WO2020177733A1 - 双功能融合蛋白及其医药用途 - Google Patents

双功能融合蛋白及其医药用途 Download PDF

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WO2020177733A1
WO2020177733A1 PCT/CN2020/077907 CN2020077907W WO2020177733A1 WO 2020177733 A1 WO2020177733 A1 WO 2020177733A1 CN 2020077907 W CN2020077907 W CN 2020077907W WO 2020177733 A1 WO2020177733 A1 WO 2020177733A1
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seq
sirpγ
antibody
variable region
fusion protein
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PCT/CN2020/077907
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English (en)
French (fr)
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顾晓玲
叶鑫
胡兵
葛虎
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to JP2021551978A priority Critical patent/JP2022523543A/ja
Priority to BR112021017399A priority patent/BR112021017399A2/pt
Priority to AU2020233058A priority patent/AU2020233058A1/en
Priority to CN202211303320.5A priority patent/CN116239698A/zh
Priority to CA3132078A priority patent/CA3132078A1/en
Priority to MX2021010531A priority patent/MX2021010531A/es
Priority to US17/436,384 priority patent/US20230012428A1/en
Priority to KR1020217030362A priority patent/KR20210137477A/ko
Priority to CN202080005976.1A priority patent/CN112969719B/zh
Priority to EP20766028.3A priority patent/EP3936526A4/en
Publication of WO2020177733A1 publication Critical patent/WO2020177733A1/zh

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Definitions

  • the present disclosure relates to a bifunctional fusion protein that specifically binds PD-L1 and CD47, a pharmaceutical composition containing the bifunctional fusion protein, and its use as an anticancer drug.
  • Programmed death-1 (programmed death-l, PD-l) is a protein receptor expressed on the surface of T cells discovered in 1992, which participates in the process of cell apoptosis.
  • PD-1 belongs to the CD28 family and has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (CTLA-4), but its expression is different from CTLA-4, and is mainly expressed in activated 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 (DC), and the expression on cells can be up-regulated after activation.
  • the expression of PD-L2 is relatively limited, and it is mainly expressed on antigen-presenting cells, such as activated macrophages and dendritic cells.
  • PD-L1 protein expression has been detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, bowel cancer, kidney cancer, and melanoma, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients . Since PD-L1 acts as a second signal pathway to inhibit T cell proliferation, blocking the combination of PD-L1/PD-1 has become a very potential emerging target in the field of tumor immunotherapy.
  • the cell surface protein CD47 is expressed or overexpressed on many tumor types, including acute myeloid leukemia, various subtypes of B-cell non-Hodgkin’s lymphoma, and many human solid tumor cells.
  • CD47 binds to signal regulatory protein ⁇ (SIRP ⁇ ) on macrophages and is a "don't eat me" signal on the surface of tumor cells.
  • SIRP ⁇ signal regulatory protein ⁇
  • anti-CD47 antibodies also help to increase the effective anti-tumor T cell response in immune tolerant mice. Therefore, anti-CD47 antibodies are a new class of immune checkpoint inhibitors that regulate the innate immune system and the adaptive immune system.
  • CD47 patents such as WO2016065329, WO2016109415, WO2014087248, WO2014093678, CN107849143A, CN108350048, CN106535914, WO2016023001A, CN107459578A, CN2017110167989, etc.
  • WO2016023001A describes a multispecific PD-1 mimetic peptide containing a high-affinity PD-1 mimetic peptide and a high-affinity SIRP- ⁇ that specifically binds CD47 and its use;
  • CN107459578A describes a multispecific PD-1 mimetic peptide containing SIRP ⁇ Mutants and anti-PD-L1 antibodies targeting CD47 molecules and recombinant fusion proteins of PD-L1 molecules;
  • CN201711016798.9 discloses a multifunctional fusion protein comprising the extracellular part of SIRP ⁇ and the extracellular part of PD-1.
  • SIRP ⁇ is expressed on T cells and activated NK cells, and compared with SIRP ⁇ , SIRP ⁇ binds CD47 with a 10-fold lower affinity.
  • the CD47-SIRP ⁇ interaction participates in the contact between antigen presenting cells and T cells, co-stimulates T cell activation and promotes T cell proliferation (Piccio et al., Blood 2005, 105, 2421-2427).
  • the CD47-SIRP ⁇ interaction plays a role in the transendothelial migration of T cells (Stefanisakis et al., Blood 2008, 112, 1280-1289).
  • the present disclosure provides bifunctional fusion proteins comprising SIRP gamma peptide variants. Compared with the wild-type SIRP ⁇ peptide, the SIRP ⁇ peptide variant has significantly improved CD47 affinity activity.
  • a bifunctional fusion protein comprising a SIRP ⁇ peptide variant and an anti-human PD-L1 antibody, the SIRP ⁇ peptide variant being linked to the anti-human PD-L1 antibody Polypeptide chain,
  • the SIRP ⁇ peptide variant is a SIRP ⁇ peptide variant with substitution mutation at position N51 corresponding to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20.
  • the aforementioned SIRP ⁇ peptide variant has the activity of binding to CD47 on the surface of tumor cells.
  • the SIRP ⁇ peptide variant has the activity of binding to CD47 on the surface of tumor cells more enhanced than the wild-type SIRP ⁇ peptide.
  • a bifunctional fusion protein comprising a human SIRP ⁇ peptide variant and an anti-human PD-L1 antibody, the SIRP ⁇ peptide variant being linked to the anti-human PD-L1 antibody Of the polypeptide chain,
  • the SIRP ⁇ peptide variant is a SIRP ⁇ peptide variant with a substitution mutation at position N51 of the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20.
  • the SIRP ⁇ peptide variant has the activity of binding to CD47 on the surface of tumor cells.
  • the SIRP ⁇ peptide variant has the activity of binding to CD47 on the surface of tumor cells more than wild-type SIRP ⁇ peptide.
  • the carboxyl terminal of the SIRP ⁇ peptide variant is connected to the amino terminal of the light chain variable region of the anti-human PD-L1 antibody,
  • the carboxyl terminal of the heavy chain of the anti-human PD-L1 antibody is connected to the amino terminal of the SIRP ⁇ peptide variant
  • the carboxyl terminal of the light chain of the anti-human PD-L1 antibody is connected to the amino terminal of the SIRP ⁇ peptide variant.
  • SIRP ⁇ peptide variant is further in K19, K53, N101, L31, Q52, E54, H56, relative to the wild-type SIRP ⁇ peptide.
  • the SIRP ⁇ peptide variant with substitution mutation at N51 does not substantially bind to CD47 on the surface of red blood cells, preferably, the SIRP ⁇ peptide variant with substitution mutation at N51 Peptide variants are SIRP gamma peptide variants with N51F, N51I, N51L, N51M, or N51V substitution mutations.
  • the bifunctional fusion protein as described above, wherein the SIRP ⁇ peptide variant further has K19E, K53G and N101D substitution mutations relative to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20 The SIRP gamma peptide variant.
  • the SIRP ⁇ peptide variant has K19E, N51V, Q52S, K53G, E54R, K19E, N51V, Q52S, K53G, E54R, and the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20. M72K and N101D mutations.
  • the SIRP ⁇ peptide variant has K19E, N51M, Q52S, K53G, E54R, K19E, N51M, Q52S, K53G, E54R, and the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20. M72K and N101D mutations.
  • the bifunctional fusion protein as described above, wherein the amino acid sequence (general formula I) of the SIRP ⁇ peptide variant is shown in SEQ ID NO:1:
  • X 1 is selected from L or W
  • X 2 is selected from M
  • X 3 is selected from Q
  • S or T is selected from E
  • T or R is selected from H
  • X 6 is selected from D
  • N or E is selected from I, V, M, R or K
  • X 8 is selected from M or V.
  • the amino acid sequence (general formula II) of the SIRP ⁇ peptide variant is shown in SEQ ID NO: 2:
  • X 1 is selected from L or W
  • X 3 is selected from Q
  • S or T is selected from E
  • T or R is selected from E
  • T or R is selected from H or R
  • X 6 is selected from D, N or E
  • X 7 Is selected from I, V, M, R or K
  • X 8 is selected from M or V.
  • the bifunctional fusion protein as described above, wherein the SIRP ⁇ peptide variants are as 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 26 or 27.
  • 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.
  • 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
  • the bifunctional fusion protein as described above, wherein the anti-human PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes the HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region includes the light chain variable region shown in SEQ ID NO: 7
  • the heavy chain variable region includes the HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region includes the light chain variable region shown in SEQ ID NO: 9.
  • the heavy chain variable region includes the HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region includes the light chain variable region shown in SEQ ID NO: 113.
  • the chain variable region has LCDR1, LCDR2 and LCDR3 regions of the same sequence.
  • the HCDR1, HCDR2, and HCDR3 regions and LCDR1, LCDR2, and LCDR3 regions are defined by the Kabat numbering rules.
  • the heavy chain variable region of the anti-human PD-L1 antibody comprises the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID NOs: 97, 98, and 99
  • the light chain variable regions of the anti-human PD-L1 antibody respectively comprise the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOs: 100, 101 and 102, or
  • the heavy chain variable regions of the anti-human PD-L1 antibody respectively comprise the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 103, 104 and 105, and the light chain variable regions of the anti-human PD-L1 antibody respectively comprise The LCDR1, LCDR2 and LCDR3 areas shown in SEQ ID NO: 106, 107 and 108;
  • the heavy chain variable regions of the anti-human PD-L1 antibody respectively comprise the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 103, 104 and 105 and the light chain variable regions of the anti-human PD-L1 antibody respectively Contains LCDR1, LCDR2, and LCDR3 regions as shown in SEQ ID NO: 106, 112, and 108.
  • the anti-human PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • 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
  • 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;
  • 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.
  • 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 SEQ ID NO: As shown in 10 or 11, the light chain constant region is shown in SEQ ID NO: 12.
  • 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 shown in SEQ ID NO: 13 or 15, the The light chain is shown in SEQ ID NO: 14; or
  • the heavy chain is shown in SEQ ID NO: 16 or 18, and the light chain is shown in SEQ ID NO: 17; or
  • the heavy chain is shown in SEQ ID NO: 16 or 18, and the light chain is shown in SEQ ID NO: 111.
  • the bifunctional fusion protein as described above, wherein the bifunctional fusion protein has a first polypeptide and a second polypeptide, wherein:
  • the first polypeptide is selected from SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
  • the polypeptide shown in any one of 60, 61 and 62, and the second polypeptide is selected from the polypeptide shown in SEQ ID NO: 14; or
  • the first polypeptide is selected from SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
  • the polypeptide shown in any one of 82 and 109, and the second polypeptide is selected from the polypeptide shown in SEQ ID NO: 17; or
  • the first polypeptide is selected from SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
  • the polypeptide shown in any one of 82 and 109, and the second polypeptide are selected from the polypeptide shown in SEQ ID NO: 111.
  • a SIRP ⁇ peptide variant is provided, which is a SIRP ⁇ peptide variant with a substitution mutation at position N51 equivalent to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20 body.
  • the SIRP ⁇ peptide variant has the activity of binding to CD47 on the surface of tumor cells.
  • the SIRP ⁇ peptide variant has the activity of binding to CD47 on the surface of tumor cells more than wild-type SIRP ⁇ peptide.
  • the SIRP ⁇ peptide variant as described above wherein the SIRP ⁇ peptide variant is at K19, K53, N101, L31, Q52, E54, H56, N70 relative to the wild-type SIRP ⁇ peptide.
  • the SIRP ⁇ peptide variant as described above wherein the SIRP ⁇ peptide variant is a SIRP ⁇ peptide variant with N51R substitution mutation relative to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20 .
  • the SIRP ⁇ peptide variant as described above, wherein the SIRP ⁇ peptide variant with substitution mutation at position N51 does not substantially bind to CD47 on the surface of red blood cells preferably, the SIRP ⁇ peptide variant with substitution mutation at position N51 does not substantially bind to CD47 on the surface of red blood cells.
  • Peptide variants are SIRP gamma peptide variants with N51F, N51I, N51L, N51M, or N51V substitution mutations.
  • the SIRP ⁇ peptide variant as described above wherein the SIRP ⁇ peptide variant is SIRP ⁇ with K19E, K53G and N101D substitution mutations relative to the wild-type SIRP ⁇ shown in SEQ ID NO: 20 Peptide variants.
  • the SIRP ⁇ peptide variant as described above wherein the SIRP ⁇ peptide has K19E, N51V, Q52S, K53G, E54R, M72K relative to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20 And N101D mutation.
  • the SIRP ⁇ peptide variant as described above wherein the SIRP ⁇ peptide has K19E, N51M, Q52S, K53G, E54R, M72K relative to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20 And N101D mutation.
  • the SIRP ⁇ peptide variant as described above wherein the SIRP ⁇ peptide variant is further defined in M6, V27, L30, V33, V36, L37, V42, E47, L66, T67, V92 Or S98 peptide variant with amino acid substitution at one or more positions.
  • X 1 is selected from L or W
  • X 2 is selected from M
  • X 3 is selected from Q
  • S or T is selected from E
  • T or R is selected from H
  • X 6 is selected from D
  • N or E is selected from I, V, M, R or K
  • X 8 is selected from M or V.
  • the SIRP ⁇ peptide variant is shown in SEQ ID NO: 2:
  • X 1 is selected from L or W
  • X 3 is selected from Q
  • S or T is selected from E
  • T or R is selected from E
  • T or R is selected from H or R
  • X 6 is selected from D, N or E
  • X 7 Selected from I, V, M, R or K
  • X 8 is selected from M or V.
  • the SIRP ⁇ peptide variant as described above, wherein the SIRP ⁇ peptide variant is as SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
  • a fusion protein comprising a SIRP ⁇ peptide variant and an antibody Fc fragment.
  • the SIRP ⁇ peptide variant is the SIRP ⁇ peptide variant described in any one of the foregoing; in some embodiments, the antibody The Fc fragment is a human antibody Fc fragment; in some preferred embodiments, the antibody Fc fragment sequence is the same as 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 SEQ ID NO: 86, 110, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 Or as shown in 131.
  • an anti-human PD-L1 antibody which comprises the light chain variable region and the heavy chain variable region of the antibody, and the heavy chain variable region includes the sequence as SEQ ID NO: 103, 104, respectively.
  • the HCDR1, HCDR2, and HCDR3 regions shown in and 105, and the light chain variable region comprise the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID NOs: 106, 112, and 108, respectively.
  • 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 .
  • 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 Human IgG1, IgG2, IgG3 and IgG4 constant regions, the light chain constant region of the antibody is selected from human antibody ⁇ and ⁇ chain constant regions, more preferably the full-length antibody comprises the heavy chain shown in SEQ ID NO: 10 or 11 The constant region and the light chain constant region shown in SEQ ID NO: 12.
  • 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 .
  • the present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the bifunctional fusion protein as described above, or the SIRP ⁇ peptide variant as described above, or the fusion as described above.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg of the bifunctional fusion protein as described above, or according to the aforementioned SIRP ⁇ peptide variant, or according to the aforementioned The fusion protein, or the anti-human PD-L1 antibody as described above.
  • the present disclosure also provides an isolated nucleic acid molecule, which encodes the bifunctional fusion protein as described above, or encodes the SIRP ⁇ peptide variant as described above.
  • the present disclosure also provides an isolated nucleic acid molecule encoding the anti-human PD-L1 antibody as described above.
  • the present disclosure also provides a recombinant vector, which contains the isolated nucleic acid molecule as described above.
  • the present disclosure also provides a host cell transformed with the recombinant vector as described above, the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or Insect cells.
  • the present disclosure also provides a method for producing the bifunctional fusion protein as described above, or the method for producing the SIRP ⁇ peptide variant as described above, or the method for producing the fusion protein as described above, or According to the anti-human PD-L1 antibody method as described above, the method comprises culturing the host cell as described above in a culture medium to form and accumulate the bifunctional fusion protein as described above, or according to The aforementioned SIRP ⁇ peptide variant, and the recovery of the bifunctional fusion protein or SIRP ⁇ peptide variant from the culture, or according to the aforementioned fusion protein, or according to the aforementioned anti-human PD-L1 antibody.
  • the present 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 aforementioned SIRP ⁇ peptide variant, or according to the aforementioned fusion protein, or according to the aforementioned anti-human PD-L1 antibody, or the aforementioned pharmaceutical composition, or the aforementioned isolated nucleic acid Molecule, preferably, the therapeutically effective amount is a unit dose of the composition containing 0.1-3000mg of the bifunctional fusion protein as described above, or according to the SIRP ⁇ peptide variant as described above, or according to the aforementioned The anti-human PD-L1 antibody.
  • about 10 ⁇ g/kg, about 50 ⁇ g/kg, about 100 ⁇ g/kg, about 200 ⁇ g/kg, about 300 ⁇ g/kg, about 400 ⁇ g/kg, about 500 ⁇ g/kg are administered to the individual in a single or cumulative application.
  • the present disclosure also provides the use of the aforementioned bifunctional fusion protein, or according to the aforementioned SIRP ⁇ peptide variant, or according to the aforementioned fusion protein, or according to the aforementioned anti- Use of the human PD-L1 antibody, 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, the unit dose of the medicament
  • the composition contains 0.1-3000mg of the aforementioned bifunctional fusion protein, or the aforementioned SIRP ⁇ peptide variant, or the aforementioned anti-human PD-L1 antibody.
  • the present disclosure also provides a bifunctional fusion protein as described above, or a variant of the SIRP ⁇ peptide as described above, for use as a drug for eliminating immunosuppression-related diseases in a subject, or according to the aforementioned
  • the content contains 0.1-3000mg of the aforementioned bifunctional fusion protein, or the aforementioned SIRP ⁇ peptide variant, or the aforementioned anti-human PD-L1 antibody.
  • the present disclosure also provides a bifunctional fusion protein as described above, or a variant of SIRP ⁇ peptide as described above, or according to the fusion protein as described above, or according to The anti-human PD-L1 antibody, or the aforementioned pharmaceutical composition, or the aforementioned isolated nucleic acid molecule, preferably, the pharmaceutical unit dose composition contains 0.1-3000 mg of the aforementioned The bifunctional fusion protein, or the SIRP ⁇ peptide variant as described above, or the anti-human PD-L1 antibody as described above.
  • the elimination of immunosuppression-related diseases in a subject as described above includes cancer, bacterial or viral infections.
  • the cancer includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • this cancer includes 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 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), T-cell/histiocytic-rich Large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tract) cancer,
  • Figure 1 Schematic diagram of the PD-L1-CD47 bifunctional fusion protein in some embodiments.
  • Figure 2A-2C The binding ability test of the PD-L1-CD47 bifunctional fusion protein with CD47 on the surface of human red blood cells, the rightmost negative control (control) is both cells + secondary antibodies.
  • Figure 2A and Figure 2B show the binding ability test of different PD-L1-CD47 bifunctional fusion proteins (10 ⁇ g/ml) and CD47 on the surface of red blood cells;
  • Figure 2C shows the binding ability of different PD-L1-CD47 bifunctional fusion proteins (10 ⁇ g/ml and 1 ⁇ g/ml).
  • Figure 3 The binding ability test of PD-L1-CD47 bifunctional fusion protein with CD47 on the surface of Raji cells, the negative control on the far right is cell + secondary antibody.
  • Figure 4 PD-L1-CD47 bifunctional fusion protein-mediated red blood cell phagocytosis.
  • Figure 5A-5B PD-L1-CD47 bifunctional fusion protein-mediated phagocytosis of tumor cells (Molp-8 cells).
  • Figures 5A and 5B show the phagocytosis of tumor cells mediated by different PD-L1-CD47 bifunctional fusion proteins tested in different batches of experiments.
  • Figure 6 Red blood cell coagulation mediated by PD-L1-CD47 bifunctional fusion protein.
  • Figure 7A-7E PD-L1-CD47 bifunctional fusion protein mediated IFN- ⁇ secretion.
