WO2022048616A1

WO2022048616A1 - SIRPγ VARIANT AND FUSION PROTEIN THEREOF

Info

Publication number: WO2022048616A1
Application number: PCT/CN2021/116340
Authority: WO
Inventors: 顾晓玲; 叶鑫; 金薪盛; 陶维康
Original assignee: 江苏恒瑞医药股份有限公司; 上海恒瑞医药有限公司
Priority date: 2020-09-04
Filing date: 2021-09-03
Publication date: 2022-03-10
Also published as: TW202219069A; CN115885044A

Abstract

Provided are a SIRPγ variant and a fusion protein thereof. Moreover, also provided are preparation for a polypeptide containing the SIRPγ variant, and an application of the polypeptide.

Description

SIRPγ variants and their fusion proteins

technical field

The present disclosure relates to SIRPγ variants, fusion proteins comprising SIRPγ variants, bifunctional fusion proteins targeting PD-1 and CD47, and their use as medicaments.

Background technique

The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

CD47 is expressed or overexpressed on many tumor types, including acute myeloid leukemia, various subtypes of B-cell non-Hodgkin lymphoma, and many human solid tumor cells (Cell. 2009 Jul 23;138(2):286 -99; Res. 2011;71:1374-84.). CD47 binds to signal regulatory protein alpha (SIRPα) on macrophages, resulting in a "don't eat me" signal that prevents host cells from engulfing tumor cells, allowing them to be removed by the innate immune system (Mol Ther. 2017 Feb 1;25(2) ): 523-533). There have been data showing that anti-CD47 antibodies contribute to an efficient anti-tumor T cell response in immune-tolerant mice (Nat Med. 2015 Oct;21(10):1209-15). Currently, there are many therapies targeting the CD47/SIRPα interaction, including anti-CD47 antibodies, engineered SIRPα receptor proteins, anti-SIRPα antibodies, and bispecific antibodies.

Antibodies to CD47 disrupt inhibitory signals of phagocytosis and can synergize with Fc-mediated prophagocytic signals to effectively eliminate tumor cells. However, CD47 is also expressed in normal cells, so therapeutic CD47 antibodies may be toxic (2017Feb;9(2):E168-E174). The new anti-CD47 humanized monoclonal antibody Hu5F9 currently in clinical use is 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg and 30 mg/kg in non-human primates. A single dose can cause transient and dose-dependent anemia. Use of Hu5F9 at doses of 1 mg/kg, 3 mg/kg or 10 mg/kg resulted in a nadir of hemoglobin levels below 10 g/dL between 5-7 days. Use of Hu5F9 at a dose of 30 mg/kg caused severe anemia in non-human primates with hemoglobin levels below 8 g/dL (J Clin Oncol 2016;34:abstr 3019).

The signal regulatory protein family (SIRP) plays an important role in regulating immune responses. This family contains three type I transmembrane glycoproteins: SIRPα, SIRPβ and SIRPγ. Among them, both SIRPα and SIRPγ can bind to CD47, but the binding capacity of SIRPγ and CD47 is about 10 times weaker than that of SIRPα and CD47. Studies have shown that the binding KD value of SIRPγ to CD47 is about 23 μM (The Journal of Immunology, 2004, 173:2562–2570.). SIRPγ is expressed on T cells and activated NK cells, and the CD47-SIRPγ interaction is involved in the contact between antigen-presenting cells and T cells, co-stimulates T cell activation and promotes T cell proliferation (Piccio et al., Blood 2005, 105, 2421 -2427; BMC Struct Biol. 2013 Jul 4;13:13.).

Patents related to CD47 signaling pathway include WO2016065329, WO2016109415, WO2014087248, WO2014093678, CN107849143A, CN108350048 and so on.

SUMMARY OF THE INVENTION

The present disclosure provides various SIRPγ variants, and fusion proteins comprising these SIRPγ variants, wherein the SIRPγ variants have higher CD47 affinity activity than wild-type, and the fusion proteins thereof have higher stability.

The SIRPγ variant in the present disclosure, through the structural analysis of SIRPγ, uses the Disulfide Scan module of the MOE (Molecular Operating Environment) software to design intrachain disulfide bonds, and introduces cysteine mutations at appropriate positions in the SIRPγ sequence to form Intrachain disulfide bonds to increase the stability of SIRPγ variant sequences. An exemplary introduction of two cysteine mutations forms a pair of disulfide bonds within the SIRPγ sequence. In some embodiments, cysteine mutations are introduced at positions 14 and 115, and 8 and 107 of SIRPγ, respectively.

In some embodiments, the SIRPγ variant comprises at least one amino acid mutation relative to the wild-type SIRPγ peptide, wherein the amino acid mutation site is selected from the group consisting of: Q8, L13, L14, N51, H56, N70, R77, S79, G107, M112 and G115.

In some embodiments, the SIRPγ variant comprises at least one amino acid mutation relative to the wild-type SIRPγ peptide, wherein the amino acid mutation site is selected from the group consisting of: M6, Q8, L13, L14, K19, N51, Q52, K53, E54, H56, N70, M72, R77, S79, N101, G107, M112 and G115.

In some embodiments, the SIRPγ variant comprises at least 3 amino acids selected from M6I, L13V, K19E, N51M or N51A, Q52S, K53G, E54R, H56Q, N70E, M72K, R77K, S79Q, N101D and M112V mutation.

In some embodiments, the SIRPγ variant, relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 14, comprises the amino acid mutations shown below:

i) L14C and G115C; or

ii) Q8C and G107C.

In some embodiments, wherein the SIRPγ variant binds CD47 with up to 10-fold greater affinity than wild-type SIRPγ (which has the sequence of SEQ ID NO: 14). In some embodiments, the SIRPγ variant binds CD47 with up to 100-fold greater affinity than wild-type SIRPγ. In some embodiments, the SIRPγ variant binds CD47 with up to 1000-fold greater affinity than wild-type SIRPγ. In some embodiments, the SIRPγ variant is less than 1 x ^10-8 M, less than 5 x ^10-9 M, less than 2 x ^10-9 M, less than 1 x ^10-9 M, less than 6 x ^10-10 M, or The KD value of less than 5×10 ^-10 M binds to CD47; the affinity can be detected by the BIAcore method, for details, please refer to Test Example 1. In some embodiments, the SIRPγ variant is at least 80% homologous to the wild-type SIRPγ shown in the sequence of SEQ ID NO:14.

In some embodiments, the SIRPγ variants of the present disclosure have a lower affinity for CD47 on the surface of erythrocytes.

In some embodiments, the SIRPγ variants of the present disclosure bind little to CD47 on the surface of red blood cells.

In some embodiments, the SIRPγ variants of the present disclosure have a higher affinity for CD47 expressed by tumor cells.

In some embodiments, the SIRPγ variant comprises an amino acid mutation selected from one or more of K19E, Q52S, K53G, E54R, M72K and N101D.

In some embodiments, the SIRPγ variant comprises K19E, Q52S, K53G, E54R, M72K and N101D amino acid mutations.

In some embodiments, the SIRPγ variant comprises an N51A or N51M mutation.

In some embodiments, the SIRPγ variant comprises L13V and/or M6I amino acid mutations.

In some embodiments, the SIRPγ variant comprises one or more amino acid mutations selected from the group consisting of H56Q, N70E, R77K, S79Q, and M112V.

In some embodiments, the SIRPγ variant comprises an amino acid mutation selected from any of the following:

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

i) M6I, L13V, N51M and R77K;

j) M6I, L13V, N51A and R77K;

k) M6I, L13V, N51A and N70E;

l) N51A and R77K;

m) L13V, N51M and R77K;

n) L13V, N51M and N70E;

o) N51A;

p) L13V and N51M; and

r) N51M.

In some embodiments, the SIRPγ variant comprises N51A and M112V amino acid mutations.

In some embodiments, the SIRPγ variant comprises: L14C, G115C, K19E, Q52S, K53G, E54R, M72K, and N101D amino acid mutations; and amino acid mutations selected from any of the following:

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

j) M6I, L13V, N51M and R77K;

k) M6I, L13V, N51A and R77K;

l) M6I, L13V, N51A and N70E;

m) N51A and R77K;

n) L13V, N51M and R77K;

o) L13V, N51M and N70E;

p)N51A;

q) L13V and N51M; and

r) N51M.

In some embodiments, the SIRPγ variant comprises Q8C, G107C, K19E, Q52S, K53G, E54R, M72K and N101D amino acid mutations; and amino acid mutations selected from any of the following:

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V and N51M;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

j) M6I, L13V, N51M and R77K;

k) M6I, L13V, N51A and R77K;

l) M6I, L13V, N51A and N70E;

m) N51A and R77K;

n) L13V, N51M and N70E; and

o) N51A; and

p) N51A and M112V.

In some embodiments, the SIRPγ variant comprises L14C, G115C, K19E, Q52S, K53G, E54R, M72K, N101D, L13V, and N51M amino acid mutations.

In some embodiments, the SIRPγ variant comprises L14C, G115C, M6I, K19E, Q52S, N51A, K53G, E54R, N70E, M72K, and N101D amino acid mutations.

In some embodiments, the SIRPγ variant comprises Q8C, G107C, K19E, N51A, Q52S, K53G, E54R, M72K, N101D, and M112V amino acid mutations.

In some embodiments, the SIRPγ variant comprises Q8C, G107C, L13V, K19E, N51A, Q52S, K53G, E54R, M72K, R77K, and N101D amino acid mutations.

In some embodiments, the aforementioned SIRPγ variant comprises the amino acid sequence shown below:

EEELQX ₁ IX ₂ PEKLX ₃ X ₄ VTVGX ₅ TATLHCTVTSLLPVGPVLWFRGVGPGRELIYX ₆ X ₇ X ₈ X ₉ GX ₁₀ FPRVTTVSDLTKRX ₁₁ NX ₁₂ DFSIX ₁₃ IX ₁₄ SITPADVGTYYCVKFRKGSPEX ₁₅ VEFKSX ₁₆ PGTEX ₁₇ ALX ₁₈ AKPS;

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₂ is selected from Q or C; X ₃ is selected from L or V; X ₄ is selected from L or C; X ₅ is selected from K or E; X ₆ is selected from N, M or A; X ₇ is selected from Q or S; X ₈ is selected from K or G; X ₉ is selected from E or R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is selected from M or K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is selected from N or D; X ₁₆ is selected from G or C; X ₁₇ is selected from M or V; X ₁₈ is selected from G or C, and

i) when _X4 is C, _X18 is C; or

ii) When X ₂ is C, X ₁₆ is C.

In some embodiments of the SIRPγ variant set forth in the aforementioned SEQ ID NO: 13, wherein:

a) X ₅ is E;

b) X ₇ is S;

c) X ₈ is G;

d) X ₉ is R;

e) X ₁₂ is K; or

f) X ₁₅ is D.

In some embodiments of the aforementioned _SIRPγ variant set forth in SEQ ID NO: ₁₃ , wherein: X5 is E; X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D.

In some embodiments of the aforementioned _SIRPγ variant set forth in SEQ ID NO: 13, wherein X6 is A or M.

In some embodiments of the aforementioned SIRPγ variant set forth in SEQ ID NO: ₁₃ , wherein _X3 is V and/or X1 is I.

a) X ₁₀ is Q;

b) X ₁₁ is E;

c) X ₁₃ is K;

d) X ₁₄ is Q; or

e) X ₁₇ is V.

a) X ₃ is V, X ₆ is A and X ₁₄ is Q;

b) X ₃ is V, X ₆ is A and X ₁₃ is K;

c) X ₃ is V, X ₆ is A and X ₁₇ is V;

d) X ₃ is V, X ₆ is M, X ₁₀ is Q and X ₁₁ is E;

e) _X3 is V and X6 is M _;

f) X ₁ is 1, X ₆ is M and X ₁₃ is K;

g) X ₁ is 1, X ₆ is A and X ₁₃ is K;

h) X ₁ is 1, X ₆ is A and X ₁₁ is E;

i) X ₁ is 1, X ₃ is V, X ₆ is M and X ₁₃ is K;

j) X ₁ is 1, X ₃ is V, X ₆ is A and X ₁₃ is K;

k) X ₁ is 1, X ₃ is V, X ₆ is A and X ₁₁ is E;

1) X ₆ is A and X ₁₃ is K;

m) _X3 is V, X6 is M and _X13 is K _;

n) X ₃ is V, X ₆ is M and X ₁₁ is E;

o) _X3 is V, X6 is _A and _X11 is E;

p) X ₆ is A;

q) _X3 is V and X6 is M _; or

r) X ₆ is M.

