GENETICALLY ENGINEERED SIRPα VARIANTS AND METHODS OF USE THEREOF

Abstract

Signal regulatory protein alpha (SIRPα) is a regulatory membrane glycoprotein from the SIRP family. It is expressed primarily by myeloid cells, but also by stem cells and neurons. SIRPα acts as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47. The present disclosure relates to engineered SIRPα variants and methods of use thereof. TIFF2025501430000013.tif168133

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 292,267, filed December 21, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named 52246-0004WO1_SL_ST26.xml. The XML file, created on December 5, 2022, is 65,094 bytes in size. The material in the XML file is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION The present disclosure relates to engineered SIRPα variants and methods of use thereof. [Background technology]

[0004] background Signal regulatory protein alpha (SIRPα) is a regulatory membrane glycoprotein from the SIRP family. It is mainly expressed by myeloid cells, but also by stem cells or neurons. SIRPα acts as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47. This interaction negatively regulates the effector functions of innate immune cells, such as host cell phagocytosis. SIRPα diffuses laterally on the macrophage membrane and accumulates at the phagocytic synapse, where it binds to CD47, which inhibits the cytoskeleton-centred process of phagocytosis by macrophages.

[0005] CD47 provides a "do not eat" signal by binding to the N-terminus of signal regulatory protein alpha (SIRPα), which has been found to be overexpressed in many different tumor cells. Targeting CD47 and / or SIRPα may be useful for cancer immunotherapy. However, the interaction between CD47 and SIRPα is necessary to protect red blood cells, platelets, and lymphocytes from rapid elimination by splenic macrophages. There is a need to develop cancer therapies that target the CD47 / SIRPα pathway with limited toxicity. Summary of the Invention

[0006] overview The present disclosure relates to engineered SIRPα variants and methods of use thereof.

[0007] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1, in some embodiments the engineered SIRPα polypeptide comprises one or more amino acid mutations in the BC loop, the C'D loop, and / or the DE loop. In some embodiments, the engineered SIRPα polypeptide described herein comprises one or more of the following: (a) the amino acid corresponding to E54 of SEQ ID NO:1 is A, H, N, I, R, G, S, D, or L, (b) the amino acid corresponding to G55 of SEQ ID NO:1 is W, F, Q, L, D, K, R, A, or P, and (c) the amino acid corresponding to H56 of SEQ ID NO:1 is P, I, T, N, V, R, L, S, G, or Q.

[0008] In some embodiments, the amino acid corresponding to H56 in SEQ ID NO:1 is I, T, N, V, L, S, G, or Q. In some embodiments, the amino acid corresponding to I31 in SEQ ID NO:1 is W, K, Y, L, A, N, or T. In some embodiments, the amino acid corresponding to I31 in SEQ ID NO:1 is W, K, Y, L, A, or N. In some embodiments, the engineered SIRPα polypeptides described herein comprise one or more of the following: (a) the amino acid corresponding to S66 in SEQ ID NO:1 is Q or N, and (b) the amino acid corresponding to T67 in SEQ ID NO:1 is G. In some embodiments, the engineered SIRPα polypeptides described herein comprise one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is N or T, (b) the amino acid corresponding to T26 in SEQ ID NO:1 is I.

[0009] In some embodiments, the engineered SIRPα polypeptides described herein comprise one or more of the following: (a) the amino acid corresponding to E70 in SEQ ID NO:1 is G, F, R, A, L, or T; (b) the amino acid corresponding to M72 in SEQ ID NO:1 is R or Y; and (c) the amino acid corresponding to D73 in SEQ ID NO:1 is I.

[0010] In some embodiments, the amino acid corresponding to K53 in SEQ ID NO:1 is R. In some embodiments, the amino acid corresponding to K53 in SEQ ID NO:1 is not R.

[0011] In some embodiments, the engineered SIRPα polypeptides described herein comprise one or more of the following: (a) the amino acid corresponding to position 27 of SEQ ID NO:1 is V or L, (b) the amino acid corresponding to position 63 of SEQ ID NO:1 is V, and (c) the amino acid corresponding to position 68 of SEQ ID NO:1 is K.

[0012] In some embodiments, the engineered SIRPα polypeptides described herein comprise an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

[0013] In one aspect, the disclosure relates to engineered SIRPα polypeptides comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:2, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is W, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is A, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is W, and (d) the amino acid corresponding to H56 in SEQ ID NO:1 is P. In some embodiments, the engineered SIRPα polypeptides described herein comprise an amino acid sequence at least 90% identical to SEQ ID NO:2.

[0014] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:3, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is W, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is A, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is F, (e) the amino acid corresponding to H56 in SEQ ID NO:1 is I, and (f) the amino acid corresponding to E70 in SEQ ID NO:1 is G. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:3.

[0015] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:4, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is N, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is H, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is Q, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T, (e) the amino acid corresponding to S66 in SEQ ID NO:1 is Q, and (f) the amino acid corresponding to M72 in SEQ ID NO:1 is R. In some embodiments the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:4.

[0016] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:5, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to E54 in SEQ ID NO:1 is N, (b) the amino acid corresponding to G55 in SEQ ID NO:1 is L, (c) the amino acid corresponding to H56 in SEQ ID NO:1 is I, and (d) the amino acid corresponding to T67 in SEQ ID NO:1 is G. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:5.

[0017] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:6, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to T26 in SEQ ID NO:1 is I, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is I, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is L, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T, and (e) the amino acid corresponding to M72 in SEQ ID NO:1 is Y. In some embodiments the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:6.

[0018] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:7, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is K, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is R, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is D, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T, and (e) the amino acid corresponding to M72 in SEQ ID NO:1 is R. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:7.

[0019] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:8, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is W, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is G, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is Q, (e) the amino acid corresponding to H56 in SEQ ID NO:1 is N, and (f) the amino acid corresponding to E70 in SEQ ID NO:1 is F. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:8.

[0020] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:9, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is Y, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is R, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is Q, (e) the amino acid corresponding to H56 in SEQ ID NO:1 is T, and (f) the amino acid corresponding to E70 in SEQ ID NO:1 is F. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:9.

[0021] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:10, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is L, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is S, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is K, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is V, and (e) the amino acid corresponding to E70 in SEQ ID NO:1 is R. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:10.

[0022] In one aspect, the disclosure relates to engineered SIRPα polypeptides comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:11, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is Y, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is G, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is R, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is R, and (e) the amino acid corresponding to E70 in SEQ ID NO:1 is A. In some embodiments the engineered SIRPα polypeptides described herein comprise an amino acid sequence at least 90% identical to SEQ ID NO:11.

[0023] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:12, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is L, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is D, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is F, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is L, and (e) the amino acid corresponding to M72 in SEQ ID NO:1 is R. In some embodiments, the engineered SIRPα polypeptides described herein comprise an amino acid sequence at least 90% identical to SEQ ID NO:12.

[0024] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:13, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is A, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is L, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is D, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is S, and (e) the amino acid corresponding to N71 in SEQ ID NO:1 is S. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:13.

[0025] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:14, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is T, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is A, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is K, (e) the amino acid corresponding to H56 in SEQ ID NO:1 is Q, and (f) the amino acid corresponding to D73 in SEQ ID NO:1 is I. In some embodiments the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:14.

[0026] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:33, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is R, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is A, and (d) the amino acid corresponding to H56 in SEQ ID NO:1 is P. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:33.

[0027] In one aspect, the disclosure relates to engineered SIRPα polypeptides comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:34, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to E54 in SEQ ID NO:1 is S, (b) the amino acid corresponding to G55 in SEQ ID NO:1 is P, (c) the amino acid corresponding to H56 in SEQ ID NO:1 is P, and (d) the amino acid corresponding to E70 in SEQ ID NO:1 is L. In some embodiments the engineered SIRPα polypeptides described herein comprise an amino acid sequence at least 90% identical to SEQ ID NO:34.

[0028] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:35, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is W, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is S, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is P, and (e) the amino acid corresponding to E70 in SEQ ID NO:1 is R. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:34.

[0029] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:36, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to I31 in SEQ ID NO:1 is W, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is R, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is A, (d) the amino acid corresponding to H56 in SEQ ID NO:1 is G, and (e) the amino acid corresponding to E70 in SEQ ID NO:1 is T. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:36.

[0030] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:37, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S29 of SEQ ID NO:1 is Q, (b) the amino acid corresponding to E54 of SEQ ID NO:1 is H, (c) the amino acid corresponding to G55 of SEQ ID NO:1 is R, (d) the amino acid corresponding to H56 of SEQ ID NO:1 is T, and (e) the amino acid corresponding to S66 of SEQ ID NO:1 is N. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:37.

[0031] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:38, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to E54 in SEQ ID NO:1 is S, (b) the amino acid corresponding to G55 in SEQ ID NO:1 is P, (c) the amino acid corresponding to H56 in SEQ ID NO:1 is R, and (d) the amino acid corresponding to E70 in SEQ ID NO:1 is L. In some embodiments the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:38.

[0032] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:39, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to V27 in SEQ ID NO:1 is L, (b) the amino acid corresponding to G55 in SEQ ID NO:1 is D, (c) the amino acid corresponding to H56 in SEQ ID NO:1 is R, and (d) the amino acid corresponding to M72 in SEQ ID NO:1 is R. In some embodiments, the engineered SIRPα polypeptides described herein comprise an amino acid sequence at least 90% identical to SEQ ID NO:39.

[0033] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:40, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to V27 in SEQ ID NO:1 is L, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is T, (c) the amino acid corresponding to H56 in SEQ ID NO:1 is P, and (d) the amino acid corresponding to E70 in SEQ ID NO:1 is G. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:40.

[0034] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:41, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is Y, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is R, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is Q, and (e) the amino acid corresponding to H56 in SEQ ID NO:1 is T. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:41.

[0035] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:42, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is N, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is R, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is Q, and (e) the amino acid corresponding to H56 in SEQ ID NO:1 is T. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:42.

[0036] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:43, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is Y, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is A, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is K, and (e) the amino acid corresponding to H56 in SEQ ID NO:1 is Q. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:43.

[0037] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:44, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, (b) the amino acid corresponding to I31 in SEQ ID NO:1 is N, (c) the amino acid corresponding to E54 in SEQ ID NO:1 is A, (d) the amino acid corresponding to G55 in SEQ ID NO:1 is K, and (e) the amino acid corresponding to H56 in SEQ ID NO:1 is Q. In some embodiments, the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:44.

[0038] In one aspect, the disclosure relates to an engineered SIRPα polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:45, in some embodiments the polypeptide comprises one or more of the following: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, (b) the amino acid corresponding to E54 in SEQ ID NO:1 is A, (c) the amino acid corresponding to G55 in SEQ ID NO:1 is K, and (d) the amino acid corresponding to H56 in SEQ ID NO:1 is Q. In some embodiments the engineered SIRPα polypeptide described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO:45.

[0039] In some embodiments, the engineered SIRPα polypeptide further comprises a CH2 domain and a CH3 domain. In some embodiments, the engineered SIRPα polypeptide further comprises a hinge region. In some embodiments, the CH2 domain is an IgG CH2 domain and the CH3 domain is an IgG CH3 domain. In some embodiments, the engineered SIRPα polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 15-28 and 46-58.

[0040] In one aspect, the disclosure relates to a protein construct comprising an engineered SIRPα polypeptide as described herein. In some embodiments, a protein construct as described herein comprises two or more engineered SIRPα polypeptides. In some embodiments, at least two engineered SIRPα polypeptides are identical. In some embodiments, at least two engineered SIRPα polypeptides are different. In some embodiments, a protein construct as described herein further comprises an Fc region. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region (e.g., having a LALA mutation or a LALA-PG mutation).

[0041] In one aspect, the disclosure relates to a protein construct comprising an engineered SIRPα polypeptide as described herein, a first fusion polypeptide comprising a first CH2 domain and a first CH3 domain, and a second fusion polypeptide comprising a second CH2 domain and a second CH3 domain. In some embodiments, the first fusion polypeptide and the second fusion polypeptide associate with each other to form a dimer. In some embodiments, the second fusion polypeptide further comprises a second engineered SIRPα polypeptide.

[0042] In one aspect, the disclosure relates to a pharmaceutical composition comprising an engineered SIRPα polypeptide as described herein or a protein construct as described herein and a pharma- ceutically acceptable carrier.

[0043] In one aspect, the disclosure relates to a nucleic acid encoding an engineered SIRPα polypeptide described herein or a protein construct described herein. In one aspect, the disclosure relates to a vector comprising a nucleic acid described herein. In one aspect, the disclosure relates to a cell comprising a nucleic acid described herein. In some embodiments, the cell is a CHO cell.