  • Fig. 7A, Fig. 7B, Fig. 7C, Fig. 7D and Fig. 7E are the results of different PD-L1-CD47 bifunctional fusion proteins in mediating the secretion of IFN- ⁇ .
  • Figure 8 The effect of different PD-L1-CD47 bifunctional fusion proteins on the tumor volume of B-hCD274/hCD47/hSIRP ⁇ mouse transplanted tumor MC38/H-11-hCD47 (#5-4) model.
  • Figure 9 The effect of different PD-L1-CD47 bifunctional fusion proteins on the tumor volume of C57/BL-6 mouse transplanted tumor MC38-hPD-L1-hCD47 model.
  • Figure 10 The effect of different PD-L1-CD47 bifunctional fusion proteins on the tumor volume of C57/BL-6 mouse transplanted tumor MC38-hPD-L1 model.
  • Figure 11 The effect of different PD-L1-CD47 bifunctional fusion proteins on the tumor volume of Molp-8 tumor-bearing nude mice. This model focuses on investigating the anti-tumor effect of the CD47 target pathway in the bifunctional fusion protein.
  • bifunctional fusion protein refers to a protein molecule that can bind to two target proteins or target antigens.
  • the bifunctional fusion protein in the present disclosure mainly includes PD-L1 and CD47 that can bind to the cell surface, which is composed of anti-PD-L1 antibodies and The SIRP ⁇ polypeptide variants are linked to the proteins formed after the fusion.
  • PD-L1 refers to programmed death ligand 1, also known as CD274 or B7H1.
  • the amino acid sequence of human full-length PD-L1 is provided in GenBank under the accession number NP_054862.1. Unless specified to be from a non-human species, the term “PD-L1” means human PD-L1.
  • Anti-human PD-L1 antibody refers to an antibody that can bind to human PD-L1 and can block the binding of PD-1 and PD-L1.
  • Anti-human PD-L1 antibody 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.
  • the anti-human PD-L1 antibody in the present disclosure can also be selected from full-length antibodies h1830, h1831, or anti-PD-L1 antibodies or antigen-binding fragments thereof that have the same CDR combination as the h1830 and h1831 antibodies, respectively.
  • SIRP ⁇ peptide refers to human SIRP ⁇ -D1 domain peptide (the amino acid sequence of the wild-type SIRP ⁇ peptide is shown in SEQ ID NO: 20), which has the activity of binding to human CD47.
  • SIRP ⁇ peptides may also include human SIRP ⁇ -D1 domain peptide mutants, or “SIRP ⁇ peptide variants”, which have amino acid substitutions at one or more positions equivalent to wild-type SIRP ⁇ peptides, so The number of amino acid substitution mutations is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2.
  • the SIRP ⁇ peptide variants have enhanced CD47 binding activity on the surface of tumor cells relative to wild-type SIRP ⁇ peptide (the affinity activity of wild-type SIRP ⁇ binding to CD47 is micromolar level). Further, in some specific embodiments, the SIRP ⁇ peptide variant has the property of not binding or (relative to tumor cell surface CD47 binding activity) reducing binding to CD47 on the surface of human red blood cells.
  • the S58 peptide is equivalent to the wild-type SIRP ⁇ peptide shown in SEQ ID NO: 20 for K19E, N51 for N51M, Q52 for Q52S, K53 for K53G, and E54 for E54R.
  • N101 is a substitution mutant of N101D.
  • the optional sites for amino acid substitution mutations may include K19, K53, N101, L31, N51, Q52, E54, H56, N70, M72, M112, M6, V27, L30, V33, V36
  • One or more of, L37, V42, E47, L66, T67, V92 or S98 undergoes amino acid substitution mutations.
  • the SIRP gamma peptide variant is shown in SEQ ID NO: 1:
  • X1 is selected from L or W
  • X2 is selected from M
  • X3 is selected from Q
  • S or T is selected from E
  • T or R is selected from H or R
  • X6 is selected from D
  • N or E is selected from I, V, M, R or K
  • X8 is selected from M or V.
  • the SIRP gamma peptide variant is shown in SEQ ID NO: 2:
  • X1 is selected from L or W
  • X3 is selected from Q
  • S or T is selected from E
  • T or R is selected from H or R
  • X6 is selected from D
  • N or E is selected from I, V, M, R or K
  • X8 are selected from M or V.
  • the following table shows the amino acid substitution mutation sites and exemplary substituted amino acid residues of different SIRP ⁇ peptide variants relative to the wild-type SIRP ⁇ peptide.
  • antibody (Ab) includes any antigen-binding molecule that includes at least one complementarity determining region (CDR) that specifically binds or interacts with a specific antigen (or its epitope, such as PD-L1 antigen or its epitope) Or molecular complexes.
  • CDR complementarity determining region
  • the term “antibody” includes: immunoglobulin molecules including four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (for example, IgM).
  • Each heavy chain includes a heavy chain variable region (abbreviated as HCVR or VH in the text) and a heavy chain constant region (CH). This heavy chain constant region contains three regions (domains), CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (abbreviated as LCVR or VL in the text) 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 (CDR), interspersed with more conservative regions, called framework regions (FR, also called framework regions, framework regions).
  • CDR complementarity determining regions
  • FR also called framework regions, framework regions.
  • Each VH and VL are composed of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FR of the anti-PD-L1 antibody may be the same as the human germline sequence, or may be modified naturally or artificially.
  • the antibodies may be antibodies of different subclasses, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subclass), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • full-length antibody refers to an antibody in a substantially intact form, as distinguished from the antigen-binding fragments defined below.
  • the term specifically refers to an antibody in which the heavy chain sequentially includes the VH region, CH1 region, hinge region and Fc region from the amino terminus to the carboxy terminus, and the light chain sequentially includes the VL region and the CL region from the amino terminus to the carboxy terminus.
  • 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 (such as isolated complementarity determining regions (CDR), such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides composed of amino acid residues mimicking the hypervariable regions of antibodies.
  • CDR complementarity determining regions
  • engineered molecules such as region-specific antibodies, single-domain antibodies, region-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent Nanobodies, double Nanobodies, etc.), small modular immunopharmaceuticals (SMIP) and shark variable IgNAR regions are also included in the term "antigen-binding fragment" as used in the text.
  • SMIP small modular immunopharmaceuticals
  • shark variable IgNAR regions are also included in the term "antigen-binding fragment" as used in the text.
  • the antigen-binding fragment of an antibody will typically contain at least one variable region.
  • the variable region can be a region of any size or amino acid composition, and will generally contain CDRs adjacent to or within one or more framework sequences.
  • the VH and VL regions can be positioned opposite to each other in any suitable arrangement.
  • the variable region may be dimerized and contain VH-VL or VL-VH dimers.
  • variable region and the constant region of the antigen-binding fragment in any configuration of the variable region and the constant region of the antigen-binding fragment, can be directly connected to each other or can be connected through a complete or partial hinge or linker region.
  • the hinge region can be composed of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, so that it is produced between adjacent variable and/or constant regions in a single polypeptide molecule Flexible and semi-flexible connection.
  • the antigen-binding fragments of the present disclosure may include non-covalently linked to each other and/or linked to one or more monomeric VH or VL regions (for example, disulfide bonds) with variable regions and constant regions. Homodimer or heterodimer (or other multimer).
  • a "murine antibody” in this disclosure is a mouse or rat-derived monoclonal antibody prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated. When the injected test subject is a mouse, the antibody produced is a mouse-derived antibody, and when the injected test subject is In the case of rats, the antibodies produced are of rat origin.
  • a "chimeric antibody” is an antibody formed by fusing the variable region of an antibody of the first species (e.g., mouse) with the constant region of an antibody of the second species (e.g., human). To establish a chimeric antibody, it is necessary to establish a hybridoma secreting the monoclonal antibody of the first species, and then clone the variable region genes from the hybridoma cells, and then clone the constant region genes of the second species antibody as needed, and convert the first species variable region The gene and the constant region gene of the second species are connected to form a chimeric gene and then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations (such as YTE mutation, back mutation, L234A and/or L235A mutation, or S228P mutation) of IgG1, IgG2 or IgG4 heavy chain constant region variants.
  • humanized antibody refers to the transplantation of the CDR sequence of an animal-derived antibody, such as a murine antibody, into a human antibody variable region framework (or framework).
  • Humanized antibodies can overcome the heterologous reaction induced by chimeric antibodies that carry a large amount of heterologous protein components.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet http://www.vbase2.org/), as well as in Kabat, EA, etc., 1991Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • a small amount of back mutation can be performed on the human antibody variable region framework sequence to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.
  • the grafting of the CDR may result in the weakened affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of hypermutation of somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of the humanized antibody.
  • the amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of animal monoclonal antibodies. Residues in the CDR donor framework that are different from the germline can be considered related.
  • the sequence can be compared with the consensus sequence of a subclass or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of hypermutation of somatic cells and thus play an important role in binding.
  • the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine ⁇ , ⁇ chain or a variant thereof, or further comprise human or murine IgG1 , IgG2, IgG3, IgG4 or variants of the heavy chain constant region.
  • inventions of the human antibody heavy chain constant region and human antibody light chain constant region refer to the heavy chain constant region or light chain constant region that has been disclosed in the prior art and does not change the structure and function of the antibody variable region.
  • exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions.
  • the specific substitutions are YTE mutations known in the art, L234A and/ Or L235A mutation, or S228P mutation, or mutation to obtain the knob-into-hole structure (making the antibody heavy chain have a combination of knob-Fc and hole-Fc), these mutations have been confirmed to give the antibody new properties, but do not change the antibody The function of the variable region.
  • Human antibody and “human antibody” can be used interchangeably. It can be an antibody derived from humans or an antibody obtained from a genetically modified organism that has been "engineered” to produce in response to antigen stimulation
  • Specific human antibodies can be produced by any method known in the art. In some technologies, elements of human heavy and light chain loci are introduced into cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted for destruction.
  • Transgenic organisms can synthesize human antibodies specific to antigens, and the organisms 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 by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.
  • “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, except for possible variant antibodies (for example, containing naturally occurring mutations or mutations generated during the manufacture of monoclonal antibody preparations, these variants are usually Except in a small amount), the individual antibodies constituting the population recognize the same and/or bind the same epitope.
  • Each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal” indicates the characteristics of the antibody as obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring any specific method to manufacture the antibody.
  • the monoclonal antibodies used in accordance with the present disclosure can be prepared by various techniques including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and the use of transgenic animals containing all or part of human immunoglobulin loci Methods of, such methods, and other exemplary methods for preparing monoclonal antibodies are described herein.
  • single-chain Fv single-chain Fv
  • scFv single-chain Fv
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies.
  • the antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulin.
  • Antigen-binding fragments can also be incorporated into single-chain molecules comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al. , 1995 Protein Eng. 8(10): 1057-1062; and US Patent US5641870).
  • Fab is an antibody fragment with a molecular weight of about 50,000 Da and antigen-binding activity obtained by treating an IgG antibody with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and The entire L chain is held together by disulfide bonds.
  • F(ab')2 is obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with pepsin. It has a molecular weight of about 100,000 Da and has antigen binding activity, and contains two Fab regions connected at the hinge position. Antibody fragments.
  • Fab' is an antibody fragment obtained by cleaving the disulfide bond of the hinge region of F(ab')2 and having a molecular weight of about 50,000 Da and having antigen-binding activity.
  • Fab' can be produced by treating F(ab')2 that specifically recognizes and binds antigen with a reducing agent such as dithiothreitol.
  • Fab' can be expressed by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector, and introducing the vector into a prokaryotic organism or eukaryotic organism.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules.
  • Such scFv molecules may have the general formula: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 (including 1, 2, 3 or 4) repeated variants (Holliger et al. (1993), Proc Natl Acad Sci USA. 90: 6444-6448).
  • linkers that can be used in the present 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.
  • Anti-human PD-L1 antibody includes a full-length antibody capable of specifically binding to human PD-L1, as well as an antigen-binding fragment comprising the light chain variable region and heavy chain variable region of the full-length antibody, including but not limited to A single chain antibody (scFv), Fab fragment, or other antigen-binding fragment containing scFv or Fab comprising the light chain variable region and heavy chain variable region of the full-length antibody.
  • scFv single chain antibody
  • Fab fragment or other antigen-binding fragment containing scFv or Fab comprising the light chain variable region and heavy chain variable region of the full-length antibody.
  • connection in which the SIRP ⁇ peptide is connected to the polypeptide chain of the anti-human PD-L1 antibody refers to an effective connection between polypeptides, including, for example, connection via a peptide bond, or connection using a linker. The connection will not lose the functions of the SIRP ⁇ peptide and the anti-human PD-L1 antibody.
  • Linker refers to a connecting peptide sequence used to connect protein domains or different proteins or different polypeptides. It usually has a certain degree of flexibility. The use of linkers will not make the original protein domains Loss of function. Exemplary linkers are shown in the table below.
  • anti-PD-L1 antibodies can be linked to SIRP ⁇ peptide variants using linkers.
  • Some exemplary bifunctional fusion proteins include the following fusion proteins:
  • Diabodies are antibody fragments in which scFv is dimerized, and are antibody fragments with bivalent antigen binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
  • dsFv is obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to the known method (Protein Engineering. 7:697 (1994)) based on the three-dimensional structure prediction of the antibody.
  • the antigen-binding fragment can be produced by the following steps: obtain the VH and/or VL of the monoclonal antibody of the present disclosure that specifically recognizes and bind to the antigen, and cDNA encoding other domains as required, and construct the encoding antigen
  • the DNA of the binding fragment is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryote to express the antigen-binding fragment.
  • the "Fc region” can be a native sequence Fc region or a variant Fc region.
  • the Fc region of an immunoglobulin heavy chain is usually defined as extending from the amino acid residue at position Cys226 or extending from Pro230 to its carboxy terminus.
  • the numbering of residues in the Fc region is as the numbering of the EU index in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
  • the Fc region of immunoglobulin usually has two constant region domains, CH2 and CH3.
  • amino acid difference or “amino acid mutation” means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including the insertion of one or more amino acids on the basis of the original protein or polypeptide , Missing or substituted.
  • variable region of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination.
  • VL variable region
  • VH variable region
  • the variable regions of the heavy and light chains each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDR) (also called hypervariable regions).
  • FR framework regions
  • CDR complementarity determining regions
  • the CDRs in each chain are held together tightly by 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 (ie, Kabat et al.
  • CDR may refer to a CDR determined by either method or a combination of both methods.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • CDR is defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-35 in human VL.
  • 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute.
  • the CDR amino acid residue numbers in VH are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1) ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
  • Antibody constant region domains refer to the domains derived from the constant regions of the light and heavy chains of an antibody, including CL and CH1, CH2, CH3, and CH4 domains derived from different types of antibodies.
  • Epitope or “antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996).
  • 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 through weak non-covalent forces; the greater the interaction, the stronger the affinity.
  • an antibody or antigen-binding fragment thereof e.g. Fab fragments
  • high affinity generally refers to having a K D 1E -9 M in K D or less (e.g.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibody binds to the antigen with a dissociation equilibrium constant (KD) less than about 1E -8 M, for example, less than about 1E -9 M, 1E -10 M, or 1E -11 M or less, for example, as using surface plasmon resonance (SPR) technology is measured in BIACORE instrument.
  • KD dissociation equilibrium constant
  • SPR surface plasmon resonance
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be a single-stranded or double-stranded DNA molecule or RNA molecule, for example, a double-stranded DNA or mRNA.
  • a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • vector means a construct capable of delivering one or more target genes or sequences and preferably expressing them in a host cell.
  • examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic flocculants, DNA or DNA encapsulated in liposomes RNA expression vectors and certain eukaryotic cells such as producer cells.
  • mice can be immunized with antigens or fragments thereof, and the obtained antibodies can be renatured and purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to non-human CDR regions.
  • the human FR germline sequence can be obtained from the website http://www.imgt.org/ by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the family Enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus (Streptococcus) and Haemophilus influenzae (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-limiting examples such as HEK293E cells), and NSO cells.
  • the engineered antibody or antigen-binding fragment can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a protein A or protein G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostics
  • agent, composition, or human manipulation such as “euthanasia” in the examples
  • administration can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a patient (or subject), for example, a composition comprising any one of the compounds of the embodiments of the present disclosure, the patient (or subject) has (or is suspected of having , Or susceptible to) one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to relieve one or more symptoms of the disease in the treated patient (or subject) or population, so as to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent .
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the disease state, age and weight of the patient (or subject), and the presence of the drug in the patient (or subject). Subject) the ability to produce the required therapeutic effect. Any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can assess whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients (or subjects).
  • amino acid conservative modification or “amino acid conservative substitution” refers to the substitution of amino acids in a protein or polypeptide with other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), thereby This allows frequent changes without changing the biological activity or other desired characteristics of the protein or polypeptide (such as antigen affinity and/or specificity).
  • amino acid conservative modification or “amino acid conservative substitution” refers to the substitution of amino acids in a protein or polypeptide with other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), thereby This allows frequent changes without changing the biological activity or other desired characteristics of the protein or polypeptide (such as antigen affinity and/or specificity).
  • Those skilled in the art recognize that, generally, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change biological activity (see, for example, Watson et al.,
  • Effective amount and “effective dose” refer to the amount of the drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the dosage of other agents required to treat the disorder , Enhance the efficacy of another agent, and/or delay the progression of the patient (or subject) of the target antigen-related disorder of the present disclosure.
  • Exogenous refers to substances produced outside organisms, cells, or humans according to circumstances.
  • Endogenous refers to substances produced in cells, organisms, or human bodies according to circumstances.
  • isolated refers to a purified state, and in this case means that the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers or salts unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described in the present disclosure or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thereby exert the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc.
  • Suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffer, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol and sodium chloride.
  • Another aspect of the present disclosure relates to methods for immunological detection or determination of target antigens, reagents for immunological detection or determination of target antigens, methods for immunological detection or determination of cells expressing target antigens, and methods for diagnosis and target antigen
  • a diagnostic agent for a disease associated with a positive cell which contains the monoclonal antibody or antibody fragment, or fusion protein, or bifunctional fusion protein of the present disclosure that specifically recognizes and binds to the target antigen as an active ingredient.
  • cancer refers to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth and are used interchangeably in this disclosure.
  • cancers or malignancies include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • this cancer includes 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 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), T-cell/histiocytic-rich Large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tract) cancer,
  • Inflammatory disorder refers to any disease, disorder, or syndrome in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, host tissue damage, or loss of tissue function.
  • Inflammatory disease also refers to a pathological state mediated by the pooling of leukocytes or neutrophil chemotaxis.
  • Inflammation refers to a local protective response caused by tissue damage or destruction, which is used to destroy, weaken or eliminate (isolate) harmful substances and injured tissues. Inflammation is significantly related to the pooling of leukocyte or neutrophil chemotaxis. Inflammation can be caused by pathogenic organisms and viruses as well as non-infectious causes such as trauma or reperfusion or stroke after myocardial infarction, immune response to foreign antigens, and autoimmune response.
  • the aforementioned diseases related to the target antigen-positive cells can be diagnosed by detecting or measuring cells expressing the target antigen using the monoclonal antibody or antibody fragment of the present disclosure.
  • the living body sample used to detect or measure the target antigen there is no particular limitation on the living body sample used to detect or measure the target antigen, as long as it has the possibility of containing cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, Stool, tissue fluid or culture fluid.
  • cells expressing the target antigen such as tissue cells, blood, plasma, serum, pancreatic juice, urine, Stool, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • the reagents used to perform the antigen-antibody reaction include buffers, salts and the like.
  • the reagents used for detection include reagents commonly used in immunological detection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label.
  • CD47 human CD47 protein, Uniprot number: Q08722
  • CD47-ECD-His The extracellular domain of CD47 protein with His tag (CD47-ECD-His): (SEQ ID NO: 3)
  • CD47 extracellular domain and human IgG1Fc fusion protein (CD47-ECD-Fc) as a detection reagent: (SEQ ID NO: 4)
  • the underlined part is human IgG1-Fc part.
