WO2022002036A1 - 一种双特异性抗体及其用途 - Google Patents

一种双特异性抗体及其用途 Download PDF

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WO2022002036A1
WO2022002036A1 PCT/CN2021/103058 CN2021103058W WO2022002036A1 WO 2022002036 A1 WO2022002036 A1 WO 2022002036A1 CN 2021103058 W CN2021103058 W CN 2021103058W WO 2022002036 A1 WO2022002036 A1 WO 2022002036A1
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seq
nos
hcdr2
hcdr3
hcdr1
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PCT/CN2021/103058
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English (en)
French (fr)
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石磊
钟琛
吴晓东
何云
陈飞
吕小成
谢金莉
戎一平
黄冰
杜芳芳
赵建勋
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和铂医药(上海)有限责任公司
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Priority to CA3183389A priority Critical patent/CA3183389A1/en
Priority to MX2022016407A priority patent/MX2022016407A/es
Priority to EP21832260.0A priority patent/EP4155318A1/en
Priority to CN202180005437.2A priority patent/CN114667296B/zh
Priority to US18/002,499 priority patent/US20230235090A1/en
Priority to BR112022026114A priority patent/BR112022026114A2/pt
Application filed by 和铂医药(上海)有限责任公司 filed Critical 和铂医药(上海)有限责任公司
Priority to JP2022579106A priority patent/JP2023530766A/ja
Priority to IL299232A priority patent/IL299232A/en
Priority to KR1020227044792A priority patent/KR20230015957A/ko
Priority to AU2021301921A priority patent/AU2021301921A1/en
Publication of WO2022002036A1 publication Critical patent/WO2022002036A1/zh
Priority to ZA2022/13707A priority patent/ZA202213707B/en

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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular to a bispecific antibody, in particular to a bispecific antibody targeting B7-H4 and 4-1BB and use thereof.
  • B7-H4 (VTCN1, B7h.5, B7S1, B7x) is a transmembrane protein belonging to the B7/CD28 superfamily.
  • the B7-H4 protein is expressed in activated T cells and B cells, monocytes and dendritic cells, and may negatively regulate the immune response of T cells.
  • B7-H4 mRNA is widely expressed, B7-H4 protein is not expressed in most normal tissues.
  • B7-H4 is overexpressed on the tumor cell surface of breast cancer, ovarian cancer, and endometrial cancer.
  • Breast cancer is the second most common malignant tumor worldwide, and its incidence is increasing.
  • Breast cancer accounts for about a quarter of female cancer patients. There were 320,000 new cases of breast cancer in the United States in 2019, with a total number of about 3.8 million, and about 300,000 new cases of breast cancer in China each year, and the number of patients is estimated to reach 2.5 million in 2021.
  • Ovarian cancer and endometrial cancer are common malignant tumors of the female reproductive system.
  • ovarian cancer In China, about 50,000 women are diagnosed with ovarian cancer every year, and about 20,000 die from ovarian cancer. The mortality rate of ovarian cancer ranks first among gynecological malignancies. Endometrial cancer has nearly 200,000 new cases every year, and ranks third among gynecological malignancies that cause death.
  • B7-H4 is also overexpressed in non-small cell lung cancer and renal cancer.
  • Lung cancer is one of the most common cancers, among which non-small cell lung cancer accounts for nearly 80% of lung cancers. Both in the world and in China, lung cancer has the highest morbidity and mortality. In 2018, there were about 2 million new cases of lung cancer worldwide and 1.76 million deaths; in China, there were about 780,000 new cases and 630,000 deaths each year. The incidence of kidney cancer is relatively low, and the annual incidence of kidney cancer in China is about 70,000.
  • B7-H4 As an emerging target for these tumors, B7-H4 has received attention in recent years.
  • B7-H4 antibody can be applied to tumor cells through various mechanisms, but the research and development direction of B7-H4 antibody mainly focuses on monoclonal antibody and ADC, and there is currently no bispecific antibody therapy.
  • 4-1BB (TNFRSF9, CD137) is a transmembrane protein belonging to the TNF receptor superfamily.
  • 4-1BB is a costimulatory molecule expressed on a variety of cells and is a multifunctional regulator of immune activity. It is induced and expressed in activated T cells, NK cells and monocytes, dendritic cells, macrophages, tumor-associated vascular endothelial cells, and the like.
  • Its ligand is 4-1BBL, which is mainly expressed on professional APC cells (such as mononuclear macrophages, DC cells, B cells), activated T cells and some tumor cells.
  • Anti-4-1BB agonistic antibodies can inhibit tumors, but the research and development of 4-1BB antibodies is mainly focused on monoclonal antibodies and antibodies targeting both PD-L1 and 4-1BB. Currently, there are no bispecific antibodies targeting B7-H4 and 4-1BB in clinical development.
  • BMS-663513 Bristol-Myers Squibb's (BMS-663513) is a fully human IgG4 antibody against 4-1BB antibody and is the first anti-4-1BB antibody to enter clinical trials.
  • the initial clinical results of Urelumab were published in 2008, and although encouraging efficacy was observed in some patients, the data showed that Urelumab was hepatotoxic and was target- and dose-related.
  • two patients in the clinical trial died due to hepatotoxicity, leading to the termination of the clinical trial of Urelumab as a single agent.
  • the bispecific antibody targeting PD-L1 and 4-1BB is still in the first clinical stage, and the expression of PD-L1 on tumor cells overlaps little with that of B7-H4. Therefore, there is an urgent need to develop a more safe and effective bispecific antibody that simultaneously targets human B7-H4 and 4-1BB and can bind to cynomolgus monkey B7-H4 and 4-1BB.
  • the technical problem to be solved by the present invention is to overcome the lack of safe and effective bispecific antibodies in the prior art that simultaneously target human B7-H4 and 4-1BB and can bind to cynomolgus monkey B7-H4 and 4-1BB
  • a bispecific antibody, especially a bispecific antibody targeting B7-H4 and 4-1BB, and its application are provided.
  • the present invention provides the following technical solutions.
  • a first aspect of the present invention provides a bispecific antibody comprising an antigen binding domain targeting B7-H4 and an antigen binding domain targeting 4-1BB.
  • the antigen binding domain of described targeting B7-H4 contains HCDR1 whose sequence is as shown in SEQ ID NO:16, HCDR2 whose sequence is as shown in SEQ ID NO:46, and HCDR2 whose sequence is as shown in SEQ ID NO:84.
  • the antigen binding domain of described targeting B7-H4 also contains LCDR1, LCDR2 and LCDR3, the aminoacid sequence of described LCDR1 is as shown in SEQ ID NO.112 or 113, the aminoacid sequence of described LCDR2 As shown in SEQ ID NO.118, the amino acid sequence of the LCDR3 is shown in any one of SEQ ID NO.131 ⁇ 133;
  • the antigen binding domain of described targeting B7-H4 also contains LCDR1 whose sequence is as shown in SEQ ID NO:112, LCDR2 whose sequence is as shown in SEQ ID NO:118, and LCDR2 whose sequence is as shown in SEQ ID NO:118: LCDR3 shown in 131; LCDR1 whose sequence is shown in SEQ ID NO:113, LCDR2 whose sequence is shown in SEQ ID NO:118, and LCDR3 whose sequence is shown in SEQ ID NO:132; or LCDR3 whose sequence is shown in SEQ ID NO:112
  • the LCDR1 shown, the LCDR2 whose sequence is shown in SEQ ID NO: 118 and the LCDR3 whose sequence is shown in SEQ ID NO: 133; preferably also contains the VL whose sequence is shown in any one of SEQ ID NO: 166 to 169; more preferably Also contains a light chain whose sequence is shown in any one of SEQ ID NOs: 198-201.
  • the antigen binding domain of described targeting B7-H4 preferably contains HCDR1 whose sequence is as shown in SEQ ID NO:16, HCDR2 whose sequence is as shown in SEQ ID NO:46, and HCDR2 whose sequence is as shown in SEQ ID NO:84 HCDR3, LCDR1 whose sequence is as shown in SEQ ID NO:112, LCDR2 whose sequence is as shown in SEQ ID NO:118, and LCDR3 whose sequence is as shown in SEQ ID NO:131; HCDR1 whose sequence is as shown in SEQ ID NO:23 , HCDR2 whose sequence is as shown in SEQ ID NO:59 and HCDR3 whose sequence is as shown in SEQ ID NO:98, LCDR1 whose sequence is as shown in SEQ ID NO:113, LCDR2 whose sequence is as shown in SEQ ID NO:118 and sequence LCDR3 as shown in SEQ ID NO:132; or, HCDR1 whose sequence is as shown in SEQ ID NO:16, HCDR2
  • the antigen binding domain of described targeting B7-H4 contains sequence as shown in SEQ ID NO:142 VH and sequence as shown in SEQ ID NO:166 VL; sequence as shown in SEQ ID NO:159 The VH shown and the VL shown in SEQ ID NO:167; the VH shown in SEQ ID NO:160 and the VL shown in SEQ ID NO:168; or the sequence shown in SEQ ID NO:159 The VH and VL of the sequence are shown in SEQ ID NO: 169.
  • the antigen binding domain of the targeting B7-H4 contains a heavy chain with a sequence as shown in SEQ ID NO:174 and a light chain with a sequence as shown in SEQ ID NO:198; the sequence is as shown in SEQ ID NO: The heavy chain shown in 191 and the light chain shown in SEQ ID NO: 199; the heavy chain shown in SEQ ID NO: 192 and the light chain shown in SEQ ID NO: 200; The heavy chain set forth in SEQ ID NO:191 and the light chain whose sequence is set forth in SEQ ID NO:201.
  • the form thereof is preferably a single VH, tandem VH, ScFv, Fab or IgG form.
  • the VH in series is preferably two or more VHs in series, such as 2, 3 or 4.
  • it When in IgG format, it preferably contains constant regions derived from human IgG1 containing mutations L234A, L235A and P329G or human IgG1 containing mutations L234A and L235A.
  • said HCDR1, HCDR2 and HCDR3 are preferably as follows:
  • the sequence of described HCDR1 is as shown in SEQ ID NO: 19 or its variant 1, or SEQ ID NO: 14, and the sequence of described HCDR2 is as shown in SEQ ID NO: 49 or its variant 2, SEQ ID NO: 51 Or shown in SEQ ID NO:43, the sequence of the HCDR3 is shown in SEQ ID NO:86 or its variant, SEQ ID NO:96, SEQ ID NO:89 or SEQ ID NO:81; wherein:
  • the mutation of the variant 1 includes one or more of T3I, S6N/R and Y7F; preferably, the sequence of the variant 1 is preferably as SEQ ID NO: 17-18 and SEQ ID NO in the sequence listing : any one of 20 to 22;
  • the mutation of the variant 2 includes one or more of S1N/D, G2S/A, S3D/G, G5D/F/S/V and S6T/N/D; the sequence of the variant 2 is preferably in the order shown in any one of SEQ ID NO:47 ⁇ 48, SEQ ID NO:50, SEQ ID NO:52 ⁇ 58 and SEQ ID NO:61 in the list;
  • the mutations of variant 3 include G2R/D/A/K, S3A/T, S4G/N/A/T/H, E5T/V/M/G, T6A, D7G/S, H9Y/S, Y10H One or more of , Y11F, N12G/D and V13I/M/T; the amino acid sequence of the variant 3 is preferably as SEQ ID NO: 85, SEQ ID NO: 87 ⁇ 88 and SEQ ID NO: Any one of 90 to 95 is shown.
  • the antigen binding domain targeting 4-1BB contains: HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 17, 47 and 85, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 48 and 86, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 49 and 87, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 19, 50 and 88, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 20, 51 and 89, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 52 and 90, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 49 and 90, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 21, 53 and 91, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 21, 54 and 92, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 19, 55 and 93, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 49 and 86, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 19, 49 and 94, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 22, 56 and 86, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 57 and 95, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 19, 58 and 96, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 61 and 95, respectively;
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 14, 43 and 81, respectively;
  • the antigen binding domain targeting 4-1BB contains one or more heavy chain variable regions whose sequences are shown in SEQ ID NOs: 143-157, 139, 284 or 161.
  • the antigen binding domain targeting 4-1BB comprises a heavy chain whose sequence is shown in SEQ ID NO: 175-189, 193, 285 or 171.
  • the antigen binding domain of described targeting 4-1BB also comprises LCDR1, LCDR2 and LCDR3 whose sequence is shown in SEQ ID NO: 109, 118 and 128 respectively; The light chain variable region shown in NO:163; more preferably, the light chain comprising the sequence shown in SEQ ID NO:195.
  • the antigen-binding domain targeting 4-1BB in the present invention can be in a conventional form in the field, such as: a single VH or a tandem VH form, an HCAb form or a ScFv form; the tandem VH is relatively Preferably, two or more VHs are connected in series, such as two, three or four.
  • the antigen-binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NO: 112, 118 and 131, respectively; HCDR1, HCDR2 and HCDR3 shown in: 16, 46 and 84; and the antigen binding domains targeting 4-1BB comprise LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 109, 118 and 128, respectively and HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 14, 43 and 81, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NOs: 113, 118 and 132, and LCDR3 as shown in SEQ ID NOs: 23, 59 and 98, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen-binding domain of said targeting 4-1BB comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 109, 118 and 128, respectively; : HCDR1, HCDR2 and HCDR3 shown in 14, 43 and 81;
  • the antigen binding domains of described targeting B7-H4 comprise LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 131 and sequences shown in SEQ ID NOs: 16, 46 and 84 respectively HCDR1, HCDR2 and HCDR3; and the antigen binding domains targeting 4-1BB comprise HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 17, 47 and 85, respectively;
  • the antigen binding domains of described targeting B7-H4 comprise LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 131 and sequences shown in SEQ ID NOs: 16, 46 and 84 respectively HCDR1, HCDR2 and HCDR3; and the antigen binding domains targeting 4-1BB comprise HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 48 and 86, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively.
  • the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 49 and 87, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 19, 50 and 88, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 20, 51 and 89, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 52 and 90, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 21, 53 and 91, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 21, 54 and 92, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 19, 55 and 93, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 49 and 86, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 19, 49 and 94, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 22, 56 and 86, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NOs: 112, 118 and 131, respectively, and LCDR3 whose sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 57 and 95, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 133, and LCDR3 with sequences shown in SEQ ID NOs: 16, 60 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 17, 47 and 85, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 133, and LCDR3 with sequences shown in SEQ ID NOs: 16, 60 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 48 and 86, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 133, and LCDR3 with sequences shown in SEQ ID NOs: 16, 60 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 49 and 86, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 133, and LCDR3 with sequences shown in SEQ ID NOs: 16, 60 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 19, 49 and 94, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NOs: 112, 118 and 133, and LCDR3 with sequences shown in SEQ ID NOs: 16, 60 and 84, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 61 and 95, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NOs: 113, 118 and 132, and LCDR3 as shown in SEQ ID NOs: 23, 59 and 98, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 48 and 86, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NOs: 113, 118 and 132, and LCDR3 as shown in SEQ ID NOs: 23, 59 and 98, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 19, 58 and 96, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NOs: 113, 118 and 132, and LCDR3 as shown in SEQ ID NOs: 23, 59 and 98, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 57 and 95, respectively;
  • the antigen binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NOs: 113, 118 and 132, and LCDR3 as shown in SEQ ID NOs: 23, 59 and 98, respectively HCDR1, HCDR2 and HCDR3 shown; and the antigen binding domain targeting 4-1BB comprises HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NOs: 18, 49 and 90, respectively.
  • bispecific antibodies of the present invention in specific embodiments of the present invention:
  • the antigenic domain targeting B7-H4 is in the form of IgG, and the antigen binding domain targeting 4-1BB is in the form of a single VH;
  • the antigenic domain targeting B7-H4 is in the form of IgG, and the antigen-binding domain targeting 4-1BB is in the form of ScFv;
  • the antigenic domain targeting B7-H4 is in the form of IgG, and the antigen binding domain targeting 4-1BB is in the form of 2 or 3 VHs in series;
  • the antigenic domain targeting B7-H4 is in the form of Fab
  • the antigen binding domain targeting 4-1BB is in the form of HCAb or in the form of HCAb and VH (HCAb-VH form);
  • the antigenic domain targeting B7-H4 is in the form of Fab
  • the antigen binding domain targeting 4-1BB is in the form of VH
  • the form of the VH is preferably a single VH, 2 or 3 VHs concatenated form.
  • the bispecific antibody is in the following form:
  • ScFv is connected to the C-terminus of IgG, and the VH of the ScFv is connected to the C-terminus; preferably, the polypeptide chain 1 of the bispecific antibody in this form is shown in the formula: VL B7-H4- CL, Polypeptide chain 2 is shown in the formula: VH B7-H4- CH1-hinge-CH2-CH3-linker- VH 4-1BB -linker-VL 4-1BB ;
  • the C-terminus of IgG is connected with VH or tandem VH; preferably, the polypeptide chain 1 of this form of bispecific antibody is shown in the formula: VL B7-H4- CL, and the polypeptide chain 2 is shown in the formula: VH B7- H4 - CH1 -hinge-CH2-CH3-joint-(VH4-1BB) n -shown;
  • Fab is connected to the N-terminus of the HCAb; preferably, the polypeptide chain 1 of the bispecific antibody in this form is shown in the formula: VH B7-H4- CH1, and the polypeptide chain 2 is shown in the formula: VL B7-H4- CL - Connector-VH 4-1BB - Connector-CH2-CH3 shown;
  • the N-termini of the two CH2 of Fc are respectively connected with Fab, and VH or tandem VH; preferably, the bispecific antibody in this form contains three polypeptide chains:
  • Polypeptide chain 1 is shown in the formula: VL B7-H4- CL;
  • Polypeptide chain 2 is shown in the formula: VH B7-H4- CH1-hinge-CH2-CH3;
  • Polypeptide chain 3 is shown in the formula: VH 4-1BB -linker-CH2 -CH3 or (VH 4-1BB ) n -linker-CH2-CH3;
  • linker sequences connecting different domains are the same or different.
  • the Fc of the IgG or HCAb contains a mutation, preferably one of the following mutations:
  • L234A and L235A optionally also contain P329G, YTE or DHS; wherein, YTE refers to M252Y/S254T/T256E, DHS refers to the mutation of L at position 309 to D, and the mutation at position 311 Q is H, and the N at position 434 is mutated to S.
  • FALA refers to the mutation of F at position 234 to A, and the mutation of L at position 235 to A;
  • the linker is selected from the group consisting of SEQ ID NOs: 241-261, 282 and 288-289; wherein:
  • the linker of form (1) preferably contains the sequence shown in SEQ ID NO: 245;
  • the linker of form (2) preferably contains the sequence shown in any of SEQ ID NOs: 243, 245-247 and SEQ ID NOs: 288-289;
  • the linker of form (3) preferably contains the sequence shown in SEQ ID NO: 250;
  • the linker of form (4) preferably contains the sequence shown in SEQ ID NO: 282;
  • the linker of form (5) preferably contains the sequence shown in SEQ ID NO:245.
  • amino acid sequence of the polypeptide chain 1 is shown in SEQ ID NO: 198, and the amino acid sequence of the polypeptide chain 2 is shown in any one of SEQ ID NO: 202-215;
  • amino acid sequence of the polypeptide chain 1 is shown in SEQ ID NO: 201
  • amino acid sequence of the polypeptide chain 2 is shown in SEQ ID NO: 217, 230, 238, 240, 226, 262 or 239;
  • amino acid sequence of the polypeptide chain 1 is shown in SEQ ID NO: 200
  • amino acid sequence of the polypeptide chain 2 is shown in SEQ ID NOs: 218-225, 235-237, 263-268, 274-281, 286 and any one of 287;
  • amino acid sequence of the polypeptide chain 1 is shown in SEQ ID NO: 201
  • amino acid sequence of the polypeptide chain 2 is shown in SEQ ID NO: 227
  • amino acid sequence of the polypeptide chain 3 is shown in SEQ ID NO: 228, 229, 231 or 232;
  • amino acid sequence of the polypeptide chain 1 is shown in SEQ ID NO: 233
  • amino acid sequence of the polypeptide chain 2 is shown in any one of SEQ ID NOs: 234 and 269-273.
  • a second aspect of the present invention provides an isolated nucleic acid encoding a bispecific antibody as described in the first aspect above.
