I or T, X9 is H, R, or L, X 10 is A, V, I, or L Yes, X 11 is I, T, S, or F, X 12 is A or G, and X 13 is E, V or L, X 14 is K or R, X 15 is E or Q, and X 16 teeth H, P, or R, X 17 is D or E, X 18 is S, L, T, or G Yes, X 19 is K or R, X 20 is E or N, X 21 is S or P ri, X 22 is S or R, X 23 is S or G, X 24 is any amino acid Yes, X 25 is any amino acid, X 26 is V or I, X 27 is F, L, ma ta is V, X 28 is either D or does not exist, 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 , in the wild-type SIRP-α D1 domain having one of the sequences of sequence numbers 1 to 10 In contrast, the polypeptide has at least two amino acid substitutions, and (b) two This includes an Fc variant comprising an Fc domain dimer having an Fc domain monomer, and here Then, each Fc domain monomer independently contains (i) the N297A mutation in human IgG1 (ii) Human IgG containing mutations in the Fc region, (ii) L234A, L235A, and G237A (iii) Mutations in the Fc region of 1, L234A, L235A, G237A, and N297A The Fc region of human IgG1 containing (iv)N297A mutation, and the Fc region of human IgG2 containing (iv)N297A mutation. (v)Fc region of human IgG2 including A330S and P331S mutations, (vi)A The Fc region of human IgG2, including mutations 330S, P331S, and N297A, (vii ) Includes mutations in S228P, E233P, F234V, L235A, and delG236 Fc region of human IgG4, or (viii)S228P, E233P, F234V, L The Fc region of human IgG4 contains mutations in 235A, delG236, and N297A. .

[0169] In some embodiments, the polypeptide includes the amino acid sequence of SEQ ID NO: 47. RP-α D1 variant, Fc domain dimer having two Fc domain monomers Includes an Fc variant containing, in this case the Fc domain mono of the Fc domain dimer One of the MARs is a human variant containing the L234A, L235A, G237A, and N297A mutations. Includes the Fc region of IgG1.

[0170] Dimerization of Fc domain monomers

[0171] In some embodiments, SIRP-α D1 variant polypeptides (e.g., Table 2) are used. (Any of the variants shown in 5 and 6) has the N-terminus attached to the first Fc domain monomer. or is fused at the C-terminus. In some embodiments, the first Fc domain monomer is It is not possible to form an Fc domain or dimer. In some embodiments, the One Fc domain monomer binds to a second Fc domain monomer, and the Fc domain also The first and second Fc domain monomers form a dimer. In some embodiments, the first and second Fc domain monomers - Amino acid substitutions that promote 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 the 2 The present invention includes amino acid substitutions that promote heterodimerization of two monomers. For example, a SIRP-α construct is fused to a first Fc domain monomer. -α D1 variant polypeptide is included in the first subunit and the second Fc domain molecule Nomers are included (for example, SIRP-α D1 variant polypeptide as well as any other polypeptides) It is formed from a second subunit (not including the petit). In some embodiments, it is constructed The substance has a single SIRP-α D1 variant polypeptide linked to the Fc domain. (For example, a single arm). In some embodiments, the structure is connected to the Fc domain. It has two SIRP-α D1 variant polypeptides (for example, double α (m). In some embodiments, K D SIRP-α D1 varian with a pulse strength of approximately 500 nM This is particularly useful for double-arm structures. In some embodiments, K D Approximately 50n The SIRP-α D1 variant with M is particularly useful for double-arm structures. In some embodiments, K D A SIRP-α D1 variant with approximately 5 nM was found to be a dubious product. Useful for double-arm and single-arm structures. In some embodiments, K D SIRP-α D1 variants with approximately 500 pM are used in double-arm structures and single Useful for arm structures. In some embodiments, K D SI with approximately 100 pM The RP-α D1 variant is useful for double-arm and single-arm structures. In some embodiments, K D The SIRP-α D1 variant, which has approximately 50 pM It is useful for double-arm and single-arm structures. In some embodiments, , K D A SIRP-α D1 variant with approximately 10 pM was found in a double-arm structure and It is useful for single-arm structures.

[0173] In some embodiments, heterodimerization of the Fc domain monomer involves two Fc domains The main monomer can be substituted with different but compatible substitutions, such as "knob and hole" residue pairs and charge This is facilitated by introducing residue pairs. The interaction between the knob and the hole is facilitated by the heterodimer While formation is prioritized, the interaction between knobs and between holes is due to three-dimensional collision, and The loss of favorable interactions hinders homodimer formation. The "hole" is the building block of the protein. This refers to the space created when an amino acid is substituted with a different amino acid that has a smaller side chain volume. A knob is a molecule in which the original amino acid of a protein is replaced by a different amino acid that has a larger volume in its side chain. This refers to the protrusions that form when this occurs. 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 It is involved in dimerization. In some embodiments, the amino acids of the knob and hole are two Fc dimers. One acts to promote or preferentially promote the heterodimerization of the main monomer. The hole in the constant domain of the CH3 antibody is shaped to accommodate the knob of another constant domain of the CH3 antibody. This is accomplished. In some embodiments, the hole in one CH3 antibody constant domain is removed from another CH3 It is formed to better accommodate the original amino acids of the antibody constant domain. In the application method, a knob in one CH3 antibody constant domain acts as the base of another CH3 antibody constant domain. It is formed to produce further interactions with the amino acids.

[0174] In some embodiments, the holes are made of large side chains such as tyrosine and tryptophan. The amino acid is replaced with an amino acid having a small side chain, such as alanine, valine, or threonine. For example, it can be produced by a mutation in the Y407V constant domain of the CH3 antibody. Similarly, in some embodiments, the knob is used to connect amino acids with small side chains to larger ones. By substituting with amino acids having side chains, for example, the T3 of the constant domain of the CH3 antibody. It is produced by the 66W mutation. In some embodiments, one of the Fc domain monomers It contains the knob mutation T366W, while the other Fc domain monomer is a hole mutation. This includes T366S, L358A, and Y407V. In some embodiments, high affinity The polypeptides of this disclosure, including the SIRP-α D1 variant, are T36 variants of the knob. It fuses to the Fc domain monomer containing 6W, suppressing the formation of unwanted knob-to-knob homodimers. Examples of knob and hole amino acid pairs are included in Table 9, but are not limited to these. 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 knob and hole method, in some embodiments, dimers of Fc domain monomers To control the turbulence, electrostatic steering is also used. Electrostatic steering is a more advanced steering system. To control the formation of the next protein molecule, peptides, protein domains, and proteins This refers to utilizing the favorable electrostatic interactions between amino acids that are charged in opposite directions. CH uses electrostatic steering to control the dimerization of Fc domain monomers. One or more amino acid residues constituting the interface between 3 and CH3 are the specific interactions introduced into the interface. Depending on the charged amino acid, it is positively or negatively charged to be electrostatically advantageous or disadvantageous. It is substituted with an amino acid residue. In some embodiments, the positively charged amino acid at the interface, For example, lysine, arginine, or histidine are negatively charged amino acids, for example, Substituted with spartic acid or glutamic acid. In some embodiments, the negative of the interface Charged amino acids are substituted with positively charged amino acids. In some embodiments, Charged amino acids are introduced into one or both of the constant domains of the interacting CH3 antibody. In some embodiments, the interaction of two Fc domain monomers with a CH3 antibody stationary state The introduction of charged amino acids into the main molecule results in electrostatic stearin due to the interaction between charged amino acids. Selective formation of heterodimers of Fc domain monomers, as controlled by the lig effect. It promotes electrostatic steering. Examples of amino acid pairs are included in Table 11, but are not limited to these. I can't. [Table 11]

[0176] Other methods used to control heterodimerization of Fc domain monomers include, It 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 human IgG1 sequence, 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 is: (a) relative to 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 mutation in the Fc region of human IgG1 with N297A, (ii) L234A, L235A, and G237A are applied to the Fc region of human IgG1. Mutations, (iii) L234A, L235A, G237 in the Fc region of human IgG1 (iv) Mutations of A and N297A in the Fc region of human IgG2 (v) Mutations of A330S and P331S in the Fc region of human IgG2, (vi ) Mutations A330S, P331S, and N297A in the Fc region of human IgG2, (vii) S228P, E233P, F234V, L Mutations in 235A and delG236, or (viii) the Fc region of human IgG4 In contrast, S228P, E233P, F234V, L235A, delG236, and N2 Includes the 97A mutation. In some embodiments, the Fc domain monomer is: (a) wild type For human IgG1, one of the following amino acid substitutions: 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 (b) comprising 09T or K409I, and further (i) in the Fc region of human IgG1 In contrast, the N297A mutation, (ii) L234A, L in the Fc region of human IgG1 (iii) mutations in the Fc region of human IgG1, L23 Mutations of 4A, L235A, G237A, and N297A, (iv) Fc region of human IgG2 (v) For the region, the N297A mutation, and for the Fc region of human IgG2, A330S and (vi) mutations in the Fc region of human IgG2, A330S, P331 S and N297A mutations, (vii) S228P for the Fc region of human IgG4, Mutations of E233P, F234V, L235A, and delG236, or (viii) S228P, E233P, F234V, L235A, for the Fc region of human IgG4 This includes mutations in delG236 and N297A.

[0179] In some embodiments, the first and second Fc domain monomers are different amino acids Substitutions are included. In some embodiments, the first Fc domain monomer is T366W Includes. In some embodiments, the second Fc domain monomer is T366S, L36 This includes 8A and Y407V. In some embodiments, the first Fc domain monomer This includes D399K. In some embodiments, the second Fc domain monomer is K Includes 409D.

[0180] IV. Serum Albumin In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0181] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0182] Fusion to serum albumin can improve the pharmacokinetics of protein formulations, and several In that embodiment, the present invention includes the high affinity SIRP-α D1 variant described herein. The polypeptide shown is linked to serum albumin.

[0183] Serum albumin is a globular protein abundant in the blood of mammals. It is produced in the liver and can make up about half of serum proteins. It is a monomer and is present in the blood. It is soluble. Some of the most important functions of serum albumin include hormones, fatty acids, and other Protein transport within the body, pH neutralization, and proper distribution of body fluids between blood vessels and tissues. This includes maintaining the necessary osmotic pressure. In a preferred embodiment, serum albumin is derived from human serum. Albumin (HSA) is used in some embodiments. It is attached to the C-terminus of the polypeptide, extending its serum half-life. In some embodiments, The N-terminus of the HSA is linked to the C-terminus of the polypeptide of this disclosure. Several embodiments In this state, HSA is either directly or via a linker at the C-terminus of the polypeptide. They are connected. In some embodiments, the HSA is connected either directly or via a linker. It is then attached to the N-terminus of the polypeptide.

[0184] In some embodiments, human serum albumin is used with the UniProt identification number shown in Table 12. Contains amino acid (aa) sequence 25-609 (sequence number 12) of product P02768. In this embodiment, a high-affinity SIRP-α D1 variant (see, for example, Tables 2, 5, and 6) is used. An HSA linked to any SIRP-α D1 variant shown is UniProt identified. It contains amino acids 25-609 (SEQ ID NO: 12) of the sequence number P02768. In the application form, the HSA includes substitution of C34S or K573P for sequence number 12. In some embodiments, the HSA is C34S and K57 for SEQ ID NO: 12. Includes substitution of 3P. [Table 12]

[0185] In some embodiments, serum albumin is genetically fused to the polypeptide of the present disclosure. or by chemical means, for example, by chemical bonding, the polypeptide In some embodiments, a spacer is inserted between the polypeptide and the HSA. Some examples of pacers are described in detail elsewhere in this specification. In some embodiments The spacer is A or AAAL. In some embodiments, the polypeptide of the present disclosure The fusion of HSA in the polypeptide not only results in the long-term retention of the polypeptide, but also halves it. The term will also be extended.

[0186] SIRP-α D1 variant polypeptide and polypeptide containing fused HSA Examples include, but are not limited to, sequence numbers 150-159 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 used for any variant 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% It contains a high-affinity SIRP-α D1 domain (or has 100% sequence identity).

[0188] In some embodiments, the polypeptide is represented by SEQ ID NOs. 154, 155, and in Table 13. For 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 with 98%, 99%, or 100% sequence identity Includes the main course.

[0189] V. Albumin-binding peptide In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0190] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0191] Binding to serum proteins can improve the pharmacokinetics of protein formulations, particularly, In some embodiments, the polypeptides described herein are serum protein-conjugated peptides It is fused to a drug or protein.

[0192] As used herein, the term "albumin-binding peptide" refers to serum albumin protein Approximately 12 to 16 amino acids that have affinity for and binding function to the substance. This refers to a sequence. In some embodiments, the albumin-binding peptide is human, mouse, and It is derived from rats.

[0193] In some embodiments, a high-affinity SIRP-α D1 variant (e.g., Tables 2 and 5) is used. The polypeptides of this disclosure, including any variant shown in 6, are used against serum albumin. It is fused to an albumin-binding peptide that exhibits binding activity, thereby extending the half-life of the polypeptide. Various albumin-binding compounds that can be used in the methods and compositions described herein. Butydops are available. In some embodiments, the albumin-binding peptide is DI Includes the sequence CLPRWGCLW (sequence number 160). In some embodiments, album The mine-bound peptide is genetically fused to the polypeptide of this disclosure, or chemically fused to it. The polypeptide is bound by a method, for example, chemical bonding.