[0044] In one aspect, the disclosure relates to a method for producing an engineered SIRPα polypeptide or a protein construct comprising an engineered SIRPα polypeptide, the method comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the engineered SIRPα polypeptide or protein construct; and (b) recovering the engineered SIRPα polypeptide or protein construct produced by the cell.

[0045] In one aspect, the disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an engineered SIRPα polypeptide as described herein or a protein construct as described herein. In some embodiments, the subject has a solid tumor or a hematological cancer. In some embodiments, the cancer is an acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, or small cell lung cancer tumor.

[0046] In one aspect, the disclosure relates to a method of reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of a composition comprising an engineered SIRPα polypeptide described herein or a protein construct described herein.

[0047] In one aspect, the disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an engineered SIRPα polypeptide described herein or a protein construct described herein.

[0048] As used herein, the term "engineered SIRPα polypeptide" refers to a polypeptide derived from a wild-type SIRPα polypeptide or a portion thereof (e.g., the extracellular region of SIRPα, or the IgV domain of SIRPα) having one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, the engineered SIRPα polypeptide comprises or consists of the extracellular region of SIRPα. In some embodiments, the engineered SIRPα polypeptide comprises or consists of the IgV domain of SIRPα. In some embodiments, the engineered SIRPα polypeptide is a modified IgV domain.

[0049] As used herein, the term "protein construct" refers to a complex having one or more polypeptides. In some embodiments, a protein construct has two or more polypeptides, which can associate with each other to form dimers or multimers.

[0050] As used herein, the term "cancer" refers to cells that have the capacity for uncontrolled and autonomous proliferation. Examples of such cells include cells that have an abnormal stage or state characterized by rapidly proliferating cell growth. The term is meant to include cancerous proliferations, e.g., tumors, tumorigenic processes, regardless of histopathological type or invasive stage, metastatic tissues, and malignantly transformed cells, tissues, or organs. Also included are malignant tumors of various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, digestive, and endocrine systems, as well as adenocarcinomas, including many colon, renal cell, prostate, and / or testicular tumors, non-small cell lung, and small intestine cancers. "Spontaneously occurring" cancers include any cancer that is not experimentally induced by the implantation of cancer cells into a subject, including, for example, spontaneous cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from the insertion of a transgenic cancer gene or the knockout of a tumor suppressor gene, and cancers caused by infectious diseases, e.g., viral infections. The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic diseases can arise from the myeloid, lymphoid or erythroid lineages, or from their precursor cells. Hematological cancers are cancers that begin in hematopoietic tissues, such as the bone marrow, or in cells of the immune system. Examples of hematological cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0051] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification to describe an animal (human or non-human) to which treatment according to the method of the present invention is provided. Veterinary and non-veterinary applications are contemplated in this disclosure. Human patients can be adults or juveniles (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swines (e.g., pigs, minipigs), horses, dogs, cats, cows, and other domestic, livestock, and zoo animals.

[0052] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to polymers of amino acids of any length, being at least two amino acids.

[0053] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0054] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will govern.

[0055] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0056] [Figure 1] 3D protein structure of the IgV domain of human SIRPα. The nine β-strands (ABC-C'-DEFG-G2) and five loop regions (BC loop, CC' loop, C'D loop, DE loop, and FG loop) are indicated. [Diagram 2] The amino acid residues of the IgV domain of human SIRPα are shown. This sequence (SEQ ID NO:1) is identical to amino acids 31-148 of GenBank Accession No. CAA71403.1 (SEQ ID NO:30). Residues in the BC, C'D, and DE loops are underlined. [Diagram 3] A to D show the results of residue scans of K53, G55, E54, and H56. [Figure 4A] FIG. 13 is a table showing data for expression levels (absorbance at 450 nm or OD450), hCD47 / hSIRPα blocking (absorbance at 450 nm or OD450), hCD47 / hSIRPα blocking capacity, human CD47 binding (absorbance at 450 nm or OD450), human CD47 binding capacity (B) / expression (E) ratio, and mouse CD47 binding (absorbance at 450 nm or OD450) for 56 selected clones. [Figure 4B] FIG. 13 is a table showing data for expression levels (absorbance at 450 nm or OD450), hCD47 / hSIRPα blocking (absorbance at 450 nm or OD450), hCD47 / hSIRPα blocking capacity, human CD47 binding (absorbance at 450 nm or OD450), human CD47 binding capacity (B) / expression (E) ratio, and mouse CD47 binding (absorbance at 450 nm or OD450) for 56 selected clones. [Diagram 5] Schematic structure of the hSIRPα-Fc protein: Two SIRPα IgV domains are attached to the N-terminus of a human IgG4 hinge and Fc region. [Figure 6] Specific amino acid mutations within the BC, C'D, and DE loop regions of the wild-type SIRPα IgV domain and 13 selected clones (mt3 to mt15) are shown. [Figure 7] The results of HPLC-SEC purification of hSIRPα-Fc mutant protein are shown. [Figure 8] 1 shows the binding affinity results of hSIRPα-Fc muteins to CD47-ECD-His using the Octet® system. [Figure 9A] Shown are the results of whole cell binding of hSIRPα-Fc mutant proteins to CD47-transfected CHO-S cells, Jurkat cells, and Raji cells, respectively. [Figure 9B] Shown are the results of whole cell binding of hSIRPα-Fc mutant proteins to CD47-transfected CHO-S cells, Jurkat cells, and Raji cells, respectively. [Figure 9C] Shown are the results of whole cell binding of hSIRPα-Fc mutant proteins to CD47-transfected CHO-S cells, Jurkat cells, and Raji cells, respectively. [Figure 9D] Shown are the results of whole cell binding of hSIRPα-Fc muteins to CD47 tf CHO-S and Raji cells, respectively. [Figure 9E] Shown are the results of whole cell binding of hSIRPα-Fc muteins to CD47 tf CHO-S and Raji cells, respectively. [Figure 10A] Figure 1 shows RBC binding results of hSIRPα-Fc muteins. Human red blood cells were collected from two donors. [Figure 10B] Figure 1 shows RBC binding results of hSIRPα-Fc muteins. Human red blood cells were collected from two donors. [Figure 10C] Figure 1 shows RBC binding results of hSIRPα-Fc muteins. Human red blood cells were collected from two donors. [Figure 10D]Figure 1 shows RBC binding results of hSIRPα-Fc muteins. Human red blood cells were collected from two donors. [Figure 11A] Platelet binding results of hSIRPα-Fc muteins are shown. Human platelets were collected from two donors. [Figure 11B] Platelet binding results of hSIRPα-Fc muteins are shown. Human platelets were collected from two donors. [Figure 11C] Platelet binding results of hSIRPα-Fc muteins are shown. Human platelets were collected from two donors. [Figure 11D] Platelet binding results of hSIRPα-Fc muteins are shown. Human platelets were collected from two donors. [Figure 12A] 1 shows the results of whole cell binding of hSIRPα-Fc mutant proteins to cynoCD47 tf CHO-S cells or LLC-MK2 cells. [Figure 12B] 1 shows the results of whole cell binding of hSIRPα-Fc mutant proteins to cynoCD47 tf CHO-S cells or LLC-MK2 cells. [Figure 12C] 1 shows the results of whole cell binding of hSIRPα-Fc mutant proteins to cynoCD47 tf CHO-S cells or LLC-MK2 cells. [Figure 13A] 1 shows the results of whole cell binding of hSIRPα-Fc mutant proteins to EMT-6 cells. [Figure 13B] FIG. 13B is a magnified view of the low MFI region of FIG. 13A. [Figure 14A] 1 shows the results of an RBC hemagglutination assay using hSIRPα-Fc mutant proteins. Human RBC cells were collected from three donors. Hu5F9-G4 was used as a positive control to induce hemagglutination. [Figure 14B] 1 shows the results of an RBC hemagglutination assay using hSIRPα-Fc mutant proteins. Human RBC cells were collected from three donors. Hu5F9-G4 was used as a positive control to induce hemagglutination. [Figure 14C]1 shows the results of an RBC hemagglutination assay using hSIRPα-Fc mutant proteins. Human RBC cells were collected from three donors. Hu5F9-G4 was used as a positive control to induce hemagglutination. [Figure 15A] The hCD47 / hSIRPα blocking ability of hSIRPα-Fc mutant proteins is shown using CD47 tf CHO-S cells, FaDu cells, and Raji cells, respectively. [Figure 15B] The hCD47 / hSIRPα blocking ability of hSIRPα-Fc mutant proteins is shown using CD47 tf CHO-S cells, FaDu cells, and Raji cells, respectively. [Figure 15C] The hCD47 / hSIRPα blocking ability of hSIRPα-Fc mutant proteins is shown using CD47 tf CHO-S cells, FaDu cells, and Raji cells, respectively. [Figure 15D] 1 shows the ability of hSIRPα-Fc mutant proteins to block hCD47 / hSIRPα using CD47 tf CHO-S cells. [Figure 15E] 1 shows the ability of hSIRPα-Fc mutant proteins to block hCD47 / hSIRPα using CD47 tf CHO-S cells. [Figure 16A] 1 shows the phagocytosis induced by hSIRPα-Fc mutant proteins by Raw264.7 mouse macrophages against Jurkat, FaDu, and Raji cells, respectively. [Figure 16B] 1 shows the phagocytosis induced by hSIRPα-Fc mutant proteins by Raw264.7 mouse macrophages against Jurkat, FaDu, and Raji cells, respectively. [Figure 16C] 1 shows the phagocytosis induced by hSIRPα-Fc mutant proteins by Raw264.7 mouse macrophages against Jurkat, FaDu, and Raji cells, respectively. [Figure 16D] 1 shows the phagocytosis induced by hSIRPα-Fc mutant proteins by Raw264.7 mouse macrophages against DLD1 and Raji cells, respectively. [Figure 16E] 1 shows the phagocytosis induced by hSIRPα-Fc mutant proteins by Raw264.7 mouse macrophages against DLD1 and Raji cells, respectively. [Figure 17A] 1 shows the phagocytosis induced by hSIRPα-Fc mutant proteins by Raw264.7 mouse macrophages against human RBC cells. [Figure 17B] 1 shows the phagocytosis of human platelets by Raw264.7 mouse macrophages induced by hSIRPα-Fc mutant proteins. [Figure 18A] 1 shows phagocytosis induced by hSIRPα-Fc mutant proteins by human monocyte-derived macrophages (MDMs) against Raji, DLD1, and Jurkat cells, respectively. [Figure 18B] 1 shows phagocytosis induced by hSIRPα-Fc mutant proteins by human monocyte-derived macrophages (MDMs) against Raji, DLD1, and Jurkat cells, respectively. [Figure 18C] 1 shows phagocytosis induced by hSIRPα-Fc mutant proteins by human monocyte-derived macrophages (MDMs) against Raji, DLD1, and Jurkat cells, respectively. [Figure 18D] 1 shows the phagocytosis of DLD1 cells by human monocyte-derived macrophages (MDMs) induced by hSIRPα-Fc mutant proteins. [Figure 19A] 1 shows the phagocytosis of RBC cells by human monocyte-derived macrophages (MDMs) induced by hSIRPα-Fc mutant proteins. Human RBC cells were collected from two donors. [Figure 19B] 1 shows the phagocytosis of RBC cells by human monocyte-derived macrophages (MDMs) induced by hSIRPα-Fc mutant proteins. Human RBC cells were collected from two donors. [Figure 19C] 1 shows the phagocytosis of RBC cells by human monocyte-derived macrophages (MDMs) induced by hSIRPα-Fc mutant proteins. Human RBC cells were collected from two donors. [Figure 20A] Tumor growth curves in NOD / SCID mice bearing Raji xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium TTI-622), or Hu5F9-IgG4. Control mice received a placebo. [Figure 20B] Individual tumor volumes at 18 days post-inoculation in Raji xenograft-bearing NOD / SCID mice treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium TTI-622), Hu5F9-IgG4, or placebo are shown. [Figure 20C] 1 shows survival curves of Raji xenograft-bearing NOD / SCID mice treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium TTI-622), Hu5F9-IgG4, or placebo. [Figure 21A] Tumor growth curves in NOD / SCID mice bearing NCI_H82 xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium TTI-622), or Hu5F9-IgG4. Control mice received a placebo. [Figure 21B] Individual tumor volumes at day 25 post-inoculation in NCI_H82 xenograft-bearing NOD / SCID mice treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium TTI-622), Hu5F9-IgG4, or placebo are shown. [Figure 21C] 1 shows survival curves of NCI_H82 xenograft-bearing NOD / SCID mice treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium TTI-622), Hu5F9-IgG4, or placebo. [Figure 22-1] The amino acid sequences of the wild-type IgV domain or mutant IgV domain of human SIRPα are listed. [Figure 22-2] The amino acid sequences of the wild-type IgV domain or mutant IgV domain of human SIRPα are listed. [Figure 23-1] Listed below are the protein sequences described in this disclosure. [Figure 23-2] Listed below are the protein sequences described in this disclosure. [Figure 23-3] Listed below are the protein sequences described in this disclosure. [Figure 23-4] Listed below are the protein sequences described in this disclosure. [Figure 23-5] Listed below are the protein sequences described in this disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description Signal regulatory protein alpha (SIRPα, SIRPa, or CD172A) is a transmembrane protein. It has an extracellular region containing three Ig-like domains and a cytoplasmic region containing an immunoreceptor tyrosine-based inhibitory motif that mediates binding of the protein tyrosine phosphatases SHP1 and SHP2. Tyrosine phosphorylation of SIRPα is regulated by various growth factors and cytokines, as well as by integrin-mediated cell adhesion to extracellular matrix proteins. SIRPα is particularly abundant in myeloid cells such as macrophages and dendritic cells, but is expressed at low levels in T, B, NK, and NKT cells.