  • the underlined part is human IgG1-Fc part.
  • the cell expression supernatant was centrifuged at high speed to remove impurities, the buffer was replaced with PBS, and imidazole was added to a final concentration of 5mM. Equilibrate the nickel column with a PBS solution containing 5mM imidazole, and wash 2-5 times the column volume. The supernatant sample after replacement was applied to the IMAC column. Wash the column with a PBS solution containing 5 mM imidazole until the A280 reading drops to baseline. Then rinse the chromatography column with PBS+10mM imidazole to remove non-specifically bound contaminated proteins, and collect the effluent.
  • the target protein was eluted with a PBS solution containing 300 mM imidazole, and the elution peak was collected.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE, 28-9893-35), and the mobile phase was PBS. Remove the aggregate peak and collect the elution peak. After the obtained protein was identified as correct by electrophoresis, peptide map, and LC-MS, it was divided into equipment for use.
  • CD47-ECD-His (SEQ ID NO: 3) with His tag is used as an immunogen or detection reagent for the antibody of the present disclosure.
  • CD47-ECD-His can also be used as an immunogen to stimulate mouse immunity after coupling reaction with KLH protein by in vitro chemical methods.
  • the cell expression supernatant was centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography.
  • the MabSelect Sure chromatography column was first regenerated with 0.1M NaOH, washed with pure water and then equilibrated with PBS. After the supernatant was combined, washed with PBS until the A280 reading dropped to the baseline.
  • the target protein was eluted with 0.1M acetate buffer at pH 3.5, and neutralized with 1M Tris-HCl. After the eluted sample is appropriately concentrated, it is further purified by PBS-balanced gel chromatography Superdex200 (GE, 28-9893-35), and the receiving tube where the target protein is collected is concentrated to an appropriate concentration.
  • This method is used to purify CD47-ECD-Fc (SEQ ID NO: 4) and SIRP ⁇ -Fc (SEQ ID NO: 5) fusion proteins. This method can also be used to purify the humanized antibody protein involved in the present disclosure.
  • variable regions of the light and heavy chains of the anti-PD-L1 antibody are modified from the anti-PD-L1 antibody of WO2017084495A1, and its sequence and related properties are recorded in the PCT application with application number PCT/CN2019/070982, and the entire content of the PCT application is cited in This application.
  • Anti-PD-L1 antibody 24D5H12L61 iso-PD-L1 antibody 24D5H12L61
  • the heavy chain constant region sequence of IgG4-S228P is as follows: (SEQ ID NO: 10)
  • the heavy chain constant region sequence of IgG1 is as follows: (SEQ ID NO: 11)
  • the light chain (Kappa chain) constant region sequence of the antibody is as follows: (SEQ ID NO: 12)
  • the constructed full-length antibodies are as follows:
  • h1830G1 heavy chain (SEQ ID NO: 15)
  • h1830G1 light chain (same as h1830 light chain, SEQ ID NO: 14):
  • h1830G1 heavy chain (SEQ ID NO: 18)
  • h1831G1 light chain (same as h1831 light chain, SEQ ID NO: 17)
  • phage library design degenerate primers, and introduce the designed mutant amino acids into the SIRP ⁇ phage mutant library by PCR.
  • the size of each library is about 10 9 .
  • SIRP ⁇ phage mutant library Add the packaged SIRP ⁇ phage mutant library (1 ⁇ 10 12 -1 ⁇ 10 13 ) and 100 ⁇ l streptocin beads (Mi1envi Biotec, Auburn, CA) into 1 ml containing 2% skimmed milk -Incubate in phosphate buffered saline (abbreviated MPBS) for 1 hour at room temperature, place on a magnetic stand, and take the supernatant.
  • MPBS phosphate buffered saline
  • the SIRP ⁇ phage mutant library uses biotinylated human CD47-ECD-his protein antigen, and after 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen), a phage mutant monoclonal with higher affinity than wild-type SIRP ⁇ is finally obtained Perform sequencing verification. The sequenced clones were compared and analyzed. After removing redundant sequences, the non-redundant sequences were converted into PDL1-CD47 bifunctional fusion protein for mammalian cell expression.
  • affinity maturation of the CD47 receptor SIRP ⁇ -D1 domain was carried out through the yeast display platform technology, and an affinity mature yeast library for the CD47 binding domain was designed and prepared on the basis of SIRP ⁇ -D1, and Screen new SIRP ⁇ mutants.
  • yeast libraries degenerate primers, amino acid mutations introduced by PCR to design SIRP ⁇ mutant libraries, each library size is about 109, the constructed yeast library sequencing verification method II The diversity of the library.
  • Collect cells with a high affinity library for CD47-Fc and then in SDCAA medium (20g dextrose, 6.7g Difco yeast nitrogen source-no amino acids, 5g Bacto casamino acids, 5.4g Na2HP O 4 and 8.56g NaH 2 PO 4 ⁇ H 2 O, dissolved in 1L of distilled water) in 250rpm and 30°C for 24 hours. Then, the culture was dissolved in SGCAA medium (20g galactose, 6.7g Difco yeast nitrogen source-no amino acids, 5g Bacto casamino acids, 5.4g Na2HP O 4 and 8.56g NaH 2 PO 4 ⁇ H 2 O) In 1L distilled water), induce 18 hours at 250rpm and 20°C.
  • SDCAA medium 20g dextrose, 6.7g Difco yeast nitrogen source-no amino acids, 5g Bacto casamino acids, 5.4g Na2HP O 4 and 8.56g NaH 2 PO 4 ⁇ H 2 O
  • the obtained enriched library was subjected to the second round of screening for binding to biotinylated recombinant human CD47-Fc.
  • 100 times the size of the library from the previous round was used as the number of input cells.
  • the library cells from the previous round were combined with 1 ⁇ g/ml biotinylated human CD47-Fc protein and 10 ⁇ g/ml Mouse Anti-cMyc (9E10, sigma) antibody in 0.1% PBSA After incubating at room temperature for 1 hour, the mixture was washed three times with 0.1% PBSA to remove unbound antibody fragments. Add Goat anti-mouse-Alexa488 (A-11001, life technologies) and Strepavidin-PE (S-866, Life technologies) and incubate for 1 hour at 4°C. Wash the mixture three times with 0.1% PBSA to remove unbound antibody fragments . Finally, through FACS screening (BD FACSAriaTM FUSION), high affinity SIRP ⁇ mutants were found.
  • FACS screening BD FACSAriaTM FUSION
  • the SIRP ⁇ mutant library uses biotinylated human CD47-Fc antigen, and has undergone 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and 2-3 rounds of FACS screening (BD FACSAriaTM FUSION). Then select about 400 yeast monoclonals to culture and induce expression, use FACS (BD FACSCanto II) to detect the binding of yeast monoclonals to human CD47-Fc antigen, and select yeast monoclonals with higher affinity than wild-type SIRP ⁇ for sequencing verification. The sequenced clones were compared and analyzed. After removing redundant sequences, the non-redundant sequences were converted into PDL1-CD47 bifunctional fusion protein for mammalian cell expression.
  • MACS screening streptomycin magnetic beads, Invitrogen
  • FACS screening BD FACSAriaTM FUSION
  • SIRP ⁇ peptide variants are as follows:
  • the obtained anti-PD-L1 antibody is connected with SIRP ⁇ to form a fusion protein, which is expressed and purified by conventional methods to obtain the PD-L1-CD47 bifunctional fusion protein.
  • Anti-CD47 antibody hu5F9 (sequence from US09017675B)
  • Hu5F9 heavy chain (SEQ ID NO: 83)
  • Hu5F9 light chain (SEQ ID NO: 84)
  • SIRP ⁇ -CV (refer to Engineered SIRP ⁇ Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies synthesis, Science.2013Jul 5;341(6141):88-91, SEQ ID NO:85)
  • TTI-621 (The sequence is from WO2014094122A1, SEQ ID NO: 133)
  • Anti-CD47 antibody Hu167-IgG4 AA prepared according to the method disclosed in WO2018095428A1 patent application.
  • Hu167-IgG4 AA heavy chain (SEQ ID NO: 87)
  • Hu167-IgG4 AA light chain (SEQ ID NO: 88)
  • the h1831 antibody was subjected to CDR mutation modification, and 36 mutants were obtained, and finally the N53K (position determined according to the Kabat numbering rule) mutant h1831K was screened. That is, the h1831 light chain LCDR2 was mutated from AAS N LES to AAS K LES, and a new antibody h1831K was obtained.
  • LCDR1 is RASESVSIHGTHLMH (SEQ ID NO: 106)
  • LCDR2 is AASKLES (SEQ ID NO: 112)
  • LCDR3 is QQSFEDPLT (SEQ ID NO: 108).
  • h1831K heavy chain (same sequence as h1831 heavy chain, SEQ ID NO: 16)
  • the IgG4 control is an antibody against a target that has nothing to do with PD-L1 and CD47.
  • IgG4-Fc and IgG1-Fc contain only the Fc segment, and do not contain the variable region segment against any antigen.
  • Test Example 1 ELISA experiment of PD-L1-CD47 bifunctional fusion protein binding CD47-his protein
  • the binding force of the PD-L1-CD47 bifunctional fusion protein is detected by the binding amount of the bifunctional fusion protein and human CD47 or cyno CD47 immobilized on the ELISA plate.
  • each PD-L1-CD47 bifunctional fusion protein has a strong affinity with free human CD47 protein, and at the same time, it has a strong cross-affinity with monkey CD47.
  • Test Example 2 ELISA of PD-L1-CD47 bifunctional fusion protein binding PD-L1-his protein
  • the binding capacity of the PD-L1-CD47 bifunctional fusion protein is detected by the amount of antibody binding to PD-L1 of different species immobilized on the ELISA plate. Dilute different germline PD-L1-his antigens (see Table 9) with PBS to 1 ⁇ g/ml and coat them on a 96-well ELISA plate (Costar, CAT#3590).
  • each PD-L1-CD47 bifunctional fusion protein has a strong affinity with free human PD-L1 protein, and at the same time, it has a strong cross-affinity activity with monkey PD-L1.
  • the PD-L1-CD47 bifunctional fusion protein containing h1830 antibody also has a strong cross-affinity activity with mouse PD-L1.
  • Dilute PD-L1-Fc (prepared in-house) with PBS to 1 ⁇ g/ml, add 100 ⁇ l/well to a 96-well plate, and place it at 4°C for 16h-20h. Aspirate the PBS buffer from the 96-well plate, wash the plate once with PBST (pH 7.4 PBS containing 0.05% tween20) buffer, add 120 ⁇ l/well PBST/1% milk, and incubate at room temperature for 1 hour for blocking.
  • PBST pH 7.4 PBS containing 0.05% tween20
  • test results show that all bifunctional fusion proteins can also effectively produce blocking bifunctional fusion proteins on the PD-L1/PD-1 and PD-L1/B7.1 pathways.
  • Fresh healthy human blood was mixed with PBS in equal volume and centrifuged at 300g for 5 minutes to obtain cell clusters. After washing with PBS for 3-5 times, red blood cells were obtained. Resuspend in FACS buffer (PBS+5% BSA), adjust the cell density to 2 ⁇ 10 6 cells/ml, plant to a 96-well round bottom plate (3795#, corning) at 100 ⁇ l/well, and then add different concentrations of antibodies and The bifunctional fusion protein was incubated at 4°C for 1 hour.
  • FACS buffer PBS+5% BSA
  • Raji cells were cultured in RPMI medium (Hyclone, CAT#SH30809.01B) (containing 10% fetal bovine serum), 1 ⁇ 10 6 cells/ml Raji cells were blocked with 5% BSA, and the bifunctional fusion protein sample was added to 10 ⁇ g /ml, after washing twice, add Alexa Fluor 488-goat anti-human (H+L) antibody (Invitrogen, CAT#A11013), after washing twice, the fluorescence signal value is read by flow cytometer.
  • RPMI medium Hyclone, CAT#SH30809.01B
  • Alexa Fluor 488-goat anti-human (H+L) antibody Invitrogen, CAT#A11013
  • the FACS test results show that the PD-L1-CD47 bifunctional fusion protein involved has a strong binding ability to the natural CD47 on the surface of Raji cells, which is equivalent to the binding ability of the control antibody Hu5F9.
  • the results are shown in Figure 3.
  • PBMC peripheral blood mononuclear cells
  • CD14+ monocytes were sorted using Human CD14 MicroBeads (130-050-201#, Miltenyi Biotec). These CD14+ monocytes were cultured in macrophage differentiation medium (1640+10% FBS+50ng/ml M-CSF) for 9 days to differentiate into macrophages. These monocyte-derived macrophages (MDM) become adhesive and have antennae. On the day of the experiment, the macrophages were trypsinized for 5 minutes, scraped off gently with a spatula, and spread on a 96-well round bottom plate (3795#).
  • MDM monocyte-derived macrophages
  • Phagocytosis was measured by selecting CFSE+ positive cells in the APC+ positive living cell gate and then evaluating the percentage of CSFE+ positive cells (see Figure 4).
  • Test Example 7 PD-L1-CD47 bifunctional fusion protein in vitro cell-mediated cell phagocytosis (ADCP) experiment
  • PBMC peripheral blood mononuclear cells
  • CD14+ monocytes were sorted using Human CD14 MicroBeads (130-050-201#, Miltenyi Biotec). These CD14+ monocytes were cultured in macrophage differentiation medium (1640+10% FBS+50ng/ml M-CSF) for 9 days to differentiate into macrophages. These monocyte-derived macrophages (MDM) become adhesive and have antennae. On the day of the experiment, the macrophages were trypsinized for 5 minutes, scraped off gently with a spatula, and spread on a 96-well round bottom plate (3795#).
  • MDM monocyte-derived macrophages
  • Test Examples 6 and 7 are shown in Figure 4 and Figures 5A-5B, showing that the added bifunctional fusion protein can effectively promote the phagocytosis of tumor cells.
  • the bifunctional fusion protein has no phagocytic effect on red blood cells, suggesting the potential safety advantages of the bifunctional fusion protein antibody of the present disclosure.
  • the control antibody hu5F9 can effectively engulf red blood cells.
  • Test Example 8 Red blood cell agglutination experiment of PD-L1-CD47 bifunctional fusion protein
  • Fresh healthy human blood was diluted 100 times with PBS (B320#, Shanghai Yuanpei Biotechnology Co., Ltd.). The diluted whole blood was spread on a 96-well round bottom plate (3795#, corning), 30 ⁇ l/well. Then add antibodies or bifunctional fusion proteins with different concentration gradients in equal volumes. After mixing, let it stand at 37°C for 4-6h. Use a high-content microscope to observe the red blood cell sedimentation. No blood coagulation is a clear red spot, and blood coagulation is a diffuse sample.
  • Each sample is diluted from the first column (0.5mg/ml) to the 11th column, 1:3 dilution.
  • the 12th column is the PBS blank well without antibody.
  • Test Example 9 BIAcore detects the affinity experiment of PD-L1-CD47 bifunctional fusion protein
  • the Biacore T200 instrument detects the dual-function fusion protein and different antigen reaction signals in real time to obtain the binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with 10mM Glycine-HCl pH1.5 buffer.
  • the experimental buffer system is 1 ⁇ HBS-EP buffer solution (Cat#BR-1001-88, GE).
  • PBMC peripheral blood mononuclear cells
  • Test Example 11 The effect of PD-L1-CD47 bifunctional fusion protein in mouse colon cancer model MC38/H-11-hCD47
  • B-hCD274/hCD47/hSIRP ⁇ mice were used to inoculate artificially modified murine colon cancer MC38 cells: MC38/H-11-hCD47 (transformed into human PD-L1 and human CD47, knockout mouse CD47 and PDL1), established Mouse tumor-bearing model, and evaluate the in vivo pharmacodynamic effects of different doses of PD-L1-CD47 bifunctional fusion protein h1830-S85, SIRP ⁇ protein S58-Fc and PD-L1 monoclonal antibody h1830 on the growth of mouse colon cancer xenograft tumors.
  • B-hCD274/hCD47/hSIRP ⁇ mice were purchased from Biocytometer experimental animals, SPF grade; body weight: 22.0 ⁇ 3.0 g; gender: female.
  • MC38/H-11-hCD47(#5-4) cells were inoculated subcutaneously into B-hCD274/hCD47/hSIRPa mice at an inoculum of 1 ⁇ 10 6 cells/100 ⁇ l/mouse.
  • the tumor volume was measured .
  • Tumor volume (TV) calculation formula: TV 1/2 ⁇ a ⁇ b 2 , where a and b respectively represent the long diameter and short diameter of the measured tumor.
  • the experimental data were statistically analyzed by Excel and GraphPad, and the animal body weight, tumor volume, and tumor weight of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and graphed with Graphpad Prism 6 software.
  • This experiment aims to evaluate the inhibitory effect of different doses of PD-L1-CD47 bifunctional fusion protein on tumor growth in B-hCD274/hCD47/hSIRPa mouse colon cancer xenograft model.
  • different antibodies or bifunctional fusion proteins were given at the same time in groups.
  • the results in Table 13 show that different doses of bifunctional fusion protein (h1830-S85) experimental group, PD-L1 monoclonal antibody (h1830) experimental group and SIRP ⁇ protein S58-Fc experimental group have smaller tumor volumes than the PBS control group ; PD-L1-CD47 dual-function fusion protein high-dose experimental group has better antitumor effect than the same dose of PD-L1 monoclonal antibody experimental group and SIRP ⁇ protein experimental group, and there is a dose-dependent relationship between the different doses of h1830-S85 experimental group .
  • Test Example 12 The effect of PD-L1-CD47 bifunctional fusion protein in mouse colon cancer model MC38-hPD-L1-hCD47
  • MC38-hPD-L1-hCD47 cells (transferred human PD-L1 and human CD47 into MC38 cells, knockout mouse CD47) were inoculated subcutaneously into C57/BL-6 mice at 5.8 ⁇ 10 5 cells/100 ⁇ l/mouse, After the tumor-bearing model was established, the tumor volume was measured, and the body weight, too large and too small animals were removed.
  • each drug was intraperitoneally administered three times a week for a total of 10 times.
  • the administration period was 18 days.
  • the monitoring of tumor-bearing mice was ended two days after the drug was stopped.
  • the tumor volume was measured twice a week, the weight was weighed, and the data was recorded. See the table below for grouping and administration.
  • different antibodies were administered in groups at the same time. Starting on the 14th day after administration, the dose of all experimental groups was halved; starting on the 25th day after administration, all experimental groups stopped administration.
  • i.p means intraperitoneal injection
  • q.o.d means once every other day.
  • the animal body weight, tumor volume, and tumor weight of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and graphed with Graphpad Prism 6 and Excel software, and statistical analysis was performed using student test.
  • Tumor volume (TV) 1/2 ⁇ L long ⁇ L short 2
  • Tumor growth rate T/C% (T-T0)/(C-C0) ⁇ 100%
  • the tumor volume of the PD-L1-CD47 bifunctional fusion protein h1830-S58 experimental group and PD-L1 monoclonal antibody (h1830) experimental group that cross-reacted with mouse PD-L1 was smaller than that of the control group and TTI- The 621 experimental group, and there was a statistical difference between the control group and the control group about one week after the administration; the TTI-621 experimental group did not show tumor suppression effect in this experiment.
  • the tumor inhibition rate reached 128.51% 7 days after the administration. By the end of the experiment, the tumor inhibition rate remained at a high level.
  • the tumor-bearing mice were euthanized, and the tumor was stripped and weighed.
  • the results of tumor weight were similar to the tumor volume.
  • Test Example 13 The effect of PD-L1-CD47 bifunctional fusion protein in mouse colon cancer model MC38-hPD-L1
  • each drug was administered to the abdominal cavity three times a week, a total of 12 times, the administration cycle was 28 days, and the monitoring of tumor-bearing mice was ended two days after the drug was stopped.
