Engineered SIRP alpha variants and methods of use thereof

Abstract

The present invention relates to engineered SIRP alpha variants and methods of use thereof. Signal regulatory protein a (SIRPa) is a regulatory membrane glycoprotein of the SIRP family. The protein is mainly expressed by bone marrow cells and also expressed by stem cells or neurons. SIRPa is used as an inhibitory receptor and interacts with a widely expressed transmembrane protein CD47. The present disclosure relates to engineered SIRPa variants and methods of use thereof.

Description

[0001] This application is a divisional application of patent application No. 202280041663.0 (International Application No. PCT / US2022 / 052373) entitled "Engineering modification of SIRPα variant and method of use thereof". International application date: December 9, 2022.

[0002] Cross-reference to related applications

[0003] This disclosure claims priority and benefit to U.S. Provisional Patent Application Serial No. 63 / 292,267, filed December 21, 2021, which is incorporated herein by reference in its entirety.

[0004] sequence list

[0005] This application contains a sequence list, which has been submitted electronically as an XML file named 52246-0004WO1_SL_ST26.xml. This 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. Technical Field

[0006] This disclosure relates to SIRPα variants for engineering modifications and methods of using them. Background Technology

[0007] Signal regulatory protein α (SIRPα) is a regulatory membrane glycoprotein from the SIRP family. It is primarily expressed 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. This interaction negatively controls effector functions of innate immune cells, such as host cell phagocytosis. SIRPα diffuses laterally across the macrophage membrane and accumulates at phagocytic synapses to bind CD47, which inhibits cytoskeleton-intensive processes via macrophage phagocytosis.

[0008] CD47 provides a "don't eat" signal by binding to the N-terminus of signal regulatory protein α (SIRPα). It has been found to be overexpressed in many different tumor cells. Targeting CD47 and / or SIRPα could be used in cancer immunotherapy. However, the interaction between CD47 and SIRPα is essential to protect erythrocytes, platelets, and lymphocytes from rapid clearance by splenic macrophages. There is a need to develop cancer therapies targeting the CD47 / SIRPα pathway with limited toxicity. Summary of the Invention

[0009] This disclosure relates to SIRPα variants for engineering modifications and methods of using them.

[0010] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% the same amino acid sequence as SEQ ID NO:1, wherein in some embodiments, the engineered SIRPα polypeptide comprises one or more amino acid mutations in the BC ring, C'D ring, and / or DE ring. 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.

[0011] In some embodiments, the amino acid corresponding to H56 of SEQ ID NO:1 is I, T, N, V, L, S, G, or Q. In some embodiments, the amino acid corresponding to I31 of SEQ ID NO:1 is W, K, Y, L, A, N, or T. In some embodiments, the amino acid corresponding to I31 of SEQ ID NO:1 is W, K, Y, L, A, or N. In some embodiments, the engineered SIRPα polypeptide described herein includes one or more of the following: (a) the amino acid corresponding to S66 of SEQ ID NO:1 is Q or N; and (b) the amino acid corresponding to T67 of SEQ ID NO:1 is G. In some embodiments, the engineered SIRPα polypeptide described herein includes one or more of the following: (a) the amino acid corresponding to H24 of SEQ ID NO:1 is N or T; and (b) the amino acid corresponding to T26 of SEQ ID NO:1 is I.

[0012] In some embodiments, the engineered SIRPα polypeptide described herein includes one or more of the following: (a) the amino acid corresponding to E70 of SEQ ID NO:1 is G, F, R, A, L or T; (b) the amino acid corresponding to M72 of SEQ ID NO:1 is R or Y; and (c) the amino acid corresponding to D73 of SEQ ID NO:1 is I.

[0013] 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.

[0014] In some embodiments, the engineered SIRPα polypeptide described herein includes 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.

[0015] In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 85%, 90%, 95%, or 100% of the same amino acid sequence as 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.

[0016] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:2, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid W corresponding to I31 of SEQ ID NO:1; (b) amino acid A corresponding to E54 of SEQ ID NO:1; (c) amino acid W corresponding to G55 of SEQ ID NO:1; and (d) amino acid P corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:2.

[0017] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:3, wherein in some embodiments, the polypeptide comprises one or more of the following: (a) amino acid R corresponding to H24 of SEQ ID NO:1; (b) amino acid W corresponding to I31 of SEQ ID NO:1; (c) amino acid A corresponding to E54 of SEQ ID NO:1; (d) amino acid F corresponding to G55 of SEQ ID NO:1; (e) amino acid I corresponding to H56 of SEQ ID NO:1; and (f) amino acid G corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:3.

[0018] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:4, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid N corresponding to H24 of SEQ ID NO:1; (b) amino acid H corresponding to E54 of SEQ ID NO:1; (c) amino acid Q corresponding to G55 of SEQ ID NO:1; (d) amino acid T corresponding to H56 of SEQ ID NO:1; (e) amino acid Q corresponding to S66 of SEQ ID NO:1; and (f) amino acid R corresponding to M72 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:4.

[0019] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:5, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid N corresponding to E54 of SEQ ID NO:1; (b) amino acid L corresponding to G55 of SEQ ID NO:1; (c) amino acid I corresponding to H56 of SEQ ID NO:1; and (d) amino acid G corresponding to T67 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:5.

[0020] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:6, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid I corresponding to T26 of SEQ ID NO:1; (b) amino acid I corresponding to E54 of SEQ ID NO:1; (c) amino acid L corresponding to G55 of SEQ ID NO:1; (d) amino acid T corresponding to H56 of SEQ ID NO:1; and (e) amino acid Y corresponding to M72 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:6.

[0021] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:7, wherein in some embodiments, the polypeptide comprises one or more of the following: (a) amino acid K corresponding to I31 of SEQ ID NO:1; (b) amino acid R corresponding to E54 of SEQ ID NO:1; (c) amino acid D corresponding to G55 of SEQ ID NO:1; (d) amino acid T corresponding to H56 of SEQ ID NO:1; and (e) amino acid R corresponding to M72 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:7.

[0022] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:8, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid T corresponding to H24 of SEQ ID NO:1; (b) amino acid W corresponding to I31 of SEQ ID NO:1; (c) amino acid G corresponding to E54 of SEQ ID NO:1; (d) amino acid Q corresponding to G55 of SEQ ID NO:1; (e) amino acid N corresponding to H56 of SEQ ID NO:1; and (f) amino acid F corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:8.

[0023] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:9, wherein in some embodiments, the polypeptide comprises one or more of the following: (a) amino acid T corresponding to H24 of SEQ ID NO:1; (b) amino acid Y corresponding to I31 of SEQ ID NO:1; (c) amino acid R corresponding to E54 of SEQ ID NO:1; (d) amino acid Q corresponding to G55 of SEQ ID NO:1; (e) amino acid T corresponding to H56 of SEQ ID NO:1; and (f) amino acid F corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:9.

[0024] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:10, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid L corresponding to I31 of SEQ ID NO:1; (b) amino acid S corresponding to E54 of SEQ ID NO:1; (c) amino acid K corresponding to G55 of SEQ ID NO:1; (d) amino acid V corresponding to H56 of SEQ ID NO:1; and (e) amino acid R corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:10.

[0025] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:11. In some embodiments, the polypeptide comprises one or more of the following: (a) amino acid Y corresponding to I31 of SEQ ID NO:1; (b) amino acid G corresponding to E54 of SEQ ID NO:1; (c) amino acid R corresponding to G55 of SEQ ID NO:1; (d) amino acid R corresponding to H56 of SEQ ID NO:1; and (e) amino acid A corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:11.

[0026] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:12, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid L corresponding to I31 of SEQ ID NO:1; (b) amino acid D corresponding to E54 of SEQ ID NO:1; (c) amino acid F corresponding to G55 of SEQ ID NO:1; (d) amino acid L corresponding to H56 of SEQ ID NO:1; and (e) amino acid R corresponding to M72 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:12.

[0027] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:13, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid A corresponding to I31 of SEQ ID NO:1; (b) amino acid L corresponding to E54 of SEQ ID NO:1; (c) amino acid D corresponding to G55 of SEQ ID NO:1; (d) amino acid S corresponding to H56 of SEQ ID NO:1; and (e) amino acid S corresponding to N71 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:13.

[0028] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:14, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid R corresponding to H24 of SEQ ID NO:1; (b) amino acid T corresponding to I31 of SEQ ID NO:1; (c) amino acid A corresponding to E54 of SEQ ID NO:1; (d) amino acid K corresponding to G55 of SEQ ID NO:1; (e) amino acid Q corresponding to H56 of SEQ ID NO:1; and (f) amino acid I corresponding to D73 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:14.

[0029] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:33, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid T corresponding to H24 of SEQ ID NO:1; (b) amino acid R corresponding to E54 of SEQ ID NO:1; (c) amino acid A corresponding to G55 of SEQ ID NO:1; and (d) amino acid P corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:33.

[0030] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:34, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid S corresponding to E54 of SEQ ID NO:1; (b) amino acid P corresponding to G55 of SEQ ID NO:1; (c) amino acid P corresponding to H56 of SEQ ID NO:1; and (d) amino acid L corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:34.

[0031] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:35, wherein in some embodiments, the polypeptide comprises one or more of the following: (a) amino acid T corresponding to H24 of SEQ ID NO:1; (b) amino acid W corresponding to I31 of SEQ ID NO:1; (c) amino acid S corresponding to E54 of SEQ ID NO:1; (d) amino acid P corresponding to H56 of SEQ ID NO:1; and (e) amino acid R corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:34.

[0032] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:36, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid W corresponding to I31 of SEQ ID NO:1; (b) amino acid R corresponding to E54 of SEQ ID NO:1; (c) amino acid A corresponding to G55 of SEQ ID NO:1; (d) amino acid G corresponding to H56 of SEQ ID NO:1; and (e) amino acid T corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:36.

[0033] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:37, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid Q corresponding to S29 of SEQ ID NO:1; (b) amino acid H corresponding to E54 of SEQ ID NO:1; (c) amino acid R corresponding to G55 of SEQ ID NO:1; (d) amino acid T corresponding to H56 of SEQ ID NO:1; and (e) amino acid N corresponding to S66 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:37.

[0034] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:38, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid S corresponding to E54 of SEQ ID NO:1; (b) amino acid P corresponding to G55 of SEQ ID NO:1; (c) amino acid R corresponding to H56 of SEQ ID NO:1; and (d) amino acid L corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:38.

[0035] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:39, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid L corresponding to V27 of SEQ ID NO:1; (b) amino acid D corresponding to G55 of SEQ ID NO:1; (c) amino acid R corresponding to H56 of SEQ ID NO:1; and (d) amino acid R corresponding to M72 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:39.

[0036] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:40, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid L corresponding to V27 of SEQ ID NO:1; (b) amino acid T corresponding to I31 of SEQ ID NO:1; (c) amino acid P corresponding to H56 of SEQ ID NO:1; and (d) amino acid G corresponding to E70 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:40.

[0037] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:41. In some embodiments, the polypeptide comprises one or more of the following: (a) amino acid T corresponding to H24 of SEQ ID NO:1; (b) amino acid Y corresponding to I31 of SEQ ID NO:1; (c) amino acid R corresponding to E54 of SEQ ID NO:1; (d) amino acid Q corresponding to G55 of SEQ ID NO:1; and (e) amino acid T corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:41.

[0038] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:42, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid T corresponding to H24 of SEQ ID NO:1; (b) amino acid N corresponding to I31 of SEQ ID NO:1; (c) amino acid R corresponding to E54 of SEQ ID NO:1; (d) amino acid Q corresponding to G55 of SEQ ID NO:1; and (e) amino acid T corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:42.

[0039] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:43, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid R corresponding to H24 of SEQ ID NO:1; (b) amino acid Y corresponding to I31 of SEQ ID NO:1; (c) amino acid A corresponding to E54 of SEQ ID NO:1; (d) amino acid K corresponding to G55 of SEQ ID NO:1; and (e) amino acid Q corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:43.