  • a third aspect of the present invention provides an expression vector comprising the isolated nucleic acid as described in the second aspect.
  • the fourth aspect of the present invention provides a host cell comprising the expression vector according to the third aspect; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.
  • a fifth aspect of the present invention provides a method for preparing a bispecific antibody, which comprises culturing the host cell according to the fourth aspect, and obtaining the bispecific antibody from the culture.
  • a sixth aspect of the present invention provides a pharmaceutical composition comprising the bispecific antibody according to the first aspect of the present invention.
  • the seventh aspect of the present invention provides the bispecific antibody according to the first aspect and the pharmaceutical composition according to the sixth aspect in the preparation of preventing (preventing and/or treating) 4-1BB and/or B7-H4 related diseases application in medicines.
  • the disease is preferably cancer
  • the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer, Gallbladder cancer, bladder cancer, sarcoma, or colorectal cancer.
  • the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer or bile duct cancer. More preferably, the cancer is preferably breast cancer.
  • the eighth aspect of the present invention provides a chimeric antigen receptor comprising the bispecific antibody of the first aspect.
  • the ninth aspect of the present invention provides an antibody drug conjugate comprising a cytotoxic agent, and the bispecific antibody according to the first aspect; preferably, the cytotoxic agent is MMAF or MMAE.
  • a tenth aspect of the present invention provides a kit comprising the bispecific antibody described in the first aspect, the chimeric antigen receptor described in the eighth aspect, the antibody-drug conjugate described in the ninth aspect and/or or the pharmaceutical composition of the sixth aspect;
  • the kit further comprises (i) a device for administering the antibody, or antigen-binding fragment thereof, or antibody-drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
  • An eleventh aspect of the present invention provides a set of medicine kits, which comprises a medicine box A and a medicine box B, wherein:
  • the kit A contains the bispecific antibody described in the first aspect, the chimeric antigen receptor described in the eighth aspect, the antibody drug conjugate described in the ninth aspect and/or the drug described in the sixth aspect combination;
  • the kit B contains other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies, and/or is composed of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • a twelfth aspect of the present invention relates to a method of diagnosing, treating and/or preventing a 4-1BB and/or B7-H4 mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of a
  • the bispecific antibody of the first aspect, the chimeric antigen receptor of the eighth aspect, the antibody drug conjugate of the ninth aspect and/or the pharmaceutical composition of the sixth aspect, or the use of the The medicine kit of the eleventh aspect treats a patient in need.
  • the disease or condition is preferably a tumor, preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer cancer, gallbladder cancer, bladder cancer, sarcoma or colorectal cancer; preferably, the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer or bile duct cancer; more preferably, the cancer is preferably breast cancer cancer.
  • the thirteenth aspect of the present invention relates to a method for immunodetection or determination of 4-1BB or B7-H4, comprising using the bispecific antibody described in the first aspect, the chimeric antigen receptor described in the eighth aspect, The antibody drug conjugate described in the ninth aspect and/or the pharmaceutical composition described in the sixth aspect detects the sample; preferably, the detection is for non-diagnostic and/or therapeutic purposes.
  • the fourteenth aspect of the present invention relates to a combination therapy comprising administering the bispecific antibody of the first aspect, the chimeric antigen receptor of the eighth aspect, the chimeric antigen receptor of the eighth aspect, and the ninth aspect, respectively, to a patient in need thereof
  • the antibody drug conjugate and/or the pharmaceutical composition of the sixth aspect, and the second therapeutic agent; the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical combination comprising the other anti-tumor antibodies drugs, and/or by hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitory molecules One or more of the group consisting of inhibitors and vaccines.
  • the B7-H4 ⁇ 4-1BB bispecific antibody of the present invention is a unique B7-H4 ⁇ 4-1BB bispecific antibody with one or two or three binding sites for 4-1BB; bispecific Both protein functional regions of the antibody have good binding activity to cynomolgus monkeys.
  • the 4-1BB antibody of the present invention is a completely new fully human antibody containing only "heavy chain", and has the activity of binding to human 4-1BB and cynomolgus monkey 4-1BB.
  • the size of the 4-1BB heavy chain antibody is only half of that of traditional IgG antibodies. Due to the absence of light chains, this antibody can be used in bispecific antibodies and solves light chain mismatches and heterodimerization. The problem.
  • the B7-H4 antibody of the present invention is a completely new fully human antibody, which has the activity of binding to human B7-H4 and cynomolgus monkey B7-H4.
  • the bispecific antibody of the present invention has one or two or three binding sites for 4-1BB, which optimizes the activity of the 4-1BB end.
  • the present invention specifically binds to tumor cells by targeting B7-H4, thereby reducing the toxicity caused by 4-1BB activation.
  • the present invention is a bispecific antibody structure with a human Fc fragment, which retains the binding effect of Fc and FcRn, thereby having a longer half-life.
  • FIG. 1 (A) is the FACS detection of the in vitro binding of B7-H4 mAb on the SK-BR-3 cell line with high expression of B7-H4; (B) is the FACS detection of B7-H4 mAb in cynomolgus monkeys with high expression In vitro binding of B7-H4 on CHO-K1-cynomolgus monkey B7-H4 cell line; (C) FACS detection of B7-H4 mAb in CHO-K1-mouse B7-H4 cell line with high expression of mouse B7-H4 in vitro binding.
  • Figures 2-(A)-(I) are FACS detection of the in vitro binding of 4-1BB mAb on the CHO-K1-human 4-1BB cell line that highly expresses human 4-1BB.
  • Figures 3-(A)-(H) are FACS detection of in vitro binding of 4-1BB monoclonal antibody on CHO-K1-cynomolgus monkey 4-1BB cell line highly expressing cynomolgus monkey 4-1BB.
  • Figures 4-(A)-(E) show that 4-1BB mAb activates the 4-1BB pathway and induces the function of activated T cells.
  • Figures 5-(A)-(I) are schematic diagrams of the structure of the B7-H4 ⁇ 4-1BB bispecific molecule.
  • Figures 6-(A)-(L) are FACS detection of the in vitro binding of B7-H4 ⁇ 4-1BB bispecific antibody on CHO-K1 cell line overexpressing human 4-1BB.
  • Figures 7-(A)-(C) are FACS detection of in vitro binding of B7-H4 ⁇ 4-1BB bispecific antibody on CHO-K1 cell line overexpressing cynomolgus monkey 4-1BB.
  • Figures 8-(A)-(K) are FACS detection of the in vitro binding of B7-H4 ⁇ 4-1BB bispecific antibody on SK-BR-3 cell line with high expression of B7-H4.
  • Figure 9-(A)-(E) are the T cell activation experiments and the release of cytokine IFN- ⁇ by the B7-H4 ⁇ 4-1BB bispecific antibody in the presence of the B7-H4 highly expressing cell line SK-BR-3 .
  • Figure 10-(A)-(N) shows the T cell activation experiment and the release of cytokine IL-2 by B7-H4 ⁇ 4-1BB bispecific antibody in the presence of B7-H4 highly expressing cell line SK-BR-3 .
  • FIGS 11-(A)-(B) show that the activation of T cells by the B7-H4 ⁇ 4-1BB bispecific antibody is independent of the presence of Fc ⁇ R.
  • Figure 12-(A)-(B) is the expression of B7-H4 on the surface of tumor cells detected by FACS.
  • Figure 13-(A)-(H) shows that B7-H4 ⁇ 4-1BB double antibody specifically depends on the expression of B7-H4 for T cell activation.
  • Figures 14-(A)-(H) are the serum stability of B7-H4 ⁇ 4-1BB dual antibodies PR003334, PR003335 and PR004282.
  • Figures 15-(A)-(C) are the in vitro binding of B7-H4 ⁇ 4-1BB double antibody PR004282 to human, monkey and mouse 4-1BB.
  • Figures 16-(A)-(C) show the in vitro binding of B7-H4 ⁇ 4-1BB double antibody PR004282 to human, monkey and mouse B7H4.
  • Figure 17 shows the simultaneous binding of B7-H4 ⁇ 4-1BB double antibody PR004282 to SK-BR-3 and CHO-K1/4-1BB in vitro.
  • Figure 18-(A)-(B) shows the in vitro binding of B7-H4 ⁇ 4-1BB double antibody PR004282 to human and monkey Pan T cells activated in vitro.
  • Figure 19-(A)-(B) are the cross-reaction results of B7-H4 ⁇ 4-1BB double antibody PR004282 with B7 family members or other members of the TNFR family
  • Figure 20-(A)-(C) is the ADCC effect result of B7-H4 ⁇ 4-1BB double antibody PR004282.
  • Figures 21-(A)-(C) are the detection results of non-specific cytokine release of B7-H4 ⁇ 4-1BB bispecific antibody PR004282.
  • 22-(A)-(F) are the pharmacokinetic data of B7-H4 ⁇ 4-1BB bispecific antibodies PR003334 and PR003335 and PR004282 in wild-type mice.
  • Figure 23 shows the pharmacokinetic data (single dose) of the B7-H4x4-1BB bispecific antibody PR004282 in normal monkeys.
  • FIG. 24(A)-(B) shows the antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody PR003334 in BALB/c-hCD137/CT26-B7-H4 mouse model.
  • Figure 25-(A)-(B) is the antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody PR003338 in MDA-MB-468 xenograft mouse model.
  • Figure 26-(A)-(B) shows the antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 in OVCAR3 xenograft mouse model.
  • Figure 27-(A)-(I) are the results of antitumor efficacy and memory immune effect of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 in BALB/c-hCD137/CT26-B7-H4 mouse model .
  • 28-(A)-(B) are the specific results of the antitumor efficacy of the B7-H4 ⁇ 4-1BB bispecific antibody PR004282.
  • the term “antibody” generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone portion that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antibody to the antigen.
  • An antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both may typically be included.
  • VL variable region
  • VH antibody heavy chain variable region
  • the "heavy chain antibody” in this application does not contain a VL region, but only contains a VH region.
  • the VH or VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH or VL can consist of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • antibodies include, but are not limited to, full-length antibodies, heavy chain antibodies (HCAbs), antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and/or or dAb), immunoconjugates, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • HCAbs heavy chain antibodies
  • Fab antigen-binding fragments
  • Fab' antigen-binding fragments
  • F(ab)2 fragment fragments
  • F(ab')2 fragments
  • scFv di-scFv and/or or dAb
  • immunoconjugates eg, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity
  • variable generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the variable domains of the light and heavy chains, called the CDRs or hypervariable regions (HVRs), with the FRs being the more highly conserved parts of the variable domains.
  • the variable domains of native heavy and light chains each comprise four FR regions, mostly in a ⁇ -sheet configuration, connected by three CDRs, forming loops connecting, and in some cases forming part of, a ⁇ -sheet structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody, the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions , eg involved in antibody-dependent cytotoxicity of antibodies.
  • the term "fully human antibody” generally refers to an antibody obtained by transferring all the human antibody-encoding genes into a genetically engineered antibody gene-deficient animal, so that the animal expresses the antibody. All parts of an antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, and the like.
  • nucleic acid refers to DNA molecules and RNA molecules. It may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • the term “specifically binds” generally refers to the binding of an antibody to an epitope through its antigen binding domain, and that binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an antigen when it is more likely to bind to an epitope through its antigen-binding domain than to a random, unrelated epitope than to a random, unrelated epitope.
  • Fab generally refers to the antigen-binding portion of a conventional antibody (eg, IgG), including the heavy chain variable region VH, light chain variable region VL and heavy chain constant region domain CH1 and light chain variable region of the antibody.
  • Chain constant region CL In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region of the heavy chain and other constant The domains are linked to form the heavy chain.
  • Fab also refers to variant structures of Fab.
  • the C-terminus of VH is linked to the N-terminus of CL to form a polypeptide chain
  • the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH/VL) structure
  • the CH1 of the Fab is not linked to the hinge region, but the C-terminus of the CL is linked to the hinge region of the heavy chain to form a Fab (cross Fd/LC) structure.
  • VH generally refers to the heavy chain variable region VH domain of an antibody, that is, it can be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals, or it can be Camelidae, etc.
  • the heavy chain variable region VHH of an animal heavy chain antibody (HCAb structure) can also be the heavy chain variable region VH of a fully human heavy chain antibody (HCAb structure) produced by using Harbour HCAb transgenic mice.
  • the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997).
  • the present application can also determine amino acid residues in variable domain sequences and full-length antibody sequences using the Combined definition rule comprising the Kabat definition and the Chothia definition (Table 1). In the present invention, each sequence is determined according to the Chothia definition.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rule) to bb position (Chothia coding rule);
  • Haa-Hbb can refer to starting from the N-terminus of the antibody heavy chain , the amino acid sequence from position aa (Chothia coding rule) to bb position (Chothia coding rule).
  • L24-L34 may refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain according to the Chothia coding rules Amino acid sequence from position 26 to position 32.
  • the present invention uses the Chothia coding rules to define the CDRs of antibodies.
  • the effector functions mediated by the Fc domain of antibodies also have very important biological functions.
  • Different IgG subtypes have different ADCC or CDC functions.
  • IgG1 and IgG3 have strong ADCC and CDC functions, while IgG2 and IgG4 are relatively weak.
  • altering the binding ability of Fc to Fc receptors by amino acid mutation or modification can also modulate the original effector function of Fc.
  • the "LALA" double mutant (L234A/L235A) in IgG1 can significantly reduce the affinity for Fc ⁇ RIIIA (CD16A), thereby reducing ADCC effect.
  • the P329G mutation can significantly reduce binding to various Fc ⁇ receptors (see, Schlothauer T, Herter S, Koller CF, Grau-Richards S, Steinhart V, Spick C, Kubbies M, Klein C, P, E. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein EngDes Sel. 2016 Oct;29(10):457-466.doi:10.1093/protein/gzw040.Epub 2016 Aug 29.PubMed PMID: 27578889).
  • the Fc of these antibodies introduced the "LALA” double mutant (L234A/L235A) or the “LALAPG” triple mutant (L234A) /L235A/P329G).
  • the heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic hypermutation of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline genes V and J gene segments of the light chain gene group. Gene rearrangements and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene segment may also yield different sequences, but overall the similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • PTMs post-translational modifications
  • some PTM sites are very conserved, for example, the conserved amino acid Asparagine Asn at position 297 (EU numbering) in the constant domain of human IgG1 antibodies is usually glycosylated to form sugar chains, which are critical for antibody structure and related effector functions.
  • EU numbering conserved amino acid Asparagine Asn at position 297
  • the presence of these PTMs may have a greater impact on antigen binding, and may also affect the physicochemical properties of antibodies. to change.
  • glycosylation, deamidation, isomerization, oxidation, etc. may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development.
  • PTMs are very important for the development of therapeutic antibodies.
  • some PTMs are highly correlated with the "pattern" of amino acid sequence composition, especially the composition of adjacent amino acids, so that potential PTMs can be predicted from the protein's primary amino acid sequence.
  • the sequence pattern of N-x-S/T (asparagine in the first position, any amino acid other than proline in the second position, and serine or threonine in the third position) predicts N-linked glycosylation sites.
  • the amino acid sequence pattern that causes PTM may be derived from germline gene sequences, such as the human germline gene fragment IGHV3-33, which naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic hypermutation.
  • NGS or NLT may be the glycosylation site
  • NS may be the deamidation site
  • DG may cause isomerization of aspartic acid.
  • the formation of a particular PTM can be reduced or eliminated by disrupting the amino acid sequence pattern of a PTM by amino acid mutation.
  • mutation design there are different approaches to mutation design.
  • One approach is to replace "hot spot" amino acids (eg, N or S in the NS pattern) with amino acids of similar physicochemical properties (eg, mutating N to Q). If the PTM sequence pattern is derived from somatic hypermutation and is not present in the germline gene sequence, another approach could be to replace the sequence pattern with the corresponding germline gene sequence.
  • multiple mutational design methods may be used for the same PTM sequence pattern.
  • This example describes a general method for antibody preparation using mammalian host cells (eg, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives), transient transfection expression, and affinity capture isolation.
  • mammalian host cells eg, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives
  • transient transfection expression e.g., transient transfection expression
  • affinity capture isolation e.g., affinity capture isolation.
  • This method is suitable for target antibody containing Fc region; the target antibody can be composed of one or more protein polypeptide chains; it can be derived from one or more expression plasmids.
  • the amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence by a codon optimization method; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with host cells.
  • the plasmid encoding the antibody polypeptide chain is simultaneously transfected into mammalian host cells according to a specific ratio, and the recombinant antibody with correct folding and polypeptide chain assembly can be obtained by using conventional recombinant protein expression and purification techniques. Specifically, FreeStyle TM 293-F cells (Thermo, #R79007) were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504).
  • the cell concentration was adjusted to 6-8 x 10 5 cells/ml and cultured at 37°C in an 8% CO 2 shaker for 24 hours at a cell concentration of 1.2 x 10 6 cells/ml.
  • a total of 30 ⁇ g of plasmids encoding antibody polypeptide chains were mixed in a certain ratio (the ratio of plasmid to cells was 1 ⁇ g: 1 ml), dissolved in 1.5 ml of Opti-MEM reduced serum medium (Thermo, #31985088), and filtered with a 0.22 ⁇ m filter. bacteria.
  • Opti-MEM 1.5 ml of Opti-MEM was dissolved in 120 ⁇ l of 1 mg/ml PEI (Polysciences, #23966-2), and it was allowed to stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop the plasmid PEI mixed solution while shaking the culture flask, and culture at 37°C in an 8% CO 2 shaker for 5 days. Cell viability was observed after 5 days. The culture was collected, centrifuged at 3300g for 10 minutes, and the supernatant was taken; then the supernatant was centrifuged at high speed to remove impurities.
  • PEI Polysciences, #23966-2
  • This example uses analytical size exclusion chromatography (SEC) to analyze protein samples for purity and aggregate form.
  • An analytical column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8 mm ⁇ 30 cm) was connected to a high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer for at least 1 hour at room temperature.
  • HPLC Analogent Technologies, Agilent 1260 Infinity II
  • An appropriate amount of protein sample (at least 10 ⁇ g) was filtered with a 0.22 ⁇ m filter and injected into the system, and the HPLC program was set: the sample was flowed through the column with PBS buffer at a flow rate of 1.0 ml/min for a maximum time of 25 minutes.
  • the HPLC will generate an analytical report reporting the retention times of components of different molecular sizes within the sample.
  • DSF Differential Scanning Fluorimetry
  • the spleen cells of the mouse are taken out.
  • the monoclonal antibody obtained by screening is further identified, according to its binding ability to human B7-H4, cynomolgus monkey B7-H4 binding ability, etc. parameters, 2 clones 80C8-2E9 and 1025_B-1H11 were selected.
  • the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence are obtained by conventional sequencing methods.
  • the candidate antibody molecules are then subjected to sequence analysis and optimization, resulting in several variant sequences.
  • the VL and VH sequences of the antibody are fused and expressed with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules.
  • This example is to study the activity of the anti-human B7-H4 H2L2 monoclonal antibody in extra-binding human/cynomolgus/mouse B7-H4.
  • CHO-K1 cell line overexpressing cynomolgus monkey B7-H4 (CHO-K1/cyno B7-H4, and platinum self-made)
  • CHO-K1 cell line overexpressing mouse B7-H4 (CHO-K1/m B7 -H4, and platinum self-made)
  • high expression of human B7-H4 cell line SK-BR-3 ( HTB-30) for antibody binding experiments at the cellular level.
  • CHO-K1/cyno B7-H4 cells CHO-K1/m B7-H4 cells or SK-BR-3 cells were digested and resuspended in PBS containing 2% BSA. The cell density was adjusted to 1 ⁇ 10 6 cells/mL, respectively. 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by adding 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration of the antibody to be tested. The cells were placed at 4°C and incubated in the dark for 2 hours.
  • the Harbour HCAb mouse (Harbour Antibodies BV, WO2010/109165A2) is a transgenic mouse carrying a human immunoglobulin immune repertoire that produces heavy chain-only antibodies that are half the size of traditional IgG antibodies. It produces antibodies with only human antibody heavy chain variable domains and mouse Fc constant domains.
  • the human VH gene was amplified from plasma cells by conventional molecular biology methods, and the amplified human VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the Fc region sequence of the heavy chain of human IgG1 antibody.