In some embodiments of the aforementioned _SIRPγ variant set forth in SEQ ID NO: ₁₃ , wherein X6 is A and X17 is V.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) is selected from any one of a) to k):

a) _X3 is V and X6 is M _;

b) X ₁ is 1, X ₆ is M and X ₁₃ is K;

c) X ₁ is 1, X ₆ is A and X ₁₃ is K;

d) X ₁ is I, X ₆ is A and X ₁₁ is E;

e) X ₁ is 1, X ₃ is V, X ₆ is M and X ₁₃ is K;

f) X ₁ is 1, X ₃ is V, X ₆ is A and X ₁₃ is K;

g) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₁ is E;

h) X6 is _A and _X13 is K;

j) _X3 is V, X6 is _A and _X13 is K; or

k) _X3 is V, _X6 is M and _X11 is E.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) is selected from any one of a) to f):

a) X ₃ is V, X ₆ is A and X ₁₄ is Q;

b) X ₃ is V, X ₆ is A and X ₁₇ is V;

c) X ₃ is V, X ₆ is M, X ₁₀ is Q and X ₁₁ is E;

d) X ₃ is V, X ₆ is M and X ₁₃ is K;

e) _X3 is V, X6 is _A and _X11 is E; or

f) X ₆ is A.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X ₆ is M.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) _X3 is V and _X6 is M

i) X ₂ is C, and X ₁₆ is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) is selected from any one of a) to o):

a) X ₃ is V, X ₆ is A and X ₁₄ is Q;

b) X ₃ is V, X ₆ is A and X ₁₃ is K;

c) X ₃ is V, X ₆ is A and X ₁₇ is V;

d) X ₃ is V, X ₆ is M, X ₁₀ is Q and X ₁₁ is E;

e) X ₃ is V, X ₆ is A and X ₁₁ is E;

f) X ₁ is 1, X ₆ is M and X ₁₃ is K;

g) X ₁ is 1, X ₆ is A and X ₁₃ is K;

h) X ₁ is 1, X ₆ is A and X ₁₁ is E;

j) X ₁ is 1, X ₃ is V, X ₆ is M and X ₁₃ is K;

k) X ₁ is 1, X ₃ is V, X ₆ is A and X ₁₃ is K;

1) X ₁ is 1, X ₃ is V, X ₆ is A and X ₁₁ is E;

m) X6 is _A and _X13 is K;

n) X6 is _A ; and;

o) _X3 is V and _X6 is M.

i) X ₂ is C, and X ₁₆ is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X6 is _A and _X17 is V.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X ₁ is I, X ₆ is A and X ₁₁ is E; and

iv) X ₂ is selected from Q; X ₃ is selected from L or V; X ₆ is selected from N, M or A; X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₆ is selected from G; X ₁₇ is selected from M or V.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) _X3 is V and X6 is M _; and

iv) X ₁ is selected from M or I; X ₂ is selected from Q; X ₄ is selected from L; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₆ is selected from G; X ₁₇ is selected from M or V.

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X ₆ is A; X ₁₇ is V; and

iv) X ₁ is selected from M or I; X ₃ is selected from L or V; X ₂ is selected from Q; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₆ is selected from G.

i) X ₂ is C, and X ₁₆ is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X6 is _A ; _X17 is V; and

iv) X ₁ is selected from M or I; X ₃ is selected from L or V; X ₄ is selected from L; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₈ is selected from G.

i) X ₂ is C, and X ₁₆ is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) _X3 is V _; X6 is A; and _X13 is K

iv) X ₁ is selected from M or I; X ₄ is selected from L; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₇ is selected from V or M; and X ₁₈ is selected from G.

In some embodiments of the SIRPγ variant shown in the aforementioned SEQ ID NO: 13, wherein the SIRPγ variant has a higher affinity for CD47 than the wild type; preferably, the SIRPγ variant is KD values of less than 2 x ^10-9 M bind to human CD47.

In some embodiments of the aforementioned SIRPγ variant shown in SEQ ID NO: 13, wherein said SIRPγ variant is the same as the amino acid sequence shown in any one of SEQ ID NO: 15-46 or SEQ ID NO: 105-106 SIRPγ variants have at least 85% sequence identity (eg, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% or 100% sequence identity).

In some embodiments of the aforementioned SIRPγ variant set forth in SEQ ID NO: 13, wherein said SIRPγ variant comprises an amino acid sequence selected from any one of SEQ ID NO: 15-46.

In some embodiments of the aforementioned SIRPγ variant set forth in SEQ ID NO: 13, wherein the SIRPγ variant comprises an amino acid sequence selected from any one of SEQ ID NO: 105-106.

In some embodiments, the present disclosure provides a fusion protein, wherein the fusion protein comprises the SIRPγ variant of any one of the preceding.

In some embodiments, the aforementioned fusion protein is a PD-1-SIRPγ fusion protein comprising an anti-PD-1 antibody. In some embodiments, the PD-1 antibody described in the aforementioned PD-1-SIRPγ fusion protein is a full-length antibody, which can be selected from any anti-PD-1 antibody, including but not limited to tislelizumab, camrelizumab, toripalimab, sintilimab, cemiplimab, pembrolizumab, nivolumab, prolgolimab, genolimzumab, dostarlimab, zimberelimab, AK-105, sasanlimab, MGD-013, HLX-10, spartalizumab, SCT-I10A, CS-1003, retifanlimab, or MEDI-0680, etc.

In some embodiments, the anti-PD-1 antibody in the aforementioned PD-1-SIRPγ fusion protein comprises:

a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 1, 2 and 3, respectively; and

A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NOs: 4, 5 and 6, respectively.

A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 95, 96 and 97, respectively; and

A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NOs: 98, 99 and 100, respectively.

In some embodiments, the anti-PD-1 antibody in the aforementioned PD-1-SIRPγ fusion protein comprises the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8.

In some embodiments, the anti-PD-1 antibody in the aforementioned PD-1-SIRPγ fusion protein comprises a heavy chain variable region shown in SEQ ID NO:101 and a light chain variable region shown in SEQ ID NO:102 variable area.

In some embodiments, the anti-PD-1 antibody in the aforementioned PD-1-SIRPγ fusion protein comprises a constant region; preferably, it comprises the heavy chain constant region of SEQ ID NO:9 and the light chain of SEQ ID NO:10 constant region.

A heavy chain comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11; and

A light chain comprising the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:12.

In some embodiments, the anti-PD-1 antibody in the aforementioned PD-1-SIRPγ fusion protein comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:103, or having at least 95% with SEQ ID NO:103, amino acid sequences of 96%, 97%, 98% or 99% sequence identity; and

A light chain comprising the amino acid sequence of SEQ ID NO:104, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:104.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises L14C, K19E, Q52S, K53G, E54R, M72K, N101D, and G115C amino acid mutations.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises Q8C, K19E, Q52S, K53G, E54R, M72K, N101D, and G107C amino acid mutations.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises an N51A or N51M amino acid mutation.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein further comprises one or more amino acid mutations selected from M6I, L13V, N70E, and R77K.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein further comprises the M112V amino acid mutation.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises an amino acid mutation selected from any of the following:

a) L13V and N51M;

b) M6I, N51M and R77K;

c) M6I, N51A and R77K;

d) M6I, N51A and N70E;

e) N51A and R77K;

f) M6I, L13V, N51M and R77K;

g) M6I, L13V, N51A and R77K;

h) M6I, L13V, N51A and N70E;

i) L13V, N51A and R77K;

j) L13V, N51M and R77K; and

k) L13V, N51M and N70E.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises N51A and M112V amino acid mutations.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises: L14C, K19E, Q52S, K53G, E54R, M72K, N101D, and G115C amino acid mutations; and selected from the group consisting of any one of the following Amino acid mutation:

a) L13V and N51M;

b) M6I, N51M and R77K;

c) M6I, N51A and R77K;

d) M6I, N51A and N70E;

e) N51A and R77K;

f) M6I, L13V, N51M and R77K;

g) M6I, L13V, N51A and R77K;

h) M6I, L13V, N51A and N70E;

i) L13V, N51A and R77K;

j) L13V, N51M and R77K; and

k) L13V, N51M and N70E.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises L14C, G115C, K19E, Q52S, K53G, E54R, M72K, N101D, L13V, and N51M amino acid mutations.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises L14C, G115C, M6I, K19E, Q52S, N51A, K53G, E54R, N70E, M72K, and N101D amino acid mutations.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises Q8C, G107C, K19E, N51A, Q52S, K53G, E54R, M72K, N101D, and M112V amino acid mutations.

In some embodiments, the SIRPγ variant described in the aforementioned PD-1-SIRPγ fusion protein comprises the amino acid sequence shown below:

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₂ is selected from Q or C; X ₃ is selected from L or V; X ₄ is selected from L or C; X ₅ is selected from K or E; X ₆ is selected from N, M or A; X ₇ is selected from Q or S; X ₈ is selected from K or G; X ₉ is selected from E or R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is selected from M or K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is selected from N or D; X 16 is selected from G or C; X ₁₇ is selected from M or V; X ₁₈ is selected from G or C, and

i) when _X4 is C and _X18 is C; or

ii) When X ₂ is C, X ₁₆ is C.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein comprises the amino acid sequence shown below:

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₂ is Q; X ₃ is selected from L or V; X ₄ is C; X ₅ is selected from K or E; X ₆ is selected from N, M or A; X ₇ is selected from Q or S; X ₈ is selected from K or G; X ₉ is selected from E or R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is selected from M or K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is selected from N or D; X ₁₆ is G; X ₁₇ is selected from M or V; X ₁₈ is C.

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₂ is Q; X ₃ is selected from L or V; X ₄ is C; X ₆ is selected from N, M or A; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₆ is G; X ₁₇ is selected from M or V; X ₁₈ is C; and:

a) X ₅ is E;

b) X ₇ is S;

c) X ₈ is G;

d) X ₉ is R;

e) X ₁₂ is K; or

f) X ₁₅ is D.

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₂ is Q; X ₃ is selected from L or V; X ₄ is C; X ₅ is E; X ₆ is selected from N, M or A; X ₇ is S; X ₈ is G X ₉ is R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is D; X ₁₆ is G; X ₁₇ is selected from M or V; X ₁₈ is C.

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₂ is Q; X ₃ is selected from L or V; X ₄ is C; X ₅ is E; X ₆ is selected from M or A; X ₇ is S; X ₈ is G; X ₉ is R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is D; X ₁₆ is G ; X ₁₇ is selected from M or V; X ₁₈ is C.

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

ii) X2 is Q _; X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X10 is H; _X12 is K; _X15 is D; _X16 is G; and _X17 is M; and

iii) is selected from any of the following:

a) _X3 is V and X6 is M _; and X1 is selected from M or _I ; X11 is selected from N or E; _X13 is selected from R or K; and _X14 _is selected from S or Q;

b) X ₁ is I, X ₆ is M and X ₁₃ is K; and X ₃ is selected from L or V, X ₁₁ is selected from N or E; and X ₁₄ is selected from S or Q;

c) X ₁ is I, X ₆ is A and X ₁₃ is K; and X ₃ is selected from L or V, X ₁₁ is selected from N or E; and X ₁₄ is selected from S or Q;

d) X ₁ is I, X ₆ is A and X ₁₁ is E; and X ₃ is selected from L or V, X ₁₃ is selected from R or K and X ₁₄ is selected from S or Q

e) X1 is _I , _X3 is V, X6 is M and _X13 is K _; and X11 is selected from N or E; and _X14 _is selected from S or Q;

f) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₃ is K; and X ₁₁ is selected from N or E; and X ₁₄ is selected from S or Q;

g) X1 is _I , _X3 is V, X6 is _A and X11 is E; and _X13 is selected from R or K; and _X14 _is selected from S or Q;

h) X6 is _A and _X13 is K; and X1 is selected from M or _I ; _X3 is selected from L or V, X11 is selected from N or E; and _X14 _is selected from S or Q;

j) _X3 is V, X6 is _A and _X13 is K; and X1 is selected from M or _I ; X11 is selected from N or E; and _X14 _is selected from S or Q; and

k) X ₃ is V, X ₆ is M and X ₁₁ is E; and X ₁ is selected from M or I; X ₁₃ is selected from R or K; and X ₁₄ is selected from S or Q.

EEELQX ₁ IX ₂ PEKLX ₃ X ₄ VTVGX ₅ TATLHCTVTSLLPVGPVLWFRGVGPGRELI YX ₆ X ₇ X ₈ X ₉ GX ₁₀ FPRVTTVSDLTKRX ₁₁ NX ₁₂ DFSIX ₁₃ IX ₁₄ SITPADVGTYYCVKFRKGSPEX ₁₅ VEFKSX ₁₆ PGTEX ₁₇ ALX ₁₈ AKPS;

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

iii) _X3 is V, X6 is M and _X13 is K, and X1 is selected from M or _I ; _X11 is selected from N or E; and _X14 _is selected from S or Q.

SEQ ID NO: 13

in:

i) X ₂ is C, and X ₁₆ is C;

ii) _X4 is L, X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X10 is H; _X12 is K; _X15 is D; and _X18 is G; and

iii) X1 is _I or M, _X3 is L or V, X6 is _A , X11 is N or E, and _X13 is R or K, _X14 is selected from S or _Q , and _X17 is V.