[0194] In some embodiments, a linker (e.g., a spacer) is provided for the polypeptide and the aluminum Inserted between bumin-binding peptides, providing further structural and spatial flexibility to the fusion protein. This makes it possible. The specific linkers (e.g., spacers) and their amino acid sequences are as follows: Further details are provided in the specification. In some embodiments, the albumin-binding peptide is It is fused to the N-terminus or C-terminus of the polypeptide of this disclosure. In one example, albumin fusion. The N-terminus of the combined peptide is directly fused to the C-terminus of the polypeptide of this disclosure via a peptide bond. In another example, the C-terminus of an albumin-binding peptide is connected via a peptide bond in this disclosure. It is directly fused to the N-terminus of the polypeptide. In some embodiments, albumin-bound polypeptide The fusion of the plutide to the polypeptide of this disclosure enhances the long-term retention of the polypeptide in its serum alcohol This is brought about by binding to bumin.

[0195] VI. Polyethylene glycol (PEG) polymer In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0196] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0197] In some embodiments, a high affinity SIRP-α D1 domain (e.g., Table 2, 5) is used. Polypeptides containing any variant shown in 6 are polymers (e.g., polyethylene It is fused to a glycol (PEG). In some embodiments, the polymer protein Binding to the formulation "masks" the protein formulation from the host's immune system. In some embodiments, a particular polymer, for example, a hydrophilic polymer, is hydrophobic. To impart water solubility to proteins and drugs. For example, in some embodiments, such polymers Examples include PEG, polysialic acid chains, and PAS chain molecules. Several implementations In this state, polymers such as PEG are covalently bonded with cysteine ​​substitution or addition to polypeptides. In some embodiments, the cysteine ​​substitution of the polypeptide is as shown in Tables 2, 5, and 6. For any one of the sequences shown, I7C, A16C, S20C, T20C, A These are 45C, G45C, G79C, S79C, or A84C. In some embodiments... The addition of cysteine ​​residues to the polypeptide is involved in peptide synthesis, genetic engineering, and molecular engineering. It is introduced using ronning, or any combination thereof. In some embodiments, The polymer, for example PEG, uses a cysteine-maleimide bond to form a cysteine ​​residue. It is bonded to polymers such as PEG by conventional methods such as chemical bonding. Using chemical methods, a polypeptide containing the high-affinity SIRP-α D1 variant was subjected to N The covalent bond is formed at either the terminal or C-terminus, or at an internal position.

[0198] VII. Bispecific constructs In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0199] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0200] In some embodiments, a high-affinity SIRP-α D1 variant (e.g., Tables 2 and 5) is used. Polypeptides having any variant shown in 6 include bispecific constructs. A bispecific construct refers to a construct that has two target interaction domains. In this embodiment, the dual specificity construct comprises an Fc domain and two target interaction domains. (1) SIRP-α D1 domain or its variant (e.g., Tables 2, 5, and (1) Any variant shown in (2) and (3) Antibody variable domain. Several embodiments So, a bispecific construct contains a first polypeptide and a second polypeptide. How many? In that embodiment, the first polypeptide has the formula ALB, in which case A is SI RP-α D1 domain or a variant thereof, where L is the linker and B is the first It contains an Fc domain monomer. In some embodiments, the second polypeptide is of formula A It has '-L'-B', in which case A' contains the antibody variable domain and L' is the linker. Furthermore, B' contains a second Fc domain monomer. In some embodiments, the first and The orientations of the two polypeptides are BLA and B'-L'-A', respectively. In that embodiment, the first and second Fc domain monomers are bonded to construct the bispecific structure. It forms the Fc domain of the substance. In some embodiments, the bispecific construct is any immunosuppressant. These are disease globulin antibody isotypes (e.g., IgG, IgE, IgM, IgA) , and IgD). The variant of the SIRP-α D1 domain is wild-type human SIRP-α The D1 domain and the wild-type D1 domain include one or more amino acid substitutions (for example) (For example, any SIRP-α D1 variant shown in Tables 2, 5, and 6). Several implementations In this state, the SIRP-α D1 variant is derived from the wild-type human SIRP-α D1 domain. It binds to CD47 with high binding affinity. In some embodiments, the bispecific construct is The antibody variable domain targets cellular antigens (for example, cellular antigens of cancer cells).

[0201] The antibody variable domain is a complementarity-determining region (CDR, e.g., CDR L1, CDR L2) (CDR L3, CDR H1, CDR H2, and CDR H3) and frame w This refers to the light and heavy chain portions of an antibody that contain the amino acid sequence of the FR region. The main purpose is to confer the ability of the antibody to bind to a specific antigen. Antibody variable domain molecules can be constructed. In some embodiments, the antibody used Examples of variable domain molecules include, but are not limited to, single-stranded Fv.

[0202] In some embodiments, the antibody variable domain in the bispecific construct is a cell antigen It targets (for example, cellular antigens of cancer cells or immune cells). Some proteins , it is expressed at higher levels in cancer cells than in non-cancer cells. For example, cancer antigens, It is a protein that is preferentially expressed by cancer cells (for example, it is expressed more in non-cancer cells than in cancer cells). It is expressed at higher levels in cancer cells, and in some cases it is expressed in cancer cells It is expressed only in certain embodiments. In some embodiments, the protein is expressed as, for example, high affinity SIRP-α By an antibody variable domain that generates an Fc domain along with the domain or its variants Proteins expressed by cancer cells that are targeted include 5T4 and 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 Examples include, but are not limited to, -1 or CSF-1R. In some embodiments, Therefore, the antibody variable domain in the bispecific construct binds to human proteins. It is not operated.

[0203] In some embodiments, the first and second in the Fc domain of the bispecific construct Each of the Fc domain monomers is a heterodimer of the first and second Fc domain monomers. Contains one or more amino acid substitutions that promote ionization. Heterodimer of Fc domain monomer. Methods for promoting transformation are described in more detail herein, for example, the knob and hole method. See also the electrostatic steering method.

[0204] In some embodiments, the Fc domain of the bispecific construct is a "dead Fc domain". It is mutated to lack one or more effect functions, which is unique to "n". In the application form, the Fc domain of the bispecific construct is derived from the IgG1 antibody, and the sequence number is The sequence 161 (Table 14) contains amino acid substitutions L14A, L15A, and G17A. This reduces the interaction or binding between the Fc domain and the Fcγ receptor. In the application form, the Fc domain monomer is derived from the IgG1 antibody, and the amino acid substitution L234 A, L235A, G237A, and N297A (Kabat et al., 1991) Therefore, it includes one or more of the following (as shown in the EU numbering system). In some embodiments, The Fc variants described herein are minimally glycosylated or glycosylated It is decreasing. In some embodiments, deglycosylation is due to the N297A mutation, Alternatively, N297 can be any amino acid other than N (Kabat et al. (1991) This is achieved by mutating it to (as shown in the EU numbering system). In several embodiments, the bispecific construct is expressed by different cell types. Designed to selectively bind to proteins (e.g., receptors such as Fc receptors). The antibody hinge, constant domain (e.g., CH2 and CH3 constant domains), or Amino acid substitutions in the hinge and constant domains can affect different cell types (e.g., regulatory T). For specific receptors (e.g., Fc receptors) expressed on cells and effector T cells Studies have shown that it is possible to effectively modify the binding affinity of antibodies. IgG having no acid substitutions A111S and P112S (for SEQ ID NO 162 in Table 14) Compared to wild-type IgG2, 2 showed significant binding to FcγRIIIa 131 H. It shows a decrease. In some embodiments, the Fc variants herein minimize glycosylation. In some embodiments, deglycosylation occurs. This is a mutation of N297A, or N297 is any amino acid other than N (Kabat, (As shown in the EU numbering system by et al. (1991)) This is achieved by the following. In some embodiments, the bispecific construct is IgG2 or It contains the Fc domain of the IgG4 subclass. In some embodiments, the IgG2 subclass The bispecific construct containing the Fc domain of Lass is, for SEQ ID NO: 162 (Table 14), A Includes mino acid substituted A111S and P112S. In some embodiments, the Fc Varian The amino acid substitutions are A330S, P331S, and N297A (Kabat, et al.). (as shown in the EU numbering system by al. (1991)) containing one or more It contains the Fc sequence of IgG2. [Table 14]

[0205] The SIRP-α D1 domain is linked to the first Fc domain monomer via a linker. The first and second Fc domain monomers include a variant thereof and a second Fc domain monomer. An example of a SIRP-α construct in which domain monomers bind to form an Fc domain is shown in Figure 1. In some embodiments, the protein bound to the second Fc monomer is shown. There is no quality or antibody variable domain. In some embodiments, the SIRP-α construct is The SIRP-α D1 domain is linked to the first Fc domain monomer via a linker. or its variant, and the antibody linked to the second Fc domain monomer via the linker It contains a variable domain, and the first and second Fc domain monomers are bound to the Fc domain It forms (as shown in Figure 2). In some embodiments, the SIRP-α structure is phosphorus The SIRP-α D1 domain is linked to the first Fc domain monomer via the Kerr, and This variant, and the therapeutic linkage to the second Fc domain monomer via the linker, Proteins used (e.g., cytokines, interleukins, antigens, steroids, anti-inflammatory drugs) A compound comprising (or an immunomodulator), wherein the first and second Fc domain monomers are bound to the Fc domain The main structure is formed (as shown in Figure 3). In some embodiments, the aforementioned SIRP-α structure is used. Two Fc domains of a structure (for example, the SIRP-α structure shown in Figures 1-3) Each monomer contains an amino acid substitution that promotes heterodimerization of the two monomers. Hmm. Different methods to promote heterodimerization of two Fc domain monomers (for example, no The methods of bu and hole, electrostatic steering, and amino acid substitution of the Fc domain are described herein. It is described in detail. For example, Figure 4A contains a knob mutation, e.g., T366W. SIR linked to the essential knob and the Fc domain monomer that restricts homodimer formation of the knob. Figure 4B shows a SIRP-α construct containing the P-α D1 domain or a variant thereof. This includes hole mutations, such as T366S, L358A, and Y407V, which are Fc domain mutations. SIRP having a SIRP-α D1 domain or a variant thereof linked to a nomer -α construct is shown. In some embodiments, similar heterodimerization methods of the Fc domain are shown. The law applies to the Fc domain in the structures shown in Figures 2 and 3. Several implementations In this state, the SIRP-α construct is SIRP-α D1 linked to the Fc domain monomer. It contains a fusion protein of the domain or a variant thereof (as shown in Figure 5A). In this embodiment, the fusion protein forms a homodimer (as shown in Figure 5B).

[0206] The Fc variants of this specification bonded to the fusion partner are preferably natural or wild. Compared to similar polypeptide constructs containing the bio-type (non-mutant) antibody Fc region, at least 1 One Fcγ receptor, CD16a, CD32a, CD32b, CD32c, and CD64 This indicates a reduction or removal of the bond. In some cases, the Fc b described herein The riant or fusion partner is CD16a, CD32a, CD32b, CD32c, This also indicates a decrease or removal of CD64 binding to the Fcγ receptor.

[0207] In some embodiments, the Fc variant of the present disclosure coupled to the fusion partner is, Compared to similar polypeptide constructs containing natural or wild-type (non-mutant) Fc regions, complement composition It shows a decrease in binding to C1q and CDC. In some cases, the Fc barrier The compound is at least 5% to 10% more concentrated than polypeptide constructs containing the wild-type Fc region. , 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or so It shows a decrease in C1q bonds beyond this. In some cases, the Fc variant is Compared to polypeptide constructs containing natural or wild-type (non-mutant) Fc regions, the reduction in CDC. This shows that in some embodiments, the Fc variant includes a polypeptide containing a wild-type Fc region. Compared to the Chido structure, at least 5%, 10%, 15%, 20%, 30%, 40%, 5 This indicates a decrease in CDC of 0%, 60%, 70%, 80%, 90%, or more.

[0208] VIII. Linker In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0209] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0210] In this disclosure, the linker is a polypeptide or protein domain or related Used to indicate binding or connection between non-protein portions. Several embodiments So, the linker is an Fc domain monomer, an albumin-binding peptide, or an HSA. This is a binding or connection between and the high affinity SIRP-α D1 variant. In the application configuration, the linker is such that two polypeptides are connected in series with each other. The C-terminus of the SIRP-α D1 variant and the N-terminus of the Fc domain monomer, the album A mine-bound peptide or the HSA is attached to it.

[0211] In some embodiments, the linker is a simple covalent bond, such as a peptide bond, or a synthetic bond. Polymers, such as polyethylene glycol (PEG) polymers, or formed from chemical reactions. Any type of bond that can be formed, such as a chemical bond. In some embodiments, the carboxylic acid group at the C-terminus of one protein domain is a different one. 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 are synthesized by conventional organic chemical reactions. Or they are formed by spontaneous production from host cells, in which case both proteins in series (e.g.) For example, the DNA sequence of the Fc domain monomer and the high-affinity SIRP-α D1 variant. Nucleic acid molecules that encode essential molecular mechanisms in host cells (e.g., DNA polymerase and By ribosomes, both proteins are directly transcribed into the adjacent polypeptide encoding them. It can be translated.

[0212] In some embodiments, when the linker is a synthetic polymer (e.g., PEG polymer), The polymer reacts with terminal amino acids at the connecting ends of the two proteins, It is functionalized with reactive chemical functional groups.

[0213] When the linker (other than the peptide bond mentioned above) consists of a chemical reaction, there are several embodiments. So, chemical functional groups (e.g., amines, carboxylic acids, esters, azides, or other functional groups) ) is synthetically bound 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 chemical means to form a chemical compound. They form a bond, and thereby connect the two proteins.

