Bifunctional fusion proteins and medical uses thereof

Abstract

The application relates to a bifunctional fusion protein and its medical use. Specifically, the application relates to a bifunctional fusion protein containing a SIRP gamma peptide variant and an anti-human PD-L1 antibody, the SIRP gamma peptide variant and its medical use. According to the bifunctional fusion protein, specific binding of PD-L1 and CD47 can be blocked, and the combination of PD-L1 or CD47 with its receptor or ligand is blocked. Meanwhile, the preparation, application and treatment of cancer and immune-related diseases of the bifunctional fusion protein are also provided.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080005976.1, filed on March 5, 2020, entitled "Bifunctional Fusion Protein and Its Pharmaceutical Use". Technical Field

[0002] This disclosure relates to a bifunctional fusion protein that specifically binds to PD-L1 and CD47, a pharmaceutical composition comprising the bifunctional fusion protein, and its use as an anticancer drug. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] Programmed death molecule 1 (PD-1) is a protein receptor expressed on the surface of T cells, discovered in 1992, and is involved in the apoptosis process. PD-1 belongs to the CD28 family and shares 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (CTLA-4), but its expression differs from CTLA-4, primarily being expressed on activated T cells, B cells, and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DCs), and its expression is upregulated in activated cells. PD-L2 expression is relatively limited, mainly expressed on antigen-presenting cells, such as activated macrophages and dendritic cells.

[0005] New research has revealed high expression of PD-L1 protein in human tumor tissues such as breast cancer, lung cancer, gastric cancer, colorectal cancer, kidney cancer, and melanoma, and the expression level of PD-L1 is closely related to the patient's clinical condition and prognosis. Since PD-L1 plays a role in inhibiting T cell proliferation through a second signaling pathway, blocking the binding between PD-L1 and PD-1 has become a very promising emerging target in the field of tumor immunotherapy.

[0006] The cell surface protein CD47 is expressed or overexpressed in many tumor types, including acute myeloid leukemia, various subtypes of B-cell non-Hodgkin lymphoma, and many human solid tumor cells. CD47 binds to signal regulatory protein α (SIRPα) on macrophages, acting as a "don't eat me" signal on the surface of tumor cells. Recent data suggest that anti-CD47 antibodies also help enhance effective anti-tumor T-cell responses in immune-tolerant mice. Therefore, anti-CD47 antibodies represent a novel class of immune checkpoint inhibitors that modulate both the innate and adaptive immune systems.

[0007] There are already related CD47 patents, such as WO2016065329, WO2016109415, WO2014087248, WO2014093678, CN107849143A, CN108350048, CN106535914, WO2016023001A, CN107459578A, CN2017110167989, etc. For example, WO2016023001A describes a multispecific PD-1 mimic peptide containing a high-affinity PD-1 mimic peptide and a high-affinity SIRP-α that specifically binds to CD47, and its uses; CN107459578A describes a recombinant fusion protein containing a SIRPα mutant and an anti-PD-L1 antibody that targets CD47 and PD-L1 molecules; CN201711016798.9 discloses a multifunctional fusion protein containing the extracellular portion of SIRPα and the extracellular portion of PD-1.

[0008] However, many therapies currently in preclinical and clinical studies target the CD47 / SIRPα interaction, including anti-CD47 antibodies, SIRPα receptor proteins and engineered SIRPα receptor proteins, anti-SIRPα antibodies and bispecific antibodies, etc. There are no reports on multispecific fusion proteins containing SIRPγ peptides.

[0009] SIRPγ is expressed on T cells and activated NK cells, and binds to CD47 with a 10-fold lower affinity compared to SIRPα. CD47-SIRPγ interaction is involved in the contact between antigen-presenting cells and T cells, co-stimulating T cell activation and promoting T cell proliferation (Piccio et al., Blood 2005, 105, 2421-2427). Furthermore, CD47-SIRPγ interaction plays a role in the transendothelial migration of T cells (Stefanisakis et al., Blood 2008, 112, 1280-1289). Summary of the Invention

[0010] This disclosure provides a bifunctional fusion protein comprising a SIRPγ peptide variant. The SIRPγ peptide variant exhibits significantly enhanced CD47 affinity activity compared to the wild-type SIRPγ peptide.

[0011] In some embodiments, a bifunctional fusion protein is provided, comprising a SIRPγ peptide variant and an anti-human PD-L1 antibody, wherein the SIRPγ peptide variant is linked to the polypeptide chain of the anti-human PD-L1 antibody.

[0012] The SIRPγ peptide variant is a SIRPγ peptide variant with a substitution mutation at position N51, corresponding to the wild-type SIRPγ peptide shown in SEQ ID NO: 20. In some embodiments, the SIRPγ peptide variant as described above has activity in binding to CD47 on the surface of tumor cells, preferably, the SIRPγ peptide variant has enhanced activity in binding to CD47 on the surface of tumor cells compared to the wild-type SIRPγ peptide.

[0013] In some embodiments, a bifunctional fusion protein is provided, comprising a human SIRPγ peptide variant and an anti-human PD-L1 antibody, wherein the SIRPγ peptide variant is linked to the polypeptide chain of the anti-human PD-L1 antibody.

[0014] The SIRPγ peptide variant is a SIRPγ peptide variant with a substitution mutation at position N51 of the wild-type SIRPγ peptide as shown in SEQ ID NO: 20. In some embodiments, the SIRPγ peptide variant as described above has activity in binding to CD47 on the surface of tumor cells; preferably, the SIRPγ peptide variant has enhanced activity in binding to CD47 on the surface of tumor cells compared to the wild-type SIRPγ peptide. In some embodiments, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant is directly linked to the polypeptide chain of the anti-human PD-L1 antibody by a peptide bond or covalently linked via a linker. Preferably, the linker may be selected from any of the linkers shown in SEQ ID NO: 89-96 and (GGGGS)n, (GGGES)n, and (GKPGS)n, where n = 2-7.

[0015] In some embodiments, as described above, the bifunctional fusion protein wherein the carboxyl terminus of the SIRPγ peptide variant is linked to the amino terminus of the heavy chain variable region of the anti-human PD-L1 antibody,

[0016] Alternatively, the carboxyl terminus of the SIRPγ peptide variant may be linked to the amino terminus of the light chain variable region of the anti-human PD-L1 antibody.

[0017] Alternatively, the carboxyl terminus of the heavy chain of the anti-human PD-L1 antibody may be linked to the amino terminus of the SIRPγ peptide variant.

[0018] Alternatively, the carboxyl terminus of the light chain of the anti-human PD-L1 antibody may be linked to the amino terminus of the SIRPγ peptide variant.

[0019] In some preferred embodiments, as described above, the bifunctional fusion protein, wherein the SIRPγ peptide variant is a SIRPγ peptide variant having amino acid substitutions at one or more sites of K19, K53, N101, L31, Q52, E54, H56, N70, M72, and M112 relative to the wild-type SIRPγ peptide.

[0020] In some preferred embodiments, as described above, the bifunctional fusion protein wherein the SIRPγ peptide variant is a SIRPγ peptide variant having amino acid substitutions at one or more sites of K19, K53, and N101 relative to the wild-type SIRPγ peptide.

[0021] In some preferred embodiments, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant is a SIRPγ peptide variant having an N51R substitution mutation relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 20.

[0022] In some preferred embodiments, as described above, the bifunctional fusion protein wherein the SIRPγ peptide variant with an alternative mutation at N51 position substantially does not bind to CD47 on the surface of erythrocytes, preferably, the SIRPγ peptide variant with an alternative mutation at N51 position is a SIRPγ peptide variant with an alternative mutation of N51F, N51I, N51L, N51M or N51V.

[0023] In some preferred embodiments, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant is a SIRPγ peptide variant further having K19E, K53G and N101D substitution mutations relative to the wild-type SIRPγ peptide shown as SEQ ID NO: 20.

[0024] In some preferred embodiments, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant has K19E, N51V, Q52S, K53G, E54R, M72K and N101D mutations relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 20.

[0025] In some preferred embodiments, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant has K19E, N51M, Q52S, K53G, E54R, M72K and N101D mutations relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 20.

[0026] In some preferred embodiments, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant is a SIRPγ peptide variant having amino acid substitutions at one or more sites of M6, V27, L30, V33, V36, L37, V42, E47, L66, T67, V92, or S98.

[0027] In some preferred embodiments, the bifunctional fusion protein as described above, wherein the amino acid sequence (general formula I) of the SIRPγ peptide variant is shown in SEQ ID NO: 1:

[0028] EEELQMIQPE KLLLVTVGET ATLHCTVTSL

[0029] Among them, X1 is selected from L or W, X2 is selected from M, V, F, I or L, X3 is selected from Q, S or T, X4 is selected from E, T or R, X5 is selected from H or R, X6 is selected from D, N or E, X7 is selected from I, V, M, R or K, and X8 is selected from M or V.

[0030] In some embodiments, as described above, the bifunctional fusion protein has the amino acid sequence (formula II) of the SIRPγ peptide variant as shown in SEQ ID NO: 2:

[0031] EEELQMIQPE KLLLVTVGET ATLHCTVTSL

[0032] Among them, X1 is selected from L or W, X3 is selected from Q, S or T, X4 is selected from E, T or R, X5 is selected from H or R, X6 is selected from D, N or E, X7 is selected from I, V, M, R or K, and X8 is selected from M or V.

[0033] In a further preferred embodiment, the bifunctional fusion protein as described above, wherein the SIRPγ peptide variant is as shown in SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, preferably as shown in 26 or 27.

[0034] In some embodiments, the bifunctional fusion protein as described above, wherein the anti-human PD-L1 antibody is selected from Avelumab, Atezolizumab, Durvalumab, JS-003, CS-1001, LY-3300054, KD-033, CK-301, CCX-4503, CX-072, KN-035, HRP00052, HRP00049, FAZ-053, GR-1405, KD-005, HLX-20, KL-A167, CBT-502, STI-A1014, REMD-290, BGB-A333, BCD-135, and MCLA-145.

[0035] In some embodiments, as described above, the bifunctional fusion protein, wherein the anti-human PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0036] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 regions with the same sequence as the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions with the same sequence as the light chain variable region shown in SEQ ID NO: 7; or

[0037] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 regions with the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions with the same sequence as the light chain variable region shown in SEQ ID NO: 9; or

[0038] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 regions with the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions with the same sequence as the light chain variable region shown in SEQ ID NO: 113. Furthermore, in some embodiments, the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions are defined by the Kabat numbering rules.

[0039] In some embodiments, as described above, the bifunctional fusion protein, wherein the variable region of the anti-human PD-L1 antibody heavy chain comprises HCDR1, HCDR2, and HCDR3 regions as shown in SEQ ID NO: 97, 98, and 99, and the variable region of the anti-human PD-L1 antibody light chain comprises LCDR1, LCDR2, and LCDR3 regions as shown in SEQ ID NO: 100, 101, and 102, or

[0040] The variable region of the heavy chain of the anti-human PD-L1 antibody includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 103, 104 and 105, and the variable region of the light chain of the anti-human PD-L1 antibody includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 106, 107 and 108, respectively.

[0041] Alternatively, the variable region of the heavy chain of the anti-human PD-L1 antibody may include HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 103, 104 and 105, and the variable region of the light chain of the anti-human PD-L1 antibody may include LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 106, 112 and 108.

[0042] In some embodiments, as described above in the bifunctional fusion protein, the anti-human PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0043] The heavy chain variable region is shown in SEQ ID NO: 6, and the light chain variable region is shown in SEQ ID NO: 7; or

[0044] The heavy chain variable region is shown in SEQ ID NO: 8, and the light chain variable region is shown in SEQ ID NO: 113;

[0045] or

[0046] The heavy chain variable region is shown in SEQ ID NO: 8, and the light chain variable region is shown in SEQ ID NO: 9.

[0047] In some embodiments, the bifunctional fusion protein as described above, wherein the anti-human PD-L1 antibody further includes a heavy chain constant region and a light chain constant region, preferably, the heavy chain constant region is as shown in SEQ ID NO:10 or 11, and the light chain constant region is as shown in SEQ ID NO:12.