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

ii) X2 is Q _; X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X10 is H; _X12 is K; _X15 is D; _X16 is G; _X14 is S, and X ₁₇ is M; and

iii) is selected from any one of the following mutations:

a) X ₃ is V and X ₆ is M; and X ₁ is selected from M or I; X ₁₁ is selected from N or E; and X ₁₃ is selected from R or K;

b) X ₁ is 1, X ₆ is M and X ₁₃ is K; and X ₃ is selected from L or V, and X ₁₁ is selected from N or E;

c) X ₁ is I, X ₆ is A and X ₁₃ is K; and X ₃ is selected from L or V, and X ₁₁ is selected from N or E;

d) X ₁ is I, X ₆ is A and X ₁₁ is E; and X ₃ is selected from L or V, and X ₁₃ is selected from R or K;

e) X ₁ is I, X ₃ is V, X ₆ is M and X ₁₃ is K; and X ₁₁ is selected from N or E;

f) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₃ is K; and X ₁₁ is selected from N or E;

g) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₁ is E; and X ₁₃ is selected from R or K;

h) X6 is _A and _X13 is K; and X1 is selected from M or _I ; _X3 is selected from L or V, and _X11 is selected from N or E;

j) X ₃ is V, X ₆ is A and X ₁₃ is K; and X ₁ is selected from M or I; and X ₁₁ is selected from N or E; and

k) X ₃ is V, X ₆ is M and X ₁₁ is E; and X ₁ is selected from M or I; and X ₁₃ is selected from R or K.

In some embodiments, the aforementioned PD-1-SIRPγ fusion protein, wherein the SIRPγ variant comprises the amino acid sequence shown below:

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

iii) X ₃ is V, X ₆ is M and X ₁₃ is K, and X ₁ is selected from M or I; and X ₁₁ is selected from N or E.

In some embodiments, the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein is linked to the heavy or light chain of the anti-PD-1 antibody, either directly or through a linker.

In some embodiments, the SIRPγ variant described in the aforementioned PD-1-SIRPγ fusion protein is linked to the heavy or light chain of the anti-PD-1 antibody through a linker, wherein the linker is in the art A well-known linker; preferably, the linker is selected from (G ₄ S)xGn, wherein x is selected from 1-10, and n is selected from 0-10; more preferably, the linker comprises as SEQ ID NO : the sequence shown at 47 or 48.

In some embodiments, the N-terminus of the SIRPγ variant in the aforementioned PD-1-SIRPγ fusion protein is linked to the C-terminus of the anti-PD-1 antibody heavy chain.

In some embodiments, the aforementioned PD-1-SIRPγ fusion protein comprises:

A first peptide chain comprising any amino acid sequence selected from SEQ ID NOs: 49-59; and/or

The second peptide chain, comprising the amino acid sequence of SEQ ID NO:12.

In some embodiments, the aforementioned PD-1-SIRPγ fusion protein comprises:

A first peptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 107 or 108; and/or

A second peptide chain comprising the amino acid sequence of SEQ ID NO:104.

In some embodiments, the aforementioned PD-1-SIRPγ fusion protein comprises two identical first and second peptide chains, wherein:

The second peptide chain, comprising the amino acid sequence of SEQ ID NO:12.

The first peptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 107 or 108; and/or

The second peptide chain comprises the amino acid sequence of SEQ ID NO: 104;

The first peptide chain comprises the amino acid sequence of SEQ ID NO: 49; and

The second peptide chain comprises the amino acid sequence of SEQ ID NO:12.

The first peptide chain comprises the amino acid sequence of SEQ ID NO:52; and the second peptide chain comprises the amino acid sequence of SEQ ID NO:12.

The first peptide chain comprises the amino acid sequence of SEQ ID NO:108; and the second peptide chain comprises the amino acid sequence of SEQ ID NO:104.

In some embodiments, there is provided a fusion protein comprising a human Fc domain monomer fused to the SIRPγ variant, which is a SIRPγ variant-Fc fusion protein.

In some embodiments, the Fc domain monomer described in the aforementioned SIRPγ variant-Fc fusion protein is the Fc region of human IgGl, IgG2, or IgG4.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises:

i) L14C and G115C; or

ii) Q8C and G107C.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises any amino acid mutation selected from the group consisting of K19E, Q52S, K53G, E54R, M72K, and N101D.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises K19E, Q52S, K53G, E54R, M72K, and N101D amino acid mutations.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises an N51A or N51M amino acid mutation.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein further comprises one or more amino acid mutations selected from the group consisting of M6I, L13V, H56Q, N70E, R77K, S79Q, and M112V.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises an amino acid mutation selected from any of the following:

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E; e) L13V and N51M;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

i) M6I, L13V, N51M and R77K;

j) M6I, L13V, N51A and R77K;

k) M6I, L13V, N51A and N70E;

l) L13V, N51A and N70E; and

m) N51A.

n) N51A and R77K; or

o) N51A and M112V.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises an amino acid mutation selected from the group consisting of:

p) N51M.

i) L14C and G115C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) is selected from the amino acid mutations described in any of the following:

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

j) M6I, L13V, N51M and R77K;

k) M6I, L13V, N51A and R77K;

l) M6I, L13V, N51A and N70E;

m) N51A; and

n) L13V and N51M.

i) L14C and G115C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

o) N51A and R77K; and

p) N51A and M112V.

i) L14C and G115C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) N51M.

i) L14C and G115C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

a) L13V, N51A and S79Q;

b) L13V, N51A and M112V;

c) L13V, N51M, H56Q and N70E;

d) L13V, N51A and N70E;

e) N51A; and

f) N51A and R77K.

i) Q8C and G107C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

j) M6I, L13V, N51M and R77K;

k) M6I, L13V, N51A and R77K;

l) M6I, L13V, N51A and N70E;

m) N51A; and

n) L13V and N51M.

i) Q8C and G107C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) N51A and R77K.

i) L14C and G115C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) L13V and N51M.

i) L14C and G115C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) M6I, N51A and N70E.

i) Q8C and G107C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) L13V, N51A and R77K.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein, wherein the SIRPγ variant comprises:

i) Q8C and G107C;

ii) K19E, Q52S, K53G, E54R, M72K and N101D; and

iii) N51A and M112V.

In some embodiments, the SIRPγ variant in the aforementioned SIRPγ variant-Fc fusion protein comprises the amino acid sequence shown below:

SEQ ID NO: 13

in:

i) when _X4 is C and _X18 is C; or

ii) When X ₂ is C, X ₁₆ is C.

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₃ is selected from L or V; X ₅ is selected from K or E; X ₆ is selected from N, M or A; X ₇ is selected from Q or S; X ₈ is selected from K or G; X ₉ is selected from E or R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is selected from M or K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is selected from N or D; X _is selected from M or V; and:

i) _X4 is C, and _X18 is C; or

ii) X ₂ is C, and X ₁₆ is C.

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₃ is selected from L or V; X ₅ is E; X ₆ is selected from N, M or A; X ₇ is S; X ₈ is G; X ₉ is R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is D; X ₁₇ is selected from M or V; and:

i) _X4 is C, and _X18 is C; or

ii) X ₂ is C, and X ₁₆ is C.

SEQ ID NO: 13

in:

X ₁ is selected from M or I; X ₃ is selected from L or V; X ₅ is E; X ₆ is selected from M or A; X ₇ is S; X ₈ is G; X ₉ is R; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₅ is D; X ₁₇ is selected from M or V; and:

i) _X4 is C, and _X18 is C; or

ii) X ₂ is C, and X ₁₆ is C.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein, wherein the SIRPγ variant comprises the amino acid sequence shown below:

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

ii) X2 is Q _; X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; _X15 is D; and _X16

is G; and iii) is selected from any of the following a) to n):

a) X ₃ is V, X ₆ is A, X ₁₄ is Q; and X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; and X ₁₇ is selected from M or V;

b) X ₃ is V, X ₆ is A, X ₁₃ is K; and X ₁ is selected from M or I; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V;

c) X ₃ is V, X ₆ is A, X ₁₇ is V; and X ₁ is selected from M or I; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K and X ₁₄ is selected from S or Q;

d) X ₃ is V, X ₆ is M, X ₁₀ is Q and X ₁₁ is E; and X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₃ is selected from R or K; X ₁₄ is selected from from S or Q; and X ₁₇ from M or V;

e) X ₃ is V and X ₆ is M; and X ₁ is selected from M or I; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V;

f) X ₁ is I, X ₆ is M and X ₁₃ is K; and X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V; and

g) X ₁ is I, X ₆ is A and X ₁₃ is K; and X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V;

h) X ₁ is I, X ₆ is A and X ₁₁ is E; and X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V;

j) X1 is _I , _X3 is V, X6 is M and _X13 is K _; and _X10 is selected from H or Q; X11 is selected from N or E; _X14 is selected from S or _Q ; and _X17 selected from M or V;

k) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₃ is K; and X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V; and

1) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₁ is E; and X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ selected from M or V;

m) X ₃ is V, X ₆ is A and X ₁₁ is E; X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V; and

n) X ₆ is A; and X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ and X ₁₇ is selected from M or V.

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

ii) X2 is Q _; X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X15 is D; and _X16 is G; and

iii) X ₆ is selected from M, and X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₂ is selected from M or K; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V.

SEQ ID NO: 13

in:

i) _X4 is C, and _X18 is C;

is G; and iii) is selected from any one of o) to r):

o) X6 is _A and _X13 is K, and _X10 is selected from H or Q; _X13 is selected from R or K; _X14 is selected from S or Q; and _X17 is selected from M or V;

p) X ₃ is V, X ₆ is M and X ₁₃ is K, and X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V ;

q) _X3 is V, X6 is M and X11 is E, and _X10 is selected from H or Q; _X13 is selected from R or K; _X14 _is selected from S or _Q ; and _X17 is selected from M or V ;and

r) X6 is _A and _X17 is V, and _X10 is selected from H or Q; _X13 is selected from R or K; _X14 is selected from S or Q; and _X17 is selected from M or V;

SEQ ID NO: 13

in:

i) X ₂ is C, and X ₁₆ is C;

ii) X2 is Q _; _X4 is L _; X5 is E; _X7 is S; _X8 is G; _X9 is R; _X12 is K; _X15 is D; _X16 is G and _X18 is G; and

iii) is selected from any one of a) to n):

e) X ₃ is V, X ₆ is A and X ₁₁ is E; X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V;

f) X ₁ is I, X ₆ is M and X ₁₃ is K; and X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V;

k) X ₁ is I, X ₃ is V, X ₆ is A and X ₁₃ is K; and X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₄ is selected from S or Q; and X ₁₇ selected from M or V;

1) X ₁ is 1, X ₃ is V, X ₆ is A and X ₁₁ is E and X ₁₀ is selected from H or Q; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from from M or V;

m) X ₆ is A; and X ₁ is selected from M or I; X ₃ is selected from L or V; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X _is selected from M or V; and

n) X ₃ is V and X ₆ is M; and X ₁ is selected from M or I; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; and X ₁₇ is selected from M or V.

SEQ ID NO: 13

in:

i) X ₂ is C, and X ₁₆ is C;

iii) is selected from any one of o) or p):

o) X6 is _A , _X13 is K; and X1 is selected from M or _I ; _X10 is selected from H or Q; _X11 is selected from N or E; _X14 is selected from S or Q; and _X17 is selected from M or V; or

p) X ₆ is A, X ₁₇ is V; and X ₁ is selected from M or I; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K and X ₁₄ is selected from S or Q;

SEQ ID NO: 13

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X ₁ is I, X ₆ is A and X ₁₁ is E; and

SEQ ID NO: 13

i) _X4 is C, and _X18 is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) _X3 is V and X6 is M _; and

iv) X ₁ is selected from M or I; X ₂ is selected from Q; X ₄ is selected from L or C; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₆ is selected from G; X ₁₇ is selected from M or V.

SEQ ID NO: 13

i) X ₂ is C, and X ₁₆ is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X ₆ is A; X ₁₇ is V; and

iv) X ₁ is selected from M or I; X ₃ is selected from L or V; X ₄ is selected from L or C; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₃ is selected from R or K; X ₁₄ is selected from S or Q; X ₁₈ is selected from G.

SEQ ID NO: 13

i) X ₂ is C, and X ₁₆ is C;

ii) X5 is E _; _X7 is S; _X8 is G; _X9 is R; _X12 is K; and _X15 is D; and

iii) X ₃ is V; X ₆ is A and X ₁₃ is K;

iv) X ₁ is selected from M or I; X ₃ is selected from L or V; X ₄ is selected from L or C; X ₁₀ is selected from H or Q; X ₁₁ is selected from N or E; X ₁₄ is selected from S or Q; X ₁₇ is selected from M or V; and X ₁₈ is selected from G.