[0058] The extracellular region of SIRPα can interact with its ligand CD47. Interaction of SIRPα on macrophages with CD47 on erythrocytes prevents phagocytosis of Ig-opsonized erythrocytes by macrophages in vitro and in vivo. Ligation of SIRPα on phagocytes by CD47 expressed on neighboring cells leads to phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory (ITIM) motif of SIRPα, resulting in recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention of myosin-IIA accumulation at the phagocytic synapse and the consequent inhibition of phagocytosis. Thus, CD47-SIRPα interaction functions as a negative immune checkpoint that sends a "don't eat me" signal to ensure that healthy self-cells are not inappropriately phagocytosed. However, overexpression of CD47 has also been found in almost all types of tumors, including acute myeloid leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer. Such negative regulation of macrophages can be minimized by blocking the binding of CD47 to SIRPα.

[0059] Blocking CD47 / SIRPα interaction can promote cell phagocytosis and therefore can be used to treat various cancers. It induces the recognition and elimination of cancer cells by innate immunity. Drugs that target CD47 or SIRPα can be used to treat various tumors and cancers, such as solid tumors, hematological malignancies (e.g., relapsed or refractory hematological malignancies), acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer tumors.

[0060] A detailed description of SIRPα and its functions can be found, for example, in Yanagita et al., "Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy." JCI insight 2.1 (2017); Seiffert et al., "Signal-regulatory protein α (SIRPα) but not SIRPβ is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34+ CD38- hematopoietic cells." Blood 97.9 (2001): 2741-2749; which are incorporated by reference in their entireties.

[0061] Furthermore, SIRPα acts to inhibit in vivo clearance of CD47-expressing host cells, including red blood cells and platelets, by macrophages. CD47-SIRPα interaction also appears to be essential for hematopoietic stem cell engraftment. Blockade of CD47 / SIRPα interaction can cause accidental killing of normal red blood cells, potentially resulting in anemia and inducing inflammation. Therefore, it is important to modulate the interaction of SIRPα targeting agents with CD47, for example, with limited or controlled effects on red blood cells.

[0062] The present disclosure provides engineered SIRPα variants that can be used to target the CD47 / SIRPα pathway, but in which the interaction of the engineered SIRPα variants with CD47 is carefully regulated.

[0063] Engineered SIRPα variants Human SIRPα is a member of the signal regulatory proteins (SIRPs). Signal regulatory proteins are cell surface Ig superfamily proteins that mediate essential cell surface protein interactions and signal transduction. All SIRPs contain an N-terminal extracellular region, a single transmembrane domain, and a C-terminal intracellular region.

[0064] The extracellular region of human SIRPα (UniProt identifier: P78324) contains an IgV domain, an Ig-like C1 type 1 domain, and an Ig-like C1 type 2 domain. These correspond to amino acids 32-137, 148-247, and 254-348 of the human SIRPα protein (SEQ ID NO: 31, NP_542970:1). Amino acids 1-30 are the signal peptide. Human SIRPα also contains a long intracellular domain that contains two putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Activation of SIRPα ITIMs delivers inhibitory signals that negatively regulate cellular responses.

[0065] Binding of SIRPα to CD47 is mediated through the extracellular IgV domain of SIRPα. The IgV domain of hSIRPα belongs to the immunoglobulin superfamily and contains nine β-strands including ABC-C'-DEFG-G2. The helix is ​​located between the E and F strands (Figure 1).

[0066] Based on the structure of human CD47 (hCD47) complexed with hSIRPα, the residues that interact with CD47 are determined. The analysis shows that multiple interacting residues located within the BC loop (corresponding to amino acids 24-36 of SEQ ID NO:1), C'D loop (corresponding to amino acids 53-56 of SEQ ID NO:1), and DE loop (corresponding to amino acids 61-78 of SEQ ID NO:1) of the hSIRPα IgV domain are highly conserved. In FIG. 1B, amino acid residues within these loop regions are underlined. These regions are targets for mutation. Thus, in some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more amino acid substitutions in the BC loop, the C'D loop, and / or the DE loop.

[0067] Further analysis indicates that Leu30, Gly34, Gln52, Lys53, Glu54, His56, Ser66, Thr67, Arg69, Lys93, Lys96, Gly97, and Asp100 of hSIRPα are involved in the interaction with CD47. Thus, in some embodiments, an engineered SIRPα polypeptide may contain one or more amino acid mutations at Leu30, Gly34, Gln52, Lys53, Glu54, His56, Ser66, Thr67, Arg69, Lys93, Lys96, Gly97, and / or Asp100.

[0068] In addition, it has been determined that Lys53 and Ser66 may be important for increasing the binding affinity of hSIRPα to CD47. Lys53 is located on the C'D loop, and S66 is located on the DE loop. Mutation of Lys at position 53 may be important for increasing the binding affinity of hSIRPα to CD47. Thus, in some embodiments, the amino acid corresponding to K53 in SEQ ID NO:1 is R. In addition, the elimination of steric hindrance at position 54 by structural change also suggests a structural change in the C'D loop, which may also increase the chance of increasing the binding affinity of hSIRPα to CD47. Mutation of the C'D loop provides a more flexible approach to screen for unique mutations that can provide increased binding affinity to CD47.

[0069] Residue scan results show that Lys53 can increase stability when substituted with amino acids having long side chains or side chains with aromatic rings, such as arginine, leucine, phenylalanine, tryptophan, and tyrosine. Thus, in some embodiments, the amino acid corresponding to K53 in SEQ ID NO:1 is arginine, leucine, phenylalanine, tryptophan, or tyrosine. In some embodiments, the amino acid corresponding to K53 in SEQ ID NO:1 is not R.

[0070] On the other hand, it may be more flexible and advantageous to direct mutations at Glu54, Gly55, and His56. In some embodiments, the engineered SIRPα polypeptide may have one or more of the following mutations: (a) the amino acid corresponding to E54 of SEQ ID NO:1 is A, H, N, I, R, G, S, D, or L, (b) the amino acid corresponding to G55 of SEQ ID NO:1 is W, F, Q, L, D, K, R, A, or P, (c) the amino acid corresponding to H56 of SEQ ID NO:1 is P, I, T, N, V, R, L, S, G, or Q.

[0071] Thr67 is a residue directly involved in the interaction, and decreasing the distance of Thr67 to CD47 may increase the binding affinity of hSIRPα to CD47. Substitution of residues Ser66 and Thr67 may bring the DE loop closer to CD47. Thus, in some embodiments, an engineered SIRPα polypeptide may have one or more of the following: (a) the amino acid corresponding to S66 in SEQ ID NO:1 is Q or N, (b) the amino acid corresponding to T67 in SEQ ID NO:1 is G.

[0072] Several other interfaces of the CD47 / hSIRPa complex are also observed in the present disclosure. Residues Ile31, Val33, and Arg69 can form a positively charged pocket. The FG loop of CD47 is completely buried in the positively charged pocket due to the charge and shape complementarity of the two regions. Thus, in some embodiments, the amino acid corresponding to I31 in SEQ ID NO:1 is W, K, Y, L, A, N, or T. In some embodiments, the amino acid corresponding to I31 in SEQ ID NO:1 is W, K, Y, L, A, or N.

[0073] In addition, the analysis in the present disclosure indicates that Val27, Val63, and Lys68 may be important for maintaining the structure of hSIRPα. Thus, in some embodiments, these amino acid residues are retained. In summary, Ile31, Glu54, Gly55, His56, Ser66, and Thr67 are identified as candidate amino acids for hSIRPα mutation screening.

[0074] Thus, in one aspect, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

[0075] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43. In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44.In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:45.

[0076] In some embodiments, the genetically engineered SIRPα variant may have at least or about one (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) amino acid insertion, deletion, or substitution compared to any one of SEQ ID NOs: 1-14 and 33-45.

[0077] In some embodiments, the engineered SIRPα polypeptide has one or more of the following mutations: (a) the amino acid corresponding to E54 in SEQ ID NO:1 is A, H, N, I, R, G, S, D, or L; (b) the amino acid corresponding to G55 in SEQ ID NO:1 is W, F, Q, L, D, K, R, A, or P; (c) The amino acid corresponding to H56 in SEQ ID NO: 1 is P, I, T, N, V, R, L, S, G, or Q.

[0078] In some embodiments, the amino acid corresponding to H56 in SEQ ID NO:1 is I, T, N, V, L, S, G, or Q. In some embodiments, the amino acid corresponding to I31 in SEQ ID NO:1 is W, K, Y, L, A, N, I, or T. In some embodiments, the amino acid corresponding to I31 in SEQ ID NO:1 is W, K, Y, L, A, N, or I.

[0079] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to S66 in SEQ ID NO: 1 is Q or N; (b) The amino acid corresponding to T67 in SEQ ID NO:1 is G.

[0080] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, N, or T; (b) The amino acid corresponding to T26 in SEQ ID NO:1 is I.

[0081] In some embodiments, the amino acid corresponding to H24 in SEQ ID NO:1 is N or T.

[0082] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to E70 in SEQ ID NO:1 is G, F, R, A, L, or T; (b) the amino acid corresponding to M72 in SEQ ID NO:1 is R or Y; and (c) The amino acid corresponding to D73 in SEQ ID NO: 1 is I.

[0083] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to position 27 of SEQ ID NO: 1 is V or L; (b) the amino acid corresponding to position 63 of SEQ ID NO:1 is V; and (c) the amino acid corresponding to position 68 of SEQ ID NO:1 is K.

[0084] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is R, N, or T; (b) the amino acid corresponding to T26 in SEQ ID NO: 1 is I; (c) the amino acid corresponding to V27 in SEQ ID NO:1 is L; (d) the amino acid corresponding to S29 in SEQ ID NO: 1 is Q; (e) the amino acid corresponding to I31 in SEQ ID NO: 1 is W, K, Y, L, A, or N; (f) the amino acid corresponding to E54 in SEQ ID NO:1 is A, H, N, I, R, G, S, D, or L; (g) the amino acid corresponding to G55 in SEQ ID NO:1 is W, F, Q, L, D, K, R, A, or P; (h) the amino acid corresponding to H56 in SEQ ID NO: 1 is P, I, T, N, V, R, L, S, G, or Q; (i) the amino acid corresponding to S66 in SEQ ID NO: 1 is Q or N; (j) the amino acid corresponding to T67 in SEQ ID NO: 1 is G; (k) the amino acid corresponding to E70 in SEQ ID NO: 1 is G, F, R, A, L, or T; (l) the amino acid corresponding to N71 in SEQ ID NO:1 is S; (m) the amino acid corresponding to M72 in SEQ ID NO:1 is R or Y; and (n) The amino acid corresponding to D73 in SEQ ID NO: 1 is I.

[0085] In some embodiments, the engineered SIRPα polypeptide does not comprise, or consist of, one or more of the following mutations: (a) the amino acid corresponding to V27 in SEQ ID NO: 1 is I or L; (b) the amino acid corresponding to I31 in SEQ ID NO:1 is F, S, or T; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is Q; (d) the amino acid corresponding to H56 in SEQ ID NO: 1 is P or R; (e) the amino acid corresponding to S66 in SEQ ID NO: 1 is T or G; (f) the amino acid corresponding to K68 in SEQ ID NO:1 is K or R; and (g) The amino acid corresponding to E70 in SEQ ID NO: 1 is N.

[0086] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is W; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is A; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is W; (d) The amino acid corresponding to H56 in SEQ ID NO: 1 is P.

[0087] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:2).

[0088] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is R; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is W; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is A; (d) the amino acid corresponding to G55 in SEQ ID NO:1 is F; (e) the amino acid corresponding to H56 in SEQ ID NO: 1 is I; (f) The amino acid corresponding to E70 in SEQ ID NO: 1 is G.