[0048] In some embodiments, the bifunctional fusion protein as described above, wherein the anti-human PD-L1 antibody comprises a heavy chain and a light chain, wherein: the heavy chain is as shown in SEQ ID NO: 13 or 15, and the light chain is as shown in SEQ ID NO: 14; or

[0049] The heavy chain is as shown in SEQ ID NO: 16 or 18, and the light chain is as shown in SEQ ID NO: 17; or

[0050] The heavy chain is shown as SEQ ID NO: 16 or 18, and the light chain is shown as SEQ ID NO: 111.

[0051] In some embodiments, as described above, a bifunctional fusion protein is wherein the bifunctional fusion protein has a first polypeptide and a second polypeptide, wherein:

[0052] The first polypeptide is selected from any one of the polypeptides shown in SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62, and the second polypeptide is selected from the polypeptide shown in SEQ ID NO: 14; or

[0053] The first polypeptide is selected from any of the polypeptides shown in SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, and 109, and the second polypeptide is selected from the polypeptide shown in SEQ ID NO: 17; or

[0054] The first polypeptide is selected from any of the polypeptides shown in SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 and 109, and the second polypeptide is selected from the polypeptide shown in SEQ ID NO: 111.

[0055] In other embodiments of this disclosure, a SIRPγ peptide variant is provided, said SIRPγ peptide variant having a substitution mutation at the N51 position corresponding to the wild-type SIRPγ peptide shown in SEQ ID NO: 20. In some embodiments, the SIRPγ peptide variant as described above has activity in binding to CD47 on the surface of tumor cells, preferably, said SIRPγ peptide variant has enhanced activity in binding to CD47 on the surface of tumor cells compared to the wild-type SIRPγ peptide.

[0056] In some preferred embodiments, the SIRPγ peptide variants as described above are SIRPγ peptide variants having amino acid substitutions at one or more sites of K19, K53, N101, L31, Q52, E54, H56, N70, M72, and M112 relative to the wild-type SIRPγ peptide.

[0057] In some preferred embodiments, SIRPγ peptide variants as described above are SIRPγ peptide variants having amino acid substitutions at one or more sites in K19, K53, and N101 relative to the wild-type SIRPγ peptide.

[0058] In some preferred embodiments, the SIRPγ peptide variants as described above are SIRPγ peptide variants that have an N51R substitution mutation relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 20.

[0059] In some preferred embodiments, SIRPγ peptide variants as described above, wherein the SIRPγ peptide variants with the N51 substitution mutation substantially do not bind to CD47 on the surface of erythrocytes, preferably, the SIRPγ peptide variants with the N51 substitution mutation are SIRPγ peptide variants with the N51F, N51I, N51L, N51M or N51V substitution mutations.

[0060] In some preferred embodiments, the SIRPγ peptide variants described above are SIRPγ peptide variants having K19E, K53G and N101D substitution mutations relative to wild-type SIRPγ as shown in SEQ ID NO: 20.

[0061] In some preferred embodiments, the SIRPγ peptide variants described above are provided, wherein the SIRPγ peptide has K19E, N51V, Q52S, K53G, E54R, M72K and N101D mutations relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 20.

[0062] In some preferred embodiments, the SIRPγ peptide variants described above are provided, wherein the SIRPγ peptide has K19E, N51M, Q52S, K53G, E54R, M72K and N101D mutations relative to the wild-type SIRPγ peptide shown in SEQ ID NO: 20.

[0063] In some preferred embodiments, the SIRPγ peptide variants as described above are further SIRPγ peptide variants having amino acid substitutions at one or more sites of M6, V27, L30, V33, V36, L37, V42, E47, L66, T67, V92, or S98.

[0064] In some preferred embodiments, SIRPγ peptide variants as described above, wherein the SIRPγ peptide variant is shown in SEQ ID NO: 1,

[0065] EEELQMIQPE KLLLVTVGET ATLHCTVTSL

[0066] Among them, X1 is selected from L or W, X2 is selected from M, V, F, I or L, X3 is selected from Q, S or T, X4 is selected from E, T or R, X5 is selected from H or R, X6 is selected from D, N or E, X7 is selected from I, V, M, R or K, and X8 is selected from M or V.

[0067] In some preferred embodiments, SIRPγ peptide variants as described above, such as SEQ ID NO: 2:

[0068] EEELQMIQPE KLLLVTVGET ATLHCTVTSL

[0069] Among them, X1 is selected from L or W, X3 is selected from Q, S or T, X4 is selected from E, T or R, X5 is selected from H or R, X6 is selected from D, N or E, X7 is selected from I, V, M, R or K, and X8 is selected from M or V.

[0070] In some preferred embodiments, SIRPγ peptide variants as described above, wherein the SIRPγ peptide variants are as shown in SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0071] Other aspects of this disclosure also provide a fusion protein comprising a SIRPγ peptide variant and an antibody Fc fragment, wherein the SIRPγ peptide variant is any of the SIRPγ peptide variants described above; in some embodiments, the antibody Fc fragment is a human antibody Fc fragment; in some preferred embodiments, the sequence of the antibody Fc fragment is identical to the Fc fragment sequence in the heavy chain constant region shown in SEQ ID NO: 10 or 11; in some preferred embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 86, 110, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131.

[0072] Other aspects of this disclosure also provide an anti-human PD-L1 antibody comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 regions with sequences as shown in SEQ ID NO: 103, 104 and 105, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions with sequences as shown in SEQ ID NO: 106, 112 and 108, respectively.

[0073] In some embodiments, such as the anti-human PD-L1 antibody described above, the heavy chain variable region is shown in SEQ ID NO: 8, and the light chain variable region is shown in SEQ ID NO: 113.

[0074] In some embodiments, the anti-human PD-L1 antibody as described above is a full-length antibody, further comprising an antibody constant region. Preferably, the heavy chain constant region of the antibody is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4, and the light chain constant region of the antibody is selected from the constant regions of human antibody κ and λ chains. More preferably, the full-length antibody comprises the heavy chain constant region shown in SEQ ID NO: 10 or 11 and the light chain constant region shown in SEQ ID NO: 12.

[0075] In some preferred embodiments, the anti-human PD-L1 antibody as described above comprises a heavy chain as shown in SEQ ID NO: 16 or 18, and a light chain as shown in SEQ ID NO: 111.

[0076] In other aspects, this disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of the bifunctional fusion protein as described above, or the SIRPγ peptide variant as described above, or the fusion protein as described above, or the anti-human PD-L1 antibody as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the therapeutically effective amount is 0.1-3000 mg of the bifunctional fusion protein as described above, or the SIRPγ peptide variant as described above, or the fusion protein as described above, or the anti-human PD-L1 antibody as described above, per unit dose of the composition.

[0077] In other aspects, this disclosure also provides an isolated nucleic acid molecule that encodes a bifunctional fusion protein as described above, or a SIRPγ peptide variant as described above.

[0078] In other aspects, this disclosure also provides an isolated nucleic acid molecule that encodes the anti-human PD-L1 antibody as described above.

[0079] In other aspects, this disclosure also provides a recombinant vector comprising the isolated nucleic acid molecules as described above.

[0080] In other aspects, this disclosure also provides a host cell transformed according to the recombinant vector as described above, said host cell being selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or insect cells.

[0081] In other aspects, this disclosure also provides methods for producing the bifunctional fusion protein as described above, or for producing the SIRPγ peptide variant as described above, or for producing the fusion protein as described above, or for producing the anti-human PD-L1 antibody as described above, the methods comprising culturing the host cells as described above in a culture medium to form and accumulate the bifunctional fusion protein as described above, or the SIRPγ peptide variant as described above, and recovering the bifunctional fusion protein or SIRPγ peptide variant, or the fusion protein as described above, or the anti-human PD-L1 antibody as described above from the culture.

[0082] In other aspects, this disclosure also provides a method for eliminating immunosuppression-related diseases in a subject, the method comprising administering to the subject a therapeutically effective amount of the bifunctional fusion protein as described above, or according to the SIRPγ peptide variant as described above, or according to the fusion protein as described above, or according to the anti-human PD-L1 antibody as described above, or the pharmaceutical composition as described above, or the isolated nucleic acid molecule as described above, preferably, the therapeutically effective amount being 0.1-3000 mg of the bifunctional fusion protein as described above, or according to the SIRPγ peptide variant as described above, or according to the anti-human PD-L1 antibody as described above, per unit dose of the composition.

[0083] In some implementations, approximately 10 μg / kg, approximately 50 μg / kg, approximately 100 μg / kg, approximately 200 μg / kg, approximately 300 μg / kg, approximately 400 μg / kg, approximately 500 μg / kg, approximately 600 μg / kg, approximately 700 μg / kg, approximately 800 μg / kg, approximately 900 μg / kg, approximately 1000 g / kg, approximately 1100 g / kg, approximately 1200 g / kg, 1300 g / kg, 1400 g / kg, 1500 g / kg, 1600 g / kg, 1700 g / kg, 1800 g / kg, 1900 g / kg, approximately 2000 g / kg, approximately 3000 g / kg, approximately 4000 g / kg, approximately 5000 g / kg, approximately 6000 g / kg, and approximately 7 μg / kg are administered to an individual in a single or cumulative application. The PD-L1-CD47 bifunctional fusion protein, SIRPγ variant peptide, or the fusion protein as described above, or the anti-human PD-L1 antibody as described above, at doses of approximately 000 g / kg, approximately 8000 g / kg, approximately 9000 g / kg, approximately 10 mg / kg, approximately 20 mg / kg, approximately 30 mg / kg, approximately 40 mg / kg, approximately 50 mg / kg, approximately 60 mg / kg, approximately 70 mg / kg, approximately 80 mg / kg, approximately 90 mg / kg, approximately 100 mg / kg, approximately 200 mg / kg, approximately 300 mg / kg, approximately 400 mg / kg, approximately 500 mg / kg, approximately 600 mg / kg, approximately 700 mg / kg, approximately 800 mg / kg, approximately 900 mg / kg, or approximately 1000 mg / kg.

[0084] In other aspects, this disclosure also provides the use of the bifunctional fusion protein as described above, or according to the SIRPγ peptide variant as described above, or according to the fusion protein as described above, or according to the anti-human PD-L1 antibody as described above, or the pharmaceutical composition as described above, or the isolated nucleic acid molecule as described above, in the preparation of a medicament for eliminating immunosuppression-related diseases in a subject, preferably, wherein a unit dose of the medicament composition contains 0.1-3000 mg of the bifunctional fusion protein as described above, or the SIRPγ peptide variant as described above, or the anti-human PD-L1 antibody as described above.

[0085] In other aspects, this disclosure also provides a bifunctional fusion protein as described above, or a SIRPγ peptide variant as described above, or a fusion protein as described above, or an anti-human PD-L1 antibody as described above, or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above, preferably, wherein a unit dose of the pharmaceutical composition contains 0.1-3000 mg of the bifunctional fusion protein as described above, or the SIRPγ peptide variant as described above, or the anti-human PD-L1 antibody as described above.

[0086] In another aspect, this disclosure also provides a bifunctional fusion protein as described above, or a SIRPγ peptide variant as described above, or according to the fusion protein as described above, or according to the anti-human PD-L1 antibody as described above, or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above, preferably, wherein a unit dose of the pharmaceutical composition contains 0.1-3000 mg of the bifunctional fusion protein as described above, or the SIRPγ peptide variant as described above, or the anti-human PD-L1 antibody as described above.

[0087] In some implementations, as previously described, the elimination of immunosuppression-related diseases in the subject includes cancer, bacterial or viral infections. The cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), and large B-cell lymphoma rich in T-cells / histocytes. B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancer. Cancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer, and skin cancer. Attached Figure Description

[0088] Figure 1 : Schematic diagram of the structure of the PD-L1-CD47 bifunctional fusion protein in some implementation schemes.

[0089] Figures 2A-2C The binding ability of the PD-L1-CD47 bifunctional fusion protein to CD47 on the surface of human erythrocytes was tested. The rightmost negative control (control) consisted of cells plus secondary antibody. Figure 2A and Figure 2B To test the binding ability of different PD-L1-CD47 bifunctional fusion proteins (10 μg / ml) to CD47 on the surface of erythrocytes; Figure 2C The binding ability of different PD-L1-CD47 bifunctional fusion proteins (10 μg / ml and 1 μg / ml) to CD47 on the surface of erythrocytes was tested.

[0090] Figure 3 The binding ability of the PD-L1-CD47 bifunctional fusion protein to CD47 on the surface of Raji cells was tested. The rightmost negative control was cells + secondary antibody.