[0040] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:44, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid R corresponding to H24 of SEQ ID NO:1; (b) amino acid N corresponding to I31 of SEQ ID NO:1; (c) amino acid A corresponding to E54 of SEQ ID NO:1; (d) amino acid K corresponding to G55 of SEQ ID NO:1; and (e) amino acid Q corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:44.

[0041] In one aspect, this disclosure relates to an engineered SIRPα polypeptide comprising at least 80% of the same amino acid sequence as SEQ ID NO:1 or SEQ ID NO:45, wherein in some embodiments the polypeptide comprises one or more of the following: (a) amino acid R corresponding to H24 of SEQ ID NO:1; (b) amino acid A corresponding to E54 of SEQ ID NO:1; (c) amino acid K corresponding to G55 of SEQ ID NO:1; and (d) amino acid Q corresponding to H56 of SEQ ID NO:1. In some embodiments, the engineered SIRPα polypeptide described herein comprises at least 90% of the same amino acid sequence as SEQ ID NO:45.

[0042] In some embodiments, the engineered SIRPα peptide further includes a CH2 domain and a CH3 domain. In some embodiments, the engineered SIRPα peptide further includes 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 peptide 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.

[0043] In one aspect, this disclosure relates to a protein construct comprising the engineered SIRPα peptide described herein. In some embodiments, the protein construct described herein comprises two or more engineered SIRPα peptides. In some embodiments, at least two engineered SIRPα peptides are identical. In some embodiments, at least two engineered SIRPα peptides are different. In some embodiments, the protein construct 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).

[0044] In one aspect, this disclosure relates to a protein construct comprising a first fusion polypeptide comprising an engineered SIRPα polypeptide as described herein, 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 and second fusion polypeptides associate with each other to form a dimer. In some embodiments, the second fusion polypeptide further comprises a second engineered SIRPα polypeptide.

[0045] In one aspect, this disclosure relates to a pharmaceutical composition comprising an engineered SIRPα polypeptide or a protein construct as described herein; and a pharmaceutically acceptable carrier.

[0046] In one aspect, this disclosure relates to a nucleic acid encoding an engineered SIRPα polypeptide or a protein construct as described herein. In one aspect, this disclosure relates to a vector comprising the nucleic acid as described herein. In one aspect, this disclosure relates to a cell comprising the nucleic acid as described herein. In some embodiments, the cell is a CHO cell.

[0047] In one aspect, this disclosure relates to a method for producing an engineered SIRPα peptide or a protein construct comprising an engineered SIRPα peptide, the method comprising (a) culturing cells as described herein under conditions sufficient to produce the engineered SIRPα peptide or protein construct; and (b) collecting the engineered SIRPα peptide or protein construct produced by the cells.

[0048] In one aspect, this disclosure relates to a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an engineered SIRPα peptide as described herein or a protein construct as described herein. In some embodiments, the subject has a solid tumor or a blood 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.

[0049] In one aspect, this disclosure relates to a method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an engineered SIRPα peptide as described herein or a protein construct as described herein.

[0050] In one aspect, this disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an engineered SIRPα peptide as described herein or a protein construct as described herein.

[0051] As used herein, the term "engineered SIRPα peptide" refers to a peptide derived from a wild-type SIRPα peptide or a portion thereof (e.g., the extracellular region of SIRPα or the IgV domain of SIRPα) having one or more mutations (e.g., insertion, deletion, or substitution). In some embodiments, the engineered SIRPα peptide includes or is composed of the extracellular region of SIRPα. In some embodiments, the engineered SIRPα peptide includes or is composed of the IgV domain of SIRPα. In some embodiments, the engineered SIRPα peptide is a modified IgV domain.

[0052] 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, wherein the polypeptides can associate with each other to form dimers or polymers.

[0053] As used herein, the term “cancer” refers to cells with the capacity for uncontrolled, autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapidly proliferating cell growth. The term implies the presence of cancerous growth, such as tumors; cells, tissues, or organs involved in carcinogenic processes, metastatic tissues, and malignant transformations, regardless of histopathological type or stage of invasion. It also includes malignant tumors of various organ systems, such as those of the respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, which include malignant tumors such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. “Spontaneously occurring” cancer includes any cancer not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by patient exposure to carcinogens, cancer caused by the insertion of transgenic oncogenes or the knockout of tumor suppressor genes, and cancer caused by infections such as viral infections. The term “cancer” is generally accepted in the art and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to cancer originating from glandular tissue or cancer in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted in the art and refers to malignant tumors of mesenchymal origin. The term "hematopoietic neoplasia" includes diseases involving proliferative / neoplastic cells of hematopoietic origin. Hematopoietic neoplasia can be caused by myeloid, lymphoid, or erythroid cells or their precursor cells. Hematologic cancers are cancers that originate in hematopoietic tissues (such as bone marrow) or cells of the immune system. Examples of hematologic cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0054] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals, humans, or non-humans to whom the methods according to the invention are administered treatment. Veterinary and non-veterinary applications are considered in this disclosure. Human patients can be adults or adolescents (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), equines, canines, felines, bovines, and other domesticated, farm, and zoo animals.

[0055] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to a polymer of amino acids of any length, consisting of at least two amino acids.

[0056] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and refer to a polymer of nucleotides of any length of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and their modifications.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0058] Other features and advantages of the invention will become clear from the following detailed description, accompanying drawings, and claims. Attached Figure Description

[0059] Figure 1 The 3D protein structure of the IgV domain of human SIRPα is shown. Nine β chains (ABC-C'-DEFG-G2) and five loop regions (BC loop, CC' loop, C'D loop, DE loop, and FG loop) are labeled.

[0060] Figure 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 number CAA71403.1 (SEQ ID NO:30). Residues in the BC, C'D, and DE rings are underlined.

[0061] Figures 3A-3D The residue scan results for K53, G55, E54, and H56 are shown.

[0062] Figures 4A-4B This shows the expression levels (absorbance at 450 nm, or OD) of 56 selected clones. 450 hCD47 / hSIRPα blocking (absorbance at 450 nm, or OD) 450 ), hCD47 / hSIRPα blocking ability, human CD47 binding (absorbance at 450 nm, or OD) 450 The ratio of human CD47 binding capacity (B) to expression (E) and mouse CD47 binding (absorbance at 450 nm, or OD) 450 (A table of data.)

[0063] Figure 5 A schematic structure of the hSIRPα-Fc protein is shown. Two SIRPαIgV domains are attached to the N-terminus of the human IgG4 hinge region and the Fc region.

[0064] Figure 6 Specific amino acid mutations in the BC, C'D, and DE ring regions of the wild-type SIRPαIgV domain and 13 selected clones (mt3-mt15) are shown.

[0065] Figure 7 The purification results of the hSIRPα-Fc mutant protein by HPLC-SEC are shown.

[0066] Figure 8 It shows the way Results of the binding affinity between the system's hSIRPα-Fc mutant protein and CD47-ECD-His.

[0067] Figures 9A-9C The results of whole-cell binding of hSIRPα-Fc mutant protein to CD47-transfected CHO-S, Jurkat, and Raji cells are shown.

[0068] Figure 9D-9E The results of whole-cell binding of hSIRPα-Fc mutant protein to CD47 tf CHO-S and Raji cells are shown.

[0069] Figure 10A-10D The RBC binding results of the hSIRPα-Fc mutant protein are shown. Human red blood cells were collected from two donors.

[0070] Figure 11A-11D Platelet binding results for the hSIRPα-Fc mutant protein are shown. Human platelets were collected from two donors.

[0071] Figures 12A-12C The results show the whole-cell binding of the hSIRPα-Fc mutant protein to cynoCD47 tf CHO-S cells or LLC-MK2 cells.

[0072] Figure 13A The results of whole-cell binding of the hSIRPα-Fc mutant protein to EMT-6 cells are shown.

[0073] Figure 13B yes Figure 13A A magnified view in the low MFI range.

[0074] Figures 14A-14CThe results of an RBC hemagglutination assay using the hSIRPα-Fc mutant protein are shown. Human RBCs were collected from three donors. Hu5F9-G4 was used as a positive control to induce hemagglutination.

[0075] Figures 15A-15C The blocking ability of hCD47 / hSIRPα using the hSIRPα-Fc mutant protein in CD47 tf CHO-S cells, FaDu cells, and Raji cells was demonstrated.

[0076] Figure 15D-15E The ability of the hCD47 / hSIRPα blocking protein of the hSIRPα-Fc mutant protein in CD47 tf CHO-S cells was demonstrated.

[0077] Figures 16A-16C The phagocytic capacity of Raw264.7 mouse macrophages against hSIRPα-Fc mutant proteins induced by Jurkat, FaDu, and Raji cells was demonstrated.

[0078] Figure 16D-16E The phagocytic capacity of hSIRPα-Fc mutant proteins induced by Raw264.7 mouse macrophages against DLD1 and Raji cells was demonstrated, respectively.

[0079] Figure 17A The phagocytic ability of the hSIRPα-Fc mutant protein induced by Raw264.7 mouse macrophages against human RBC cells was demonstrated.

[0080] Figure 17B The phagocytic capacity of the hSIRPα-Fc mutant protein induced by Raw264.7 mouse macrophages against human platelets was demonstrated.

[0081] Figures 18A-18C The phagocytic capacity induced by human monocyte-derived macrophages (MDM) against the hSIRPα-Fc mutant protein of Raji, DLD1, and Jurkat cells was demonstrated.

[0082] Figure 18D This study demonstrates the phagocytic ability of human monocyte-derived macrophages (MDM) against the hSIRPα-Fc mutant protein of DLD1 cells.

[0083] Figures 19A-19C This study demonstrates the phagocytic ability of human monocyte-derived macrophages (MDMs) against the hSIRPα-Fc mutant protein in RBC cells. Human RBC cells were collected from two donors.

[0084] Figure 20ATumor growth curves are shown in NOD / SCID mice carrying Raji xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium truncatum TTI-622), or Hu5F9-IgG4. Control mice were given a placebo.

[0085] Figure 20B Individual tumor volumes are shown on day 18 post-inoculation in NOD / SCID mice carrying Raji xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium truncatum TTI-622), Hu5F9-IgG4, or placebo.

[0086] Figure 20C Survival curves of NOD / SCID mice carrying Raji xenografts are shown. They were treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium truncatum TTI-622), Hu5F9-IgG4, or placebo.

[0087] Figure 21A Tumor growth curves are shown in NOD / SCID mice carrying NCI_H82 xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium truncatum TTI-622), or Hu5F9-IgG4. Control mice were given a placebo.

[0088] Figure 21B Individual tumor volumes are shown on day 25 post-inoculation in NOD / SCID mice carrying NCI_H82 xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium truncatum TTI-622), Hu5F9-IgG4, or placebo.

[0089] Figure 21C Survival curves of NOD / SCID mice carrying NCI_H82 xenografts treated with hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-wt (Trillium tsao-TTI-622), Hu5F9-IgG4, or placebo are shown.

[0090] Figure 22 The amino acid sequences of the wild-type IgV domain or IgV domain mutants of human SIRPα are listed.

[0091] Figure 23 The protein sequences discussed in this disclosure are listed. Detailed Implementation

[0092] Signal regulatory protein α (SIRPα, SIRPa, or CD172A) is a transmembrane protein. It possesses an extracellular region comprising three Ig-like domains and a cytoplasmic region containing tyrosine-based inhibitory motifs that mediate the binding of protein tyrosine phosphatases SHP1 and SHP2. Tyrosine phosphorylation of SIRPα is regulated by various growth factors and cytokines, as well as integrin-mediated cell-extracellular matrix protein adhesion. SIRPα is particularly abundant in myeloid cells such as macrophages and dendritic cells, while it is expressed at low levels in T, B, NK, and NKT cells.