  • the plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the supernatant of fully human HCAb antibody is obtained.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the binding of HCAb antibody supernatant to CHO-K1 cell CHO-K1/hu4-1BB highly expressing human 4-1BB was tested by FACS, and positive HCAb antibody was identified.
  • HCAb antibodies were further identified, and several candidate HCAb antibody molecules were selected according to their binding ability to human 4-1BB, cynomolgus monkey 4-1BB binding ability, T cell activation ability and other parameters.
  • the candidate HCAb antibody molecules were then sequenced and optimized, resulting in several variant sequences.
  • the VH sequence of the HCAb antibody and the human IgG1 heavy chain Fc sequence are fused and expressed to obtain a fully human recombinant HCAb antibody molecule.
  • PR004469 is a PTM variant of PR001838, and PR007381 is a germline variant of PR004469.
  • the specific mutation sites are shown in Table 5.
  • variable region mutation Recombinant antibody isotype PR001838 PR004469 G53A human IgG1 PR001838 PR007381 F37V,P40A,E42G,T43K,K46E,G53A human IgG1
  • Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune repertoire, which produces antibodies with intact human antibody variable domains and rat constant domains.
  • Harbour H2L2 mice were immunized with multiple rounds of soluble recombinant human 4-1BB-Fc fusion protein.
  • the titer of 4-1BB-specific antibody in the mouse serum reached a certain level, the spleen cells of the mouse were taken out and fused with the myeloma cell line to obtain hybridoma cells; after multiple rounds of screening and cloning of the hybridoma cells , and isolated the hybridoma cell line expressing anti-4-1BB monoclonal antibody molecule.
  • the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence are obtained by conventional hybridoma sequencing methods.
  • the candidate antibody molecules are then subjected to sequence analysis and optimization, resulting in several variant sequences.
  • the VL and VH sequences of the antibody are fused and expressed with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules.
  • This example is to study the activity of anti-human 4-1BB HCAb and H2L2 monoclonal antibody in extra-binding human and cynomolgus monkey 4-1BB.
  • CHO-K1 cell line overexpressing human 4-1BB (CHO-K1-hu 4-1BB, Genescript)
  • CHO-K1 cell line overexpressing cynomolgus monkey 4-1BB (CHO-K1-cyno 4-1BB, Genescript) to perform antibody binding experiments at the cellular level.
  • cells CHO-K1-hu 4-1BB and CHO-K1-cyno 4-1BB cells were digested and resuspended in DMEM complete medium to adjust the cell density to 1 ⁇ 10 6 cells/mL, respectively.
  • 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #: 3894), and then 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration of the antibody to be tested, was added. The cells were placed at 4°C and incubated in the dark for 1 hour. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g and 4° C. for 5 minutes, and the supernatant was discarded.
  • the anti-4-1BB antibodies of the present invention can all bind to human 4-1BB, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration.
  • These antibodies bind human 4-1BB more sensitively at lower concentrations than the reference antibodies (Urelumab and Utomilumab), with EC50s comparable to the reference antibodies Utomilumab and Urelumab.
  • all the anti-4-1BB antibodies of the present invention can bind to monkey 4-1BB, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration.
  • These antibodies bind monkey 4-1BB more sensitively at lower concentrations than the reference antibody Tab (Utomilumab), with EC50s comparable to or better than the reference antibody Utomilumab.
  • the reference antibody Urelumab did not have cross-binding activity with monkey 4-1BB.
  • Antigen-binding proteins can activate the 4-1BB pathway in vitro
  • CHO-K1 cells (CHO-K1/CD32b, Genscript, #M00600) overexpressing human CD32b were treated with 10 ⁇ g/ml of mitomycin (Beijing Zhongsheng Ruitai Technology, #10107409001) for 30 minutes at 37°C. It was then washed 4 times with F-12K medium containing 10% FBS. The treated cells were placed in a 96-well plate, 1.5 ⁇ 10 4 per well, and cultured overnight in a 37°C incubator. The next day, human CD3 positive T cells were isolated from human PBMCs using the MACS kit (Miltenyi Biotec, #130-096-535).
  • 4-1BB antibody has the function of activating 4-1BB pathway and inducing activation of T cells.
  • PR001758, PR001760, PR001764, PR001771, PR001774, PR001780, PR001781, PR001830, PR001833, PR001836, PR001838, PR001840, and PR000448 all had stronger activation than utomilumab, and showed higher IFN- ⁇ signal than utomilumab.
  • the combination of the anti-B7-H4 antibody selected from Example 2 and the anti-4-1BB antibody selected from Example 3 is used to prepare a bispecific antibody that can simultaneously bind to two targets, one of which can recognize tumor cell surface specificity Expressed B7-H4, while the other end can bind to 4-1BB molecules on T cells.
  • B7-H4 ⁇ 4-1BB double antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing the tumor cells.
  • the B7-H4 ⁇ 4-1BB bispecific antibody prepared in this example includes a variety of molecular structures:
  • Figures 7-(A)-(I) are schematic diagrams of the structures of the bispecific molecules of the present invention.
  • the bispecific antibody of IgG-scFv tetravalent symmetrical structure was constructed by using anti-B7-H4 H2L2 antibody and anti-4-1BB H2L2 antibody.
  • the binding protein of IgG-scFv tetravalent symmetrical structure (as shown in Figure 5-(A)) contains two polypeptide chains: polypeptide chain 1, also known as short chain, from the amino terminus to the carboxyl terminus, which contains VL_A-CL; polypeptide Chain 2, also called the long chain, from the amino terminus to the carboxy terminus, comprises VH_A-CH1-h-CH2-CH3-L1-VH_B L2-VL_B.
  • h is the hinge region or derived sequence of an IgG antibody.
  • the linker peptide L1 and linker peptide L2 of the polypeptide chain 2 may be the sequences listed in Table 8.
  • the bispecific antibody of IgG-VH tetravalent symmetrical structure was constructed by using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody.
  • the binding protein of IgG-VH tetravalent symmetrical structure (as shown in Figure 5-(B)) contains two polypeptide chains: polypeptide chain 1, also known as short chain, from the amino terminus to the carboxyl terminus, which contains VL_A-CL; Chain 2, also called the long chain, from the amino terminus to the carboxy terminus, comprises VH_A-CH1-h-CH2-CH3-L-VH_B.
  • h is the hinge region or derived sequence of an IgG antibody.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, i.e. the length of L is 0.
  • CH3 of polypeptide chain 2 is linked to VH_B via a linker peptide L; L may be the sequence listed in Table 8.
  • the bispecific antibody of IgG-VH-VH hexavalent symmetrical structure was constructed by anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody.
  • the binding protein of IgG-VH-VH hexavalent symmetrical structure (as shown in Figure 5-(C)) contains two polypeptide chains: polypeptide chain 1, also called short chain, from the amino terminus to the carboxyl terminus, which contains VL_A-CL ; Polypeptide chain 2, also known as the long chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B.
  • h is the hinge region or derived sequence of an IgG antibody.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L is 0.
  • the linking peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 8.
  • Fab-HCAb symmetrical structure bispecific antibody including two different structures Fab(CL)-VH-Fc (as shown in Figure 5-(D) shown) Fab(CH)-VH-Fc (as shown in Figure 5-(E)).
  • the binding protein of Fab(CL)-VH-Fc contains two polypeptide chains, polypeptide chain 1, also known as short chain, from the amino terminus to the carboxyl terminus, which contains VH_A-CH1; polypeptide chain 2, also known as long chain, from the amino terminus.
  • Linking peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 8.
  • the binding protein of Fab(CH)-VH-Fc contains two polypeptide chains, polypeptide chain 1, also known as short chain, from the amino terminus to the carboxyl terminus, which contains VL_A-CL; End to carboxyl terminus, it contains VH_A-CH1-L1-VH_B-L2-CH2-CH3.
  • Linking peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 8.
  • a Fab-Fc-VH(n) asymmetric bispecific antibody was constructed using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody.
  • the binding protein of Fab-Fc-VH(n) asymmetric structure contains three polypeptide chains, polypeptide chain 1, also known as short chain, from the amino terminus to the carboxyl group End, which contains VL_A-CL; Polypeptide chain 2, also called long chain, from amino terminus to carboxy terminus, which contains VH_A-CH1-h-CH2-CH3; Polypeptide chain 3, from amino terminus to carboxy terminus, which contains VH_B -h-CH2-CH3, or polypeptide chain 3, from amino terminus to carboxy terminus, which contains VH_B-L-VH_B-h-CH2-CH3, or polypeptide chain 3, from amino terminus to carboxy terminus, which contains VH_B-L1 -VH_B-L2-
  • mutated heterodimers were used Fc regions, which carry "knob-hole” mutated and engineered disulfide bridges, as described in WO2009080251 and WO2009080252.
  • a bispecific antibody with Fab-VH-Fc-VH symmetrical structure was constructed using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody.
  • the binding protein of the Fab-VH-Fc-VH symmetrical structure (as shown in Figure 5-(I)) contains two polypeptide chains, polypeptide chain 1, also called short chain, from the amino terminus to the carboxyl terminus, which contains VL_A-CL ; Polypeptide chain 2, also called the long chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1-L1-VH_B-CH1-h-CH2-CH3-VHB.
  • h is the hinge region or derived sequence of an IgG antibody.
  • Linking peptides L1 and L2 of polypeptide chain 3 may be the sequences listed in Table 8.
  • PR002408 (H:gm) involved in PR007379 in Table 11-(B) is the same as the CDR of PR002408, and there are 2 mutations in FR2 of the variable region VH, namely T69I and E101G.
  • amino acid sequence of the polypeptide chain of the diabody molecule obtained in the present invention is shown in the sequence listing as follows.
  • PR003489 200 225 PR004158 201 226 PR004358 201 239 PR004160 201 227 228 PR004161 201 227 229 PR004181 201 227 231 PR004182 201 227 232 PR004279 233 234 PR004995 201 262 PR005183 200 263 PR005184 200 264 PR005185 200 265 PR005186 200 266 PR005187 200 267 PR005188 200 268 PR005189 233 269 PR005190 233 270 PR005827 200 274 PR005828 200 275 PR005829 200 276 PR005830 200 277 PR005649 233 271 PR005650 233 272 PR005651 233 273 PR005838 200 278 PR005839 200 279 PR005866 200 280 PR007165 200 281 PR007379 200 286 PR007380 200 287
  • the CDR sequence numbers of the antigenic domains of the B7-H4 ⁇ 4-1BB bispecific antibody are shown in the table below.
  • the sequence number 1# is the antigenic domain that binds to B7-H4
  • the sequence number 2# is the antigenic domain that binds to 4-1BB. antigenic domain.
  • Table 11-(A)-(F) is the molecular structure information of the bispecific antibody of the present invention.
  • Table 12-(A)-(F) is the protein expression and physicochemical properties of the bispecific antibodies of the present invention.
  • Example 5 FACS detection of in vitro binding of B7-H4 ⁇ 4-1BB bispecific antibody on CHO-K1 cell line overexpressing human and cynomolgus monkey 4-1BB
  • CHO-K1 cell line overexpressing human 4-1BB (CHO-K1/hu 4-1BB, Genscript, #M00538) and CHO-K1 cell line overexpressing cynomolgus 4-1BB (CHO-K1/cyno 4 -1BB, Genscript, #M00569) for antibody binding experiments at the cellular level. Briefly, cells CHO-K1/hu 4-1BB and CHO-K1/cyno 4-1BB cells were digested and resuspended in F12K complete medium and washed once with PBS.
  • the cell density was adjusted to 1 ⁇ 10 6 cells/ml with PBS, respectively.
  • 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by adding 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration of the antibody to be tested.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g and 4° C. for 5 minutes, and the supernatant was discarded.
  • the cells were washed twice with 100 ⁇ L/well of pre-cooled PBS, centrifuged at 500 g and 4° C. for 5 minutes, and the supernatant was discarded. Finally, 200 ⁇ L/well of pre-cooled PBS was used to resuspend the cells, and the fluorescence signal value was read by BD FACS CANTOII or ACEA NovoCyte series flow cytometer.
  • the B7-H4 ⁇ 4-1BB bispecific antibodies in this example all specifically bound to CHO-K1 cells overexpressing human 4-1BB.
  • the specific performance is that the Span value of the binding curve of antibody PR003335 is about 1.5 times that of Urelumab, and the maximum MFI value is higher than that of Urelumab.
  • the B7-H4 ⁇ 4-1BB bispecific antibodies in this example all specifically bound to CHO-K1 cells overexpressing monkey 4-1BB.
  • the reference antibody Urelumab did not have cross-binding activity with monkey 4-1BB.
  • This example is to investigate the human B7-H4 binding activity of the B7-H4 arm of the B7-H4 ⁇ 4-1BB bispecific antibody.
  • the SK-BR-3 cell line with high expression of B7-H4 was used to conduct the horizontal binding experiment with human B7-H4 cells. Briefly, SK-BR-3 cell suspensions were collected and the cell density was adjusted to 2 x 106 cells/mL, respectively. 50 ⁇ L cells/well were seeded in 96-well V-bottom plate (Corning, #: 3894), and then 50 ⁇ L/well, 2 times the final concentration of 3 times the concentration of the antibody to be tested, was added. The cells were placed at 4°C and incubated in the dark for 2 hours.
  • the B7-H4 ⁇ 4-1BB bispecific antibodies in this example all specifically bind to human B7-H4 on SK-BR-3 cell line, and the binding curve EC50 Usually 0.1-10 nM.
  • Example 7 T cell activation experiment and cytokine release by B7-H4 ⁇ 4-1BB bispecific antibody in the presence of B7-H4 highly expressing cell line SK-BR-3
  • a 96-well flat bottom plate (Corning, #3599) was coated with OKT3, the density of pan T was adjusted to 2 ⁇ 10 6 cells/mL, and the density of SK-BR-3 was adjusted to 2 ⁇ 10 5 cells/mL , inoculate 50 ⁇ L cells/well of the two cell suspensions in 96-well flat bottom plates (Corning, #3599), and then add 50 ⁇ L/well, 3 times the final concentration of different gradient dilutions of the antibody to be tested, of which the antibody has the highest final concentration It is 100nM or 30nM or 20nM or 6nM, each antibody has a total of 6 or 3 or 2 concentrations, and the final effect-to-target ratio is 10:1, and two replicates are set.
  • an isotype IgG control group was set in the plate.
  • the 96-well plate was incubated in a 37°C carbon dioxide incubator for 2 or 3 days. After incubation, the supernatant was taken, added to a 96-well plate (Corning, #3599), and centrifuged at 500 g and 4°C for 5 minutes. The supernatant incubated for 2 days was used to detect the release of cytokine IL-2 or the supernatant incubated for 3 days was used to detect the release of IFN-gamma.
  • ELISA detection method refers to IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit and IFN gamma (IFN gamma Human Uncoated ELISA Kit, Thermo, #88-7316-88) operating instructions.
  • IL-2 IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88
  • IFN gamma IFN gamma Human Uncoated ELISA Kit, Thermo, #88-7316-88
  • FIG. 10 B7-H4 ⁇ 4-1BB bispecific antibody activates the 4-1BB pathway in the presence of the B7-H4 high expressing cell line SK-BR-3 and induces the release of IL from activated T cells -2.
  • PR002790, PR002791, PR002802, PR002804, PR002806, PR003334, PR003335, PR003336, PR003337, PR003338, PR003487, PR003488, PR003489, PR004158, PR004160, PR004161, PR004162, PR004181, PR004182, PR004279, PR004280, PR004281, PR004282, PR004357, PR004358 , PR004359 is better than or equivalent to Urelumab in activation of 4-1BB pathway. Specifically, the production of cytokines IFN- ⁇ or IL-2 was higher than that of Urelumab.
  • PR004281 and PR004282 outperformed PR003338 and Urelumab (Fig. 10-K).
  • the B7H4 arm with different epitopes can affect the activation of T-cells by diabodies, such as PR004281 and PR004282 outperforming PR004995 and Urelumab (Fig. 10-L).
  • Example 8 Activation of T cells by B7-H4 ⁇ 4-1BB bispecific antibody is independent of Fc ⁇ R cells
  • the ELISA detection method refers to the operation instructions of the IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88).
  • B7-H4 ⁇ 4-1BB bispecific antibody PR004282-mediated T cell activation was comparable to isotype IgG controls in the presence of CHO-K1/CD32b cells, a cell line highly expressing Fc ⁇ RIIb.
  • B7-H4 ⁇ 4-1BB bispecific antibody activity is dependent on B7-H4 expression.
  • Human pan T cells were used as effector cells, cell lines SK-BR-3 and MDA-MB-468 cells with high B7-H4 expression and COV644 and JIMT-1 and MDA-MB-231 cells without B7-H4 expression were used as mediators
  • the cross-linked cells were subjected to in vitro activation experiments and cytokine release assays. Specifically, a 96-well flat bottom plate (Corning, #3599) was coated with OKT3, the density of pan T was adjusted to 2 ⁇ 10 6 cells/mL, and the density of tumor cells was adjusted to 2 ⁇ 10 5 cells/mL.
  • cytokine IL-2 ELISA detection method refers to IL -2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit operating instructions.
  • Figure 12 FACS detection of B7-H4 expression on the surface of tumor cells.
  • Figure 12-(A) The B7-H4 expression on the surface of tumor cells was detected with B7-H4 positive control antibody 1.
  • Figure 12-(B) B7-H4 expression on the surface of tumor cells was detected by B7-H4 ⁇ 4-1BB bispecific antibody.
  • SK-BR-3 and MDA-MB-468 are tumor cells that highly express B7-H4, and JIMT-1 and COV644 and MDA-MB-231 are tumor cells that do not express B7-H4.
  • B7-H4 ⁇ 4-1BB double antibody specifically depends on the expression of B7-H4 for T cell activation.
  • A B7-H4 ⁇ 4-1BB bispecific antibody PR003334-mediated T cell activation was superior to B7-H4 ⁇ 4-1BB bispecific antibody PR003334 in the presence of high B7-H4 expressing cell lines SK-BR-3 or MDA-MB-468 cells positive control.
  • C B7-H4 expressing cell lines SK-BR-3 or MDA-MB-468 cells positive control.
  • C In the presence of cell lines COV644 or JIMT-1 cells that do not express B7-H4, PR003334-mediated T cell activation was comparable to isotype IgG controls.
  • PR0004282 was serially diluted with 95% human serum, divided into 5 tubes, incubated at 37°C for 0 days, 1 day, 2 days, 4 days, 7 days, and 14 days, and then quickly frozen in liquid nitrogen and stored at -80°C.
  • the in vitro binding of PR004282 to B7H4+ cells and 4-1BB+ cells was detected by FACS after being placed in high concentration human serum at 37 degrees for different times. The process is as follows:
  • Digest CHO-K1/h4-1BB, CHO-K1/cyno4-1BB, CHO-K1/hB7H4 and CHO-K1/cynoB7H4 cells resuspend in DMEM complete medium, wash with PBS and adjust the cell density to 1 ⁇ 10 6 cells/mL.
  • 100 ⁇ L cells/well were seeded in 96-well V-bottom plate (Corning, #3894), and then 100 ⁇ L/well, 2 times the final concentration and 3 times the final concentration of the human serum to be tested were treated with the PR004282 antibody (antibodies with different treatment times). contains 4.5% human serum in each dose).
  • the cells were placed at 4°C and incubated in the dark for 2 hours.
  • the binding of PR003334 and PR003335 to SK-BR-3 cells with high expression of B7-H4 and CHO-K1-human 4-1BB cells in 90% serum did not change at different incubation times with human serum, indicating that PR003334 and PR003335 have strong Good serum stability.
  • FIG. 14-(E-H) Serum stability of B7-H4x4-1BB dual anti-PR004282. At different serum incubation times, the binding of PR004282 to CHO-K1 cells highly expressing B7H4 or 4-1BB in 95% serum did not change, indicating that PR004282 has good serum stability.
  • This example is to study the in vitro binding activity of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 to human/cynomolgus/mouse 4-1BB.
  • CHO-K1 cell line overexpressing human 4-1BB (CHO-K1/h4-1BB, Gensript, #M00538)
  • CHO-K1 cell line of cynomolgus monkey 4-1BB (CHO-K1/cyno4-1BB, Genscript , #M00569)
  • the CHO-K1 cell line (CHO-K1/m4-1BB, Genscript, #M00568) overexpressing mouse 4-1BB was subjected to antibody binding experiments at the cellular level.