[0091] Figure 4 : PD-L1-CD47 bifunctional fusion protein-mediated erythrocyte phagocytosis.

[0092] Figures 5A-5B Phagocytosis of tumor cells (Molp-8 cells) mediated by the PD-L1-CD47 bifunctional fusion protein. Figure 5A and Figure 5B The study investigated the phagocytosis of tumor cells mediated by different PD-L1-CD47 bifunctional fusion proteins in different batches of experiments.

[0093] Figure 6 : PD-L1-CD47 bifunctional fusion protein-mediated erythrocyte agglutination.

[0094] Figures 7A-7E : PD-L1-CD47 bifunctional fusion protein-mediated IFN-γ secretion. Figure 7A , Figure 7B , Figure 7C , Figure 7D and Figure 7E The results show the effects of different PD-L1-CD47 bifunctional fusion proteins in mediating IFN-γ secretion.

[0095] Figure 8 Effects of different PD-L1-CD47 bifunctional fusion proteins on tumor volume in the B-hCD274 / hCD47 / hSIRPα mouse xenograft MC38 / H-11-hCD47(#5-4) model.

[0096] Figure 9 Effects of different PD-L1-CD47 bifunctional fusion proteins on tumor volume in the C57 / BL-6 mouse xenograft MC38-hPD-L1-hCD47 model.

[0097] Figure 10 Effects of different PD-L1-CD47 bifunctional fusion proteins on tumor volume in the C57 / BL-6 mouse xenograft MC38-hPD-L1 model.

[0098] Figure 11 The effect of different PD-L1-CD47 bifunctional fusion proteins on tumor volume in a Molp-8 tumor-bearing nude mouse model. This model focuses on investigating the tumor-suppressive effect of the CD47 target pathway in the bifunctional fusion proteins. Detailed Implementation

[0099] the term

[0100] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0101] The term "bifunctional fusion protein" refers to a protein molecule that can bind to two target proteins or target antigens. In this disclosure, the bifunctional fusion protein mainly includes PD-L1 and CD47 that can bind to the cell surface, and is formed by the fusion of an anti-PD-L1 antibody and a SIRPγ peptide variant.

[0102] The term "PD-L1" refers to programmed death ligand 1, also known as CD274 or B7H1. The amino acid sequence of the full-length human PD-L1 is available in GenBank with accession number NP_054862.1. Unless otherwise specified from a non-human species, the term "PD-L1" refers to human PD-L1.

[0103] Anti-human PD-L1 antibodies are antibodies that can bind to human PD-L1 and block the binding of PD-1 to PD-L1. Anti-human PD-L1 antibodies can be selected from Avelumab, Atezolizumab, Durvalumab, JS-003, CS-1001, LY-3300054, KD-033, CK-301, CCX-4503, CX-072, KN-035, HRP00052, HRP00049, FAZ-053, GR-1405, KD-005, HLX-20, KL-A167, CBT-502, STI-A1014, REMD-290, BGB-A333, BCD-135, MCLA-145, etc. In addition, the anti-human PD-L1 antibody in this disclosure may also be selected from full-length antibodies h1830, h1831, or anti-PD-L1 antibodies or their antigen-binding fragments that have the same CDR combination with h1830 and h1831 antibodies, respectively.

[0104] "SIRPγ peptide" refers to a human SIRPγ-D1 domain peptide (the amino acid sequence of its wild-type SIRPγ peptide is shown in SEQ ID NO: 20), which has the activity of binding human CD47. SIRPγ peptide may also include human SIRPγ-D1 domain peptide mutants, or "SIRPγ peptide variants," which have amino acid substitutions at one or more sites corresponding to the wild-type SIRPγ peptide. The number of such amino acid substitution mutations is at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. These SIRPγ peptide variants have enhanced activity relative to the wild-type SIRPγ peptide in binding to CD47 on the surface of tumor cells (the affinity of wild-type SIRPγ for CD47 is in the micromolar range). Furthermore, in some specific embodiments, the SIRPγ peptide variant acquires the property of not binding to or (relative to tumor cell surface CD47 binding activity) reducing binding to human erythrocyte surface CD47. As shown in Table 1 below, the S58 peptide is an alternative mutant corresponding to the wild-type SIRPγ peptide shown in SEQ ID NO: 20, with K19E at position K19, N51M at position N51, Q52S at position Q52, K53G at position K53, E54R at position E54, and N101D at position N101.

[0105] In some specific embodiments, the optional sites for the amino acid substitution mutation may include one or more of the following: K19, K53, N101, L31, N51, Q52, E54, H56, N70, M72, M112, M6, V27, L30, V33, V36, L37, V42, E47, L66, T67, V92, or S98.

[0106] In some specific implementations, the SIRPγ peptide variant is shown in SEQ ID NO: 1:

[0107] EEELQMIQPE KLLLVTVGET ATLHCTVTSL

[0108] Among them, X1 is selected from L or W, X2 is selected from M, V, F, I or L, X3 is selected from Q, S or T, X4 is selected from E, T or R, X5 is selected from H or R, X6 is selected from D, N or E, X7 is selected from I, V, M, R or K, and X8 is selected from M or V.

[0109] In some specific implementations, the SIRPγ peptide variant is shown in SEQ ID NO: 2:

[0110] EEELQMIQPE KLLLVTVGET ATLHCTVTSL

[0111] Among them, X1 is selected from L or W, X3 is selected from Q, S or T, X4 is selected from E, T or R, X5 is selected from H or R, X6 is selected from D, N or E, X7 is selected from I, V, M, R or K, and X8 is selected from M or V.

[0112] The table below shows the amino acid substitution mutation sites and exemplary substituted amino acid residues of different SIRPγ peptide variants relative to the wild-type SIRPγ peptide.

[0113] Table 1

[0114]

[0115]

[0116] The term "antibody (Ab)" includes any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific antigen (or its epitope, such as the PD-L1 antigen or its epitope). The term "antibody" includes: an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and its polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region (CH). This heavy chain constant region contains three regions (domains): CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region (CL). The light chain constant region contains one region (domain, CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of this disclosure, the FRs of the anti-PD-L1 antibody (or its antigen-binding fragment) may be identical to human germline sequences or may be naturally or artificially modified. The antibody may be a different subclass antibody, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subclasses), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0117] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably in this document to refer to an antibody in essentially its complete form, as defined below as an antigen-binding fragment. Specifically, this term refers to antibodies in which the heavy chain, from the amino terminus to the carboxyl terminus, contains the VH, CH1, hinge, and Fc regions, respectively, and the light chain, from the amino terminus to the carboxyl terminus, contains the VL and CL regions, respectively.

[0118] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as region-specific antibodies, single-domain antibodies, region-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and squalane-variable IgNAR regions, are also included in the term "antigen-binding fragment" as used herein.

[0119] Antigen-binding fragments of antibodies typically contain at least one variable region. The variable region can be of any size or amino acid composition and generally contains a CDR adjacent to or within one or more frame sequences. In antigen-binding fragments having both VH and VL regions, the VH and VL regions can be arranged opposite each other in any suitable configuration. For example, the variable region can be dimerized and contain VH-VL or VL-VH dimers.

[0120] In some embodiments, in any configuration of variable and constant regions, the antigen-binding fragment may have the variable and constant regions directly linked to each other or linked by complete or partial hinge or linker regions. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60, or more), such that it creates flexible and semi-flexible linkages between adjacent variable and / or constant regions within a single polypeptide molecule. Furthermore, the antigen-binding fragment of this disclosure may comprise a homodimer or heterodimer (or other multimer) having variable and constant regions, non-covalently linked to each other and / or linked (e.g., by disulfide bonds) to one or more monomeric VH or VL regions.

[0121] In this disclosure, "mouse-derived antibody" refers to a monoclonal antibody derived from a mouse or rat and prepared in accordance with the knowledge and skills in the art. Preparation involves injecting an antigen into the test subject, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. When the injected test subject is a mouse, the resulting antibody is a mouse-derived antibody; when the injected test subject is a rat, the resulting antibody is a rat-derived antibody.

[0122] A chimeric antibody is an antibody formed by fusing the variable region of a first-species (e.g., mouse) antibody with the constant region of a second-species (e.g., human) antibody. To create a chimeric antibody, a hybridoma secreting a first-species monoclonal antibody is first established. Then, the variable region gene is cloned from the hybridoma cells. Next, the constant region gene of the second-species antibody is cloned as needed. The first-species variable region gene and the second-species constant region gene are linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of this disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain, or a variant thereof. The heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a heavy chain constant region of human IgG1, IgG2 or IgG4, or using a variant of the heavy chain constant region of IgG1, IgG2 or IgG4 with amino acid mutations (such as YTE mutation, reversion mutation, L234A and / or L235A mutation, or S228P mutation).

[0123] The term "humanized antibody," including CDR-grafted antibodies, refers to antibodies produced by grafting the CDR sequence of an animal-derived antibody, such as a murine antibody, into the variable region (or framework region) of a human antibody. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies due to the large amount of heterologous protein components they carry. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available at http: / / www.vbase2.org / ) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity due to reduced immunogenicity, a small amount of reversion mutation can be performed on the aforementioned human antibody variable region framework sequence to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies that have undergone affinity maturation of CDR by phage display.

[0124] Due to contact residues with the antigen, CDR transplantation can lead to a decrease in the affinity of the resulting antibody or its antigen-binding fragment for the antigen due to the framework residues in contact with the antigen. Such interactions may be a result of high somatic mutations. Therefore, it may still be necessary to transplant such donor framework amino acids into the framework of humanized antibodies. The amino acid residues involved in antigen binding from non-human antibodies or their antigen-binding fragments can be identified by examining the variable region sequence and structure of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the species can be considered relevant. If the closest species cannot be determined, the sequence can be compared with a subclass common sequence or a common sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be a result of high somatic mutations, thus playing an important role in binding.

[0125] In one embodiment of this disclosure, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0126] "Conventional variants" of the human antibody heavy chain constant region and human antibody light chain constant region refer to variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region, as disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions include YTE mutations, L234A and / or L235A mutations, or S228P mutations known in the prior art, or mutations that obtain a knock-in-hole structure (resulting in the antibody heavy chain having a knock-Fc and hole-Fc combination). These mutations have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0127] "Human antibody" and "human-derived antibody" are used interchangeably. It can be an antibody derived from a human being or obtained from a transgenic organism "modified" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into cell lines in which endogenous heavy and light chain loci are targeted and disrupted. The transgenic organism can synthesize antigen-specific human antibodies and can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0128] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or mutations generated during the manufacture of a monoclonal antibody preparation, which are typically present in small amounts), the individual antibodies constituting said population recognize and / or bind to the same epitopes. Each monoclonal antibody in a monoclonal antibody preparation (formulation) targets a single determinant cluster on an antigen. Therefore, the modifier "monoclonal" indicates the characteristics of an antibody as obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the manufacture of the antibody by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, as well as other exemplary methods for preparing monoclonal antibodies, are described herein.

[0129] Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. The antigen-binding moiety can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0130] The antigen-binding fragment can also be incorporated into a single-chain molecule containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10): 1057-1062; and US Patent US5641870).

[0131] Fab is an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity obtained by treating IgG antibodies with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0132] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000 Da and antigen-binding activity, containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with pepsin.

[0133] Fab' is an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to antigens with a reducing agent such as dithiothreitol.

[0134] In addition, Fab' can be expressed by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote.

[0135] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules comprising a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have the general formula: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (including 1, 2, 3, or 4) (Holliger et al. (1993), Proc Natl Acad Sci USA. 90:6444-6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur J Immuno. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol Immunother. 50:51-59.

[0136] "Anti-human PD-L1 antibody" includes a full-length antibody that can specifically bind to human PD-L1, as well as antigen-binding fragments containing the light chain variable region and heavy chain variable region of the full-length antibody, including but not limited to single-chain antibodies (scFv), Fab fragments, or other antigen-binding fragments containing the light chain variable region and heavy chain variable region of the full-length antibody.

[0137] The term "link" in the polypeptide chain of the SIRPγ peptide to the anti-human PD-L1 antibody refers to an effective link between the polypeptides, including, for example, linking via peptide bonds or using a linker. This link does not result in the loss of function of either the SIRPγ peptide or the anti-human PD-L1 antibody.

[0138] A "linker" is a linking polypeptide sequence used to connect protein domains or different proteins or polypeptides. Linkers typically possess a degree of flexibility, and their use does not cause the original function of the protein domain to be lost. Exemplary linkers are shown in the table below.