[0093] The extracellular domain of SIRPα can interact with its ligand CD47. The interaction of SIRPα on macrophages with CD47 on erythrocytes prevents phagocytosis of Ig-opsonized erythrocytes by macrophages both in vitro and in vivo. SIRPα is linked to phagocytes by CD47 expressed on neighboring cells, leading to phosphorylation of the immune receptor tyrosine-based inhibitory (ITIM) motif in the SIRPα cytoplasm, resulting in the recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention of myosin-IIA accumulation at phagocyte synapses, thereby inhibiting phagocytosis. Thus, the CD47-SIRPα interaction acts as a negative immune checkpoint, sending a "don't eat me" signal to ensure that healthy autologous cells are not improperly phagocytosed. However, overexpression of CD47 is also found in almost all types of tumors, including acute myeloid leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer. This negative regulation of macrophages can be minimized by blocking the binding of CD47 to SIRPα.

[0094] Blocking the CD47 / SIRPα interaction can promote phagocytosis, thus potentially being used to treat various cancers. It triggers the recognition and elimination of cancer cells through the innate immune system. Agents targeting CD47 or SIRPα can be used to treat various tumors and cancers, such as solid tumors, hematologic malignancies (e.g., relapsed or refractory hematologic malignancies), acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer.

[0095] Detailed descriptions of SIRPα and its functions can be found, for example, Yanagita et al., “Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy,” JCI Insight 2.1 (2017); and 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; these are incorporated herein by reference in their entirety.

[0096] In addition, SIRPα is used to inhibit the in vivo clearance of CD47-expressing host cells (including erythrocytes and platelets) by macrophages. CD47-SIRPα interaction also appears to be essential for hematopoietic stem cell transplantation. Blocking the CD47 / SIRPα interaction can lead to the unintended killing of normal erythrocytes, potentially causing anemia and triggering inflammation. Therefore, modulating the interaction between SIRPα targets and CD47 is important, for example, to have limited or controlled effects on erythrocytes.

[0097] This disclosure provides engineered SIRPα variants. These engineered SIRPα variants can be used to target the CD47 / SIRPα pathway, and the interaction between the engineered SIRPα variants and CD47 is carefully modulated.

[0098] SIRPα variants for engineering modification

[0099] Human SIRPα is a member of the signal regulatory protein (SIRP) family. Signal regulatory proteins are cell surface Ig superfamily proteins that mediate essential cell surface protein interactions and signal transduction. SIRPs all contain an N-terminal extracellular domain, a single transmembrane domain, and a C-terminal intracellular domain.

[0100] The extracellular region of human SIRPα (UniProt identifier: P78324) contains an IgV domain, an Ig-like C11 domain, and an Ig-like C12 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 signal peptides. Human SIRPα also possesses long intracellular domains comprising two putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Activation of the SIRPα ITIMs delivers an inhibitory signal that negatively regulates the cellular response.

[0101] SIRPα binding to CD47 is mediated by the extracellular IgV domain of SIRPα. The IgV domain of hSIRPa belongs to the immunoglobulin superfamily and contains nine β chains, comprising ABC-C'-DEFG-G2. The helix is ​​located between the E and F chains. Figure 1 ).

[0102] Based on the structure of the human CD47 (hCD47) and hSIRPα complex, the residues interacting with CD47 were identified. Analysis showed that multiple interacting residues are located within the BC ring (corresponding to amino acids 24-36 of SEQ ID NO:1), C'D ring (corresponding to amino acids 53-56 of SEQ ID NO:1), and DE ring (corresponding to amino acids 61-78 of SEQ ID NO:1) of the hSIRPα IgV domain, and are highly conserved. Figure 2 In this text, the amino acid residues within these ring regions are underlined. These regions are targets for mutation. Therefore, in some embodiments, the engineered SIRPα polypeptide includes or consists of one or more amino acid mutations at the BC ring, C'D ring, and / or DE ring.

[0103] Further analysis revealed that Leu30, Gly34, Gln52, Lys53, Glu54, His56, Ser66, Thr67, Arg69, Lys93, Lys96, Gly97, and Asp100 in hSIRPα are involved in the interaction with CD47. Therefore, in some embodiments, the engineered SIRPα peptide may include one or more amino acid mutations at Leu30, Gly34, Gln52, Lys53, Glu54, His56, Ser66, Thr67, Arg69, Lys93, Lys96, Gly97, and / or Asp100.

[0104] Furthermore, Lys53 and Ser66 have been identified as potentially important for increasing the binding affinity of hSIRPa to CD47. Lys53 is located on the C'D ring, and Ser66 is located on the DE ring. A mutation at position 53 (Lys) is likely important for increasing the binding affinity of hSIRPa to CD47. Therefore, in some embodiments, the amino acid corresponding to K53 in SEQ ID NO:1 is R. Additionally, the elimination of steric hindrance at position 54 due to the structural change also suggests that altering the structure of the C'D ring could increase the chance of increased binding affinity of hSIRPa to CD47. The mutation at the C'D ring provides a more flexible approach to screening for unique mutations that may provide increased binding affinity to CD47.

[0105] Based on the results from residue scanning, Lys53 can exhibit increased stability when substituted with amino acids having long side chains or aromatic ring side chains, such as arginine, leucine, phenylalanine, tryptophan, and tyrosine. Therefore, 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.

[0106] Meanwhile, guiding mutations at Glu54, Gly55, and His56 may be more flexible and potentially more advantageous. In some embodiments, the engineered SIRPα peptide 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.

[0107] Thr67 is a residue directly involved in the interaction; reducing the distance between Thr67 and CD47 may increase the binding affinity of hSIRPa to CD47. Substitution of residues Ser66 and Thr67 can bring the DE ring closer to CD47. Therefore, in some embodiments, the 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.

[0108] Other interfaces of the CD47 / hSIRPa complex were also observed in this disclosure. Residues Ile31, Val33, and Arg69 can form a positively charged pocket. Due to the complementary charge and shape of the two regions, the FG ring of CD47 is completely embedded in the positively charged pocket. Therefore, 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.

[0109] Furthermore, the analysis in this disclosure shows that Val27, Val63, and Lys68 are likely important for maintaining the structure of hSIRPa. Therefore, in some embodiments, these amino acid residues are retained. In summary, Ile31, Glu54, Gly55, His56, Ser66, and Thr67 were identified as candidate amino acids for hSIRPa mutation screening.

[0110] Therefore, in one aspect, the engineered SIRPα polypeptide comprises or is composed of at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as or constitutes thereof with SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

[0111] In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:9. In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:14. In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:41. In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:42. In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:43. In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:44. In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as SEQ ID NO:45.

[0112] In some embodiments, compared to any one of SEQ ID NOs:1-14 and 33-45, the engineered SIRPα variant may have at least 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.

[0113] In some embodiments, the engineered SIRPα peptide has one or more of the following mutations:

[0114] (a) The amino acid corresponding to E54 of SEQ ID NO:1 is A, H, N, I, R, G, S, D or L;

[0115] (b) The amino acid corresponding to G55 of SEQ ID NO:1 is W, F, Q, L, D, K, R, A or P;

[0116] (c) The amino acid corresponding to H56 of SEQ ID NO:1 is P, I, T, N, V, R, L, S, G or Q.

[0117] In some embodiments, the amino acid corresponding to H56 of SEQ ID NO:1 is I, T, N, V, L, S, G, or Q. In some embodiments, the amino acid corresponding to I31 of SEQ ID NO:1 is W, K, Y, L, A, N, I, or T. In some embodiments, the amino acid corresponding to I31 of SEQ ID NO:1 is W, K, Y, L, A, N, or I.

[0118] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0119] (a) The amino acid corresponding to S66 of SEQ ID NO:1 is Q or N;

[0120] (b) The amino acid corresponding to T67 in SEQ ID NO:1 is G.

[0121] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0122] (a) The amino acid corresponding to H24 of SEQ ID NO:1 is R, N or T;

[0123] (b) The amino acid corresponding to T26 of SEQ ID NO:1 is I.

[0124] In some embodiments, the amino acid corresponding to H24 of SEQ ID NO:1 is N or T.

[0125] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0126] (a) The amino acid corresponding to E70 of SEQ ID NO:1 is G, F, R, A, L or T;

[0127] (b) The amino acid corresponding to M72 in SEQ ID NO:1 is R or Y; and

[0128] (c) The amino acid corresponding to D73 of SEQ ID NO:1 is I.

[0129] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0130] (a) The amino acid corresponding to position 27 of SEQ ID NO:1 is V or L;

[0131] (b) The amino acid corresponding to position 63 of SEQ ID NO:1 is V; and

[0132] (c) The amino acid corresponding to position 68 of SEQ ID NO:1 is K.

[0133] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0134] (a) The amino acid corresponding to H24 of SEQ ID NO:1 is R, N or T;

[0135] (b) The amino acid corresponding to T26 of SEQ ID NO:1 is I;

[0136] (c) The amino acid corresponding to V27 of SEQ ID NO:1 is L;

[0137] (d) The amino acid corresponding to S29 of SEQ ID NO:1 is Q;

[0138] (e) The amino acid corresponding to I31 of SEQ ID NO:1 is W, K, Y, L, A or N;

[0139] (f) The amino acid corresponding to E54 of SEQ ID NO:1 is A, H, N, I, R, G, S, D or L;

[0140] (g) The amino acid corresponding to G55 of SEQ ID NO:1 is W, F, Q, L, D, K, R, A or P;

[0141] (h) The amino acid corresponding to H56 of SEQ ID NO:1 is P, I, T, N, V, R, L, S, G or Q.

[0142] (i) The amino acid corresponding to S66 of SEQ ID NO:1 is Q or N;

[0143] (j) The amino acid corresponding to T67 in SEQ ID NO:1 is G;

[0144] (k) The amino acid corresponding to E70 of SEQ ID NO:1 is G, F, R, A, L or T;

[0145] (l) The amino acid corresponding to N71 in SEQ ID NO:1 is S;

[0146] (m) The amino acid corresponding to M72 in SEQ ID NO:1 is R or Y; and

[0147] (n) The amino acid corresponding to D73 of SEQ ID NO:1 is I.

[0148] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0149] (a) The amino acid corresponding to V27 of SEQ ID NO:1 is I or L;

[0150] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is F, S or T;

[0151] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is Q;

[0152] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is P or R;

[0153] (e) The amino acid corresponding to S66 in SEQ ID NO:1 is T or G;

[0154] (f) The amino acid corresponding to K68 in SEQ ID NO:1 is K or R; and

[0155] (g) The amino acid corresponding to E70 of SEQ ID NO:1 is N.

[0156] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0157] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is W;

[0158] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is A;

[0159] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is W;

[0160] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is P.

[0161] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:2).

[0162] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0163] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is R;

[0164] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is W;

[0165] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is A;

[0166] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is F;

[0167] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is I;

[0168] (f) The amino acid corresponding to E70 of SEQ ID NO:1 is G.

[0169] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:3).

[0170] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0171] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is N;

[0172] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is H;

[0173] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is Q;

[0174] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is T;

[0175] (e) The amino acid corresponding to S66 in SEQ ID NO:1 is Q;

[0176] (f) The amino acid corresponding to M72 of SEQ ID NO:1 is R.

[0177] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:4).

[0178] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0179] (a) The amino acid corresponding to E54 of SEQ ID NO:1 is N;

[0180] (b) The amino acid corresponding to G55 of SEQ ID NO:1 is L;

[0181] (c) The amino acid corresponding to H56 of SEQ ID NO:1 is I;

[0182] (d) The amino acid corresponding to T67 in SEQ ID NO:1 is G.

[0183] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:5).

[0184] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0185] (a) The amino acid corresponding to T26 of SEQ ID NO:1 is I;

[0186] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is I;

[0187] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is L;

[0188] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is T;

[0189] (e) The amino acid corresponding to M72 in SEQ ID NO:1 is Y.

[0190] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:6).

[0191] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0192] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is K;

[0193] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is R;

[0194] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is D;

[0195] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is T;

[0196] (e) The amino acid corresponding to M72 in SEQ ID NO:1 is R.

[0197] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:7).

[0198] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0199] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is T;

[0200] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is W;

[0201] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is G;

[0202] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is Q;

[0203] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is N;

[0204] (f) The amino acid corresponding to E70 of SEQ ID NO:1 is F.