  • CHO-K1/h4-1BB cells, CHO-K1/cyno 4-1BB cells and CHO-K1/m 4-1BB cells were digested and resuspended in PBS containing 2% FBS. The cell density was adjusted to 1 ⁇ 10 6 cells/mL, respectively. 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by adding 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration of the antibody to be tested. The cells were placed at 4°C and incubated in the dark for 2 hours.
  • This example is to study the in vitro binding activity of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 to human/cynomolgus/mouse B7-H4.
  • the CHO-K1 cell line (CHO-K1/hB7-H4, made by Heplatin) overexpressing human B7H4
  • the CHO-K1 cell line (CHO-K1/cynoB7-H4, made by Heplatin) of cynomolgus monkey B7-H4
  • CHO-K1 cell line (CHO-K1/mB7-H4, made with platinum) overexpressing mouse B7-H4 was used for antibody binding experiments at the cellular level.
  • CHO-K1/hB7H4 cells, CHO-K1/cyno B7-H4 cells and CHO-K1/mB7-H4 cells were digested and resuspended in PBS containing 2% FBS. The cell density was adjusted to 1 ⁇ 10 6 cells/mL, respectively. 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by adding 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration of the antibody to be tested. The cells were placed at 4°C and incubated in the dark for 2 hours.
  • Example 13 FACS detection of the ability of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 to simultaneously bind to CHO-K1/h4-1BB and SK-BR-3
  • This example is to investigate the ability of the B7-H4 ⁇ 4-1BB bispecific antibody PR004282 to simultaneously bind CHO-K1/h4-1BB and SK-BR-3. Briefly, digestion of CHO-K1 / h4-1BB cells and SK-BR-3 cells with PBS and were adjusted to a cell density of 1 ⁇ 10 6 cells / mL, with 0.5 ⁇ M Far-red (lifetechnologies, # C34572) of SK-BR-3, CHO-K1/h4-1BB cells were stained with 0.5 ⁇ M CFSE (lifetechnologies, #C34544) for 5 min at room temperature. Stained cells were centrifuged and washed once with >20 ml of medium containing 1% FBS.
  • the washed cells were resuspended in FACS buffer, and the cell density was adjusted to 2*10 6 /ml.
  • 50 ⁇ l SK-BR-3 cells (1*10 5 cells per well) and 25 ⁇ l CHO-K1/h4-1BB (1 cell per well) were added to each well of a 96-well V-plate (Corning, #3894). *10 5 ) and 25ul of the antibody to be tested diluted in 4-fold gradient FACS buffer, mix thoroughly. Incubate for 1 hour at 4°C.
  • Figure 17 shows that only PR004282 has the ability to bind both CHO-K1/h4-1BB and SK-BR-3. Monoclonal antibodies do not have this ability.
  • Example 14 FACS detection of the binding ability of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 at the level of primary cells
  • This example is to study the activity of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 in vitro binding to activated human/cynomolgus monkey primary T cells.
  • Monkey CD3+ T cells were isolated from cryopreserved monkey PBMCs (PharmaLegacy, #SC1702051 ) using the non-human primate CD3 isolation kit (Miltenyi, #130-092-012) in medium RPMI1640+10% FBS+1% Sodium pyruvate (Thermo, #11360-070) + 1% non-essential amino acid solution (Thermo, #11140-050) was resuspended to 2 ⁇ 10 6 /ml, respectively took 1ml into a 6-well plate, and added 1ml containing 20ng/ml ml PMA (Sigma, # P1585-1MG) and 5nM Ionomycin (Sigma, # 407952-5MG) culture medium, 37 °C CO 2 incubator for 16h.
  • Human CD3+ T cells were isolated from cryopreserved human PBMCs (Saily, XFB-HP100B) using a human CD3 isolation kit (Miltenyi, #130-096-535), and other operations were the same as the activation of monkey CD3+ T cells.
  • the activated monkey T cell density was adjusted to 1 ⁇ 10 6 cells/mL with PBS containing 2% FBS, and 100 ⁇ L CD3+ T cells/well were seeded in 96-well V-bottom plates (Corning, #3894), followed by adding 100 ⁇ L/well , 2 times the final concentration of 3 times the concentration of the antibody to be tested PR004282/hIgG1/Urelumab/hIgG4, the cells were placed at 4°C, and incubated in the dark for 2 hours. After that, 100 ⁇ L/well of pre-cooled PBS containing 2% FBS was added to rinse the cells twice, centrifuged at 500 g and 4° C. for 5 minutes, and the supernatant was discarded.
  • the activated human T cells were incubated with Zombie NIR TM dye (Biolegend, #77184) for 15-30 minutes at room temperature in the dark, the cells were washed once with FACS buffer, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL, respectively.
  • 100 ⁇ L CD3+ T cells/well were seeded in 96-well V-bottom plate (Corning, #3894), and then 100 ⁇ L/well, 2 times the final concentration of the 3-fold dilution of the test antibody PR004282/hIgG1 was added, and the cells were placed in Incubate at 4°C for 2 hours in the dark.
  • B7-H4/4-1BB bispecific antibody PR004282 can bind to activated human or monkey CD3+ T cells.
  • Example 15 ELISA detection of cross-reactivity of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 with other members of B7 family or TNFR family
  • the proteins of the B7 family were diluted with PBS to 1 ⁇ g/ml, respectively, added to a 96-well plate (Corning, #9018), 100 ⁇ l per well, and incubated overnight at 4°C. After discarding the liquid, wash the plate three times with PBST buffer (pH 7.4, containing 0.05% tween-20), add 250 ⁇ l of 2% BSA blocking solution, and incubate at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed three times with PBST buffer.
  • the antigen-binding protein to be tested was diluted to 3 concentrations of 100 nM, 10 nM and 1 nM, and 100 ⁇ l was added to each well, and incubated at 37°C for 1 hour. After washing three times with PBST buffer, 5000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added, and incubated at 37°C for 1 hour in the dark.
  • Figure 19-(A) illustrates that the antibody PR004282 of the present invention does not cross-react with other member proteins of the B7 family.
  • the proteins of the TNFR family were diluted with PBS to 1 ⁇ g/ml, added to a 96-well plate (Corning, #9018), 100 ⁇ l per well, and incubated overnight at 4°C. After discarding the liquid, wash the plate three times with PBST buffer (pH 7.4, containing 0.05% tween-20), add 250 ⁇ l of 2% BSA blocking solution, and incubate at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed three times with PBST buffer.
  • PBST buffer pH 7.4, containing 0.05% tween-20
  • the antigen-binding protein to be tested was diluted to 3 concentrations of 100 nM, 10 nM and 1 nM, and 100 ⁇ l was added to each well, and incubated at 37°C for 1 hour.
  • the isotype antibody was used as a control. After washing three times with PBST buffer, 5000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added, and incubated at 37°C for 1 hour in the dark.
  • Figure 19-(B) illustrates that the antibody PR004282 of the present invention does not cross-react with other member proteins of the TNFR family.
  • CHO-K1/h4-1BB or SK-BR-3 were centrifuged at 300g for 5 minutes and then resuspended in RPMI1640+4% FBS serum medium. The density of cells was adjusted to 6 ⁇ 10 5 cells/ml, 50 ⁇ l of the cell suspension was added to each well of a 96-well plate, and the cells were incubated at 37° C. overnight.
  • Jurkat Fc ⁇ RIIIa-V158/NFAT-Luc cells were centrifuged at 400g for 4 minutes and then resuspended in RPMI1640+4% FBS serum medium. The density of cells was adjusted to 3 ⁇ 10 6 cells/ml, and 50 ⁇ l of the cell suspension was added to each well of a 96-well plate.
  • Antibodies were diluted with RPMI1640+4% FBS medium, the highest final concentration was 100 nM, each antibody had 4 concentrations in total, 5-fold dilution, and two replicates were set up, and 50 ⁇ l of antibody dilution was added to each well of a 96-well plate. At the same time, an isotype IgG control group and a blank medium control group were set in the plate.
  • PR004469 and PR003369 (B7H4 mAbs of affinity matured variants, with ADCC-enhancing effect, heavy chain sequence shown in SEQ ID NO: 290, light chain sequence shown in SEQ ID NO: 291) were used as positive controls, human Iso IgG1 ( CrownBio, #C0001-4) antibody served as a negative control.
  • Figures 20-(A)-(B) show that PR004282 has no significant ADCC activity on CHO-K1/h4-1BB and SK-BR-3 cells. However, the positive controls PR004469 and PR003369 had strong ADCC effects on CHO-K1/4-1BB or SK-BR-3 in a dose-dependent manner, respectively.
  • NK was used as effector cells
  • the cell line SK-BR-3 with high expression of B7H4 was used as target cells.
  • the 96-well E-plate was first equilibrated with 50 ⁇ l of complete medium. Digestion of SK-BR-3 cells were resuspended in complete RPM1640 medium containing 10% fetal bovine serum, diluted to 4 * 10 5 / ml, were plated 50 ⁇ l / hole in plate E-plate 96, i.e. 2 * 104 /well, incubate overnight at 37°C.
  • NK was sorted by EasySepTM Human CD56 Positive Selection Kit (Stem cell, #17855), and 50 ⁇ l of fresh culture medium containing 1*10 5 PBMCs was added to each well, followed by 50 ⁇ l of 4 ⁇ concentration gradient diluted antibodies.
  • the highest final concentration of antibody was 100 nM, and each antibody had a total of 4 concentrations, 5-fold dilution, and two replicates were set.
  • PR003369 was used as a positive control
  • human Iso IgG1 (CrownBio, C0001-4) antibody was used as a negative control.
  • the assay was performed using an Octet Red 96e Molecular Interaction Analyzer, and the assay buffer was kinetic buffer (ForteBio, #18-1105) diluted to 1x.
  • Example 18 Detection of non-specific cytokine release by B7-H4 ⁇ 4-1BB bispecific antibody
  • Monocytes were isolated using a human CD14 isolation kit (Meltenyi, #130-050-201) and resuspended in RPM1640 complete medium containing 10% fetal bovine serum, or PBMCs from the same donor, diluted to 2E6/ml , plate 100 ⁇ l/well in a flat-bottom 96 plate (Costar, #3599), that is, 2E5/well, then add 100 ⁇ l of 2x final concentration of the serially diluted antibody, the highest concentration of the antibody is 300nM, 10-fold dilution, a total of three concentrations, set Two replicates were incubated overnight at 37°C.
  • Urelumab was used as a positive control, and Utomilumab and human Iso IgG1 (CrownBio, C0001-4) antibody were used as negative controls. After 24h incubation, the supernatant was collected to detect the release of TNF-a (Thermo, 88-7066-88) and IL-6 (Thermo, 88-7346-77), and the supernatant was collected after 72h of incubation to detect IFN-gamma (Thermo, 88-7316- 77) release.
  • TNF-a Thermo, 88-7066-88
  • IL-6 Thermo, 88-7346-77
  • FIG. 21-(A)-(C) Cytokine release after co-incubation of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 with PBMC or monocytes.
  • Urelumab showed strong release of TNF- ⁇ and IL-6 regardless of PBMC or monocytes, while IFN-gamma was only shown in the case of co-incubation with PBMC.
  • PR004282 showed a good safety profile like Utomilumab, showing significant cytokine release whether incubated with PBMC or monocytes.
  • This example tests the pharmacokinetic properties of the fusion protein.
  • the method is as follows, selecting 6 female C57BL/6 mice with a body weight of 18-22 grams, and giving the bispecific antibody drug by intravenous injection at the dose of 5mg/kg PR003334 or 5mg/kg PR003335 or 5mg/kg PR004282; Whole blood was collected from 3 animals before administration and 15 minutes, 24 hours (1 day), 4 days, and 10 days after administration, and the other group of 3 animals was collected before administration and 5 hours after administration, Whole blood was collected on days 2, 7, and 14. Whole blood was allowed to stand for 30 minutes to clot, then centrifuged at 2,000 rpm for 5 minutes at 4°C and isolated serum samples were frozen at -80°C until analysis.
  • ELISA method 1 the overall detection method, captures the fusion protein containing human Fc in mouse serum by goat anti-human Fc polyclonal antibody coated on a 96-well plate, and then adds HRP-labeled goat anti-human Fc secondary antibody ELISA method 2
  • the functional domain detection method captures the bispecific antibody containing human 4-1BB HCAb in mouse serum by coating human 4-1BB protein in a 96-well plate, and then adds HRP labeling
  • the goat anti-human Fc secondary antibody was used for detection.
  • NCA non-compartmental model
  • Figures 22-(A)-(F) and Table 19 show pharmacokinetic data for B7-H4x4-1BB bispecific antibodies PR003334 and PR003335 and PR004282.
  • Figure 22-(A) The results show that under the overall detection method, the half-life of PR003334 in mice is about 8 days.
  • Figure 22-(B) The functional domain detection method shows that the half-life of PR003334 in mice is about 9 days.
  • Figure 22-(C) Under the overall detection method, the half-life of PR003335 in mice is about 14 days.
  • Figure 22-(D) The functional domain detection method shows that the half-life of PR003335 in mice is about 12 days.
  • Figure 22-(E) Under the overall detection method, the half-life of PR004282 in mice is about 11 days.
  • Figure 22-(F) The functional domain detection method shows that the half-life of PR004282 in mice is about 8 days.
  • This example tests the pharmacokinetic process of B7-H4/4-1BB bispecific antibody in cynomolgus monkeys.
  • PR004282 was diluted with 0.9% normal saline and injected intravenously into male cynomolgus monkeys at a dose of 1 mg/kg.
  • the samples were collected before and after administration at 0.5, 1, 2, 4, 8, 12, 24, 48 , 72, 168, 336, 504, 672, 840, 1008 hours of blood samples.
  • the blood samples were left standing at room temperature for 30 minutes, and then centrifuged to obtain serum, and the content of B7-H4/4-1BB bispecific antibody in serum was detected by the established specific ELISA method (overall detection method).
  • the B7-H4/4-1BB bispecific antibody displayed a typical IgG-like pharmacokinetic profile after intravenous administration to cynomolgus monkeys.
  • the half-life of PR004282 in male monkeys is approximately 3 days.
  • Example 21 Evaluation of antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody in BALB/c-hCD137/CT26-B7-H4 mouse model
  • FIG 24 shows the antitumor efficacy of the B7-H4 ⁇ 4-1BB bispecific antibody in the BALB/c-hCD137/CT26-B7-H4 mouse model.
  • the B7-H4 ⁇ 4-1BB bispecific antibody PR003334 had the effect of inhibiting tumor growth in mice at the concentration of 18mpk and 6mpk.
  • the body weight changes of the mice in each group were within the normal range.
  • Example 22 Evaluation of antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody in MDA-MB-468 xenograft mouse model
  • mice 6-8 week old female NCG mice were used, and then 5 ⁇ 10 6 MDA-MB-468 cells were subcutaneously inoculated in each experimental mouse on the day of tumor cell inoculation. Mice were randomized into groups of 6 mice when the mean tumor volume reached 120 mm3. After grouping, human 5 ⁇ 10 6 PBMC cells were inoculated, and on the second day, drugs with specific concentrations diluted in PBS were administered by intraperitoneal injection (ip) twice a week for a total of 6 doses (BIW*3). , with the isotype control IgG as the control group.
  • ip intraperitoneal injection
  • FIG 25 shows the antitumor efficacy of the B7-H4x4-1BB bispecific antibody in the MDA-MB-468 xenograft mouse model.
  • the B7-H4 ⁇ 4-1BB bispecific antibody PR003338 has the effect of inhibiting tumor growth in mice at a concentration of 18mpk, which is better than that of Urelumab at a concentration of 15mpk.
  • the body weight changes of the mice in each group were within the normal range.
  • Example 23 Evaluation of antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody in OVCAR3 xenograft mouse model
  • Figure 26 shows the antitumor efficacy of the B7-H4x4-1BB bispecific antibody PR004282 in the OVCAR3 xenograft mouse model.
  • the B7-H4 ⁇ 4-1BB bispecific antibody PR004282 had the effect of inhibiting tumor growth in mice at a concentration of 18mpk.
  • the body weight changes of the mice in each group were within the normal range.
  • Example 24 Evaluation of antitumor efficacy and memory immune effect of B7-H4 ⁇ 4-1BB bispecific antibody in BALB/c-hCD137/CT26-hB7-H4 mouse model
  • mice 6-8 week old female BALB/c-hCD137 mice were used, and then 5 ⁇ 10 5 CT26-B7-H4 cells were subcutaneously inoculated in each experimental mouse on the day of tumor cell inoculation. Mice were randomized into groups of 6 mice when the mean tumor volume reached 80 mm3. After grouping, the specific concentration of the drug diluted in PBS was administered by intraperitoneal injection (ip), twice a week for a total of 6 doses (BIW*3), with PBS as the blank control group and Urelumab as the positive control Group.
  • ip intraperitoneal injection
  • BiW*3 twice a week for a total of 6 doses
  • Figure 27 shows the antitumor efficacy and memory immune effect of B7-H4 ⁇ 4-1BB bispecific antibody PR004282 in BALB/c-hCD137/CT26-B7-H4 mouse model.
  • the B7-H4 ⁇ 4-1BB bispecific antibody PR004282 has the effect of inhibiting tumor growth in mice at a concentration of 1mpk, which is comparable to that of Urelumab.
  • the body weight changes of the mice in each group were within the normal range.
  • Example 25 Specificity assessment of antitumor efficacy of B7-H4 ⁇ 4-1BB bispecific antibody
  • PR004282 has no effect on inhibiting tumor growth in mice at a concentration of 18mpk, while Urelumab shows a certain effect at a concentration of 15mpk.
  • JIMT-1 is a cell that does not express B7H4, indicating that PR004282 cannot kill Tumor cells expressing B7H4.