[0139] Table 2. Exemplary Connector Subsequences

[0140]

[0141] In some implementations, anti-PD-L1 antibodies can be linked to SIRPγ peptide variants using a linker; some exemplary bifunctional fusion proteins include the fusion proteins shown below:

[0142] Table 3. PD-L1-CD47 bifunctional fusion protein

[0143]

[0144]

[0145] A diabody is an antibody fragment in which scFv is dimerized; it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0146] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0147] In some embodiments of this disclosure, the antigen-binding fragment can be produced by the following steps: obtaining cDNA encoding the VH and / or VL of the monoclonal antibody that specifically recognizes and binds to the antigen, as well as other desired domains; constructing DNA encoding the antigen-binding fragment; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.

[0148] The "Fc region" can be a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region in the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Residues in the Fc region are numbered using the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD., 1991. The Fc region of immunoglobulins typically has two constant region domains, CH2 and CH3.

[0149] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0150] The “variable region” of an antibody refers to the variable region (VL) of the antibody light chain alone or in combination, or the variable region (VH) of the antibody heavy chain. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together tightly by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, a CDR may refer to a CDR determined by either method or a combination of both methods.

[0151] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0152] The terms "complementarity-determining region" and "CDR" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGapAlign procedure.

[0153] "Antibody constant region domains" refer to the domains derived from the constant regions of the light and heavy chains of antibodies, including CL and CH1, CH2, CH3, and CH4 domains derived from different classes of antibodies.

[0154] An epitope, or antigenic determinant, is a site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0155] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

[0156] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single epitope. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple amino acid sites via weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" for an antibody or its antigen-binding fragment (e.g., the Fab fragment) generally refers to an antibody with a 1E affinity. -9 M or smaller K D (e.g., 1E) -10 M or smaller K D 1E -11 M or smaller K D 1E -12 M or smaller K D 1E -13 M or smaller K D 1E -14 M or smaller K D Antibody or antigen-binding fragments (such as antibodies or antigens).

[0157] The term "KD" or "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, antibodies dissociate at a rate less than approximately 1E. -8 M, for example, less than approximately 1E -9 M, 1E -10 M or 1E -11An antigen is bound by a dissociation equilibrium constant (KD) of M or smaller, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR) technology. The smaller the KD value, the greater the affinity.

[0158] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded DNA or RNA molecules, such as double-stranded DNA or mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0159] The term "vector" refers to a construct capable of delivering one or more target genes or sequences and preferably expressing them in host cells. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0160] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with antigens or fragments thereof, and the resulting antibodies can be refolded, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this disclosure are genetically engineered to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained from websites by comparing against the IMGT Human Antibody Variable Region Germplasm Database and MOE software. http: / / www.imgt.org / Obtained, or from the Immunoglobulin Journal, 2001 ISBN012441351.

[0161] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), HEK293 cells (non-restrictive implementations such as HEK293E cells), and NSO cells.

[0162] Engineered antibodies or antigen-binding fragments can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As an alternative existing technology, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified using conventional techniques, such as using a Sepharose FF column with adjusted buffer for protein A or protein G. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0163] "Administration," "dosage," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, composition, or human intervention to an animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving" and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0164] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds described in this disclosure, to a patient (or subject) who has (or is suspected of having, or is susceptible to) one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient (or subject) or population in an amount that effectively relieves one or more symptoms of a disease to induce the regression of such symptoms or inhibit the progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's (or subject's) disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient (or subject). Whether the disease symptoms have been relieved can be evaluated using any clinical testing method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may not be effective in alleviating symptoms of every target disease, they should reduce symptoms of the target disease in a statistically significant number of patients (or subjects) according to any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0165] "Conservative amino acid modification" or "conservative amino acid substitution" refers to the substitution of an amino acid in a protein or polypeptide with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), thereby allowing for frequent alterations without changing the protein or polypeptide's biological activity or other desired properties (e.g., antigen affinity and / or specificity). Those skilled in the art will recognize that, generally, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conserved substitutions are described in the table "Exemplary Conservative Amino Acid Substitutions" below.

[0166] Table 4. Exemplary Conserved Substitutions of Amino Acids

[0167] Original residues Conservative replacement Ala(A) Gly;Ser Arg(R) Lys;His Asn(N) Gln; His; Asp Asp(D) Glu;Asn Cys(C) Ser;Ala;Val Gln(Q) Asn; Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn;Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0168] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this disclosure or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in a patient (or subject).

[0169] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the circumstances.

[0170] "Endogenous" refers to substances that are produced in cells, organisms, or the human body, depending on the circumstances.

[0171] "Separated" refers to a purified state, and in this context means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compound as described herein.

[0172] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0173] "Pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0174] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0175] Furthermore, this disclosure also relates to methods for immunodetection or determination of target antigens, reagents for immunodetection or determination of target antigens, methods for immunodetection or determination of cells expressing target antigens, and diagnostic agents for diagnosing diseases associated with target antigen-positive cells, comprising the monoclonal antibody or antibody fragment, or fusion protein, or bifunctional fusion protein of this disclosure that specifically recognizes and binds to the target antigen as an active ingredient.

[0176] The terms “cancer,” “carcinoma,” or “malignant tumor” refer to or describe a physiological condition in mammals generally characterized by unregulated cell growth and are used interchangeably in this disclosure. Examples of cancer or malignant tumors include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), and large B-cell lymphomas rich in T-cells / histocytes. Lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancer. Cancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer, and skin cancer.

[0177] "Inflammatory syndrome" refers to any disease, condition, or syndrome in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, damage to host tissues, or loss of tissue function. "Inflammatory disease" also refers to a pathological state mediated by the chemotactic aggregation of leukocytes or neutrophils.

[0178] Inflammation is a localized protective response resulting from tissue damage or destruction, designed to destroy, weaken, or isolate harmful substances and injured tissue. Inflammation is significantly associated with the aggregation of leukocytes or neutrophils through chemotaxis. Inflammation can be caused by pathogenic organisms and viruses, as well as non-infectious causes such as trauma, reperfusion after myocardial infarction or stroke, immune responses to exogenous antigens, and autoimmune responses.

[0179] The aforementioned diseases associated with cells positive for the target antigen can be diagnosed by detecting or measuring cells expressing the target antigen using the monoclonal antibody or antibody fragment disclosed herein.

[0180] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0181] In this disclosure, there are no particular limitations on the live sample used for detecting or measuring the target antigen, as long as it has the potential to contain cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0182] Depending on the required diagnostic method, diagnostic reagents containing the monoclonal antibody or antibody fragment thereof of this disclosure may also contain reagents for performing an antigen-antibody reaction or for detecting the reaction. Reagents for performing the antigen-antibody reaction include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragment, or conjugates, and substrates corresponding to the labeled antibodies.

[0183] Details of one or more embodiments of the invention have been set forth in the foregoing description. While this disclosure may be practiced or tested using any methods and materials similar to or the same as those described herein, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless clearly indicated otherwise in the context. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this invention pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to more fully illustrate preferred embodiments of the invention disclosed herein. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0184] Example

[0185] Example 1. Preparation of CD47 antigen and detection protein

[0186] Using UniProt leukocyte surface antigen CD47 (human CD47 protein, UniProt No.: Q08722) as a template for CD47, the amino acid sequences of the antigen and detection protein involved in this disclosure are designed. Optionally, different tags such as his tag or Fc tag are fused to the CD47 protein.

[0187] 1. His-tagged extracellular domain of CD47 protein (CD47-ECD-His): (SEQ ID NO: 3)

[0188] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE HHHHHH

[0189] Note: The underlined part is a 6×his tag.

[0190] 2. CD47 extracellular domain fusion protein with human IgG1 Fc (CD47-ECD-Fc) as a detection reagent: (SEQ ID NO: 4)

[0191] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE EPKSSDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0192] Note: The underlined part is the human IgG1-Fc portion.

[0193] 3. Human SIRPα and human IgG1Fc fusion protein (SIRPα-Fc) as a binding and blocking detection reagent: (SEQ ID NO: 5)

[0194] EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS EPKSSDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0195] Note: The underlined part is the human IgG1-Fc portion.

[0196] 4. The extracellular domain of the SIRPα protein with a His tag (SIRPα-His)

[0197] EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS HHHHHH (SEQ ID NO: 132)

[0198] Example 2. Purification of CD47 and SIRPα-related recombinant proteins

[0199] 1. Purification steps for His-tagged recombinant proteins:

[0200] Cell expression supernatant was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. Imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS containing 5 mM imidazole and washed 2-5 column volumes. The supernatant sample was loaded onto an IMAC column. The column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound proteins, and the eluent was collected. The target protein was then eluted with PBS containing 300 mM imidazole, and the elution peak was collected. The collected eluent was concentrated and further purified by gel chromatography using a Superdex 200 (GE, 28-9893-35) with PBS as the mobile phase. The aggregate peak was removed, and the elution peak was collected. The obtained protein was identified by electrophoresis, peptide mapping, and LC-MS, and then aliquoted for use.

[0201] The His-tagged CD47-ECD-His (SEQ ID NO: 3) was used as an immunogen or detection reagent for the antibody disclosed herein. CD47-ECD-His can also be used as an immunogen to stimulate mouse immunization after being coupled with KLH protein in vitro using a chemical method.

[0202] 2. Purification steps of CD47-ECD-Fc and SIRPα-Fc fusion proteins:

[0203] Cell expression supernatant was centrifuged at high speed to remove impurities, and then subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography. The MabSelect Sure column was regenerated with 0.1M NaOH, washed with pure water, and then equilibrated with PBS. After binding the supernatant, the column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with 0.1M acetate buffer at pH 3.5 and neutralized with 1M Tris-HCl. After appropriate concentration, the eluted sample was further purified using a PBS-equilibrated Superdex 200 gel chromatography system (GE, 28-9893-35). The collected target protein samples were then concentrated to an appropriate concentration.

[0204] This method is used to purify the CD47-ECD-Fc (SEQ ID NO: 4) and SIRPα-Fc (SEQ ID NO: 5) fusion proteins. This method can also be used to purify the humanized antibody proteins involved in this disclosure.

[0205] Example 3. Construction and expression of anti-PD-L1 humanized antibody (IgG4-S228P form)

[0206] The light and heavy chain variable regions of the anti-PD-L1 antibody are modified from the anti-PD-L1 antibody WO2017084495A1, the sequence of which and related properties are described in PCT application with application number PCT / CN2019 / 070982, the entire contents of which are incorporated herein by reference.

[0207] Anti-PD-L1 antibody 9-2H5L11:

[0208] 9-2H5 heavy chain variable region (SEQ ID NO: 6)

[0209] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS DGSAYWS WIRQHPGKGLEYIG FISRAGSTYNTPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS

[0210] 9-2L11 Light Chain Variable Region (SEQ ID NO: 7)

[0211] DIVMTQSPDSLAVSLGERATINC KSSQSLFYHSNQKHSLA WYQQKPGQPPKLLIY GASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYGYPYT FGGGTKVEIK

[0212] Anti-PD-L1 antibody 24D5 H12L61:

[0213] 24D5 H12 heavy chain variable region (SEQ ID NO: 8)

[0214] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RITPSSGFAMYNEKFKN RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

[0215] 24D5 L61 light chain variable region (SEQ ID NO: 9)

[0216] DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLES GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIK

[0217] Note: The underlined CDRs in the light and heavy chain variable regions derived from antibodies 9-2 and 24D5 above are CDRs as defined by the Kabat numbering rules.

[0218] Table 5. Antibody CDRs defined by the Kabat numbering rules

[0219]

[0220] Primers were designed, and humanized antibody VH / VK gene fragments were constructed by PCR. These fragments were then homologously recombinated with the expression vector pHr (which contains a signal peptide and a constant region gene (CH1-FC / CL) fragment) to construct the full-length antibody expression vector VH-CH1-FC-pHr / VK-CL-pHr.