[0214] Spacer In some embodiments of this disclosure, Fc domain monomers, albumin-bound particles The linker between the peptide or HSA and the polypeptides of this disclosure is approximately 1 to 200 ammonium. It is an amino acid spacer containing anoacids. Suitable peptide spacers are flexible amino acids. Examples include peptide linkers containing residues, such as glycine and serine. Linker sequence Examples are shown in Table 15. In some embodiments, the spacer is a motif, for example, G S, GG, GGS, GGG, GGGGS (Sequence ID 163), GGSG (Sequence ID 164) ), or includes multiple or repeating motifs of SGGG (sequence number 165). In the application form, the spacer is a GS motif, for example, GS, GSGS (sequence number 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGS Includes GSGS (sequence number 169) or GSGSGSGSGSGS (sequence number 170) It contains 2 to 12 amino acids. In some embodiments, the spacer is a GGS motif. For example, GGS, GGSGGS (sequence number 171), GGSGGSGGS (sequence number 171) 72), and 3 to 12 amino acids including GGSGGSGGSGGS (SEQ ID NO: 173) Includes. In some embodiments, the spacer is the motif of GGSG (SEQ ID NO: 164), For example, GGSG (sequence number 164), GGSGGGSG (sequence number 174), or G Contains 4 to 12 amino acids, including GSGGGSGGGSG (Sequence No. 175). How many? In that embodiment, the spacer is the motif of GGGGS (Sequence ID 163), for example, GG Includes GGSGGGGSGGGGS (Sequence ID 176). In some embodiments, Spec -sa contains amino acids other than glycine and serine, for example, AAS (SEQ ID NO: 177), AAAL (sequence number 178), AAAK (sequence number 179), AAAAR (sequence number 180) ),EGKSSGSGSESKST (Sequence No. 181),GSAGSAAGSGEF (Distribution Column number 182), AEAAAKEAAAKA (Sequence number 183), KESGSVSSEQ LAQFRSLD (Sequence ID 184), GGGGAGGGG (Sequence ID 185), GEN LYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGS AGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG(Sequence No. 19) 0), AAANSSIDLISVPVDSR (Sequence ID 191), or GGSGGGS Includes EGGGSEGGGSEGGGSEGGGSEGGGSGGGS (Sequence No. 192) nothing.

[0215] In some embodiments, the spacer is a motif, for example, EAAAK (Sequence ID 193) ) includes multiple or repeating motifs. In some embodiments, the spacer is a motif, e.g. For example, multiple or repeating motifs of proline-rich sequences, e.g., X is any amino acid (e.g.) For example, (XP)n is A, K, or E) and n is 1 to 5, and PAPAP (array). Includes number 194). [Table 15-1] [Table 15-2]

[0216] In some embodiments, the length of the peptide spacer and amino acid used is involved. Depending on the desired degree of flexibility in the two proteins and the final protein fusion polypeptide It is adjusted accordingly. In some embodiments, the length of the spacer is adjusted to allow for appropriate protein folding. It is adjusted to ensure folding and avoid the formation of aggregates. In some embodiments, H Spacers such as spacers between SA and the polypeptides disclosed herein are A or AAAL (Sequence number 178).

[0217] IX. Vectors, host cells, and protein production In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0218] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0219] In some embodiments, the polypeptides of this disclosure are produced from host cells. The polypeptides and fusion polypeptides described herein are derived from their corresponding nucleic acids. This refers to a medium containing cellular components necessary for the expression of a substance, such as organelles. Morphologically, the nucleic acid undergoes transformation, transfection, electroporation, and phosphorus It is introduced into host cells by calcium acid precipitation, direct microinjection, infection, etc. It is contained in the nucleic acid vector. In some embodiments, the selection of the nucleic acid vector is used. It depends on the host cell. In some embodiments, the host cell is a prokaryote (e.g., bacteria). It is either of the following origins: ) or eukaryotes (e.g., mammals).

[0220] In some embodiments, polypeptides, for example, SIRP-α D1 variants (e.g.) For example, any variant shown in Tables 2, 5, and 6, as well as a fusion partner, e.g., F Polypeptide constructs containing c variants, HSA, and albumin-binding peptides are nucleic acids Preferably, the polypeptide construct (e.g., Fc variant, linker, and fusion compound) Host cells transformed with an expression vector containing nucleic acid encoding (the Culturing under conditions suitable for inducing or triggering the expression of polypeptide constructs. It is produced by. In some embodiments, the conditions suitable for expression are selected expression beds. It changes depending on the host cell. In some embodiments, mammalian cells, bacteria, insects A wide variety of suitable host cells can be used, including, but not limited to, cells and yeast. For example, the various cell lines for which applications are found in this disclosure include American type cell lines. Listed in the ATCC® cell line catalog available from the Char Collection. In some embodiments, the Fc variant of the present disclosure is used to genetically modify the cell line. Alternatively, by changing cell culture conditions such as adding kifunensin, or by prokaryotes (Ec By using a host that does not naturally undergo glycosylation, such as oli (or similar), the cells can be... The expressed protein is optimized not to glycosylate in the cells, and several In that case, modification of the glycosylation sequence of Fc is not necessary.

[0221] nucleic acid vector constructs and host cells The nucleic acid sequence encoding the amino acid sequence of the polypeptide disclosed herein can be prepared by various methods. These can be manufactured using oligonucleotide-mediated (or site-specific) methods. Examples include, but are not limited to, heterologous mutation generation and PCR mutation generation. In some embodiments, the nucleic acid molecules encoding the polypeptides of this disclosure are standard techniques, e.g. For example, it can be obtained using gene synthesis. Another method is to obtain the wild-type SIRP-α D1 domain. Nucleic acid molecules that encode , are created using standard technologies, such as QuikChange (trademark). Mutation is performed using mutagenesis to include specific amino acid substitutions. In some cases Nucleic acid molecules are synthesized using nucleotide synthesizers or PCR technology.

[0222] In some embodiments, polypeptide constructs, such as SIRP-α D1 varian, are used. (For example, any variant shown in Tables 2, 5, and 6) and fusion partners, e.g. If, then, a polypeptide construct containing an Fc variant, HSA, and albumin-binding peptide The encoding nucleic acid is incorporated into an expression vector to express the protein. Various expression vectors can be used for protein expression. Expression vectors are self This may include vectors for replication, extrachromosomal vectors, or integration into the host genome. Furthermore, it may also contain various components or elements. For example, in some embodiments... The vector components include transcription and translation regulatory sequences, such as promoter sequences and ribodynamic sequences. Somal binding site, signal sequence, transcription start and stop sequence, translation start and stop sequence, 3' and The 5' untranslated region (UTR), as well as the enhancer or activator sequence, replicate A point, a selection marker gene, and a nucleic acid sequence encoding the target polypeptide, and transcription. Examples include, but are not limited to, stop sequences. In some embodiments, expression vectors This includes control or regulating arrays, selectable markers, any fusion partners, and further elements. Or includes proteins operably linked to any combination thereof. The term "operable" "Linked to" means that the nucleic acid is positioned in a functional relationship with another nucleic acid sequence. Generally, these expression vectors are operably linked to nucleic acids encoding Fc variants. Containing linked transcriptional and translational regulatory nucleic acids, typically used to express the protein. These are appropriate for host cells. Examples include antibiotic resistance genes and fluorescent protein genes, etc. Selected genes or markers, not limited to those mentioned above, can be expressed in host cells containing the expression vector, for example, It can be used for selection by antibiotics or fluorescence expression. Various selection genes The child is available.

[0223] In some embodiments, the components or elements of the vector are such that the expression vector is host cell type and It is optimized to fit. Expression vectors for which applications are found in this disclosure include mammalian cells. Examples include bacteria, insect cells, yeast, and systems that enable protein expression in vitro. However, this is not limited to these.

[0224] In some embodiments, mammalian cells host to produce the polypeptides of the present disclosure. It is used as a principal cell. An example of a mammalian cell type is human embryonic kidney (HEK) (for example, 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 (mouse myeloma cell line that intrinsically does not produce any immunoglobulin chains), CRL7 Examples include, but are not limited to, O3O and HsS78Bst cells. In this application, E. coli cells act as host cells to produce the polypeptide of this disclosure. It is used. An example of an E. coli strain is E. coli 294 (ATCC (registered trademark)). )31,446), E. coli λ 1776 (ATCC (registered trademark) 31,537, E.coli BL21(DE3)(ATCC(registered trademark) BAA-1025), and E .coli RV308 (ATCC(registered trademark) 31,608) is one example, but these include Not limited.

[0225] Different host cells perform post-translational processing and modification of protein products (e.g., glycosylation). It has a characteristic and specific mechanism of (polymerization). In some embodiments, the expressed polypeptide To ensure accurate modification and processing of cytoplasm, an appropriate cell line or host system is selected. When the vector is introduced into a host cell for protein production, the host cell promotes Depending on the induction of a transformer, the selection of a transformant, or the amplification of the gene encoding the desired sequence, the transformation may be modified. They are cultured in a decorated, standard nutrient medium.

[0226] In some embodiments, polypeptide constructs, such as SIRP-α D1 varian, are used. (For example, any variant shown in Tables 2, 5, and 6) and fusion partners, e.g. However, the polypeptide construct containing the Fc variant, HSA, and albumin-binding peptide is The expression construct is created using viruses such as retroviruses and adenoviruses in the mammal. It is expressed in mammalian expression systems, including systems in which it is introduced into cells. In some embodiments, Cat, mouse, rat, hamster, or primate cells can be used. Suitable cells include: This includes known research cells, including Jurkat T cells, NIH3T3, CHO, COS, and 293 cells are mentioned, but are not limited to these. Alternatively, several implementations In this context, proteins are expressed in bacterial cells. The bacterial expression system is well known in the relevant field. , Escherichia coli (E.coli), Bacillus subti lis, Streptococcus cremoris, and Streptococcus This includes us lividans. In some cases, it includes Fc variants. Lipeptide constructs include, but are not limited to, insect cells such as Sf9 and Sf21 cells. , or Saccharomyces, Pichia, Kluyveromyces, H This yeast is derived from, but is not limited to, microorganisms of the genera Ansenula and Yarrowia. It is produced in the parent cell. In some cases, it is used to construct a polypeptide containing the Fc variant. The substance is expressed in vitro using a cell-free translation system. (e.g., E. coli) In vitro translation from both eukaryotic cells (e.g., wheat germ, rabbit reticulocytes) The system is available, and in some embodiments, the expression level and function of the target protein can be determined. Selected based on characteristics. For example, in vitro translation as understood by those skilled in the art. This is necessary for several display technologies, such as ribosome displays. In some embodiments, the Fc variant is used for liquid-phase peptide synthesis and solid-phase peptide synthesis. It is produced by chemical synthesis methods, including but not limited to those mentioned above. Non-chemical methods such as bacterial extracts. In the case of in vitro transcription using a glycosylation system, the Fc has a natural glycosylation site. Even if present, glycosylation does not occur, and consequently, the inactivation of Fc is obtained to the same extent.

[0227] In some embodiments, polypeptide constructs are similar to naturally occurring amino acids. Non-natural amino acids, amino acid analogs, amino acid mimes, or their functions in a non-natural way Includes any combination. Naturally coded amino acids are typically 20 common amino acids. Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glut Taminic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, fe Nylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine ) as well as pyrrolicin and selenocysteine. Amino acid analogs are naturally occurring compounds. It has the same basic chemical structure as amino acids, for example, a carbon atom bonded to hydrogen, a carboxyl group, and an amino acid. Compounds having a group and an R group, for example, homoserine, norleucine, methionine sulfopropyl alcohol The side refers to methionine methylsulfonium. In some embodiments, such analogs , having a modified R group (e.g., norleucine) or a modified peptide skeleton, Normally, they retain the same basic chemical structure as naturally occurring amino acids.

[0228] Protein production, recovery, and purification In some embodiments, host cells used to produce the polypeptides of this disclosure The cells are grown in a culture medium suitable for the selected host cells. Examples include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), E xpi293(trademark) expression medium, DMEM supplemented with fetal bovine serum (FBS), and R PMI-1640 is one example. Examples of culture media suitable for bacterial host cells include those containing selective agents, etc. Essential nutrient supplements include, for example, Luria Broth (LB) containing ampicillin. In some embodiments, the host cells are kept at an appropriate temperature, for example, about 20°C to about 39°C, for example , approximately 25°C to approximately 37°C, preferably 37°C, and CO2 level, for example, approximately 5% to 10% It is cultured in a medium. In some embodiments, the pH of the medium is about pH 6, mainly depending on the host organism. pH 0.8 to 7.4, for example, pH 7.0. The inducible promoter is used in the expression vector. If so, protein expression can be induced under conditions suitable for the activation of the promoter. .

[0229] In some embodiments, protein recovery is performed, for example, by osmotic shock, sonication, This involves the destruction of host cells by lysis. Once the cells are destroyed, the cellular debris is separated by centrifugation. It is removed by filtration. The protein can then be further purified. In the application form, the polypeptides disclosed herein can be purified by various protein purification methods, for example, by chromium. Mathography (e.g., ion exchange chromatography, affinity chromatography) Fees and size exclusion column chromatography, centrifugation, solubility difference chromatography, or tanning It is purified by any other standard technique for protein purification. For example, in several embodiments. So, the protein in question is an affinity column, for example, a protein A column (for example, POROS Protein A chromatography) and chromatography column (e.g., P By appropriately selecting and combining OROS HS-50 cation exchange chromatography, Then, it is isolated and purified by filtration, ultrafiltration, desalting, and dialysis procedures. In this embodiment, the polypeptide is linked to a marker sequence such as a peptide to facilitate purification. They are combined. An example of a marker amino acid sequence is nickel-functionalized agarose affinity color. Hexahistidine peptide (His6) can bind to the molecule with micromolar affinity. (Tag) Another method is to use epidos derived from influenza hemagglutinin protein. The hemagglutinin "HA" tag corresponding to the link can be used.