  • the tumor volume was measured twice a week, the weight was weighed, and the data was recorded. See the table below for grouping and administration.
  • the animal body weight, tumor volume, and tumor weight of each group are expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and graphed with Graphpad Prism 5 and Excel software, and statistical analysis is performed using student test.
  • Tumor volume (TV) 1/2 ⁇ L long ⁇ L short 2
  • Tumor growth rate T/C% (T-T0)/(C-C0) ⁇ 100%
  • This experiment aims to detect the inhibitory effect of different IgG forms of PD-L1-CD47 bifunctional fusion protein on tumor growth in C57/BL-6 mouse colon cancer xenograft model.
  • different antibodies were administered in groups at the same time. Starting on the 14th day after administration, the dose of all experimental groups was halved; starting on the 25th day after administration, all experimental groups stopped administration.
  • the tumor volume of all bifunctional fusion protein administration groups and PD-L1 monoclonal antibody h1830 administration groups was smaller than that of IgG4 control group and SIRP ⁇ -CV (TTI-621) experiment Group, and there are statistical differences between the control group.
  • the control group and the SIRP ⁇ -CV (TTI-621) experimental group were euthanized due to the large tumor size, while the remaining experimental groups were stopped and continued observation.
  • the results showed that the tumor volume of the PD-L1 monoclonal antibody h1830 experimental group showed a trend of rapid recovery over time, and the tumor volume of the bifunctional fusion protein h1830-19-S79 and h1830G1-19-S79 experimental groups did not change significantly, and There is also no significant difference between these two different IgG forms of double antibodies.
  • the tumor-bearing mice were euthanized, and the tumor was stripped and weighed.
  • the tumor weight results were similar to the size of the tumor. There was no significant difference in body weight between the administration group and the control group, and the mice were tolerant to each administration antibody good.
  • Test Example 14 The efficacy of PD-L1-CD47 bifunctional fusion protein on MOLP-8 xenograft nude mice
  • mice Balb/c nude mice were inoculated with MOLP-8 cells (5 ⁇ 10 6 +50% matrigel/mouse) subcutaneously on the right ribs, a total of 120 mice. After 10 days, the average tumor volume was about 214.89 ⁇ 6.75mm 3 .
  • the tumor volume was measured twice a week, the weight was weighed, and the data was recorded.
  • the animal body weight, tumor volume and tumor weight of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and graphed with Graphpad Prism 6 and Excel software, and statistically analyzed by student t test.
  • V tumor volume
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV )/C RTV (%)
  • V 0 and V T are the tumor volume at the beginning and end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volumes of the blank control group (Blank) and the experimental group at the end of the experiment, respectively.
  • the results of this experiment show (see Figure 11), intraperitoneal injection, once every other day, 10 consecutive administrations. Statistics are based on the data from the 21st day of the experiment.
  • the PD-L1-CD47 dual-function fusion protein h1830-S37 (30mpk) has a tumor inhibition rate of 34.98% (P ⁇ 0.05);
  • the dual-function fusion protein h1831K-19-S37 (30mpk) has a tumor inhibition rate of 54.18% (P ⁇ 0.01) ; H1830 (25mpk) did not inhibit the growth of the tumor.
  • Test Example 15 PD-L1-CD47 bifunctional fusion protein blocking CD47/SIRP ⁇ binding
  • Test Example 16 The efficacy of PD-L1-CD47 bifunctional fusion protein on human breast cancer cell MDA-MB-231 xenograft
  • MDA-MB-231 cells (ATCC) 3 ⁇ 10 6 cells/200 ⁇ l/mouse (containing 50% Matrigel) were inoculated subcutaneously on the right ribs of NOD/SCID mice, when the average tumor volume of tumor-bearing mice reached about 145mm 3
  • mice were randomly divided into 4 groups: PBS, h1831K-19-S37-30mpk, h1831K-19-S37-10mpk, h1831K-25mpk (maintain equimolar concentration with h1831K-19-S37 high dose), 8 mice in each group , And define the grouping day as Day0 of the experiment.
  • the PBMCs of two volunteers stimulated by CD3 antibody for 3 days were mixed at a ratio of 1:1 and injected into the mouse tumor tissue at 5 ⁇ 10 5 cells/100 ⁇ l/mouse. The remaining PBMCs stopped stimulation and continued to be cultured.
  • 5 ⁇ 10 6 cells/100 ⁇ l/mouse were injected into the tumor-bearing mice intraperitoneally, which was regarded as the first round of injection.
  • a total of two rounds of PBMCs were injected. Starting from Day0, each antibody to be tested was injected intraperitoneally three times a week. The tumor volume and animal weight were monitored twice a week and the data was recorded. When the tumor volume exceeds 1000 mm 3 or most tumors are ulcerated or have a weight loss of 20%, the tumor-bearing animals are euthanized as the experimental endpoint.
  • V tumor volume
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100
  • T, C are the tumor volumes of the treatment group and the control group at the end of the experiment
  • T 0 , C 0 are the tumors at the beginning of the experiment volume.
  • TGI (%) 1-T/C (%).
  • the PD-L1-CD47 bispecific antibody h1831K-19-S37 (30, 10mg/kg) can significantly inhibit the growth of human breast cancer MDA-MB-231 mice subcutaneously transplanted tumors, and there is a dose-dependent relationship between high and low doses. From 3 days after administration to the end of the experiment (Day23), whether in the high-dose group or the low-dose group, the tumor suppressor effect of h1831K-19-S37 is always better than that of the high-dose PD-L1 monoclonal antibody control h1831K (25mg/kg ) (p ⁇ 0.001), and there are also statistical differences between high and low doses (p ⁇ 0.01) (Table 17).
  • mice tolerated the PDL1-CD47 bispecific antibody and its monoclonal antibody well, and there was only a slight fluctuation in body weight during the entire administration process, and no obvious drug-induced weight loss and other symptoms occurred.

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Abstract

涉及双功能融合蛋白及其医药用途。具体地，涉及一种包含SIRPγ肽变体和抗人PD-L1抗体的双功能融合蛋白、SIRPγ肽变体以及其医药用途。根据双功能融合蛋白能够特异性结合PD-L1和CD47，来阻断PD-L1或CD47与其受体或配体的结合。同时，还提供所述双功能融合蛋白的制备、应用及癌症和免疫相关疾病的治疗。

Description

双功能融合蛋白及其医药用途 技术领域
本公开涉及一种特异性结合PD-L1和CD47的双功能融合蛋白、包含所述双功能融合蛋白的药物组合物，以及其作为抗癌药物的用途。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
程序性死亡分子1(programmed death-l,PD-l)是1992年发现的表达在T细胞表面的一个蛋白受体，参与细胞的凋亡过程。PD-l属于CD28家族，与细胞毒性T淋巴细胞抗原4(cytotoxic T Iymphocyte antigen 4,CTLA-4)具有23％的氨基酸同源性，但其表达却与CTLA-4不同，主要表达在活化的T细胞、B细胞和髓系细胞上。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1的主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell,DC)上，在活化后细胞上的表达能够进行上调。而PD-L2的表达相对较局限，主要表达在抗原呈递细胞上，如活化的巨噬细胞和树突状细胞。
新的研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤等人类肿瘤组织中检测到高PD-L1蛋白的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。由于PD-L1起到着第二信号通路抑制T细胞增殖的作用，所以阻断PD-L1/PD-1之间结合成为了肿瘤免疫治疗领域一个非常有潜力的新兴靶点。
细胞表面蛋白CD47在许多肿瘤类型上表达或过度表达，包括急性骨髓性白血病，B细胞非霍奇金淋巴瘤的多种亚型，以及许多人类实体瘤细胞。CD47与巨噬细胞上的信号调节蛋白α(SIRPα)结合，是肿瘤细胞表面的“不吃我”信号。最近的数据表明抗CD47抗体也有助于提高免疫耐受性小鼠中有效的抗肿瘤T细胞应答。因此，抗CD47抗体是调节先天免疫系统和适应性免疫系统的新一类免疫检查点抑制剂。
目前已有相关的CD47专利，如WO2016065329、WO2016109415、WO2014087248、WO2014093678、CN107849143A、CN108350048、CN106535914、WO2016023001A、CN107459578A、CN2017110167989等。例如，WO2016023001A中描述了包含高亲和性PD-1模拟肽和特异性结合CD47的高亲和性SIRP-α的多特异性PD-1模拟肽及其用途；CN107459578A中描述了一种包含SIRPα突变体和抗PD-L1抗体的靶向CD47分子与PD-L1分子的重组融合蛋白；CN201711016798.9公开了包含SIRPα的胞外部分和PD-1的胞外部分的多功能融合蛋白。
然而，目前临床前和临床研究许多疗法是针对CD47/SIRPα相互作用的，包括抗CD47抗体、SIRPα受体蛋白和工程SIRPα受体蛋白、抗SIRPα抗体和双特异 性抗体等，并没有关于包含SIRPγ肽的多特异性融合蛋白的相关报道。
SIRPγ在T细胞和活化的NK细胞上表达，并且与SIRPα相比，SIRPγ以低10倍的亲和力结合CD47。CD47-SIRPγ相互作用参与抗原呈递细胞与T细胞之间的接触，共刺激T细胞活化并促进T细胞增殖(Piccio等人，Blood 2005，105，2421-2427)。此外，CD47-SIRPγ相互作用在T细胞的跨内皮迁移中发挥作用(Stefanisakis等人，Blood 2008，112，1280-1289)。
发明内容
本公开提供了包含SIRPγ肽变体的双功能融合蛋白。其中SIRPγ肽变体相较于野生型SIRPγ肽具有显著大幅提高的CD47亲和活性。
在一些实施方案中，提供一种双功能融合蛋白，所述双功能融合蛋白包含SIRPγ肽变体和抗人PD-L1抗体，所述SIRPγ肽变体连接至所述抗人PD-L1抗体的多肽链，
所述SIRPγ肽变体是在相当于如SEQ ID NO：20所示的野生型SIRPγ肽的N51位具有替代突变的SIRPγ肽变体。在一些实施方案中，如前所述的SIRPγ肽变体具有结合肿瘤细胞表面CD47的活性，优选地，所述SIRPγ肽变体具有较野生型SIRPγ肽增强地结合肿瘤细胞表面CD47的活性。
在一些实施方案中，提供一种双功能融合蛋白，所述双功能融合蛋白包含人SIRPγ肽变体和抗人PD-L1抗体，所述SIRPγ肽变体连接至所述抗人PD-L1抗体的多肽链，
所述SIRPγ肽变体是在如SEQ ID NO：20所示的野生型SIRPγ肽的N51位具有替代突变的SIRPγ肽变体。在一些实施方案中，如前所述SIRPγ肽变体具有结合肿瘤细胞表面CD47的活性，优选地，所述SIRPγ肽变体具有较野生型SIRPγ肽增强地结合肿瘤细胞表面CD47的活性。在一些实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体与所述抗人PD-L1抗体的多肽链之间由肽键直接连接或通过连接子共价连接。优选地连接子可选自SEQ ID NO：89-96和(GGGGS)n、(GGGES)n和(GKPGS)n，其中n＝2-7的整数中任一所示的连接子。
在一些实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体的羧基端与所述抗人PD-L1抗体的重链可变区的氨基端连接，
或所述SIRPγ肽变体的羧基端与所述抗人PD-L1抗体的轻链可变区的氨基端连接，
或所述抗人PD-L1抗体的重链的羧基端与所述SIRPγ肽变体的氨基端连接，
或所述抗人PD-L1抗体的轻链的羧基端与所述SIRPγ肽变体的氨基端连接。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体是相对于所述野生型SIRPγ肽进一步地在K19、K53、N101、L31、Q52、E54、H56、N70、M72和M112中的一个或更多个位点具有氨基酸替代的SIRPγ肽变 体。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体是相对于所述野生型SIRPγ肽进一步地在K19、K53和N101中的一个或更多个位点具有氨基酸替代的SIRPγ肽变体。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体是相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有N51R替代突变的SIRPγ肽变体。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述N51位具有替代突变的SIRPγ肽变体基本不结合红细胞表面的CD47，优选地，所述N51位具有替代突变的SIRPγ肽变体是具有N51F、N51I、N51L、N51M或N51V替代突变的SIRPγ肽变体。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽是进一步地具有K19E、K53G和N101D替代突变的SIRPγ肽变体。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、N51V、Q52S、K53G、E54R、M72K和N101D突变。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、N51M、Q52S、K53G、E54R、M72K和N101D突变。
在一些优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体是进一步地在M6、V27、L30、V33、V36、L37、V42、E47、L66、T67、V92或S98的一个或更多个位点具有氨基酸替代的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的双功能融合蛋白，其中所述SIRPγ肽变体的氨基酸序列(通式I)如SEQ ID NO：1所示：
Figure PCTCN2020077907-appb-000001
其中，X 1选自L或W，X 2选自M、V、F、I或L，X 3选自Q、S或T，X 4选自E、T或R，X 5选自H或R，X 6选自D、N或E，X 7选自I、V、M、R或K，和X 8选自M或V。
在一些实施方案中，如前所述双功能融合蛋白，所述SIRPγ肽变体的氨基酸序列(通式II)如SEQ ID NO：2所示：
Figure PCTCN2020077907-appb-000002
其中，X 1选自L或W，X 3选自Q、S或T，X 4选自E、T或R，X 5选自H或R，X 6选自D、N或E，X 7选自I、V、M、R或K，和X 8选自M或V。
在进一步优选的实施方案中，如前所述双功能融合蛋白，其中所述SIRPγ肽变体如SEQ ID NO：21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40所示，优选如26或27所示。
在一些实施方案中，如前所述双功能融合蛋白，其中所述抗人PD-L1抗体选自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和MCLA-145。
在一些实施方案中，如前所述双功能融合蛋白，其中所述抗人PD-L1抗体包含重链可变区和轻链可变区，其中：
所述重链可变区包含与SEQ ID NO：6所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：7所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；或
所述重链可变区包含与SEQ ID NO：8所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：9所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；或
所述重链可变区包含与SEQ ID NO：8所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：113所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区。更进一步地，在一些实施方案中，所述HCDR1、HCDR2和HCDR3区和LCDR1、LCDR2和LCDR3区是kabat编号规则定义的。
在一些实施方案中，如前所述双功能融合蛋白，所述抗人PD-L1抗体重链可变区分别包含如SEQ ID NO：97、98和99所示的HCDR1、HCDR2和HCDR3区和所述抗人PD-L1抗体轻链可变区分别包含如SEQ ID NO：100、101和102所示的LCDR1、LCDR2和LCDR3区，或
所述抗人PD-L1抗体重链可变区分别包含如SEQ ID NO：103、104和105所示的HCDR1、HCDR2和HCDR3区和所述抗人PD-L1抗体轻链可变区分别包含如SEQ ID NO：106、107和108所示的LCDR1、LCDR2和LCDR3区；