In some embodiments, the Fc domain monomer described in the aforementioned SIRPγ variant-Fc fusion protein comprises an Fc variant, wherein the Fc variant comprises at least one amino acid mutation.

In some embodiments, the Fc variants described in the aforementioned SIRPγ variant-Fc fusion proteins exhibit abolished or reduced binding to Fcγ receptors as compared to the wild-type version of a human IgG Fc region. In some embodiments, the Fc variant exhibits abolished or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcy receptors compared to the wild-type version of a human IgG Fc region. In some embodiments, the Fc variant exhibits abolished or reduced binding to C1q compared to the human IgG Fc wild-type version. In some embodiments, the Fc variant exhibits abolished or reduced binding to Fcγ receptors compared to the wild-type human IgG4 Fc region.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein, wherein the Fc domain monomer comprises one or more amino acid breaks in L234A, L235A, G237A, and N297A.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein, wherein the Fc domain monomer comprises L234A, L235A, G237A, and N297A amino acid mutations.

In some embodiments, two Fc domain monomers can form an Fc domain dimer, wherein each Fc domain monomer is independently selected from (i) a human IgGl Fc region comprising mutations L234A, L235A, G237A, and N297A (ii) a human IgG2 Fc region comprising mutations A330S, P331S and N297A; (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A and N297A; or (iv) a human IgG4 Fc region comprising mutations F234A, L235A; above For the amino acid mutation sites of the Fc domain, the EU numbering convention is used.

In some embodiments, the two Fc domain monomers are identical (ie, homodimers). In some embodiments, the two Fc domain monomers are different (i.e., heterodimers). In some embodiments, at least one of the Fc domain monomers in the Fc domain dimer is a human IgGl Fc region comprising the L234A, L235A, G237A, and N297A mutations. In some embodiments, at least one of the Fc domain monomers in the Fc domain dimer is a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations. In some embodiments, at least one of the Fc domain monomers in the Fc domain dimer is a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and N297A mutations. In some embodiments, at least one of the Fc domain monomers in the Fc domain dimer is a human IgG4 Fc region comprising F234A, L235A.

In some embodiments, the Fc domain monomer in the aforementioned SIRPγ variant-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 60 or 61.

In some embodiments, the Fc domain monomer in the aforementioned SIRPγ variant-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:109.

In some embodiments, the SIRPγ variant described in the aforementioned SIRPγ variant-Fc fusion proteins is linked to the N-terminus or C-terminus of the Fc domain monomer by a peptide bond or linker.

In some embodiments, a linker (eg, a spacer) is inserted between the SIRPγ variant and the Fc domain monomer. In some embodiments, a high-affinity SIRPγ variant comprising the present disclosure is fused to an Fc domain monomer that is incapable of dimerizing. In some embodiments, the SIRPγ variants of the present disclosure are fused to an Fc domain monomer capable of forming a dimer (eg, a heterodimer) with another Fc domain monomer. In some embodiments, a SIRPγ variant of the present disclosure is fused to an Fc domain monomer, and the fusion protein forms a homodimer. In some embodiments, the SIRPγ variants of the present disclosure are fused to a first Fc domain monomer, and a different protein or peptide (eg, an antibody variable region) is fused to a second Fc domain monomer. In some embodiments, a SIRPγ variant is linked to a first Fc domain monomer, and a therapeutic protein (eg, a cytokine, interleukin, antigen, steroid, anti-inflammatory or immunomodulatory agent) is linked to a second Fc domain monomer. In some embodiments, the first and second Fc domain monomers form a heterodimer.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein comprises an Fc domain dimer, wherein the SIRPγ variant C-terminus is linked to the N-terminus of the Fc domain monomer via a linker; preferably , the linker Gm(G ₄ S)×Gn, wherein x, m, n are selected from integers of 0-10, and x, m, n are not 0 at the same time.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein comprises an Fc domain dimer, wherein the SIRPγ variant C-terminus is linked by a peptide bond to the N-terminus of the Fc domain monomer.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein comprises an Fc domain dimer, wherein the N-terminus of the SIRPγ variant is linked to the C-terminus of the Fc domain monomer by a peptide bond.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein comprises an Fc domain dimer, wherein the SIRPγ variant N-terminus is linked to the C-terminus of the Fc domain monomer by a linker.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein comprises any one of the amino acid sequences selected from SEQ ID NOs: 62-90.

In some embodiments, the aforementioned SIRPγ variant-Fc fusion protein comprises any amino acid sequence selected from the group consisting of SEQ ID NOs: 110-113.

In some embodiments, the SIRPγ variant-Fc fusion protein described in the aforementioned SIRPγ variant-Fc fusion protein is a homodimer comprising two identical pairs of any one of SEQ ID NOs: 62-90. A shows the amino acid sequence.

In some embodiments, the SIRPγ variant-Fc fusion protein described in the aforementioned SIRPγ variant-Fc fusion proteins is a homodimer comprising two identical pairs of any one of SEQ ID NOs: 110-113 A shows the amino acid sequence.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the SIRPγ variant of any of the foregoing, or the fusion protein of any of the foregoing, and one or more pharmaceutical agents acceptable carrier, diluent, buffer or excipient.

In some embodiments, the therapeutically effective amount is a unit dose of the composition comprising 0.1-3000 mg of the SIRPγ variant of any of the foregoing, or the fusion protein of any of the foregoing.

In some embodiments, the present disclosure provides a nucleic acid molecule encoding a polypeptide according to any of the foregoing, or a fusion protein of any of the foregoing.

In some embodiments, the present disclosure provides an expression vector comprising the aforementioned nucleic acid molecule.

In some embodiments, the present disclosure provides a host cell comprising the aforementioned expression vector. In some embodiments, the host cell may be selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells. In some embodiments, wherein the host cell is a non-human mammalian cell. In some embodiments, wherein the host cells do not include human embryonic cells, wherein the mammalian cells include, but are not limited to, CHO, 293, NSO.

In some embodiments, the present disclosure provides a method of treating an individual with a disease or disorder, the method comprising administering to the individual with the disease or disorder a therapeutically effective amount of the SIRPγ variant of any of the foregoing, Or the fusion protein described in any one of the foregoing, or the foregoing pharmaceutical composition, or the foregoing nucleic acid molecule, or the foregoing carrier.

In some embodiments, the aforementioned methods further comprise administering to the individual suffering from the disease or disorder a therapeutically effective amount of a CD38 antagonist.

In some embodiments, the aforementioned methods further comprise administering to the individual suffering from the disease or disorder a therapeutically effective amount of a CD38 antagonist, wherein the CD38 antagonist is an anti-CD38 antibody.

In some embodiments, the anti-CD38 antibody of the aforementioned methods comprises:

A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 119, 120 and 121, respectively; and a light chain variable region comprising set forth in SEQ ID NOs: 122, 123 and 124, respectively of LCDR1, LCDR2 and LCDR3.

In some embodiments, the anti-CD38 antibody described in the preceding methods comprises a heavy chain variable region as set forth in SEQ ID NO:117 and a light chain variable region as set forth in SEQ ID NO:118.

In some embodiments, the anti-CD38 antibodies described in the aforementioned methods comprise a heavy chain as set forth in SEQ ID NO: 115 and a light chain as set forth in SEQ ID NO: 116.

In some embodiments, the present disclosure provides the SIRPγ variant of any of the foregoing, or the fusion protein of any of the foregoing, or the foregoing pharmaceutical composition, or the foregoing nucleic acid molecule, or the foregoing expression vector Use in the manufacture of a medicament for the treatment of a disease or condition.

In some embodiments, the present disclosure provides the SIRPγ variant of any of the foregoing, or the fusion protein of any of the foregoing, or the foregoing pharmaceutical composition, or the foregoing nucleic acid molecule, or the foregoing expression vector Use in combination with a CD38 antagonist (eg, an anti-CD38 antibody) in the manufacture of a medicament for the treatment of a disease or disorder.

In some embodiments, the present disclosure provides a SIRPγ variant of any of the foregoing, or a fusion protein of any of the foregoing, or a pharmaceutical composition of the foregoing, or the foregoing for use as a medicament for the treatment of a disease or disorder Nucleic acid molecules, or the aforementioned expression vectors.

In some embodiments, the aforementioned disease or disorder is cancer, an autoimmune disease, or an inflammatory disease.

In some embodiments, the aforementioned disease or disorder, wherein the cancer is selected from the group consisting of: solid tumors, hematological cancers, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Non-Hodgkin Lymphoma, Hodgkin Lymphoma, Multiple Myeloma, Bladder Cancer, Pancreatic Cancer, Cervical Cancer, Endometrial Cancer, Lung Cancer, Small Cell Lung Cancer, Non-Small Cell Lung Cancer, Bronchial Cancer, Liver Cancer, Ovarian Cancer , colon and rectal cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, Merkel cell carcinoma, virus induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma and brain tumor; wherein said autoimmune disease or said inflammation The disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune disease, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerosis sclerosis, Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia-reperfusion, Crohn's disease, endometriosis, glomerulonephritis, Myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis. In some embodiments, the aforementioned disease or disorder is associated with CD47 and/or PD-1.

Exemplary anti-PD-1 antibody sequences, preparation methods and related properties in the present disclosure have been described in PCT application No. PCT/CN2020/074098, the entire contents of which are incorporated herein by reference.

Exemplarily, the anti-PD-1 antibody is Hu23-11, the sequence of which is as follows:

Table 1. CDRs of anti-PD-1 antibodies

>Hu23-11 heavy chain variable region:

>Hu23-11 light chain variable region:

>Hu23-11 heavy chain constant region:

>Hu23-11 light chain constant region:

>Hu23-11 antibody heavy chain:

>Hu23-11 light chain

>Hu33-5 heavy chain variable region:

>Hu33-5 light chain variable region:

>Hu33-5 heavy chain:

>Hu33-5 light chain:

Description of drawings

Figure 1 shows the experimental results of SIRPγ fusion protein binding to human erythrocytes;

Fig. 2A-Fig. 2F respectively show the experimental results of different SIRPγ fusion proteins binding to tumor cell Karpass 299;

Figure 3 shows the experimental results of the pharmacodynamics of PD-1-SIRPγ fusion protein in the mouse Karpas299 model reconstituted by human PBMC;

Figure 4A shows the schematic structure of PD-1-SIRPγ-fusion protein; Figure 4B shows the structure diagram of SIRPγ linked to the C-terminus of Fc; Figure 4C shows the structure diagram of SIRPγ linked to the N-terminus of Fc.

Figure 5 shows the results of fusion proteins of the present disclosure in hemagglutination experiments.

Figure 6A shows the effect of fusion protein 4656 on stimulating PBMC to release IFNγ; Figure 6B shows the effect of

fusion proteins

4658 and 4646 on stimulating PBMC to release IFNγ.

Figure 7 shows the therapeutic effect of the fusion protein combined with anti-CD38 antibody on the xenograft of MOLP-8 cells in mice.

Figure 8 shows the therapeutic effects of different fusion proteins on the xenografts of MDA-MB-231 cells in mice.

detailed description

the term

The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. 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 disclosure belongs.

As used in the specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," and the like, should be construed in an inclusive rather than an exclusive or exhaustive sense; that is, " including but not limited to".

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

The term "SIRPγ" refers to any SIRPγ polypeptide or fragment thereof capable of binding CD47, and the amino acid sequence of the wild-type SIRPγ peptide is set forth in SEQ ID NO:14.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Conservatively modified variants thereof are also implicitly encompassed by a particular polypeptide sequence unless otherwise stated.

"SIRPy variants" comprise amino acid substitutions, deletions or insertions (or combinations thereof) compared to wild-type SIRPy peptides, which SIRPy variants have enhanced cell surface CD47 binding activity relative to wild-type SIRPy peptides. In some embodiments, the fusion protein formed by the SIRPγ variant is more stable and less prone to breakage. In some embodiments, wherein said amino acid mutation occurs in a selected from M6, Q8, L13, L14, K19, N51, Q52, K53, E54, H56, N70, M72, R77, S79, N101, G107, M112 and one or more of the amino acid residues in G115. In some embodiments, "SIRPγ variant" comprises at least 3 selected from M6I, Q8C, L13V, L14C, K19E, N51M or N51A, Q52S, K53G, E54R, H56Q, N70E, M72K, R77K, S79Q, N101D, G107C , M112V or G115C amino acid mutation. In some embodiments, the aforementioned SIRPγ variant, relative to the wild-type SIRPγ peptide set forth in SEQ ID NO: 14, comprises the amino acid mutations shown below:

i) L14C and G115C;

ii) Q8C and G107C; or

iii) Q8C and G115C.

The term "high affinity SIRPγ variant" refers to a polypeptide comprising a SIRPγ mutant that binds CD47 with a higher affinity for CD47 than wild-type SIRPγ.