[0205] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:8).

[0206] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0207] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is T;

[0208] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is Y;

[0209] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is R;

[0210] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is Q;

[0211] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is T;

[0212] (f) The amino acid corresponding to E70 of SEQ ID NO:1 is F.

[0213] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:9).

[0214] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0215] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is L;

[0216] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is S;

[0217] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is K;

[0218] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is V;

[0219] (e) The amino acid corresponding to E70 of SEQ ID NO:1 is R.

[0220] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:10).

[0221] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0222] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is Y;

[0223] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is G;

[0224] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is R;

[0225] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is R;

[0226] (e) The amino acid corresponding to E70 of SEQ ID NO:1 is A.

[0227] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:11).

[0228] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0229] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is L;

[0230] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is D;

[0231] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is F;

[0232] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is L;

[0233] (e) The amino acid corresponding to M72 in SEQ ID NO:1 is R.

[0234] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:12).

[0235] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0236] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is A;

[0237] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is L;

[0238] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is D;

[0239] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is S;

[0240] (e) The amino acid corresponding to N71 of SEQ ID NO:1 is S.

[0241] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:13).

[0242] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0243] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is R;

[0244] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is T;

[0245] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is A;

[0246] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is K;

[0247] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is Q;

[0248] (f) The amino acid corresponding to D73 of SEQ ID NO:1 is I.

[0249] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:14).

[0250] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0251] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is T;

[0252] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is R;

[0253] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is A;

[0254] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is P.

[0255] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:33).

[0256] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0257] (a) The amino acid corresponding to E54 of SEQ ID NO:1 is S;

[0258] (b) The amino acid corresponding to G55 of SEQ ID NO:1 is P;

[0259] (c) The amino acid corresponding to H56 of SEQ ID NO:1 is P;

[0260] (d) The amino acid corresponding to E70 of SEQ ID NO:1 is L.

[0261] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:34).

[0262] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0263] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is T;

[0264] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is W;

[0265] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is S;

[0266] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is P;

[0267] (e) The amino acid corresponding to E70 of SEQ ID NO:1 is R.

[0268] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:35).

[0269] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0270] (a) The amino acid corresponding to I31 in SEQ ID NO:1 is W;

[0271] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is R;

[0272] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is A;

[0273] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is G;

[0274] (e) The amino acid corresponding to E70 of SEQ ID NO:1 is T.

[0275] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:36).

[0276] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0277] (a) The amino acid corresponding to S29 of SEQ ID NO:1 is Q;

[0278] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is H;

[0279] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is R;

[0280] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is T;

[0281] (e) The amino acid corresponding to S66 of SEQ ID NO:1 is N.

[0282] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:37).

[0283] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0284] (a) The amino acid corresponding to E54 of SEQ ID NO:1 is S;

[0285] (b) The amino acid corresponding to G55 of SEQ ID NO:1 is P;

[0286] (c) The amino acid corresponding to H56 of SEQ ID NO:1 is R;

[0287] (d) The amino acid corresponding to E70 of SEQ ID NO:1 is L.

[0288] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:38).

[0289] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0290] (a) The amino acid corresponding to V27 of SEQ ID NO:1 is L;

[0291] (b) The amino acid corresponding to G55 of SEQ ID NO:1 is D;

[0292] (c) The amino acid corresponding to H56 of SEQ ID NO:1 is R;

[0293] (d) The amino acid corresponding to M72 of SEQ ID NO:1 is R.

[0294] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:39).

[0295] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0296] (a) The amino acid corresponding to V27 of SEQ ID NO:1 is L;

[0297] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is T;

[0298] (c) The amino acid corresponding to H56 of SEQ ID NO:1 is P;

[0299] (d) The amino acid corresponding to E70 of SEQ ID NO:1 is G.

[0300] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:40).

[0301] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0302] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is T;

[0303] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is Y;

[0304] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is R;

[0305] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is Q;

[0306] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is T.

[0307] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:41).

[0308] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0309] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is T;

[0310] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is N;

[0311] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is R;

[0312] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is Q;

[0313] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is T.

[0314] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:42).

[0315] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0316] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is R;

[0317] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is Y;

[0318] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is A;

[0319] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is K;

[0320] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is Q.

[0321] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:43).

[0322] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0323] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is R;

[0324] (b) The amino acid corresponding to I31 in SEQ ID NO:1 is N;

[0325] (c) The amino acid corresponding to E54 of SEQ ID NO:1 is A;

[0326] (d) The amino acid corresponding to G55 of SEQ ID NO:1 is K;

[0327] (e) The amino acid corresponding to H56 of SEQ ID NO:1 is Q.

[0328] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:44).

[0329] In some embodiments, the engineered SIRPα peptide comprises or consists of one or more of the following mutations:

[0330] (a) The amino acid corresponding to H24 in SEQ ID NO:1 is R;

[0331] (b) The amino acid corresponding to E54 of SEQ ID NO:1 is A;

[0332] (c) The amino acid corresponding to G55 of SEQ ID NO:1 is K;

[0333] (d) The amino acid corresponding to H56 of SEQ ID NO:1 is Q.

[0334] In some embodiments, the engineered SIRPα polypeptide comprises or consists of at least 80%, 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NOs:1-14 and 33-45 (e.g., SEQ ID NO:1 or SEQ ID NO:45).

[0335] In some embodiments, the engineered SIRPα peptide includes peptides having, for example, the following characteristics: Figure 6 Any one of the SEQ ID NOs:1-14 and 33-45 of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations shown (e.g. SEQ ID NO:1 or SEQ ID NO:44) has at least 80%, 85%, 90% or 95% identical amino acid sequences or is composed of them.

[0336] The engineered SIRPα peptide may have additional modifications. In some embodiments, the engineered SIRPα peptide may have an Fc CH2 domain and / or a CH3 domain. In some embodiments, the engineered SIRPα peptide 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 peptide 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, and CH3 domain have a sequence that is at least 80%, 85%, 90%, 95%, and 100% identical to SEQ ID NO:29.

[0337] In some embodiments, the engineered SIRPα polypeptide comprises or is composed of 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% of the same amino acid sequence as or constitutes thereof.

[0338] SIRPα protein construct

[0339] This 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, thereby enhancing the immune response. In some embodiments, these protein constructs can initiate phagocytosis.

[0340] In some embodiments, the engineered SIRPα protein construct may include any engineered SIRPα variant as described herein. In some embodiments, the engineered SIRPα protein construct may have 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 sequences to any sequence in SEQ IDNOs:1-14 and 33-45. In some embodiments, the engineered SIRPα protein construct may include or consist of 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 sequences to or composed of any of the sequences in SEQ ID NOs:15-28 and 46-58.

[0341] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising 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 sequences to any sequence of SEQ ID NOs:1-14 and 33-45 or SEQ ID NOs:15-28 and 46-58.

[0342] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are considered identical at that position when 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 percentage of identity between two sequences is a function of the number of shared identical positions, taking into account the number of vacancies and the length of each vacancy, which needs to be introduced for optimal alignment of the two sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0343] The engineered SIRPα protein construct may further include an Fc region of an antibody. 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 the IgG4 Fc region (e.g., the human IgG4 Fc region).

[0344] In some embodiments, the engineered SIRPα variant is linked to the Fc region via an antibody hinge region (e.g., an IgG or IgE hinge region). Furthermore, the Fc region can be modified to provide desired effector function or serum half-life.

[0345] The engineered SIRPα variants and protein constructs described herein can block the binding between CD47 and endogenous SIRPα expressed on immune cells. In some embodiments, by binding to CD47, the engineered SIRPα variants and protein constructs can inhibit the binding of CD47 (e.g., CD47 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.

[0346] 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., myeloid 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.

[0347] In some implementations, engineered SIRPα variants and protein constructs can be delivered in less than 0.1 s. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Or less than 0.00001s -1 dissociation rate (k off ) binds to SIRPα (e.g., human SIRPα, monkey SIRPα (e.g., cynomolgus monkey, mouse SIRPα). In some embodiments, the dissociation rate (k off (greater than 0.01s) -1 Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 or greater than 0.000001s -1 .

[0348] In some embodiments, the dynamic association rate (k on Greater than 1 x 10 2 / Ms, greater than 1x 10 3 / Ms, greater than 1x10 4 / Ms, greater than 1x 10 5 / Ms or greater than 1x 10 6 / Ms. In some embodiments, the kinetic association rate (k on Less than 1x10 5 / Ms, less than 1x 10 6 / Ms or less than 1x 10 7 / Ms.

[0349] Affinity can be derived from the quotient of the kinetic rate constant (KD = k off / k on This is derived. In some embodiments, KD is less than 1 x 10. -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M or less than 1 x 10 -10M. In some embodiments, 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, KD is greater than 1 x 10^6 pM. -7 M, greater than 1 x 10 - 8 M, greater than 1 x 10 -9 M, greater than 1 x 10 -10 M, greater than 1 x 10 -11 M or greater than 1 x 10 -12 M.

[0350] Common techniques for measuring affinity include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, engineered SIRPα variants and protein constructs may bind to monkey SIRPα and / or mouse SIRPα. In some embodiments, engineered SIRPα variants and protein constructs may not bind to monkey SIRPα and / or mouse SIRPα.

[0351] In some embodiments, thermal stability is determined. The engineered SIRPα variants and protein constructs described herein may have a Tm greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0352] In some embodiments, the engineered SIRPα variants and / or protein constructs as described herein have a tumor growth inhibition percentage (TGI%) 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 as described herein have a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be determined, for example, on days 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 after the start of treatment, or on months 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 after the start of treatment. As used herein, the percentage of tumor growth inhibition (TGI%) is calculated using the following formula:

[0353] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0354] 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 zero. 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 zero.

[0355] In some embodiments, the tumor-suppressive effects of engineered SIRPα variants and / or protein constructs as described herein are comparable to those of anti-CD47 reference antibodies (e.g., Hu5F9-G4) or anti-SIRPa antibodies (e.g., CC-95251). Hu5F9-G4 is described, for example, 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 herein by reference in its entirety. CC-95251. In some embodiments, the tumor-suppressive effects of the engineered SIRPα variants and / or protein constructs as described herein are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold higher than those of anti-CD47 reference antibodies (e.g., Hu5F9-G4) or anti-SIRPa antibodies (e.g., CC-95251). In some embodiments, the tumor-suppressive effects of the engineered SIRPα variants and / or protein constructs as described herein are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold higher than those of *Trillium truncatum*. Details of hSIRPα-Fc-wt (*Trillium truncatum*), also known as TTI-622, are also described. The amino acid sequence of hSIRPα-Fc-wt (*Trillium truncatum*) is shown in SEQ ID NO:15.

[0356] In some embodiments, the protein construct as described herein has 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 functions of the functional Fc region are both ADCC and phagocytosis. In some embodiments, the protein construct as described herein has an Fc region without effector function. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (L234A and L235A mutations in EU numbers) or an LALA-PG mutation (L234A, L235A, P329G mutations in EU numbers).

[0357] Other modifications can be made to the Fc region. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in that region. The resulting homodimeric fusion protein can have any increased half-life in vitro and / or in vivo.

[0358] In some embodiments, IgG4 has an S228P mutation (EU number). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0359] In some embodiments, an Fc region is provided having a carbohydrate structure lacking fucose linkage (directly or indirectly) to the Fc region. For example, the amount of fucose in such Fc region compositions can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located approximately at position 297 of the Fc region (Eu number of the Fc region residue; or position 314 in the Kabat number); however, due to minor sequence variations in the Fc region sequence, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between position 294 and position 300. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0360] In some embodiments, the binding affinity between CD47 (e.g., human CD47, monkey CD47, mouse CD47, or their extracellular domains) and engineered SIRPα variants and / or protein constructs as described herein is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times that between CD47 and wild-type SIRPα or its protein constructs.

[0361] In some embodiments, the engineered SIRPα variants and / or protein constructs as 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 Exceeding the expression OD 450 In some cases, the B / E ratio is greater than 0.4.