或所述抗人PD-L1抗体重链可变区分别包含如SEQ ID NO：103、104和105所示的HCDR1、HCDR2和HCDR3区和所述抗人PD-L1抗体轻链可变区分别包含如SEQ ID NO：106、112和108所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述双功能融合蛋白，所述的抗人PD-L1抗体包含重链可变区和轻链可变区，其中：
所述重链可变区为SEQ ID NO：6所示，和所述轻链可变区为SEQ ID NO：7 所示；或
所述重链可变区为SEQ ID NO：8所示，和所述轻链可变区为SEQ ID NO：113所示；
或
所述重链可变区为SEQ ID NO：8所示，和所述轻链可变区为SEQ ID NO：9所示。
在一些实施方案中，如前所述双功能融合蛋白，其中所述抗人PD-L1抗体还包括重链恒定区和轻链恒定区，优选地，所述重链恒定区如SEQ ID NO:10或11所示，所述轻链恒定区如SEQ ID NO:12所示。
在一些实施方案中，如前所述双功能融合蛋白，其中所述抗人PD-L1抗体包含重链和轻链，其中：所述重链如SEQ ID NO：13或15所示，所述轻链如SEQ ID NO：14所示；或
所述重链如SEQ ID NO：16或18所示，所述轻链如SEQ ID NO：17所示；或
所述重链如SEQ ID NO：16或18所示，所述轻链如SEQ ID NO：111所示。
在一些实施方案中，如前所述双功能融合蛋白，其中所述双功能融合蛋白具有第一多肽和第二多肽，其中：
所述第一多肽选自如SEQ ID NO：41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61和62任一所示的多肽，和所述第二多肽选自如SEQ ID NO：14所示的多肽；或
所述第一多肽选自如SEQ ID NO：63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82和109任一所示的多肽，和所述第二多肽选自如SEQ ID NO：17所示的多肽；或
所述第一多肽选自如SEQ ID NO：63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82和109中任一所示的多肽，和所述第二多肽选自如SEQ ID NO：111所示的多肽。
在本公开的另一些实施方案，提供一种SIRPγ肽变体，所述SIRPγ肽变体是在相当于如SEQ ID NO：20所示的野生型SIRPγ肽的N51位具有替代突变的SIRPγ肽变体。在一些实施方案中，如前所述SIRPγ肽变体具有结合肿瘤细胞表面CD47的活性，优选地，所述SIRPγ肽变体具有较野生型SIRPγ肽增强地结合肿瘤细胞表面CD47的活性。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变体是相对于所述野生型SIRPγ肽在K19、K53、N101、L31、Q52、E54、H56、N70、M72和M112中的一个或多个位点具有氨基酸替代的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变体是相对于所述野生型SIRPγ肽在K19、K53和N101中的一个或多个位点具有氨 基酸替代的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变体是相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有N51R替代突变的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述N51位替代突变的SIRPγ肽变体基本不结合红细胞表面的CD47，优选地，所述具有N51位替代突变的SIRPγ肽变体是具有N51F、N51I、N51L、N51M或N51V替代突变的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变体是相对于如SEQ ID NO：20所示的野生型SIRPγ具有K19E、K53G和N101D替代突变的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、N51V、Q52S、K53G、E54R、M72K和N101D突变。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、N51M、Q52S、K53G、E54R、M72K和N101D突变。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变体是进一步地在M6、V27、L30、V33、V36、L37、V42、E47、L66、T67、V92或S98的一个或更多个位点具有氨基酸替代的SIRPγ肽变体。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变体如SEQ ID NO：1所示，
Figure PCTCN2020077907-appb-000003
其中，X 1选自L或W，X 2选自M、V、F、I或L，X 3选自Q、S或T，X 4选自E、T或R，X 5选自H或R，X 6选自D、N或E，X 7选自I、V、M、R或K，和X 8选自M或V。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，所述SIRPγ肽变体如SEQ ID NO：2所示：
Figure PCTCN2020077907-appb-000004
其中，X 1选自L或W，X 3选自Q、S或T，X 4选自E、T或R，X 5选自H或R，X 6选自D、N或E，X 7选自I、V、M、R或K，和X 8选自M或V。
在一些优选的实施方案中，如前所述的SIRPγ肽变体，其中所述SIRPγ肽变 体如SEQ ID NO：21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40所示。
本公开另一些方面还提供一种融合蛋白，包含SIRPγ肽变体和抗体Fc片段，所述SIRPγ肽变体为前述任一项所述的SIRPγ肽变体；在一些实施方案中，所述抗体Fc片段为人抗体Fc片段；在一些优选的实施方案中，所述抗体Fc片段序列与SEQ ID NO：10或11所示的重链恒定区中的Fc片段序列相同；在一些优选的实施方案中，所述融合蛋白的氨基酸序列如SEQ ID NO：86、110、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130或131所示。
本公开另一些方面还提供一种抗人PD-L1抗体，其包含抗体的轻链可变区和重链可变区，所述重链可变区包含序列分别如SEQ ID NO：103、104和105所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含序列分别如SEQ ID NO：106、112和108所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述的抗人PD-L1抗体，所述重链可变区为SEQ ID NO：8所示，和所述轻链可变区为SEQ ID NO：113所示。
在一些实施方案中，如前所述的抗人PD-L1抗体，所述抗人PD-L1抗体为全长抗体，进一步包括抗体恒定区，优选地，所述抗体的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区，所述抗体的轻链恒定区选自人抗体κ和λ链恒定区，更优选所述全长抗体包含SEQ ID NO:10或11所示的重链恒定区和SEQ ID NO:12所示的轻链恒定区。
在一些优选的实施方案中，如前所述的抗人PD-L1抗体，所述抗体包含如SEQ ID NO：16或18所示的重链，和如SEQ ID NO：111所示的轻链。
在另一些方面，本公开还提供一种药物组合物，其含有治疗有效量的根据如前所述的双功能融合蛋白，或如前所述的SIRPγ肽变体，或如前所述的融合蛋白，或如前所述的抗人PD-L1抗体，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。在一些实施方案中，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的双功能融合蛋白，或根据如前所述的SIRPγ肽变体，或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体。
在另一些方面，本公开还提供一种分离的核酸分子，其编码如前所述的双功能融合蛋白，或编码如前所述的SIRPγ肽变体。
在另一些方面，本公开还提供一种分离的核酸分子，其编码如前所述的抗人PD-L1抗体。
在另一些方面，本公开还提供一种重组载体，其包含如前所述的分离的核酸分子。
在另一些方面，本公开还提供一种用根据如前所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物 细胞或昆虫细胞。
在另一些方面，本公开还提供用于生产如前所述的双功能融合蛋白，或生产根据如前所述的SIRPγ肽变体的方法，或生产根据如前所述的融合蛋白，或生产根据如前所述的抗人PD-L1抗体的方法，所述方法包括将如前所述的宿主细胞在培养基中进行培养以形成并积累如前所述的双功能融合蛋白，或根据如前所述的SIRPγ肽变体，以及从培养物回收所述双功能融合蛋白或SIRPγ肽变体，或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体。
在另一些方面，本公开还提供一种消除受试者免疫抑制相关疾病的方法，所述方法包括向受试者施用治疗有效量的如前所述的双功能融合蛋白，或根据如前所述的SIRPγ肽变体，或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体，或如前所述的药物组合物，或如前所述的分离的核酸分子，优选地，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的双功能融合蛋白，或根据如前所述的SIRPγ肽变体，或根据如前所述的抗人PD-L1抗体。
在一些实施方案中，在单次或累积的施加中向个体施用约10μg/kg、约50μg/kg、约100μg/kg、约200μg/kg、约300μg/kg、约400μg/kg、约500μg/kg、约600μg/kg、约700μg/kg、约800μg/kg、约900μg/kg、约1000g/kg、约1100g/kg、1200g/kg、1300g/kg、1400g/kg、1500g/kg、1600g/kg、1700g/kg、1800g/kg、1900g/kg、约2000g/kg、约3000g/kg、约4000g/kg、约5000g/kg、约6000g/kg、约7000g/kg、约8000g/kg、约9000g/kg、约10mg/kg、约20mg/kg、约30mg/kg、约40mg/kg、约50mg/kg、约60mg/kg、约70mg/kg、约80mg/kg、约90mg/kg、约100mg/kg、约200mg/kg、约300mg/kg、约400mg/kg、约500mg/kg、约600mg/kg、约700mg/kg、约800mg/kg、约900mg/kg或约1000mg/kg剂量的本公开所述的PD-L1-CD47双功能融合蛋白、SIRPγ变体肽、或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体。
在另一些方面，本公开还提供利用如前所述的双功能融合蛋白，或根据如前所述的SIRPγ肽变体，或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体，或如前所述的药物组合物，或如前所述的分离的核酸分子制备消除受试者免疫抑制相关疾病的药物中的用途，优选地，所述药物的单位剂量的组合物中含有0.1-3000mg的如前所述的双功能融合蛋白，或如前所述的SIRPγ肽变体，或如前所述的抗人PD-L1抗体。
在另一些方面，本公开还提供一种用作消除受试者免疫抑制相关疾病的药物的如前所述的双功能融合蛋白，或如前所述的SIRPγ肽变体，或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体，或如前所述的药物组合物，或如前所述的分离的核酸分子，优选地，所述药物单位剂量的组合物中含有0.1-3000mg的如前所述的双功能融合蛋白，或如前所述的SIRPγ肽变体，或如前所述的抗人PD-L1抗体。
在另一方面，本公开还提供一种用作药物的如前所述的双功能融合蛋白，或 如前所述的SIRPγ肽变体，或根据如前所述的融合蛋白，或根据如前所述的抗人PD-L1抗体，或如前所述的药物组合物，或如前所述的分离的核酸分子，优选地，所述药物单位剂量的组合物中含有0.1-3000mg的如前所述的双功能融合蛋白，或如前所述的SIRPγ肽变体，或如前所述的抗人PD-L1抗体。
在一些实施方案中，如前所述的消除受试者免疫抑制相关疾病包括癌症、细菌或病毒感染。所述癌症包括但不限于癌瘤，淋巴瘤，胚细胞瘤(blastoma)，肉瘤，和白血病或淋巴样恶性。这种癌症的更具体的例子包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
附图说明
图1：部分实施方案中的PD-L1-CD47双功能融合蛋白结构示意图。
图2A-图2C：PD-L1-CD47双功能融合蛋白与人红细胞表面CD47的结合能力测试，最右侧阴性对照(control)均为细胞+二抗。图2A和图2B为不同PD-L1-CD47双功能融合蛋白(10μg/ml)与红细胞表面CD47的结合能力测试；图2C为不同PD-L1-CD47双功能融合蛋白(10μg/ml和1μg/ml)与红细胞表面CD47的结合能力测试。
图3：PD-L1-CD47双功能融合蛋白与Raji细胞表面CD47的结合能力测试，最右侧阴性对照为细胞+二抗。
图4：PD-L1-CD47双功能融合蛋白介导的红细胞吞噬作用。
图5A-图5B：PD-L1-CD47双功能融合蛋白介导的肿瘤细胞(Molp-8细胞)的吞噬作用。图5A和图5B为不同批次试验检测的不同PD-L1-CD47双功能融合蛋白介导的肿瘤细胞的吞噬作用。
图6：PD-L1-CD47双功能融合蛋白介导的红细胞凝结作用。
图7A-图7E：PD-L1-CD47双功能融合蛋白介导的IFN-γ的分泌作用。图7A、图7B、图7C、图7D和图7E为不同PD-L1-CD47双功能融合蛋白在介导IFN-γ的分泌作用的结果。
图8：不同PD-L1-CD47双功能融合蛋白对B-hCD274/hCD47/hSIRPα小鼠移植瘤MC38/H-11-hCD47(#5-4)模型肿瘤体积的影响。
图9：不同PD-L1-CD47双功能融合蛋白对C57/BL-6小鼠移植瘤MC38-hPD-L1-hCD47模型肿瘤体积的影响。
图10：不同PD-L1-CD47双功能融合蛋白对C57/BL-6小鼠移植瘤MC38-hPD-L1模型肿瘤体积的影响。
图11：不同PD-L1-CD47双功能融合蛋白对Molp-8荷瘤裸鼠体内模型肿瘤体积的影响。该模型重点考察双功能融合蛋白中CD47靶点通路的抑瘤作用。
具体实施方式
术语
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“双功能融合蛋白”指能够与两个目标蛋白或目标抗原结合的蛋白分子，在本公开双功能融合蛋白主要包含能够结合细胞表面的PD-L1和CD47，其由抗PD-L1抗体和SIRPγ多肽变体连接融合后形成的蛋白。
术语“PD-L1”是指程序性死亡配体1，也称为CD274或B7H1。人全长PD-L1的氨基酸序列在GenBank中以登录号NP_054862.1提供。除非指定来自非人物种，否则术语“PD-L1”意指人PD-L1。
抗人PD-L1抗体指能够结合人PD-L1，并且能够阻断PD-1与PD-L1的结合的抗体。抗人PD-L1抗体可选自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等。除此之外，本公开中的抗人PD-L1抗体还可选自全长抗体h1830、h1831或分别与h1830、h1831抗体具有相同CDR组合的抗PD-L1抗体或其抗原结合片段。
“SIRPγ肽”指人SIRPγ-D1结构域肽(其野生型SIRPγ肽的氨基酸序列如SEQ ID NO：20所示)，其具有结合人CD47的活性。SIRPγ肽还可以包括人SIRPγ-D1结构域肽突变体，或称“SIRPγ肽变体”，所述SIRPγ肽变体在相当于野生型SIRPγ肽的一个或更多个位点具有氨基酸替代，所述氨基酸替代突变数量至多不超过20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个，2个，所述SIRPγ肽变体相对于野生型SIRPγ肽具有增强的结合肿瘤细胞表面CD47的活性(野生型SIRPγ结合CD47的 亲和活性为微摩尔级)。进一步地，在一些具体实施方案中，所述SIRPγ肽变体获得不结合或(相对于肿瘤细胞表面CD47结合活性)降低结合人红细胞表面CD47的特性。如下表1所示，如S58肽是在相当于SEQ ID NO：20所示的野生型SIRPγ肽的K19位进行K19E，N51位进行N51M，Q52位进行Q52S，K53位进行K53G，E54位进行E54R，N101位进行N101D的替代突变体。
在一些具体实施方案中，所述氨基酸替代突变的可选位点可以包括K19、K53、N101、L31、N51、Q52、E54、H56、N70、M72、M112、M6、V27、L30、V33、V36、L37、V42、E47、L66、T67、V92或S98中的一个或更多个进行氨基酸替代突变。
在一些具体实施方案中，SIRPγ肽变体如SEQ ID NO：1所示：
Figure PCTCN2020077907-appb-000005
其中，X1选自L或W，X2选自M、V、F、I或L，X3选自Q、S或T，X4选自E、T或R，X5选自H或R，X6选自D、N或E，X7选自I、V、M、R或K，和X8选自M或V。
在一些具体实施方案中，SIRPγ肽变体如SEQ ID NO：2所示：
Figure PCTCN2020077907-appb-000006
其中，X1选自L或W，X3选自Q、S或T，X4选自E、T或R，X5选自H或R，X6选自D、N或E，X7选自I、V、M、R或K，和X8选自M或V。
下表显示了不同SIRPγ肽变体相对于野生型SIRPγ肽的氨基酸替代突变位点及示例性替代氨基酸残基。
表1
Figure PCTCN2020077907-appb-000007
Figure PCTCN2020077907-appb-000008
术语“抗体(antibody，Ab)”包含任何包括至少一个与具体抗原(或其表位，例如PD-L1抗原或其表位)特异性结合或相互作用的互补决定区(CDR)的抗原结合分子或分子复合物。术语“抗体”包含：包括通过双硫键相互连接的四条多肽链，两条重(H)链和两条轻(L)链的免疫球蛋白分子以及其多聚体(例如IgM)。各重链包含重链可变区(文中缩写为HCVR或VH)和重链恒定区(CH)。这一重链恒定区包含三个区(结构域)，CH1、CH2和CH3。各轻链包含轻链可变区(文中缩写为LCVR或VL)和轻链恒定区(CL)。轻链恒定区包含一个区(结构域，CL)。VH和VL区可进一步细分为高变区，称为互补决定区(CDR)，其间散布着较保守性区域，称为框架区(framework region,FR，也称骨架区、构架区)。各VH和VL是由三个CDR和四个FR所组成，以下列顺序由氨基端排列到羧基端：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在本公开的不同实施例中，抗PD-L1抗体(或其抗原结合片段)的FR可与人种系序列相同，或可经自然或人工修饰。抗体可以是不同亚类(subclass)的抗体，例如，IgG(例如：IgG1、IgG2、IgG3、或IgG4亚类)、IgA1、IgA2、IgD、IgE或IgM抗体。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指重链由氨基端至羧基端依次包含VH区、CH1区、铰链区和Fc区，轻链由氨基端至羧基端依次包含VL区和CL区的抗体。
抗原结合片段的非限定示例包含：(i)Fab片段；(ii)F(ab′)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)由模拟抗体高变区的氨基酸残基所组成的最小识别单位(例如分离的互补决定区(CDR)，例如CDR3肽)或限制性FR3-CDR3-FR4肽。其它工程改造分子，例如区域特异性抗体、单域抗体、区域删除抗体、嵌合抗体、CDR-植入抗体、双抗体、三抗体、四抗体、微抗体、纳米抗体(例如单价纳米抗体、双价纳米抗体等)、小模块免疫医药(SMIP)和鲨可变IgNAR区，也涵盖在文中所用的“抗原结合片段”的词语内。
抗体的抗原结合片段典型地将包含至少一个可变区。可变区可以是任何大小或氨基酸组成的区域，且一般将包含与一个或多个框架序列相邻或在其框架内的 CDR。在具有VH区与VL区的抗原结合片段中，VH和VL区可以任何适合的排列位于彼此相对处。例如可变区可为二聚化并含有VH-VL或VL-VH二聚体。
在某些实施例中，抗原结合片段在任何可变区和恒定区的配置中，可变区和恒定区可直接彼此相连接或可通过完整或部分的绞链或连接子区相连接。绞链区可由至少2个(例如5、10、15、20、40、60或更多个)氨基酸所组成，使得其在单一多肽分子中在相邻的可变和/或恒定区之间产生柔性和半柔性连结。再者，在本公开抗原结合片段可包含以非共价彼此相互连结和/或与一个或多个单体VH或VL区相连结(例如以双硫键)的具有可变区和恒定区的同源二聚体或异源二聚体(或其它多聚体)。
“鼠源抗体”在本公开中为根据本领域知识和技能制备的来源于小鼠或大鼠的单克隆抗体。制备时用抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤，当所注射的试验对象为小鼠时，所产生的抗体为小鼠来源抗体，当所注射的试验对象为大鼠时，所产生的抗体为大鼠来源抗体。
“嵌合抗体(chimeric antibody)”，是将第一物种(如：鼠)抗体的可变区与第二物种(如：人)抗体的恒定区融合而成的抗体。建立嵌合抗体，要先建立分泌第一物种单抗的杂交瘤，然后从杂交瘤细胞中克隆可变区基因，再根据需要克隆第二物种抗体的恒定区基因，将第一物种可变区基因与第二物种恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(如YTE突变，回复突变，L234A和/或L235A突变，或S228P突变)的IgG1、IgG2或IgG4重链恒定区变体。
术语“人源化抗体(humanized antibody)”，包括CDR移植抗体(CDR-grafted antibody)，是指将动物来源抗体，例如鼠源抗体的CDR序列移植到人的抗体可变区框架区(或构架区，framework region)中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网http://www.vbase2.org/获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降引起活性的下降，可对所述的人抗体可变区框架序列进行量少的回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
由于抗原的接触残基，CDR的移植可由于与抗原接触的构架残基而导致产生的抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以可能是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体 的构架。来自非人抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查动物单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚类共有序列或具有高相似性百分数的动物抗体序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在本公开一个的实施方案中，所述的抗体或其抗原结合片段，可进一步包含人源或鼠源κ、λ链或其变体的轻链恒定区，或进一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。
人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，或S228P突变，或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”与“人源抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座的元件引入到细胞株中，这些细胞株中的内源性重链和轻链基因座被靶向破坏。转基因生物可以合成对抗原特异的人抗体，并且该生物可以用于产生人抗体-分泌杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
“单克隆抗体”是指从基本上均质抗体的群体获得的抗体，即除可能的变体抗体(例如含有天然存在的突变或在制造单克隆抗体制剂的期间产生的突变，这些变体通常以少量存在)之外，构成所述群体的个别抗体识别相同和/或结合相同表位。单克隆抗体制备物(制剂)的每个单克隆抗体针对抗原上的单一决定簇。因此，修饰语“单克隆”指示如从基本上均质抗体群体获得的抗体的特性，且不应解释为需要通过任何特定方法来制造抗体。例如，根据本公开使用的单克隆抗体可通过各种技术制备，所述技术包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法以及利用含有全部或部分人免疫球蛋白基因座的转基因动物的方法，此类方法以及用于制备单克隆抗体的其他示例性方法在本文中进行描述。
此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人 (1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选的片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。