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Abstract

提供一种双特异性抗体及其用途。所述的双特异性抗体包含靶向B7-H4的抗原结合结构域和靶向4-1BB的抗原结合结构域。双特异性抗体具有一个或两个或三个与4-1BB结合的位点，同时具有全新的全人B7-H4抗体；所述双特异性抗体通过靶向B7-H4特异性结合肿瘤细胞，降低4-1BB激活引起的毒性；另外，所述双特异性抗体带有人Fc片段，保留了Fc与FcRn的结合作用，具有较长的半衰期。

Description

一种双特异性抗体及其用途
本申请要求申请日为2020/6/30的中国专利申请202010618149.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物制药领域，尤其涉及一种双特异性抗体，特别是靶向B7-H4和4-1BB的双特异性抗体及其用途。
背景技术
B7-H4(VTCN1,B7h.5,B7S1,B7x)是一种隶属于B7/CD28超家族的跨膜蛋白。B7-H4蛋白表达于激活的T细胞和B细胞、单核细胞和树突状细胞，有可能负调控T细胞的免疫应答。
尽管B7-H4 mRNA表达广泛，B7-H4蛋白在大多数正常组织中无表达。但是，B7-H4过表达于乳腺癌、卵巢癌、子宫内膜癌的肿瘤细胞表面。乳腺癌是全球范围第二大的恶性肿瘤，且发病率逐渐上升。女性癌症患者中乳腺癌患者约占四分之一。美国乳腺癌患者2019年新增病例32万，总人数约380万，中国乳腺癌患者每年新增病例约30万，预估2021年患者人数达250万。卵巢癌、子宫内膜癌为女性生殖系统常见的恶性肿瘤。在中国，每年约5万女性确诊为卵巢癌，约2万死于卵巢癌，卵巢癌的死亡率占妇科恶性肿瘤之首。子宫内膜癌每年近20万的新发病例，在导致死亡的妇科恶性肿瘤中排三位。
B7-H4还过表达于非小细胞肺癌和肾癌。肺癌为最常见的癌症之一，其中，非小细胞肺癌占肺癌的近80％。无论在全球还是在中国，肺癌的发病率和致死率都居首位。2018年全球肺癌新增病例约200万，死亡人数176万；中国每年新增病例约78万，死亡人数约63万。肾癌发病率较低，中国肾癌年发病患者约7万。
B7-H4作为这些肿瘤的一个新兴靶点，近年来受到关注。B7-H4抗体可以通过多种机制作用于肿瘤细胞，但是B7-H4抗体的研发方向主要集中在单克隆抗体和ADC上，目前尚无双特异性抗体疗法。
4-1BB(TNFRSF9，CD137)是一种隶属于TNF受体超家族的跨膜蛋白。4-1BB是在多种细胞上表达的共刺激分子，为免疫活性的多功能调节剂。其诱导表达于活化的T细胞、NK细胞和单核细胞、树突状细胞、巨噬细胞、肿瘤相关血管内皮细胞等。其配体为4-1BBL，主要表达在专职APC细胞(如单核巨噬细胞、DC细胞、B细胞)、活化的T细 胞及一些肿瘤细胞上。抗4-1BB激动性抗体可抑制肿瘤，但是4-1BB抗体的研发方向主要集中在单克隆抗体以及同时靶向PD-L1和4-1BB的抗体。目前，尚无处于临床开发阶段的靶向B7-H4和4-1BB的双特异性抗体。
百时美施贵宝(BMS)公司的urelumab(BMS-663513)为抗4-1BB抗体的全人源IgG4抗体，是最早进入临床试验的抗4-1BB抗体。Urelumab最初的临床结果发表于2008年，尽管有在部分患者上观察到令人鼓舞的疗效，但数据显示Urelumab具有肝毒性，且与靶标和剂量有关。严重的是，临床试验中有两位患者因肝毒性死亡，导致Urelumab作为单药的临床试验被终止。而靶向PD-L1和4-1BB的双特异性抗体目前还在临床一期阶段，且在肿瘤细胞上PD-L1的表达与B7-H4的表达重叠很少。因此，急需研发更加安全有效的同时靶向人的B7-H4和4-1BB、并能结合食蟹猴的B7-H4和4-1BB的双特异性抗体。
发明内容
本发明所要解决的技术问题是为克服现有技术中缺乏安全有效的同时靶向人的B7-H4和4-1BB、并能结合食蟹猴的B7-H4和4-1BB的双特异性抗体的缺陷，提供一种双特异性抗体，特别是靶向B7-H4和4-1BB的双特异性抗体及其应用。
为解决上述技术问题，本发明提供以下技术方案。
本发明的第一方面提供一种双特异性抗体，其包括靶向B7-H4的抗原结合结构域和靶向4-1BB的抗原结合结构域。
较佳地，所述的靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:46所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3；序列如SEQ ID NO:23所示的HCDR1、序列如SEQ ID NO:59所示的HCDR2以及序列如SEQ ID NO:98所示的HCDR3；或者，序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:60所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3；优选包含序列如SEQ ID NO:142所示、如SEQ ID NO:159所示或者如SEQ ID NO:160所示的VH；更优选包含序列如SEQ ID NO:174所示、如SEQ ID NO:191所示或者如SEQ ID NO:192所示的重链；
更佳地，所述的靶向B7-H4的抗原结合结构域还含有LCDR1、LCDR2和LCDR3，所述的LCDR1的氨基酸序列如SEQ ID NO.112或者113所示，所述的LCDR2的氨基酸序列如SEQ ID NO.118所示，所述的LCDR3的氨基酸序列如SEQ ID NO.131～133中任一个所示；
进一步更佳地，所述的靶向B7-H4的抗原结合结构域还含有序列如SEQ ID NO:112 所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:131所示的LCDR3；序列如SEQ ID NO:113所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:132所示的LCDR3；或者序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:133所示的LCDR3；优选还含有序列如SEQ ID NO:166～169任一个所示的VL；更优选还含有序列如SEQ ID NO:198～201任一个所示的轻链。
在如上所述的双特异性抗体中：
所述的靶向B7-H4的抗原结合结构域较佳地含有序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:46所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3，序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:131所示的LCDR3；序列如SEQ ID NO:23所示的HCDR1、序列如SEQ ID NO:59所示的HCDR2以及序列如SEQ ID NO:98所示的HCDR3，序列如SEQ ID NO:113所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:132所示的LCDR3；或者，序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:60所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3，序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:133所示的LCDR3。
更佳地，所述的靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:142所示的VH和序列如SEQ ID NO:166所示的VL；序列如SEQ ID NO:159所示的VH和序列如SEQ ID NO:167所示的VL；序列如SEQ ID NO:160所示的VH和序列如SEQ ID NO:168所示的VL；或者序列如SEQ ID NO:159所示的VH和序列如SEQ ID NO:169所示的VL。
进一步更佳地，所述靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:174所示的重链和序列如SEQ ID NO:198所示的轻链；序列如SEQ ID NO:191所示的重链和序列如SEQ ID NO:199所示的轻链；序列如SEQ ID NO:192所示的重链和序列如SEQ ID NO:200所示的轻链；或者，序列如SEQ ID NO:191所示的重链和序列如SEQ ID NO:201所示的轻链。
关于所述靶向B7-H4的抗原结合结构域，其形式优选单个VH、串联VH、ScFv、Fab或者IgG的形式。其中，所述的串联VH较佳地为2个以上VH串联，如2个、3个或者4个。当为IgG形式时，其含有的恒定区较佳地来自含有突变L234A、L235A和P329G的人IgG1或者含有突变L234A和L235A的人IgG1。
关于如上所述的靶向4-1BB的抗原结合结构域，其含有HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3优选如下所述：
所述的HCDR1的序列如SEQ ID NO:19或其变体1、或者SEQ ID NO:14所示，所述的HCDR2的序列如SEQ ID NO:49或其变体2、SEQ ID NO:51或者SEQ ID NO:43所示，所述的HCDR3的序列如SEQ ID NO:86或其变体、SEQ ID NO:96、SEQ ID NO:89或者SEQ ID NO:81所示；其中：
所述的变体1的突变包括T3I、S6N/R以及Y7F中的一个或多个；较佳地，所述变体1的序列优选如序列表中SEQ ID NO:17～18以及SEQ ID NO:20～22中任一个所示；
所述变体2的突变包括S1N/D、G2S/A、S3D/G、G5D/F/S/V以及S6T/N/D中的一个或多个；所述变体2的序列优选如序列表中SEQ ID NO:47～48、SEQ ID NO:50、SEQ ID NO:52～58以及SEQ ID NO:61中的任一个所示；
所述的变体3的突变包括G2R/D/A/K、S3A/T、S4G/N/A/T/H、E5T/V/M/G、T6A、D7G/S、H9Y/S、Y10H、Y11F、N12G/D以及V13I/M/T中的一个或多个；所述变体3的氨基酸序列优选如序列表中SEQ ID NO:85、SEQ ID NO:87～88以及SEQ ID NO:90～95中的任一个所示。
在具体实施方案中，所述的靶向4-1BB的抗原结合结构域含有：序列分别如SEQ ID NO:17、47和85所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、49和87所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:19、50和88所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:20、51和89所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、52和90所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、49和90所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:21、53和91所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:21、54和92所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:19、55和93所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、49和86所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:19、49和94所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:22、56和86所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、57和95所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:19、58和96所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:18、61和95所示的HCDR1、HCDR2和HCDR3；
或者，序列分别如SEQ ID NO:14、43和81所示的HCDR1、HCDR2和HCDR3；
较佳地，所述的靶向4-1BB的抗原结合结构域含有一个或者多个序列如SEQ ID NO:143～157、139、284或者161所示的重链可变区。
更佳地，所述的靶向4-1BB的抗原结合结构域包含序列如SEQ ID NO:175～189、193、285或者171所示的重链。
进一步更佳地，所述的靶向4-1BB的抗原结合结构域还包含序列分别如SEQ ID NO:109、118和128所示的LCDR1、LCDR2和LCDR3；较佳地，包含序列如SEQ ID NO:163所示的轻链可变区；更佳地，包含序列如SEQ ID NO:195所示的轻链。
本发明中如上所述的靶向4-1BB的抗原结合结构域可为本领域中的常规形式，例如：单个VH或者串联VH的形式、HCAb的形式或者ScFv的形式；所述的串联VH较佳地为2个以上VH串联，如2个、3个或者4个。
在本发明一具体实施方案中，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:109、118和128所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:14、43和81所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:109、118和128所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:14、43和81所示的HCDR1、HCDR2和HCDR3；
所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:17、47和85所示的HCDR1、HCDR2和HCDR3；
所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118 和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和87所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、50和88所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:20、51和89所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、52和90所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:21、53和91所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:21、54和92所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、55和93所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和86所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、49和94所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:22、56和86所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、57和95所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:17、47和85所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和86所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、49和94所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如 SEQ ID NO:18、61和95所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、58和96所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、57和95所示的HCDR1、HCDR2和HCDR3；
或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和90所示的HCDR1、HCDR2和HCDR3。
关于本发明所述的双特异性抗体，在本发明的具体实施方案中：
所述靶向B7-H4的抗原结构域为IgG的形式，所述靶向4-1BB的抗原结合结构域为单个VH的形式；
或者，所述靶向B7-H4的抗原结构域为IgG的形式，所述靶向4-1BB的抗原结合结构域为ScFv的形式；
或者，所述靶向B7-H4的抗原结构域为IgG的形式，所述靶向4-1BB的抗原结合结构域为2个或者3个VH串联的形式；
或者，所述靶向B7-H4的抗原结构域为Fab的形式，所述靶向4-1BB的抗原结合结构域为HCAb的形式或者为HCAb的形式和VH的形式(HCAb-VH形式)；
或者，所述靶向B7-H4的抗原结构域为Fab的形式，所述靶向4-1BB的抗原结合结构域为VH的形式，所述VH的形式优选单个VH、2个或者3个VH串联的形式。
更具体地：
所述的双特异性抗体为如下形式：
(1)IgG的C端连接有ScFv，所述ScFv的VH连接于所述C端；较佳地，该形式 的双特异性抗体的多肽链1如式：VL B7-H4-CL所示，多肽链2如式：VH B7-H4-CH1-铰链-CH2-CH3-接头-VH 4-1BB-接头-VL 4-1BB所示；
(2)IgG的C端连接有VH或者串联VH；较佳地，该形式的双特异性抗体的多肽链1如式：VL B7-H4-CL所示，多肽链2如式：VH B7-H4-CH1-铰链-CH2-CH3-接头-(VH 4- 1BB) n-所示；
(3)HCAb的N端连接有Fab；较佳地，该形式的双特异性抗体的多肽链1如式：VH B7-H4-CH1所示，多肽链2如式：VL B7-H4-CL-接头-VH 4-1BB-接头-CH2-CH3所示；
(4)HCAb的N端连接有Fab，所述Fab的C端连接有VH；较佳地，该形式的双特异性抗体的多肽链1如式：VH B7-H4-CH1所示，多肽链2如式：VL B7-H4-CL-接头-VH 4- 1BB-接头-CH2-CH3-VH 4-1BB所示；
或者，(5)Fc的2个CH2的N端分别连接有Fab，以及VH或者串联VH；较佳地，该形式的双特异性抗体含三条多肽链：
多肽链1如式：VL B7-H4-CL所示；
多肽链2如式：VH B7-H4-CH1-铰链-CH2-CH3所示；
多肽链3如式：VH 4-1BB-接头-CH2-CH3或者(VH 4-1BB) n-接头-CH2-CH3所示；
其中，连接不同结构域的所述接头序列相同或者不同。
其中：所述IgG或者HCAb的Fc含有突变，优选含有如下突变之一：
1)为IgG1时，根据EU编号的L234A和L235A，可选地还含有P329G、YTE或者DHS；其中，YTE指的是M252Y/S254T/T256E，DHS指309位L突变为D，311位Q突变为H，434位N突变为S。
2)为IgG1时，根据EU编号的第233～235位的缺失；
3)为IgG1时，根据EU编号的L234A和G237A；
4)为IgG4时，根据EU编号的S228P和FALA；FALA指234位F突变位A，235位L突变为A；
5)为IgG1时，根据EU编号的N297A。
如上所述的铰链和所述的接头可为本领域常规，较佳地：所述的接头选自由SEQ ID NO:241～261、282以及288～289构成的群组；其中：
形式(1)的接头优选含有如SEQ ID NO:245所示的序列；
形式(2)的接头优选含有如SEQ ID NO:243、245～247以及SEQ ID NO:288～289任一所示的序列；
形式(3)的接头优选含有如SEQ ID NO:250所示的序列；
形式(4)的接头优选含有如SEQ ID NO:282所示的序列；
形式(5)的接头优选含有如SEQ ID NO:245所示的序列。
在本发明一较佳实施例中，所述多肽链1的氨基酸序列如SEQ ID NO:198所示，且所述多肽链2的氨基酸序列如SEQ ID NO:202～215中任一个所示；
或者，所述多肽链1的氨基酸序列如SEQ ID NO:201所示，且所述多肽链2的氨基酸序列如SEQ ID NO:217、230、238、240、226、262或者239所示；
或者，所述多肽链1的氨基酸序列如SEQ ID NO:200所示，且所述多肽链2的氨基酸序列如SEQ ID NO:218～225、235～237、263～268、274～281、286以及287中任一个所示；
或者，所述多肽链1的氨基酸序列如SEQ ID NO:201所示，所述多肽链2的氨基酸序列如SEQ ID NO:227所示，且所述多肽链3的氨基酸序列如SEQ ID NO:228、229、231或者232所示；
或者，所述多肽链1的氨基酸序列如SEQ ID NO:233所示，且所述多肽链2的氨基酸序列如SEQ ID NO:234、269～273中的任一个所示。
本发明的第二方面提供一种分离的核酸，其编码如上第一方面所述的双特异性抗体。
本发明的第三方面提供一种包含如第二方面所述的分离的核酸的表达载体。
本发明的第四方面提供一种宿主细胞，其包含如第三方面所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞。
本发明的第五方面提供一种双特异性抗体的制备方法，其包含培养如第四方面所述的宿主细胞，从培养物中获得所述双特异性抗体。
本发明的第六方面提供一种药物组合物，其包含如本发明第一方面所述的双特异性抗体。
本发明的第七方面提供如第一方面所述的双特异性抗体、第六方面所述的药物组合物在制备防治(预防和/或治疗)4-1BB和/或B7-H4相关的疾病的药物中的应用。
其中所述的疾病优选癌症，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤或者结直肠癌。较佳地，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌或胆管癌。更佳地，所述的癌症优选乳腺癌。
本发明的第八方面提供一种嵌合抗原受体，其包含第一方面所述的双特异性抗体。
本发明的第九方面提供一种抗体药物偶联物，其包含细胞毒性剂，以及如第一方面所述的双特异性抗体；优选地，所述细胞毒性剂为MMAF或MMAE。
本发明的第十方面提供一种试剂盒，其包括第一方面所述的双特异性抗体、第八方面所述的嵌合抗原受体、第九方面所述的抗体药物偶联物和/或第六方面所述的药物组合物；
优选地，所述试剂盒还包括(i)施用抗体或其抗原结合片段或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
本发明的第十一方面提供一种套装药盒，其包含药盒A和药盒B，其中：
所述药盒A含有第一方面所述的双特异性抗体、第八方面所述的嵌合抗原受体、第九方面所述的抗体药物偶联物和/或第六方面所述的药物组合物；
所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明的第十二方面涉及一种诊断、治疗和/或预防4-1BB和/或B7-H4介导的疾病或病症的方法，所述方法包括向有需要的患者施用治疗有效量的如第一方面所述的双特异性抗体、第八方面所述的嵌合抗原受体、第九方面所述的抗体药物偶联物和/或第六方面所述的药物组合物，或者使用第十一方面所述的套装药盒治疗有需要的患者。
其中，所述的疾病或病症较佳地为肿瘤，优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤或者结直肠癌；较佳地，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌或胆管癌；更佳地，所述的癌症优选乳腺癌。
本发明的第十三方面涉及一种免疫检测或者测定4-1BB或者B7-H4的方法，其包括使用第一方面所述的双特异性抗体、第八方面所述的嵌合抗原受体、第九方面所述的抗体药物偶联物和/或第六方面所述的药物组合物检测样品；优选地，所述检测为非诊断和/或治疗目的的。
本发明的第十四方面涉及一种联合疗法，其包括分别向有需要的患者施用第一方面所述的双特异性抗体、第八方面所述的嵌合抗原受体、第九方面所述的抗体药物偶联物和/或第六方面所述的药物组合物，和第二治疗剂；所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
需知：本发明中关于“变体1”、“变体2”和“变体3”中的数字没有特殊含义，仅 为区分相同术语。
本发明的B7-H4×4-1BB双特异性抗体是一种特有的B7-H4×4-1BB双特异性抗体，具有一个或两个或三个与4-1BB结合的位点；双特异性抗体的两个蛋白功能区均具有良好的结合食蟹猴的活性。
本发明的积极进步效果在于：