[0230] In some embodiments, the polypeptides of the present disclosure, for example, the SIRP-α D1 barrier, are used. nt (for example, any variant shown in Tables 2, 5, and 6) and fusion partner, e.g. For example, polypeptide constructs containing Fc variants, HSA, and albumin-binding peptides. This refers to the cells of the target (e.g., human), and, for example, in relation to gene therapy, the present disclosure Viral vectors containing nucleic acid molecules encoding lipeptides (e.g., retroviral vectors) Virus vectors, adenovirus vectors, poxvirus vectors (e.g., mutant vaccinia) Vaccinia virus vectors such as Ilu-Ankara (MVA), adeno-associated virus vectors It is produced by administering vectors such as vir and alphavirus vectors. The vector, once it enters the target cell (for example, transformation, transfection), , electroporation, calcium phosphate precipitation, direct microinjection, It can be used to express the polypeptides disclosed herein (due to infection, etc.). In some cases, polypeptides are secreted from cells. In some embodiments, If the treatment of the illness or disorder yields a desirable outcome, no further action is necessary. In terms of form, if recovery of the protein is desired, blood is collected from the subject and collected by various methods. The protein is then purified from the blood.

[0231] X. Pharmaceutical compositions and pharmaceuticals In some embodiments, the wild-type SIRP-α D1 is disclosed herein. Compared to the main protein, there is an amino acid mutation at residue 80, as well as a wild-type signal regulatory protein α( For the SIRP-α)D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0232] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0233] This disclosure relates to polypeptides described herein, for example, high affinity SIRP-α D1 variant. The pharmaceutical composition is characterized by comprising a polypeptide having an ant. In some embodiments, The pharmaceutical compositions of this disclosure include the polypeptides of this disclosure as therapeutic proteins. In the embodiments described herein, the pharmaceutical composition of this disclosure comprising the polypeptide described herein is used for treatment. and other drugs or compositions (e.g., therapeutic agents, biologics, small molecules, or any of them) Used in combination with (a combination of). In some embodiments, one or more further treatments A therapeutic agent, for example, a small molecule, a chemical compound, or a biocompound, for example, siR Polynuclear compounds include, but are not limited to, NA, short polypeptides, and therapeutically effective antibodies. Cleotides and polypeptides may optionally be used in the polypeptide pharmaceutical compositions described herein. It is formulated. In some embodiments, the formulation of the polypeptide construct described herein is For preservation purposes, the polypeptide constructs described herein, having the desired purity, are to be used in any pharmaceutical or pharmaceutical product. In the form of a lyophilized formulation or aqueous solution, together with a suitably acceptable carrier, excipient, or stabilizer. It is prepared by mixing. In some embodiments, the pharmaceutical compositions of the present disclosure are Nucleic acid molecules (DNA or RNA, e.g., mRNA) encoding the disclosed polypeptide. or a vector containing such nucleic acid molecules.

[0234] Acceptable carriers, excipients, or stabilizers in pharmaceutical compositions are preferably administered It is nontoxic to the recipient at the appropriate dose and concentration. In some embodiments, it is acceptable. Examples of carriers, excipients, and stabilizers include buffering agents such as phosphoric acid, citric acid, and HEPE. S, TAE, and other organic acids, antioxidants such as ascorbic acid and methionine, preservatives (For example, hexamethonium chloride, octadecyldimethylbenzylammonium chloride, salt) Benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol Alkylparabens, for example, methyl or propylparaben, catechol, resorcinol (e.g., cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (e.g.) For example, polypeptides (less than approximately 10 residues), proteins, such as human serum albumin, etc. Latin, dextran, and immunoglobulins, hydrophilic polymers, such as polyvinyl pyropropyl alcohol. Lydone, amino acids such as glycine, glutamine, histidine, and lysine, monosaccharides, two Sugars and other carbohydrates, such as glucose, mannose, sucrose, and sorbitol Chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose. Rose or sorbitol, sweeteners and other flavoring and odor-modifying agents, fillers, for example, microcrystalline cereals Lullose, lactose, corn and other starches, binders, additives, colorants, salts Pair ions, e.g., sodium, metal complexes (e.g., Zn-protein complexes), non-ions Potency surfactants, such as TWEEN (trademark), PLURONICS (trademark), and P Examples include ethylene glycol (PEG) or any combination thereof.

[0235] In some embodiments, the pharmaceutical composition comprising the polypeptide described herein is water-soluble. It is a pharmaceutically acceptable form, for example, intended to contain both an acid and a base addition salt. It exists as a salt that is pharmaceutically acceptable. The term "pharmaceutically acceptable acid addition salt" refers to a free base that is pharmaceutically acceptable. Maintaining academic validity and being otherwise appropriate, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, etc. Acids, nitric acid, phosphoric acid, etc., and organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid. Malonic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid Cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid This refers to salts formed from benzoic acid, salicylic acid, etc. The term is "medically acceptable base addition salt." For example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron Examples include those derived from inorganic bases such as zinc, copper, manganese, and aluminum salts. Particularly preferred are ammonium, potassium, sodium, calcium, and magnesium It is a salt. As a salt derived from a pharmaceutically acceptable organic non-toxic base, it is classified as primary or secondary. , and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and A basic ion exchange resin, for example, isopropylamine, trimethylamine, diethylamine Examples include salts of mine, triethylamine, tripropylamine, and ethanolamine. The formulation used for in vivo administration is preferably sterile. This is achieved by filtration through a sterile membrane. Alternatively, it can be achieved by other means.

[0236] In some embodiments, the pharmaceutical compositions of this 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 solution. It is prescribed using a liquid as a medium. Medically acceptable mediums include sterile water. Physiological saline and cell culture medium (e.g., Dulbecco's modified Eagle medium (DMEM), α-modified) Examples include, but are not limited to, Eagle Medium (α-MEM) and F-12 Medium. Various formulation methods are available.

[0237] In some embodiments, the polypeptides described herein are used as immunoliposomes. Liposomes are various types of lipids that are useful for delivering therapeutic drugs to mammals. Liposomes are vesicles containing lipids or surfactants. Liposomes containing antibodies or Fc fusions are They can be prepared by various methods known in the art. In some embodiments The components of liposomes are arranged in a bilayer structure similar to the lipid arrangement in biological membranes. In one embodiment, the liposome contains phosphatidylcholine, cholesterol, and PE. A lipid composition containing PEG-PE derivatized phosphatidylethanolamine was used. They are produced by reverse-phase evaporation. In some embodiments, the liposomes have a desired diameter. To obtain liposomes, they are extruded through a filter with a predetermined pore size. In several embodiments, the chemotherapeutic agent or other therapeutic agent may optionally be liposome It is included within.

[0238] In some embodiments, polypeptide constructs described herein and other therapeutic effects The drugs used include coacervation technology and interfacial polymerization (for example, hydroxymethylcellulose). Alternatively, using gelatin microcapsules, or poly(methyl methacrylate) microcapsules. (Using capsules), colloidal drug delivery systems (e.g., liposomes, albumin microsulfates) Faires, microemulsions, nanoparticles, and nanocapsules, as well as macroemuls Microcapsules prepared by methods including, but not limited to, this method It will be enclosed.

[0239] In some embodiments, sustained-release formulations are prepared. A preferred example of a sustained-release formulation is: Examples include semipermeable matrices of solid hydrophobic polymers, and these matrices are used in molded products. The product may take the form of a film or microcapsules. Examples of sustained-release matrices are also included. For example, polyester, hydrogel (for example, poly(2-hydroxyethyl methacrylate) ) or poly(vinyl alcohol), polylactide, L-glutamic acid and L-glutamic acid Gamma ethyl acetate copolymer, non-degradable ethylene vinyl acetate, LUPRON DEP OT (trademark) (Injectable microparticle consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) Degradable lactic acid-glycolic acid copolymers such as Rothfair, poly-D-(-)-3-hydro xybutyric acid and poly-DL-lactide-co-glycolide (PLG) within the matrix ProL is a microsphere-based delivery system consisting of incorporated desired bioactive molecules. One example is ease (registered trademark) (commercially available from Alkermes). Several others While sustained-release formulations allow for the release of molecules over several months, for example, 1 to 6 months, other formulations... The formulation releases the pharmaceutical composition of this disclosure for a shorter period, for example, from a few days to several weeks.

[0240] In some embodiments, the concentration of the polypeptide described herein in the formulation is about 0 It varies from 0.1% to 100% by weight. In some cases, the polyp The concentration of butyl is in the range of 0.003 to 1.0 mole. In some cases, the formulation The concentration of the polypeptide in the prescription is approximately 5 mg / mL to approximately 50 mg / mL (for example, approximately 10 It varies from mg / mL to about 40 mg / mL or from about 20 mg / mL to about 30 mg / mL. Yes. In some embodiments, the polypeptides described herein are used to treat a patient. A therapeutically effective dose is administered. The term "therapeutic dose" refers to the dose that produces the desired effect. This refers to the quantity. The exact dose depends on the purpose of the treatment. In some embodiments, the dose is 0.01 to 100 mg / kg body weight or more, for example, 0.1, 1, 5, 10, 1 The doses range from 5, 20, 25, 30, 35, 40, 45, or 50 mg / kg body weight. In the embodiment, the degradation of polypeptide constructs, systemic delivery vs. local delivery, and novel protea The rate of synthesis, as well as age, weight, general condition, sex, diet, administration time, drug interactions, and The treatment needs to be adjusted according to the severity of the condition.

[0241] In some embodiments, the compositions and formulations described herein are used for subjects requiring them. It is administered to [the target]. In some embodiments, such administration is performed in vivo. In some embodiments, such administration is performed exovivo. The administration of the pharmaceutical composition containing the polypeptide described in the specification may be by oral, subcutaneous, intravenous, or nasal cavity. Intraotically, transdermally, topically (e.g., gels, ointments, lotions) (Cream formulations, etc.), intraperitoneal, intramuscular, pulmonary (for example, commercially available from Aradigm) Inhalable technology AERx®, or Inhale Therapeutics Commercially available lung delivery systems include Inhance®, transvaginal, parenteral, rectal, and This can be performed in various ways, including, but not limited to, intraocularly. Several embodiments The pharmaceutical composition is then formulated appropriately according to the method of introduction.

[0242] In some embodiments, the pharmaceutical composition for gene therapy is in an acceptable diluent. or including a sustained-release matrix in which a gene transfer medium is embedded. Several implementations In this context, retroviral vectors are used as vectors for in vivo gene transfer. Vaccineurs, adenovirus vectors, poxvirus vectors (e.g., mutant vaccinia) Vaccinia virus vectors such as Virus Ankara, adeno-associated virus vectors, and Examples include, but are not limited to, alphavirus vectors.

[0243] XI. Route, Dosage, and Administration In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0244] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0245] In some embodiments, a pharmaceutical compound comprising the polypeptide of this disclosure as a therapeutic protein is used. The product can be administered, for example, intravenously, parenterally, subcutaneously, intramuscularly, intra-arterially, or intrathecally. It is formulated for administration or intraperitoneal administration. In some embodiments, the pharmaceutical composition is administered via It is prescribed for oral, nasal, spray, aerosol, rectal, or vaginal administration, or it It is administered via a carrier. Various effective drug carriers are available for injectable formulations.

[0246] In some embodiments, nucleic acid molecules or such nuclei encoding the polypeptide of the present disclosure Pharmaceutical compositions containing acid molecules and vectors are administered by gene transfer methods. Gene introduction methods are available. In some embodiments, in vivo gene introduction and expression are used. The vectors used include retroviral vectors, adenovirus vectors, and poppers. Vaccinia virus vectors (for example, mutated vaccinia virus Ankara (MVA) and other vaccinia Near-virus vectors, adeno-associated virus vectors, and alphavirus vectors Examples include, but are not limited to, the polypeptides of the present disclosure. The mRNA molecule encoding it is administered directly to the target.

[0247] The dosage of the pharmaceutical composition disclosed herein depends 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 overall health. In some embodiments, a single The amount of polypeptide of this disclosure included in the administration is effective in treating the disease without inducing significant toxicity. A quantity that specifically prevents, delays, or treats. In some embodiments, the pharmaceutical composition of the present disclosure The substance contains the polypeptide of this disclosure in an amount of 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), in more specific embodiments, about 0.1 to about 50 The dosage includes mg / kg, and in more specific embodiments, doses ranging from approximately 1 to approximately 30 mg / kg. In some embodiments, the dosage is determined by a physician according to the severity of the disease and different parameters. It is applied by.

[0248] In some embodiments, the toxicity of the therapeutic agents and polypeptides described herein is standard By a pharmaceutical procedure, in cell culture or experimental animals, for example, LD 50 (50 in the group) (Lethal dose for % of cases) or LD 100 (A lethal dose for 100% of the population) This is determined by measurement. In some embodiments, these cell culture assays Furthermore, data obtained from animal studies can be used to formulate a dosage range that is non-toxic for human use. It is used for this purpose. The protein doses described herein are preferably those that are almost toxic. It is within the range of circulating concentrations that include an effective dose of or none. In some embodiments, The amount varies within this range depending on the dosage form used and the route of administration utilized. In this embodiment, the exact prescription, route of administration, and dosage are determined individually, taking into consideration the patient's condition. Selected by a doctor.