[0362] In some embodiments, after purification by a protein A column, the main peak of HPLC-SEC accounts for at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the engineered SIRPα variant and / or protein construct as described herein.

[0363] In some embodiments, engineered SIRPα variants and / or their protein constructs, as described herein, can bind to tumor cells expressing human CD47 (e.g., human CD47 tf CHO-S cells, Jurkat cells, or Raji cells) with an affinity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times that of wild-type SIRPα or its protein constructs. In some embodiments, the EC50 value of the engineered SIRPα variants and / or their protein constructs binding to tumor cells expressing human CD47 (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 described herein can bind to cells expressing monkey CD47 (e.g., cynoCD47 tf CHO-S cells or LLC-MK2 cells) with an affinity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times that of wild-type SIRPα or its protein constructs, compared to wild-type SIRPα or its protein constructs. In some embodiments, the engineered SIRPα variants and / or protein constructs described herein can bind to cells expressing mouse CD47 (e.g., EMT-6 cells) with an affinity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times that of wild-type SIRPα or its protein constructs.

[0364] In some embodiments, the engineered SIRPα variants and / or their protein constructs as described herein can bind to CD47-expressing tumor cells (e.g., CD47 tf CHO-S cells, Jurkat cells, or Raji cells) with an affinity of 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).

[0365] In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein can bind to RBC cells or platelets (e.g., from human donors) with an affinity of 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% compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4). In some embodiments, the EC50 values ​​of engineered SIRPα variants and / or their protein constructs bound to RBC cells are 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 engineered SIRPα variant and / or its protein construct binds to platelets with an EC50 value of 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.

[0366] In some embodiments, the engineered SIRPα variants and / or their protein constructs as described herein do not induce hemagglutination. In some embodiments, the engineered SIRPα variants and / or their protein constructs as described herein induce hemagglutination at minimum concentrations greater than 500-fold, 2000-fold, 5000-fold, 20000-fold, or 50000-fold compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0367] In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein can block the interaction between human CD47 and human SIRPα. In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein can block the interaction between cells expressing human CD47 (e.g., CD47 tf CHO-S cells, FaDu cells, or Raji cells) and human SIRPα. In some embodiments, the blocking ability of engineered SIRPα variants and / or their protein constructs as 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 ability of the engineered SIRPα variants and / or their protein constructs as described herein is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times that of wild-type SIRPα or its protein constructs. In some embodiments, the IC50 value of the engineered SIRPα variants and / or their protein constructs 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.

[0368] In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein can induce phagocytosis of CD47-expressing tumor cells (e.g., Jurkat cells, FaDu cells, or Raji cells) by mouse macrophages (e.g., Raw264.7 cells). In some embodiments, the EC50 value of phagocytosis induced by engineered SIRPα variants and / or their protein constructs in 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 of phagocytosis induced by engineered SIRPα variants and / or their protein constructs in CD47-expressing tumor cells is comparable to the EC50 value of an anti-CD47 reference antibody (e.g., Hu5F9-G4) (e.g., at least 80%, 85%, 90%, or 95%). In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein have a weaker ability to induce phagocytosis of RBC cells via mouse macrophages (e.g., Raw264.7 cells) than anti-CD47 reference antibodies (e.g., Hu5F9-G4). In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein have a weaker ability to induce phagocytosis of platelets via mouse macrophages (e.g., Raw264.7 cells) than anti-CD47 reference antibodies (e.g., Hu5F9-G4).

[0369] In some embodiments, engineered SIRPα variants and / or their protein constructs as 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 ability of engineered SIRPα variants and / or their protein constructs to induce phagocytosis is comparable to that of an anti-CD47 reference antibody (e.g., Hu5F9-G4) (at least 80%, 85%, 90%, or 95%). In some embodiments, the ability of engineered SIRPα variants and / or their protein constructs to induce phagocytosis is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times that of wild-type SIRPα or its protein constructs. In some embodiments, engineered SIRPα variants and / or their protein constructs as described herein have a weaker ability to induce phagocytosis of RBC cells via human macrophages (e.g., MDM cells) than anti-CD47 reference antibodies (e.g., Hu5F9-G4).

[0370] In some embodiments, engineered SIRPα variants and / or their protein constructs, as described herein, can inhibit tumor growth. In some embodiments, Raji cells or NCI-H82 cells are injected into immunodeficient mice (e.g., NOD / SCID mice) to generate xenograft models.

[0371] Methods for preparing engineered SIRPα variants and protein constructs

[0372] The 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 in the amino acid sequence.

[0373] Screening is possible. Within this population of variants, some engineered SIRPα variants will have increased affinity for CD47. Any combination of deletions, insertions, and / or combinations can be made to obtain variants with increased binding affinity to the target. Amino acid changes introduced into variants can also alter or introduce new post-translational modifications into the peptide, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are attached by enzymes present in the cell), or introducing new glycosylation sites.

[0374] Engineered SIRPα variants can be derived from any animal species, including mammals. Non-restrictive examples of SIRPα variants include those derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and vicuñas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0375] This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., vectors that cause the host cells to contain polynucleotides and / or include polynucleotides), and the production of recombinant polypeptides or fragments thereof by recombinant technology.

[0376] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when introduced into the host cell. An “expression vector” is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell that has been introduced into the expression vector. Thus, in an expression vector, the polynucleotide of interest is operatively positioned for expression in the vector by means of regulatory elements such as promoters, enhancers, and / or poly-A tails located within the vector or in the genome of the host cell, at or near the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell introduced into the expression vector.

[0377] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection and infection, and / or transduction (e.g., with recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0378] In some embodiments, a viral expression system (e.g., vaccinia virus or other poxviruses, retroviruses, or adenoviruses) is used to introduce the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein). This may involve the use of a non-pathogenic (deficient), replicating virus, or a replication-deficient virus. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can also be “naked.” The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are then efficiently transported into cells.

[0379] For expression, DNA inserts containing polynucleotides encoding polypeptides disclosed herein can be operatively linked to suitable promoters (e.g., heterologous promoters), such as the bacteriophage λPL promoter, E. coli lac, trp, and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, to name just 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 contain sites for transcription initiation and termination, and ribosome-binding sites for translation within the transcribed region. The coding portion of the mature transcript expressed by the construct may contain translation initiated at the start and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0380] As described above, the expression vector may contain at least one selectable biomarker. Such biomarkers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes for cultures in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli* cells, *Streptomyces* cells, and *Salmonella typhimurium* cells; fungal cells such as yeast cells; insect cells such as *Drosophila S2* and *Noctus Sf9* cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

[0381] Non-restrictive vectors for bacteria include pQE70, pQE60, and pQE-9, available from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5, available from Pharmacia. Non-restrictive 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 skilled in the art.

[0382] Suitable non-restrictive bacterial promoters include the *E. coli* lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs (such as the promoter of Rous Sarcoma Virus (RSV)), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0383] In Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH can be used.

[0384] Constructs can be introduced into host cells via calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods 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.

[0385] Transcription of DNA encoding the disclosed polypeptides in higher eukaryotes can be increased by inserting enhancer sequences into a vector. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, that increase the transcriptional activity of the promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located posterior to the origin of replication at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer posterior to the origin of replication, and the adenovirus enhancer.

[0386] To enable translated proteins to be secreted into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. This signal can be endogenous to the polypeptide or it can be a heterologous signal.

[0387] Peptides (e.g., SIRPα variants) can be expressed in modified forms, such as fusion proteins (e.g., GST fusion proteins) or with histidine tags, and can include not only secretion signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of a peptide to improve its stability and persistence in host cells during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to peptides to facilitate purification. Such regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to induce secretion or excretion, improve stability, and facilitate purification (among other things) is a well-known and conventional technique in the art.

[0388] Treatment

[0389] The engineered SIRPα variants and protein constructs disclosed herein can be used for a variety of therapeutic purposes.

[0390] In one aspect, this disclosure provides methods for treating cancer in a subject, methods for reducing the rate at which the volume of a tumor increases over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may reduce the number, severity, and / or duration of one or more cancer symptoms in a subject.

[0391] In one aspect, this disclosure is characterized by a method comprising administering a therapeutically effective amount of the engineered SIRPα variant and protein construct disclosed herein to a subject in need of such treatment (e.g., a subject who has or has been identified or diagnosed with cancer), such as breast cancer (e.g., triple-negative breast cancer), carcinoid tumor, 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, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant 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, cancer is melanoma, pancreatic cancer, mesothelioma, hematologic malignancies, especially non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0392] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0393] As used herein, “effective amount” means an amount or dose sufficient to produce a beneficial or desired result, including stopping, slowing, delaying, or inhibiting the progression of a disease, such as cancer.

[0394] The effective dose will vary depending on factors such as the age and weight of the subject, the severity of symptoms, and the route of administration, including, for example, the administration of the engineered SIRPα variant and protein construct, the vector containing the polynucleotide encoding the engineered SIRPα variant and protein construct, and / or combinations thereof, and therefore can be determined on an individual basis.

[0395] An effective dose can be administered in one or more applications. For example, an effective dose 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 in vitro proliferation of cells (e.g., biopsy cells, any cancer cells or cell lines described herein, e.g., cancer cell lines)). As understood in the art, the effective dose can vary, particularly depending on patient history and other factors such as the type (and / or dose) of the engineered SIRPα variant and protein construct used.

[0396] The effective amount and schedule for administering the engineered SIRPα variants and protein constructs disclosed herein, the polynucleotides and / or compositions encoding the engineered SIRPα variants and protein constructs, can be determined empirically, and such determination is within the scope of the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal to which the engineered SIRPα variants and protein constructs will be received, the polynucleotides and / or compositions disclosed herein, the route of administration, the specific type of polynucleotides and / or compositions disclosed herein, and other drugs administered to the mammal.

[0397] The typical daily dose of an effective amount of engineered SIRPα variants and / or protein constructs is 0.1 mg / kg to 100 mg / kg (mg / kg patient body weight). In some embodiments, the dose 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 dose 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 dose 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 to 10 mg / kg, about 1 to 5 mg / kg, or about 2 to 5 mg / kg.

[0398] In any of the methods described herein, the engineered SIRPα variant and protein construct may be administered to the 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).

[0399] In some embodiments, one or more additional therapeutic agents may be administered to the subject before or after the administration of the engineered SIRPα variant and protein construct. In some embodiments, the administration of one or more additional therapeutic agents to the subject is such that there is an overlap in the biological activity period of the one or more additional therapeutic agents and the engineered SIRPα variant and protein construct in the subject.

[0400] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of: inhibitors of B-Raf, EGFR, MEK, ERK, K-Ras, c-Met, anaplastic lymphoma kinase (ALK), phosphatidylinositol 3-kinase (PI3K), Akt, mTOR, dual PI3K / mTOR, Bruton's tyrosine kinase (BTK), and 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., icardolstat).

[0401] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0402] In some embodiments, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, and sorafenib. IB), pazopanib (Votrient), pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0403] In some embodiments, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapeutically targeted CX3CL1, therapeutically targeted CXCL9, therapeutically targeted CXCL10, therapeutically targeted CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0404] In some embodiments, the subject is administered carboplatin, nalbupivacaine, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0405] 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).

[0406] Pharmaceutical Compositions and Routes of Administration

[0407] This document also provides pharmaceutical compositions containing the engineered SIRPα variant and protein construct described herein. The pharmaceutical compositions can be formulated in any manner known in the art.

[0408] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may contain a sterile diluent (e.g., sterile water or saline), non-volatile 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, etc., antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and isotonic agents such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers. The formulation of the composition may be formulated and packaged in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., in injectable formulations), adequate flowability can be maintained by using, for example, a coating (such as lecithin) or a surfactant. Drug absorption can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved through implants and microencapsulated delivery systems that may contain biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0409] Compositions containing the engineered SIRPα variant and protein constructs described herein can be formulated for parenteral (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dose units (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and uniform dosage).