1、本发明的4-1BB抗体是一种全新的仅含“重链”的全人抗体，具有与人4-1BB和食蟹猴4-1BB结合的活性。该4-1BB重链抗体的大小只有传统IgG抗体的一半，由于不含轻链的这一特点，使得该抗体可以用于双特异性抗体，并解决了轻链错配和异源二聚化的问题。
2、本发明的B7-H4抗体是一种全新的全人抗体，具有与人B7-H4和食蟹猴B7-H4结合的活性。
3、本发明的双特异性抗体具有一个或两个或三个与4-1BB结合的位点，优化了4-1BB端的活性。
4、本发明通过靶向B7-H4特异性结合肿瘤细胞，降低4-1BB激活引起的毒性。
5、本发明是一种带有人Fc片段的双特异性抗体结构，保留了Fc与FcRn的结合作用，从而具有较长的半衰期。
附图说明
图1中(A)为FACS检测B7-H4单抗在高表达B7-H4的SK-BR-3细胞系上的体外结合；(B)为FACS检测B7-H4单抗在高表达食蟹猴B7-H4的CHO-K1-食蟹猴B7-H4细胞系上的体外结合；(C)为FACS检测B7-H4单抗在高表达鼠B7-H4的CHO-K1-鼠B7-H4细胞系上的体外结合。
图2-(A)-(I)为FACS检测4-1BB单抗在高表达人4-1BB的CHO-K1-人4-1BB细胞系上的体外结合。
图3-(A)-(H)为FACS检测4-1BB单抗在高表达食蟹猴4-1BB的CHO-K1-食蟹猴4-1BB细胞系上的体外结合。
图4-(A)-(E)为4-1BB单抗激活4-1BB通路并诱导激活T细胞的功能。
图5-(A)-(I)为B7-H4×4-1BB双特异性分子的结构示意图。
图6-(A)-(L)为FACS检测B7-H4×4-1BB双特异性抗体在过表达人4-1BB的CHO-K1细胞株上的体外结合。
图7-(A)-(C)为FACS检测B7-H4×4-1BB双特异性抗体在过表达食蟹猴4-1BB的 CHO-K1细胞株上的体外结合。
图8-(A)-(K)为FACS检测B7-H4×4-1BB双特异性抗体在高表达B7-H4的SK-BR-3细胞系上的体外结合。
图9-(A)-(E)为B7-H4×4-1BB双特异性抗体在B7-H4高表达细胞系SK-BR-3存在时的T细胞激活实验以及细胞因子IFN-γ的释放。
图10-(A)-(N)为B7-H4×4-1BB双特异性抗体在B7-H4高表达细胞系SK-BR-3存在时的T细胞激活实验以及细胞因子IL-2的释放。
图11-(A)-(B)为B7-H4×4-1BB双特异性抗体对T细胞的激活不依赖FcγR的存在。
图12-(A)-(B)为FACS检测肿瘤细胞表面的B7-H4表达量。
图13-(A)-(H)为B7-H4×4-1BB双抗对T细胞激活特异性依赖B7-H4的表达。
图14-(A)-(H)为B7-H4×4-1BB双抗PR003334和PR003335及PR004282的血清稳定性。
图15-(A)-(C)为B7-H4×4-1BB双抗PR004282与人、猴及小鼠的4-1BB的体外结合。
图16-(A)-(C)为B7-H4×4-1BB双抗PR004282与人、猴及小鼠的B7H4的体外结合。
图17为B7-H4×4-1BB双抗PR004282与SK-BR-3及CHO-K1/4-1BB的体外同时结合。
图18-(A)-(B)为B7-H4×4-1BB双抗PR004282与体外激活的人、猴Pan T细胞的体外结合。
图19-(A)-(B)为B7-H4×4-1BB双抗PR004282与B7家族成员或TNFR家族其他成员的交叉反应结果
图20-(A)-(C)为B7-H4×4-1BB双抗PR004282的ADCC效应结果。
图21-(A)-(C)为B7-H4×4-1BB双特异性抗体PR004282的非特异性细胞因子的释放检测结果。
图22-(A)-(F)为B7-H4×4-1BB双特异性抗体PR003334和PR003335及PR004282在野生型小鼠的药代动力学数据。
图23为B7-H4×4-1BB双特异性抗体PR004282在正常猴体内的药代动力学数据(单剂量)。
图24-(A)-(B)为B7-H4×4-1BB双特异性抗体PR003334在BALB/c-hCD137/CT26-B7-H4小鼠模型中的抗肿瘤药效。
图25-(A)-(B)为B7-H4×4-1BB双特异性抗体PR003338在MDA-MB-468异种移植小鼠模型中的抗肿瘤药效。
图26-(A)-(B)为B7-H4×4-1BB双特异性抗体PR004282在OVCAR3异种移植小鼠模型中的抗肿瘤药效。
图27-(A)-(I)为B7-H4×4-1BB双特异性抗体PR004282在BALB/c-hCD137/CT26-B7-H4小鼠模型中的抗肿瘤药效和记忆性免疫效应结果。
图28-(A)-(B)为B7-H4×4-1BB双特异性抗体PR004282的抗肿瘤药效的特异性结果。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
在本申请中，术语“抗体”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗体与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。例如本申请中的“重链抗体”不含VL区，仅含VH区。VH或VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH或VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的实例包括但不限于全长抗体、重链抗体(HCAb)、抗原结合片段(Fab，Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，称为CDR或高变区(HVR)，FR为可变域中更高度保守的部分。天然重链和轻链的可变结构域各自包含四个FR区，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点，恒定区不直接参与抗体与 抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术等。
在本申请中，术语“核酸”是指DNA分子和RNA分子。其可以是单链或双链的，但优选是双链DNA。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。
在本申请中，术语“特异性结合”通常是指抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。
在本申请中，术语“Fab”通常指常规抗体(例如IgG)中与抗原结合的部分，包括抗体的重链可变区VH、轻链可变区VL和重链恒定区结构域CH1以及轻链恒定区CL。在常规抗体中，VH的C端与CH1的N端联结形成重链Fd片段，VL的C端与CL的N端联结形成轻链，CH1的C端又进一步与重链的铰链区和其他恒定区结构域联结形成重链。在一些实施例中，“Fab”也指Fab的变体结构。例如，在某些实施例中，VH的C端与CL的N端联结形成一个多肽链，VL的C端与CH1的N端联结形成另一个多肽链，形成Fab(cross VH/VL)的结构；在某些实施例中，Fab的CH1不与铰链区联结，而是CL的C端与重链的铰链区联结，形成Fab(cross Fd/LC)的结构。
在本申请中，术语“VH”通常指抗体的重链可变区VH结构域，即可以是人或者其他动物的常规抗体(H2L2结构)的重链可变区VH，也可以是骆驼科等动物的重链抗体(HCAb结构)的重链可变区VHH，还可以是利用Harbour HCAb转基因小鼠产生的全人源重链抗体(HCAb结构)的重链可变区VH。
在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)。本申请还可使用包含Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基(表1)。在本发 明中，根据Chothia定义确定各序列。
表1.本申请抗体可用的CDR定义方法(可参见http://bioinf.org.uk/abs/)
CDR区 Kabat定义 Chothia定义 Combined定义
LCDR1 L24--L34 L24--L34 L24--L34
LCDR2 L50--L56 L50--L56 L50--L56
LCDR3 L89--L97 L89--L97 L89--L97
HCDR1 H31--H35 H26--H32 H26--H35
HCDR2 H50--H65 H52--H56 H50--H65
HCDR3 H95--H102 H95--H102 H95--H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。本发明使用Chothia编码规则来定义抗体的CDR。
抗体Fc结构域介导的效应子功能如ADCC和CDC也有非常重要的生物学功能，不同的IgG亚型有着不同的ADCC或CDC功能，例如IgG1和IgG3有较强的ADCC和CDC作用，而IgG2和IgG4的作用相对较弱。另外，通过氨基酸突变或者修饰来改变Fc与Fc受体的结合能力也可以调节Fc原有的效应子功能。例如，IgG1中的“LALA”双突变体(L234A/L235A)能够显著降低与FcγRIIIA(CD16A)的亲和力，进而降低ADCC作用。另外，P329G突变能够显著降低与多种Fcγ受体的结合(参见，Schlothauer T,Herter S,Koller CF,Grau-Richards S,Steinhart V,Spick C,Kubbies M,Klein C,
Figure PCTCN2021103058-appb-000001
P,
Figure PCTCN2021103058-appb-000002
E.Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions.Protein EngDes Sel.2016 Oct；29(10):457-466.doi:10.1093/protein/gzw040.Epub 2016 Aug 29.PubMed PMID:27578889)。在申请中，为了减少B7-H4×4-1BB双特异性抗体与Fcγ受体的结合，这些抗体的Fc引入了“LALA”双突变体(L234A/L235A)或者“LALAPG”三突变体(L234A/L235A/P329G)。
实施例1.抗体的序列分析、表达纯化、和理化性质表征分析
1.1 抗体的序列分析和优化
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系 基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。
蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰，称为翻译后修饰(PTM)。对于抗体而言，一些PTM的位点是非常保守的，例如，在人的IgG1抗体的恒定结构域的第297位(EU编号)的保守的氨基酸天冬酰胺Asn通常会发生糖基化修饰形成糖链，而该糖链结构对于抗体结构和相关的效应功能是至关重要的。但是，如果在抗体的可变结构域尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM是和氨基酸序列的组成尤其是相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。例如，N-x-S/T(第一位是天冬酰胺，第二位是非脯氨酸以外的任意氨基酸，第三位是丝氨酸或者苏氨酸)的序列模式预测出N-连接糖基化位点。引起PTM的氨基酸序列模式有可能来源于胚系基因序列，例如人胚系基因片段IGHV3-33天然地在FR3区域存在糖基化模式NST；也可能来源于体细胞高频突变。例如，NGS或NLT可能是糖基化位点，NS可能是脱酰胺位点，DG可能引起天冬氨酸的异构化。
可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
1.2 抗体的表达和纯化
本实施例介绍了利用哺乳动物宿主细胞(例如，人胚肾细胞HEK293或中国仓鼠卵巢细胞CHO及其衍生细胞)、瞬时转染表达和亲和捕获分离等技术来制备抗体的一般方法。本方法适用于含有Fc区的目标抗体；目标抗体可以由一条或多条蛋白质多肽链组成； 可以来源于一个或多个表达质粒。
将抗体多肽链的氨基酸序列通过密码子优化方法转换成核苷酸序列；合成编码的核苷酸序列并克隆到与宿主细胞兼容的表达载体上。将编码抗体多肽链的质粒按照特定比例同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到具有正确折叠和多肽链组装的重组抗体。具体地，将FreeStyle TM 293-F细胞(Thermo,#R79007)在FreeStyle TM F17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6-8×10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将编码抗体多肽链的质粒按照一定比例混合共计30μg质粒(质粒与细胞的比例为1μg:1ml)溶解于1.5ml Opti-MEM减血清培养基(Thermo,#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS pH7.4缓冲液平衡含有MabSelect TM(GE Healthcare,#71-5020-91)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS缓冲液冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，随后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,#UFC901024)浓缩换液至PBS缓冲液或者含有其他成分的缓冲液，得到纯化的重组抗体溶液。最后用NanoDrop(Thermo,NanoDrop TM One)测定浓度，分装、存储备用。
1.3 利用SEC-HPLC分析蛋白纯度和聚体
本实施例使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience,#08541,5μm,7.8mm×30cm)连接到高压液相色谱仪HPLC(Agilent Technologies,Agilent 1260Infinity II)，用PBS缓冲液室温下平衡至少1小时。适量蛋白样品(至少10μg)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用PBS缓冲液将样品以1.0ml/分钟的流速流过色谱柱，最长时间为25分钟。HPLC将生成分析报告，报告出样品内不同分子尺寸组份的滞留时间。
1.4 利用DSF测定蛋白分子的热稳定性
差示扫描荧光法(Differential Scanning Fluorimetry,DSF)是一种常用的高通量的测定蛋白质热稳定性的方法。它使用实时荧光定量PCR仪器通过监测与去折叠的蛋白分子结合的染料的荧光强度的变化，来反映蛋白质的变性的过程，从而反映出蛋白分子的热 稳定性。本实施例利用DSF方法来测定蛋白分子热变性温度(Tm)。10μg蛋白加入96-孔PCR板(Thermo,#AB-0700/W)，接着加入2μl 100×稀释的染料SYPROTM(Invitrogen,#2008138)，然后加入缓冲液使得终体积为40μl每孔。将PCR板密封，放置于实时荧光定量PCR仪器(Bio-Rad CFX96 PCR System)，先于25℃孵育5分钟，然后以0.2℃/0.2分钟的梯度逐渐从25℃升温至95℃，在测试结束时将温度降至25℃。使用FRET扫描模式并使用Bio-Rad CFX Maestro软件进行数据分析并计算出样品的Tm。
实施例2.抗B7-H4全人源抗体的获得
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人B7-H4-ECD-mFc融合蛋白(Sino Biological，#10738-H05H)对Harbour H2L2小鼠进行多轮免疫，或者用过表达人B7-H4的CHO-K1细胞转染mCD40L后，与免疫佐剂混合成免疫原试剂后对Harbour H2L2小鼠进行多轮免疫。
当检测小鼠血清中B7-H4特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出。通过杂交瘤融合技术，B细胞克隆技术和噬菌体库技术进行筛选，对筛选获得的单克隆抗体进行进一步的鉴定，根据其对人B7-H4的结合能力、食蟹猴B7-H4的结合能力等参数，选出2个克隆80C8-2E9和1025_B-1H11。利用常规的测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。
本实例中抗B7-H4的重组全人源IgG抗体列于表2。
表2.抗B7-H4H2L2抗体的序列编号(CDR都是按照Chothia规则定义)
Figure PCTCN2021103058-appb-000003
另外根据专利查询，(来自专利WO2016040724A1)，又称1D11.v1.9varC2，设计了对照抗体1，在后续实验中作为对照：
表3.B7-H4对照抗体的序列编号(SEQ ID NO:)
Figure PCTCN2021103058-appb-000004
2.1 FACS检测抗人B7-H4H2L2单抗在细胞水平的结合能力
本实施例是为了研究抗人B7-H4的H2L2单抗体外结合人/食蟹猴/小鼠B7-H4的活性。采用过表达食蟹猴B7-H4的CHO-K1细胞株(CHO-K1/cyno B7-H4，和铂自制)、过表达小鼠B7-H4的CHO-K1细胞株(CHO-K1/m B7-H4，和铂自制)和高表达人B7-H4的细胞系SK-BR-3(
Figure PCTCN2021103058-appb-000005
HTB-30)进行细胞水平上的抗体结合实验。简言之，消化CHO-K1/cyno B7-H4细胞、CHO-K1/m B7-H4细胞或SK-BR-3细胞，并用含2％BSA的PBS重悬。将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,#109-545-06,1:500稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％BSA的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％BSA的PBS重悬细胞，使用ACEA Novocyte3000流式细胞仪读取荧光发光信号值。
如图1-(A)所示：B7-H4抗体在高表达B7-H4的SK-BR-3细胞系上的体外结合；PR001476和PR002408与SK-BR-3细胞系结合活性优于对照抗体1。
如图1-(B)所示：B7-H4抗体在高表达食蟹猴B7-H4的CHO-K1-食蟹猴B7-H4细胞系上的体外结合；PR001476和PR002408与CHO-K1-食蟹猴B7-H4细胞系结合活性优于对照抗体1。
如图1-(C)所示：B7-H4抗体在高表达鼠B7-H4的CHO-K1-鼠B7-H4细胞系上的体外结合；PR002410与CHO-K1-鼠B7-H4细胞系结合活性与对照抗体1没有显著差异。
实施例3. 4-1BB全人源抗体的获得
3.1 抗4-1BB全人源HCAb抗体的获得
Harbour HCAb小鼠(Harbour Antibodies BV，WO2010/109165A2)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生仅有重链的抗体，该抗体的大小只有传统IgG 抗体的一半。其产生的抗体仅具有人的抗体重链可变结构域和小鼠Fc恒定结构域。
用可溶的重组人4-1BB-Fc融合蛋白(睿智化学)或者过表达了人4-1BB的NIH-3T3细胞(睿智化学)对Harbour HCAb小鼠进行多轮免疫。当检测小鼠血清中4-1BB特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用BD FACS AriaII Cell Sorter分选CD138阳性的浆细胞和人4-1BB抗原阳性的B细胞群。用常规的分子生物学手段从浆细胞中扩增人VH基因，并将扩增的人VH基因片段构建到编码人IgG1抗体重链Fc区域序列的哺乳动物细胞表达质粒pCAG载体中。质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，得到全人源HCAb抗体上清。用FACS测试HCAb抗体上清与高表达人4-1BB的CHO-K1细胞CHO-K1/hu4-1BB的结合，鉴定出阳性HCAb抗体。对这些HCAb抗体进行进一步的鉴定，根据其对人4-1BB的结合能力、食蟹猴4-1BB的结合能力、T细胞激活能力等参数，优选出数个候选HCAb抗体分子。然后对候选HCAb抗体分子进行序列分析和优化，得到数个变体序列。将HCAb抗体的VH序列和人的IgG1重链Fc序列进行融合表达，得到全人源重组HCAb抗体分子。其中，PR004469是PR001838的PTM的变体，PR007381为PR004469的胚系化变体，具体突变位点见表5。
表4.抗4-1BB的重组全人源HCAb抗体的序列表
抗体编号 重链 VH HCDR1 HCDR2 HCDR3
PR001758 175 143 17 47 85
PR001760 176 144 18 48 86
PR001763 177 145 18 49 87
PR001764 178 146 19 50 88
PR001767 179 147 20 51 89
PR001768 180 148 18 52 90
PR001771 181 149 18 49 90
PR001774 182 150 21 53 91
PR001780 183 151 21 54 92
PR001781 184 152 19 55 93
PR001830 185 153 18 49 86
PR001833 186 154 19 49 94
PR001836 187 155 22 56 86
PR001838 188 156 18 57 95
PR001840 189 157 19 58 96
PR001842 190 158 19 49 97
PR004469 193 161 18 61 95
PR007381 285 284 18 61 95
表5. 4-1BB HCAb序列的突变位点设计
初始抗体 变体 可变区突变 重组抗体亚型
PR001838 PR004469 G53A 人IgG1
PR001838 PR007381 F37V,P40A,E42G,T43K,K46E,G53A 人IgG1
3.2 抗4-1BB全人源H2L2抗体的获得
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。用可溶的重组人4-1BB-Fc融合蛋白对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中4-1BB特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，分离出表达抗-4-1BB单克隆抗体分子的杂交瘤细胞株。利用常规的杂交瘤测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。
表6.抗4-1BB的重组全人源IgG抗体序列表
Figure PCTCN2021103058-appb-000006
另外根据专利查询，设计了对照抗体，在后续实验中作为对照：
表7.对照抗体序列表
Figure PCTCN2021103058-appb-000007
3.3 FACS检测抗人4-1BB单抗在细胞水平的结合能力
本实施例是为了研究抗人4-1BB的HCAb和H2L2单抗体外结合人和食蟹猴4-1BB的活性。采用过表达人4-1BB的CHO-K1细胞株(CHO-K1-hu 4-1BB,Genescript)、过表达食蟹猴4-1BB的CHO-K1细胞株(CHO-K1-cyno 4-1BB,Genescript)进行细胞水平上的 抗体结合实验。简言之，消化细胞CHO-K1-hu 4-1BB和CHO-K1-cyno 4-1BB细胞，并用DMEM完全培养基重悬，将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#:3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,#:109-545-06,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值。
图2-(A)-(I)：4-1BB抗体在高表达人4-1BB的CHO-K1-人4-1BB细胞系上的体外结合；
图3-(A)-(H)：4-1BB抗体在高表达食蟹猴4-1BB的CHO-K1-食蟹猴4-1BB细胞系上的体外结合。
如图2-(A)-(I)所示，本发明的抗4-1BB的抗体均能结合人4-1BB，且检测到的抗体结合能力与抗体浓度成正相关关系递增。与参照抗体(Urelumab和Utomilumab)相比，这些抗体能以较低的浓度更灵敏地结合人4-1BB，与参照抗体Utomilumab和Urelumab的EC50相当。
如图3-(A)-(H)所示，本发明的抗4-1BB的抗体均能结合猴4-1BB，且检测到的抗体结合能力与抗体浓度成正相关关系递增。与参照抗体Tab(Utomilumab)相比，这些抗体能以较低的浓度更灵敏地结合猴4-1BB，与参照抗体Utomilumab的EC50相当或更优。而参照抗体Urelumab不具有与猴4-1BB交叉结合的活性。
3.4 抗原结合蛋白能在体外激活4-1BB通路