In some specific embodiments, the SIRPγ variant comprises the sequence set forth in SEQ ID NO: 13:

SEQ ID NO: 13

in:

i) when X ₄ is C, X ₁₈ is C;

ii) when _X2 is C, _X16 is C; or

iii) When X ₂ is C, X ₁₈ is C.

Exemplary amino acid substitution sites included in the SIRPγ variants of the present disclosure are shown in Table 2:

The term "amino acid mutation" encompasses amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct so long as the final construct possesses the desired properties. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and amino acid insertions. Particular amino acid mutations are amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, replacing one amino acid with another amino acid with different structural and/or chemical properties. Amino acid substitutions include substitution by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (eg, 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) . Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modifications, may also be available. Various names may be used herein to refer to the same amino acid mutation.

"Conservative" amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxyl side chains are serine and threonine; a The amino acids with amide side chains are asparagine and glutamine; the amino acids with aromatic side chains are phenylalanine, tyrosine and tryptophan; the amino acids with basic side chains are lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid- aspartic acid, and asparagine-glutamine.

The term "fusion protein" generally refers to a protein resulting from the fusion of two or more proteins or polypeptides. The genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or fused nucleic acid molecule, which can encode the fusion protein. Translation of the fusion gene produces a single polypeptide having the properties of at least one, if not each, of the two or more proteins or polypeptides prior to fusion. The terms fusion protein and recombinant fusion protein are used herein with the same meaning. The fusion proteins described herein generally comprise at least two domains (A and B), and optionally a third component, a linker between the two domains. The generation of recombinant fusion proteins is known in the art and typically involves removal of stop codons from the cDNA sequence encoding a first protein or polypeptide, followed by ligation or overlap extension PCR to in-frame attachment of the cDNA for the second protein sequence. This DNA sequence is then expressed by the cell as a single protein. The protein can be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion of either. A fusion protein in the present disclosure refers to a fusion protein comprising a SIRPγ variant in the present disclosure. In some embodiments, wherein the fusion protein is a PD-1-SIRPγ variant fusion protein, which is a tetrapeptide structure comprising an anti-PD-1 antibody and a SIRPγ variant, wherein the N-terminus of the SIRPγ variant passes through A linker is attached to the C-terminus of the anti-PD-1 antibody heavy chain. In other embodiments, wherein the fusion protein is a SIRPγ-Fc fusion protein, wherein the C-terminus of the SIRPγ variant can be linked to the N-terminus of the Fc through a peptide bond or linker.

The "linkage" in which the SIRPγ variant is attached to the polypeptide chain of the anti-PD-1 antibody refers to an operative linkage between the polypeptides, including, for example, via peptide bonds, or linkage using a linker. The linkage does not result in loss of the respective functions of the SIRPγ peptide and the anti-PD-1 antibody.

"Linker" or "linker" refers to a linking polypeptide sequence used to connect protein domains or different proteins or different polypeptides, usually with a certain flexibility, the use of linkers will not cause loss of the original function of the protein domains.

The terms "and/or" such as "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide explicit support for either or both meanings.

The terms "programmed cell death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PDCD1" and "hPD-1" are used interchangeably and include human PD-1 Variants, isoforms, species homologues, and analogs that share at least one epitope with PD-1. The complete PD-1 sequence is available in GenBank Accession No. U64863.

The term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T when bound to PD-1 Cell activation and cytokine secretion. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologues of hPD-L1, and 5 species having at least one of hPD-L1 Analogues of common epitopes. The complete hPD-L1 sequence can be found in GenBank Accession No. Q9NZQ7.

The terms "CD47", "integrin-associated protein (IAP)", "ovarian cancer antigen OA3" and "Rh-associated antigen" are synonymous and used interchangeably to refer to human CD47 comprising CD47 shown in NP_001768.1, and Any natural polymorphism of human CD47, eg comprising a single nucleotide polymorphism (SNP) or a splice variant.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

"Native antibody" refers to naturally-occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains joined by disulfide bonds. From N to C-terminus, each heavy chain has a variable domain (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or light chain variable domain, followed by a constant light (CL) domain.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Among them, the terms "complementarity determining region" and "CDR" refer to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated by screening a library of complementary VL or VH, respectively, using VH or VL from antibodies that bind to that antigen.

The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP, et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. The relationship between numbering systems including, for example, the Kabat numbering and the IMGT unique numbering system is well known to those skilled in the art and is as follows shown in Table 3.

Table 3. Relationship between CDR numbering systems

	Kabat+ChothiaKabat+Chothia	IMGTIMGT	KabatKabat	ChothiaChothia
HCDR1HCDR1	26-3526-35	26-3526-35	31-3531-35	26-3226-32
HCDR2HCDR2	50-6550-65	51-5751-57	50-6550-65	52-5652-56
HCDR3HCDR3	95-10295-102	93-10293-102	95-10295-102	95-10295-102
LCDR1LCDR1	24-3424-34	27-3227-32	24-3424-34	26-3226-32
LCDR2LCDR2	50-5650-56	50-5250-52	50-5650-56	50-5250-52
LCDR3LCDR3	89-9789-97	89-9789-97	89-9789-97	91-9691-96

Unless otherwise stated, the "Kabat" numbering convention applies to the variable regions and CDR sequences in the examples of the present disclosure.

The "class" of an antibody refers to the type of constant region possessed by its heavy chain. Antibody light chains include two types, kappa (κ) and lambda (λ), according to their constant region amino acid sequences. According to the different amino acid composition and arrangement sequence of the constant region of the antibody heavy chain, antibodies can be divided into five categories, or antibody isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chains respectively , delta chains, gamma chains, alpha chains, and epsilon chains. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Each of the five classes of Ig can have a kappa chain or a lambda chain. The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region, exemplary variants include IgG1, IgG2, IgG3, IgG4 heavy chain constant region variants with site-directed engineering and amino acid substitutions in the heavy chain constant region. In some embodiments, the substitution is a YTE mutation, a L234A and/or L235A mutation, an S228P mutation, and/or a mutation that obtains a knob-into-hole structure. These mutations have been shown to confer new properties to the antibody without altering the function of the variable region of the antibody. In some embodiments, the antibody is of the IgGl isotype with P329, P234 and P235 mutations in the hinge region to reduce effector function. In some embodiments, the antibody is of the IgG2 isotype. In some embodiments, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody.

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, diabodies, linear antibodies, single chain antibody molecules (eg, scFv); formed multispecific antibodies.

The terms "Fc domain", "Fc region" or "fragment crystallizable region" are used to define the C-terminal region of an antibody heavy chain, including native sequence Fc regions and variant Fc regions. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The boundaries of the Fc region of an antibody heavy chain may also vary, for example deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region or deletion of the C-terminal glycine and lysine (according to the EU numbering system) of the Fc region. residues 446 and 447). Thus, in some embodiments, compositions of complete fusion proteins may include a population of fusion proteins with all K447 residues and/or G446+K447 residues removed. In some embodiments, compositions of complete fusion proteins may include a population of fusion proteins without removal of K447 residues and/or G446+K447 residues. In some embodiments, the composition of complete fusion proteins has a population of fusion proteins with and without antibody mixtures of K447 residues and/or G446+K447 residues. Suitable native sequence Fc regions for the fusion proteins described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service , National Institutes of Health, Bethesda, MD, 1991.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, and the remainder of the heavy and/or light chain is derived from a different source or species.

The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (ie, the constant regions and framework portions of the variable regions).

The term "human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived using a non-human source from a collection of human antibodies or other human antibody coding sequences The amino acid sequences of the antibodies correspond. The meaning of human antibody expressly excludes humanized antibodies comprising non-human antigen-binding residues.

The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (ie kd/ka) and expressed as molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Methods for determining antibody KD include measuring surface plasmon resonance using biosensing systems such as systems, or measuring affinity in solution by solution equilibrium titration (SET).

The term "effector function" refers to those biological activities attributable to an antibody Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors) ; and B cell activation.

The term "antigen" refers to a molecule or molecular portion capable of being bound by a selective binding agent such as an antigen-binding protein (including, for example, an antibody), and otherwise capable of being used in an animal to generate an antibody capable of binding the antigen. Antigens can have one or more epitopes capable of interacting with different antigen binding proteins (eg, antibodies).

The term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. In general, binding affinity refers to the combined strength of non-covalent interactions between a molecule and its binding partner, such as high affinity SIRP-gamma peptide variants and CD47. Unless otherwise indicated, binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is usually described by the dissociation constant (KD) or the association constant (KA).

The term "KD less than" refers to a numerically smaller KD value and increased binding affinity relative to the recited KD value. As used herein, the term "KD greater than" refers to a numerically greater KD value and reduced binding affinity relative to the recited KD value.

The terms "anti-PD-1 antibody" and "anti-PD-1 binding antibody" refer to an antibody capable of binding PD-1 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD-1 . In one embodiment, the degree of binding of an anti-PD-1 antibody to an unrelated, non-PD-1 protein is less than about 10% of the binding of the antibody to PD-1, eg, by

measured by surface plasmon resonance assay. In certain embodiments, the antibody that binds to PD-1 has the following dissociation constants (KD) < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM or < About 0.001 nM (eg, 10-8M or less, eg, 10-8M to 10-12M, eg, 10-9M to 10-10M). In certain embodiments, anti-PD-1 antibodies bind PD-1 epitopes that are conserved among PD-1 from different species.

"About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, ie, the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless expressly stated otherwise in the Examples or elsewhere in the specification, "about" means within one standard deviation, or up to 5%, according to convention in the art .

The term "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a mechanism of inducing cell death that relies on antibody coating of target cells with lytically active effector cells ( Interactions such as natural killer cells (NK), monocytes, macrophages and neutrophils) occur via Fcy receptors (FcyRs) expressed on effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcyRIIIa. ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on the release of labels (eg, radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are eliminated by the internalization of phagocytic cells, such as macrophages or dendritic cells.

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-binding antibody binds and activates the complement component C1q, which in turn activates the complement cascade leading to target cell death. Activation of complement can also lead to deposition of complement components on the surface of target cells that promote CDC by binding to complement receptors (eg, CR3) on leukocytes.

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA) ). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. In particular, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which one or more selected (or all) codons are substituted at the third position by mixed bases and/or deoxy Inosine residue substitution (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

The term sequence "identity" means that when two sequences are optimally aligned, gaps are introduced as necessary to obtain the maximum percent sequence identity, and no conservative substitutions are considered part of the sequence identity, the two The degree (percent) to which amino acids/nucleic acids of sequences are identical at equivalent positions. To determine percent sequence identity, alignment can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). )software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term "conservatively modified variant" or "conservative substitution" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (eg, charge, side chain size, hydrophilicity/hydrophobicity, backbone structure, rigidity, etc.) , so that such changes can often be made without altering the biological activity of the protein. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). The term "conservatively modified variant" when applied to nucleic acid sequences refers to those nucleic acids encoding the same or substantially the same amino acid sequence, or in the case of the nucleic acid not encoding an amino acid sequence, substantially the same the sequence of. Due to the degeneracy of the genetic code, any given protein can be encoded by multiple functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where a codon specifies an alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. The skilled artisan will recognize that every codon in a nucleic acid (except AUG--usually the only codon for methionine; and TGG--usually the only codon for tryptophan) can be modified to produce function the same molecule. Thus, each silent variation of the nucleic acid encoding the polypeptide is implied in each such sequence.

The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome.

The term "expression vector" or "expression construct" refers to a device suitable for transforming a host cell and containing one or more heterologous coding regions that direct and/or control (along with the host cell) the expression of which is operably linked to it. Vectors of nucleic acid sequences. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and when introns are present, RNA splicing of the coding region to which they are operably linked.

As used herein, "operably linked" means that the components to which the term applies are in a relationship that allows them to perform their inherent functions under suitable conditions. For example, a control sequence in a vector that is "operably linked" to a protein-coding sequence is linked thereto such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamsters Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermolerans, Pichia salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomyces cerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum , Physcomitrella patens and Neurospora crassa. Pichia, any Saccharomyces cerevisiae, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Luc Mold (Chrysosporium lucknowense), any Fusarium spp., Yarrowia lipolytica and Neurospora crassa.

As used in this application, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include primary subject cells and cultures derived therefrom, regardless of the number of passages. It will also be appreciated that not all progeny will have exactly the same DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cells from which they were screened are included.

"Optional" or "optionally" means that the subsequently described event or circumstance can, but need not, occur, and that the description includes instances where the event or circumstance occurs or instances where it does not.

The term "pharmaceutical composition" refers to a mixture comprising one or more of the antibodies or antigen-binding fragments thereof described herein and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is distinct from the active ingredient and that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products, which contain information on indications, usage, dosage, administration, combination therapy, contraindications and/or warnings on the use of such therapeutic products .