[0089] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:3).

[0090] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is N; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is H; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is Q; (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T; (e) the amino acid corresponding to S66 in SEQ ID NO:1 is Q; (f) The amino acid corresponding to M72 in SEQ ID NO: 1 is R.

[0091] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:4).

[0092] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to E54 in SEQ ID NO: 1 is N; (b) the amino acid corresponding to G55 in SEQ ID NO: 1 is L; (c) the amino acid corresponding to H56 in SEQ ID NO: 1 is I; (d) The amino acid corresponding to T67 in SEQ ID NO: 1 is G.

[0093] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:5).

[0094] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to T26 in SEQ ID NO: 1 is I; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is I; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is L; (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T; (e) The amino acid corresponding to M72 in SEQ ID NO: 1 is Y.

[0095] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:6).

[0096] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is K; (b) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (c) the amino acid corresponding to G55 in SEQ ID NO:1 is D; (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T; (e) The amino acid corresponding to M72 in SEQ ID NO: 1 is R.

[0097] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:7).

[0098] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is W; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is G; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is Q; (e) the amino acid corresponding to H56 in SEQ ID NO: 1 is N; (f) The amino acid corresponding to E70 in SEQ ID NO: 1 is F.

[0099] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:8).

[0100] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is Y; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is Q; (e) the amino acid corresponding to H56 in SEQ ID NO: 1 is T; (f) The amino acid corresponding to E70 in SEQ ID NO: 1 is F.

[0101] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:9).

[0102] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is L; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is S; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is K; (d) the amino acid corresponding to H56 in SEQ ID NO: 1 is V; (e) The amino acid corresponding to E70 in SEQ ID NO: 1 is R.

[0103] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:10).

[0104] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is Y; (b) the amino acid corresponding to E54 in SEQ ID NO: 1 is G; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is R; (d) the amino acid corresponding to H56 in SEQ ID NO: 1 is R; (e) The amino acid corresponding to E70 in SEQ ID NO: 1 is A.

[0105] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:11).

[0106] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is L; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is D; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is F; (d) the amino acid corresponding to H56 in SEQ ID NO: 1 is L; (e) The amino acid corresponding to M72 in SEQ ID NO: 1 is R.

[0107] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:12).

[0108] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is A; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is L; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is D; (d) the amino acid corresponding to H56 in SEQ ID NO:1 is S; (e) The amino acid corresponding to N71 in SEQ ID NO:1 is S.

[0109] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:13).

[0110] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is R; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is T; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is A; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is K; (e) the amino acid corresponding to H56 in SEQ ID NO: 1 is Q; (f) The amino acid corresponding to D73 in SEQ ID NO: 1 is I.

[0111] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:14).

[0112] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T; (b) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is A; (d) The amino acid corresponding to H56 in SEQ ID NO: 1 is P.

[0113] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:33).

[0114] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to E54 in SEQ ID NO:1 is S; (b) the amino acid corresponding to G55 in SEQ ID NO: 1 is P; (c) the amino acid corresponding to H56 in SEQ ID NO: 1 is P; (d) The amino acid corresponding to E70 in SEQ ID NO: 1 is L.

[0115] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:34).

[0116] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is W; (c) the amino acid corresponding to E54 in SEQ ID NO:1 is S; (d) the amino acid corresponding to H56 in SEQ ID NO: 1 is P; (e) The amino acid corresponding to E70 in SEQ ID NO: 1 is R.

[0117] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:35).

[0118] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to I31 in SEQ ID NO: 1 is W; (b) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is A; (d) the amino acid corresponding to H56 in SEQ ID NO: 1 is G; (e) The amino acid corresponding to E70 in SEQ ID NO: 1 is T.

[0119] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:36).

[0120] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to S29 in SEQ ID NO: 1 is Q; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is H; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is R; (d) the amino acid corresponding to H56 in SEQ ID NO:1 is T; (e) The amino acid corresponding to S66 in SEQ ID NO:1 is N.

[0121] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:37).

[0122] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to E54 in SEQ ID NO:1 is S; (b) the amino acid corresponding to G55 in SEQ ID NO: 1 is P; (c) the amino acid corresponding to H56 in SEQ ID NO: 1 is R; (d) The amino acid corresponding to E70 in SEQ ID NO: 1 is L.

[0123] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:38).

[0124] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to V27 in SEQ ID NO:1 is L; (b) the amino acid corresponding to G55 in SEQ ID NO:1 is D; (c) the amino acid corresponding to H56 in SEQ ID NO: 1 is R; (d) The amino acid corresponding to M72 in SEQ ID NO: 1 is R.

[0125] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:39).

[0126] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to V27 in SEQ ID NO:1 is L; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is T; (c) the amino acid corresponding to H56 in SEQ ID NO: 1 is P; (d) The amino acid corresponding to E70 in SEQ ID NO: 1 is G.

[0127] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:40).

[0128] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is Y; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is Q; (e) The amino acid corresponding to H56 in SEQ ID NO: 1 is T.

[0129] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:41).

[0130] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO:1 is T; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is N; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is Q; (e) The amino acid corresponding to H56 in SEQ ID NO: 1 is T.

[0131] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:42).

[0132] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is R; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is Y; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is A; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is K; (e) The amino acid corresponding to H56 in SEQ ID NO:1 is Q.

[0133] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:43).

[0134] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is R; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is N; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is A; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is K; (e) The amino acid corresponding to H56 in SEQ ID NO:1 is Q.

[0135] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:44).

[0136] In some embodiments, the engineered SIRPα polypeptide comprises or consists of one or more of the following mutations: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is R; (b) the amino acid corresponding to E54 in SEQ ID NO:1 is A; (c) the amino acid corresponding to G55 in SEQ ID NO: 1 is K; (d) The amino acid corresponding to H56 in SEQ ID NO:1 is Q.

[0137] In some embodiments, the genetically engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:45).

[0138] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 1-14 and 33-45 (e.g., SEQ ID NO: 1 or SEQ ID NO: 44) having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations, as shown in FIG.

[0139] The engineered SIRPα polypeptide may have further modifications. In some embodiments, the engineered SIRPα polypeptide may have an Fc CH2 domain and / or CH3 domain. In some embodiments, the engineered SIRPα polypeptide may be linked to the N-terminus of the CH2 domain (e.g., via an optional hinge region or GS linker). In some embodiments, the engineered SIRPα polypeptide may be linked to the C-terminus of the CH3 domain (e.g., via an optional GS linker). In some embodiments, the hinge region is an IgG hinge region (e.g., an IgG4 hinge region). In some embodiments, the CH2 domain is an IgG CH2 domain (e.g., an IgG4 CH2 domain). In some embodiments, the CH3 domain is an IgG CH3 domain (e.g., an IgG4 CH3 domain). In some embodiments, the hinge region, CH2 domain, CH3 domain have a sequence that is at least 80%, 85%, 90%, 95%, 100% identical to SEQ ID NO:29.

[0140] In some embodiments, the engineered SIRPα polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58.

[0141] SIRPα protein constructs The present disclosure provides engineered SIRPα protein constructs that can specifically bind to CD47. In some embodiments, these protein constructs can block the CD47 / SIRPα signaling pathway and thus increase the immune response. In some embodiments, these protein constructs can initiate phagocytosis.

[0142] In some embodiments, the engineered SIRPα protein construct can comprise any of the engineered SIRPα variants described herein. In some embodiments, the engineered SIRPα protein construct can have a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 1-14 and 33-45. In some embodiments, the engineered SIRPα protein construct may comprise or consist of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 15-28 and 46-58.

[0143] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the sequences of SEQ ID NOs: 1 to 14 and 33 to 45, or SEQ ID NOs: 15 to 28 and 46 to 58.

[0144] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences depends on the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For example, sequence comparison and percent identity determination between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0145] The engineered SIRPα protein construct can further comprise an antibody Fc region. These antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is an IgG4 Fc region (e.g., a human IgG4 Fc region).

[0146] In some embodiments, the engineered SIRPα variant is linked to the Fc region via an antibody hinge region (e.g., IgG, IgE hinge region). Further, the Fc region can be modified to provide desired effector functions or serum half-life.

[0147] The engineered SIRPα variants and protein constructs described herein can block binding between endogenous SIRPα expressed on immune cells and CD47. In some embodiments, by binding to CD47, the engineered SIRPα variants and protein constructs can inhibit binding of CD47 (e.g., expressed on tumor cells) to endogenous SIRPα expressed on immune cells (e.g., myeloid cells, macrophages, and dendritic cells), thereby blocking the CD47 / SIRPα pathway, upregulating the immune response and promoting phagocytosis.

[0148] In some embodiments, the engineered SIRPα variants and protein constructs described herein can increase the immune response, activity or number of immune cells (e.g., bone marrow cells, macrophages, dendritic cells, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or 20-fold.

[0149] In some implementations, the engineered SIRPα variants and protein constructs are -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or equal to 0.00001s -1 Dissociation rate (k off ) can bind to SIRPα (e.g., human SIRPα, monkey SIRPα (e.g., Macaca fascicularis, mouse SIRPα). In some embodiments, the dissociation rate (k off ) is 0.01s -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Greater than or equal to 0.000001s -1 It's super.

[0150] In some embodiments, the kinetic association rate (k on ) is 1 × 102 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1×10 6 In some embodiments, the kinetic association rate (k on ) is 1 × 10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0151] The affinity can be estimated from the quotient of the kinetic rate constants (KD=k off / k on In some embodiments, the KD is 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1×10 -10 In some embodiments, the KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1×10 -12 It's super M.

[0152] Common techniques for measuring affinity include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the engineered SIRPα variants and protein constructs are capable of binding to monkey SIRPα and / or mouse SIRPα. In some embodiments, the engineered SIRPα variants and protein constructs are not capable of binding to monkey SIRPα and / or mouse SIRPα.

[0153] In some embodiments, thermal stability is determined. The engineered SIRPα variants and protein constructs described herein may have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C. In some embodiments, the Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0154] In some embodiments, the engineered SIRPα variants and / or protein constructs described herein have a tumor growth inhibition (TGI%) of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the engineered SIRPα variants and / or protein constructs described herein have a tumor growth inhibition that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after initiation of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after initiation of treatment. As used herein, tumor growth inhibition (TGI%) is calculated using the following formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100 Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0155] In some embodiments, the tumor inhibitory effect of the engineered SIRPα variants and / or protein constructs described herein is comparable to an anti-CD47 reference antibody, e.g., Hu5F9-G4, or an anti-SIRPα antibody, e.g., CC-95251. Hu5F9-G4 is described, e.g., in Sikic et al., "First-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers." Journal of Clinical Oncology 37.12(2019):946, which is incorporated by reference herein in its entirety. CC-95251. In some embodiments, the tumor inhibitory effect of the engineered SIRPα variants and / or protein constructs described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than an anti-CD47 reference antibody, e.g., Hu5F9-G4, or an anti-SIRPα antibody, e.g., CC-95251. In some embodiments, the tumor inhibitory effect of the engineered SIRPα variants and / or protein constructs described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than Trillium. Details of hSIRPα-Fc-wt(Trillium), also named TTI-622. The amino acid sequence of hSIRPα-Fc-wt(Trillium) is shown in SEQ ID NO: 15.

[0156] In some embodiments, the protein constructs described herein have a functional Fc region. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the protein constructs described herein have an Fc region that does not have an effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).

[0157] Several other modifications to the Fc region can be made. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may optionally have an increased half-life in vitro and / or in vivo.

[0158] In some embodiments, the IgG4 has a S228P mutation (EU numbering), which prevents IgG4 Fab arm exchange in vivo and in vitro.

[0159] In some embodiments, Fc Regions are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc Region. For example, the amount of fucose in such Fc Region compositions can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (position 314 in the Eu numbering, or Kabat numbering, of Fc Region residues) of the Fc Region, although Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., positions 294-300, due to minor sequence variations in the Fc Region sequence. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region can be further engineered to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0160] In some embodiments, the binding affinity between CD47 (e.g., human CD47, monkey CD47, mouse CD47, or an extracellular domain thereof) and an engineered SIRPα variant and / or protein construct described herein is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater than the binding affinity between CD47 and wild-type SIRPα or a protein construct thereof.

[0161] In some embodiments, the engineered SIRPα variants and / or protein constructs described herein have a B / E ratio (CD47 binding OD) of 1 to 1.25, 1.25 to 1.5, 1.5 to 1.75, 1.75 to 2, or greater than 2. 450 Vs. expression OD 450 In some cases, the B / E ratio is greater than 0.4.

[0162] In some embodiments, the main HPLC-SEC peak represents at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the engineered SIRPα variant and / or protein construct described herein after purification on a Protein A column.