使用10μg/ml的丝裂霉素(北京中生瑞泰科技，#10107409001)处理过表达人CD32b的CHO-K1细胞(CHO-K1/CD32b,Genscript,#M00600)，30分钟37℃。然后用含10％FBS的F-12K培养液，洗涤4次。把处理过的细胞放入96孔板里，每孔1.5×10 4个，37℃保温箱培养过夜。第二天，使用MACS试剂盒(Miltenyi Biotec,#130-096-535)从人PBMC里分离人CD3阳性T细胞。首先确定细胞数量，然后根据细胞数量加入相应量的MACS缓冲液和Pan-T细胞生物素抗体，混匀，4℃静置5分钟。然后加入相应量微磁珠，4℃静置10分钟。通过LS柱的是CD3阳性的T细胞。将前一天96孔板的培养液洗掉，加入纯化的T细胞，每孔1×10 5个。然后加入相应浓度的4-1BB抗体或者是对照抗体，加入OKT3(eBiosciences，#16-0037-85)并且使其最终浓度达到0.3μg/ml。37℃保温箱培 养72小时。72小时后，收取上清液，使用ELISA试剂盒(Invitrogen，#88-7316-88)来检测IFN-γ的含量。在96平底板里加入包被抗体，4℃过夜。第二天加入ELISA缓冲液，室温1小时。加入收取的上清液，室温培养2小时。洗板2次，加入检测抗体，室温1小时。洗板两次，加入HRP-链霉亲和素，室温孵育1小时。然后加入TMB底物，稍后加入ELISA终止液(BBI，#E661006-0200)。酶标仪(PerkinElemer，#Enspire)读取450nm和570nm吸光值(OD 450-OD 570)，计算IFN-γ浓度。
结果如图4-(A)-(E)：4-1BB抗体具有激活4-1BB通路，并诱导激活T细胞的功能。PR001758，PR001760，PR001764，PR001771，PR001774，PR001780，PR001781，PR001830，PR001833，PR001836，PR001838，PR001840,PR000448激活作用均比utomilumab更强，表现为IFN-γ的信号高于utomilumab。
实施例4.B7-H4×4-1BB双特异性抗体的制备
自实施例2所选的抗B7-H4抗体和自实施例3所选的抗4-1BB抗体组合用于制备双特异性抗体，可以同时结合两个靶点，其中一端可以识别肿瘤细胞表面特异表达的B7-H4，而另一端可以结合T细胞上的4-1BB分子。当B7-H4×4-1BB双抗分子结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，从而杀死肿瘤细胞。
本实施例制备的B7-H4×4-1BB双特异性抗体包括多种分子结构：
图7-(A)-(I)是本发明的双特异性分子的结构示意图。
4.1 构建IgG-scFv四价对称结构的双特异性抗体
利用抗B7-H4的H2L2抗体和抗4-1BB的H2L2抗体构建IgG-scFv四价对称结构的双特异性抗体。IgG-scFv四价对称结构(如图5-(A)所示)的结合蛋白包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L1-VH_B L2-VL_B。h是IgG抗体的铰链区或衍生序列。多肽链2的连接肽L1和连接肽L2可以是表8中所列序列。
4.2 构建IgG-VH四价对称结构的双特异性抗体
利用抗B7-H4的H2L2抗体和抗4-1BB的HCAb抗体构建IgG-VH四价对称结构的双特异性抗体。IgG-VH四价对称结构(如图5-(B)所示)的结合蛋白包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L-VH_B。h是IgG抗体的铰链区或衍生序列。在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L的长 度为0。在另一个实施方案中，多肽链2的CH3经由连接肽L联结到VH_B；L可以是表8中所列序列。
4.3 构建IgG-VH-VH六价对称结构的双特异性抗体
利用抗B7-H4的H2L2抗体和抗4-1BB的HCAb抗体构建IgG-VH-VH六价对称结构的双特异性抗体。IgG-VH-VH六价对称结构(如图5-(C)所示)的结合蛋白包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B。h是IgG抗体的铰链区或衍生序列。在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链2的连接肽L1和L2可以是表8中所列序列。
4.4 构建Fab-HCAb结构的双特异性抗体分子
利用抗B7-H4的H2L2抗体和抗4-1BB的HCAb抗体构建Fab-HCAb对称结构的双特异性抗体，包含两种不同的结构Fab(CL)-VH-Fc(如图5-(D)所示)Fab(CH)-VH-Fc(如图5-(E)所示)。Fab(CL)-VH-Fc的结合蛋白包含两条多肽链，多肽链1，也称短链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2，也称长链，从氨基末端到羧基末端，其包含VL_A-CL-L1-VH_B-L2-CH2-CH3。多肽链2的连接肽L1和L2可以是表8中所列序列。Fab(CH)-VH-Fc的结合蛋白包含两条多肽链，多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-L1-VH_B-L2-CH2-CH3。多肽链2的连接肽L1和L2可以是表8中所列序列。
4.5 构建Fab-Fc-VH(n)非对称结构
利用抗B7-H4的H2L2抗体和抗4-1BB的HCAb抗体构建Fab-Fc-VH(n)非对称结构的双特异性抗体。Fab-Fc-VH(n)非对称结构(如图5-(F)(G)(H)所示)的结合蛋白包含三条多肽链，多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3；多肽链3，从氨基末端到羧基末端，其包含VH_B-h-CH2-CH3，或者多肽链3，从氨基末端到羧基末端，其包含VH_B-L-VH_B-h-CH2-CH3，或者多肽链3，从氨基末端到羧基末端，其包含VH_B-L1-VH_B-L2-VH_B-h-CH2-CH3。h是IgG抗体的铰链区或衍生序列。多肽链3的连接肽L1和L2可以是表8中所列序列。
为了将具有错配的重链(例如抗4-1BB抗体的两条重链或者抗B7-H4的两条重链的 错配)的副产物形成最小化，使用了突变的异源二聚体Fc区，其携带“knob-hole”突变和改造的二硫键，如WO2009080251和WO2009080252中所述。
4.6 构建Fab-VH-Fc-VH对称结构
利用抗B7-H4的H2L2抗体和抗4-1BB的HCAb抗体构建Fab-VH-Fc-VH对称结构的双特异性抗体。Fab-VH-Fc-VH对称结构(如图5-(I)所示)的结合蛋白包含两条多肽链，多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-L1-VH_B-CH1-h-CH2-CH3-VHB。h是IgG抗体的铰链区或衍生序列。多肽链3的连接肽L1和L2可以是表8中所列序列。表11-(B)中的PR007379涉及到的PR002408(H:gm)与PR002408的CDR相同，可变区VH的FR2有2个突变，分别为T69I，E101G。
表8.连接肽序列表
Figure PCTCN2021103058-appb-000008
Figure PCTCN2021103058-appb-000009
本发明所得双抗分子的多肽链的氨基酸序列在序列表中如下所示。
表9.本发明所得双抗分子序列表
抗体编号 多肽链1 多肽链2 多肽链3
PR002789 198 202  
PR002972 201 217  
PR002790 198 203  
PR002791 198 204  
PR002792 198 205  
PR002793 198 206  
PR002794 198 207  
PR002795 198 208  
PR002798 198 209  
PR002800 198 210  
PR002801 198 211  
PR002802 198 212  
PR002804 198 213  
PR002805 198 214  
PR002806 198 215  
PR002808 198 216  
PR003334 200 218  
PR003335 200 219  
PR003336 200 220  
PR003337 200 221  
PR003338 200 222  
PR004162 201 230  
PR004357 201 238  
PR004359 201 240  
PR004280 200 235  
PR004281 200 236  
PR004282 200 237  
PR003487 200 223  
PR003488 200 224  
PR003489 200 225  
PR004158 201 226  
PR004358 201 239  
PR004160 201 227 228
PR004161 201 227 229
PR004181 201 227 231
PR004182 201 227 232
PR004279 233 234  
PR004995 201 262  
PR005183 200 263  
PR005184 200 264  
PR005185 200 265  
PR005186 200 266  
PR005187 200 267  
PR005188 200 268  
PR005189 233 269  
PR005190 233 270  
PR005827 200 274  
PR005828 200 275  
PR005829 200 276  
PR005830 200 277  
PR005649 233 271  
PR005650 233 272  
PR005651 233 273  
PR005838 200 278  
PR005839 200 279  
PR005866 200 280  
PR007165 200 281  
PR007379 200 286  
PR007380 200 287  
B7-H4×4-1BB双特异性抗体为的抗原结构域的CDR序列编号如下表所示，表10中1#序号为结合B7-H4的抗原结构域，2#序号为结合4-1BB的抗原结构域。
表10.双抗的抗原结合结构域的CDR序列编号(SEQ ID NO:)
Figure PCTCN2021103058-appb-000010
Figure PCTCN2021103058-appb-000011
Figure PCTCN2021103058-appb-000012
表11-(A)-(F)是本发明的双特异性抗体分子结构信息。
表11-(A):具有IgG-scFv对称结构的B7-H4×4-1BB双抗分子
Figure PCTCN2021103058-appb-000013
表11-(B):具有IgG-VH四价对称结构的B7-H4×4-1BB双抗分子
Figure PCTCN2021103058-appb-000014
Figure PCTCN2021103058-appb-000015
Figure PCTCN2021103058-appb-000016
表11-(C):具有IgG-VH-VH六价对称结构的B7-H4x 4-1BB双抗分子
Figure PCTCN2021103058-appb-000017
表11-(D):具有Fab-HCAb四价对称结构的B7-H4x 4-1BB双抗分子
Figure PCTCN2021103058-appb-000018
Figure PCTCN2021103058-appb-000019
表11-(E):具有Fab-Fc-VH(n)非对称结构的B7-H4x 4-1BB双抗分子
Figure PCTCN2021103058-appb-000020
表11-(F):具有Fab-VH-Fc-VH结构的B7-H4x 4-1BB双抗分子
Figure PCTCN2021103058-appb-000021
表12-(A)-(F)是本发明的双特异性抗体的蛋白表达和物理化学性质。
表12-(A):IgG-scFv对称结构的双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021103058-appb-000022
表12-(B):IgG-VH对称结构的双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021103058-appb-000023
表12-(C):IgG-VH-VH六价对称结构的双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021103058-appb-000024
表12-(D):Fab-HCAb四价对称结构的双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021103058-appb-000025
表12-(E):Fab-Fc-VH(n)非对称结构的双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021103058-appb-000026
表12-(F):具有Fab-VH-Fc-VH对称结构的双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021103058-appb-000027
实施例5.FACS检测B7-H4×4-1BB双特异性抗体在过表达人和食蟹猴4-1BB的CHO-K1细胞株上的体外结合
本实施例为了研究B7-H4×4-1BB双特异性抗体4-1BB臂体外结合人和食蟹猴4-1BB 的活性。采用过表达人4-1BB的CHO-K1细胞株(CHO-K1/hu 4-1BB,Genscript，#M00538)和过表达食蟹猴4-1BB的CHO-K1细胞株(CHO-K1/cyno 4-1BB,Genscript，#M00569)进行细胞水平上的抗体结合实验。简言之，消化细胞CHO-K1/hu 4-1BB和CHO-K1/cyno 4-1BB细胞，并用F12K完全培养基重悬，用PBS洗涤一次。用PBS将细胞密度分别调整为1×10 6细胞/ml。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch,#:109-545-06,1:500稀释)或者(Alexa 
Figure PCTCN2021103058-appb-000028
647,Goat Anti-Human IgG,Fcγfragment specific，Jackson ImmunoResearch,#:109-605-098,1:1000稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII或艾森ACEA NovoCyte系列流式细胞仪读取荧光发光信号值。
图6-(A)-(L):B7-H4×4-1BB双特异性抗体在过表达人4-1BB的CHO-K1细胞株上的体外结合；
图7-(A)-(C):B7-H4×4-1BB双特异性抗体在过表达食蟹猴4-1BB的CHO-K1细胞株上的体外结合；
如图6-(A)-(L)所示，本实例中的B7-H4×4-1BB双特异性抗体均与过表达人4-1BB的CHO-K1细胞特异性结合。PR002789，PR002972，PR003334，PR003335,PR003336,PR003337，PR003338，PR004160，PR004161，PR004158，PR004162，PR004357，PR004359，PR004279,PR004280,PR004281,PR004282,PR005829等与人4-1BB的结合强于阳性对照Urelumab。具体表现为，抗体PR003335结合曲线的Span值约为Urelumab的1.5倍，MFI最大值高于Urelumab。
如图7-(A)-(C)所示，本实例中的B7-H4×4-1BB双特异性抗体均与过表达猴4-1BB的CHO-K1细胞特异性结合。而参照抗体Urelumab不具有与猴4-1BB交叉结合的活性。
实施例6.FACS检测B7-H4×4-1BB双特异性抗体在高表达B7-H4的SK-BR-3细胞系上的体外结合
本实施例为了研究B7-H4×4-1BB双特异性抗体B7-H4臂的结合人B7-H4的活性。采用高表达B7-H4的SK-BR-3细胞系进行与人B7-H4细胞水平结合实验。简言之，收集SK-BR-3细胞悬液，将细胞密度分别调整为2×10 6细胞/mL。以50μL细胞/孔接种于96 孔V底板(Corning,#:3894)，随后加入50μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson ImmunoResearch,#:109-605-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μL/孔预冷FACS重悬细胞，使用ACEA Novocyte3000读取荧光发光信号值。
图8-(A)-(K):B7-H4×4-1BB双特异性抗体在高表达B7-H4的SK-BR-3细胞系上的体外结合；
如图8-(A)-(K)中所示，本实例中B7-H4×4-1BB双特异性抗体均与SK-BR-3细胞系上人B7-H4特异性结合，结合曲线EC50大多为0.1-10nM。
实施例7.B7-H4×4-1BB双特异性抗体在B7-H4高表达细胞系SK-BR-3存在时的T细胞激活实验以及细胞因子的释放
为了研究B7-H4×4-1BB双特异性抗体体外介导靶T细胞的激活能力，采用人pan T细胞作为效应细胞，高表达B7-H4的细胞系SK-BR-3作为介导交联的细胞进行体外激活实验以及细胞因子释放的检测。具体的，将96孔平底板(Corning,#3599)用OKT3包板，将pan T的密度调整为2×10 6细胞/mL，SK-BR-3的密度调整为2×10 5细胞/mL，以两种细胞悬液各50μL细胞/孔接种于96孔平底板(Corning,#3599)，随后加入50μL/孔，3倍于终浓度的不同梯度稀释的待测抗体，其中抗体最高终浓度为100nM或30nM或20nM或6nM，每个抗体共6或3个或2个浓度，最终效靶比为10:1，设置两个重复。同时，在板内设置同型IgG对照组。96孔板置于37℃二氧化碳培养箱孵育2天或3天。孵育完成后，取上清液，加入96孔板(Corning,#3599)，于500g、4℃下离心5分钟。孵育2天的上清，用于检测细胞因子IL-2的释放或者孵育3天的上清检测IFN-gamma的释放。ELISA检测方法参照IL-2(IL-2 Human Uncoated ELISA Kit,Thermo,#88-7025-88)试剂盒和IFN gamma(IFN gamma Human Uncoated ELISA Kit,Thermo,#88-7316-88)操作说明。
图9-(A)-(E)：B7-H4×4-1BB双特异性抗体在B7-H4高表达细胞系SK-BR-3存在时激活4-1BB通路，并诱导激活T细胞释放IFN-γ。
图10-(A)-(N)：B7-H4×4-1BB双特异性抗体在B7-H4高表达细胞系SK-BR-3存在时激活4-1BB通路，并诱导激活T细胞释放IL-2。
如图9-(A)-(E)和图10-(A)-(N)所示，在高表达B7-H4的细胞系SK-BR-3存在时，B7-H4×4-1BB双特异性抗体能够介导的T细胞激活。并且PR002790、PR002791、PR002802、PR002804、PR002806、PR003334、PR003335、PR003336、PR003337、PR003338、PR003487、PR003488、PR003489、PR004158、PR004160、PR004161、PR004162、PR004181、PR004182、PR004279、PR004280、PR004281、PR004282、PR004357、PR004358、PR004359对4-1BB通路的激活优于或相当于Urelumab。具体表现为产生的细胞因子IFN-γ或IL-2高于Urelumab。
不同的linker长度也会影响双抗对T细胞的激活，比如PR004281和PR004282表现优于PR003338和Urelumab(图10-K)。
带有不同抗原表位的B7H4臂会影响双抗对T细胞的激活，比如PR004281和PR004282表现优于PR004995和Urelumab(图10-L)。
带有不同的Fc突变位点会影响双抗对T细胞的激活，如PR005185、PR005186及PR004282优于Urelumab(图10-M)。
实施例8.B7-H4×4-1BB双特异性抗体对T细胞的激活不依赖于FcγR细胞
为了研究B7-H4×4-1BB双特异性抗体活性是否依赖于FcγR的表达。采用人pan T细胞作为效应细胞，高表达FcγRIIb的细胞系CHO-K1/CD32b或高表达FcγRI的细胞系CHO-K1/CD64作为介导交联的细胞进行体外激活实验以及细胞因子释放的检测。具体的，将96孔平底板(Corning,#3599)用OKT3包板，将pan T的密度调整为2×10 6细胞/mL，FcγR细胞的密度调整为2×10 5细胞/mL，以两种细胞悬液各50μL细胞/孔接种于96孔平底板(Corning,#3599)，随后加入100μL/孔，2倍于终浓度的不同梯度稀释的待测抗体，最终效靶比为10:1，设置两个重复。同时，在板内设置同型IgG对照组。96孔板置于37℃二氧化碳培养箱孵育2天。孵育完成后，取100μl上清液，加入96孔板(Corning,#3894)，于500g、4℃下离心5分钟，取上清，用于检测细胞因子IL-2的释放。ELISA检测方法参照IL-2(IL-2 Human Uncoated ELISA Kit,Thermo,#88-7025-88)试剂盒操作说明。
图11-(A)-(B)：B7-H4×4-1BB双抗对T细胞激活特异性不依赖FcγR的表达。(A)高表达FcγRIIb的细胞系CHO-K1/CD32b细胞存在时，B7-H4×4-1BB双特异性抗体PR004282介导的T细胞激活和同型IgG对照相当。(B)高表达FcγRI的细胞系CHO-K1/CD64细胞存在时，B7-H4×4-1BB双特异性抗体PR004282介导的T细胞激活和同型IgG对照相当。
实施例9.B7-H4×4-1BB双特异性抗体对T细胞的激活依赖于B7-H4的表达
为了研究B7-H4×4-1BB双特异性抗体活性是否依赖于B7-H4表达。采用人pan T细胞作为效应细胞，高表达B7-H4的细胞系SK-BR-3和MDA-MB-468细胞以及不表达B7-H4的COV644和JIMT-1及MDA-MB-231细胞作为介导交联的细胞进行体外激活实验以及细胞因子释放的检测。具体的，将96孔平底板(Corning,#3599)用OKT3包板，将pan T的密度调整为2×10 6细胞/mL，肿瘤细胞的密度调整为2×10 5细胞/mL，以两种细胞悬液各50μL细胞/孔接种于96孔平底板(Corning,#3599)，随后加入50μL/孔，3倍于终浓度的待测抗体，最终效靶比为10：1，设置两个重复。同时，在板内设置同型IgG对照组。96孔板置于37℃二氧化碳培养箱孵育2天。孵育完成后，取100μl上清液，加入96孔板(Corning,#3894)，于500g、4℃下离心5分钟，取上清，用于检测细胞因子IL-2的释放ELISA检测方法参照IL-2(IL-2 Human Uncoated ELISA Kit,Thermo,#88-7025-88)试剂盒操作说明。
图12：FACS检测肿瘤细胞表面的B7-H4表达量。图12-(A)用B7-H4阳性对照抗体1检测肿瘤细胞表面的B7-H4表达量。图12-(B)用B7-H4×4-1BB双特异性抗体检测肿瘤细胞表面的B7-H4表达量。如图所示，SK-BR-3和MDA-MB-468是高表达B7-H4的肿瘤细胞，JIMT-1和COV644及MDA-MB-231是不表达B7-H4的肿瘤细胞。
图13-(A)-(H)：B7-H4×4-1BB双抗对T细胞激活特异性依赖B7-H4的表达。(A),(B)高表达B7-H4的细胞系SK-BR-3或MDA-MB-468细胞存在时，B7-H4×4-1BB双特异性抗体PR003334介导的T细胞激活优于阳性对照。(C),(D)不表达B7-H4的细胞系COV644或JIMT-1细胞存在时,PR003334介导的T细胞激活程度与同型IgG对照相当。(E),(F)高表达B7-H4的细胞系SK-BR-3细胞存在时，B7-H4×4-1BB双特异性抗体PR004282介导的T细胞激活优于阳性对照Urelumab。(G),(H)不表达B7-H4的细胞系JIMT-1或MDA-MB-231细胞存在时,PR004282介导的T细胞激活程度与同型IgG对照相当。
实施例10.B7-H4×4-1BB双特异性抗体的血清稳定性研究
为了研究B7-H4×4-1BB双特异性抗体在高浓度人血清中的稳定性，PR0003334和PR0003335用90％人血清进行梯度稀释，初始浓度为100nM,1:3梯度稀释8个浓度点。分成6份样本，分别在37度孵育0天、1天、2天、4天、7天、14天后，液氮速冻后放置于-70度保存。FACS检测B7-H4×4-1BB双特异性抗体在高浓度血清中37度放置不同时间后，与高表达B7-H4的SK-BR-3细胞系和CHO-K1-hu4-1BB细胞的体外结合，参照实施例3.3和实施例6。
PR0004282用95％人血清进行梯度稀释，分成5管，分别在37度孵育0天、1天、2天、4天、7天、14天后，液氮速冻后放置于-80度保存。在高浓度人血清中37度放置不同时间后的PR004282用FACS方法检测与B7H4+细胞和4-1BB+细胞的体外结合，过程如下：