The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless indicated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

"Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means that an exogenous drug, therapeutic agent, diagnostic agent, or composition interacts with the animal, human, or biological fluid. , subject, cell, tissue, organ or biological fluid contact.

The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , fluids in the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditions culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ cultures or cell cultures.

"Treatment or treat" (and grammatical variants thereof) refers to clinical interventions that attempt to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or amelioration of Prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of a disease or slow the progression of a disease.

An "effective amount" is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or underlying causes, prevent the appearance of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with a disease state (eg lung disease). In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or to otherwise prevent, retard, delay or reverse the disease state or any other irreversible disorder in any way associated with the disease state Amount of progression of desired symptoms. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms . Full therapeutic or prophylactic effect does not necessarily occur after administration of one dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations. A "therapeutically effective amount" and a "prophylactically effective amount" may vary depending on factors such as the individual's disease state, age, sex and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status in a patient.

The term "disease associated with CD47 and/or PD-1 activity" refers to any disease or disorder caused by and/or associated with CD47 and/or PD-1 activity, eg, caused by CD47 and/or PD-1 activity and/or any disease or disorder caused by and/or associated with an increase or decrease in CD47 and/or PD-1 activity.

The terms "cancer" and "cancerous" refer to or describe a physiological disorder that is typically characterized by unregulated cell growth in mammals. Benign and malignant cancers are included in this definition. "Early stage cancer" or "early stage tumor" refers to cancer that is either non-invasive or metastatic, or classified as stage 0, stage I, or stage II cancer. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastric secretinomas and islet cell carcinomas), mesothelioma, Schwann cell tumors (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemic or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous cell carcinoma of the lung , peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastric or stomach cancer) including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer or hepatic cancer), bladder cancer, liver tumor ( hepatoma), breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer and multiple myeloma.

"Autoimmune disease" or "inflammatory disease" refers to any disease, disorder or syndrome in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, host tissue damage, or loss of tissue function. In some embodiments of the present disclosure, wherein said autoimmune disease or said inflammatory disease is selected from multiple sclerosis, rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, antibody-mediated inflammation, or Autoimmune disease, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia-reperfusion , Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmunity myositis.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the native CD38 polypeptide. Suitable antagonist molecules include, inter alia, antagonist antibodies or antibody fragments (eg, antigen-binding fragments), fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like.

Example

The present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. The experimental methods that do not specify specific conditions in the examples of this disclosure generally follow conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions suggested by raw material or commodity manufacturers. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example 1. SIRPγ mutant screening and preparation

In order to obtain a high-affinity CD47 receptor, the CD47 receptor (SIRPγ) was affinity matured by yeast display and phage display technology, and an affinity-matured library for CD47 binding was designed and prepared on the basis of SIRPγ, and high-affinity receptors were screened from them. SIRPγ mutants. At the same time, according to the crystal structure of SIRPγ, the intrachain disulfide bond was designed by computer simulation (Molecular Operating Environment software).

>SIRPγ peptide wild type

After screening, the SIRPγ variants were finally obtained as follows:

>S-46:

>S-56:

>S-57:

>S-58:

>S-59:

>S-60:

>S-61:

>S-62:

>S-63:

>S-64:

>S-65:

>S-66:

>S-8Q:

>S-10Q:

>S-12Q:

>S-14Q:

>S-15Q:

>S-20Q

>S-21Q

>S-8L

>S-12L

>S-14L

>S-15L

>S-20L

>S-30

>S-31

>S-32

>S-33

>S-34

>S-35

>S-36

>S-37

Further mutation of S-10Q and S-12Q resulted in the following mutants:

>S-10Qm:

>S-12Qm

Example 2. Construction and expression of PD-1-SIRPγ fusion protein

The SIRPγ mutant is linked directly or through a linker to the anti-PD-1 antibody, wherein the SIRPγ mutant can be linked to the C-terminal or N-terminal of the heavy chain or light chain of the anti-PD-1 antibody to construct different forms of fusion proteins. Wherein, the linker can be: (G ₄ S)nGx, wherein x and n are independently selected from integers from 1-10.

Exemplary linkers are as follows:

Table 4. Linker sequences

Exemplarily, the SIRPγ mutant is linked to the C-terminus of the heavy chain of the anti-PD-1 antibody through a linker, and then the resulting heavy chain of the anti-PD-1 antibody comprising the SIRPγ mutant is linked to the light chain of the anti-PD-1 antibody. After recombination, a tetrapeptide fusion protein containing two identical polypeptides was obtained (see Figure 4A for the structure).

Based on the following sequences, the heavy chain of the fusion protein containing the SIRPγ mutant and the light chain gene fragment of the anti-PD-1 antibody were synthesized, and the heavy chain gene and light chain gene were cloned into eukaryotic expression vectors, respectively, to form a heavy chain plasmid and light chain plasmid. The expression vector is then introduced into eukaryotes to express the antibody fusion protein. The cell culture supernatant was purified with MabSelect Sure affinity column to obtain the expression product.

The amino acid sequence of the light chain of an exemplary anti-PD-1 antibody fusion protein in the present disclosure is as follows:

(1) The relevant sequence of the fusion protein obtained by fusion with Hu-23-11 antibody:

>Antibody fusion protein light chain amino acid sequence (fusion protein second chain)

The amino acid sequence of an exemplary anti-PD-1 antibody fusion protein heavy chain in the present disclosure is as follows:

>4646 heavy chain amino acid sequence (fusion protein first chain)

>4656 heavy chain amino acid sequence (fusion protein first chain)

>4657 heavy chain amino acid sequence (fusion protein first chain)

>4658 heavy chain amino acid sequence (fusion protein first chain)

>4659 heavy chain amino acid sequence (fusion protein first chain)

>4660 heavy chain amino acid sequence (first chain of fusion protein)

>4661 heavy chain amino acid sequence (fusion protein first chain)

>4662 heavy chain amino acid sequence (fusion protein first chain)

>4663 heavy chain amino acid sequence (fusion protein first chain)

>4664 heavy chain amino acid sequence (fusion protein first chain)

>4665 heavy chain amino acid sequence (fusion protein first chain)

(2) The relevant sequence of the fusion protein obtained by fusion with Hu-33-5 antibody:

>Fusion protein second chain (light chain):

>5048 first strand sequence (fusion protein second strand):

>5047 first strand sequence:

Example 3. Construction and expression of SIRPγ-Fc fusion proteins

The SIRPγ mutant was linked to Fc to construct a SIRPγ-Fc fusion protein. In some embodiments, wherein the Fc comprises an Fc variant containing at least one amino acid mutation.

Exemplary Fc domain monomer sequences are as follows:

Fc-1:

Fc-2:

Fc-3:

Exemplarily, a SIRPγ-Fc fusion protein is a dipeptide structure comprising two identical polypeptide chains, wherein the N-terminus of the SIRPγ mutant is fused directly or through a linker to the C-terminus of the Fc, or the The C-terminus of the SIRPγ mutant was directly fused to the N-terminus of Fc, resulting in a SIRPγ-Fc fusion protein (see Figure 4B and Figure 4C for the structure).

Different gene fragments were synthesized based on the following sequences, and the genes were cloned into eukaryotic expression vectors. Then, the expression vector was introduced into eukaryotes to express the SIRPγ-Fc fusion protein, and the supernatant after cell culture was purified by MabSelect Sure affinity column to obtain the expression product.

The exemplary SIRPγ-Fc fusion protein amino acid sequence of the present disclosure is as follows:

>4666

>8QCGC

>10QCGC

>12QCGC

>14QCGC

>15QCGC

>20QCGC

>21QCGC

>8LCGC

>10LCGC

>12LCGC

>14LCGC

>15LCGC

>20LCGC

>21LCGC

>56-Fc

>57-Fc

>58-Fc

>60-Fc

>61-Fc

>62-Fc

>30-Fc

>31-Fc

>32-Fc

>33-Fc

>34-Fc

>35-Fc

>36-Fc

>37-Fc

>4854(10QCGCm)

>4855(12QCGCm)

>4924

>4845

The following proteins were also prepared and purified by conventional methods as control proteins:

HX009 is an anti-CD47 and anti-PD-1 bispecific antibody, prepared with reference to WO2019109357, and its sequence is as follows:

>HX009 heavy chain amino acid sequence

>HX009 light chain amino acid sequence

ALX148 is a SIRPα-Fc fusion protein, prepared with reference to US10259859, and its sequence is as follows;

>ALX148

>SIRPγWT-Fc

>CD47-Fc

The present disclosure also uses an unrelated antibody against HIV, C25, as a negative control.

The anti-CD38 antibody used in the test example is the hu11E antibody disclosed in the WO2020052546 patent, and its sequence is as follows:

>hu11E antibody heavy chain:

>hu11E antibody light chain:

> hu11E antibody heavy chain variable region:

> hu11E antibody light chain variable region:

CDR region of hu11E antibody:

>HCDR1:DYGMH (SEQ ID NO: 119)

>HCDR2: FISSGSSSIYYADTVKG (SEQ ID NO: 120)

>HCDR3:NYVSSYGYFDY (SEQ ID NO: 121)

>LCDR1:RASENVDNYGISFMH (SEQ ID NO: 122)

>LCDR2:RASNLES (SEQ ID NO: 123)

>LCDR3:QQSNKDPLT (SEQ ID NO: 124).

test case

Test Example 1. Affinity test of PD-1-SIRPγ fusion protein detected by BIAcore

Affinity capture of IgG with Protein A biosensor chip (Cat.#29127556, GE) makes different antigens (hCD47: Cat.#12283-H08H, Lot.#LC12OC2201, SB; hPD-1: Cat.#10377-H08H , Lot. #LC13AU2111, SB) flowed over the chip surface, and the Biacore T200 instrument detected the fusion protein and different antigen reaction signals in real time to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl pH 1.5 buffer. The experimental buffer system was 1×HBS-EP buffer solution (Cat#BR-1001-88, GE). After the experiment, GE Biacore T200 Evaluation version 3.0 software was used to fit the data with the (1:1) Langmuir model, and the affinity values were obtained. The results are shown in Table 5.

The results show that the affinity of the SIRPγ variant fusion protein in this application with human CD47 is significantly higher than that of the wild-type SIRPγ peptide (the affinity of wild-type SIRPγ and CD47 is about 23 μM), and the PD-1-SIRPγ fusion protein has the same affinity with human PD-1-SIRPγ. The affinity of 1 was comparable to that of PD-1 naked anti-hu23-11, and higher than that of the bispecific antibody HX009 against CD47 and PD-1; the affinity of SIRPγ-Fc fusion protein to human CD47 was significantly higher than that of wild-type SIRPγ peptide.

Table 5. Affinities of fusion proteins

Test Example 2. Experiment of fusion protein binding to human erythrocytes

Fresh healthy human blood was mixed with equal volume of PBS, centrifuged at 300 g for 5 min to obtain cell clusters, and washed with PBS for 3-5 times to obtain red blood cells. Resuspend with FACS buffer (PBS+5%BSA), adjust the cell density to 2×10 ⁶ cells/mL, and seed them into 96-well round bottom plates (3795#, corning) according to 100 μL/well, and then add different concentrations of antibodies Or fusion protein, with C25 as negative control. Incubate for 1 hour at 4°C. Then, after washing twice with FACS buffer (PBS+2% FBS), secondary antibody (Alexa 488 goat anti-human IgG antibody: Invitrogen, CAT#A11013) was added and incubated on ice for 30 minutes in the dark. Finally, the cells were resuspended after washing twice with FACS buffer. Plates were read in a FACS Cantoll. The results are shown in Figure 1.

FACS test results show that the control CD47 antibody hu5F9 (prepared with reference to US9017675) and the SIRPα-Fc fusion protein ALX148 have strong binding ability to the natural CD47 on the surface of human erythrocytes, while the PD-1-SIRPγ fusion protein in this application The native CD47 on the surface of human erythrocytes has little or no binding ability. The native CD47 binding of SIRPγ-Fc to the surface of erythrocytes was weaker than that of the control antibody and fusion protein, suggesting the advantages of the above-mentioned SIRPγ-Fc and PD-1-SIRPγ fusion proteins in terms of safety.

Test Example 3. Fusion protein binding to tumor cells experiment

Karpas 299 cells (Shanghai Zeye Biotechnology Co., Ltd.) were cultured in DMEM F12 medium (containing 10% fetal bovine serum), 1×10 ⁶ cells/mL Karpas 299 cells were blocked with 5% BSA, and the sample was added to 10 μg After washing twice, Alexa Fluor 488-goat anti-human (H+L) antibody (Invitrogen, CAT#A11013) was added. After washing twice, the fluorescence signal value was read by flow cytometer. The results are shown in Figures 2A-2F.

The FACS test results showed that the PD-1-SIRPγ fusion protein and the SIRPγ-Fc fusion protein in the present disclosure have strong binding ability to the native CD47 on the surface of Karpas 299 cells, which is better than the control bispecific antibody HX009.