[0163] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can bind to human CD47-expressing tumor cells (e.g., human CD47 tf CHO-S cells, Jurkat cells, or Raji cells) with at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater affinity than wild-type SIRPα or protein constructs thereof. In some embodiments, the EC50 value of the engineered SIRPα variants and / or protein constructs thereof for binding to human CD47-expressing tumor cells (e.g., human CD47 tf CHO-S cells) is less than 5 nM, less than 4 nM, less than 3.5 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, or less than 1 nM. In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can bind to monkey CD47-expressing cells (e.g., cynoCD47 tf CHO-S cells, or LLC-MK2 cells) with at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater affinity than wild-type SIRPα or protein constructs thereof. In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can bind to mouse CD47-expressing cells (e.g., EMT-6 cells) with at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater affinity than wild-type SIRPα or protein constructs thereof.

[0164] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can bind to CD47-expressing tumor cells (e.g., CD47 tf CHO-S cells, Jurkat cells, or Raji cells) with an affinity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% greater than that of an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0165] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can bind to RBC cells or platelets (e.g., from a human donor) with less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, or less than 1% affinity compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4). In some embodiments, the EC50 value of the engineered SIRPα variants and / or protein constructs thereof binding to RBC cells is less than 5 nM, less than 4 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1 nM, or less than 0.5 nM. In some embodiments, the EC50 value of the engineered SIRPα variant and / or protein construct thereof that binds to platelets is less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM.

[0166] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein do not induce hemagglutination, hi some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein induce hemagglutination at a minimum concentration that is greater than 500-fold, 2000-fold, 5000-fold, 20000-fold, or 50000-fold greater than an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0167] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein are capable of blocking the interaction between human CD47 and human SIRPα. In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein are capable of blocking the interaction between human CD47 expressing cells (e.g., CD47 tf CHO-S cells, FaDu cells, or Raji cells) and human SIRPα. In some embodiments, the blocking ability of the engineered SIRPα variants and / or protein constructs thereof described herein is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4). In some embodiments, the blocking potency of the engineered SIRPα variants and / or protein constructs thereof described herein is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater than wild-type SIRPα or protein constructs thereof. In some embodiments, the IC50 value for the engineered SIRPα variants and / or protein constructs thereof for blocking CD47 / SIRPα interaction is less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM.

[0168] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can induce phagocytosis of CD47-expressing tumor cells (e.g., Jurkat, FaDu, or Raji cells) by mouse macrophages (e.g., Raw264.7 cells). In some embodiments, the EC50 value for the engineered SIRPα variants and / or protein constructs thereof to induce phagocytosis of CD47-expressing tumor cells is less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, the EC50 value for the engineered SIRPα variants and / or protein constructs thereof to induce phagocytosis of CD47-expressing tumor cells is comparable (e.g., at least 80%, 85%, 90%, or 95%) to an anti-CD47 reference antibody (e.g., Hu5F9-G4). In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein have a weaker ability to induce phagocytosis of RBC cells by mouse macrophages (e.g., Raw264.7 cells) than an anti-CD47 reference antibody (e.g., Hu5F9-G4). In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein have a weaker ability to induce phagocytosis of platelets by mouse macrophages (e.g., Raw264.7 cells) than an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0169] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can induce phagocytosis of CD47-expressing tumor cells (e.g., Raji cells, DLD1 cells, or Jurkat cells) by human macrophages (e.g., MDM cells). In some embodiments, the phagocytosis-inducing ability of the engineered SIRPα variants and / or protein constructs thereof is comparable (at least 80%, 85%, 90%, or 95%) to an anti-CD47 reference antibody (e.g., Hu5F9-G4). In some embodiments, the phagocytosis-inducing ability of the engineered SIRPα variants and / or protein constructs thereof is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater than that of wild-type SIRPα or protein constructs thereof. In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein have a weaker ability to induce phagocytosis of RBC cells by human macrophages (e.g., MDM cells) than an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0170] In some embodiments, the engineered SIRPα variants and / or protein constructs thereof described herein can inhibit tumor growth. In some embodiments, Raji or NCI-H82 cells are injected into immunodeficient mice (e.g., NOD / SCID mice) to generate xenograft models.

[0171] Methods for producing engineered SIRPα variants and protein constructs Variants of SIRPα described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the SIRPα peptide or a portion thereof, or by peptide synthesis, such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence.

[0172] Screening can be performed. In such a population of variants, some engineered SIRPα variants have increased affinity for CD47. Any combination of deletions, insertions and / or combinations can be performed to arrive at variants with increased binding affinity for the target. The amino acid changes introduced into the variants can also alter or introduce new post-translational modifications to the polypeptide, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in the cell), or introducing new glycosylation sites.

[0173] The genetically engineered SIRPα variants may be derived from any species of animal, including mammals. Non-limiting examples of SIRPα variants include those derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0174] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cell contains the polynucleotide and / or the vector comprising the polynucleotide), and the production of the recombinant polypeptides or fragments thereof by recombinant techniques.

[0175] As used herein, a "vector" is any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" can deliver and express one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to a regulatory element, such as a promoter, enhancer, and / or polyA tail, at or near or adjacent to the integration site of the polynucleotide of interest, either within the vector or in the genome of the host cell, such that the polynucleotide of interest is translated in the host cell into which the expression vector is introduced.

[0176] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0177] In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may include the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-defective virus. Techniques for incorporating DNA into such expression systems are well known to those of skill in the art. The DNA may be "naked." Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0178] For expression, the DNA insert comprising a polynucleotide encoding a polypeptide disclosed herein may be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs, to name a few. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct may further comprise sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon at the beginning of the polypeptide to be translated, and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0179] As indicated, the expression vector may contain at least one selection marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline resistance or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma and HK293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

[0180] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene, and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0181] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs, such as the Rous sarcoma virus (RSV) promoter, and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0182] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used.

[0183] Introduction of the construct into the host cell can be by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods, which are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0184] Transcription of DNA encoding the polypeptides of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located at base pairs 100-270 on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0185] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0186] The polypeptides (e.g., SIRPα variants) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification, among others, is a well-known and routine technique in the art.

[0187] Treatment method The engineered SIRPα variants and protein constructs of the present disclosure can be used for a variety of therapeutic purposes.

[0188] In one aspect, the present disclosure provides a method for treating cancer in a subject, a method for reducing the rate of increase in tumor volume over time in a subject, a method for reducing the risk of developing metastasis, or a method for reducing the risk of developing further metastasis in a subject. In some embodiments, the treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0189] In one aspect, the disclosure features a method that includes administering a therapeutically effective amount of the engineered SIRPα variants and protein constructs disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer (e.g., breast cancer (e.g., triple negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematological malignancy). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0190] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0191] As used herein, an "effective amount" refers to an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, delaying, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight, severity of symptoms, and route of administration of the subject to which the engineered SIRPα variants and protein constructs, vectors comprising polynucleotides encoding engineered SIRPα variants and protein constructs, and / or compositions thereof are administered, and thus dosing can be determined on an individual basis.

[0192] An effective amount can be administered in one or more doses. By way of example, an effective amount of an engineered SIRPα variant and / or protein construct is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow and / or delay the progression of cancer in a patient, or an amount sufficient to improve, stop, stabilize, reverse, slow and / or delay the proliferation of a cell (e.g., a biopsy cell, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount can vary depending on other factors, such as the patient's medical history and the type (and / or dosage) of engineered SIRPα variant and protein construct used, among others.

[0193] Effective amounts and schedules for administering the engineered SIRPα variants and protein constructs, polynucleotides encoding the engineered SIRPα variants and protein constructs, and / or compositions disclosed herein may be determined empirically, and making such determinations is within the skill of the art. One of ordinary skill in the art will understand that the dosages that must be administered will vary depending, for example, on the mammal receiving the engineered SIRPα variants and protein constructs, polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of polynucleotide and / or composition disclosed herein that is used, and other drugs administered to the mammal.

[0194] A typical daily dosage of an effective amount of an engineered SIRPα variant and / or protein construct is 0.1 mg / kg to 100 mg / kg (mg per kg patient body weight). In some embodiments, the dosage may be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg, In some embodiments, the dosage is about 1-10 mg / kg, about 1-5 mg / kg, or about 2-5 mg / kg.

[0195] In any of the methods described herein, the engineered SIRPα variants and protein constructs may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0196] In some embodiments, one or more additional therapeutic agents may be administered to the subject before or after administration of the engineered SIRPα variants and protein constructs. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that there is an overlap in the period of biological activity of the one or more additional therapeutic agents and the engineered SIRPα variants and protein constructs in the subject.

[0197] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

[0198] In some embodiments, the additional therapeutic agent can include one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0199] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, cyclosporine ... The therapeutic agents may include one or more therapeutic agents selected from the group consisting of Zantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temazolomide, IL-2, IFNa, Vinblastine, Thalomid, Dacarbazine, Cyclophosphamide, Lenalidomide, Azacitidine, Lenalidomide, Bortezomib, Amrubicin, Carfilzomib, Pralatrexate, and Enzastaurin.

[0200] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0201] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0202] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, an anti-CD47 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some embodiments, the additional therapeutic agent is an anti-CD20 antibody (e.g., rituximab) or an anti-EGF receptor antibody (e.g., cetuximab).

[0203] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing the engineered SIRPα variants and protein constructs described herein. Pharmaceutical compositions can be formulated in any manner known in the art.

[0204] Pharmaceutical compositions are formulated to suit their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonicity agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions may also be used as pharma-ceutically acceptable carriers. The preparation of the composition may be formulated and enclosed in ampoules, disposable syringes, or multiple dose vials. Where required (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of the drug can be prolonged by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems which can include biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0205] Compositions containing the engineered SIRPα variants and protein constructs described herein may be formulated in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration.

[0206] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dose for a single administration). Pharmaceutical compositions can be formulated with one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. For injection, the engineered SIRPα variants and protein constructs can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce discomfort at the injection site. The solution can contain compounding agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the engineered SIRPα variants and protein constructs can be in lyophilized form for dissolution in a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0207] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0208] Exemplary doses include milligram or microgram amounts of any of the engineered SIRPα variants and protein constructs described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, about 1 μg / kg to about 50 μg / kg, about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg). While these doses cover a wide range, one of skill in the art will understand that therapeutic agents may vary in their potency and that effective amounts may be determined by methods known in the art. Typically, a relatively low dose is administered initially, and the attending medical or veterinary professional (for therapeutic applications) or researcher (if still in development) may then gradually increase the dose until an appropriate response is obtained. It will further be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the particular compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and in vivo half-life of the engineered SIRPα variant and protein construct.

[0209] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods of producing the engineered SIRPα variants and protein constructs for the various uses described herein. EXAMPLES

[0210] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0211] Example 1. Design of an engineered IgV domain of human SIRPα The IgV domain of human SIRPα (hSIRPα) belongs to the immunoglobulin superfamily, which contains nine β-strands with the structure ABC-C'-DEFG-G2. The helix is ​​located between the E and F strands. The 3D structure of the hSIRPα IgV domain is shown in Figure 1.

[0212] A detailed analysis of the CD47 / hSIRPa complex structure was performed. The structure revealed that Leu30, Gly34, Gln52, Lys53, Glu54, His56, Ser66, Thr67, Arg69, Lys93, Lys96, Gly97, and Asp100 in hSIRPα were involved in the interaction with CD47. These interacting residues were mainly located on the loop structure of hSIRPa, including the BC loop, C'D loop, DE loop, and FG loop. The results indicated that hSIRPa interacts with CD47 mainly through its loop regions (Figure 2).

[0213] Furthermore, the structure of hSIRPa-FD6 has been elucidated (Weiskopf, Kipp, et al. Science 2013 July 5;341(6141)). Based on the CD47 / hSIRPa-FD6 complex structure (PDB ID: 4KJY), the interacting residues of hSIRPa-FD6 with CD47 were determined to include Leu30, Gly34, Gln52, Arg53, Glu54, His56, Thr66, Thr67, Arg69, Lys93, Lys96, Gly97, and Asp100. These residues are the same as those shown in the CD47 / hSIRPa complex. Only two interacting residues in the wild type, Lys53 and Ser66, are mutated to Arg53 and Thr66, respectively, in hSIRPa-FD6, resulting in a dramatic increase in binding affinity to CD47. The other changed residues from hSIRPa to hSIRPa-FD6 are not involved in CD47 interaction. This finding suggested that Lys53 and Ser66 may be important for increasing the binding affinity of hSIRPa to CD47.