[0249] In some embodiments, the pharmaceutical composition is used in a manner that is compatible with the dosage formulation to treat a disease or It is administered in a dose that is therapeutically effective in improving or correcting the symptoms of the disorder. In several embodiments, the pharmaceutical composition is administered in various dosage forms, such as intravenous or subcutaneous. It is administered in various forms and in oral administration forms (e.g., ingestible solutions, drug-releasing capsules). Generally, therapeutic proteins are administered in doses of 0.1 to 100 mg / kg, for example, 1 to 50 mg / kg. It is administered in g. In some embodiments, a pharmaceutical composition comprising the polypeptide of the present disclosure is For those who need it, for example, daily, weekly, monthly, every six months, once a year or more. For example, it may be administered 1 to 10 times or more, or as medically necessary. The dosage may be a single dose or It can be administered in any of multiple drug regimens. In some embodiments, The interval between episodes decreases as the patient's condition improves, or depending on the patient's health status. It increases as the condition worsens.

[0250] XII. Treatment method In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0251] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0252] In some embodiments, further disclosed herein is wild-type SIRP-α A mutation in the D1 domain at residue 80, as well as a wild-type signal regulatory protein For the α(SIRP-α)D1 domain, residues 6, 27, 31, and 47, A residue selected from the group consisting of residues 53, 54, 56, 66, and 92. The SIRP-α D1 domain has at least one further amino acid mutation, The administration of a polypeptide containing the SIRP-α D1 variant, which includes the fragment thereof, is included. This is a treatment method.

[0253] In some embodiments, this disclosure relates to diseases associated with SIRP-α or CD47 activity. or suffering from disorders, such as cancer and immune diseases (e.g., autoimmune diseases and inflammatory diseases) The present invention provides pharmaceutical compositions and treatment methods used to treat patients who have suffered from certain conditions. In the embodiments described herein, the polypeptide is used to target the target cells of the subject ( For example, it is administered in a manner that increases the phagocytic activity of cancer cells. In some embodiments, The polypeptide is administered to the target in a manner that kills the target's cancer cells. In several embodiments, the polypeptide removes the regulatory T cells of the target. It is administered by the method described herein. In some embodiments, the polypeptide described herein is the target The method is administered to the target in a manner that increases the engraftment of hematopoietic stem cells, and the method is administered to the target This includes regulating the interaction between SIRP-α and CD47. In some embodiments, The polypeptides described herein alter the immune response of a target (for example) It is administered in a manner that suppresses the immune response. In some embodiments, the aforementioned method is Other methods of treating the disease are disclosed herein. In some embodiments, the following are disclosed herein: This involves a combination of a lipeptide (e.g., SIRP-α D1 variant) and a second therapeutic agent. In some embodiments, the combination is a polypeptide (e.g., SIRP-α D1 variant). The present invention comprises an antidote and a second therapeutic agent, the second therapeutic agent being an antibody. In some embodiments, This combination involves an amino acid mutation at residue 80 compared to the wild-type SIRP-α D1 domain. Furthermore, compared to the wild-type SIRP-α D1 domain, residues 6, 27, 31, and remainder Selected from the group consisting of group 47, residue 53, residue 54, residue 56, residue 66, and residue 92. SIRP-α D1 domain has at least one further amino acid mutation in the residue. The SIRP-α D1 variant, or a fragment thereof, and the antibody. In that embodiment, the combination is sequence numbers 78-85, 98-104, 107-113, It has one of the following sequences: 116-122, 135-137, or 152-159. It includes polypeptides and antibodies.

[0254] In some embodiments, the aforementioned method is used for diseases in which polypeptide administration is a therapeutic option. It is used in conjunction with treatment methods. Non-exclusive examples include antibodies or protein fragments. The use of is one example. For example, in some embodiments, the antibody or protein fragment is It is administered in combination with the Fc variant polypeptides disclosed herein. In embodiments, the polypeptide constructs disclosed herein improve the phagocytic action of other drugs. It is used for that purpose.

[0255] The treatment method is (i) SIRP-α D1 for subjects with a disease (e.g., cancer). The procedure includes administering a polypeptide containing a variant and optionally (ii) an antibody. In this embodiment, before treating the disease (e.g., cancer) in the subject, SIRP- The amino acid sequence(s) of α can be, for example, two pairs that encode the SIRP-α gene. It is determined from each gene. In this treatment method, the S in the biological sample derived from the subject is The amino acid sequence(s) of the IRP-α polypeptide is determined first. The target then proceeds as follows: A therapeutically effective amount of the polypeptide of this disclosure is administered. In some embodiments, The high-affinity SIRP-α D1 variant has a CD47 in the SIRP-α polypeptide. Except for introducing amino acid changes that enhance affinity, the SIR in the biological sample of the subject It has the same amino acid sequence as the P-α polypeptide. The sexual SIRP-α D1 variant is preferably used in the target after administration of the polypeptide. It exhibits minimal immunogenicity.

[0256] In some embodiments, the antibody is administered in addition to the polypeptide disclosed herein. In some embodiments, the antibody is administered simultaneously with the polypeptide. In this embodiment, the antibody is simultaneously, for example, a medical device having both the polypeptide and the antibody. It is administered in a drug composition. Alternatively, the antibody may be administered before or after the administration of the polypeptide. It is administered to one of the following. In some embodiments, the polypeptide and the antibody are substantially Simultaneously (for example, 1 week, 6, 5, 4, 3, 2, 1 day, 12, 6, 3, 2, 1) The antibody is administered (within a few hours, or substantially simultaneously), and then administered alone. In this embodiment, the antibody is administered first, and then the polypeptide and the antibody are administered substantially simultaneously. (That is, each other for 1 week, 6, 5, 4, 3, 2, 1 day, 12, 6, 3, 2, 1 hour) Administer (intravenously or substantially simultaneously).

[0257] Co-administered, supplied in a composition, or supplied in a manner disclosed herein Antibodies target cells such as cancer cells and immune system cells such as T cells (e.g., regulatory T cells). This refers to an antibody that is an immunoglobulin antibody isotype, for example, IgG. It may be IgE, IgM, IgA, or IgD. In some embodiments, The antibody is a human IgG1 isotype antibody. In some embodiments, the antibody is a human IgG1 isotype antibody. It is an IgG2 isotype antibody. In some embodiments, the antibody is human IgG4 It is an isotype antibody.

[0258] The term "antibody" is used in its broadest sense herein, as long as they exhibit the desired activity. Monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) It contains, but is not limited to, antibody fragments and antibody-like proteins, encompassing a variety of antibody structures. Includes. The "antibody fragment" is a part of an intact antibody, preferably an intact antibody. It contains an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', and F. (ab')2, and Fv fragment, bispecific antibody, linear antibody, single-chain antibody molecule, and multiplex Specific antibodies are one example. Monoclonal antibodies are obtained from a population of substantially identical antibodies. This refers to antibodies that may exist in trace amounts, for example, individual antibodies in that population may be present in small quantities. They have the same primary sequence except for spontaneously occurring mutations. Monoclonal antibodies are highly specific. It is targeted and can target a single antigen site (e.g., an epitope of a cancer antigen). In contrast to polyclonal antibody preparations, which typically contain different antibodies against specific epitopes, Each monoclonal antibody is generally targeted at a single epitope on an antigen. The modifier "noclonal" refers to the characteristic of antibodies that are obtained from a population of essentially identical antibodies. This indicates a symptom and is interpreted as requiring the production of antibodies by any specific method. It should not. In some embodiments, the antibodies in the compositions of this disclosure are antibody-dependent molecules. It causes vesicular phagocytosis (ADCP) or antibody-dependent cytotoxicity (ADCC). Non-exclusive examples of diseases treated under this law include cancers such as blood cancers, for example, leukemia. (For example, acute myeloid leukemia), immune disorders (for example, those with the disorder or reduced immune function) (To enhance the immune response, or to limit the overactive immune response of the target), and pathogenicity Infection is one example.

[0259] In some embodiments, the methods disclosed herein are polypeptides described herein. (For example, SIRP-a D1 variant), and antibodies targeting cancer antigens are administered. This includes being targeted by an antibody or antibody-like protein. Cancer antigens are complexes with human leukocyte antigen (HLA) class I molecules on the surface of the cell. It is an exposed peptide derived from ulcer-associated antigens (TAAs) (also known as an MHC / peptide complex). (known). Such cancer antigens, for example, antibodies or antibody-like proteins in the compositions of this disclosure Peptides that form complexes with HLA molecules exposed on the surface of cancer cells targeted by the substance. Non-exclusive examples include: NY-ESO-1 / LAGE1, SSX-2, MAGE Fami Lee (MAGE-A3), gp100 / pmel17, Melan-A / MART-1, gp 75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminaria Nin receptor, MOK / RAGE-1, WT-1, Her2 / neu, EphA3, SAP -1, BING-4, Ep-CAM, MUC1, PRAME, Survivalbin, Mesothelin, BRCA1 / 2 (mutant), CDK4, CML66, MART-2, p53 (mutant), Ra s (mutation), β-catenin (mutation), TGF-βRII (mutation), HPV E6, E7 Examples of such antibodies include ESK1(WT-1) and RL1B(Her2-E). 75) Examples include Pr20(PRAME) and 3.2G1(hCGβ).

[0260] In some embodiments, the methods disclosed herein involve wild-type SIRP-α D1 dormancy In addition to the mutation in residue 80 of the in, the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, At least one additional amino acid in a residue selected from the group consisting of residues 66 and 92. SIRP-α D1 domain containing a mutation, or SIRP-α D1 containing a fragment thereof. This includes administering polypeptides containing variants, as well as antibodies targeting cancer antigens. In some embodiments, the methods disclosed herein are for wild-type SIRP-α D1 The main molecule has an amino acid mutation at residue 80, as well as a wild-type SIRP-α D1 domain. In contrast, residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, remain At least one additional amino acid is added to a residue selected from the group consisting of group 66 and residue 92. SIRP-α D1 domain containing an acid mutation, or SIRP-α D containing a fragment thereof Polypeptides containing one variant, as well as NY-ESO-1 / LAGE1, SSX-2 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 (mutant), CDK4, CML66, MART-2, p5 3 (mutation), Ras (mutation), β-catenin (mutation), TGF-βRII (mutation), HP V includes administering antibodies targeting E6 and E7. In some embodiments, the present invention The method disclosed in the document involves adding an amino acid to residue 80 of the wild-type SIRP-α D1 domain. Mutations in the wild-type SIRP-α D1 domain, as well as residues 6, 27, and 3 A group consisting of residues 31, 47, 53, 54, 56, 66, and 92. SIRP-α D having at least one further amino acid mutation in the selected residues Polypeptides containing a SIRP-α D1 variant, which includes one domain or a fragment thereof. Also, ESK1 (WT-1), RL1B (Her2-E75), Pr20 (PRAME This includes administering an antibody that is ), and 3.2G1(hCGβ).

[0261] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 Sequences 04, 107-113, 116-122, 135-137, or 152-159 This includes administering polypeptides having and antibodies targeting cancer antigens. In embodiments, the methods disclosed herein include Sequence IDs 78-85, 98-104, and 107. One of the following distributions: ~113, 116~122, 135~137, or 152~159 Polypeptides having columns, and NY-ESO-1 / LAGE1, SSX-2, MAGE Family (MAGE-A3), gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor, MOK / RAGE-1, WT-1, Her2 / neu, EphA3, S AP-1, BING-4, Ep-CAM, MUC1, PRAME, Survival, Mesoteri N, BRCA1 / 2 (mutation), CDK4, CML66, MART-2, p53 (mutation), Ras (mutation), β-catenin (mutation), TGF-βRII (mutation), HPV E6, E This includes administering an antibody that targets 7. In some embodiments, the following are disclosed herein. The method is for sequence numbers 78-85, 98-104, 107-113, 116-122, 13 Polypeptides having one of the sequences 5-137 or 152-159, and ESK1 (WT-1), RL1B (Her2-E75), Pr20 (PRAME), and This includes administering an antibody that is 3.2G1(hCGβ).

[0262] In some embodiments, the antibody is, for example, a protein expressed by cancer cells. By binding, it targets cancer cells. Some proteins are more effective than non-cancer cells. It is also expressed at high levels in cancer cells. For example, cancer antigens are predominantly expressed by cancer cells. It is a protein that is expressed first (for example, it is expressed more in cancer cells than in non-cancer cells). (expressed at high levels), and in some cases, it is expressed only in cancer cells. Protein Quality, for example, expressed by cancer cells targeted by the antibody in the composition of this disclosure Non-specific examples of proteins that can be affected 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 Examples include -1, CSF-1 R, or any combination thereof. Several embodiments Therefore, the polypeptides described herein are checkpoint inhibitors, for example, CTLA- Antibody inhibitors of 4 (e.g., ipilimumab, tremelimumab), antibody inhibitors of PD-1 (e.g.) For example, nivolumab, pidilizumab, MK3475, also known as pembrolizumab, BMS93 6559, and MPDL3280A), as well as antibody inhibitors of LAG-3 (e.g., BM It is administered in combination with S986016.