抗原结合片段还可并入至包含一对串联Fv片段(VH-CH1-VH-CH1)的单链分子中，该对串联Fv片段连同互补轻链多肽一起形成一对抗原结合区(Zapata等人,1995 Protein Eng.8(10):1057-1062；及美国专利US5641870)。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体所获得的具有约50,000Da的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000Da并具有抗原结合活性，并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000Da并具有抗原结合活性的抗体片段。Fab'可以通过用还原剂例如二硫苏糖醇处理特异性识别并结合抗原的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中，并将载体导入到原核生物或真核生物中以表达Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般通式：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个(包括1个、2个、3个或4个)重复的变体(Holliger等人(1993)，Proc Natl Acad Sci USA.90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur J Immuno.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J Mol Biol.293:41-56和Roovers等人(2001)，Cancer Immunol Immunother.50:51-59.描述。
“抗人PD-L1抗体”包括能够特异性结合人PD-L1的全长抗体，也包括包含该全长抗体的轻链可变区和重链可变区的抗原结合片段，包括但不限于包含该全长抗体的轻链可变区和重链可变区的单链抗体(scFv)、Fab片段或包含scFv或Fab的其他抗原结合片段。
SIRPγ肽连接至所述抗人PD-L1抗体的多肽链中的“连接”指多肽之间的有效连接，包括例如经过肽键连接，或使用连接子连接。所述连接不会使SIRPγ肽和抗人PD-L1抗体各自的功能丧失。
“Linker”或“接头”或“连接子”指用于连接蛋白质结构域或不同蛋白或不 同多肽的连接性多肽序列，通常具有一定的柔性，连接子的使用不会使蛋白质结构域原有的功能丧失。示例性的连接子如下表所示。
表2.示例性连接子序列
Figure PCTCN2020077907-appb-000009
在一些实施方案中，抗PD-L1抗体可以与SIRPγ肽变体使用接头连接起来，一些示例性的双功能融合蛋白包括以下所示的融合蛋白：
表3.PD-L1-CD47双功能融合蛋白
Figure PCTCN2020077907-appb-000010
Figure PCTCN2020077907-appb-000011
双抗体(diabody)是指scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering.7:697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开一些实施例中抗原结合片段可以通过以下步骤来生产：获得本公开的特异性识别并结合抗原的单克隆抗体的VH和/或VL及所需的其他结构域的编码cDNA，构建编码抗原结合片段的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达抗原结合片段。
"Fc区"可以是天然序列Fc区或变体Fc区。虽然免疫球蛋白重链的Fc区的边界可能变化，但人IgG重链Fc区通常被定义成从位置Cys226上的氨基酸残基或从Pro230延伸至其羧基端。Fc区中的残基的编号为如Kabat中的EU索引的编号。Kabat等，Sequences of Proteins of Immunological Interest，第5版Public Health Service,National Institutes of Health,Bethesda,Md.,1991。免疫球蛋白的Fc区通常具有两个恒定区结构域CH2和CH3。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个或更多个氨基酸的插入、缺失或替代。
抗体的“可变区”是指单独的或组合的抗体轻链的可变区(VL)或抗体重链的可变区(VH)。如在本领域中已知的，重链和轻链的可变区各自由通过3个互补决定区(CDR)(也称为高变区)连接的4个框架区(FR)组成。每一条链中的CDR通过FR紧密地保持在一起并且与来自另一条链的CDR一起促成抗体的抗原结合部位的形成。存在至少2个用于确定CDR的技术：(1)基于跨种序列变异性的方法(即，Kabat等Sequences of Proteins of Immunological Interest,(第5版，1991,National Institutes of Health,Bethesda MD))；和(2)基于抗原-抗体复合物的晶体学研究的方法(Al-Lazikani等，J.Molec.Biol.273:927-948(1997))。如本文中所用，CDR可指由任一方法或由两种方法的组合确定的CDR。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”和“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)和每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1 991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist,7,132-136(1999)；Lefranc,M.P.等，Dev.Comp.Immunol.,27,55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
“抗体恒定区结构域”指来源于抗体的轻链和重链的恒定区的结构域，包括CL和来源于不同类抗体的CH1、CH2、CH3和CH4结构域。
“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。
术语“亲和力”是指在单一表位处，抗体与抗原之间相互作用的强度。在各抗原位点内，抗体“臂”的可变区通过弱非共价力与抗原在多个氨基酸位点处相互作用；相互作用愈大，亲和力愈强。如本文所用，抗体或其抗原结合片段(例如Fab片段)的术语“高亲和力”通常是指具有1E -9M或更小的K D(例如1E -10M或更小的K D、1E -11M或更小的K D、1E -12M或更小的K D、1E -13M或更小的K D、1E -14M或更小的 K D等)的抗体或抗原结合片段。
术语"KD"或“K D”是指特定抗体-抗原相互作用的解离平衡常数。通常，抗体以小于大约1E -8M，例如小于大约1E -9M、1E -10M或1E -11M或更小的解离平衡常数(KD)结合抗原，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。KD值越小，亲和力越大。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的DNA分子或RNA分子，例如是双链DNA或mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语"载体"意指能够递送一个或多个目标基因或序列并且优选地在宿主细胞中表达其的构建体。载体的示例包括，但不限于，病毒载体、裸露DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂缔合的DNA或RNA表达载体、包封在脂质体中的DNA或RNA表达载体和某些真核生物细胞诸如生产细胞。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用抗原或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从网站 http://www.imgt.org/得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、HEK293细胞(非限制性实施例如HEK293E细胞)和NS0细胞。
工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种可选的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的蛋白A或蛋白G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再 用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给药”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指将外源性药物、治疗剂、诊断剂、组合物或者人为操作(比如实施例中的“安乐死”)提供给动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者(或受试者)内用或外用治疗剂，例如包含本公开实施例的任一种化合物的组合物，所述患者(或受试者)具有(或疑似患有、或易感于)一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者(或受试者)或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床有测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者(或受试者)的疾病状态、年龄和体重，以及药物在患者(或受试者)产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)可能无法在缓解每个目标疾病症状方面都有效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者(或受试者)中应当减轻目标疾病症状。
“氨基酸保守修饰”或“氨基酸保守取代”指蛋白质或多肽中的氨基酸被具有相似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其他氨基酸取代，从而使得在不改变蛋白质或多肽的生物活性或其他所需特性(例如抗原亲和力和/或特异性)的情况下，可以经常进行改变。本领域技术人员认识到，通常，多肽的非必需区域中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson等人，(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页(第4版))。此外，结构上或功能上相似的氨基酸的取代不太可能破坏生物活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
表4.示例性氨基酸保守取代
原始残基 保守取代
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
“有效量”、“有效剂量”是指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者(或受试者)的本公开靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。
“内源性”指根据情况在细胞、生物或人体内产生的物质。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本公开所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而 发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本公开另一方面涉及用于免疫检测或测定目标抗原的方法、用于免疫检测或测定目标抗原的试剂、用于免疫检测或测定表达目标抗原的细胞的方法和用于诊断与目标抗原阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别并结合目标抗原的单克隆抗体或抗体片段、或融合蛋白、或双功能融合蛋白作为活性成分。
术语“癌症”、“癌的”或“恶性肿瘤”指或描述哺乳动物中一般以不受调节的细胞生长为特征的生理状况，在本公开中可以互换使用。癌症或恶性肿瘤的例子包括但不限于癌瘤，淋巴瘤，胚细胞瘤(blastoma)，肉瘤，和白血病或淋巴样恶性。这种癌症的更具体的例子包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
“炎性病症”是指其中过度或不受调节的炎症反应导致过度炎性症状、宿主组织损伤或组织功能丧失的任何疾病、病症或综合征。“炎性疾病”也是指由白细胞或嗜中性粒细胞趋化性的汇集而介导的病理状态。
“炎症”是指由于组织损伤或破坏而引起的局部保护性反应，其用来破坏、削弱或杜绝(隔离)有害物质和受伤组织。炎症与白细胞或中性粒细胞趋化性的汇集显著相关。炎症可以由病原生物体和病毒以及非感染性原因引起，所述非感染性原因 如创伤或心肌梗塞后的再灌注或中风、对外源性抗原的免疫应答和自身免疫应答。
上述与目标抗原阳性细胞相关的疾病可以通过用本公开的单克隆抗体或抗体片段检测或测定表达目标抗原的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原的活体样品没有特别限制，只要它具有包含表达目标抗原的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本发明一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本公开，但是以下描述优选的方法和材料。通过说明书和权利要求书，本公开的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本发明所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本发明公开的优选实施方式。这些实施例不应以任何方式理解为限制本公开的范围，本公开的范围由权利要求书限定。
实施例
实施例1.CD47抗原及检测用蛋白的制备
以UniProt白细胞表面抗原CD47(人CD47蛋白，Uniprot号：Q08722)作为CD47的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列，可选的在CD47蛋白基础上融合不同的标签如his标签或Fc等。
1.带His标签的CD47蛋白胞外域(CD47-ECD-His)：(SEQ ID NO：3)
Figure PCTCN2020077907-appb-000012
注释：划线部分为6×his标签。
2.CD47胞外域与人IgG1Fc融合蛋白(CD47-ECD-Fc)作为检测试剂：(SEQ ID NO：4)
Figure PCTCN2020077907-appb-000013
注释：划线部分为人IgG1-Fc部分。
3.人SIRPα与人IgG1Fc融合蛋白(SIRPα-Fc)作为结合及阻断检测试剂：(SEQ ID NO：5)
Figure PCTCN2020077907-appb-000014
注释：划线部分为人IgG1-Fc部分。
4.带His标签的SIRPα蛋白胞外域(SIRPα-His)
Figure PCTCN2020077907-appb-000015
实施例2.CD47、SIRPα相关重组蛋白的纯化
1.带His标签的重组蛋白的纯化步骤：
将细胞表达上清高速离心去除杂质，并将缓冲液置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品上IMAC柱。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE，28-9893-35)进一步纯化，流动相为PBS。去除聚体峰，收集洗脱峰。所得到的蛋白经电泳、肽图、LC-MS鉴定为正确后，分装备用。
得到带His标签的CD47-ECD-His(SEQ ID NO：3)用于本公开抗体的免疫原或检测试剂。CD47-ECD-His也可以通过体外化学法与KLH蛋白进行偶联反应后作为免疫原刺激小鼠免疫。
2.CD47-ECD-Fc及SIRPα-Fc融合蛋白的纯化步骤：
将细胞表达上清高速离心去除杂质，上清进行MabSelect Sure(GE，17-5438-01) 亲和层析。MabSelect Sure层析柱先用0.1M NaOH再生，利用纯水冲洗后用PBS平衡柱子，将上清结合后，利用PBS进行洗涤至A280读数降至基线。用0.1M醋酸缓冲液在pH3.5条件下洗脱目的蛋白，用1M Tris-HCl中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE，28-9893-35)进一步纯化，合并收集目的蛋白所在接收管浓缩至适当浓度。
此方法用来纯化CD47-ECD-Fc(SEQ ID NO：4)及SIRPα-Fc(SEQ ID NO：5)融合蛋白，该方法也可以用来纯化本公开中涉及的人源化抗体蛋白。
实施例3.构建和表达抗PD-L1人源化抗体(IgG4-S228P形式)
抗PD-L1抗体的轻重链可变区改造自WO2017084495A1的抗PD-L1抗体，其序列和相关性能记载在申请号为PCT/CN2019/070982的PCT申请中，该PCT申请中的全部内容引用至本申请。
抗PD-L1抗体9-2H5L11：
9-2 H5重链可变区(SEQ ID NO：6)
Figure PCTCN2020077907-appb-000016
9-2 L11轻链可变区(SEQ ID NO：7)
Figure PCTCN2020077907-appb-000017
抗PD-L1抗体24D5H12L61：
24D5 H12重链可变区(SEQ ID NO：8)
Figure PCTCN2020077907-appb-000018
24D5 L61轻链可变区(SEQ ID NO：9)
Figure PCTCN2020077907-appb-000019
注释：上述抗体9-2和24D5来源的轻重链可变区中下划线所示CDR为Kabat编号规则所定义的CDR。
表5.Kabat编号规则定义的抗体CDR
Figure PCTCN2020077907-appb-000020
Figure PCTCN2020077907-appb-000021
设计引物，PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pHr/VK-CL-pHr。
IgG4-S228P的重链恒定区序列如下：(SEQ ID NO：10)
Figure PCTCN2020077907-appb-000022
IgG1的重链恒定区序列如下：(SEQ ID NO：11)
Figure PCTCN2020077907-appb-000023
抗体的轻链(Kappa链)恒定区序列如下：(SEQ ID NO：12)
Figure PCTCN2020077907-appb-000024
构建的全长抗体如下：
抗PD-L1 IgG4抗体h1830
h1830重链(SEQ ID NO：13)
Figure PCTCN2020077907-appb-000025
Figure PCTCN2020077907-appb-000026
h1830轻链(SEQ ID NO：14)
Figure PCTCN2020077907-appb-000027
抗PD-L1 IgG1抗体h1830G1
h1830G1重链：(SEQ ID NO：15)
Figure PCTCN2020077907-appb-000028
h1830G1轻链(同h1830轻链，SEQ ID NO：14)：
Figure PCTCN2020077907-appb-000029
PD-L1抗体h1831：
h1831重链(SEQ ID NO：16)
Figure PCTCN2020077907-appb-000030
Figure PCTCN2020077907-appb-000031
h1831轻链(SEQ ID NO：17)
Figure PCTCN2020077907-appb-000032
抗PD-L1 IgG1抗体h1831G1
h1830G1重链：(SEQ ID NO：18)
Figure PCTCN2020077907-appb-000033
h1831G1轻链(同h1831轻链，SEQ ID NO：17)
Figure PCTCN2020077907-appb-000034
实施例4.SIRPγ突变体筛选和制备
4.1 SIRPγ的亲和力成熟噬菌体文库的构建以及文库的筛选
为获得高亲和力的CD47受体，通过噬菌体展示平台技术对CD47的受体SIRPγD1结构域进行亲和力成熟，在野生型SIRPγ的基础上设计并制备针对CD47结合结构域的亲和力成熟噬菌体文库，并从中筛选新的SIRPγ突变体。野生型SIRPγD1结构域具体序列如下：
野生型SIRPγ肽的DNA编码序列：(SEQ ID NO：19)
Figure PCTCN2020077907-appb-000035
Figure PCTCN2020077907-appb-000036
野生型SIRPγ肽：(SEQ ID NO：20)
Figure PCTCN2020077907-appb-000037
噬菌体文库的构建：设计简并引物，通过PCR的方法将设计的突变氨基酸引入到SIRPγ噬菌体突变体文库中，每个文库的大小在10 9左右。
SIRPγ噬菌体突变体文库的筛选：将包装好的SIRPγ噬菌体突变体文库(1×10 12-1×10 13)与100μl链菌素微珠(Mi1envi Biotec，Auburn，CA)加入1m1含2％脱脂牛奶-磷酸盐缓冲液(缩写MPBS)中于室温下孵育1小时，放置在磁力架上，取上清。上清加10μg/ml生物素化的人CD47-ECD-his蛋白(购自Sino Biological)，于室温下孵育1小时，再加入100μl链霉亲和素包被的磁珠(1ml MPBS预孵育)于室温下孵育1小时。并使其负载于磁力架系统上用于分选，吸去上清。加入1ml PBST(含0.1％Tween-20的磷酸盐缓冲液)，翻转多次，吸尽后再加入新鲜洗液，重复11次，以去除未结合的突变体，加入0.5ml洗脱液(50μl 10mg/ml trypsin stock solution(存储液)加入450μl PBS中)。室温下摇晃15min。放置在磁力架上，吸出上清至一新EP管中。TG1接入2YT培养基中，扩增至细菌密度OD600＝0.4时。每管加入1.75ml TG1(OD600＝0.4)，并加入250μl洗脱后phage(噬菌体)，37℃水浴中静置孵育30min，梯度稀释涂板，用于测试滴度。其余TG1溶液离心，涂板，37℃过夜孵育。
SIRPγ噬菌体突变体文库利用生物素化的人CD47-ECD-his蛋白抗原，经过2-3轮MACS筛选(链霉素磁珠，Invitrogen)，最终获得比野生型SIRPγ亲和力高的噬菌体突变体单克隆进行测序验证。对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成PDL1-CD47双功能融合蛋白后进行哺乳动物细胞表达。
4.2 SIRPγ的亲和力成熟酵母文库的构建以及文库的筛选
为获得更高亲和力的CD47受体，通过酵母展示平台技术对CD47的受体SIRPγ-D1 domain进行亲和力成熟，在SIRPγ-D1的基础上设计并制备针对CD47结合的domain的亲和力成熟酵母文库，并从中筛选新的SIRPγ突变体。
酵母文库的构建：设计简并引物，通过PCR的方法将设计的突变氨基酸引入到SIRPγ突变体文库中，每个文库的大小在10 9左右，构建好的酵母菌文库通过二代测序的方法验证文库的多样性。
在第一轮筛选中，将来自SIRPγ突变体文库的约5×10 10个细胞与10μg/ml生物素化的人CD47-Fc蛋白在50m1含0.1％牛血清白蛋白(BSA)-磷酸盐缓冲液(称为PBSA)中于室温下孵育1小时。然后，混合物用0.1％PBSA洗涤三次，以去除未结合的抗体片段。然后往结合生物素化人CD47-Fc蛋白的SIRPγ突变体文库中加入100μl链菌素微珠(Mi1envi Biotec,Auburn,CA)，并使其负载于AutoMACS系统上用于分选。收集具有对CD47-Fc的高亲和力的文库的细胞，然后在SDCAA培养基(20g右旋糖，6.7g Difco酵母菌氮源-无氨基酸，5g Bacto酪蛋白氨基酸，5.4gNa2HP O 4和8.56g NaH 2PO 4·H 2O，溶解于1L蒸榴水中)中于250rpm和30℃下扩增24小时。然后，将培养物在SGCAA培养基(20g半乳糖，6.7g Difco酵母菌氮源-无氨基酸，5g Bacto酪蛋白氨基酸，5.4g Na2HP O 4和8.56g NaH 2PO 4·H 2O，溶解于1L蒸榴水中)中，于250rpm和20℃下诱导18个小时。将所得到的富集文库进行针对与生物素化的重组人CD47-Fc的结合的第二轮筛选。为了确保用于第二和/或第三轮筛选的抗体文库的充分多样性，将来自前一轮的文库大小的100倍用作输入的细胞数量。
对于第三轮和第四轮的筛选，将来自前一轮的文库细胞与1μg/ml生物素化的人CD47-Fc蛋白和10μg/ml Mouse Anti-cMyc(9E10,sigma)抗体在0.1％PBSA中于室温下孵育1小时，混合物用0.1％PBSA洗涤三次，以去除未结合的抗体片段。加入Goat anti-mouse-Alexa488(A-11001,life technologies)和Strepavidin-PE(S-866,Life technologies)于4℃下孵育1小时，混合物用0.1％PBSA洗涤三次，以去除未结合的抗体片段。最后，通过FACS筛选(BD FACSAriaTM FUSION)出亲和力高的SIRPγ突变体。