[0221] The heavy chain constant region sequence of IgG4-S228P is as follows: (SEQ ID NO: 10)

[0222] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP CPAPE FL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0223] The heavy chain constant region sequence of IgG1 is as follows: (SEQ ID NO: 11)

[0224] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0225] The constant region sequence of the light chain (Kappa chain) of the antibody is as follows: (SEQ ID NO: 12)

[0226] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0227] The full-length antibody constructed is as follows:

[0228] Anti-PD-L1 IgG4 antibody h1830

[0229] The heavy chain of h1830 (SEQ ID NO: 13)

[0230] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS DGSAYWS WIRQHPGKGLEYIG FISRAGSTYNTPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP CPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0231] h1830 light chain (SEQ ID NO: 14)

[0232] DIVMTQSPDSLAVSLGERATINC KSSQSLFYHSNQKHSLA WYQQKPGQPPKLLIY GASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYGYPYT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0233] Anti-PD-L1 IgG1 antibody h1830G1

[0234] h1830G1 heavy chain: (SEQ ID NO: 15)

[0235] QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSAYWSWIRQHPGKGLEYIGFISRAGSTYNTPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0236] h1830G1 light chain (same as h1830 light chain, SEQ ID NO: 14):

[0237] DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0238] PD-L1 antibody h1831:

[0239] h1831 heavy chain (SEQ ID NO: 16)

[0240] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RITPSSGFAMYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP CPAPE FL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0241] Light chain of h1831 (SEQ ID NO: 17)

[0242] DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLES GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0243] Anti-PD-L1 IgG1 antibody h1831G1

[0244] Heavy chain of h1830G1: (SEQ ID NO: 18)

[0245] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RITPSSGFAMYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKh1831G1 light chain (same as h1831 light chain, SEQ ID NO:17)

[0246] DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLES GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0247] Example 4. Screening and preparation of SIRPγ mutants

[0248] 4.1 Construction and screening of SIRPγ affinity maturation phage libraries

[0249] To obtain a high-affinity CD47 receptor, the SIRPγD1 domain of the CD47 receptor was maturated using phage display technology. Based on wild-type SIRPγ, an affinity-matured phage library targeting the CD47 binding domain was designed and prepared, and new SIRPγ mutants were screened from it. The specific sequence of the wild-type SIRPγD1 domain is as follows:

[0250] DNA coding sequence of wild-type SIRPγ peptide: (SEQ ID NO: 19)

[0251] GAGGAGGAGCTACAGATGATTCAGCCTGAGAAGCTCCTGTTGGTCACAGTTGGAAAGACAGCCACTCTGCACTGCACTGTGACCTCCCTGCTTCCCGTGGGACCCGTCCTGTGGTTCAGAGGAGTTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCAGGG TAACAACAGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCAGTAGCATCACCCCAGCAGATGTCGGCACATACTACTGTGTGAAGTTTCGAAAAGGGAGCCCTGAGAACGTGGAGTTTAAGTCTGGACCAGGCACTGAGATGGCTTTGGGTGCCAAACCCTCT

[0252] Wild-type SIRPγ peptide: (SEQ ID NO: 20)

[0253] EEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPS

[0254] Construction of phage libraries: Degenerate primers were designed, and designed mutant amino acids were introduced into SIRPγ phage mutant libraries using PCR. Each library was approximately 10^6 nucleotides in size. 9 about.

[0255] Screening of SIRPγ phage mutant libraries: Packaged SIRPγ phage mutant libraries (1×10⁻⁶) 12 -1×10 13Add 100 μl of streptavidin-coated magnetic beads (Milenvi Biotec, Auburn, CA) to 1 ml of 2% skim milk-phosphate buffer (MPBS) and incubate at room temperature for 1 hour. Place on a magnetic rack and collect the supernatant. Add 10 μg / ml biotinylated human CD47-ECD-his protein (purchased from Sino Biological) to the supernatant and incubate at room temperature for 1 hour. Then add 100 μl of streptavidin-coated magnetic beads (pre-incubated in 1 ml MPBS) and incubate at room temperature for 1 hour. Load the beads onto a magnetic rack system for sorting and remove the supernatant. Add 1 ml of PBST (phosphate buffer containing 0.1% Tween-20), invert several times, aspirate thoroughly, and then add fresh wash buffer. Repeat 11 times to remove unbound mutants. Add 0.5 ml of elution buffer (50 μl of 10 mg / ml trypsin stock solution added to 450 μl of PBS). Shake at room temperature for 15 min. Place on a magnetic rack and aspirate the supernatant into a new EP tube. Inoculate TG1 into 2YT medium and amplify to a bacterial density of OD600 = 0.4. Add 1.75 ml of TG1 (OD600 = 0.4) to each tube, along with 250 μl of eluted phage. Incubate at 37°C for 30 min, then perform serial dilutions and plate for titer testing. Centrifuge the remaining TG1 solution, plate, and incubate overnight at 37°C.

[0256] SIRPγ phage mutant libraries were used to obtain phage mutant single clones with higher affinity than wild-type SIRPγ through 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen) after biotinylated human CD47-ECD-his protein antigen. Sequencing validation followed by sequencing of the sequenced clones. After alignment analysis and removal of redundant sequences, the non-redundant sequences were converted into a PDL1-CD47 bifunctional fusion protein for expression in mammalian cells.

[0257] 4.2 Construction and screening of mature yeast libraries for SIRPγ affinity

[0258] To obtain CD47 receptors with higher affinity, the CD47 receptor SIRPγ-D1 domain was matured using yeast display platform technology. Based on SIRPγ-D1, an affinity-matured yeast library targeting the CD47 binding domain was designed and prepared, and new SIRPγ mutants were screened from it.

[0259] Construction of yeast libraries: Degenerate primers were designed, and the designed mutant amino acids were introduced into SIRPγ mutant libraries by PCR. The size of each library was 10^6 nucleotides. 9The constructed yeast library was then validated for diversity using next-generation sequencing.

[0260] In the first round of screening, approximately 5 × 10⁶ samples from the SIRPγ mutant library were selected. 10 Cells were incubated with 10 μg / ml biotinylated human CD47-Fc protein in 50 ml of 0.1% bovine serum albumin (BSA)-phosphate buffer (PBSA) at room temperature for 1 hour. The mixture was then washed three times with 0.1% PBSA to remove unbound antibody fragments. 100 μl of streptokinin beads (Milenvi Biotec, Auburn, CA) were then added to a SIRPγ mutant library bound to biotinylated human CD47-Fc protein and loaded onto an AutoMACS system for sorting. Cells with libraries exhibiting high affinity for CD47-Fc were collected and amplified for 24 hours at 250 rpm and 30°C in SDCAA medium (20 g dextrose, 6.7 g Difco yeast nitrogen source - amino acid-free, 5 g Bacto casein amino acids, 5.4 g Na2HP O4 and 8.56 g NaH2PO4·H2O dissolved in 1 L distilled water). The cultures were then induced for 18 hours at 250 rpm and 20°C in SGCAA medium (20 g galactose, 6.7 g Difco yeast nitrogen source - amino acid-free, 5 g Bacto casein amino acids, 5.4 g Na2HP O4 and 8.56 g NaH2PO4·H2O, dissolved in 1 L distilled water). The resulting enriched libraries were subjected to a second round of screening for binding to biotinylated recombinant human CD47-Fc. To ensure sufficient diversity of antibody libraries used in the second and / or third rounds of screening, 100 times the size of the libraries from the previous round were used as the input cell number.

[0261] For the third and fourth rounds of screening, the library cells from the previous round were incubated with 1 μg / ml biotinylated human CD47-Fc protein and 10 μg / ml Mouse Anti-cMyc (9E10, Sigma) antibody in 0.1% PBSA at room temperature for 1 hour. The mixture was washed three times with 0.1% PBSA to remove unbound antibody fragments. Goat anti-mouse-Alexa488 (A-11001, Life Technologies) and Strepavidin-PE (S-866, Life Technologies) were added and incubated at 4°C for 1 hour. The mixture was washed three times with 0.1% PBSA to remove unbound antibody fragments. Finally, high-affinity SIRPγ mutants were selected by FACS screening (BD FACSAria™ FUSION).

[0262] The SIRPγ mutant library was prepared using biotinylated human CD47-Fc antigen and underwent 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and 2-3 rounds of FACS screening (BD FACSAria™ FUSION). Approximately 400 yeast clones were then selected, cultured, and induced to express the protein. FACS (BD FACSCanto II) was used to detect the binding of yeast clones to human CD47-Fc antigen. Yeast clones with higher affinity for SIRPγ than wild-type were selected for sequencing verification. The sequenced clones were compared and analyzed, and redundant sequences were removed. The non-redundant sequences were then converted into a PDL1-CD47 bifunctional fusion protein for expression in mammalian cells.

[0263] After screening, the following SIRPγ peptide variants were finally obtained:

[0264] S58 (SEQ ID NO:21)

[0265] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0266] S79 (SEQ ID NO:22)

[0267] EEELQMIQPEKLLLVTVGETATLHCTVTSLWPVGPVLWFRGVGPGRELIYRTGTGRFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEVALGAKPS

[0268] S15 (SEQ ID NO:23)

[0269] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSSGRGHFPRVTTVSDLTKRENRDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0270] S12 (SEQ ID NO:24)

[0271] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0272] S19(SEQ ID NO:25)

[0273] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRNNRDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0274] S85(SEQ ID NO:26)

[0275] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0276] S37(SEQ ID NO:27)

[0277] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0278] S38(SEQ ID NO:28)

[0279] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYFSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0280] S22(SEQ ID NO:29)

[0281] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYISGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0282] S29(SEQ ID NO:30)

[0283] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYLSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0284] S34(SEQ ID NO:31)

[0285] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYRSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0286] S41(SEQ ID NO:32)

[0287] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0288] S42(SEQ ID NO:33)

[0289] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNIDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0290] S43(SEQ ID NO:34)

[0291] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNRDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0292] S44(SEQ ID NO:35)

[0293] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNVDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0294] S45(SEQ ID NO:36)

[0295] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRDNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0296] S46(SEQ ID NO:37)

[0297] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0298] S47(SEQ ID NO:38)

[0299] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEVALGAKPS

[0300] S48(SEQ ID NO:39)

[0301] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYRSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0302] S49 (SEQ ID NO:40)

[0303] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYRSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0304] Example 5. Construction and expression of PD-L1-CD47 bifunctional fusion protein

[0305] The obtained anti-PD-L1 antibody was linked to SIRPγ to form a fusion protein, which was then expressed and purified using conventional methods to obtain the PD-L1-CD47 bifunctional fusion protein.

[0306] Table 6. PD-L1-CD47 bifunctional fusion protein and its sequence

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327] The following proteins were prepared and purified using conventional methods as positive or negative controls.

[0328] Anti-CD47 antibody hu5F9 (sequence from US09017675B)

[0329] Hu5F9 heavy chain (SEQ ID NO:83)

[0330] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0331] Hu5F9 light chain (SEQ ID NO:84)

[0332] DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0333] SIRPα-CV (synthesized according to Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies, Science. 2013 Jul 5;341(6141):88-91, SEQ ID NO:85)

[0334] EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0335] TTI-621: (sequence from WO2014094122A1, SEQ ID NO:133)

[0336] EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0337] S58-Fc (SEQ ID NO:86)

[0338] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0339] Anti-CD47 antibody Hu167-IgG4 AA (prepared by the method disclosed in the patent application WO2018095428A1)

[0340] Hu167-IgG4 AA heavy chain (SEQ ID NO:87)

[0341] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNIDPSDSETHYNQKFKDRVT MTRDTSISTAYMELSRLRSDDTAVYYCARWGYLGRSAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0342] Hu167-IgG4 AA light chain (SEQ ID NO:88)

[0343] DVQITQSPSSSLSASVGDRVTITCRTSKSISKFLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQHNEYPWTTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0344] S37-Fc (SEQ ID NO:110)

[0345] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0346] Antibody h1831K

[0347] The h1831 antibody underwent CDR mutation modification, yielding 36 mutants. The N53K mutant (position determined according to Kabat numbering rules) h1831K was ultimately selected. The h1831 light chain LCDR2 was then modified using AAS... N LES mutation to AAS K LES was used to obtain a new antibody, h1831K.