[0263] In some embodiments, the methods disclosed herein involve wild-type SIRP-α D1 dormancy In addition to the mutation in residue 80 of the in, the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, At least one additional amino acid in a residue selected from the group consisting of residues 66 and 92. SIRP-α D1 domain containing a mutation, or SIRP-α D1 containing a fragment thereof. Targeting polypeptides containing variants, as well as proteins expressed by cancer cells. This includes administering an antibody. In some embodiments, the method disclosed herein is Compared to the wild-type SIRP-α D1 domain, there is an amino acid mutation at residue 80, and wild For the SIRP-α D1 domain, residues 6, 27, 31, 47, and Residues selected from the group consisting of residues 53, 54, 56, 66, and 92 are less The SIRP-α D1 domain has at least one further amino acid mutation, or Polypeptides containing a fragment of SIRP-α D1 variant, as well as 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, Antibody targeting CD27L, DKK-1, CSF-1 R, or any combination thereof This includes administering the body. In some embodiments, the methods disclosed herein are wild-type The SIRP-α D1 domain has an amino acid mutation at residue 80, as well as a wild-type SIR For the P-α D1 domain, residues 6, 27, 31, 47, 53, remain At least one residue selected from the group consisting of group 54, residue 56, residue 66, and residue 92 A SIRP-α D1 domain, or a fragment thereof, having one further amino acid mutation polypeptides containing the SIRP-α D1 variant, as well as antibody inhibition of CTLA-4. Harmful agents (e.g., ipilimumab, tremelimumab), PD-1 antibody inhibitors (e.g., nibo Lumab, pidilizumab, MK3475, also known as pembrolizumab, BMS936559. (and MPDL3280A), or antibody inhibitors of LAG-3 (e.g., BMS98601) 6) This includes administering an antibody.

[0264] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 Polypeptides having any one of the sequences, and proteins expressed by cancer cells are marked. This includes administering the target antibody. In some embodiments, the method disclosed herein This includes sequence numbers 78-85, 98-104, 107-113, 116-122, 135-1 A polypeptide having one of the sequences 37 or 152-159, and 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-1 R, or any combination thereof This includes administering antibodies that target [the target]. In some embodiments, the following are disclosed herein. The method is for sequence numbers 78-85, 98-104, 107-113, 116-122, 13 Polypeptides having one of the sequences 5-137 or 152-159, and CTLA-4 antibody inhibitors (e.g., ipilimumab, tremelimumab), PD-1 antibody Body inhibitors (e.g., nivolumab, pidilizumab, MK3475, also known as pembrolizumab) , BMS936559, and MPDL3280A), or antibody inhibitors of LAG-3 (e.g.) For example, this includes administering an antibody such as BMS986016.

[0265] In some embodiments, the methods disclosed herein are polypeptides described herein. This includes administering antibodies (e.g., SIRP-a D1 variant) and immuno-oncological antibodies. In some embodiments, the antibody used in the compositions of this disclosure is cetuximab. Necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469, atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, Ipilimumab, Tremelimumab, Urelumab, PF-05082566, Enoblituzumab, vantictu mab), varlilumab, mogamalizumab zumab), SAR650984, daratumumab, trastuzumab, trastuzumab Mutansine, pertuzumab, elotuzumab, rituximab, o Fatumumab, Obinutuzumab, RG7155, FPA008, Panitumumab, Brentz Ximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, Panitumumab, Ramucirumab, Necitumumab, Nivolumab, Pembrolizumab, Avelumab B, 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 Examples include, but are not limited to, AFF antibodies, anti-VEGF antibodies, or anti-VEGFR2 antibodies. Not possible. In some embodiments, the methods disclosed herein are wild-type SIRP-α D 80 amino acid mutations in one domain, as well as wild-type SIRP-α D1 domain For 'in', residues 6, 27, 31, 47, 53, 54, and 56 At least one further A is selected from the group consisting of residue 66 and residue 92. SIRP-α containing a SIRP-α D1 domain with a mino acid mutation, or a fragment thereof. Polypeptides containing the D1 variant, as well as anti-HER2 antibodies, anti-CD20 antibodies, anti-C2 antibodies. 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 This includes administering an antibody. In some embodiments, the method disclosed herein is Compared to the wild-type SIRP-α D1 domain, there is an amino acid mutation at residue 80, and wild For the SIRP-α D1 domain, residues 6, 27, 31, 47, and Residues selected from the group consisting of residues 53, 54, 56, 66, and 92 are less The SIRP-α D1 domain has at least one further amino acid mutation, or polypeptides containing a fragment of SIRP-α D1 variant, and cetuximab Necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469, atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, Ipilimumab, Tremelimumab, Urelumab, PF-05082566, Enoblituzumab, vantictu mab), varlilumab, mogamalizumab zumab), SAR650984, daratumumab, trastuzumab, trastuzumab Mutansine, pertuzumab, elotuzumab, rituximab, o Fatumumab, Obinutuzumab, RG7155, FPA008, Panitumumab, Brentz Ximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, Panitumumab, Ramucirumab, Necitumumab, Nivolumab, Pembrolizumab, Avelumab B, atezolizumab, durvalumab, MEDI0680, pidilizumab, or BM This includes administering the antibody S-93659.

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

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

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

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

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

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

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

[0273] In some embodiments, the methods disclosed herein involve wild-type SIRP-α D1 dormancy In addition to the mutation in residue 80 of the in, the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, At least one additional amino acid in a residue selected from the group consisting of residues 66 and 92. SIRP-α D1 domain containing a mutation, or SIRP-α D1 containing a fragment thereof. Polypeptides containing variants, as well as pantimumab. This includes administering antibodies.

[0274] In some embodiments, the methods disclosed herein involve wild-type SIRP-α D1 dormancy In addition to the mutation in residue 80 of the in, the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, At least one additional amino acid in a residue selected from the group consisting of residues 66 and 92. SIRP-α D1 domain containing a mutation, or SIRP-α D1 containing a fragment thereof. The administration of a polypeptide containing a variant, as well as an antibody which is ramucirumab, is included. .

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

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

[0277] In some embodiments, the methods disclosed herein involve wild-type SIRP-α D1 dormancy In addition to the mutation in residue 80 of the in, the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, At least one additional amino acid in a residue selected from the group consisting of residues 66 and 92. SIRP-α D1 domain containing a mutation, or SIRP-α D1 containing a fragment thereof. The administration of a polypeptide containing the variant, as well as the antibody pembrolizumab. include.

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

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

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

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

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

[0283] In some embodiments, the methods disclosed herein involve wild-type SIRP-α D1 dormancy In addition to the mutation in residue 80 of the in, the wild-type SIRP-α D1 domain In contrast, residues 6, 27, 31, 47, 53, 54, 56, At least one additional amino acid in a residue selected from the group consisting of residues 66 and 92. SIRP-α D1 domain containing a mutation, or SIRP-α D1 containing a fragment thereof. Administering a polypeptide containing the variant, as well as the antibody BMS-93659. Includes.

[0284] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 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 This includes administering an antibody. In some embodiments, the method disclosed herein is Sequence numbers 78-85, 98-104, 107-113, 116-122, 135-13 Polypeptides having sequences 7 or 152-159, as well as cetuximab, necit Mumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED1 6469, Atezolizumab, Avelumab, Durvalumab, MEDI6383, RG78 88, Ipilimumab, Tremelimumab, Urelumab, PF-05082566, Enobili Tuzumab (enoblituzumab), vantictumab varlilumab, mogamalizuma b) SAR650984, daratumumab, trastuzumab, trastuzumab emtans 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 This includes administering an antibody that is 659.

[0285] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 A polypeptide having one of the sequences, and the antibody trastuzumab are administered. This includes doing so.

[0286] In some embodiments, the methods disclosed herein are shown in SEQ ID NOs: 7, 78-88. 5, 98-104, 107-113, 116-122, 135-137, or 152- A polypeptide having any one of 159 sequences, and an antibody that is rituximab This includes administering the drug.

[0287] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 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 are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 Administering a polypeptide having one of the sequences, and an antibody that is daratumumab. Includes.

[0289] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 The administration of a polypeptide having one of the sequences, and an antibody that is belimumab. include.

[0290] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 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 are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 The administration of a polypeptide having one of the sequences, and an antibody that is denosumab. include.

[0292] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 A polypeptide having any one of the sequences, and pantimumab This includes administering an antibody.

[0293] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 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 are represented by Sequence IDs 78-85, 98-1 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 are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 The antibody comprises a polypeptide having any one of the sequences, and nivolumab.

[0296] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 Administer a polypeptide having one of the sequences, and an antibody that is pembrolizumab. This includes the following.

[0297] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 The administration of a polypeptide having one of the sequences, and an antibody that is avelumab. include.

[0298] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 Administering a polypeptide having one of the sequences, as well as an antibody that is atezolizumab. This includes.

[0299] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 Administering a polypeptide having one of the sequences, as well as an antibody that is durvalumab. This includes.

[0300] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 Administer a polypeptide having one of the sequences, and an antibody that is MEDI0680. This includes the following.

[0301] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 The antibody comprises a polypeptide having any one of the sequences, and a pidilizumab antibody.

[0302] In some embodiments, the methods disclosed herein are represented by Sequence IDs 78-85, 98-1 04, 107-113, 116-122, 135-137, or 152-159 A polypeptide having one of the sequences, and an antibody that is BMS-93659 are administered. This includes doing so.

[0303] In some embodiments, the polypeptides disclosed herein are antitumor agents of rituximab. To enhance activity. In some embodiments, the polypeptide disclosed herein is Raji -Enhance the antitumor activity of rituximab in NSG xenograft models. Several implementations In this context, the polypeptides disclosed herein are used in rituxi in non-human primates (NHPs). Mab increases the depletion of B cells mediated by the drug.

[0304] In some embodiments, polypeptides and pharmaceutical compositions of the present disclosure are used in various cancer treatments. It is used. Cancers suitable for treatment according to this disclosure include solid tumor cancers, hematological cancers, and acute bone cancers. Myelin leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's disease Lymphoma, Hodgkin lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, intrauterine cancer Membrane cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colorectal cancer, gastric cancer (ST) omatous cancer, gastric cancer, gallbladder cancer, gastrointestinal interstitial cancer Tumor cancer, thyroid cancer, head and neck cancer, oral and pharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin Hmm, Merkel cell carcinoma, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer Cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lymphoma, non-epithelial malignant tumors, glioma, brain cancer Examples include, but are not limited to, epithelial malignancies. In some embodiments, Cancer conditions suitable for treatment according to this disclosure include metastatic cancer. Therefore, cancers suitable for treatment according to this disclosure are solid tumors or hematological cancers.

[0305] In some embodiments, the antibody binds to a protein expressed by cells of the immune system. This targets immune system cells such as T cells, for example, regulatory T cells. In some embodiments, the methods disclosed herein involve polypeptides described herein (for example) If, then, administer the SIRP-a D1 variant and antibodies that target immune system cells. This includes. Examples of proteins expressed by immune system cells include 41BB and 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 Examples include, but are not limited to, 83, TIGIT, CD154, CD160, and PD1H. It is not done. In some embodiments, the antibody is T against proteins (e.g., receptors) expressed by cells (e.g., regulatory T cells) Designed to have selective bonding. In some embodiments, the compositions of the present disclosure The antibodies contain the Fc domain of an IgG1, IgG2, or IgG4 subclass.

[0306] In some embodiments, the methods disclosed herein are used to alter the immune response in a subject. The method involves applying a high-affinity SIRP-α D1 variant to the target. This includes administering peptides and antibodies to alter the immune response in the subject. In some embodiments, altering the immune response is equivalent to suppressing the immune response. Includes.

[0307] In some embodiments, the polypeptides and pharmaceutical compositions of the present disclosure are used to treat immunological diseases. It is used in various therapies to treat autoimmune diseases and inflammatory diseases suitable for treatment according to this disclosure. Diseases include multiple sclerosis, rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, and antibodies. Mediated inflammatory or autoimmune diseases, graft-versus-host diseases, sepsis, diabetes, psoriasis, atherosclerosis Arteriosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, Hemorrhagic reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma These include respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis. This is not limited to these.

[0308] In some embodiments, the delivery of polypeptides to cells is performed using the compositions described herein. This involves bringing one or more of them into contact with the cells.

[0309] The effective dose of such treatment options depends on the method of administration, the target site, the physiological state of the patient, and the patient's condition. Whether it is a human or an animal, other drugs administered, and whether the treatment is preventive or therapeutic. It varies due to many different factors, including. In some embodiments, the patient is human. However, non-human mammals, such as companion animals like dogs, cats, and horses, rabbits, and maws... Experimental animals such as rats are also treated. In some embodiments, the therapeutic dose is stable. The totality and effectiveness are gradually increased to optimize them.

[0310] In some embodiments, the therapeutic dose is approximately 0.0001 to 100 mg / kg of the host's body weight. g, more typically ranging from 0.01 to 30 mg / kg. In some embodiments, for example, The dosage is 1 mg / kg body weight or 30 mg / kg body weight, or 1-30 mg / It is within the range of kg. In some embodiments, an exemplary treatment plan is once a week or The treatment involves administration every two weeks, once a month, or once every three to six months. Several implementations In this configuration, 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 is also indicated by measuring the blood levels of the therapeutic entity in the patient. Therefore, it is irregular. Alternatively, the therapeutic or polypeptide constructs described herein It is administered as a sustained-release formulation, in which case less frequent administration is possible. In the embodiment, the dose and frequency vary depending on the half-life of the polypeptide in the patient.

[0311] In prophylactic applications, in some embodiments, relatively low doses are used for relatively infrequent use. It is administered at intervals over a long period of time. In some embodiments, the patient is given for the rest of their life. Continue treatment over time. In other therapeutic applications, the progression of the disease is reduced or terminated. Until, preferably until the patient shows partial or complete remission of the symptoms of the disease. Therefore, relatively high doses are required at relatively short intervals. Subsequently, in some embodiments, The patient will be given a prophylactic treatment plan.

[0312] As used herein, terms such as "treatment" and "to treat" are used for the purpose of achieving an effect. This refers to administering a drug or performing a treatment to a person. In some embodiments, this effect It is preventive in that it completely or partially prevents a disease or its symptoms. In some embodiments, the effect is the partial or complete cure of the disease or its symptoms. It is therapeutic in that it has an effect.