SIRPγ突变体文库利用生物素化的人CD47-Fc抗原，经过2-3轮MACS筛选(链霉素磁珠，Invitrogen)和2-3轮FACS筛选(BD FACSAriaTM FUSION)。再挑选400个左右的酵母单克隆培养和诱导表达，使用FACS(BD FACSCanto II)检测酵母单克隆对人CD47-Fc抗原的结合，挑选出比野生型SIRPγ亲和力高的酵母单克隆进行测序验证，对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成PDL1-CD47双功能融合蛋白后进行哺乳动物细胞表达。
经过筛选，最终获得SIRPγ肽变体如下：
S58(SEQ ID NO:21)
Figure PCTCN2020077907-appb-000038
S79(SEQ ID NO:22)
Figure PCTCN2020077907-appb-000039
Figure PCTCN2020077907-appb-000040
S15(SEQ ID NO:23)
Figure PCTCN2020077907-appb-000041
S12(SEQ ID NO:24)
Figure PCTCN2020077907-appb-000042
S19(SEQ ID NO:25)
Figure PCTCN2020077907-appb-000043
S85(SEQ ID NO:26)
Figure PCTCN2020077907-appb-000044
S37(SEQ ID NO:27)
Figure PCTCN2020077907-appb-000045
S38(SEQ ID NO:28)
Figure PCTCN2020077907-appb-000046
S22(SEQ ID NO:29)
Figure PCTCN2020077907-appb-000047
S29(SEQ ID NO: 30)
Figure PCTCN2020077907-appb-000048
S34(SEQ ID NO: 31)
Figure PCTCN2020077907-appb-000049
S41(SEQ ID NO: 32)
Figure PCTCN2020077907-appb-000050
S42(SEQ ID NO: 33)
Figure PCTCN2020077907-appb-000051
S43(SEQ ID NO: 34)
Figure PCTCN2020077907-appb-000052
S44(SEQ ID NO:35)
Figure PCTCN2020077907-appb-000053
S45(SEQ ID NO:36)
Figure PCTCN2020077907-appb-000054
S46(SEQ ID NO:37)
Figure PCTCN2020077907-appb-000055
S47(SEQ ID NO:38)
Figure PCTCN2020077907-appb-000056
S48(SEQ ID NO:39)
Figure PCTCN2020077907-appb-000057
S49(SEQ ID NO:40)
Figure PCTCN2020077907-appb-000058
实施例5.构建和表达PD-L1-CD47双功能融合蛋白
将所获得的抗PD-L1抗体与SIRPγ连接，形成融合蛋白，经常规方法表达和纯化，获得PD-L1-CD47双功能融合蛋白。
表6.PD-L1-CD47双功能融合蛋白及序列
Figure PCTCN2020077907-appb-000059
Figure PCTCN2020077907-appb-000060
Figure PCTCN2020077907-appb-000061
Figure PCTCN2020077907-appb-000062
Figure PCTCN2020077907-appb-000063
Figure PCTCN2020077907-appb-000064
Figure PCTCN2020077907-appb-000065
Figure PCTCN2020077907-appb-000066
Figure PCTCN2020077907-appb-000067
Figure PCTCN2020077907-appb-000068
Figure PCTCN2020077907-appb-000069
Figure PCTCN2020077907-appb-000070
Figure PCTCN2020077907-appb-000071
Figure PCTCN2020077907-appb-000072
Figure PCTCN2020077907-appb-000073
Figure PCTCN2020077907-appb-000074
Figure PCTCN2020077907-appb-000075
Figure PCTCN2020077907-appb-000076
Figure PCTCN2020077907-appb-000077
Figure PCTCN2020077907-appb-000078
同样经常规方法制备纯化下列蛋白作为阳性或阴性对照。
抗CD47抗体hu5F9(序列来自US09017675B)
Hu5F9重链(SEQ ID NO:83)
Figure PCTCN2020077907-appb-000079
Hu5F9轻链(SEQ ID NO:84)
Figure PCTCN2020077907-appb-000080
SIRPα-CV(参照Engineered SIRPαVariants as Immunotherapeutic Adjuvants to Anticancer Antibodies合成，Science.2013 Jul 5；341(6141):88-91，SEQ ID NO:85)
Figure PCTCN2020077907-appb-000081
TTI-621：(序列来自WO2014094122A1，SEQ ID NO:133)
Figure PCTCN2020077907-appb-000082
S58-Fc(SEQ ID NO:86)
Figure PCTCN2020077907-appb-000083
抗CD47抗体Hu167-IgG4 AA(按照WO2018095428A1专利申请中公开的方法制备)
Hu167-IgG4 AA重链(SEQ ID NO:87)
Figure PCTCN2020077907-appb-000084
Hu167-IgG4 AA轻链(SEQ ID NO:88)
Figure PCTCN2020077907-appb-000085
S37-Fc(SEQ ID NO:110)
Figure PCTCN2020077907-appb-000086
抗体h1831K
对h1831抗体进行CDR突变改造，获得36个突变体，最终筛选到N53K(位置根据Kabat编号规则确定)突变体h1831K。即将h1831轻链LCDR2由AAS NLES突变为AAS KLES，获得新的抗体h1831K。
h1831K轻链：(SEQ ID NO:111)
Figure PCTCN2020077907-appb-000087
其中LCDR1为 RASESVSIHGTHLMH(SEQ ID NO：106)，LCDR2为 AASKLES(SEQ ID NO：112)，LCDR3为 QQSFEDPLT(SEQ ID NO：108)。
h1831K轻链可变区(SEQ ID NO：113)
Figure PCTCN2020077907-appb-000088
h1831K重链(与h1831重链序列相同，SEQ ID NO：16)
Figure PCTCN2020077907-appb-000089
>h1831K-19-S37重链(与h1831-19-S37重链序列相同，SEQ ID NO：109)
Figure PCTCN2020077907-appb-000090
>h1831K-19-S37轻链(与h1831K轻链序列相同，SEQ ID NO：111)
Figure PCTCN2020077907-appb-000091
S79-Fc(SEQ ID NO:114)
Figure PCTCN2020077907-appb-000092
S15-Fc(SEQ ID NO:115)
Figure PCTCN2020077907-appb-000093
S12-Fc(SEQ ID NO:116)
Figure PCTCN2020077907-appb-000094
S19-Fc(SEQ ID NO:117)
Figure PCTCN2020077907-appb-000095
Figure PCTCN2020077907-appb-000096
S85-Fc(SEQ ID NO:118)
Figure PCTCN2020077907-appb-000097
S38-Fc(SEQ ID NO:119)
Figure PCTCN2020077907-appb-000098
S22-Fc(SEQ ID NO:120)
Figure PCTCN2020077907-appb-000099
S29-Fc(SEQ ID NO:121)
Figure PCTCN2020077907-appb-000100
S34-Fc(SEQ ID NO:122)
Figure PCTCN2020077907-appb-000101
S41-Fc(SEQ ID NO:123)
Figure PCTCN2020077907-appb-000102
S42-Fc(SEQ ID NO:124)
Figure PCTCN2020077907-appb-000103
S43-Fc(SEQ ID NO:125)
Figure PCTCN2020077907-appb-000104
S44-Fc(SEQ ID NO:126)
Figure PCTCN2020077907-appb-000105
Figure PCTCN2020077907-appb-000106
S45-Fc(SEQ ID NO:127)
Figure PCTCN2020077907-appb-000107
S46-Fc(SEQ ID NO:128)
Figure PCTCN2020077907-appb-000108
S47-Fc(SEQ ID NO:129)
Figure PCTCN2020077907-appb-000109
S48-Fc(SEQ ID NO:130)
Figure PCTCN2020077907-appb-000110
S49-Fc(SEQ ID NO:131)
Figure PCTCN2020077907-appb-000111
作为阴性对照的IgG4对照为与PD-L1和CD47均无关的靶点的抗体，IgG4-Fc和IgG1-Fc为分别仅含Fc区段，不含针对任何抗原的可变区区段。
测试例
测试例1.PD-L1-CD47双功能融合蛋白结合CD47-his蛋白的ELISA实验
PD-L1-CD47双功能融合蛋白的结合力通过双功能融合蛋白与固定在ELISA板上人CD47或cyno CD47的结合的量来检测。用PBS稀释CD47-ECD-His(见表7)至1μg/ml包被在96孔ELISA板上，洗板封闭后，加入不同浓度的双功能融合蛋白样品，再洗板后加入辣根过氧化物酶-羊抗人(H+L)抗体(Jackson，CAT#109-035-088)，再洗板，加入四甲基联苯胺溶液显色，最后加入终止液，在酶标仪上测量OD450并计算其EC50值，结果见表8-1和表8-2。
表7.不同种系的CD47来源
CD47类型 MW/kDa Cat.No. Lot.No. 生产商
人CD47 15.2 12283-H08H N/A S.B
Cyno CD47 15.8 CD7-C52H1 2171b-76VF1-K9 ACROBiosystems
表8-1.PD-L1-CD47双功能融合蛋白和人CD47及cyno CD47结合ELISA
Figure PCTCN2020077907-appb-000112
Figure PCTCN2020077907-appb-000113
表8-2.PD-L1-CD47双功能融合蛋白和人CD47及cyno CD47结合ELISA
Figure PCTCN2020077907-appb-000114
结果显示，各PD-L1-CD47双功能融合蛋白与游离的人CD47蛋白具有很强的亲和力，同时，与猴CD47也有很强的交叉亲和活力。
测试例2.PD-L1-CD47双功能融合蛋白结合PD-L1-his蛋白的ELISA
PD-L1-CD47双功能融合蛋白的结合力通过抗体与固定在ELISA板上不同种属PD-L1结合的量来检测。用PBS稀释不同种系PD-L1-his抗原(见表9)至1μg/ml包被在96孔ELISA板(Costar，CAT#3590)上，洗板封闭后，加入不同浓度的PD-L1-CD47双功能融合蛋白样品，洗板后加入辣根过氧化物酶-羊抗人(H+L)抗体(Jackson，CAT#109-035-088)，再洗板，加入四甲基联苯胺溶液显色，最后加入终止液，在酶标仪上测量OD450并计算其EC50值，结果见表10。
表9.不同种系的PD-L1来源
PD-L1类型 MW/kDa Cat.No. Lot.No. 生产商
hPD-L1-His 26.8 10084-H08H LC11SE1203 S.B
Cyno PD-L1-His 26.7 90251-C08H LC10DE1308 S.B
mPD-L1 26.3 50010-M08H LC10NO0102 S.B
表10.PD-L1-CD47双功能融合蛋白和不同种属PD-L1结合ELISA
Figure PCTCN2020077907-appb-000115
结果显示，各PD-L1-CD47双功能融合蛋白与游离的人PD-L1蛋白具有很强的亲和力，同时，与猴PD-L1也有很强的交叉亲和活力。包含h1830抗体的PD-L1-CD47双功能融合蛋白与小鼠PD-L1也有很强的交叉亲和活力。
测试例3.PD-L1-CD47双功能融合蛋白对PD-L1/PD1，PD-L1/B7.1结合的阻断
用PBS将PD-L1-Fc(内部制备)稀释至1μg/ml，以100μl/孔的体积加于96孔板中，于4℃放置16h-20h。将96孔板中PBS缓冲液吸掉，用PBST(pH7.4PBS含0.05％tween20)缓冲液洗板1次后，加入120μl/孔PBST/1％milk，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板1次后，加入90μl用样品稀释液(pH7.4PBS含5％BSA，0.05％Tween20)稀释至合适浓度的待测PD-L1-CD47双功能融合蛋白，置4℃预孵育1h。以10μl/孔的体积加入10×浓度的生物素标记PD-1(北京义翘神州生物技术有限公司，10μg/ml)或B7-1(北京义翘神州生物技术有限公司10μg/ml)，在振荡器上振荡、混匀后，置37℃孵育1h。移去反应体系，用PBST洗板6次后，加入100μl/孔用PBST缓冲液1:400稀释的Streptavidin–Peroxidase Polymer，室温振荡孵育50分钟。用PBST洗板6次后，加入100μl/孔TMB，于室温孵育5-10min。加入100μl/孔1M H 2SO 4终止反应。用NOVOStar在酶标仪上测量OD450并计算其IC50值，结果见表11。
表11.PD-L1-CD47双功能融合蛋白的阻断ELISA
Figure PCTCN2020077907-appb-000116
试验结果显示所有双功能融合蛋白也均能有效的对PD-L1/PD-1和PD-L1/B7.1通路产生阻断双功能融合蛋白。
测试例4.PD-L1-CD47双功能融合蛋白结合人的红细胞的实验
新鲜健康人血与PBS等体积混合后，300g离心5min得到细胞团，用PBS洗涤3-5次后，得到红细胞。用FACS buffer(PBS+5％BSA)重悬，调整细胞密度为2×10 6个/ml，按照100μl/well，种到96孔圆底板(3795#，corning)，然后加入不同浓度的抗体及双功能融合蛋白，4℃孵育1小时。然后用FACS buffer(PBS+2％FBS)洗涤2次后，加入二抗(Alexa 488 goat anti-human IgG antibody(Invitrogen，CAT#A11013)，冰上避光孵育30min。最后用FACS buffer洗涤2次后再重悬细胞。FACS Cantoll中读板。
FACS检测结果显示，对照抗体hu5F9和Hu167 IgG4 AA对人的红细胞表面的天然CD47有较强的结合能力，涉及的双功能融合蛋白中，除含S79，S34和S49的双功能融合蛋白能结合人红细胞表面CD47外，其他双功能融合蛋白对人的红细胞表面的天然CD47几乎没有结合能力，提示上述双功能融合蛋白在安全性方面的优势，结果见图2A、图2B和图2C(其中图2A、图2B和图2C所做实验是三批次实验，所使用供体细胞不同)。
测试例5.PD-L1-CD47双功能融合蛋白结合肿瘤细胞的实验
Raji细胞培养在RPMI培养基中(Hyclone，CAT#SH30809.01B)(含10％胎牛血清)，1×10 6细胞/ml Raji细胞用5％BSA封闭后，加入双功能融合蛋白样品至10μg/ml，洗两次后，再加入Alexa Fluor 488-羊抗人(H+L)抗体(Invitrogen，CAT#A11013)，洗两次后，流式细胞仪读取荧光信号值。
FACS检测结果显示，涉及的PD-L1-CD47双功能融合蛋白对Raji细胞表面的天然CD47具有很强的结合能力，和对照抗体Hu5F9的结合能力相当，结果见图3。
测试例6.PD-L1-CD47双功能融合蛋白在体外细胞介导的细胞吞噬作用(ADCP)的实验
PBMC分离自新鲜人血，然后利用Human CD14 MicroBeads(130-050-201#，Miltenyi Biotec)分选CD14+单核细胞。这些CD14+单核细胞通过在巨噬细胞分化培养基(1640+10％FBS+50ng/ml M-CSF)中培养9天分化成为巨噬细胞。这些单核细胞来源的巨噬细胞(MDM)变得具有粘附力，长有触角。实验当天，将巨噬细胞用胰酶消化5min，刮刀轻轻刮下，铺在96孔圆底板(3795#)。用0.5μM CFSE(BD，货号565082#)在37℃水浴锅标记人RBC细胞(血红细胞)13分钟，随后PBS清洗2次后，并将其以每个巨噬细胞给予5个RBC细胞的比例添加至巨噬细胞中，并以多种浓度加入PD-L1-CD47双功能融合蛋白。对靶细胞进行2.5小时的吞噬作用。吞噬结束后，用PBS清洗两次，然后按照一定的比例加入人抗体Fc封闭试剂(Human Fc blocker，564219#,BD)，室温放置10分钟，以排除非特异性结合。随后加入标记了APC的CD14抗体(17-0149-42#,Ebioscience)。冰上孵育30分钟。最后清洗2次后通过流式细胞术进行分析。通过在APC+阳性活细胞门选CFSE+阳性细胞，随后评估CSFE+阳性细胞百分比的方式测量吞噬作用(见图4)。
测试例7.PD-L1-CD47双功能融合蛋白在体外细胞介导的细胞吞噬作用(ADCP)的实验
PBMC分离自新鲜人血，然后利用Human CD14 MicroBeads(130-050-201#，Miltenyi Biotec)分选CD14+单核细胞。这些CD14+单核细胞通过在巨噬细胞分化培养基(1640+10％FBS+50ng/ml M-CSF)中培养9天分化成为巨噬细胞。这些单核细胞来源的巨噬细胞(MDM)变得具有粘附力，长有触角。实验当天，将巨噬细胞用胰酶消化5min，刮刀轻轻刮下，铺在96孔圆底板(3795#)。用0.1μM CFSE在37℃水浴锅标记Molp-8细胞(南京科佰)13分钟，随后PBS清洗2次后，并将其以每个巨噬细胞给予5个Molp-8肿瘤细胞的比例添加至巨噬细胞中，并以加入多种浓度的PD-L1-CD47抗体。对靶细胞进行2.5小时的吞噬作用。吞噬结束后，用PBS清洗两次，然后按照一定的比例加入人抗体Fc封闭试剂(Human Fc blocker)， 室温放置10分钟，以排除非特异性结合。随后加入标记了APC的CD14抗体。冰上孵育30分钟。最后清洗2次后通过流式细胞术进行分析。通过在APC+阳性活细胞门选，随后评估CSFE+阳性细胞百分比的方式测量吞噬作用(见图5A和图5B)。
测试例6和7的结果见图4和图5A-图5B，显示加入的双功能融合蛋白可以有效促进对肿瘤细胞的吞噬作用。但是双功能融合蛋白对红细胞没有吞噬作用，提示本公开的双功能融合蛋白抗体在安全性方面的潜在优势。而对照抗体hu5F9可以有效的吞噬红细胞。
测试例8.PD-L1-CD47双功能融合蛋白的红细胞凝集实验
新鲜健康人血用PBS(B320#，上海源培生物科技股份有限公司)稀释100倍。稀释后的全血铺到96孔圆底板(3795#，corning)，30μl/well。然后等体积加入不同浓度梯度的抗体或双功能融合蛋白。混匀后，放在37℃静置4-6h。用高内涵显微镜观察红细胞沉降情况。未发生血凝为清晰红点，发生血凝为弥散样。
每个样品从第一列(0.5mg/ml)往后，稀释到第11列，1:3稀释。第12列是不加抗体的PBS空白孔。
结果显示(见图6)，同样条件下双功能融合蛋白h1830-S37、h1830-S19、h1830-S12、h1830-S15、h1831-19-S58、h1831-19-S79，在测试的不同浓度下均不会引起红细胞凝集，提示本公开的双功能融合蛋白在安全性方面的优势。
测试例9.BIAcore检测PD-L1-CD47双功能融合蛋白的亲和力实验
用Protein A生物传感芯片(Cat.#29127556,GE)亲和捕获IgG，不同抗原(hCD47，cyno CD47，hPD-L1，cyno PD-L1，mPD-L1来源见测试例3，4)流过芯片表面，Biacore T200仪器实时检测双功能融合蛋白和不同抗原反应信号，获得结合和解离曲线。在每个实验循环解离完成后，用10mM Glycine-HCl pH1.5的缓冲液将生物传感芯片洗净再生。实验缓冲体系为1×HBS-EP缓冲溶液(Cat#BR-1001-88，GE)。实验结束后用GE Biacore T200 Evaluation version 3.0软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，结果见表12-1和表12-2。结果显示，所有改造的SIRPγ肽变体与人CD47的亲和力均较野生型SIRPγ肽大幅度提高。
表12-1抗体对不同抗原的Biacore亲和力(KD(M))
Figure PCTCN2020077907-appb-000117
Figure PCTCN2020077907-appb-000118
表12-2双功能融合蛋白对人CD47的Biacore亲和力(KD(M))
Figure PCTCN2020077907-appb-000119
测试例10.PD-L1-CD47双功能融合蛋白在PBMC-T淋巴细胞激活实验中对细胞IFNγ的分泌作用
为了研究PD-L1-CD47双功能融合蛋白对人原代T淋巴细胞功能的影响，收集和纯化人外周血单核细胞(PBMC)，采用结核菌素(TB)体外刺激5天后，检测细胞因子IFNγ分泌水平。实验过程简单描述如下：
新鲜血液利用Ficoll-Hypaque(17-5442-02，GE)，密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI 1640(SH30809.01，GE)培养基中培养，该培养基中添加10％(v/v)FBS(10099-141，Gibco)，37℃，5％CO 2条件下培养。
新鲜分离纯化的PBMC以RPMI 1640培养基调整密度为2×10 6个/ml，20mL细胞悬液中加入40μl结核菌素(97-8800，Synbiotics)，37℃，5％CO 2培养箱培养5天。第5天，收集上述培养的细胞离心，重悬至新鲜的RPMI 1640培养基中，调 整密度为1.1×10 6个/ml，接种至96孔细胞培养板，每孔90μl。同时加入梯度稀释的抗体样品，用PBS(B320，上海源培生物科技股份有限公司)稀释，每孔10μl。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)收集细胞培养上清，采用ELISA的方法(人IFN-γ检测试剂盒：EHC102g.96，欣博盛)，检测IFN-γ的水平。具体操作参考试剂说明书。
结果(见图7A-图7E)显示，在所测试的分子中，所有双功能融合蛋白都可以激活IFN-γ的分泌，和对照抗体HRP00052相当。
测试例11.PD-L1-CD47双功能融合蛋白在小鼠结肠癌模型MC38/H-11-hCD47中的作用
本实验采用B-hCD274/hCD47/hSIRPα小鼠接种人为改造过的鼠结肠癌MC38细胞：MC38/H-11-hCD47(转入人PD-L1和人CD47，敲除鼠CD47和PDL1)，建立小鼠荷瘤模型，并评价不同剂量的PD-L1-CD47双功能融合蛋白h1830-S85，SIRPγ蛋白S58-Fc和PD-L1单抗h1830对鼠结肠癌移植瘤生长抑制的体内药效作用。B-hCD274/hCD47/hSIRPα小鼠购自百奥赛图实验动物，SPF级；体重：22.0±3.0g；性别：雌性。
将MC38/H-11-hCD47(#5-4)细胞以1×10 6个/100μl/只的接种量接种到B-hCD274/hCD47/hSIRPa小鼠皮下，荷瘤模型确立后，测量肿瘤体积，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为5组(n＝7)：PBS对照组、h1830-S85高剂量组实验组、h1830-S85低剂量组实验组、h1830实验组、S58-Fc实验组，以分组给药日期设定为D0。
1)使用测量瘤径的方法，观察并记录肿瘤的生长情况，同时观察并记录动物体重。
2)肿瘤瘤径和动物体重的测量周期为每周2次。
3)给药后第17天，PBS对照组动物瘤体积较大，遵循动物福利原则，处死PBS实验组动物；给药后第25天，处死剩余实验组动物。