[0410] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., doses for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, engineered SIRPα variants and protein constructs may be formulated in aqueous solutions, preferably in physiologically compatible buffers, to minimize injection site discomfort. The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, engineered SIRPα variants and protein constructs may be in lyophilized form for use with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0411] The toxicity and therapeutic efficacy of the composition can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. In cases where the agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined using other standard pharmaceutical procedures.

[0412] Exemplary doses comprise milligrams or micrograms per kilogram of the engineered SIRPα variants and protein constructs described herein (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, those skilled in the art will understand that the potency of the therapeutic agent can vary, and the effective amount can be determined by methods known in the art. Typically, a relatively low dose is administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the research and development phase) may subsequently and gradually increase the dose until an appropriate response is obtained. Furthermore, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health condition, sex and diet, time of administration, route of administration, excretion rate, and the in vivo half-life of the engineered SIRPα variant and protein construct.

[0413] Pharmaceutical compositions may be contained in containers, packages, or dispensers along with instructions for use. This disclosure also provides methods for manufacturing engineered SIRPα variants and protein constructs for the various uses described herein.

[0414] Example

[0415] The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0416] Example 1. Design of the IgV domain of human SIRPα gene modification

[0417] The IgV domain of human SIRPα (hSIRPα) belongs to the immunoglobulin superfamily and contains nine β chains, comprising ABC-C'-DEFG-G2. The helix is ​​located between the E and F chains. The 3D structure of the hSIRPα IgV domain is shown in... Figure 1 As shown in the image.

[0418] The structure of the CD47 / hSIRPa complex was analyzed in detail. Based on the structure, Leu30, Gly34, Gln52, Lys53, Glu54, His56, Ser66, Thr67, Arg69, Lys93, Lys96, Gly97, and Asp100 in hSIRPα were identified as participating in the interaction with CD47. These interacting residues are mainly located on the ring structure of hSIRPa, including the BC ring, C'D ring, DE ring, and FG ring. The results indicate that hSIRPa mainly interacts with CD47 through its ring regions. Figure 2 ).

[0419] Furthermore, the structure of hSIRPa-FD6 has been resolved (Weiskopf, Kipp et al., Science, July 5, 2013; 341(6141)). Based on the structure of the CD47 / hSIRPa-FD6 complex (PDB ID: 4KJY), the residues involved in the interaction between hSIRPa-FD6 and CD47 were identified as Leu30, Gly34, Gln52, Arg53, Glu54, His56, Thr66, Thr67, Arg69, Lys93, Lys96, Gly97, and Asp100. These residues are identical to those shown in the CD47 / hSIRPa complex. The two interacting residues, Lys53 and Ser66, which are unique to the wild type, are mutated to Arg53 and Thr66, respectively, in hSIRPa-FD6, resulting in a significant increase in binding affinity to CD47. Other altered residues from hSIRPa to hSIRPa-FD6 do not participate in CD47 interactions. This finding suggests that Lys53 and Ser66 may be important for increasing the binding affinity of hSIRPa to CD47.

[0420] To identify which residues exhibited good binding affinity and stability within the C'D ring, residue scans at positions 53, 54, 55, and 56 were performed using a version of the MOE assay. Based on the results of the residue scans, Lys53 showed increased stability when substituted with amino acids having long side chains or aromatic side chains, such as arginine, leucine, phenylalanine, tryptophan, and tyrosine (Fig. 3A). However, this substitution did not improve the affinity of hSIRPa. On the other hand, substitutions for Glu54, Gly55, and His56 showed greater flexibility (Figs. 3B, 3C, and 3D), and the affinity and stability of hSIRPa could be improved by changing Glu54, Gly55, and His56 to several other amino acids. Therefore, mutations were introduced into Glu54, Gly55, and His56 for screening.

[0421] In addition, Ile31, Glu54, Gly55, His56, Ser66, and Thr67 were identified as candidate amino acids for hSIRPa mutation screening via structural analysis. Val27, Val63, and Lys68 are involved in maintaining the structure of hSIRPa itself. To avoid negatively impacting the structure of hSIRPa, alterations to these residues are not recommended. Based on the structure of the human CD47 (hCD47) / hSIRPα (PDBID:2JJT) complex, interacting residues were analyzed. The results showed that several interacting residues in the BC, C'D, and DE rings of the hSIRPα IgV domain are highly conserved. Figure 2As shown, the amino acid residues within these ring regions are underlined.

[0422] To screen for hSIRPα IgV domain mutants that exhibit higher blocking activity against hCD47 compared to wild-type hSIRPα (e.g., different binding affinity for hCD47) and lower RBC binding compared to the anti-CD47 reference antibody Hu5F9-G4, selected residues within the BC, C'D, and DE rings of the hSIRPα IgV domain were mutated. These residues comprise 10 amino acids (e.g., Ile31) of His24-Val33 within the BC ring, 3 amino acids (e.g., Glu54-His56) within the C'D ring, and 8 amino acids (e.g., Ser66 and Thr67) of Ser66-Asp73 within the DE ring. The amino acid positions are based on the hSIRPα IgV domain (SEQ ID NO:1).

[0423] Example 2. Filtering and Reconfirmation

[0424] Phage expression systems were used for screening via phage display. Specifically, CD47 antigen or CD47-expressing cell lines were used for panning. The obtained phages were used to reinfect TG1 electroporated competent *E. coli* cells, which were then plated onto LB agar plates and incubated for 16 hours. Single colonies were picked and inoculated into 700 μl of 2×YT medium supplemented with 10% phosphate buffer. When OD... 600 When the temperature approached 1°C, 1 mM isopropyl β-d-1-thiogalactoside (IPTG) was added to induce protein expression at 30°C. After 16 hours, the bacterial culture was centrifuged at 3200 g, and the supernatant was collected for subsequent experiments.

[0425] Determining the level of expression

[0426] To determine the expression level of the hSIRPαIgV domain mutant, an ELISA plate coated with 2 μg / ml anti-His antibody (R&D, catalog number: MAB050-500) was prepared. 30 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 (Bethy, catalog number: A190-104P) diluted 1:10000 was added to the plate, along with 3,3',5,5'-tetramethylbenzidine (TMB) (SURMODICS, catalog number: TMBW-0100-01) for colorimetric development. Varioskan was used. TM The absorbance (OD) value measured at 450 nm using a LUX board reader (Thermo) 450 ).

[0427] Determination of human CD47 binding capacity

[0428] To determine the human CD47 binding capacity of the hSIRPαIgV domain mutant, an ELISA plate coated with a 1 μg / ml human CD47 extracellular domain (ECD) linked to the Fc region (hCD47ECD Fc) was prepared. 30 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 diluted 1:10000 was added to the plate, and TMB was added for color development. Varioskan was used. TM The absorbance value at 450 nm was measured using a LUX board reader.

[0429] Determination of CD47 binding capacity in mice

[0430] To determine the mouse CD47 binding capacity of the hSIRPαIgV domain mutant, an ELISA plate coated with 1 μg / ml mouse CD47 ECD Fc (Sino Bio, catalog number: 57231-M31H) was 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 diluted 1:10000 was added to the plate, and TMB was added for color development. Varioskan was used. TM The absorbance value at 450 nm was measured using a LUX board reader.

[0431] Determination of hCD47 / hSIRPα blocking ability

[0432] To determine the hCD47 / hSIRPα blocking ability of the hSIRPα IgV domain mutant, an ELISA plate coated with 1 μg / ml human CD47 ECD Fc was 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, catalog number: 79004) diluted 1:1000 was added to the plate, and TMB was added for color development. Varioskan was used. TM The absorbance value at 450 nm was measured using a LUX board reader.

[0433] Determination of protein thermal stability

[0434] Transfer 100 μl of the collected supernatant to a PCR tube and heat it in a PCR machine at 65°C or 70°C for 90 minutes. Simultaneously, human CD47 binding is measured using both heated and unheated supernatants. Specifically, an ELISA plate coated with 1 μg / ml human CD47 ECD Fc is prepared. Add 30 μl of either heated or unheated supernatant to the ELISA plate and incubate at 25°C for 1 hour. After incubation, add goat anti-c-Myc HRP diluted 1:10000 to the plate, and add TMB for color development. Varioskan is used. TM The absorbance value at 450 nm was measured using the LUX board data acquisition device.

[0435] Determination of the binding capacity of whole cells to CD47-TF-CHO-S, erythrocytes, and platelets

[0436] 3×10 4 10 CD47 tf CHO-S cells (CHO-S cells transfected with human CD47), 1×10 5 Individual red blood cells (RBCs) or 3 × 10 5 Individual platelets and collected supernatant were incubated at 4°C for 30 minutes. Then, anti-His PE (Abcam, catalog number: ab72467) diluted 1:100 was added, and flow cytometry (Beckman Coulter, catalog number: CytoFlex) was used to detect MFI (mean fluorescence intensity) values.

[0437] Clones with higher hCD47 / hSIRPα blocking ability than wild-type hSIRPα were isolated, and human CD47 binding OD based on them was obtained. 450 (B) / expression OD450(E) ratios were grouped. Specifically, 12 clones had hCD47 binding B / E ratios greater than 2; 5 clones had ratios between 1.75 and 2; 5 clones had ratios between 1.5 and 1.75; 7 clones had ratios between 1.25 and 1.5; and 2 clones had ratios between 1 and 1.25. Seven clones with B / E ratios smaller than the wild-type hSIRPα IgV domain were also selected. Because human SIRPα has no cross-reactivity with mouse CD47, this screening also identified 18 clones with mouse CD47 binding abilities exceeding expression ratios greater than 0.4. Therefore, a total of 56 candidate clones were selected, among which detailed expression, hCD47 / hSIRPα blockade, and CD47 binding results were within the range of... Figures 4A-4B As shown in the image.

[0438] Next, the selected clones were further screened by determining the binding capacity of whole cells to CD47 tf CHO-S, RBCs, and platelets, as well as the thermal stability of the protein at high temperatures (e.g., 65°C and 70°C). Based on the results of the whole-cell binding assay, clones with platelet and RBC binding capacities lower than those of the wild-type hSIRPαIgV domain, and clones that could bind to CD47 tf CHO-S with varying binding capacities, were selected. The thermal stability of the protein in the selected clones was verified, and no significant differences were observed compared to the wild-type SIRPαIgV 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 were constructed to express the wild-type SIRPαIgV domain or an engineered SIRPαIgV domain mutant linked to the human IgG4 hinge region and Fc region (SEQ ID NO:29). A schematic structure of the expressed protein is shown in [image / description]. Figure 5 The image shows specific amino acid mutations within the BC, C'D, and DE loops of the wild-type SIRPαIgV domain, and 26 clones. Figure 6 As shown in the image.

[0439] Example 3. Purification of hSIRPα-Fc and determination of binding affinity between hSIRPα-Fc and CD47-ECD-His by Octet

[0440] The expressed protein was purified using a protein A column, followed by HPLC-SEC (High Performance Liquid Chromatography combined with Size Exclusion Chromatography; Agilent), and the percentages of high molecular weight peaks (HMW%), main peak (main peak %), and low molecular weight peaks (LMW%) were measured. Figure 7As shown, hSIRPα-Fc mutant proteins can be harvested with high purity using the methods described above. The amino acid sequences of hSIRPα-Fc-wt and 13 hSIRPα-Fc mutant proteins (mt3-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. Immunogenicity was not identified.

[0441] The binding affinity of hSIRPα-Fc protein to CD47-ECD-His also increased from... The system has determined this. (For example...) Figure 8 As shown, two sets of experiments were conducted, with hSIRPα-Fc-wt serving as a negative control and the anti-CD47 reference antibody Hu5F9-G4 serving as a positive control. The relative binding affinity of each protein was also derived from the corresponding KD value compared to hSIRPα-Fc-wt. The results indicate that most engineered SIRPαIgV domain mutants exhibited stronger binding to CD47 compared to the wild-type SIRPαIgV domain.

[0442] Example 4. Whole-cell binding assay of CD47 tf CHO-S and CD47-expressing tumor cells.