[0214] To find out which residues are good for binding affinity and stable in the C'D loop, residue scans for positions 53, 54, 55 and 56 were performed by MOE test version. The results of residue scans showed that Lys53 can increase stability when substituted by amino acids with long side chains or side chains with aromatic rings, such as arginine, leucine, phenylalanine, tryptophan and tyrosine (Figure 3A). However, this kind of substitution cannot improve the affinity of hSIRPa. Meanwhile, substitutions of Glu54, Gly55 and His56 showed higher flexibility (Figure 3B, Figure 3C and Figure 3D), and the affinity and stability of hSIRPa could be improved by changing Glu54, Gly55 and His56 to some other amino acids. Therefore, mutations were introduced into Glu54, Gly55 and His56 for screening.

[0215] Furthermore, Ile31, Glu54, Gly55, His56, Ser66, and Thr67 were identified as candidate amino acids for hSIRPa mutation screening by structural analysis. Val27, Val63, and Lys68 are involved in maintaining the structure of hSIRPa itself. To avoid adversely affecting the structure of hSIRPa, changes in these residues are not recommended. Based on the structure of human CD47 (hCD47) complexed with hSIRPα (PDB ID: 2JJT), the interacting residues were analyzed. The analysis results show that multiple interacting residues in the BC, C'D, and DE loops of the hSIRPα IgV domain are highly conserved. As shown in Figure 2, the amino acid residues in these loop regions are underlined.

[0216] To screen for hSIRPα IgV domain variants with higher blocking activity for hCD47 than wild-type hSIRPα (e.g., with different binding affinity for hCD47) and lower RBC binding compared to the anti-CD47 reference antibody Hu5F9-G4, selected residues in the BC, C'D, and DE loops of the hSIRPα IgV domain were mutated. These residues include 10 amino acids from His24 to Val33 in the BC loop (e.g., Ile31), 3 amino acids from Glu54 to His56 in the C'D loop, and 8 amino acids from Ser66 to Asp73 in the DE loop (e.g., Ser66 and Thr67). The amino acid positions are based on the IgV domain of hSIRPα (SEQ ID NO: 1).

[0217] Example 2. Screening and revalidation A phagemid expression system was used for screening by phage display. Specifically, CD47 antigen or cell lines expressing CD47 were used for panning. The resulting phages were used to reinfect TG1 electroporation competent E. coli cells, which were then spread on LB agar plates and incubated for 16 hours. A single colony was picked and inoculated into 700 μL of 2×YT medium supplemented with 10% buffered phosphate. OD 600 When the β-d-1-thiogalactopyranoside approached 1, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce protein expression at 30 °C. After 16 h, the bacterial culture was centrifuged at 3200 g and the supernatant was collected for subsequent experiments.

[0218] Determination of expression levels To determine the expression levels of hSIRPα IgV domain mutants, ELISA plates coated with 2 μg / mL anti-His antibody (R&D, Cat. No.: MAB050-500) were prepared. Thirty μL of the collected supernatant was added to the ELISA plate and incubated at 25°C for 1 h. After incubation, goat anti-c-Myc HRP (Bethy, Cat. No.: A190-104P) was added to the plate at a dilution ratio of 1:10000, and 3,3',5,5'-tetramethylbenzidine (TMB) (SURMODICS, Cat. No.: TMBW-0100-01) was added for color development. The absorbance values ​​at 450 nm (OD 450 ) was measured using a Varioskan™ LUX plate reader (Thermo).

[0219] Determination of human CD47 binding capacity To determine the human CD47 binding ability of hSIRPα IgV domain mutants, ELISA plates coated with 1 μg / mL human CD47 extracellular domain (ECD) linked to Fc region (hCD47 ECD Fc) were prepared. 30 μL of collected supernatant was added to the ELISA plate and incubated at 25° C. for 1 hour. After incubation, goat anti-c-Myc HRP was added to the plate at a dilution ratio of 1:10000, and TMB was added for color development. Absorbance values ​​at 450 nm were measured using a Varioskan™ LUX plate reader.

[0220] Determination of mouse CD47 binding ability To determine the mouse CD47 binding ability of hSIRPα IgV domain mutants, ELISA plates coated with 1 μg / mL mouse CD47 ECD Fc (Sino Bio, Catalog No.: 57231-M31H) were prepared. 100 μL of the collected supernatant was added to the ELISA plate and incubated at 25° C. for 1 hour. After incubation, goat anti-c-Myc HRP was added to the plate at a dilution ratio of 1:10000, and TMB was added for color development. The absorbance value at 450 nm was measured using a Varioskan™ LUX plate reader.

[0221] Determination of hCD47 / hSIRPα blocking ability To determine the hCD47 / hSIRPα blocking ability of the hSIRPα IgV domain mutants, ELISA plates coated with 1 μg / mL human CD47 ECD Fc were prepared. 150 μL of collected supernatant and 20 μL of biotin-labeled SIRPα ECD His protein (final concentration 2.5 μg / mL) were added to the ELISA plate and incubated at 25° C. for 1 hour. After incubation, Avidin-HRP (BioLegend, Cat. No.: 79004) was added to the plate at a dilution ratio of 1:1000, and TMB was added for color development. Absorbance values ​​at 450 nm were measured using a Varioskan™ LUX plate reader.

[0222] Determination of protein thermostability 100 μL of the collected supernatant was transferred to a PCR tube, which was heated at 65° C. or 70° C. for 90 minutes in a PCR machine. Human CD47 binding assay was performed using heated and unheated supernatants simultaneously. Specifically, an ELISA plate coated with 1 μg / mL human CD47 ECD Fc was prepared. 30 μL of heated or unheated supernatant was added to the ELISA plate and incubated at 25° C. for 1 hour. After incubation, goat anti-c-Myc HRP was added to the plate at a dilution ratio of 1:10000, and TMB was added for color development. The absorbance value at 450 nm was measured using a Varioskan™ LUX plate reader.

[0223] Determination of total cell binding capacity for CD47 tf CHO-S, RBCs, and platelets 3×10 4 CD47 tf CHO-S cells (transfected CHO-S cells expressing human CD47), 1 × 10 5 human red blood cells (RBCs), or 3 x 10 5Human platelets were incubated with the collected supernatant for 30 minutes at 4° C. Then, anti-His PE (Abcam, Catalog No.: ab72467) was added at a dilution ratio of 1:100, and the MFI (mean fluorescence intensity) values ​​were detected using a flow cytometer (Beckman Coulter, Catalog No.: CytoFlex).

[0224] Clones with higher hCD47 / hSIRPα blocking ability than wild-type hSIRPα were isolated, and their human CD47 binding OD 450 (B) / Expression OD 450 The clones were grouped based on the hCD47 binding B / E ratio (B / E ratio). Specifically, 12 clones had a hCD47 binding B / E ratio of more than 2, 5 clones had a ratio of 1.75 to 2, 5 clones had a ratio of 1.5 to 1.75, 7 clones had a ratio of 1.25 to 1.5, and 2 clones had a ratio of 1 to 1.25. Seven clones with B / E ratios lower than the wild-type hSIRPα IgV domain were also selected. Because human SIRPα has no cross-reactivity to mouse CD47, 18 clones with mouse CD47 binding capacity to expression ratios greater than 0.4 were also selected through this screening. Thus, a total of 56 candidate clones were selected, and the detailed expression, hCD47 / hSIRPα blocking, and CD47 binding results are shown in Figure 4A-B.

[0225] The selected clones were then further screened by determining the whole cell binding ability to CD47 tf CHO-S, RBCs and platelets, as well as the protein thermostability at high temperatures (e.g., 65°C and 70°C). Based on the results of the whole cell binding assay, clones with lower platelet and RBC binding ability than the wild-type hSIRPα IgV domain and clones that could bind to CD47 tf CHO-S with different binding ability were selected. The protein thermostability of the selected clones was verified and no significant difference was observed compared to the wild-type SIRPα IgV domain. Finally, 21 clones were selected, including mt3-mt15 (SEQ ID NOs: 2-14) and mt16-mt23 (SEQ ID NOs: 33-40). Five additional clones were also generated, including mt31-mt35 (SEQ ID NOs: 41-45). Plasmids expressing the wild-type SIRPα IgV domain or the engineered SIRPα IgV domain mutants (SEQ ID NO: 29) linked to the human IgG4 hinge and Fc region were constructed. The schematic structure of the expressed protein is shown in Figure 5. The specific amino acid mutations within the BC, C'D, and DE loop regions of the wild-type SIRPα IgV domain and the 26 clones are shown in Figure 6.

[0226] Example 3. Purification and Determination of Binding Affinity of hSIRPα-Fc to CD47-ECD-His by Octet The expressed proteins were purified by Protein A column followed by HPLC-SEC (high performance liquid chromatography coupled with size exclusion chromatography, Agilent), and the percentage of high molecular weight peaks (HMW%), main peaks (Main%), and low molecular weight peaks (LMW%) were measured. As shown in Figure 7, hSIRPα-Fc mutant proteins can be recovered with high purity using the above method. The amino acid sequences of hSIRPα-Fc-wt and 13 hSIRPα-Fc mutant proteins (mt3 to mt15) were analyzed using a deimmunization tool (Immune Epitope Database And Analysis Resource; Dhanda et al. "Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity." Immunology 153.1 (2018): 118-132) to identify immunogenic regions. No immunogenicity was identified.

[0227] The binding affinity of hSIRPα-Fc protein to CD47-ECD-His was also determined by the Octet® system. Two sets of experiments were performed using hSIRPα-Fc-wt as a negative control and anti-CD47 reference antibody Hu5F9-G4 as a positive control, as shown in Figure 8. The relative binding affinity of each protein was also estimated from their respective KD values ​​compared to that of hSIRPα-Fc-wt. The results show that most of the engineered SIRPα IgV domain mutants showed stronger binding to CD47 compared to the wild-type SIRPα IgV domain.

[0228] Example 4. Whole cell binding assay for CD47 tf CHO-S and CD47-expressing tumor cells To determine the total cell binding ability of hSIRPα-Fc muteins to CD47, hSIRPα-Fc muteins were purified by protein A chromatography and serially diluted (5-fold) from 500 nM to 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.064 nM. PD1-Fc-wt (two human PD1 extracellular domains linked to the N-terminus of human IgG4, schematic structure shown in Figure 5) was used as a negative control. Hu5F9-G4 was used as a positive control. Diluted proteins were diluted to 5 × 10 4 The cells were incubated with CD47 tf CHO-S cells, Jurkat cells, or Raji cells. After incubation, anti-hFcr-PE (1:100, Invitrogen, Cat. No.: 109-115-098) was added and the MFI values ​​were measured by flow cytometry.

[0229] As shown in Figures 9A to 9C, hSIRPα-Fc-mt4, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, hSIRPα-Fc-mt11, hSIRPα-Fc-mt12, hSIRPα-Fc-mt13, and hSIRPα-Fc-15 showed similar whole cell binding ability to CD47 tf CHO-S, Jurkat, and Raji cells compared to the anti-CD47 antibody Hu5F9-G4.

[0230] As shown in Figure 9D, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt19, and hSIRPα-Fc-mt22 showed higher CD47 tf CHO-S cell binding ability than hSIRPα-Fc-mt17, hSIRPα-Fc-mt18, hSIRPα-Fc-mt19, and Hu5F9-G4. In addition, all tested hSIRPα-Fc mutant proteins showed significantly higher CD47 tf CHO-S cell binding ability than hSIRPα-Fc-wt. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0231] [Table 1]

[0232] As shown in Figure 9E, hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 showed higher Raji cell binding ability than hSIRPα-Fc-mt21, hSIRPα-Fc-mt16, and hSIRPα-Fc-mt23. All hSIRPα-Fc mutant proteins tested showed similar Raji cell binding ability compared to Hu5F9-G4 and significantly higher Raji cell binding ability compared to hSIRPα-Fc-wt.

[0233] Example 5. RBC Binding Assay To determine the RBC binding ability of the hSIRPα-Fc muteins, the hSIRPα-Fc muteins were purified by Protein A chromatography and serially diluted (5-fold) from 500 nM to 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.064 nM. PD1-Fc-wt was used as a negative control. Hu5F9-G4 was used as a positive control. The diluted proteins were diluted with 1 × 10 5 The cells were incubated with human RBC cells. After incubation, anti-hFcr-PE (1:100, Invitrogen) was added and the MFI values ​​were measured by flow cytometry.

[0234] As shown in Figures 10A-B, hSIRPα-Fc-mt12, hSIRPα-Fc-mt13, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, and hSIRPα-Fc-mt15 showed weaker RBC binding ability compared to anti-CD47 antibody Hu5F9-G4. Notably, hSIRPα-Fc-mt6, hSIRPα-Fc-mt14, and hSIRPα-Fc-wt did not show any binding to RBCs.