[0348] h1831K light chain: (SEQ ID NO:111)

[0349] DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASKLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0350] LCDR1 is RASESVSIHGTHLMH (SEQ ID NO: 106), LCDR2 is AASKLES (SEQ ID NO: 112), LCDR3 is QQSFEDPLT (SEQ ID NO: 108). h1831K light chain variable region (SEQ ID NO: 113)

[0351] DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASKLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIKh1831K heavy chain (same sequence as h1831 heavy chain, SEQ ID NO: 16)

[0352] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RITPSSGFAMYNEKFKN RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP CPAE FL GGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLG

[0353] >h1831K-19-S37 heavy chain (identical to the h1831-19-S37 heavy chain sequence, SEQ ID NO: 109) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGEEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPS

[0354] >h1831K-19-S37 light chain (identical to the h1831K light chain sequence, SEQ ID NO: 111)

[0355] DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASKLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0356] S79-Fc (SEQ ID NO:114)

[0357] EEELQMIQPEKLLLVTVGETATLHCTVTSLWPVGPVLWFRGVGPGRELIYRTGTGRFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEVALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0358] S15-Fc(SEQ ID NO:115)

[0359] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRENRDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0360] S12-Fc(SEQ ID NO:116)

[0361] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0362] S19-Fc(SEQ ID NO:117)

[0363] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRNNRDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0364] S85-Fc(SEQ ID NO:118)

[0365] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0366] S38-Fc(SEQ ID NO:119)

[0367] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYFSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0368] S22-Fc(SEQ ID NO:120)

[0369] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYISGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0370] S29-Fc(SEQ ID NO:121)

[0371] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYLSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0372] S34-Fc(SEQ ID NO:122)

[0373] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYRSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0374] S41-Fc(SEQ ID NO:123)

[0375] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYVSGRGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0376] S42-Fc(SEQ ID NO:124)

[0377] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNIDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0378] S43-Fc(SEQ ID NO:125)

[0379] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNRDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0380] S44-Fc(SEQ ID NO:126)

[0381] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRNNVDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0382] S45-Fc(SEQ ID NO:127)

[0383] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRDNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0384] S46-Fc(SEQ ID NO:128)

[0385] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0386] S47-Fc(SEQ ID NO:129)

[0387] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYMSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEVALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0388] S48-Fc(SEQ ID NO:130)

[0389] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYRSGRGHFPRVTTVSDLTKRNNKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0390] S49-Fc (SEQ ID NO:131)

[0391] EEELQMIQPEKLLLVTVGETATLHCTVTSLLPVGPVLWFRGVGPGRELIYRSGRGHFPRVTTVSDLTKRENKDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEMALGAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0392] The negative control IgG4 control is an antibody targeting targets that are not related to PD-L1 and CD47. IgG4-Fc and IgG1-Fc contain only Fc segments and do not contain variable regions targeting any antigen.

[0393] Test case

[0394] Test Example 1. ELISA assay of PD-L1-CD47 bifunctional fusion protein binding to CD47-his protein

[0395] The binding affinity of the PD-L1-CD47 bifunctional fusion protein was determined by the amount of binding between the bifunctional fusion protein and human CD47 or cyno CD47 immobilized on an ELISA plate. CD47-ECD-His (see Table 7) was diluted with PBS to 1 μg / ml and coated onto 96-well ELISA plates. After washing and blocking, different concentrations of the bifunctional fusion protein sample were added, followed by washing and adding horseradish peroxidase-goat anti-human (H+L) antibody (Jackson, CAT#109-035-088). After washing again, tetramethylbenzidine solution was added for color development, and finally, stop solution was added. OD450 was measured on a microplate reader, and EC50 values ​​were calculated. The results are shown in Tables 8-1 and 8-2.

[0396] Table 7. Sources of CD47 in different strains

[0397] CD47 type MW / kDa Cat.No. Lot No. Manufacturer Human CD47 15.2 12283-H08H N / A SB Cyno CD47 15.8 CD7-C52H1 2171b-76VF1-K9 ACROBiosystems

[0398] Table 8-1. ELISA results of PD-L1-CD47 bifunctional fusion protein binding to human CD47 and cynoCD47

[0399]

[0400]

[0401] Table 8-2. ELISA results of PD-L1-CD47 bifunctional fusion protein binding to human CD47 and cynoCD47

[0402]

[0403] The results showed that each PD-L1-CD47 bifunctional fusion protein had a strong affinity for free human CD47 protein, and also had strong cross-affinity activity with monkey CD47.

[0404] Test Example 2. ELISA of PD-L1-CD47 bifunctional fusion protein binding to PD-L1-his protein

[0405] The binding affinity of the PD-L1-CD47 bifunctional fusion protein was detected by measuring the amount of antibody binding to different species of PD-L1 immobilized on an ELISA plate. Different strains of PD-L1-his antigen (see Table 9) were diluted with PBS to 1 μg / ml and coated onto 96-well ELISA plates (Costar, CAT#3590). After washing and blocking, different concentrations of the PD-L1-CD47 bifunctional fusion protein samples were added. After washing, horseradish peroxidase-goat anti-human (H+L) antibody (Jackson, CAT#109-035-088) was added, followed by washing again. Tetramethylbenzidine solution was added for color development, and finally, stop solution was added. The OD450 was measured and the EC50 value was calculated using a microplate reader. The results are shown in Table 10.

[0406] Table 9. Sources of PD-L1 in different strains

[0407] PD-L1 type MW / kDa Cat.No. Lot No. Manufacturer hPD-L1-His 26.8 10084-H08H LC11SE1203 SB Cyno PD-L1-His 26.7 90251-C08H LC10DE1308 SB mPD-L1 26.3 50010-M08H LC10NO0102 SB

[0408] Table 10. ELISA results of PD-L1-CD47 bifunctional fusion protein and PD-L1 binding in different species

[0409]

[0410] The results showed that each PD-L1-CD47 bifunctional fusion protein had a strong affinity for free human PD-L1 protein, and also exhibited strong cross-affinity activity with monkey PD-L1. The PD-L1-CD47 bifunctional fusion protein containing the h1830 antibody also showed strong cross-affinity activity with mouse PD-L1.

[0411] Test Example 3. Blocking of PD-L1 / PD1 and PD-L1 / B7.1 binding by the PD-L1-CD47 bifunctional fusion protein.

[0412] Dilute PD-L1-Fc (prepared internally) to 1 μg / ml with PBS, and add 100 μl / well to each well of a 96-well plate. Incubate at 4°C for 16-20 h. Remove the PBS buffer from the 96-well plate, wash once with PBST (pH 7.4 PBS containing 0.05% Tween 20), and add 120 μl / well of PBST / 1% milk. Incubate at room temperature for 1 h for blocking. Remove the blocking solution, wash once with PBST buffer, and add 90 μl of the PD-L1-CD47 bifunctional fusion protein diluted to the appropriate concentration with sample dilution buffer (pH 7.4 PBS containing 5% BSA, 0.05% Tween 20). Incubate at 4°C for 1 h. Add 10 μl / well of 10× biotin-labeled PD-1 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 10 μg / ml) or B7-1 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 10 μg / ml) to each well, shake and mix, then incubate at 37°C for 1 h. Remove the reaction mixture, wash the plate 6 times with PBST, add 100 μl / well of Streptavidin–Peroxidase Polymer diluted 1:400 with PBST buffer, and incubate at room temperature with shaking for 50 min. Wash the plate 6 times with PBST, add 100 μl / well of TMB, and incubate at room temperature for 5–10 min. Terminate the reaction by adding 100 μl / well of 1M H2SO4. Measure the OD450 and calculate the IC50 value using a NOVOStar microplate reader; the results are shown in Table 11.

[0413] Table 11. Blocking ELISA of PD-L1-CD47 bifunctional fusion protein

[0414]

[0415] The experimental results showed that all bifunctional fusion proteins could also effectively block the PD-L1 / PD-1 and PD-L1 / B7.1 pathways.

[0416] Test Example 4. Experiment on the binding of PD-L1-CD47 bifunctional fusion protein to human red blood cells.

[0417] Fresh, healthy human blood was mixed with an equal volume of PBS and centrifuged at 300g for 5 minutes to obtain cell clusters. Red blood cells were then washed 3-5 times with PBS. The cells were resuspended in FACS buffer (PBS + 5% BSA) and the cell density was adjusted to 2 × 10⁻⁶ cells / mL. 6Cells were seeded at a rate of 100 μl / well into 96-well round-bottom plates (3795#, corning). Different concentrations of antibody and bifunctional fusion protein were then added, and the plates were incubated at 4°C for 1 hour. Cells were then washed twice with FACS buffer (PBS + 2% FBS), and secondary antibody (Alexa 488 goat anti-human IgG antibody (Invitrogen, CAT#A11013)) was added. The plates were incubated on ice in the dark for 30 minutes. Finally, the cells were washed twice with FACS buffer and resuspended. The plates were then read from FACS Cantoll.

[0418] FACS results showed that the control antibodies hu5F9 and Hu167 IgG4AA had a strong binding ability to native CD47 on the surface of human erythrocytes. Among the bifunctional fusion proteins involved, only those containing S79, S34, and S49 could bind to CD47 on the surface of human erythrocytes; the other bifunctional fusion proteins had almost no binding ability to native CD47 on the surface of human erythrocytes. This suggests the safety advantage of the aforementioned bifunctional fusion proteins. (See attached results). Figure 2A , Figure 2B and Figure 2C (in Figure 2A , Figure 2B and Figure 2C The experiment was conducted in three batches, using different donor cells.

[0419] Test Example 5. Experiment on the binding of PD-L1-CD47 bifunctional fusion protein to tumor cells.

[0420] Raji cells were cultured in RPMI medium (Hyclone, CAT#SH30809.01B) (containing 10% fetal bovine serum), 1×10⁻⁶ 6 After blocking Raji cells with 5% BSA, the bifunctional fusion protein sample was added to a concentration of 10 μg / ml. After washing twice, Alexa Fluor 488-goat anti-human (H+L) antibody (Invitrogen, CAT#A11013) was added, and after washing twice, the fluorescence signal value was read by flow cytometry.

[0421] FACS analysis showed that the involved PD-L1-CD47 bifunctional fusion protein had a strong binding affinity to native CD47 on the surface of Raji cells, comparable to that of the control antibody Hu5F9. (See attached results). Figure 3 .

[0422] Test Example 6. Experiment on cell-mediated phagocytosis (ADCP) of PD-L1-CD47 bifunctional fusion protein in vitro.

[0423] PBMCs were isolated from fresh human blood and then sorted into CD14+ monocytes using Human CD14 MicroBeads (130-050-201#, Miltenyi Biotec). These CD14+ monocytes were cultured for 9 days in macrophage differentiation medium (1640 + 10% FBS + 50 ng / ml M-CSF) to differentiate into macrophages. These monocyte-derived macrophages (MDMs) became adhesive and developed tentacles. On the day of the experiment, macrophages were digested with trypsin for 5 min, gently scraped off with a spatula, and seeded into 96-well round-bottom plates (3795#). Human RBCs (erythrocytes) were labeled with 0.5 μM CFSE (BD, catalog number 565082#) in a 37°C water bath for 13 min. After washing twice with PBS, the macrophages were added to the macrophages at a ratio of 5 RBCs per macrophage, and PD-L1-CD47 bifunctional fusion protein was added at various concentrations. Target cells underwent phagocytosis for 2.5 hours. After phagocytosis, the cells were washed twice with PBS, and then human Fc blocking reagent (Human Fc blocker, 564219#, BD) was added at a specific ratio. The cells were incubated at room temperature for 10 minutes to exclude non-specific binding. Subsequently, CD14 antibody labeled with APC (17-0149-42#, Ebioscience) was added. The cells were incubated on ice for 30 minutes. After two final washes, the cells were analyzed by flow cytometry. Phagocytosis was measured by gating CFSE+ positive cells to APC+ positive viable cells, followed by assessment of the percentage of CSFE+ positive cells (see [link to relevant documentation]). Figure 4 ).

[0424] Test Example 7. Experiment on cell-mediated phagocytosis (ADCP) of PD-L1-CD47 bifunctional fusion protein in vitro.

[0425] PBMCs were isolated from fresh human blood and then sorted into CD14+ monocytes using Human CD14 MicroBeads (130-050-201#, Miltenyi Biotec). These CD14+ monocytes were cultured for 9 days in macrophage differentiation medium (1640 + 10% FBS + 50 ng / ml M-CSF) to differentiate into macrophages. These monocyte-derived macrophages (MDMs) became adhesive and developed tentacles. On the day of the experiment, macrophages were digested with trypsin for 5 min, gently scraped off with a scraper, and seeded into 96-well round-bottom plates (3795#). Molp-8 cells (Nanjing Kebai) were labeled with 0.1 μM CFSE in a 37°C water bath for 13 min, followed by washing twice with PBS. Then, macrophages were added to the macrophages at a ratio of 5 Molp-8 tumor cells per macrophage, along with multiple concentrations of PD-L1-CD47 antibody. The target cells were subjected to phagocytosis for 2.5 hours. After phagocytosis, cells were washed twice with PBS, then human Fc blocker was added at a specific ratio and incubated at room temperature for 10 minutes to exclude non-specific binding. Subsequently, CD14 antibody labeled with APCs was added. Cells were incubated on ice for 30 minutes. After two final washes, analysis was performed by flow cytometry. Phagocytosis was measured by gating APC+ positive viable cells and then assessing the percentage of CSFE+ positive cells (see [link to relevant documentation]). Figure 5A and Figure 5B ).