[0443] To determine the whole-cell binding capacity of the hSIRPα-Fc mutant protein to CD47, the hSIRPα-Fc mutant protein was 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 (a two-PD1 extracellular domain linked to the N-terminus of human IgG4, its schematic structure is shown in...) Figure 5 (shown in the image) was used as a negative control. Hu5F9-G4 was used as a positive control. The diluted protein was mixed with 5 × 10⁻⁶... 4CD47tf CHO-S cells, Jurkat cells, or Raji cells were incubated together. After incubation, anti-hFcr-PE (1:100, Invitrogen, catalog number: 109-115-098) was added, and the MFI value was measured by flow cytometry.

[0444] like Figures 9A-9C As shown, compared with the anti-CD47 antibody Hu5F9-G4, hSIRPα-Fc-mt4, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, hSIRPα-Fc-mt11, hSIRPα-Fc-mt12, hSIRPα-Fc-mt13, and hSIRPα-Fc-15 exhibited similar whole-cell binding capacity against CD47 tf CHO-S cells, Jurkat cells, and Raji cells.

[0445] like Figure 9D As shown, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt19, and hSIRPα-Fc-mt22 exhibited higher CD47 tf CHO-S cell-binding capacity than hSIRPα-Fc-mt17, hSIRPα-Fc-mt18, hSIRPα-Fc-mt19, and Hu5F9-G4. Furthermore, all tested hSIRPα-Fc mutant proteins showed significantly higher CD47 tf CHO-S cell-binding capacity than hSIRPα-Fc-wt. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0446] Table 1. EC50 values ​​of CD47 tf CHO-S cell binding

[0447]

[0448]

[0449] like Figure 9E As shown, hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 exhibited higher Raji cell-binding abilities than hSIRPα-Fc-mt21, hSIRPα-Fc-mt16, and hSIRPα-Fc-mt23. Compared to Hu5F9-G4, all tested hSIRPα-Fc mutant proteins showed similar Raji cell-binding abilities, and exhibited significantly higher Raji cell-binding abilities compared to hSIRPα-Fc-wt.

[0450] Example 5. Erythrocyte binding assay

[0451] To determine the RBC binding capacity of the hSIRPα-Fc mutant protein, the hSIRPα-Fc mutant protein was 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 protein was compared with 1×10⁻⁶ protein from the two donors. 5 Individual RBC cells were incubated together. After incubation, anti-hFcr-PE (1:100, Invitrogen) was added, and MFI values ​​were measured by flow cytometry.

[0452] like Figures 10A-10B As shown, compared with the anti-CD47 antibody Hu5F9-G4, hSIRPα-Fc-mt12, hSIRPα-Fc-mt13, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, and hSIRPα-Fc-mt15 exhibited weaker RBC binding ability. In particular, hSIRPα-Fc-mt6, hSIRPα-Fc-mt14, and hSIRPα-Fc-wt showed no binding to RBCs.

[0453] like Figure 10C As shown, hSIRPα-Fc-mt18 and hSIRPα-Fc-mt19 exhibited higher RBC (from donor 1) binding capacity 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 capacity 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.

[0454] Table 2. EC50 values ​​for RBC binding

[0455] protein EC50(nM) Hu5F9-G4 0.6414 hSIRPα-Fc-wt (Trillium) - hSIRPα-Fc-mt10 0.4939 hSIRPα-Fc-mt15 2.908 hSIRPα-Fc-mt16 2.556 hSIRPα-Fc-mt17 0.5624 hSIRPα-Fc-mt18 0.1107 hSIRPα-Fc-mt19 0.125 hSIRPα-Fc-mt21 2.074 hSIRPα-Fc-mt22 0.4952

[0456] like Figure 10D As shown, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 exhibited significantly lower RBC (from donor 2) binding capacity than Hu5F9-G4. No RBC cell binding was detected by hSIRPα-Fc-wt.

[0457] Example 6. Platelet binding assay

[0458] To determine the platelet-binding capacity of the hSIRPα-Fc mutant protein, the protein was purified using 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 protein was compared with 5 × 10⁻⁶ protein from two donors. 5 Individual platelets were incubated together. After incubation, anti-hFcr-PE (1:100; Invitrogen) was added, and MFI values ​​were measured by flow cytometry.

[0459] like Figure 11A-11B As shown, compared with anti-CD47 antibody Hu5F9-G4, 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, while hSIRPα-Fc-mt14, hSIRPα-Fc-wt and hSIRPα-Fc-mt6 showed significantly lower platelet-binding ability.

[0460] like Figure 11C As shown, compared with Hu5F9-G4, 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 exhibited stronger or similar platelet (from donor 1) binding capacity. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0461] Table 3. EC50 values ​​of platelet binding

[0462] protein EC50(nM) Hu5F9-G4 0.3334 hSIRPα-Fc-wt (Trillium) 95.95 hSIRPα-Fc-mt10 0.1572 hSIRPα-Fc-mt15 0.2297 hSIRPα-Fc-mt16 0.3098 hSIRPα-Fc-mt17 0.2429 hSIRPα-Fc-mt18 0.1593 hSIRPα-Fc-mt19 0.1505 hSIRPα-Fc-mt20 0.2472 hSIRPα-Fc-mt21 0.2469 hSIRPα-Fc-mt22 0.3996

[0463] like Figure 11D As shown, compared with Hu5F9-G4, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt21, hSIRPα-Fc-mt23, and hSIRPα-Fc-mt16 exhibited stronger or similar platelet-binding abilities. The EC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0464] Table 4. EC50 values ​​of platelet binding

[0465]

[0466]

[0467] Example 7. Whole-cell binding assay of cells expressing monkey CD47

[0468] To determine the whole-cell binding ability of the hSIRPα-Fc mutant protein to monkey CD47, the hSIRPα-Fc mutant protein was purified using 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 protein was mixed with 1×10 5 One transfected CHO-S cell expressing cynomolgus monkey CD47 (cynoCD47tf CHO-S) or 1×10 5 The rhesus monkey kidney epithelial cell line LLC-MK2 was incubated together. After incubation, anti-hFcr-PE (1:100; Invitrogen) was added, and the MFI value was measured by flow cytometry. Figure 12A-12B As shown, all hSIRPα-Fc mutant proteins can bind to monkey CD47.

[0469] like Figure 12C As shown, Hu5F9-G4 exhibits stronger LLC-MK2 binding ability than hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21 and hSIRPα-Fc-mt23.

[0470] Example 8. Whole-cell binding assay of cells expressing mouse CD47

[0471] To determine the whole-cell binding ability of the hSIRPα-Fc mutant protein to mouse CD47, the hSIRPα-Fc mutant protein was purified using 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 (an anti-human mouse CD47 antibody) was used as a positive control. The diluted protein was mixed with 3 × 10⁻⁶ ppm of the protein. 4EMT-6 cells were incubated together. After incubation, anti-hFcr-PE (1:100; Invitrogen) was added, and MFI values ​​were measured by flow cytometry. For MIAP410, anti-mouse (H+L)-FITC (1:100) was used. Figures 13A-13B As shown, 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 do not bind to EMT-6 cells.

[0472] Example 9. Determination of hemagglutination (HA) activity

[0473] To determine the HA activity induced by the hSIRPα-Fc mutant protein, a 10% RBC solution was prepared from whole blood of two healthy donors by washing twice with 0.9% sodium chloride buffer and then diluting 10-fold in 0.9% sodium chloride buffer. The hSIRPα-Fc mutant protein was 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 round-bottom 96-well plates. After incubation, agglutinated RBCs uniformly covered the wells, while non-agglutinated 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. Figures 14A-14C As shown, none of the tested hSIRPα-Fc mutant proteins induced hemagglutination.

[0474] Example 10. Determination of the blocking ability of CD47 tf CHO-S cells, FaDu cells, and Raji cells.

[0475] To determine the blocking ability of the hSIRPα-Fc mutant protein against human CD47, the hSIRPα-Fc mutant protein was purified using 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 protein was mixed with 3 × 10⁻⁶... 4CD47 tf CHO-S cells (CHO-S cells transfected with human CD47), FaDu cells or Raji cells, and 1 μg / ml biotin-labeled hSIRPα-Fc-wt were incubated together. After incubation, streptavidin-PE (0.3 μl per well, eBioscience, catalog number: EBS12-4317-87) was added, and the MFI value was measured by flow cytometry.

[0476] like Figures 15A-15C As shown, all tested hSIRPα-Fc mutant proteins could block the interaction between hSIRPα and CD47 tfCHO-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 exhibited blocking abilities comparable to Hu5F9-G4.

[0477] like Figure 15D As shown, all tested hSIRPα-Fc mutant proteins exhibited 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.

[0478] Table 5. IC50 values ​​of whole-cell blockade

[0479] protein IC50(nM) Hu5F9-G4 0.6765 hSIRPα-Fc-wt (Trillium) 6.122 hSIRPα-Fc-mt10 0.3282 hSIRPα-Fc-mt15 0.3758 hSIRPα-Fc-mt16 0.5815 hSIRPα-Fc-m17 0.4449 hSIRPα-Fc-mt18 0.1529 hSIRPα-Fc-mt19 0.2129 hSIRPα-Fc-mt21 0.1975 hSIRPα-Fc-mt22 0.2757

[0480] like Figure 15E As shown, hSIRPα-Fc-mt21 exhibited the strongest whole-cell blocking ability. Compared with Hu5F9-G4, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, and hSIRPα-Fc-mt23 showed similar whole-cell blocking abilities. hSIRPα-Fc-wt exhibited the lowest CD47 / SIRPα blocking ability. The IC50 values ​​of the tested hSIRPα-Fc proteins are listed in the table below.

[0481] Table 6. IC50 values ​​of whole-cell blockade

[0482] protein IC50(nM) Hu5F9-G4 0.1035 hSIRPα-Fc-wt (Trillium) 11.56 hSIRPα-Fc-mt15 0.1132 hSIRPα-Fc-mt16 0.1199 hSIRPα-Fc-mt21 0.04067 hSIRPα-Fc-mt22 0.1125

[0483] Example 11. Induction of phagocytosis of CD47-expressing tumor cells by mouse macrophages

[0484] Phagocytosis of tumor cells expressing human CD47 (Jurkat, FaDu, or Raji) was determined by co-incubating tumor cells with Raw264.7 mouse macrophages in the presence of the hSIRPα-Fc mutant protein. The experiment was conducted as follows: Jurkat, FaDu, or Raji cells were incubated at 37°C with 5 nM CellTrace... TM CFSE (Thermo, catalog number: C34554) was labeled for 10 minutes, followed by washing with complete DMEM medium containing 10% FBS (fetal bovine serum). The hSIRPα-Fc mutant protein was 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 truncatum) were used as positive controls. PD1-Fc-wt was used as a negative control. 1 × 10 5 CFSE-labeled Jurkat, FaDu, or Raji cells (target cells) per well were incubated with diluted hSIRPα-Fc mutant protein in a low-binding 96-well U-shaped plate at 37°C for 30 minutes. Afterwards, 5 × 10⁶ cells / well were incubated... 4 One Raw264.7 cell was added to each well, and the plate was incubated at 37°C for 2 hours. Raw264.7 cells were stained with a PE-anthocyanin 7 conjugated F4 / 80 antibody (eBioscience, catalog number: 25-4801-82). The ability of the hSIRPα-Fc mutant protein to induce phagocytosis was assessed by calculating the percentage of CFSE+F4 / 80+ from macrophages (indicating macrophages phagocytosed CFSE-labeled Jurkat, FaDu, or Raji cells) relative to the total F4 / 80 signal from macrophages.

[0485] like Figures 16A-16C As shown, compared with Hu5F9-G4, hSIRPα-Fc-mt4, hSIRPα-Fc-mt8, hSIRPα-Fc-mt10, hSIRPα-Fc-mt13, and hSIRPα-Fc-mt15 exhibited comparable or even higher phagocytic capacity in inducing CD47 expression in tumor cells. The EC50 values ​​for each tumor cell were also calculated, as shown in the table below.