[0313] XIII. Kit In some embodiments, the wild-type signal control protein is disclosed herein. A mutation in the amino acid at residue 80 of the α(SIRP-α)D1 domain, as well as a wild-type mutation. For the SIRP-α D1 domain, residues 6, 27, 31, 47, and 5 3. Less than a few residues selected from the group consisting of residues 54, 56, 66, and 92. A SIRP-α D1 domain having at least one further amino acid mutation, or This polypeptide contains a SIRP-α D1 variant that includes a fragment.

[0314] In some embodiments, also disclosed herein, are Fc variants. A polypeptide wherein the Fc variant has two Fc domain monomers. The domain dimer is included, and each Fc domain monomer is independently (i) L23 The Fc region of human IgG1 consisting of mutations 4A, L235A, G237A, and N297A. (ii) Human IgG2 Fc consisting of A330S, P331S, and N297A mutations Area, or (iii) S228P, E233P, F234V, L235A, delG2 Selected from the Fc region of human IgG4, including mutations 36 and N297A, the polypept It's Chido.

[0315] Also provided is a kit containing the polypeptides described herein and instructions for their use. The kit may optionally include at least one additional reagent. As a non-limiting example, a chemotherapy agent or antitumor antibody may be used in combination with at least one additional agent. It can fulfill its role. In some embodiments, the kit is intended to be the contents of the kit. Includes labels indicating the illustrated uses. The term "label" refers to the labels supplied on the kit. Alternatively, any optional kit supplied with the kit or otherwise attached to the kit. This includes written documents or records.

[0316] In some embodiments, the kit (i) contains a high affinity SIRP-α D1 variant Polypeptides containing (ii) antibodies, and (iii) subjects with the disease. (i) and (ii) instructions for administering (if provided). Several implementations In terms of form, the kit contains (i) a polypeptide containing a high-affinity SIRP-α D1 variant. , and (ii) an antibody provided in the kit for a subject having the disease Includes instructions for administration with antibodies that are not present. In some embodiments, the kit is (i) antibodies, and (ii) high affinity SIRP-α D for subjects with the disease. Includes instructions for administration with a polypeptide containing one variant.

[0317] In some embodiments, the kit is used for, for example, solid tumors, hematological cancers, and acute myeloid cancers. Leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's disease Parkinson's cancer, Hodgkin's lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometriosis Lung cancer, bronchial cancer, liver cancer, ovarian cancer, colorectal cancer, stomach cancer (stom Gallbladder cancer, gastric cancer, gastrointestinal stromal tumor Cancer, thyroid cancer, head and neck cancer, oral and pharyngeal 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 pelvis cancer, leukemia, lymphoma, non-epithelial malignant tumor, glioma, brain cancer, etc. It is used to treat patients with cancer, including cutaneous malignancies and any combination thereof. In some embodiments, the kit is used for subjects with solid tumor cancer or hematological cancer. It is used to treat [condition].

[0318] In some embodiments, the kit is used to treat subjects with immune disorders. In some embodiments, the immune disease is an autoimmune disease or an inflammatory disease. For example, multiple sclerosis, rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, antibody-mediated Inflammatory or autoimmune diseases, graft-versus-host diseases, sepsis, diabetes, psoriasis, atherosclerosis Arteriosclerosis, 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 the above. It is a combination. [Examples]

[0319] Example 1 - SIRP-α D1 variant polypeptide Generation of polypeptides in this disclosure The polypeptides of this disclosure, including the high-affinity SIRP-α D1 variant, are compared to conventional molecular polymers. It is produced using ronning and protein expression techniques. Wild-type SIRP-α D1 domain Possible amino acid substitutions in the SIRP-α D1 variant for yin are shown in Table 2. A...

Claims

1. Residue 80 of the wild-type signal regulatory protein α(SIRP-α) D1 domain Mutations in the mino acid, as well as residues 6 and 27 of the wild-type SIRP-α D1 domain. , consisting of residues 31, 47, 53, 54, 56, 66, and 92 SIRP- has at least one further amino acid mutation in a residue selected from the group. Polypeptide containing the α D1 domain or a fragment thereof, and a SIRP-α D1 variant. Chido.

2. The wild-type SIRP-α D1 domain contains one of the sequences of sequence numbers 1 to 10. The polypeptide according to claim 1, having

3. The aforementioned SIRP-α D1 domain is, compared to the wild-type SIRP-α D1 domain, Group 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and A residue selected from the group of 92 residues contains 1 to 9 further amino acid mutations. The polypeptide described in Item 1.

4. The SIRP-α D1 variant has an 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 Includes TELSVRAKPS (SEQ ID NO: 49), where X １ is V, L, or I , X ２ is A, I, V, or L, and X ３ is I, F, S, or T, X ４ is E, V or L, X ５ is K or R, X ６ is E or Q, X ７ H, P , or R, X ８ is L, T, S, or G, X ９ A is X １０ はVma The SIRP-α D1 variant is a wild-type variant having the sequence of sequence number 1. The claim is that the SIRP-α D1 domain has at least two amino acid substitutions. The polypeptide described in 1.

5. The SIRP-α D1 variant is one of the amino acids in SEQ ID NOs. 78 to 85. The polypeptide according to claim 4, having an array.

6. The SIRP-α D1 variant has an 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 Includes FKSGAGTTELSVRAKPS (SEQ ID NO: 218), where X １ is V, L, or I, X ２ is A, V, L, or I, and X ３ is I, S, T, or F ri, X ４ is E, L, or V, and X ５ is K or R, 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 It is a amino acid, X １０ is any amino acid, X １１ is any amino acid, X １２ is any amino acid, X １３ is V or I, and the SIRP-α D1 varian The wild-type SIRP-α D1 domain having the sequence of Sequence ID No. 1 has at least The polypeptide according to claim 1, having two amino acid substitutions.

7. X ９ The polypeptide according to claim 6, wherein A.

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

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

10. X ９ N is X １０ The polypeptide according to claim 6, wherein P is

11. X ９ N is X １１ Claim 6, wherein is any amino acid other than S, T, or C. The polypeptide described.

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

13. X １１ The polypeptide according to claim 6, wherein is an amino acid other than T.

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

15. X ９ N is X １２ The polypeptide according to claim 6, wherein is any amino acid other than P. Chido.

16. The SIRP-α D1 variant has an 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 Includes AGTELSVRAKPS (SEQ ID NO: 219), where X １ is V, L, or I X ２ is A, V, L, or I, and X ３ is I, S, T, or F, and X ４ is E, L, or V, and X ５ is K or R, X ６ is E or Q, X ７ teeth H, R, or P, X ８ is S, G, L, or T, X ９ is N, X １０ is any amino acid other than P, and X １１ is V or I, and the SIRP-α D1 The variant is less than the wild-type SIRP-α D1 domain which has the sequence of SEQ ID NO:

1. The polypeptide according to claim 1, having at least two amino acid substitutions.

17. The SIRP-α D1 variant has an amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRELIYNQX ４ EGX ５ FPRVTTVSDX ６ TKRNNMDF SIRIGX ７ ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELS Includes VRAKPS (SEQ ID NO: 52), where X １ is V, L, or I, and X ２ teeth is A, I, or L, and X ３ is I, T, S, or F, and X ４ is K or R , X ５ is H, P, or R, and X ６ is L, T, or G, X ７ is A, and before The SIRP-α D1 variant is a wild-type SIRP-α D variant having the sequence of sequence number 1. The polypeptide according to claim 1, having at least two amino acid substitutions per domain Chido.

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

19. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of sequence number 1. The D1 domain has at least three amino acid substitutions, according to claim 17. Polypeptide.

20. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of sequence number 1. The D1 domain has at least four amino acid substitutions, according to claim 17. Polypeptide.

21. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of sequence number 1. The D1 domain has at least five amino acid substitutions, according to claim 17. Polypeptide.

22. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of sequence number 1. The D1 domain has at least six amino acid substitutions, according to claim 17. Polypeptide.

23. The SIRP-α D1 variant is a wild-type SIRP-α having the sequence of sequence number 1. The D1 domain has at least seven amino acid substitutions, according to claim 17. Polypeptide.

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

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

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

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

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

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

30. X １ , X ２ , X ３ , X ４ , X ５ , and X ６ Each of them is not a wild-type amino acid, claim 1 polypeptide as described in 7.

31. The SIRP-α D1 variant is one of the amino acids in sequence numbers 81 to 85. The polypeptide according to claim 17, having an array.

32. The aforementioned 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 TYYCVKFRKGSPDTEFKSGAGTTELSVRAKPS (Sequence ID 212) This includes, where X １ is V, L, or I, and X ２ is V, I, or L, and X ３ is I, T, S, or F, X ４ is K or R, X ５ is H, P, or R ri, X ６ is S, T, or G, X ７ is A, and the SIRP-α D1 varian The wild-type SIRP-α D1 domain having the sequence of sequence number 2 is at least The polypeptide according to claim 1, which also has two amino acid substitutions.

33. For human CD47, K Ｄ Approximately 5x10 －９ The polymer according to claim 1, which is bonded at a minimum of M. peptide.

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

35. The Fc domain monomer is the Fc of wild-type human IgG1, IgG2, or IgG4. The polypeptide according to claim 34, comprising at least one mutation in the region.

36. One amino acid combination of SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137 The polypeptide according to claim 35, having columns.

37. The Fc domain monomer (a) has the following amino acids relative to wild-type human IgG1 One of the replacements: 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, (b) (i) N297A mutation in the Fc region of human IgG1, (ii) human IgG Mutations of L234A, L235A, and G237A in the Fc region of 1, (iii) For the Fc region of IgG1, L234A, L235A, G237A, and N297A Mutation of (iv) human IgG2 Fc region, N297A mutation, (v) human Ig Mutations of A330S and P331S in the Fc region of G2, (vi) human IgG2 F Mutations in the c region: A330S, P331S, and N297A, (vii) Human Ig For the Fc region of G4, S228P, E233P, F234V, L235A, and de Mutations in IgG236, or (viiii) S228P in the Fc region of human IgG4 This includes mutations E233P, F234V, L235A, delG236, and N297A. The polypeptide according to claim 35.

38. In phagocytic assays, compared to the polypeptide of the Fc region of wild-type human IgG, The polypeptide according to claim 35, which exhibits a reduction in usage.

39. The Fc domain monomer is a second polypeptide comprising a second Fc domain monomer. The polypeptide according to claim 35, which is linked to and forms an Fc domain dimer.

40. Claim 39, wherein the second Fc domain monomer is linked to a further polypeptide. Polypeptides as described.

41. The further polypeptide comprises an antibody variable domain, as described in claim 40. Chido.

42. The antibody variable domain targets an antigen expressed on a cell, according to claim 41. Polypeptide.

43. The polypeptide according to claim 42, wherein the cells are cancer cells.

44. The aforementioned antibody variable domain targets cell surface proteins involved in immune cell regulation. The polypeptide according to claim 43.

45. The further polypeptide comprises a therapeutic protein, as described in claim 40. Chido.

46. The aforementioned therapeutic proteins include cytokines, interleukins, antigens, steroids, and anti-inflammatory agents. The polypeptide according to claim 45, which is a symptomatic drug or an immunomodulator.

47. The further polypeptide comprises the SIRP-α D1 variant as described in claim 45. Polypeptide.

48. Furthermore, the product according to claim 1 contains human serum albumin (HSA) (SEQ ID NO: 12). Lipeptide.

49. The HSA contains the amino acid substitution C34S or K573P in relation to SEQ ID NO:

12. The polypeptide according to claim 48.

50. The amino acid sequence of any one of sequence numbers 152 to 159, according to claim 48. Polypeptide.

51. Furthermore, the polypeptide according to claim 1, comprising an albumin-binding peptide.

52. The albumin-binding peptide is the amino acid of DICLPRWGCLW (SEQ ID NO: 160). The polypeptide according to claim 51, comprising an acid sequence.

53. Furthermore, the polyethylene glycol (PEG) polymer according to claim 1 is further comprising polyethylene glycol (PEG) polymer. Petit Do.

54. The PEG polymer is linked to the cysteine ​​substitution of the polypeptide, according to the claim. Polypeptides as described in 53.

55. Polypeptides, (a) signal regulatory protein α(SIRP-α)D1 barrier It is a marker, and also 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 Includes KPS (Sequence ID 47), where X １ is E or G , X ２ is L, I, or V, and X ３ is V, L, or I, and X ４ is S, or F X ５ is L or S, X ６ is S or T, X ７ is A or V Yes, X ８ is I or T, X ９ is H, R, or L, X １０ is A, V, I , or L, X １１ is I, T, S, or F, X １２ is A or G , X １３ is E, V, or L, and X １４ is K or R, and X １５ is E, or Q is X １６ is H, P, or R, and X １７ is D or E, 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, X ２２ is S or R, X ２３ is S or G Yes, X ２４ is any amino acid, X ２５ is any amino acid, X ２６ は V, ma ta is I, X ２７ is F, L, or V, and X ２８ is either D or does not exist , X ２９ is T or V, X ３０ is F or V, and X ３１ is A or G Yes, wild-type SIRP-α D1 domain has one of the sequences 1 to 10. The SIRP-α D1 variant having at least two amino acid substitutions relative to n , and (b) Fc domain dimer comprising two Fc domain monomers In addition to being a variant, each Fc domain monomer independently (i) N297A (ii) the Fc region of human IgG1 containing mutations, L234A, L235A, and G237A The Fc region of human IgG1 containing the following mutations: (iii) L234A, L235A, G237A , and the Fc region of human IgG1 including the N297A mutation, (iv) including the N297A mutation Human IgG2 containing mutations in the Fc region, (v)A330S and P331S Human IgG containing mutations in the Fc region, (vi)A330S, P331S, and N297A Fc area 2, (vii) S228P, E233P, F234V, L235A, and de Human IgG4 Fc region containing the IgG236 mutation, or (viiii)S228P, E2 Human I containing mutations 33P, F234V, L235A, delG236, and N297A The polypeptide comprising the Fc variant which is the Fc region of gG4.