4)肿瘤体积(tumor volume，TV)计算公式：TV＝1/2×a×b 2，其中a，b分别代表测量肿瘤的长径和短径。
5)相对肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
6)抑瘤率TGI％＝1-T/C％
应用Excel和GraphPad对实验数据进行统计分析，各组动物体重、肿瘤体积、瘤重均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 6软件作图。
本实验旨在评价不同剂量的PD-L1-CD47双功能融合蛋白对B-hCD274/hCD47/hSIRPa小鼠鼠结肠癌移植瘤模型中肿瘤生长的抑制作用。本次实验在分组同时开始给予不同抗体或双功能融合蛋白。
如图8，表13的结果显示，不同剂量的双功能融合蛋白(h1830-S85)实验组、 PD-L1单抗(h1830)实验组和SIRPγ蛋白S58-Fc实验组肿瘤体积均小于PBS对照组；PD-L1-CD47双功能融合蛋白高剂量实验组抑瘤效应优于同等剂量的PD-L1单抗实验组和SIRPγ蛋白实验组，并且不同剂量的h1830-S85实验组之间存在剂量依赖关系。
实验过程中，给药组与对照组动物体重未出现明显差异，小鼠对各给药抗体耐受良好。
表13.抗体或双功能融合蛋白对小鼠移植瘤的抑瘤作用(TGI％)
Figure PCTCN2020077907-appb-000120
测试例12.PD-L1-CD47双功能融合蛋白在小鼠结肠癌模型MC38-hPD-L1-hCD47中的作用
将MC38-hPD-L1-hCD47细胞(向MC38细胞转入人PD-L1和人CD47，敲除小鼠CD47)以5.8×10 5个/100μl/只接种到C57/BL-6小鼠皮下，荷瘤模型确立后，测量肿瘤体积，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为5组(n＝7)：IgG4对照组、h1830-S58实验组、HRP00052实验组、h1830实验组和TTI-621实验组，以分组给药日期设定为D0。分组后腹腔给与各药物，每周三次，共10次，给药周期18天，停药后两天结束对荷瘤小鼠的监测。每周测2次瘤体积，称体重，记录数据。分组及给药情况见下表。本次实验在分组同时开始给予不同抗体，给药后第14天开始，所有实验组给药剂量减半；给药后第25天开始，所有实验组停止给药。
表14.实验分组及给药情况
Figure PCTCN2020077907-appb-000121
注：i.p表示腹腔注射，q.o.d表示隔一天给药一次。
各组动物体重、肿瘤体积以及瘤重均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 6和Excel软件作图，使用student t test统计分析。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
抑瘤率％TGI＝1-T/C％
如图9结果显示，与小鼠PD-L1存在交叉反应的PD-L1-CD47双功能融合蛋白h1830-S58实验组和PD-L1单抗(h1830)实验组肿瘤体积均小于对照组和TTI-621实验组，并且在给药后一周左右与对照组之间出现统计学差异；TTI-621实验组在本次实验中未表现出抑瘤效应。h1830-S58实验组在给药后7天抑瘤率达到128.51％，至实验结束，抑瘤率维持在较高的水平。
实验结束后对荷瘤小鼠进行安乐死并剥瘤称重，瘤重结果与瘤体积大小存在一定相似性。实验过程中，给药组与对照组动物体重未出现明显差异，小鼠对各给药抗体耐受良好。
测试例13.PD-L1-CD47双功能融合蛋白在小鼠结肠癌模型MC38-hPD-L1中的作用
将MC38-hPD-L1细胞(向MC38细胞转入人PD-L1)以3.5×10 5个/100μl/只接种到C57/BL-6小鼠皮下，荷瘤模型确立后，测量肿瘤体积，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为5组(n＝7)：IgG4对照组、h1830-19-S79实验组、h1830G1-19-S79实验组、SIRPα-CV实验组和h1830实验组，以分组给药日期设定为D0。分组后腹腔给与各药物，每周三次，共12次，给药周期28天，停药后两天结束对荷瘤小鼠的监测。每周测2次瘤体积，称体重，记录数据。分组及给药情况见下表。
表15.实验分组及给药情况
Figure PCTCN2020077907-appb-000122
各组动物体重、肿瘤体积以及瘤重均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 5和Excel软件作图，使用student t test统计分析。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
抑瘤率％TGI＝1-T/C％
本实验旨在检测不同IgG形式的PD-L1-CD47双功能融合蛋白对C57/BL-6小鼠结肠癌移植瘤模型中肿瘤生长的抑制作用。本次实验在分组同时开始给予不同抗体，给药后第14天开始，所有实验组给药剂量减半；给药后第25天开始，所 有实验组停止给药。
如图10结果显示，截止至给药后第25天，所有双功能融合蛋白给药组和PD-L1单抗h1830给药组肿瘤体积均小于IgG4对照组和SIRPα-CV(TTI-621)实验组，并且与对照组之间存在统计学差异。
给药后第25天后，对照组和SIRPα-CV(TTI-621)实验组由于肿瘤体积较大，对小鼠进行安乐死处理，剩余实验组则停药继续观察。结果显示，PD-L1单抗h1830实验组肿瘤体积随着时间的推移呈现出快速回升的趋势，双功能融合蛋白h1830-19-S79和h1830G1-19-S79实验组肿瘤体积未发生明显变化，且这两种不同IgG形式的双抗之间也没有显著性差异。
实验结束后对荷瘤小鼠进行安乐死并剥瘤称重，瘤重结果与瘤体积大小存在一定相似性，给药组与对照组动物体重未出现明显差异，小鼠对各给药抗体耐受良好。
测试例14.PD-L1-CD47双功能融合蛋白对MOLP-8移植瘤裸鼠疗效
Balb/c裸鼠在右肋部皮下接种MOLP-8细胞(5×10 6+50％matrigel/只)，共120只。10d后，按照平均瘤体积约214.89±6.75mm 3。将荷瘤小鼠随机分为7组(n＝8)：PBS对照组、h1830-S37实验组、h1830-S58实验组、h1831K-19-S37、h1830实验组、S37-Fc、和Hu167 IgG4AA实验组，以分组给药日期设定为D0。分组后腹腔给与各药物，一周二次，连续给药3周。每周测2次瘤体积，称体重，记录数据。各组动物体重、肿瘤体积以及瘤重均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 6和Excel软件作图，使用student t test统计分析。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的空白对照组(Blank)及实验组的相对肿瘤体积。
本次实验结果显示(见图11)，腹腔注射、隔天一次，连续给药10次。以实验进行到21天时的数据进行统计。PD-L1-CD47双功能融合蛋白h1830-S37(30mpk)抑瘤率达到34.98％(P<0.05)；双功能融合蛋白h1831K-19-S37(30mpk)抑瘤率达到54.18％(P<0.01)；h1830(25mpk)没有抑制该肿瘤生长的作用。
给药过程中各组动物体重正常，提示各双功能融合蛋白无明显毒副作用。
测试例15.PD-L1-CD47双功能融合蛋白对CD47/SIRPα结合的阻断
用PBS将CD47-Fc稀释至1μg/ml，以100μl/孔的体积加于96孔板中，于4℃放置16h-20h。将96孔板中PBS缓冲液吸掉，用PBST(pH7.4PBS含0.05％tween20)缓冲液洗板1次后，加入120μl/孔PBST/1％milk，室温孵育1h进行封闭。移去封 闭液，用PBST缓冲液洗板1次后，加入90μl用样品稀释液(pH7.4PBS含5％BSA，0.05％Tween20)稀释至合适浓度的待测PD-L1-CD47双功能融合蛋白，置4℃预孵育1h。以10μl/孔的体积加入10×浓度的生物素标记SIRPα-his(5μg/ml)，在振荡器上振荡、混匀后，置37℃孵育1h。移去反应体系，用PBST洗板6次后，加入100μl/孔用PBST缓冲液1:400稀释的Streptavidin–Peroxidase Polymer，室温振荡孵育50分钟。用PBST洗板6次后，加入100μl/孔TMB，于室温孵育5-10min。加入100μl/孔1M H 2SO 4终止反应。用NOVOStar在酶标仪上测量OD450并计算其IC50值，结果见表16。
表16.双特异性抗体对CD47/SIRPα结合的阻断结果
样品 IC50(ng/ml)
h1831K-19-S37 251.7
S37-Fc 566
TTI-621 25985
Hu5F9 263.1
结果显示双功能融合蛋白能有效阻断CD47与SIRPα的通路。
测试例16.PD-L1-CD47双功能融合蛋白对人乳腺癌细胞MDA-MB-231移植瘤的疗效
将MDA-MB-231细胞(ATCC)3×10 6cells/200μl/mouse(含50％基质胶)接种于NOD/SCID小鼠右肋部皮下，当荷瘤小鼠平均肿瘤体积达到145mm 3左右时将小鼠随机分为4组：PBS、h1831K-19-S37-30mpk、h1831K-19-S37-10mpk、h1831K-25mpk(与h1831K-19-S37高剂量保持等摩尔浓度)，每组8只，并将分组当天定义为该实验Day0。Day0时将经CD3抗体刺激3天的两名志愿者的PBMCs以1:1比例混合，以5×10 5cells/100μl/mouse注射到小鼠肿瘤组织中，剩余的PBMCs停止刺激并继续培养，1周后以5×10 6cells/100μl/mouse腹腔注射到荷瘤鼠体内，视为第1轮注射，至实验结束共注射两轮轮PBMCs。Day0开始每周三次腹腔注射各待测抗体。每周2次监测肿瘤体积、动物重量并记录数据。当肿瘤体积超过1000mm 3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。
所有数据使用Excel和GraphPad Prism 5软件进行作图及统计分析。
肿瘤体积(V)计算公式为：V＝1/2×a×b 2其中a、b分别表示长、宽。
相对肿瘤增殖率T/C(％)＝(T-T 0)/(C-C 0)×100其中T、C为实验结束时治疗组和对照组的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
实验结果显示，在人乳腺癌MDA-MB-231小鼠皮下移植瘤模型中，PDL1-CD47 双特异性抗体h1831K-19-S37、及PDL1单抗h1831K均表现出良好的抑瘤效果(p＜0.001vs PBS)。
PD-L1-CD47双特异性抗体h1831K-19-S37(30、10mg/kg)能显著抑制人乳腺癌MDA-MB-231小鼠皮下移植瘤的生长，且高低剂量间呈剂量依赖性。从给药后3天开始至实验结束(Day23)，无论是高剂量组还是低剂量组，h1831K-19-S37的抑瘤效果始终优于高剂量的PD-L1单抗对照h1831K(25mg/kg)(p＜0.001)，且高低剂量间也存在统计学差异(p＜0.01)(表17)。
实验终点将荷瘤小鼠安乐死，剥离肿瘤并测量瘤重，结果显示离体的肿瘤重量与肿瘤体积趋势相符，所有给药组均显著优于对照组(p＜0.001)，双特异性抗体h1831K-19-S37的高低剂量组均优于高剂量的PDL1单抗对照h1831K(25mg/kg，p＜0.001)，且h1831K-19-S37的高低剂量之间存在剂量效应。
荷瘤小鼠对PDL1-CD47双特异性抗体及其单抗均耐受良好，在整个给药过程中体重只有轻微波动，无明显药物致体重减轻等症状发生。
表17.双特异性抗体对小鼠皮下移植瘤的抑制效果
Figure PCTCN2020077907-appb-000123
注：第0天:第一次给药时间。***表示p＜0.001vs PBS通过student T test测定.

Claims (35)

  1. 一种双功能融合蛋白，所述双功能融合蛋白包含SIRPγ肽变体和抗人PD-L1抗体，所述SIRPγ肽变体直接地或通过连接子连接至所述抗人PD-L1抗体的多肽链，
    所述SIRPγ肽变体是在相对于如SEQ ID NO：20所示的野生型SIRPγ肽的N51位具有替代突变的SIRPγ肽变体，优选地，所述连接子选自SEQ ID NO：89-96和(GGGGS)n、(GGGES)n和(GKPGS)n中的任一个，其中n＝2-7的整数。
  2. 根据权利要求1所述的双功能融合蛋白，其中所述SIRPγ肽变体的羧基端与所述抗人PD-L1抗体的重链可变区的氨基端连接，
    或所述SIRPγ肽变体的羧基端与所述抗人PD-L1抗体的轻链可变区的氨基端连接，
    或所述抗人PD-L1抗体的重链的羧基端与所述SIRPγ肽变体的氨基端连接，
    或所述抗人PD-L1抗体的轻链的羧基端与所述SIRPγ肽变体的氨基端连接。
  3. 根据权利要求1或2所述的双功能融合蛋白，其中所述SIRPγ肽变体相对于所述野生型SIRPγ肽进一步地在K19、K53、N101、L31、Q52、E54、H56、N70、M72和M112中的一个或更多个位点具有氨基酸替代。
  4. 根据权利要求1至3任一项所述的双功能融合蛋白，其中所述N51位具有替代突变的SIRPγ肽变体基本上不结合红细胞表面的CD47，优选地，所述具有N51位替代突变的SIRPγ肽变体具有N51F、N51I、N51L、N51M或N51V替代突变。
  5. 根据权利要求1至3任一项所述的双功能融合蛋白，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有N51R替代突变。
  6. 根据权利要求1至5任一项所述的双功能融合蛋白，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、K53G和N101D替代突变；优选地，
    所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、N51V、Q52S、K53G、E54R、M72K和N101D突变；或
    所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有K19E、N51M、Q52S、K53G、E54R、M72K和N101D突变。
  7. 根据权利要求6所述的双功能融合蛋白，其中所述SIRPγ肽变体进一步地在M6、V27、L30、V33、V36、L37、V42、E47、L66、T67、V92或S98中的一个或更多个位点具有氨基酸替代。
  8. 根据权利要求6所述的双功能融合蛋白，其中所述SIRPγ肽变体如SEQ ID NO：1所示。
  9. 根据权利要求6所述的双功能融合蛋白，其中所述SIRPγ肽变体如SEQ ID NO：2所示。
  10. 根据权利要求6所述的双功能融合蛋白，其中所述SIRPγ肽变体如SEQ ID NO：21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40中的任一所示。
  11. 根据权利要求1至10任一项所述的双功能融合蛋白，其中所述抗人PD-L1抗体选自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和MCLA-145。
  12. 根据权利要求1至10任一项所述的双功能融合蛋白，其中所述抗人PD-L1抗体包含重链可变区和轻链可变区，其中：
    所述重链可变区包含与SEQ ID NO：6所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：7所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含与SEQ ID NO：8所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：9所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；或
    所述重链可变区包含与SEQ ID NO：8所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：113所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；优选地，
    所述重链可变区包含分别如SEQ ID NO：97、98和99所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：100、101和102所示的LCDR1、LCDR2和LCDR3区；或
    所述重链可变区包含分别如SEQ ID NO：103、104和105所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：106、107和108所示的LCDR1、LCDR2和LCDR3区；或
    所述重链可变区包含分别如SEQ ID NO：103、104和105所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：106、112和108所示的LCDR1、LCDR2和LCDR3区。
  13. 根据权利要求12所述的双功能融合蛋白，其中所述的抗人PD-L1抗体包含重链可变区和轻链可变区，其中：
    所述重链可变区为SEQ ID NO：6所示，和所述轻链可变区为SEQ ID NO：7所示；或
    所述重链可变区为SEQ ID NO：8所示，和所述轻链可变区为SEQ ID NO：113所示；或
    所述重链可变区为SEQ ID NO：8所示，和所述轻链可变区为SEQ ID NO：9所示。
  14. 根据权利要求12或13所述的双功能融合蛋白，其中所述抗人PD-L1抗体还包括重链恒定区和轻链恒定区，优选地，所述重链恒定区如SEQ ID NO:10或11所示，所述轻链恒定区如SEQ ID NO:12所示。
  15. 根据权利要求14所述的双功能融合蛋白，其中所述抗人PD-L1抗体包含重链和轻链，其中所述重链如SEQ ID NO：13或15所示，所述轻链如SEQ ID NO：14所示；或
    所述重链如SEQ ID NO：16或18所示，所述轻链如SEQ ID NO：17或111所示。
  16. 根据权利要求15所述的双功能融合蛋白，其中所述双功能融合蛋白具有第一多肽和第二多肽，其中：
    所述第一多肽选自如SEQ ID NO：41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61和62中任一所示的多肽，和所述第二多肽选自如SEQ ID NO：14所示的多肽；或
    所述第一多肽选自如SEQ ID NO：63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82和109中任一所示的多肽，和所述第二多肽选自如SEQ ID NO：17所示的多肽；或
    所述第一多肽选自如SEQ ID NO：63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82和109中任一所示的多肽，和所述第二多肽选自如SEQ ID NO：111所示的多肽。
  17. 一种SIRPγ肽变体，其中所述SIRPγ肽变体是相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有N51位替代突变的SIRPγ肽变体。
  18. 根据权利要求17所述的SIRPγ肽变体，其中所述SIRPγ肽变体相对于所述野生型SIRPγ肽进一步地在K19、K53、N101、L31、Q52、E54、H56、N70、M72和M112中的一个或更多个位点具有氨基酸替代。
  19. 根据权利要求17或18所述的SIRPγ肽变体，其中所述N51位替代突变的SIRPγ肽变体基本上不结合红细胞表面的CD47，优选地，所述具有N51位替代突变的SIRPγ肽变体具有N51F、N51I、N51L、N51M或N51V替代突变。
  20. 根据权利要求17或18所述的SIRPγ肽变体，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ肽具有N51R替代突变。
  21. 根据权利要求17至20任一项所述的SIRPγ肽变体，其中所述SIRPγ肽变体相对于如SEQ ID NO：20所示的野生型SIRPγ具有K19E、K53G和N101D替代突变。
  22. 根据权利要求17至21任一项所述的SIRPγ肽变体，其中所述SIRPγ肽变体进一步地在M6、V27、L30、V33、V36、L37、V42、E47、L66、T67、V92或S98的一个或更多个位点具有氨基酸替代。
  23. 根据权利要求21所述的SIRPγ肽变体，其中所述SIRPγ肽变体如SEQ ID NO：1所示。
  24. 根据权利要求21所述的SIRPγ肽变体，其中所述SIRPγ肽变体如SEQ ID NO：2所示。
  25. 根据权利要求21所述的SIRPγ肽变体，其中所述SIRPγ肽变体如SEQ ID NO：21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40所示。
  26. 一种融合蛋白，其包含SIRPγ肽变体和抗体Fc片段，所述SIRPγ肽变体为权利要求17至25任一项所述SIRPγ肽变体，优选地，所述抗体Fc片段为人抗体Fc片段，更优选地，所述抗体Fc片段序列与SEQ ID NO：10或11所述重链恒定区中的Fc片段序列相同，最优选地，所述融合蛋白的氨基酸序列如SEQ ID NO：86、110、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130或131所示。
  27. 一种抗人PD-L1抗体，其包含轻链可变区和重链可变区，所述重链可变区包含分别如SEQ ID NO：103、104和105所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：106、112和108所示的LCDR1、LCDR2和LCDR3区。
  28. 根据权利要求27所述的抗人PD-L1抗体，其中所述重链可变区为SEQ ID NO：8所示，和所述轻链可变区为SEQ ID NO：113所示。
  29. 根据权利要求28所述的抗人PD-L1抗体，其中所述抗人PD-L1抗体为全长抗体，进一步包括抗体恒定区，优选地，所述抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区，所述抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区，更优选所述全长抗体包含SEQ ID NO:10或11所示的重链恒定区和SEQ ID NO:12所示的轻链恒定区。
  30. 根据权利要求28所述的抗人PD-L1抗体，其中所述抗体包含如SEQ ID NO：16或18所示的重链，和如SEQ ID NO：111所示的轻链。
  31. 一种药物组合物，其含有治疗有效量的根据权利要求1至16任一项所述的双功能融合蛋白，或根据权利要求17至25任一项所述的SIRPγ肽变体，或根据权利要求26所述的融合蛋白，或根据权利要求27至30任一项所述的抗人PD-L1抗体，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  32. 一种分离的核酸分子，其编码权利要求1至16任一项所述的双功能融合蛋白，或根据权利要求17至25任一项所述的SIRPγ肽变体，或根据权利要求26所述的融合蛋白，或根据权利要求27至30任一项所述的抗人PD-L1抗体。
  33. 一种重组载体，其包含权利要求32所述的分离的核酸分子。
  34. 一种消除受试者免疫抑制相关疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1至16任一项所述的双功能融合蛋白，或根据权利要求17至25任一项所述的SIRPγ肽变体，或根据权利要求26所述的融合蛋白，或根据权利要求27至30任一项所述的抗人PD-L1抗体，或权利要求31所述的药物组合物，或权利要求32所述的分离的核酸分子，优选地，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的权利要求1至16任一项所述的双功能融合蛋白，或根据权利要求17至25任一项所述的SIRPγ肽变体，或根据权利要求26所述的融合蛋白，或根据权利要求27至30任一项所述的抗人PD-L1抗体。
  35. 根据权利要求34所述的消除受试者免疫抑制相关疾病的方法，其中所述免疫抑制相关疾病包括癌症、细菌或病毒感染，优选地，所述癌症包括癌瘤，淋巴瘤，胚细胞瘤，肉瘤，和白血病或淋巴样恶性，更优选包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、头和颈鳞状细胞癌、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、急性成淋巴细胞性白血病、急性髓细胞样白血病、慢性淋巴细胞性白血病、慢性髓细胞样白血病、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白、骨髓异常增生综合征、胃肠道癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、膀胱癌、乳腺癌、结肠癌、肝细胞癌、透明细胞肾细胞癌、头和颈癌、咽喉癌、肝胆癌、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
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