[0486] Table 7. EC50 values ​​of phagocytosis by Jurkat cells

[0487]

[0488]

[0489] Table 8. EC50 values ​​of phagocytosis by FaDu cells

[0490] protein EC50(nM) Hu5F9-G4 0.19 SIRPα-Fc (Trillium, G4) 26.78 hSIRPα-Fc-mt4 0.09 hSIRPα-Fc-mt6 18.25 hSIRPα-Fc-mt8 0.22 hSIRPα-Fc-mt10 0.19 hSIRPα-Fc-mt13 0.26 hSIRPα-Fc-mt15 0.56

[0491] Table 9. EC50 values ​​of phagocytosis by Raji cells

[0492] protein EC50(nM) Hu5F9-G4 0.05 SIRPα-Fc (Trillium, G4) 23.06 hSIRPα-Fc-mt4 0.06 hSIRPα-Fc-mt6 15.87 hSIRPα-Fc-mt8 0.08 hSIRPα-Fc-mt10 0.07 hSIRPα-Fc-mt13 0.09 hSIRPα-Fc-mt15 0.13

[0493] like Figure 16D As shown, compared with Hu5F9-G4, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 exhibited comparable ability to induce phagocytosis in DLD1 cells. Figure 16E As shown, compared with Hu5F9-G4, hSIRPα-Fc-mt10, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt20, hSIRPα-Fc-mt21 and hSIRPα-Fc-mt23 showed a comparable ability to induce phagocytosis in DLD1 cells.

[0494] Example 12. Induction of RBC phagocytosis by mouse macrophages

[0495] The phagocytic activity of human RBCs was determined by co-incubating RBCs with Raw264.7 mouse macrophages in the presence of the hSIRPα-Fc mutant protein. The experiment was conducted as follows: 5 nM CellTrace was used at 37°C. TM CFSE (Thermo) labeling of RBCs was performed for 10 minutes, followed by washing with complete DMEM medium containing 10% FBS. The hSIRPα-Fc mutant protein was 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 truncatum) were used as positive controls. PD1-Fc-wt was used as a negative control. 1×10 6 CFSE-labeled RBCs (target cells) per well were incubated with diluted hSIRPα-Fc mutant protein in a low-binding 96-well U-shaped plate at 37°C for 30 minutes. Afterwards, 5 × 10⁶ cells / well were... 4One Raw264.7 cell was added to each well, and the plate was incubated at 37°C for 2 hours. Raw264.7 cells were stained with a PE-anthocyanin 7 conjugated F4 / 80 antibody (eBioscience). The ability of the hSIRPα-Fc mutant protein to induce RBC phagocytosis was assessed by flow cytometry to calculate the percentage of CFSE+F4 / 80+ (indicating that macrophages have phagocytosed CFSE-labeled erythrocytes) from macrophages to the total F4 / 80 signal from macrophages.

[0496] like Figure 17A As shown, all tested hSIRPα-Fc mutant proteins exhibited a weaker ability to induce macrophages (Raw264.7) to phagocytose RBCs.

[0497] Example 13. Induction of platelet phagocytosis by mouse macrophages

[0498] The phagocytic activity of human platelets was determined by incubating platelets with Raw264.7 mouse macrophages in the presence of the hSIRPα-Fc mutant protein. The experiment was conducted as follows: Platelets were incubated at 37°C with 5 nM CellTrace... TM CFSE (Thermo) labeling was performed for 10 minutes, followed by washing with complete DMEM medium containing 10% FBS. The hSIRPα-Fc mutant protein was 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 truncatum) were used as positive controls. PD1-Fc-wt was used as a negative control. 5 × 10⁵ 5 CFSE-labeled platelets (target cells) per well were incubated with diluted hSIRPα-Fc mutant protein in low-binding 96-well U-shaped plates at 37°C for 30 minutes. Afterwards, 5 × 10⁶ cells / well were... 4 One Raw264.7 cell was added to each well, and the plate was incubated at 37°C for 2 hours. Raw264.7 cells were stained with a PE-anthocyanin 7 conjugated F4 / 80 antibody (eBioscience). The ability of the hSIRPα-Fc mutant protein to induce platelet phagocytosis was assessed by flow cytometry to calculate the percentage of CFSE+F4 / 80+ from macrophages (indicating that macrophages phagocytosed CFSE-labeled RBCs) to the total F4 / 80 signal from macrophages.

[0499] like Figure 17BAs shown, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21 and hSIRPα-Fc-mt23 exhibited a weaker ability to induce macrophages (Raw264.7) to phagocytose platelets.

[0500] Example 14. Induction of phagocytosis of CD47-expressing tumor cells by human macrophages

[0501] The phagocytosis of CD47-expressing tumor cells (Raji, DLD1, or Jurkat) by human macrophages was determined as follows. PBMCs were isolated from human blood and differentiated into macrophages by incubating them for 10–14 days in complete RPMI medium containing 10% FBS, 1× streptomycin / penicillin, and 200 U / ml GM-CSF (BioLegend, catalog number: 576304). Monocyte-derived macrophages (MDMs) became adherent, and unattached cells were washed away. This was determined by... (eBioscience, Catalog No.: EBS00-4555-56) MDM were isolated from the plate by incubating and scraping together. Tumor cells expressing human CD47 (Raji, DLD1, or Jurkat) were incubated at 37°C with 5 nM CellTrace. TM CFSE (Thermo) labeling was performed for 10 minutes, followed by washing 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 truncatum) were used as positive controls. PD1-Fc-wt was used as a negative control. 2.4 × 10⁻⁶ 4 -6×10 4 CFSE-labeled Raji, DLD1, or Jurkat cells (target cells) per well were incubated with diluted hSIRPα-Fc mutant protein in a low-binding 96-well U-plate at 37°C for 30 minutes. Afterward, 1.2 × 10⁻⁶ cells were added to each well. 4 -3×10 4 Half the number of target cells per well of MDM cells were incubated at 37°C for 2 hours. MDM cells were stained with a PE-anthocyanin 7 conjugated CD14 antibody (eBioscience, catalog number: 25-0149-42). The ability of the hSIRPα-Fc mutant protein to induce phagocytosis was assessed by calculating the percentage of CFSE+CD14+ from macrophages (indicating that macrophages phagocytosed CFSE-labeled RBC cells) to total CD14 signal from macrophages.

[0502] like Figures 18A-18C As shown, compared with hSIRPα-Fc-wt (hSIRPα-Fc), hSIRPα-Fc-mt10, hSIRPα-Fc-mt15 and Hu5F9-G4 showed a better ability to induce phagocytosis of CD47-expressing tumor cells by MDM cells.

[0503] like Figure 18D As shown, compared with hSIRPα-Fc-wt, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 exhibited better ability to induce phagocytosis of DLD1 cells via MDM cells. Hu5F9 showed the highest ability to induce phagocytosis via MDM cells.

[0504] Example 15. Induction of RBC phagocytosis by human macrophages

[0505] The phagocytosis of RBCs by human macrophages was determined as follows. PBMCs were isolated from human blood and differentiated into macrophages by incubating them for 10–14 days in complete RPMI medium containing 10% FBS, 1× streptomycin / penicillin, and 200 U / ml GM-CSF (Biolegend). MDMs became adherent, and unattached cells were washed away. (eBioscience) Incubate together and scrape to separate MDM from the plate. Use 5 nM CellTrace at 37°C. TM CFSE (Thermo)-labeled RBCs for 10 minutes 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 truncatum) were used as positive controls. PD1-Fc-wt was used as a negative control. 4 × 10⁴ 5 CFSE-labeled RBCs (target cells) per well were incubated with diluted hSIRPα-Fc mutant protein in a low-binding 96-well U-shaped plate at 37°C for 30 minutes. Afterwards, 4 × 10⁴ cells were added to each well. 4MDM cells were collected and the plate was incubated at 37°C for 2 hours. MDM cells were stained with a PE-anthocyanin 7 conjugated CD14 antibody (eBioscience). The ability of the hSIRPα-Fc mutant protein to induce phagocytosis was assessed by calculating the percentage of CFSE+CD14+ from macrophages (indicating that macrophages phagocytosed CFSE-labeled RBC cells) to the total CD14 signal from macrophage flow cytometry.

[0506] like Figures 19A-19B As shown, hSIRPα-Fc-mt10, hSIRPα-Fc-mt13, and hSIRPα-Fc-mt15 do not induce human MDM to phagocytose RBCs. Figure 19C As shown, hSIRPα-Fc-mt15, hSIRPα-Fc-mt16, hSIRPα-Fc-mt21, and hSIRPα-Fc-mt23 do not induce human MDM to phagocytose RBCs.

[0507] Example 16. Determination of in vivo antitumor efficacy

[0508] The in vivo antitumor efficacy of the hSIRPα-Fc mutant protein was determined using Raji cells. Specifically, NOD / SCID mice were inoculated with Raji cells on day 0. On day 4, the mice were placed in 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 via intraperitoneal injection on days 7 and 14 post-inoculation. Control group mice were administered an equal volume of placebo. Tumor volume in each group was measured on days 4, 7, 11, 14, and 18 post-inoculation. The mean tumor volume in each group is shown in the table below. Tumor growth inhibition (TGI) and p-value were also determined.

[0509] Table 10.

[0510] Day 18 after vaccination dose level Mean ± SEM TGI (%) p-value hSIRPα-Fc-mt10 3.2 mg / kg 289±147 81 0.0002(***) hSIRPα-Fc-mt15 3.2 mg / kg 288±110 80 <0.0001(****) hSIRPα-Fc-wt (Trillium) 3.2 mg / kg 772±181 42 0.0295(*) Hu5F9-G4 6mg / kg 243±86 82 <0.0001(****) placebo - 1303±116 - -

[0511] Notes: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001

[0512] like Figures 20A-20BAs shown, hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 exhibited comparable antitumor efficacy compared to the 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. Survival curves for mice in each group were also determined, as shown... Figure 20C As shown, this indicates that administration of hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 significantly prolonged the percentage of surviving mice compared to the placebo control.

[0513] In different experiments, the in vivo antitumor efficacy of the hSIRPα-Fc mutant protein was determined using NCI-H82 cells. Specifically, NOD / SCID mice were inoculated with NCI-H82 cells on day 0. On day 4, the mice were placed in a control group and four treatment groups. For mice in the treatment groups, hSIRPα-Fc-mt10 (G1), hSIRPα-Fc-mt15 (G2), hSIRPα-Fc-wt (Trillium; G3), or Hu5F9-G4 (G4) were administered via intraperitoneal injection on days 4, 7, 14, 11, 14, 18, 21, 25, and 28 post-inoculation. Control group mice were administered an equal volume of placebo. Tumor volume was measured in mice in each group on days 4, 7, 14, 11, 14, 18, 21, 25, 28, and 32 post-vaccination. The mean tumor volume in each group is shown in the table below. Tumor growth inhibition (TGI) and p-value were also determined.

[0514] Table 11.

[0515]

[0516]

[0517] Notes: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001

[0518] like Figures 21A-21B As shown, hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 exhibited comparable antitumor efficacy compared to the 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. Survival curves for mice in each group were also determined, as shown... Figure 21CAs shown, this indicates that administration of hSIRPα-Fc-mt10 and hSIRPα-Fc-mt15 significantly prolonged the percentage of surviving mice compared to the placebo control.

[0519] Other embodiments

[0520] It should be understood that although the invention has been described in conjunction with its detailed description above, the description is intended to be illustrative and not to limit 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 SEQ ID NO:

9.

2. The engineered SIRPα polypeptide according to claim 1, wherein the engineered SIRPα polypeptide further comprises a CH2 domain, a CH3 domain, and an optional hinge region.

3. The engineered SIRPα polypeptide according to claim 1, wherein the engineered SIRPα polypeptide includes SEQ ID NO:

23.

4. A method of treating a human subject with cancer, said cancer comprising cancer cells expressing CD47, said method comprising administering to the human subject a therapeutically effective amount of a composition comprising the engineered SIRPα peptide according to claim 3.

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