56. One of the Fc domain monomers of the Fc domain dimer is L234A, L23 A claim comprising the Fc region of human IgG1 including mutations 5A, G237A, and N297A. Polypeptides as described in 55.

57. Sequence numbers 98-104, 107-113, 116-122, or 135-137 The polypeptide according to claim 55, comprising one of the amino acid sequences.

58. The Fc variant, compared to the wild-type version of the Fc region of human IgG, has different properties when interacting with the Fcγ receptor. The polypeptide according to claim 55, which exhibits the removal or reduction of a bond.

59. The Fc variant of IgG1 or IgG2 is the Fc variant of human IgG1 or IgG2. Compared to the wild-type version of the region, CD16a, CD32a, CD32b, CD32c, and C The polypter according to claim 55, which exhibits removal or reduction of the binding of D64 to the Fcγ receptor. Petit Do.

60. The Fc variant of IgG4 is compared to the wild-type version of the Fc region of human IgG4. Claims to show the removal or reduction of the binding of CD16a and CD32b to the Fcγ receptor. Polypeptides as described in item 55.

61. The Fc variant of IgG1 or IgG2 is the Fc variant of human IgG1 or IgG2. Claim 55 shows removal or reduction of the bond to C1q compared to the wild-type version of the fusion. The polypeptide described.

62. The Fc variant, in relation to the Fcγ receptor, Ｄ Approximately 5x10 －６ They bond in a magnitude greater than M. The polypeptide according to claim 55.

63. A polypeptide comprising an Fc variant, wherein the Fc variant comprises two Fc-domed molecules. It comprises an Fc domain dimer having an in monomer, and each Fc domain monomer independently (i) Human I consisting of mutations L234A, L235A, G237A, and N297A. Human γ1 mutations consisting of the Fc region, (ii)A330S, P331S, and N297A IgG2 Fc region, or (iii) S228P, E233P, F234V, L235 Selected from the Fc region of human IgG4, including mutations A, delG236, and N297A. The polypeptide.

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

65. At least one of the Fc domain monomers is L234A, L235A, G237A The Fc region of human IgG1 consisting of the N297A mutation, as described in claim 63. Lipeptide.

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

67. The Fc variant, compared to the wild-type version of the Fc region of human IgG, exhibits the following characteristics regarding Fcγ receptors. The polypeptide according to claim 63, which exhibits removal or reduction of binding to the body.

68. The Fc variant, compared to the wild-type version of the Fc region of human IgG, has CD16a, C Removal of binding of D32a, CD32b, CD32c, and CD64 to the Fcγ receptor. The polypeptide according to claim 67, which exhibits a decrease.

69. The Fc variant, compared to the wild-type version of the human IgG Fc fusion, has a different relationship with C1q. The polypeptide according to claim 63, which exhibits the removal or reduction of a bond.

70. At least one of the Fc domain monomers is S228P, E233P, F234V In the Fc region of human IgG4, including mutations in L235A, delG236, and N297A A polypeptide according to claim 63.

71. The Fc variant, compared to the Fc region of wild-type human IgG4, interacts with the Fcγ receptor. The polypeptide according to claim 70, which exhibits the removal or reduction of a bond.

72. The Fc variant, compared to the wild-type version of the Fc region of human IgG4, has a CD16 Claim 71 describes the removal or reduction of the binding of a and CD32b to the Fcγ receptor. Polypeptide.

73. The Fc variant, in relation to the Fcγ receptor, Ｄ Approximately 5x10 －６ They bond in a magnitude greater than M. The polypeptide according to claim 63.

74. Furthermore, the polypeptide according to claim 63, comprising a CD47-conjugated polypeptide.

75. The Fc variant, compared to the wild-type version of the Fc region of human IgG, has different properties when interacting with the Fcγ receptor. The polypeptide according to claim 74, which exhibits the removal or reduction of a bond.

76. The aforementioned CD47-binding polypeptide causes acute anemia in rodents and non-human primates. The polypeptide according to claim 74, which does not cause a reaction.

77. Claim 7, the CD47-binding polypeptide does not cause acute anemia in humans. Polypeptide as described in 4.

78. The CD47-binding polypeptide is a signal regulatory protein α(SIRP-α) polypeptide. The polypeptide according to claim 74, wherein the polypeptide is a peptide or a fragment thereof.

79. The SIRP-α polypeptide has an amino acid sequence EEELQX １ IQPDKSVLVAAGETATLRCTX ２ TSLX ３ PVGPIQ WFRGAGPGRX ４ LIYNQX ５ EGX ６ FPRVTTVSDX ７ TKRNNMD FSIRIGX ８ ITPADAGTYYCE ９ KFRKGSPDDVEFKSGAGTE Includes LSVRAKPS (SEQ ID NO: 51), where X １ is V or I, X ２ is A or I, X ３ is I or F, X ４ is E or V, X ５ is K or R X ６ is H or P, X ７ is L or T, X ８ is any mesh other than N It is an acid, X ９ Claim 78 includes a SIRP-α D1 variant which is V or I. Polypeptides as described.

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

81. A polypeptide, which is a signal regulatory protein α(SIRP-α)D1 variant. It is also a non-naturally occurring high-affinity SIRP-α D1 domain, and is human CD47 The affinity of the naturally occurring SIRP-α D1 domain that binds to it is at least 10 times higher than that of the parent. The SIRP-α D1 variant, which binds to human CD47 in a non-viable manner, and Fc Domain It is a monomer and also linked to a second polypeptide containing a second Fc domain monomer. They are connected to form an Fc domain, and the Fc domain is removed or reduced effector - The preceding Fc domain monomer having a function and a removed or reduced C1q bond Note polypeptide.

82. The aforementioned non-spontaneously occurring high-affinity SIRP-α D1 domain has an amino acid mutation at residue 80. The polypeptide according to claim 81, comprising:

83. The polypeptide contained the signal regulatory protein α(SIRP-α)D1 variant. And the SIRP-α D1 variant is K Ｄ CD4 of the first species with a mass of less than 250 nM 7 is bonded to the SIRP-α D1 variant, K Ｄ Less than 250 nM and of second species origin The CD47 is bound to the K of the first species. Ｄ And the second The K for CD47 derived from the species Ｄ The first species and the The second species is selected from the group consisting of humans, rodents, and non-human primates, the polyp Petit Do.

84. The SIRP-α D1 variant is bound to CD47 derived from at least three different species. The polypeptide according to claim 83.

85. The polypeptide according to claim 83, wherein the non-human primate is a cynomolgus macaque.

86. (a) K Ｄ Signal regulatory protein α(SI) that binds to human CD47 at a M of less than 250 nM (b) RP-α D1 domain, and (b) the N-terminus or C of the SIRP-α D1 domain. Polypeptides comprising a terminally linked Fc domain monomer, for use in rodents and non-rodents. The polypeptide that does not cause acute anemia in primates.

87. Claim 86 describes a non-naturally occurring variant of the human SIRP-α D1 domain. polypeptide.

88. In vivo administration of the aforementioned polypeptide resulted in a hemoglobin level of less than 50% during the first week after administration. The polypeptide according to claim 86, which causes a reduction.

89. In humans, administration of the aforementioned polypeptide resulted in a hemoglobin level of less than 50% during the first week after administration. The polypeptide according to claim 86, which causes a reduction.

90. Furthermore, it includes at least one Fc variant, and the Fc variant is ( i) Human IgG1 consisting of mutations L234A, L235A, G237A, and N297A Human IgG consisting of mutations in the Fc region, (ii)A330S, P331S and N297A. Fc area 2, or (iii) S228P, E233P, F234V, L235A, d Selected from the Fc region of human IgG4, including mutations elG236 and N297A. The polypeptide described in item 83.

91. The Fc variant is a mutation of L234A, L235A, G237A, and N297A. The polypeptide according to claim 90, which is the Fc region of human IgG1 consisting of the following.

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

93. The Fc variants are S228P, E233P, F234V, L235A, and delG. The Fc region of human IgG4 containing mutations 236 and N297A, as described in claim 90. polypeptide.

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

95. Claim 94, wherein the disease or disorder is cancer, an autoimmune disease, or an inflammatory disease. Method of description.

96. The disease or disorder is cancer, and the cancer is a solid tumor cancer, a hematological cancer, or acute bone marrow cancer. Myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's disease Lymphoma, Hodgkin lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometriosis Cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colorectal cancer, stomach cancer (sto (Mach cancer, gastric cancer), gallbladder cancer, gastrointestinal stromal tumor Thyroid cancer, head and neck cancer, oral and pharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer Merkel cell carcinoma, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer Hmm, renal cell carcinoma, renal pelvis cancer, leukemia, lymphoma, non-epithelial malignant tumor, glioma, brain cancer, The method according to claim 94, selected from and epithelial malignant tumors.

97. The disease or disorder is an autoimmune disease or an inflammatory disease, and the autoimmune disease or The aforementioned inflammatory diseases include multiple sclerosis, rheumatoid arthritis, spondyloarthritis, and systemic lupus erythematosus. S, antibody-mediated inflammatory or autoimmune diseases, graft-versus-host diseases, sepsis, diabetes, psoriasis, Atherosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary artery disease Syndrome, ischemic reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic lung disease selected from fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis. The method according to claim 94, which is selected.

98. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 94, having the following characteristics.

99. Furthermore, the method according to claim 98, comprising administering at least one further agent. Law.

100. The at least one further agent is an antibody, tumor-associated antigen, or non-antibody therapeutic agent. The method according to claim 99.

101. The method according to claim 100, wherein at least two further drugs are administered.

102. The method according to claim 101, wherein the at least two further agents comprise two antibodies. 。

103. Claim 101, wherein the at least two further agents include an antibody and a tumor-associated antigen. Method of description.

104. The method according to claim 100, wherein the at least one further agent is an antibody.

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

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

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

108. The aforementioned antibodies include anti-HER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, and anti-C2 antibody. 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 A selected antibody from FF antibody, anti-VEGF antibody, or anti-VEGFR2 antibody, according to claim 104. Method of description.

109. The aforementioned antibodies include anti-HER2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, and anti-C2 antibody. Selected from D38 antibody, anti-PD-1 antibody, anti-RANKL antibody, or anti-PD-L1 antibody. The method according to claim 108.

110. The said at least one further agent is at least one antibody, and the antibody is Tuximab, Necitumumab, Pembrolizumab, Nivolumab, Pidilizumab, MEDI 0680, MED16469, Atezolizumab, Avelumab, Durvalumab, MEDI 6383, RG7888, Ipilimumab, Tremelimumab, Urelumab, PF-0508 2566, Enobirituzumab, Vanchikutuzumab tictumab, varlilumab, mogamalizumab (mog amalizumab), SAR650984, daratumumab, trastuzumab, trast Tuzumab emtansine, pertuzumab, elotuzumab, rituk Simab, ofatumumab, obinutuzumab, RG7155, FPA008, panitumumab Brentuximab vedotin, MSB0010718C, belimumab, bevacizumab, Nosumab, panitumumab, ramucirumab, nesitumumab, nivolumab, pembrolizumab avelumab, atezolizumab, durvalumab, MEDI0680, pidilizumab, The method according to claim 104, or selected from BMS-93659.

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

112. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 111, comprising:

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

114. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 113, comprising:

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

116. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 115, comprising:

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

118. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 117, comprising:

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

120. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 119, comprising:

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

122. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 121, comprising:

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

124. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 123, having the following characteristics.

125. The method according to claim 110, wherein the antibody is pantimumab. 。

126. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 125, having the following characteristics.

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

128. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 127, having the following characteristics.

129. The method according to claim 110, wherein the antibody is nesitumumab.

130. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 129, comprising:

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

132. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 131, comprising:

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

134. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 133, having the following characteristics.

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

136. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 135, wherein the method is provided for.

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

138. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 137, having the following characteristics.

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

140. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 139, wherein the method is provided.

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

142. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 141, comprising:

143. The method according to claim 110, wherein the antibody is pidilyzumab.

144. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 143, wherein the method is characterized by having the following:

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

146. The aforementioned SIRP-α D1 variant is sequence numbers 78-85, 98-104, 107- One of the following sequences: 113, 116-122, 135-137, or 152-159 The method according to claim 145, wherein the method is characterized by having the following:

147. The at least one further agent is a tumor-associated antigen, and the tumor-associated antigen is immune The method according to claim 110, which induces a response.

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

149. The aforementioned antibodies include NY-ESO-1 / LAGE1, SSX-2, and the 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) Contains β-catenin (mutant), TGF-βRII (mutant), HPV E6, or E7. The method according to claim 148, which targets an HLA / peptide or MHC / peptide complex. method.

150. Claim 149, wherein the antibody is ESK1, RL1B, Pr20, or 3.2G1. Methods used.

151. The polypeptide according to claim 1 for the treatment of cancer.

152. The polypeptide according to claim 1 for the treatment of autoimmune diseases.

153. The polypeptide according to claim 1 for the treatment of inflammatory diseases.

154. Use of the polypeptide according to claim 1 for the manufacture of a drug for the treatment of cancer.

155. Use of the polypeptide according to claim 1 for the manufacture of a therapeutic agent for autoimmune diseases.

156. Use of the polypeptide according to claim 1 for the manufacture of a therapeutic agent for inflammatory diseases.

Images