Test Example 4. Pharmacodynamic experiment of SIRPγ fusion protein in mouse Karpas299 model reconstituted by human PBMC

In this test case, the Karpas299 model of NDG mice (Beijing Biositu Gene Biotechnology Co., Ltd.) reconstructed by human PBMC was used to evaluate the antitumor efficacy of PD-1-SIRPγ fusion protein in mice.

Karpas299 cells (1×10 ⁵ cells/cell/100 μL, containing 50 μL Matrigel) were subcutaneously inoculated into the right flank of female NDG mice, and the PBMCs of the two donors were mixed at a ratio of 1:1 at a rate of 5×10 ⁶ cells/ 100 μL/mouse was injected into the abdominal cavity of mice. When the tumor volume of the tumor-bearing mice reached about 160 mm ³ , the mice were randomly divided into groups of 7-8 mice. The day of the grouping was defined as the day 0 of the experiment (Day 0), and the samples to be tested were injected intraperitoneally on day 0. , 2 times a week for a total of 2 weeks, a total of 4 times, 2 times a week to monitor the tumor volume, animal weight and record the data. All data were graphed and statistically analyzed using Excel and GraphPad Prism 5 software.

The formula for calculating tumor volume (V) is: V=1/2×a×b ² where a and b represent length and width, respectively.

Relative tumor proliferation rate T/C(%)=(T-T0)/(C-C0)×100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. tumor volume.

Tumor inhibition rate TGI (%)=1-T/C (%).

The results (see Figure 3 and Table 6) showed that the tumor inhibition rate of the PD-1 antibody hu23-11-8.5mpk was only 10.24% after 11 days of administration. The tumor inhibition rates of high dose 4.4mpk and low dose 0.4mpk of Fc-SIRPγ fusion protein 4666 were 77.05% (p<0.001) and 33.04% (p<0.01), respectively. The tumor inhibition rate of bispecific antibody HX009-10mpk was 60.00% (p<0.001).

The high-dose 10mpk and low-dose 1mpk tumor inhibition rates of PD-1-SIRPγ fusion protein 4646 were 92.14% (p<0.001) and 41.59% (p<0.01), respectively, which were significantly better than high-dose PD-1 antibody hu23- 11 and the same dose of bispecific antibody HX009.

Table 6. Tumor inhibition rate of different proteins

蛋白protein	剂量dose	抑瘤率％(Day11)Tumor inhibition rate % (Day11)
hu23-11hu23-11	8.5mpk8.5mpk	10.2410.24
46664666	4.4mpk4.4mpk	77.0577.05
46664666	0.4mpk0.4mpk	33.0433.04
HX009 HX009		10mpk10mpk	6060
46464646	10mpk10mpk	92.1492.14
46464646	1mpk1mpk	41.5941.59

Test Example 5. Hemagglutination experiment of fusion protein

Fresh healthy human blood was diluted 100 times with PBS (B320#, Shanghai Yuanpei Biotechnology Co., Ltd.). The diluted whole blood was plated on a 96-well round bottom plate (3795#, corning), 30 μL/well. Then equal volumes of different concentration gradients of antibody or bifunctional fusion protein were added. After mixing, let stand at 37°C for 4-6 hours. Erythrocyte sedimentation was observed with high-content microscopy. No blood coagulation was seen as a clear red spot, and blood coagulation was diffuse.

Each sample was diluted 1:3 from the first column (0.25 mg/mL) onwards to column 10. Column 11 is a PBS blank well with no antibody added. The results are shown in Figure 5.

The results showed that under the same conditions, PD-1-SIRPγ fusion protein and SIRPγ-Fc fusion protein did not cause hemagglutination at different concentrations tested. The advantages of the PD-1-SIRPγ fusion protein and the SIRPγ-Fc fusion protein of the present disclosure are suggested in terms of safety.

Test Example 6. Experiment of PD-1-SIRPγ bifunctional fusion protein to stimulate PBMC-T lymphocytes to secrete IFNγ

In order to study the effect of PD-1-SIRPγ bifunctional fusion protein on the function of human primary T lymphocytes, human peripheral blood mononuclear cells (PBMC) were collected and purified, and after 5 days of in vitro stimulation with tuberculin (TB), cytokines were detected. IFNγ secretion levels. The experimental process is briefly described as follows:

Fresh blood was obtained by Ficoll-Hypaque (17-5442-02, GE), density gradient centrifugation (Stem Cell Technologies) to obtain PBMC, and cultured in RPMI 1640 (SH30809.01, GE) medium supplemented with 10% ( v/v) FBS (10099-141, Gibco), cultured at 37°C, 5% CO ₂ .

Freshly isolated and purified PBMCs were adjusted to a density of 2×10 ⁶ cells/mL in RPMI 1640 medium, 40 μL of tuberculin (97-8800, Synbiotics) was added to 20 mL of cell suspension, and cultured in a 37°C, 5% CO ₂ incubator for 5 sky. On the 5th day, the cultured cells were collected and centrifuged, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1.1×10 ⁶ cells/mL, and seeded into a 96-well cell culture plate with 90 μL per well. At the same time, serially diluted antibody or fusion protein samples were added, diluted with PBS (B320, Shanghai Yuanpei Biotechnology Co., Ltd.), 10 μL per well. Place the cell culture plate in a 37°C, 5% _CO2 incubator for 3 days. The cell culture plate was taken out, and the cell culture supernatant was collected by centrifugation (4000 rpm, 10 min), and the level of IFNγ was detected by ELISA (human IFN-γ detection kit: EHC102g.96, Xinbosheng). Refer to the reagent manual for specific operations. The results are shown in Tables 7-8 and Figures 6A and 6B.

Table 7. Results of fusion protein activation of PBMC to secrete IFNγ

样品sample	EC50(nM)EC50(nM)	Emax％ Emax%
46564656	0.1560.156	156.517156.517
hu23-11hu23-11	5.4315.431	117.468117.468
HX009HX009	0.1360.136	100100

Table 8. Results of fusion protein activation of PBMC to secrete IFNγ

样品sample	EC50(nM)EC50(nM)	Emax％ Emax%
46464646	0.1540.154	134.811134.811
46584658	0.2770.277	147.607147.607
HX009HX009	0.1450.145	100100

The results showed that all bifunctional fusion proteins could activate the secretion of IFN-γ and outperformed the control bispecific antibody HX009.

Test Example 7. Therapeutic effect of SIRPγ-Fc fusion protein on human multiple myeloma cell MOLP-8 xenograft tumor

MOLP-8 cells 5×10 ⁶ cells/cell/200 μL (containing 50% Matrigel) were inoculated subcutaneously in the right flank of 98 Balb/c nude mice, and when the tumor volume of the tumor-bearing mice reached 160 mm ³ The mice were randomly divided into 5 groups, with 7 mice in each group (the molecules to be tested were kept at equimolar concentration). The day of grouping was defined as the 0th day of the experiment (Day0), and each fusion protein or antibody was intraperitoneally injected twice a week. From the 8th day onwards, the frequency of administration of 4855 and 4845 was increased to three times a week, and the administration of CD38 antibody hu11E was stopped. , a total of 17 days of administration (Day17). Tumor volumes, animal weights were monitored and data recorded twice weekly. Tumor-bearing animals were euthanized as experimental endpoints when tumor volume exceeded 1500 mm ³ or when most tumors ruptured or when body weight decreased by 20%.

The CD38 antibody used in the experiment was the hu11E antibody disclosed in the WO2020052546 patent.

All data were graphed and statistically analyzed using Excel and GraphPad Prism 5 software.

Relative tumor proliferation rate T/C(%)=(TT ₀ )/(CC ₀ )×100 where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T ₀ and C ₀ are the tumors at the beginning of the experiment volume.

Tumor inhibition rate TGI (%)=1-T/C (%).

At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, and the tumor weight was measured. The results showed that the tumor weight in vitro was basically consistent with the tumor volume trend. Tumor-bearing mice tolerated each specific antibody and its monoclonal antibody well, with only slight fluctuations in body weight during the entire administration process, and no obvious drug-induced weight loss and other symptoms. The results are shown in Table 9 and Figure 7.

Table 9. Pharmacological effects of SIRPγ-Fc fusion protein on subcutaneous xenografts of MOLP-8 mice

Note: Day0: the time of the first administration; 4855 and 4845 increase the administration frequency to three times a week after administration for seven days, and stop the administration of CD38 antibody; *p<0.05vs Vehicle by student T test.

The experimental results show that in the human multiple myeloma cell MOLP-8 mouse subcutaneous tumor model, the CD38-specific antibody hu11E, SIRPγ-

Fc test molecules

4855 and 4845 alone are effective against the human multiple myeloma cell MOLP-8 small cells. The growth of subcutaneously transplanted tumors in mice has a certain inhibitory effect, and the tumor inhibition rates are 84.86%, 33.89% and 32.58%, respectively. After the combination of CD38 antibody hu11E and 4855, the tumor inhibition rate was more than 100%, and there was a significant difference compared with the CD38 antibody alone group (p<0.05).

Test Example 8. The efficacy of PD-1-SIRPγ bifunctional fusion protein on human breast cancer cell MDA-MB-231 xenograft tumor

MDA-MB-231 cells (ATCC) 3×10 ⁶ cells/200 μL/cell (containing 50% Matrigel) were inoculated subcutaneously in the right flank of NOD/SCID mice, when the average tumor volume of the tumor-bearing mice reached about 178 mm ³ The mice were randomly divided into 9 groups: PBS, 4646(10mpk), 5048(10mpk), 5047(10mpk), 4658(10mpk, 3mpk), 4924(4.4mpk), HX009(10mpk), hu23-11(8.6 mpk), 7 animals in each group, and the day of grouping was defined as Day 0 of the experiment. On Day 0, the PBMCs of the two volunteers stimulated with CD3 antibody for 3 days were mixed at a ratio of 1:1 and injected into the mouse tumor tissue at 5×10 ⁵ cells/100 μL/mouse. The remaining PBMCs were stopped stimulated and continued to be cultured. One week later, 5×10 ⁶ cells/100 μL/mice were intraperitoneally injected into tumor-bearing mice, which was regarded as the first round of injection, and two rounds of PBMCs were injected until the end of the experiment. Starting from Day 0, each fusion protein or antibody to be tested was injected intraperitoneally twice a week. Tumor volumes, animal weights were monitored and data recorded twice weekly. Tumor-bearing animals were euthanized as experimental endpoints when tumor volume exceeded 1000 ^mm3 or when most tumors ruptured or lost 20% of body weight.

Data were recorded using Excel 2007 statistical software, the mean value was calculated by avg; SD value was calculated by STDEV; SEM value was calculated by STDEV/SQRT. Graphpad Prism8 was used for graph processing and statistical analysis, and the difference P value between groups was analyzed by variance analysis.

Tumor volume: V = 1/2 × L _long × L _short ²

Relative tumor proliferation rate: T/C(%)=[(T-T0)/(C-C0)]×100%

Tumor inhibition rate: TGI (%)=1-T/C (%).

Among them, T0 and T are the tumor volumes of the treatment group at the beginning and end of the experiment, respectively. C ₀ and C are the tumor volumes of the vehicle control group at the beginning of the experiment and at the end of the experiment, respectively.

In the experiment, the test substance was administered by intraperitoneal injection twice a week for a total of four weeks, and the tumor volume data on the 32nd day after group administration was plotted and statistically analyzed. The results are shown in Table 10 and FIG. 8 .

Table 10. The efficacy of fusion protein on MDA-MB-231 xenografts

The results showed that the inhibitory effect of 4646, 5048, 5047 and 4658 on MDA-MB-231 xenograft tumor was significantly better than that of the control bifunctional molecule HX009 at the same dose of 10mpk. The tumor inhibitory effect of 4658 was dose-dependent.

Test Example 9. Aggregation degree of fusion protein

Aggregation degree monitoring was performed by SEC-HPLC. A Waters e2695 chromatograph was used, the chromatographic column was Waters Xbridge BEH 200A SEC, the mobile phase was PBS (adjusted to pH 6.8 with dilute hydrochloric acid), 50 μg of protein was injected, and the isocratic elution was performed at a flow rate of 0.5 mL/min. The results are shown in Table 11.

Table 11. Fusion protein purity

融合蛋白fusion protein	SEC单体(％)SEC monomer (%)
8QCGC8QCGC	96.4896.48
10QCGC10QCGC	97.6897.68
48544854	99.0699.06
12QCGC12QCGC	9797
48554855	98.6998.69
14QCGC14QCGC	96.7896.78
15QCGC15QCGC	98.5898.58
20QCGC20QCGC	96.9896.98

After investigation, the SEC purity of the fusion molecules of the present disclosure are all above 95%.