[0235] As shown in Figure 10C, hSIRPα-Fc-mt18 and hSIRPα-Fc-mt19 showed higher RBC (from donor 1) binding ability than Hu5F9-G4, while hSIRPα-Fc-mt22, hSIRPα-Fc-mt10, hSIRPα-Fc-mt17, hSIRPα-Fc-mt16, hSIRPα-Fc-mt15, and hSIRPα-Fc-mt21 showed lower RBC binding ability than Hu5F9-G4. No RBC cell binding was detected by hSIRPα-Fc-wt. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0236] [Table 2]

[0237] As shown in Figure 10D, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 showed significantly lower RBC (from donor 2) binding ability than Hu5F9-G4. No RBC cell binding was detected by hSIRPα-Fc-wt.

[0238] Example 6. Platelet binding assay To determine the platelet binding ability of the hSIRPα-Fc muteins, the hSIRPα-Fc muteins were purified by protein A beads and serially diluted (5-fold) from 500 nM to 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.064 nM. PD1-Fc-wt was used as a negative control. Hu5F9-G4 was used as a positive control. The diluted proteins were diluted with 5 × 10 5 After incubation, anti-hFcr-PE (1:100, Invitrogen) was added and MFI values ​​were measured by flow cytometry.

[0239] As shown in Figures 11A to 11B, hSIRPα-Fc-mt4, hSIRPα-Fc-mt10, hSIRPα-Fc-mt8, hSIRPα-Fc-mt11, hSIRPα-Fc-mt12, hSIRPα-Fc-mt13, and hSIRPα-Fc-mt15 showed stronger platelet binding ability than the anti-CD47 antibody Hu5F9-G4, whereas hSIRPα-Fc-mt14, hSIRPα-Fc-wt, and hSIRPα-Fc-mt6 showed significantly lower platelet binding ability.

[0240] As shown in Figure 11C, hSIRPα-Fc-mt19, hSIRPα-Fc-mt18, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt21, hSIRPα-Fc-mt20, hSIRPα-Fc-mt17, and hSIRPα-Fc-mt22 showed stronger or similar platelet (from donor 1) binding ability compared to Hu5F9-G4. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0241] [Table 3]

[0242] As shown in Figure 11D, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt21, hSIRPα-Fc-mt23, and hSIRPα-Fc-mt16 showed stronger or similar platelet binding ability compared to Hu5F9-G4. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0243] [Table 4]

[0244] Example 7. Whole cell binding assay for cells expressing monkey CD47 To determine the whole cell binding ability of the hSIRPα-Fc muteins to monkey CD47, the hSIRPα-Fc muteins were purified by protein A beads and serially diluted (5-fold) from 500 nM to 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.064 nM. PD1-Fc-wt was used as a negative control. Hu5F9-G4 was used as a positive control. The diluted proteins were then transferred to 1 × 10 5 1 × 10 transfected CHO-S cells (cynoCD47 tf CHO-S) or 1 × 10 5 The hSIRPα-Fc mutant proteins were incubated with rhesus monkey kidney epithelial cell line LLC-MK2. After incubation, anti-hFcr-PE (1:100, Invitrogen) was added and MFI values ​​were measured by flow cytometry. As shown in Figure 12A-B, all hSIRPα-Fc mutant proteins can bind to monkey CD47.

[0245] As shown in Figure 12C, Hu5F9-G4 showed stronger LLC-MK2 binding ability than hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23.

[0246] Example 8. Whole cell binding assay for cells expressing mouse CD47 To determine the whole cell binding ability of hSIRPα-Fc muteins to mouse CD47, hSIRPα-Fc muteins were purified by protein A beads and serially diluted (5-fold) from 1000 nM to 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.0128 nM. PD1-Fc-wt was used as a negative control. MIAP410 (anti-human mouse CD47 antibody) was used as a positive control. Diluted proteins were diluted at 3 × 10 4The IgG antibodies were incubated with 100 EMT-6 cells. After incubation, anti-hFcr-PE (1:100, Invitrogen) was added, and the MFI values ​​were measured by flow cytometry. For MIAP410, anti-mouse (H+L)-FITC (1:100) was used. As shown in Figure 13A-B, hSIRPα-Fc-mt13, hSIRPα-Fc-mt11, hSIRPα-Fc-mt4, hSIRPα-Fc-mt12, hSIRPα-Fc-mt8, and hSIRPα-Fc-mt10 can bind to cells expressing mouse CD47. However, hSIRPα-Fc-wt, hSIRPα-Fc-mt6, hSIRPα-Fc-mt14, and hSIRPα-Fc-mt15 did not bind to EMT-6 cells.

[0247] Example 9. Determination of hemagglutination (HA) activity To determine the HA activity induced by hSIRPα-Fc muteins, 10% RBC solutions were prepared from whole blood of two healthy donors by washing twice with 0.9% sodium chloride buffer and then diluted 10-fold in 0.9% sodium chloride buffer. hSIRPα-Fc muteins were serially diluted (3-fold) to final concentrations of 500 nM, 166.7 nM, 55.6 nM, 18.5 nM, 6.2 nM, 2.1 nM, 685.8 pM, 228.6 pM, 76.2 pM, 25.4 pM, or 8.4 pM and incubated overnight at room temperature (RT) with 12 μL of 10% RBC solution in a round-bottom 96-well plate. After incubation, the aggregated RBCs evenly coated the wells, whereas the non-aggregated cells formed distinct red dots at the bottom of the wells. Hu5F9-G4 was used as a positive control. hSIRPα-Fc-wt and PD1-Fc-wt were used as negative controls. As shown in Figures 14A-C, none of the hSIRPα-Fc muteins tested induced hemagglutination.

[0248] Example 10. Determination of blocking capacity against CD47 tf CHO-S, FaDu and Raji cells To determine the blocking ability of hSIRPα-Fc muteins against human CD47, hSIRPα-Fc muteins were purified by protein A beads and serially diluted (8-fold) from 1000 nM to 125 nM, 15.63 nM, 1.95 nM, 0.24 nM, 0.03 nM, 0.0038 nM, and 0.0004 nM. PD1-Fc-wt was used as a negative control. Hu5F9-G4 was used as a positive control. The diluted proteins were diluted at 3 × 10 4 CD47 tf CHO-S cells (transfected CHO-S cells expressing human CD47), FaDu cells, or Raji cells were incubated with 1 μg / mL of biotin-labeled hSIRPα-Fc-wt. After incubation, streptavidin-PE (0.3 μL / well, eBioscience, Cat. No.: EBS12-4317-87) was added, and the MFI values ​​were measured by flow cytometry.

[0249] As shown in Figures 15A-C, all tested hSIRPα-Fc muteins can block the interaction between hSIRPα and CD47 tf CHO-S cells or CD47-expressing tumor cells (FaDu and Raji). In particular, hSIRPα-Fc-mt4, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, hSIRPα-Fc-mt11, hSIRPα-Fc-mt12, hSIRPα-Fc-mt13, hSIRPα-Fc-mt15, and hSIRPα-Fc-wt showed blocking ability comparable to that of Hu5F9-G4.

[0250] As shown in Figure 15D, all tested hSIRPα-Fc mutant proteins showed similar whole cell blocking ability compared to Hu5F9-G4. hSIRPα-Fc-wt showed the lowest CD47 / SIRPα blocking ability. The IC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0251] [Table 5]

[0252] As shown in Figure 15E, hSIRPα-Fc-mt21 showed the strongest whole cell blocking ability. hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, and hSIRPα-Fc-mt23 showed similar whole cell blocking ability compared to Hu5F9-G4. hSIRPα-Fc-wt showed the lowest CD47 / SIRPα blocking ability. The IC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0253] [Table 6]

[0254] Example 11. Induction of phagocytosis of CD47-expressing tumor cells by mouse macrophages Phagocytosis of human CD47-expressing tumor cells (Jurkat, FaDu, or Raji) was determined by incubating the tumor cells with Raw264.7 mouse macrophages in the presence of hSIRPα-Fc mutant proteins. The experiments were performed as follows: Jurkat, FaDu, or Raji cells were labeled with 5 nM CellTrace™ CFSE (Thermo, Cat. No.: C34554) for 10 min at 37°C, and then washed with complete DMEM medium containing 10% FBS (fetal bovine serum). The hSIRPα-Fc mutant proteins were then serially diluted (10-fold) to final concentrations of 1 μM, 100 nM, 10 nM, 1 nM, 100 pM, and 10 pM. Hu5F9-G4 and hSIRPα-Fc-wt (Trillium) were used as positive controls. PD1-Fc-wt was used as a negative control. 1 × 10 5 Cells / well of CFSE-labeled Jurkat, FaDu, or Raji cells (target cells) were incubated with diluted hSIRPα-Fc muteins in low-binding 96-well U-shaped plates for 30 min at 37°C. Then, 5 × 10 4Raw264.7 cells were added to each well and the plate was incubated for 2 hours at 37°C. Raw264.7 cells were stained with PE-Cyanine 7 conjugated F4 / 80 antibody (eBioscience, Catalog No: 25-4801-82). The phagocytosis-inducing ability of hSIRPα-Fc muteins was assessed by flow cytometry by calculating the ratio of macrophage-derived CFSE+F4 / 80+ (indicating that macrophages had phagocytosed CFSE-labeled Jurkat, FaDu, or Raji cells) to the total macrophage-derived F4 / 80 signal.

[0255] As shown in Figures 16A to 16C, hSIRPα-Fc-mt4, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, hSIRPα-Fc-mt13, and hSIRPα-Fc-mt15 showed similar or even higher phagocytosis-inducing ability of CD47-expressing tumor cells compared to Hu5F9-G4. EC50 values ​​were also calculated for each tumor cell line, as shown in the table below.

[0256] [Table 7]

[0257] [Table 8]

[0258] [Table 9]

[0259] As shown in Figure 16D, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 showed comparable phagocytosis-inducing ability of DLD1 cells compared to Hu5F9-G4. As shown in Figure 16E, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt20, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 showed comparable phagocytosis-inducing ability of DLD1 cells compared to Hu5F9-G4.

[0260] Example 12. Induction of RBC phagocytosis by mouse microphages Phagocytosis of human RBCs was determined by incubating RBCs with Raw264.7 mouse macrophages in the presence of hSIRPα-Fc mutant proteins. The experiment was performed as follows: RBCs were labeled with 5 nM CellTrace™ CFSE (Thermo) for 10 min at 37° C. and then washed with complete DMEM medium containing 10% FBS. hSIRPα-Fc mutant proteins were then serially diluted (10-fold) to final concentrations of 1 μM, 100 nM, 10 nM, 1 nM, 100 pM, and 10 pM. Hu5F9-G4 and hSIRPα-Fc-wt (Trillium) were used as positive controls. PD1-Fc-wt was used as a negative control. 1×10 6 Cells / well of CFSE-labeled RBCs (target cells) were incubated with diluted hSIRPα-Fc muteins in low-binding 96-well U-shaped plates for 30 min at 37°C. Then, 5 × 10 4 Raw264.7 cells were added to each well and the plate was incubated at 37° C. for 2 hours. Raw264.7 cells were stained with PE-Cyanine 7 conjugated F4 / 80 antibody (eBioscience). The ability of hSIRPα-Fc mutant proteins to induce RBC phagocytosis was assessed by flow cytometry by calculating the ratio of macrophage-derived CFSE+F4 / 80+ (indicating that macrophages had phagocytosed CFSE-labeled RBC cells) to the total macrophage-derived F4 / 80 signal.

[0261] As shown in FIG. 17A, all tested hSIRPα-Fc muteins exhibited a weaker ability to induce RBC phagocytosis by macrophages (Raw264.7).

[0262] Example 13. Induction of platelet phagocytosis by mouse microphages Phagocytosis of human platelets was determined by incubating platelets with Raw264.7 mouse macrophages in the presence of hSIRPα-Fc mutant proteins. The experiment was performed as follows: Platelets were labeled with 5 nM CellTrace™ CFSE (Thermo) for 10 min at 37° C. and then washed with complete DMEM medium containing 10% FBS. hSIRPα-Fc mutant proteins were then serially diluted (10-fold) to final concentrations of 500 nM, 50 nM, 5 nM, 0.5 nM, 50 pM, 5 pM, and 0.5 pM. Hu5F9-G4 and hSIRPα-Fc-wt (Trillium) were used as positive controls. PD1-Fc-wt was used as a negative control. 5×10 5 Cells / well of CFSE-labeled platelets (target cells) were incubated with diluted hSIRPα-Fc muteins in low-binding 96-well U-shaped plates for 30 min at 37°C. Then, 5 × 10 4 Raw264.7 cells were added to each well and the plate was incubated at 37° C. for 2 hours. Raw264.7 cells were stained with PE-Cyanine 7 conjugated F4 / 80 antibody (eBioscience). The ability of hSIRPα-Fc muteins to induce platelet phagocytosis was assessed by flow cytometry by calculating the ratio of macrophage-derived CFSE+F4 / 80+ (indicating that macrophages had phagocytosed CFSE-labeled RBC cells) to the total macrophage-derived F4 / 80 signal.