[0426] The results of test examples 6 and 7 are shown in Figure 4 and Figures 5A-5B The results showed that the added bifunctional fusion protein effectively promoted phagocytosis of tumor cells. However, the bifunctional fusion protein did not phagocytose erythrocytes, suggesting a potential safety advantage for the bifunctional fusion protein antibody disclosed in this paper. In contrast, the control antibody hu5F9 effectively phagocytosed erythrocytes.

[0427] Test Example 8. Erythrocyte agglutination assay of PD-L1-CD47 bifunctional fusion protein

[0428] Fresh, healthy human blood was diluted 100-fold with PBS (B320#, Shanghai Yuanpei Biotechnology Co., Ltd.). The diluted whole blood was spread into 96-well round-bottom plates (3795#, corning), 30 μl / well. Then, equal volumes of antibodies or bifunctional fusion proteins at different concentration gradients were added. After mixing, the plates were incubated at 37°C for 4–6 hours. Erythrocyte sedimentation was observed using a high-content microscope. No hemagglutination was indicated by clear red dots, while hemagglutination was indicated by diffuse patterns.

[0429] Each sample was diluted 1:3 from the first column (0.5 mg / ml) to the 11th column. The 12th column contains PBS blank wells without antibody.

[0430] The results show (see) Figure 6 Under the same conditions, the bifunctional fusion proteins h1830-S37, h1830-S19, h1830-S12, h1830-S15, h1831-19-S58, and h1831-19-S79 did not cause erythrocyte agglutination at different tested concentrations, suggesting the safety advantages of the bifunctional fusion proteins disclosed herein.

[0431] Test Example 9. Affinity assay of PD-L1-CD47 bifunctional fusion protein detected by BIAcore.

[0432] IgG was captured using a Protein A biosensor chip (Cat.#29127556, GE) through affinity binding. Different antigens (hCD47, cynoCD47, hPD-L1, cynoPD-L1, and mPD-L1, sources of which are described in Test Examples 3 and 4) flowed across the chip surface. A Biacore T200 instrument was used to detect the bifunctional fusion protein and the reaction signals of different antigens in real time, obtaining binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated using 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, the data were fitted using a (1:1) Langmuir model with GE Biacore T200 Evaluation version 3.0 software to obtain affinity values. The results are shown in Tables 12-1 and 12-2. The results showed that all modified SIRPγ peptide variants had significantly increased affinity for human CD47 compared to wild-type SIRPγ peptide.

[0433] Table 12-1 Biacore affinity (KD(M)) of antibodies for different antigens

[0434]

[0435]

[0436] Table 12-2 Biacore affinity (KD(M)) of bifunctional fusion proteins for human CD47

[0437]

[0438] Test Example 10. Effects of PD-L1-CD47 bifunctional fusion protein on PBMC-T lymphocyte activation assay.

[0439] Secretory function of IFNγ

[0440] To investigate the effect of the PD-L1-CD47 bifunctional fusion protein on the function of human primary T lymphocytes, human peripheral blood mononuclear cells (PBMCs) were collected and purified. After in vitro stimulation with tuberculin (TB) for 5 days, the secretion level of the cytokine IFNγ was measured. The experimental procedure is briefly described below:

[0441] PBMCs were obtained from fresh blood using Ficoll-Hypaque (17-5442-02, GE) density gradient centrifugation (Stem Cell Technologies) and cultured in RPMI 1640 (SH30809.01, GE) medium supplemented with 10% (v / v) FBS (10099-141, Gibco) at 37°C and 5% CO2.

[0442] Freshly isolated and purified PBMCs were adjusted to a density of 2 × 10⁶ cells / mL using RPMI 1640 medium. 6 Cells were cultured at 20 mL of 20 mL cell suspension with 40 μl of tuberculin (97-8800, Synbiotics) added, and incubated at 37°C in a 5% CO2 incubator for 5 days. On day 5, the cultured cells were collected, centrifuged, and resuspended in fresh RPMI 1640 medium, adjusting the density to 1.1 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 90 μl / ml into 96-well cell culture plates. Serially diluted antibody samples were added simultaneously, diluted with PBS (B320, Shanghai Yuanpei Biotechnology Co., Ltd.), to 10 μl per well. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 3 days. The cell culture plates were then removed, centrifuged (4000 rpm, 10 min), and the cell culture supernatant was collected. The IFN-γ level was detected using an ELISA kit (Human IFN-γ Detection Kit: EHC102g.96, Xinbosheng). For detailed procedures, please refer to the reagent instructions.

[0443] Results (see) Figures 7A-7E The results showed that all bifunctional fusion proteins tested could activate IFN-γ secretion, comparable to the control antibody HRP00052.

[0444] Test Example 11. Role of PD-L1-CD47 bifunctional fusion protein in the mouse colon cancer model MC38 / H-11-hCD47

[0445] In this experiment, B-hCD274 / hCD47 / hSIRPα mice were inoculated with artificially modified mouse colon cancer MC38 cells: MC38 / H-11-hCD47 (transformed with human PD-L1 and human CD47, and knocked out mouse CD47 and PDL1) to establish a mouse tumor-bearing model. The in vivo efficacy of different doses of the PD-L1-CD47 bifunctional fusion protein h1830-S85, SIRPγ protein S58-Fc, and PD-L1 monoclonal antibody h1830 on inhibiting the growth of mouse colon cancer xenografts was evaluated. B-hCD274 / hCD47 / hSIRPα mice were purchased from Biocytogen Laboratory Animals, SPF grade; weight: 22.0±3.0 g; sex: female.

[0446] MC38 / H-11-hCD47(#5-4) cells were fed at a dose of 1×10⁻⁶. 6 100 μl of each mouse was injected subcutaneously into B-hCD274 / hCD47 / hSIRPa mice. After establishing the tumor-bearing model, the tumor volume was measured. Animals with excessive weight, excessively large tumors, or excessively small tumors were removed. The tumor-bearing mice were randomly divided into 5 groups (n=7) according to the size of the tumor: PBS control group, h1830-S85 high-dose experimental group, h1830-S85 low-dose experimental group, h1830 experimental group, and S58-Fc experimental group. The date of administration for each group was set as D0.

[0447] 1) Use the method of measuring tumor diameter to observe and record tumor growth, and at the same time observe and record animal weight.

[0448] 2) The tumor diameter and animal weight were measured twice a week.

[0449] 3) On day 17 after administration, the tumor volume of the animals in the PBS control group was larger. In accordance with animal welfare principles, the animals in the PBS experimental group were euthanized. On day 25 after administration, the remaining animals in the experimental group were euthanized.

[0450] 4) Formula for calculating tumor volume (TV): TV = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0451] 5) Relative tumor proliferation rate T / C% = (T-T0) / (C-C0) × 100%

[0452] 6) Tumor inhibition rate TGI% = 1 - T / C%

[0453] Statistical analysis of the experimental data was performed using Excel and GraphPad. Animal body weight, tumor volume, and tumor weight in each group were expressed as mean ± standard deviation (Mean ± SEM), and graphs were generated using GraphPad Prism 6 software.

[0454] This experiment aimed to evaluate the inhibitory effect of different doses of PD-L1-CD47 bifunctional fusion protein on tumor growth in a B-hCD274 / hCD47 / hSIRPa mouse colon cancer xenograft model. Different antibodies or bifunctional fusion proteins were administered simultaneously to each group in this experiment.

[0455] like Figure 8 The results in Table 13 show that the tumor volume of the experimental groups with different doses of bifunctional fusion protein (h1830-S85), PD-L1 monoclonal antibody (h1830), and SIRPγ protein S58-Fc was smaller than that of the PBS control group. The high-dose experimental group of PD-L1-CD47 bifunctional fusion protein showed better tumor-suppressing effect than the experimental groups with the same doses of PD-L1 monoclonal antibody and SIRPγ protein. Furthermore, there was a dose-dependent relationship between the different doses of h1830-S85.

[0456] During the experiment, there was no significant difference in body weight between the treatment group and the control group, and the mice tolerated the administered antibodies well.

[0457] Table 13. Antitumor effect of antibodies or bifunctional fusion proteins on mouse xenografts (TGI%)

[0458]

[0459] Test Example 12. PD-L1-CD47 bifunctional fusion protein in mouse colon cancer model MC38-hPD-L1-hCD47

[0460] The role of

[0461] MC38-hPD-L1-hCD47 cells (MC38 cells were transfected with human PD-L1 and human CD47, and mouse CD47 was knocked out) were fed at 5.8 × 10⁻⁶ cells per cell line. 5 100 μl of each drug was subcutaneously injected into C57 / BL-6 mice to establish the tumor-bearing model. Tumor volume was measured, and mice with excessively large or small tumors were removed. Based on tumor size, the tumor-bearing mice were randomly divided into 5 groups (n=7): IgG4 control group, h1830-S58 experimental group, HRP00052 experimental group, h1830 experimental group, and TTI-621 experimental group. The date of drug administration was designated as D0. After grouping, each drug was administered intraperitoneally three times a week for a total of 10 weeks, with an 18-day administration cycle. Monitoring of the tumor-bearing mice ended two days after drug withdrawal. Tumor volume and body weight were measured twice a week, and data were recorded. Grouping and drug administration details are shown in the table below. Different antibodies were administered simultaneously with grouping. Starting on day 14 after drug administration, the dosage for all experimental groups was halved; starting on day 25 after drug administration, drug administration was discontinued for all experimental groups.

[0462] Table 14. Experimental Groups and Drug Administration

[0463]

[0464] Note: IP means intraperitoneal injection, and qod means administration once every other day.

[0465] Animal body weight, tumor volume, and tumor weight in each group are expressed as mean ± standard deviation (Mean ± SEM), and graphs are generated using Graphpad Prism 6 and Excel software. Statistical analysis is performed using the student t-test.

[0466] Tumor volume (TV) = 1 / 2 × L 长 ×L 短 2

[0467] Tumor proliferation rate T / C% = (T-T0) / (C-C0) × 100%

[0468] Tumor inhibition rate % TGI = 1 - T / C%

[0469] like Figure 9 The results showed that the tumor volumes in the h1830-S58 experimental group (which cross-reacts with mouse PD-L1) and the PD-L1 monoclonal antibody (h1830) experimental group were smaller than those in the control group and the TTI-621 experimental group, and statistical differences appeared between them and the control group approximately one week after administration. The TTI-621 experimental group did not show any tumor-suppressing effect in this experiment. The h1830-S58 experimental group achieved a tumor inhibition rate of 128.51% seven days after administration, and maintained a high level of tumor inhibition until the end of the experiment.

[0470] After the experiment, the tumor-bearing mice were euthanized, and the tumors were removed and weighed. The tumor weight was somewhat similar to the tumor size. During the experiment, there was no significant difference in body weight between the treatment group and the control group, and the mice tolerated the administered antibodies well.

[0471] Test Example 13. The role of PD-L1-CD47 bifunctional fusion protein in the mouse colon cancer model MC38-hPD-L1

[0472] MC38-hPD-L1 cells (transferred into MC38 cells with human PD-L1) were used at a rate of 3.5 × 10⁻⁶. 5100 μl of each drug was subcutaneously injected into C57 / BL-6 mice to establish the tumor-bearing model. Tumor volume was measured, and mice with excessively large or small tumors were removed. Based on tumor size, the tumor-bearing mice were randomly divided into 5 groups (n=7): IgG4 control group, h1830-19-S79 experimental group, h1830G1-19-S79 experimental group, SIRPα-CV experimental group, and h1830 experimental group. The date of drug administration was designated as D0. After grouping, each drug was administered intraperitoneally three times a week for 12 weeks, with a dosing cycle of 28 days. Monitoring of the tumor-bearing mice ended two days after drug withdrawal. Tumor volume and body weight were measured twice a week, and data were recorded. Grouping and drug administration details are shown in the table below.