Claims

A SIRPγ variant comprising the amino acid mutation shown in any one of i) to ii) below relative to the SIRPγ peptide shown in SEQ ID NO: 14:

i) L14C and G115C; or

ii) Q8C and G107C.
The SIRPγ variant of claim 1, further comprising an amino acid mutation selected from one or more of K19E, Q52S, K53G, E54R, M72K and N101D;

Preferably, wherein the SIRPγ variant further comprises an N51A or N51M amino acid mutation.
The SIRPγ variant of claim 1 or 2, further comprising L13V and/or M6I amino acid mutations.
The SIRPγ variant of claim 3, further comprising one or more amino acid mutations selected from the group consisting of H56Q, N70E, R77K, S79Q and M112V.
The SIRPγ variant of any one of claims 1 to 4, further comprising an amino acid mutation selected from any of the following a) to r):

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

i) M6I, L13V, N51M and R77K;

j) M6I, L13V, N51A and R77K;

k) M6I, L13V, N51A and N70E;

l) N51A and R77K;

m) L13V, N51M and R77K;

n) L13V, N51M and N70E;

o) N51A;

p) L13V and N51M;

q) N51A and M112V; and

r) N51M;

Preferably, the SIRPγ variant comprises:

i) L14C and G115C mutations;

ii) K19E, Q52S, K53G, E54R, M72K and N101D mutations; and

iii) is selected from the amino acid mutations shown in any one of the following a)-r):

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) N51A;

g) M6I, N51M and R77K;

h) M6I, N51A and R77K;

j) M6I, N51A and N70E;

k) M6I, L13V, N51M and R77K;

l) M6I, L13V, N51A and R77K;

m) M6I, L13V, N51A and N70E;

n) N51A and R77K;

o) L13V, N51M and R77K;

p) L13V, N51M and N70E;

q) L13V and N51M; and

r) N51M; or

The SIRPγ variant comprises:

i) Q8C and G107C mutations;

ii) K19E, Q52S, K53G, E54R, M72K and N101D mutations; and

iii) is selected from the amino acid mutations shown in any one of the following a)-p):

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V and N51M;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

j) M6I, L13V, N51M and R77K;

k) M6I, L13V, N51A and R77K;

l) M6I, L13V, N51A and N70E;

m) N51A and R77K;

n) L13V, N51A and N70E;

o) N51A; and

p) N51A and M112V.
The SIRPγ variant of claim 1, wherein the SIRPγ variant comprises the amino acid sequence shown below:

EEELQX 1 IX 2 PEKLX 3 X 4 VTVGX 5 TATLHCTVTSLLPVGPVLWFRGVGPGRELIYX 6 X 7 X 8 X 9 GX 10 FPRVTTVSDLTKRX 11 NX 12 DFSIX 13 IX 14 SITPADVGTYYCVKFRKGSPEX 15 VEFKSX 16 PGTEX 17 ALX 18 AKPS;

SEQ ID NO: 13

in:

X 1 is selected from M or I; X 2 is selected from Q or C; X 3 is selected from L or V; X 4 is selected from L or C; X 5 is selected from K or E; X 6 is selected from N, M or A; X 7 is selected from Q or S; X 8 is selected from K or G; X 9 is selected from E or R; X 10 is selected from H or Q; X 11 is selected from N or E; X 12 is selected from M or K; X 13 is selected from R or K; X 14 is selected from S or Q; X 15 is selected from N or D; X 16 is selected from G or C; X 17 is selected from M or V; X 18 is selected from G or C; and

i) when X4 is C, X18 is C; or

ii) When X2 is C, X16 is C.
The SIRPγ variant of claim 6, wherein:

a) X 5 is E;

b) X 7 is S;

c) X 8 is G;

d) X 9 is R;

e) X 12 is K; and/or

f) X 15 is D;

Preferably, wherein X 6 is A or M.
SIRPγ variant according to claim 7 , wherein X3 is V and/or X1 is I.
The SIRPγ variant of claim 8, wherein

a) X 10 is Q;

b) X 11 is E;

c) X 13 is K;

d) X 14 is Q; or

e) X 17 is V.
The SIRPγ variant of any one of claims 6 to 9, wherein:

a) X 3 is V, X 6 is A and X 14 is Q;

b) X 3 is V, X 6 is A and X 13 is K;

c) X 3 is V, X 6 is A and X 17 is V;

d) X 3 is V, X 6 is M, X 10 is Q and X 11 is E;

e) X3 is V and X6 is M ;

f) X 1 is 1, X 6 is M and X 13 is K;

g) X 1 is 1, X 6 is A and X 13 is K;

h) X 1 is 1, X 6 is A and X 11 is E;

i) X 1 is 1, X 3 is V, X 6 is M and X 13 is K;

j) X 1 is 1, X 3 is V, X 6 is A and X 13 is K;

k) X 1 is 1, X 3 is V, X 6 is A and X 11 is E;

1) X 6 is A and X 13 is K;

m) X3 is V, X6 is M and X13 is K ;

n) X 3 is V, X 6 is M and X 11 is E;

o) X3 is V, X6 is A and X11 is E;

p) X 6 is A;

q) X6 is A and X17 is V; or

r) X 6 is M.
The SIRPγ variant of any one of claims 1 to 10, wherein the SIRPγ variant is a high-affinity SIRPγ variant;

Preferably, the SIRPγ variant binds to human CD47 with a KD of less than 2×10 −9 M.
The SIRPγ variant of any one of claims 1 to 11, comprising an amino acid sequence selected from any of SEQ ID NOs: 15-46 or SEQ ID NOs: 105-106.
A fusion protein comprising the SIRPγ variant of any one of claims 1-12.
The fusion protein according to claim 13, further comprising an anti-PD-1 antibody; preferably, wherein the anti-PD-1 antibody comprises:

A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 1, 2 and 3, respectively; and

A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NOs: 4, 5 and 6, respectively; or

A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 95, 96 and 97, respectively; and

A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NOs: 98, 99 and 100, respectively.
The fusion protein of claim 14, wherein the anti-PD-1 antibody comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8; Or comprise a heavy chain variable region as set forth in SEQ ID NO:101 and a light chain variable region as set forth in SEQ ID NO:102.
The fusion protein according to claim 15, wherein the anti-PD-1 antibody further comprises a constant region; preferably, the anti-PD-1 antibody comprises the heavy chain constant region shown in SEQ ID NO: 9 and The light chain constant region as set forth in SEQ ID NO:10.
The fusion protein of claim 16, wherein the anti-PD-1 antibody comprises:

A heavy chain comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11; and

A light chain comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12; or

A heavy chain comprising the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 103; and

A light chain comprising the amino acid sequence of SEQ ID NO:104, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:104.
The fusion protein of any one of claims 14 to 17, wherein the SIRPγ variant comprises amino acid mutations of K19E, N51A or N51M, Q52S, K53G, E54R, M72K and N101D, and is selected from i) or ii ) amino acid mutation, where:

i) L14C and G115C;

ii) Q8C and G107C.
The fusion protein of claim 18, wherein the SIRPγ variant further comprises one or more amino acid mutations selected from the group consisting of M6I, L13V, N70E, R77K and M112V.
The fusion protein according to claim 18 or 19, wherein the SIRPγ variant comprises an amino acid mutation selected from any one of the following a) to m):

a) L13V and N51M;

b) M6I, N51M and R77K;

c) M6I, N51A and R77K;

d) M6I, N51A and N70E;

e) N51A and R77K;

f) M6I, L13V, N51M and R77K;

g) M6I, L13V, N51A and R77K;

h) M6I, L13V, N51A and N70E;

i) L13V, N51A and R77K;

j) L13V, N51M and R77K;

k) L13V, N51M and N70E; and

m) N51A and M112V.
The fusion protein of any one of claims 14 to 20, wherein the SIRPγ variant is linked to the anti-PD-1 antibody directly or through a linker;

Preferably, the N-terminus of the SIRPγ variant is linked to the C-terminus of the anti-PD-1 antibody heavy chain through a linker;

More preferably, wherein said fusion protein comprises:

A first peptide chain comprising any amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59; and/or

a second peptide chain comprising the amino acid sequence of SEQ ID NO: 12; or

A first peptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 107 or 108; and/or

A second peptide chain comprising the amino acid sequence of SEQ ID NO:104.
The fusion protein according to claim 21, wherein the fusion protein comprises two identical first and second peptide chains, wherein:

A first peptide chain comprising any amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59; and a second peptide chain comprising the amino acid sequence of SEQ ID NO: 12; or

A first peptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 107 or 108; and a second peptide chain comprising the amino acid sequence of SEQ ID NO: 104;

Preferably, the fusion protein comprises two identical first and second peptide chains, wherein:

a first peptide chain comprising the amino acid sequence of SEQ ID NO:49; and a second peptide chain comprising the amino acid sequence of SEQ ID NO:12; or

a first peptide chain comprising the amino acid sequence of SEQ ID NO:52; and a second peptide chain comprising the amino acid sequence of SEQ ID NO:12; or

A first peptide chain comprising the amino acid sequence of SEQ ID NO:108; and a second peptide chain comprising the amino acid sequence of SEQ ID NO:104.
The fusion protein of claim 13, further comprising a human Fc domain monomer; preferably, wherein the Fc domain monomer comprises at least one amino acid mutation; more preferably,

wherein said Fc domain monomer comprises one or more amino acid mutations selected from the group consisting of 234A, 235A, G237A and N297A.
The fusion protein of claim 23, wherein the SIRPγ variant comprises an amino acid mutation selected from any one of i) to ii) below:

i) L14C and G115C; or

ii) Q8C and G107C;

And, wherein said SIRPγ variant further comprises amino acid mutation of K19E, N51A or N51M, Q52S, K53G, E54R, M72K and N101D;

Preferably, wherein said SIRPγ variant further comprises one or more amino acid mutations selected from M6I, L13V, H56Q, N70E, R77K, S79Q and M112V.
The fusion protein of claim 24, wherein the SIRPγ variant comprises an amino acid mutation selected from any one of the following a) to p):

a) L13V, N51A and S79Q;

b) L13V, N51A and R77K;

c) L13V, N51A and M112V;

d) L13V, N51M, H56Q and N70E;

e) L13V, N51A and N70E;

f) M6I, N51M and R77K;

g) M6I, N51A and R77K;

h) M6I, N51A and N70E;

i) M6I, L13V, N51M and R77K;

j) M6I, L13V, N51A and R77K;

k) M6I, L13V, N51A and N70E;

l) N51A;

m) L13V and N51M;

n) N51A and R77K;

o) N51A and M112V; and

p) N51M.
The fusion protein of any one of claims 23 to 25, wherein the Fc domain monomer comprises the amino acid sequence of SEQ ID NO: 60, 61 or 109;

Preferably, wherein the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 62-90 or SEQ ID NOs: 110-113;

More preferably, the fusion protein is a homodimer comprising two identical amino acid sequences selected from any one of SEQ ID NOs: 62-90 or SEQ ID NOs: 110-113.
A pharmaceutical composition comprising a therapeutically effective amount of a SIRPγ variant according to any one of claims 1 to 12, or a fusion protein according to any one of claims 13 to 26, and one or Various pharmaceutically acceptable carriers, buffers or excipients.
A nucleic acid molecule encoding the SIRPγ variant of any one of claims 1-12, or the fusion protein of any one of claims 13-26.
An expression vector comprising the nucleic acid molecule of claim 28.
A host cell comprising the expression vector of claim 29.
A method of treating an individual suffering from a disease or condition, the method comprising administering to the individual suffering from the disease or condition a therapeutically effective amount of the SIRPγ variant according to any one of claims 1 to 12, or according to the The fusion protein of any one of claims 13 to 26, or the pharmaceutical composition of claim 27, or the nucleic acid molecule of claim 28, or the expression vector of claim 29.
The method of claim 31, wherein the disease or disorder is cancer, an autoimmune disease, or an inflammatory disease;

Preferably, the cancer is selected from the group consisting of: solid tumor, hematological cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma Gold lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bronchial cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer, gallbladder Carcinoma, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, Merkel cell carcinoma, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma and brain tumor; wherein said autoimmune disease or said inflammatory disease is selected from: multiple sclerosis , rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjogren's syndrome , progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia-reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary Fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis;

More preferably, wherein said disease or disorder is associated with CD47 and/or PD-1.
The method of claim 31 or 32, further comprising administering to an individual suffering from a disease or disorder a therapeutically effective amount of a CD38 antagonist; preferably, wherein the CD38 antagonist is an anti-CD38 antibody; more preferably, The anti-CD38 antibody described therein comprises:

A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 119, 120 and 121, respectively; and a light chain variable region comprising set forth in SEQ ID NOs: 122, 123 and 124, respectively LCDR1, LCDR2 and LCDR3; most preferably, wherein said anti-CD38 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 117 and a light chain variable region as shown in SEQ ID NO: 118; Optionally, wherein said anti-CD38 antibody comprises a heavy chain as shown in SEQ ID NO:115 and a light chain as shown in SEQ ID NO:116.