[0263] As shown in Figure 17B, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 showed a weaker ability to induce platelet phagocytosis by macrophages (Raw264.7).

[0264] Example 14. Induction of phagocytosis of CD47-expressing tumor cells by human macrophages Phagocytosis of CD47-expressing tumor cells (Raji, DLD1, or Jurkat) by human microphages was determined as follows: PBMCs were isolated from human blood and monocytes were differentiated into macrophages by incubation for 10-14 days in complete RPMI medium containing 10% FBS, 1x streptomycin / penicillin, and 200 U / mL GM-CSF (BioLegend, Cat. No.: 576304). Monocyte-derived macrophages (MDMs) became adherent and non-adherent cells were washed away. MDMs were incubated with Accutase® (eBioscience, Cat. No.: EBS00-4555-56) and detached from the plate by scraping. Human CD47-expressing tumor cells (Raji, DLD1, or Jurkat) were labeled with 5 nM CellTrace™ CFSE (Thermo) for 10 min at 37° C. and then washed with complete RPMI medium containing 10% FBS. Candidate clones were then serially diluted (10-fold) to final concentrations of 200 nM, 20 nM, 2 nM, 200 pM, 20 pM, and 2 pM. Hu5F9-G4 and hSIRPα-Fc-wt (Trillium) were used as positive controls. PD1-Fc-wt was used as a negative control. 2.4×10 4 ~6×10 4 Cells / well of CFSE-labeled Raji, DLD1, or Jurkat cells (target cells) were incubated with diluted hSIRPα-Fc mutant proteins in low-binding 96-well U-shaped plates for 30 min at 37°C. Then, 1.2 × 10 4 ~3×10 4MDM cells (half the number of target cells) at 100 cells / well were added to each well and the plate was incubated at 37° C. for 2 hours. MDM cells were stained with PE-Cyanine 7 conjugated CD14 antibody (eBioscience, Catalog No.: 25-0149-42). The phagocytosis-inducing ability of hSIRPα-Fc mutant protein was evaluated by flow cytometry by calculating the ratio of macrophage-derived CFSE+CD14+ (indicating that macrophages phagocytosed CFSE-labeled RBC cells) to the total macrophage-derived CD14 signal.

[0265] As shown in Figures 18A to 18C, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, and Hu5F9-G4 showed better ability to induce phagocytosis of CD47-expressing tumor cells by MDM cells compared to hSIRPα-Fc-wt (hSIRPα-Fc).

[0266] As shown in Figure 18D, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 showed better ability to induce phagocytosis of DLD1 cells by MDM cells than hSIRPα-Fc-wt. Hu5F9 showed the highest ability to induce phagocytosis by MDM cells.

[0267] Example 15. Induction of RBC phagocytosis by human microphages Phagocytosis of RBCs by human microphages was determined as follows: PBMCs were isolated from human blood, and monocytes were differentiated into macrophages by incubation for 10-14 days in complete RPMI medium containing 10% FBS, 1x streptomycin / penicillin, and 200 U / mL GM-CSF (BioLegend). MDMs became adherent, and non-adherent cells were washed away. MDMs were incubated with Accutase® (eBioscience) and detached from the plate by scraping. RBCs were labeled with 5 nM CellTrace™ CFSE (Thermo) for 10 min at 37°C and washed with complete RPMI medium containing 10% FBS. Candidate clones were then serially diluted (10-fold) to final concentrations of 200 nM, 20 nM, 2 nM, 200 pM, 20 pM, and 2 pM. Hu5F9-G4 and hSIRPα-Fc-wt (Trillium) were used as positive controls. PD1-Fc-wt was used as a negative control. 4×10 5 Cells / well of CFSE-labeled RBCs (target cells) were incubated with diluted hSIRPα-Fc muteins in low-binding 96-well U-shaped plates for 30 min at 37°C. Then, 4 × 10 4 MDM cells were added to each well and the plates were incubated for 2 hours at 37°C. MDM cells were stained with PE-Cyanine 7 conjugated CD14 antibody (eBioscience). The phagocytosis-inducing ability of hSIRPα-Fc mutant proteins was assessed by flow cytometry by calculating the ratio of macrophage-derived CFSE+CD14+ (indicating that macrophages had phagocytosed CFSE-labeled RBC cells) to the total macrophage-derived CD14 signal.

[0268] As shown in Figures 19A-B, hSIRPα-Fc-mt10, hSIRPα-Fc-mt13, and hSIRPα-Fc-mt15 did not induce RBC phagocytosis in human MDMs, and as shown in Figure 19C, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 did not induce RBC phagocytosis in human MDMs.

[0269] Example 16. Determination of in vivo antitumor effects The in vivo antitumor effect of hSIRPα-Fc mutant proteins was determined using Raji cells. Specifically, NOD / SCID mice were inoculated with Raji cells on day 0. On day 4, the mice were divided into a control group and four treatment groups. For the treatment group mice, hSIRPα-Fc-mt10 (G1), hSIRPα-Fc-mt15 (G2), hSIRPα-Fc-wt (Trillium, G3) or Hu5F9-G4 (G4) were administered by intraperitoneal injection on days 7 and 14 after inoculation. The control group mice were administered an equal amount of placebo. The tumor volume of the mice in each group was measured on days 4, 7, 11, 14 and 18 after inoculation. The average tumor volume of the mice in each group is shown in the following table. The tumor growth inhibition (TGI) and p-value were also determined.

[0270] [Table 10] Note: * :p<0.05, ** :p<0.01, *** :p<0.001, **** :p<0.0001

[0271] As shown in Figures 20A-B, hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 showed comparable anti-tumor effects compared to anti-CD47 antibody Hu5F9-G4. Furthermore, both hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 were more effective than hSIRPα-Fc-wt in inhibiting tumor growth. As shown in Figure 20C, the survival curves of mice in each group were also determined, which indicates that administration of hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 can significantly prolong the proportion of surviving mice compared to the placebo control.

[0272] In a different experiment, the in vivo antitumor effect of hSIRPα-Fc mutant proteins was determined using NCI-H82 cells. Specifically, on day 0, NOD / SCID mice were inoculated with NCI-H82 cells. On day 4, the mice were divided into a control group and four treatment groups. For the treatment group mice, hSIRPα-Fc-mt10 (G1), hSIRPα-Fc-mt15 (G2), hSIRPα-Fc-wt (Trillium, G3) or Hu5F9-G4 (G4) were administered by intraperitoneal injection on days 4, 7, 14, 11, 14, 18, 21, 25 and 28 after inoculation. The control group mice were administered an equivalent amount of placebo. The tumor volumes of mice in each group were measured on days 4, 7, 14, 11, 14, 18, 21, 25, 28, and 32 after inoculation. The average tumor volumes of mice in each group are shown in the table below. Tumor growth inhibition (TGI) and p-values ​​were also determined.

[0273] [Table 11] Note: * :p<0.05, ** :p<0.01, *** :p<0.001, **** :p<0.0001.

[0274] As shown in Figures 21A-B, hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 showed comparable anti-tumor effects compared to anti-CD47 antibody Hu5F9-G4. Furthermore, both hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 were more effective than hSIRPα-Fc-wt in inhibiting tumor growth. As shown in Figure 21C, the survival curves of mice in each group were also determined, which indicates that administration of hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 can significantly prolong the proportion of surviving mice compared to the placebo control.

[0275] Other embodiments While the invention has been described in conjunction with the detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An engineered SIRPα polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, wherein the engineered SIRPα polypeptide comprises one or more amino acid mutations in the BC loop, the C'D loop, and / or the DE loop.

2. (a) the amino acid corresponding to E54 in SEQ ID NO: 1 is A, H, N, I, R, G, S, D, or L; (b) the amino acid corresponding to G55 in SEQ ID NO: 1 is W, F, Q, L, D, K, R, A, or P; and (c) the amino acid corresponding to H56 in SEQ ID NO: 1 is P, I, T, N, V, R, L, S, G, or Q; 2. The engineered SIRPα polypeptide of claim 1, comprising one or more mutations of:

3. 2. The engineered SIRPα polypeptide of claim 1, wherein the amino acid corresponding to I31 of SEQ ID NO: 1 is W, K, Y, L, A, N, or T.

4. (a) the amino acid corresponding to S66 in SEQ ID NO: 1 is Q or N; and (b) the amino acid corresponding to T67 in SEQ ID NO: 1 is G; 2. The engineered SIRPα polypeptide of claim 1, comprising one or more mutations of:

5. (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is N or T; and (b) the amino acid corresponding to T26 in SEQ ID NO: 1 is I; 2. The engineered SIRPα polypeptide of claim 1, comprising one or more mutations of:

6. (a) the amino acid corresponding to E70 in SEQ ID NO: 1 is G, F, R, A, L, or T; (b) the amino acid corresponding to M72 in SEQ ID NO: 1 is R or Y; and (c) the amino acid corresponding to D73 in SEQ ID NO: 1 is I; 2. The engineered SIRPα polypeptide of claim 1, comprising one or more mutations of:

7. 2. The engineered SIRPα polypeptide of claim 1, wherein the amino acid corresponding to K53 in SEQ ID NO: 1 is R or is not R.

8. (a) the amino acid corresponding to position 27 of SEQ ID NO: 1 is V or L; (b) the amino acid corresponding to position 63 of SEQ ID NO: 1 is V; and (c) the amino acid corresponding to position 68 of SEQ ID NO: 1 is K; 2. The engineered SIRPα polypeptide of claim 1, comprising one or more mutations of:

9. 2. The engineered SIRPα polypeptide of claim 1, comprising an amino acid sequence at least 85% identical to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

10. 1. An engineered SIRPα polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:1 or SEQ ID NO:9, said polypeptide comprising: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is T; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is Y; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is R; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is Q; (e) the amino acid corresponding to H56 in SEQ ID NO: 1 is T; and (f) the amino acid corresponding to E70 in SEQ ID NO: 1 is F; A genetically engineered SIRPα polypeptide comprising one or more mutations among:

11. 1. An engineered SIRPα polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:1 or SEQ ID NO:14, said polypeptide comprising: (a) the amino acid corresponding to H24 in SEQ ID NO: 1 is R; (b) the amino acid corresponding to I31 in SEQ ID NO: 1 is T; (c) the amino acid corresponding to E54 in SEQ ID NO: 1 is A; (d) the amino acid corresponding to G55 in SEQ ID NO: 1 is K; (e) the amino acid corresponding to H56 in SEQ ID NO: 1 is Q; and (f) the amino acid corresponding to D73 in SEQ ID NO: 1 is I; A genetically engineered SIRPα polypeptide comprising one or more mutations among:

12. 2. The engineered SIRPα polypeptide of claim 1, further comprising a CH2 domain and a CH3 domain.

13. A protein construct comprising the engineered SIRPα polypeptide of claim 1.

14. A protein construct comprising: a first fusion polypeptide comprising the engineered SIRPα polypeptide of claim 1, a first CH2 domain, and a first CH3 domain; a second fusion polypeptide comprising a second CH2 domain and a second CH3 domain; Including, the first fusion polypeptide and the second fusion polypeptide associate with each other to form a dimer; Protein constructs.

15. An engineered SIRPα polypeptide according to any one of claims 1 to 12 or a protein construct according to claim 13 or 14; a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

16. A nucleic acid encoding the engineered SIRPα polypeptide of any one of claims 1 to 12 or the protein construct of claim 13 or 14.

17. A vector comprising the nucleic acid of claim 16.

18. A cell comprising the nucleic acid of claim 16.

19. 20. A method for producing an engineered SIRPα polypeptide or a protein construct comprising said engineered SIRPα polypeptide, comprising culturing the cell of claim 18 under conditions sufficient for said cell to produce said engineered SIRPα polypeptide or said protein construct.

20. 15. A pharmaceutical composition comprising the engineered SIRPα polypeptide of any one of claims 1 to 12 or the protein construct of claim 13 or 14 for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of the composition.

21. 15. A pharmaceutical composition comprising an engineered SIRPα polypeptide of any one of claims 1 to 12 or a protein construct of claim 13 or 14 for use in a method of reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of the composition.

22. 15. A pharmaceutical composition comprising an engineered SIRPα polypeptide of any one of claims 1 to 12 or a protein construct of claim 13 or 14 for use in a method of killing tumor cells, the method comprising contacting tumor cells with an effective amount of the composition.

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