[0473] Table 15. Experimental Groups and Drug Administration

[0474]

[0475] Animal body weight, tumor volume, and tumor weight in each group are expressed as mean ± standard deviation (Mean ± SEM), and graphs are generated using Graphpad Prism 5 and Excel software. Statistical analysis is performed using the student t-test.

[0476] Tumor volume (TV) = 1 / 2 × L 长 ×L 短 2

[0477] Tumor proliferation rate T / C% = (T-T0) / (C-C0) × 100%

[0478] Tumor inhibition rate % TGI = 1 - T / C%

[0479] This experiment aimed to investigate the inhibitory effect of different IgG forms of PD-L1-CD47 bifunctional fusion protein on tumor growth in a C57 / BL-6 mouse colon cancer xenograft model. Different antibodies were administered to each group simultaneously. Starting on day 14 post-administration, the dosage was halved in all experimental groups; and administration was discontinued on day 25 post-administration in all experimental groups.

[0480] like Figure 10 The results showed that, up to day 25 after administration, the tumor volume in all bifunctional fusion protein administration groups and PD-L1 monoclonal antibody h1830 administration groups was smaller than that in the IgG4 control group and the SIRPα-CV (TTI-621) experimental group, and there was a statistically significant difference between them and the control group.

[0481] On day 25 after drug administration, mice in the control group and the SIRPα-CV (TTI-621) experimental group were euthanized due to their large tumor volume, while the remaining experimental groups were discontinued and observed further. Results showed that the tumor volume in the PD-L1 monoclonal antibody h1830 experimental group exhibited a rapid recovery trend over time, while the tumor volume in the bifunctional fusion protein h1830-19-S79 and h1830G1-19-S79 experimental groups did not change significantly, and there was no significant difference between these two different IgG forms of bispecific antibodies.

[0482] After the experiment, the tumor-bearing mice were euthanized and the tumors were removed and weighed. The tumor weight was similar to the tumor size. There was no significant difference in body weight between the drug-treated group and the control group. The mice tolerated the various drugs well.

[0483] Test Example 14. The therapeutic effect of PD-L1-CD47 bifunctional fusion protein on MOLP-8 xenograft nude mice

[0484] Balb / c nude mice were subcutaneously inoculated with MOLP-8 cells (5 × 10⁻⁶) in the right rib area. 6 +50% Matrigel / animal), a total of 120 animals. After 10 days, the average tumor volume was approximately 214.89 ± 6.75 mm. 3 Tumor-bearing mice were randomly divided into 7 groups (n=8): PBS control group, h1830-S37 experimental group, h1830-S58 experimental group, h1831K-19-S37, h1830 experimental group, S37-Fc, and Hu167IgG4AA experimental group. The date of administration for each group was designated as D0. After grouping, each drug was administered intraperitoneally twice a week for 3 consecutive weeks. Tumor volume and body weight were measured twice weekly, and data were recorded. Body weight, tumor volume, and tumor weight for each group are expressed as mean ± standard deviation (Mean ± SEM), and graphs were created using Graphpad Prism 6 and Excel software. Statistical analysis was performed using the student t-test.

[0485] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0486] Relative volume (RTV) = V T / V0

[0487] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[0488] Among them, V0, V T The figures represent the tumor volume at the beginning and end of the experiment, respectively. CRTV T RTV The figures represent the relative tumor volumes of the blank control group and the experimental group at the end of the experiment.

[0489] The results of this experiment show (see) Figure 11 The drug was administered intraperitoneally every other day for 10 consecutive days. Data were collected on day 21 of the experiment. The PD-L1-CD47 bifunctional fusion protein h1830-S37 (30 mpk) showed a tumor inhibition rate of 34.98% (P<0.05); the bifunctional fusion protein h1831K-19-S37 (30 mpk) showed a tumor inhibition rate of 54.18% (P<0.01); h1830 (25 mpk) did not inhibit tumor growth.

[0490] During the administration process, the weight of animals in all groups remained normal, indicating that the bifunctional fusion proteins had no obvious toxic side effects.

[0491] Test Example 15. Blocking of CD47 / SIRPα binding by the PD-L1-CD47 bifunctional fusion protein

[0492] Dilute CD47-Fc to 1 μg / ml with PBS and add 100 μl / well to each well of a 96-well plate. Incubate at 4°C for 16-20 h. Remove the PBS buffer from the 96-well plate, wash once with PBST (pH 7.4 PBS containing 0.05% Tween 20), and add 120 μl / well of PBST / 1% milk. Incubate at room temperature for 1 h for blocking. Remove the blocking buffer, wash once with PBST buffer, and add 90 μl of the target PD-L1-CD47 bifunctional fusion protein diluted to the appropriate concentration with sample dilution buffer (pH 7.4 PBS containing 5% BSA, 0.05% Tween 20). Incubate at 4°C for 1 h. Add 10 μl / well of 10× biotin-labeled SIRPα-his (5 μg / ml), vortex to mix, and incubate at 37°C for 1 h. Remove the reaction mixture, wash the plate 6 times with PBST, add 100 μl / well of Streptavidin–Peroxidase Polymer diluted 1:400 with PBST buffer, and incubate at room temperature with shaking for 50 minutes. Wash the plate 6 times with PBST, add 100 μl / well of TMB, and incubate at room temperature for 5–10 minutes. Terminate the reaction by adding 100 μl / well of 1M H₂SO₄. Measure the OD450 and calculate the IC50 value using a NOVOStar microplate reader; the results are shown in Table 16.

[0493] Table 16. Results of bispecific antibody blocking CD47 / SIRPα binding

[0494] sample IC50 (ng / ml) h1831K-19-S37 251.7 S37-Fc 566 TTI-621 25985 Hu5F9 263.1

[0495] The results showed that the bifunctional fusion protein could effectively block the CD47-SIRPα pathway.

[0496] Test Example 16. The therapeutic effect of PD-L1-CD47 bifunctional fusion protein on human breast cancer cell MDA-MB-231 xenografts.

[0497] MDA-MB-231 cells (ATCC) 3×10 6 200 μl / mouse (containing 50% matrix gel) of cells were subcutaneously injected into the right rib area of ​​NOD / SCID mice. The tumor was controlled when the average tumor volume of the tumor-bearing mice reached 145 mm². 3 Mice were randomly divided into four groups: PBS, h1831K-19-S37-30mpk, h1831K-19-S37-10mpk, and h1831K-25mpk (maintaining an equimolar concentration with the high dose of h1831K-19-S37), with eight mice in each group. The day of grouping was defined as Day 0 of the experiment. On Day 0, PBMCs from two volunteers who had been stimulated with CD3 antibody for 3 days were mixed at a 1:1 ratio and administered at 5 × 10⁻⁶ mg / L. 5 PBMCs were injected into mouse tumor tissue at a dose of 100 μl / cell. The remaining PBMCs were then cultured after stimulation was stopped. One week later, they were inoculated at a dose of 5 × 10⁻⁶ cells / mouse. 6 PBMCs were injected intraperitoneally into tumor-bearing mice at a dose of 100 μl / mouse, marking the first round of injections. Two rounds of PBMC injections were administered until the end of the experiment. Starting from Day 0, each test antibody was injected intraperitoneally three times weekly. Tumor volume and animal weight were monitored and recorded twice weekly. When the tumor volume exceeded 1000 mmHg... 3 The experimental endpoint was to euthanize the tumor-bearing animals when most tumors ulcerated or the animals lost 20% of their body weight.

[0498] All data were plotted and statistically analyzed using Excel and GraphPad Prism 5 software.

[0499] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively.

[0500] The 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.

[0501] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0502] Experimental results showed that in the human breast cancer MDA-MB-231 mouse subcutaneous xenograft model, both the PDL1-CD47 bispecific antibody h1831K-19-S37 and the PDL1 monoclonal antibody h1831K exhibited good tumor-suppressing effects (p < 0.001 vs PBS).

[0503] The PD-L1-CD47 bispecific antibody h1831K-19-S37 (30 and 10 mg / kg) significantly inhibited the growth of subcutaneous xenografts of human breast cancer MDA-MB-231 mice, and the inhibition was dose-dependent between high and low doses. From day 3 after administration until the end of the experiment (Day 23), the tumor-inhibiting effect of h1831K-19-S37 was consistently superior to that of the high-dose PD-L1 monoclonal antibody control h1831K (25 mg / kg) in both the high-dose and low-dose groups (p < 0.001), and there was also a statistically significant difference between the high and low doses (p < 0.01) (Table 17).

[0504] At the end of the experiment, tumor-bearing mice were euthanized, tumors were removed, and tumor weight was measured. The results showed that the tumor weight and tumor volume trended out of the body. All treatment groups were significantly better than the control group (p < 0.001). Both the high and low dose groups of the bispecific antibody h1831K-19-S37 were better than the high dose of the PDL1 monoclonal antibody control h1831K (25 mg / kg, p < 0.001), and there was a dose effect between the high and low doses of h1831K-19-S37.

[0505] Tumor-bearing mice tolerated both the PDL1-CD47 bispecific antibody and its monoclonal antibody well, with only slight fluctuations in body weight throughout the administration process and no obvious drug-induced weight loss or other symptoms.

[0506] Table 17. Inhibitory effect of bispecific antibodies on subcutaneous xenografts in mice

[0507]

[0508] Note: Day 0: First administration date. *** indicates p < 0.001 vs PBS, determined by student T test.

Claims

1. An anti-human PD-L1 antibody comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 103, 104 and 105, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 106, 112 and 108, respectively.

2. The anti-human PD-L1 antibody according to claim 1, wherein the heavy chain variable region is as shown in SEQ ID NO: 8, and the light chain variable region is as shown in SEQ ID NO:

113.

3. The anti-human PD-L1 antibody according to claim 2, wherein the anti-human PD-L1 antibody is a full-length antibody, including an antibody constant region.

4. The anti-human PD-L1 antibody according to claim 3, wherein the heavy chain constant region of the antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4, and the light chain constant region of the antibody constant region is selected from the constant regions of human antibody κ and λ chains.

5. The anti-human PD-L1 antibody according to claim 3, wherein the full-length antibody comprises the heavy chain constant region shown in SEQ ID NO:10 or 11 and the light chain constant region shown in SEQ ID NO:

12.

6. The anti-human PD-L1 antibody according to claim 2, wherein the antibody comprises a heavy chain as shown in SEQ ID NO: 16 or 18, and a light chain as shown in SEQ ID NO:

111.

7. A pharmaceutical composition comprising a therapeutically effective amount of the anti-human PD-L1 antibody according to any one of claims 1 to 6, and one or more pharmaceutically acceptable carriers.

8. An isolated nucleic acid molecule encoding the anti-human PD-L1 antibody as described in any one of claims 1 to 6.

9. A recombinant vector comprising the isolated nucleic acid molecule of claim 8.

10. Use of the anti-human PD-L1 antibody according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 7, or the isolated nucleic acid molecule according to claim 8 in the preparation of a medicament for treating a subject's cancer, wherein the cancer is selected from non-small cell lung cancer, head and neck squamous cell carcinoma, gastrointestinal cancer, ovarian cancer, liver cancer, endometrial cancer, prostate cancer, thyroid cancer, pancreatic cancer, cervical cancer, bladder cancer, breast cancer, clear cell renal cell carcinoma, central nervous system cancer, malignant pleural mesothelioma, neuroendocrine tumor, testicular cancer, and skin cancer.

11. Use of the anti-human PD-L1 antibody according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 7, or the isolated nucleic acid molecule according to claim 8 in the preparation of a medicament for treating a subject's cancer, wherein the cancer is selected from colon cancer, hepatocellular carcinoma, neuroblastoma, glioblastoma multiforme, head and neck cancer, Merkel cell carcinoma, small cell lung cancer, melanoma, and esophageal cancer.

12. Use of the anti-human PD-L1 antibody according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 7, or the isolated nucleic acid molecule according to claim 8 in the preparation of a medicament for treating a subject's cancer, wherein the cancer is selected from brain cancer, colorectal cancer, hepatobiliary cancer, and pharyngeal cancer.

13. Use of the anti-human PD-L1 antibody according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 7, or the isolated nucleic acid molecule according to claim 8 in the preparation of a medicament for treating a subject's cancer, wherein the cancer is selected from squamous cell carcinoma and glioma.

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