消化CHO-K1/h4-1BB、CHO-K1/cyno4-1BB、CHO-K1/hB7H4及CHO-K1/cynoB7H4细胞，并用DMEM完全培养基重悬，用PBS洗涤后将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测人血清处理后PR004282抗体(不同处理时间的抗体的每个剂量均含有4.5％人血清)。将细胞放置于4℃，避光孵育2小时。再加入100μL/孔预冷含2％FBS的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson,#109-545-06,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％FBS的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％FBS的PBS重悬细胞，使用艾森ACEA NovoCyte系列流式细胞仪读取荧光发光信号值。
图14-(A-D)：B7-H4×4-1BB双抗PR003334和PR003335的血清稳定性。在不同的人血清孵育时间，PR003334和PR003335在90％血清中与高表达B7-H4的SK-BR-3细胞和CHO-K1-人4-1BB细胞的结合没有改变，说明PR003334和PR003335具有很好的血清稳定性。
图14-(E-H)：B7-H4×4-1BB双抗PR004282的血清稳定性。在不同的血清孵育时间，PR004282在95％血清中与高表达B7H4或4-1BB的CHO-K1细胞的结合没有改变，说明PR004282具有很好的血清稳定性。
实施例11.FACS检测B7-H4×4-1BB双特异性抗体PR004282在4-1BB+细胞水平的结合能力
本实施例是为了研究B7-H4×4-1BB双特异性抗体PR004282体外结合人/食蟹猴/小鼠4-1BB的活性。采用过表达人4-1BB的CHO-K1细胞株(CHO-K1/h4-1BB，Gensript,#M00538)、食蟹猴4-1BB的CHO-K1细胞株(CHO-K1/cyno4-1BB,Genscript,#M00569)、过表达小鼠4-1BB的CHO-K1细胞株(CHO-K1/m4-1BB,Genscript,#M00568)进行细胞水平上的抗体结合实验。简言之，消化CHO-K1/h4-1BB细胞、CHO-K1/cyno 4-1BB细胞及CHO-K1/m 4-1BB细胞，并用含2％FBS的PBS重悬。将细胞密度分别调整为1×10 6 细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷含2％FBS的PBS的FACS缓冲液漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa
Figure PCTCN2021103058-appb-000029
647,Goat Anti-Human IgG,Fcγ fragment specific，Jackson ImmunoResearch,#109-605-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷FACS缓冲液洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷FACS缓冲液重悬细胞，使用ACEA Novocyte3000流式细胞仪读取荧光发光信号值。
如图15-(A)所示：B7-H4/4-1BB双特异性抗体PR004282在高表达人4-1BB的CHO-K1细胞系上的体外结合情况；PR004282与CHO-K1/h4-1BB细胞系结合活性优于对照抗体Urelumab。
如图15-(B)所示：PR004282在高表达食蟹猴4-1BB的CHO-K1细胞系上的体外结合情况；PR004282可以结合猴4-1BB，而Urelumab不具有与猴的交叉反应。
如图15-(C)所示：PR004282在高表达鼠4-1BB的CHO-K1细胞系上的体外结合情况；PR004282不具有小鼠4-1BB结合能力。
实施例12.FACS检测B7-H4×4-1BB双特异性抗体PR004282在B7H4+细胞水平的结合能力
本实施例是为了研究B7-H4×4-1BB双特异性抗体PR004282体外结合人/食蟹猴/小鼠B7-H4的活性。采用过表达人B7H4的CHO-K1细胞株(CHO-K1/hB7-H4，和铂自制)食蟹猴B7-H4的CHO-K1细胞株(CHO-K1/cynoB7-H4，和铂自制)、过表达小鼠B7-H4的CHO-K1细胞株(CHO-K1/mB7-H4，和铂自制)进行细胞水平上的抗体结合实验。简言之，消化CHO-K1/hB7H4细胞、CHO-K1/cyno B7-H4细胞及CHO-K1/mB7-H4细胞，并用含2％FBS的PBS重悬。将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷含2％FBS的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa
Figure PCTCN2021103058-appb-000030
647,Goat Anti-Human IgG,Fcγfragment specific，Jackson ImmunoResearch,#109-605-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％FBS的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％BSA的PBS重悬细胞，使用ACEA Novocyte3000流式细胞仪读取荧光发 光信号值。
如图16-(A)所示：B7-H4/4-1BB双特异性抗体PR004282在高表达人B7-H4的CHO-K1细胞系上的体外结合；PR004282与CHO-K1/hB7H4细胞系结合活性高于B7H4单抗抗体PR002408。
如图16-(B)所示：B7-H4抗体在高表达食蟹猴B7-H4的CHO-K1细胞系上的体外结合；PR004282和PR002408与CHO-K1-食蟹猴B7-H4细胞系结合活性相当。
如图16-(C)所示：B7-H4抗体在高表达鼠B7-H4的CHO-K1细胞系上的体外结合；PR004282有微弱的小鼠B7H4结合能力。
实施例13.FACS检测B7-H4×4-1BB双特异性抗体PR004282同时结合CHO-K1/h4-1BB和SK-BR-3的能力
本实施例是为了研究B7-H4×4-1BB双特异性抗体PR004282同时结合CHO-K1/h4-1BB和SK-BR-3的能力。简言之，消化CHO-K1/h4-1BB细胞和SK-BR-3细胞，并用PBS将细胞密度分别调整为1×10 6细胞/mL，用0.5μM Far-red(lifetechnologies,#C34572)对SK-BR-3，用0.5μM CFSE(lifetechnologies,#C34544)对CHO-K1/h4-1BB细胞在室温染色5分钟。离心染色后的细胞，用>20ml的含1％FBS培养基洗涤一次。重悬洗涤后的细胞于FACS缓冲液中，并调整细胞密度均为2*10 6/ml。在96孔V型板内(Corning,#3894)每孔加入50μl SK-BR-3细胞(每孔细胞数为1*10 5)和25μl CHO-K1/h4-1BB(每孔细胞数为1*10 5)以及25ul 4倍梯度FACS缓冲液稀释的待测抗体，混匀完全。4℃孵育1小时。使用ACEA Novocyte3000流式细胞仪，识别FITC+CHO-K1/h4-1BB细胞和Alexa647+SK-BR-3双阳性百分比。数据用FlowJo软件(Tree Star,Ashland,OR)分析，双染细胞的百分比用于确定双抗介导的共结合率＝(Q2/(Q1+Q2)。
图17显示仅有PR004282具有同时结合CHO-K1/h4-1BB和SK-BR-3的能力。单抗均无该能力。
实施例14.FACS检测B7-H4×4-1BB双特异性抗体PR004282在原代细胞水平的结合能力
本实施例是为了研究B7-H4×4-1BB双特异性抗体PR004282体外结合激活后的人/食蟹猴原代T细胞的活性。用非人的灵长目CD3分离试剂盒(Miltenyi，#130-092-012)从冻存的猴PBMC(PharmaLegacy，#SC1702051)分离出猴CD3+T细胞，用培养基RPMI1640+10％FBS+1％丙酮酸鈉(Thermo,#11360-070)+1％非必需氨基酸溶液 (Thermo,#11140-050)重悬至2^10 6/ml,分别取1ml至6孔板内，加入1ml含有20ng/ml PMA(Sigma,#P1585-1MG)和5nM Ionomycin(Sigma,#407952-5MG)的培养基，37℃CO 2培养箱16h。用人CD3分离试剂盒(Miltenyi，#130-096-535)从冻存的人PBMC(Saily，XFB-HP100B)分离出人CD3+T细胞，其他操作同猴CD3+T细胞的激活。
激活后的猴T细胞密度用含2％FBS的PBS调整至1×10 6细胞/mL，以100μL CD3+T细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体PR004282/hIgG1/Urelumab/hIgG4,将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷含2％FBS的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa
Figure PCTCN2021103058-appb-000031
647,Goat Anti-Human IgG,Fcγ fragment specific，Jackson ImmunoResearch,#109-605-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％FBS的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，100μL/孔预冷含2％FBS的PBS重悬细胞，使用ACEA Novocyte3000流式细胞仪读取荧光发光信号值。
Zombie NIR TM染料(Biolegend，#77184)与激活后的人T细胞室温暗处孵育15-30分钟，FACS缓冲液洗涤一次细胞，将细胞密度分别调整为1×10 6细胞/mL。以100μL CD3+T细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体PR004282/hIgG1,将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷含2％FBS的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa
Figure PCTCN2021103058-appb-000032
488,Goat Anti-Human IgG,Fcγ fragment specific，Jackson ImmunoResearch,#109-545-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％FBS的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，100μL/孔预冷含2％FBS的PBS重悬细胞，使用ACEA Novocyte3000流式细胞仪读取荧光发光信号值。
如图18-(A-B)所示：B7-H4/4-1BB双特异性抗体PR004282可以与激活后的人或猴CD3+T细胞结合。
实施例15.ELISA检测B7-H4×4-1BB双特异性抗体PR004282与B7家族或TNFR家族其他成员的交叉反应性
15.1 ELISA检测B7-H4×4-1BB双特异性抗体PR004282与B7家族其他成员的交叉反应性
将B7家族的蛋白(表13)分别用PBS稀释为1μg/ml，加至96孔板(Corning， #9018)中，每孔100μl，4℃下孵育过夜。弃去液体后用PBST缓冲液(pH 7.4，含0.05％tween-20)洗板3次，加入250μl 2％BSA封闭液，37℃条件下孵育1小时。弃去封闭液，并用PBST缓冲液洗板3次，将待测抗原结合蛋白，稀释成100nM、10nM和1nM 3个浓度，每个孔加入100μl，37℃孵育1小时。PBST缓冲液清洗3次后，加入稀释5000倍的羊抗人HRP二抗(Invitrogen，#A18805)，37℃条件下，避光孵育1小时。PBST缓冲液清洗3次后，添加100μl/孔TMB(Biopanda,#TMB-S-003)，室温避光放置约30分钟；每孔加入50μl/孔终止液(BBI life sciences，#E661006-0200)终止反应，置酶标仪(PerkinElemer，#Enspire)中测定450nm吸光度(OD450)值。
图19-(A)说明本发明的抗体PR004282不与B7家族其它成员蛋白发生交叉反应。
表13.B7家族其它成员供应商信息
Figure PCTCN2021103058-appb-000033
15.2 ELISA检测B7-H4×4-1BB双特异性抗体PR004282与TNFR家族其他成员的交叉反应性
将TNFR家族的蛋白(表14)分别用PBS稀释为1μg/ml，加至96孔板(Corning，#9018)中，每孔100μl，4℃下孵育过夜。弃去液体后用PBST缓冲液(pH 7.4，含0.05％tween-20)洗板3次，加入250μl 2％BSA封闭液，37℃条件下孵育1小时。弃去封闭液，并用PBST缓冲液洗板3次，将待测抗原结合蛋白，稀释成100nM、10nM和1nM 3个浓度，每个孔加入100μl，37℃孵育1小时，同型抗体作为对照。PBST缓冲液清洗3次后，加入稀释5000倍的羊抗人HRP二抗(Invitrogen，#A18805)，37℃条件下，避光孵育1小时。PBST缓冲液清洗3次后，添加100μl/孔TMB(Biopanda,#TMB-S-003)，室温避光放置约30分钟；每孔加入50μl/孔终止液(BBI life sciences#E661006-0200)终止反应，置酶标仪(PerkinElemer#Envision)中测定450nm吸光度(OD450)值。
表14.TNFR家族其它成员供应商信息
Figure PCTCN2021103058-appb-000034
Figure PCTCN2021103058-appb-000035
图19-(B)说明本发明的抗体PR004282不与TNFR家族其它成员蛋白发生交叉反应。
实施例16.B7-H4×4-1BB双特异性抗体PR004282的ADCC效果检测
16.1 本实施例是通过ADCC报告基因细胞系来检测PR004282的ADCC效果
使用Jurkat FcγRIIIa-V158/NFAT-Luc细胞(和铂自制)，检测PR004282对CHO-K1/h4-1BB或SK-BR-3的ADCC效应的活性。CHO-K1/h4-1BB或SK-BR-3于300g离心5分钟，然后用RPMI1640+4％FBS血清培养基重悬。将细胞的密度调整到6×10 5细胞/ml，在96孔板的各孔内加入50μl细胞悬液，于37℃孵育过夜。Jurkat FcγRIIIa-V158/NFAT-Luc细胞于400g离心4分钟，然后用RPMI1640+4％FBS血清培养基重悬。将细胞的密度调整到3×10 6细胞/ml，在96孔板的各孔内加入50μl细胞悬液。抗体用RPMI1640+4％FBS培养基稀释，最高终浓度为100nM，每个抗体共4个浓度，5倍稀释，设置两个重复，在96孔板的各孔内加入50μl抗体稀释液。同时，在板内设置同型IgG对照组和空白培养基对照组。细胞与抗体于37℃孵育5小时。将96孔板于常温静置30分钟，加入60μl/孔的室温One-Glo显色液(Promega,#E6110)。之后样品避光常温孵育10分钟。用PE Enspire进行Luminscence读值。倍数＝抗体孔的Luminscence读值/空白培养基对照组的Luminscence读值，用Prism 8软件作图。PR004469及PR003369(亲和力成熟变体的B7H4单抗，具有ADCC增强效应，重链序列如SEQ ID NO:290所示，轻链序列如SEQ ID NO:291所示)作为阳性对照，human Iso IgG1(CrownBio，#C0001-4)抗体作为阴性对照。
图20-(A)-(B)示出PR004282对CHO-K1/h4-1BB及SK-BR-3细胞无明显的ADCC活性。而阳性对照PR004469及PR003369能以剂量依赖的方式分别对CHO-K1/4-1BB或SK-BR-3产生较强ADCC效应。
16.2 本实施例是为了研究B7H4/4-1BB双抗PR004282通过ADCC效应介导NK细胞杀伤靶细胞的活性。
本实验采用人NK作为效应细胞，高表达B7H4的细胞系SK-BR-3作为靶细胞。使用ACEA公司的RTCA仪器检测靶细胞的电导率反映杀伤效率。96孔板E-plate先用50μl 完全培养基平衡。消化SK-BR-3细胞，重悬于含10％胎牛血清的RPM1640完全培养基中，稀释至4*10 5/ml，铺板50μl/孔于E-plate 96板中，即2*10 4/孔，37℃培养过夜。次日用EasySep TM Human CD56阳选试剂盒(Stem cell，#17855)分选出NK，每孔加入新鲜50μl含1*10 5PBMC的培液，随后加入50μl 4×的浓度梯度稀释的抗体，抗体最高终浓度为100nM，每个抗体共4个浓度，5倍稀释，设置两个重复。PR003369作为阳性对照，human Iso IgG1(CrownBio，C0001-4)抗体作为阴性对照。实时检测靶细胞的电导率，取24小时时间点的数据计算靶细胞杀伤效率＝(1-样品/iso对照)×100％。
图20-(C)结果显示PR004282与PR003369相比，PR004282没有明显的杀伤。说明PR004282对SK-BR-3靶细胞不具有ADCC活性。
实施例17.利用BLI方法检测抗体与人、小鼠、食蟹猴Fcγ受体蛋白的亲和力
测试使用Octet Red 96e分子相互作用分析仪，实验缓冲液为稀释至1x的动力学缓冲液(ForteBio，#18-1105)。
设置仪器转动速度为1000转/分钟，先将置于一列的FAB2G传感器(Fortebio，#18-5125)在测试缓冲液中平衡10分钟，然后用FAB2G传感器捕获抗体，捕获高度1nm；FAB2G传感器在缓冲液中平衡120s后与2倍梯度稀释的Fcγ受体蛋白结合，设置结合时间60s、解离时间30s，蛋白浓度见表15。最后将FAB2G传感器浸入10mM甘氨酸-盐酸pH 1.5溶液进行再生，以洗脱结合在传感器上的蛋白。对于抗体与FcRn的亲和力测试，在缓冲液pH6.0和7.4两个条件下分别进行。对于带有avi标签的食蟹猴CD32b和CD64，使用SA传感器(Fortebio,18-5019)捕获受体蛋白，抗体为分析物。
表15.Fcγ受体蛋白信息及分析物浓度
Figure PCTCN2021103058-appb-000036
Figure PCTCN2021103058-appb-000037
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，以0nM为参照孔，扣除参照信号(reference subtraction)，选择“1:1Global fitting”方法进行数据拟合，计算出蛋白与抗体结合的动力学参数，得到kon(1/Ms)值、kdis(1/s)值和KD(M)值；对于快结合快解离的相互作用，选用“steady state”方法拟合数据，得到KD(M)值。
从表16-18可以看出，PR004282的KD值均高于对参照抗体Urelumab，表明其与人鼠猴Fcγ受体蛋白结合较弱。
表16.PR004282与人Fcγ受体的亲和力
Figure PCTCN2021103058-appb-000038
表17.PR004282与猴Fcγ受体的亲和力
Figure PCTCN2021103058-appb-000039
表18.PR004282与小鼠Fcγ受体的亲和力
Figure PCTCN2021103058-appb-000040
实施例18.B7-H4×4-1BB双特异性抗体的非特异性细胞因子的释放检测
为了评估B7-H4×4-1BB双特异性抗体是否引起细胞因子的非特异性释放。用人CD14分离试剂盒(Meltenyi,#130-050-201)分离出单核细胞，将其或同一供体的PBMC重悬于含10％胎牛血清的RPM1640完全培养基中，稀释至2E6/ml，铺板100μl/孔于平底96板(Costar,#3599)中，即2E5/孔，随后加入100ul 2x终浓度的梯度稀释的抗体，抗体 最高浓度为300nM,10倍稀释，共三个浓度，设置两个重复，37℃培养过夜。Urelumab作为阳性对照，Utomilumab和human Iso IgG1(CrownBio，C0001-4)抗体作为阴性对照。孵育24h收取上清检测TNF-a(Thermo,88-7066-88)和IL-6(Thermo，88-7346-77)的释放,孵育72h收上清检测IFN-gamma(Thermo,88-7316-77)的释放。
图21-(A)-(C)：B7-H4×4-1BB双特异性抗体PR004282与PBMC或单核细胞共孵育后细胞因子的释放情况。不管是PBMC还是单核细胞，Urelumab均表现出较强的TNF-a和IL-6的释放，IFN-gamma则仅表现在与PBMC共孵育的情况下。而PR004282则与Utomilumab一样显示出较好的安全性，不管是与PBMC或单核细胞孵育，均为显示出明显的细胞因子释放。
实施例19.B7-H4×4-1BB双特异性抗体在正常小鼠中的药代动力学研究
本实施例测试融合蛋白的药代动力学性能。其方法如下，选取体重18～22克的雌性C57BL/6小鼠6只，按5mg/kg PR003334或5mg/kg PR003335或5mg/kg PR004282的剂量通过静脉注射给与双特异性抗体药物；一组3只于给药前以及给药后15分钟、24小时(1天)、第4天、和第10天采集全血，另一组3只于只于给药前以及给药后5小时、第2天、第7天、和第14天采集全血。将全血静置30分钟使其凝固，随后在4℃下以2,000rpm离心5分钟并将分离的血清样品在-80℃下冻存直至分析。本实施例采用两种ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法一，即总体检测方法，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的融合蛋白，然后加入HRP标记的山羊抗人Fc第二抗体来检测；ELISA方法二，即功能结构域检测方法，通过包被于96孔板的人4-1BB蛋白来捕获小鼠血清中的含有人4-1BB HCAb的双特异性抗体，然后加入HRP标记的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件8.2版，选用非房室模型(NCA)对血药浓度数据进行分析以评价其药代动力学。
图22-(A)-(F)和表19显示的是B7-H4×4-1BB双特异性抗体PR003334和PR003335和PR004282的药代动力学数据。图22-(A)结果表明，总体检测方法下，PR003334在小鼠体内的半衰期约为8天。图22-(B)功能域检测方法显示，PR003334在小鼠体内的半衰期约为9天。图22-(C)总体检测方法下，PR003335在小鼠体内的半衰期约为14天。图22-(D)功能域检测方法显示，PR003335在小鼠体内的半衰期约为12天。图22-(E)总体检测方法下，PR004282在小鼠体内的半衰期约为11天。图22-(F)功能域检测方法显示，PR004282在小鼠体内的半衰期约为8天。
表19.PR003334和PR003335和PR004282的药代动力学
Figure PCTCN2021103058-appb-000041
实施例20.B7H4/4-1BB双抗PR004282在食蟹猴体内的药代动力学研究
本实施例测试B7-H4/4-1BB双特异性抗体在食蟹猴体内的药代动力学过程进行研究。具体的，将PR004282以0.9％生理盐水稀释，按照1mg/kg的剂量静脉注射入雄性食蟹猴体内，采集给药前及给药后0.5，1，2，4，8，12，24，48，72，168，336，504，672，840，1008小时的血样。血样在室温静置30分钟后离心分离得到血清，利用建立的特异性ELISA方法(总体检测法)检测血清中B7-H4/4-1BB双特异性抗体的含量。
图23和表20所示，B7-H4/4-1BB双特异性抗体在静脉注射给予食蟹猴后显示出典型的IgG样药代动力学曲线。PR004282在雄性猴体内的半衰期约为3天。
表20.PR004282在正常猴体内的药代动力学(单剂量)
Figure PCTCN2021103058-appb-000042
实施例21.BALB/c-hCD137/CT26-B7-H4小鼠模型中B7-H4×4-1BB双特异性抗体的抗肿瘤药效评估
为了评估B7-H4×4-1BB双特异性抗体的体内抗肿瘤药效，采用6-8周雌性BALB/c-hCD137小鼠，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 5CT26/hB7-H4细 胞。当平均肿瘤体积达到80mm 3时对小鼠进行随机分组，每组6只小鼠。分组后将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、每周给药2次总共给药6次(BIW*3)的方式进行给药，以PBS为空白对照组。在初次给药当天及之后第4、7、11、14和18天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图24所示B7-H4×4-1BB双特异性抗体在BALB/c-hCD137/CT26-B7-H4小鼠模型中的抗肿瘤药效。如图24-(A)所示，B7-H4×4-1BB双特异性抗体PR003334在18mpk和6mpk浓度下对小鼠均有抑制肿瘤生长的药效。如图24-(B)所示，受试各组小鼠体重变化均在正常范围内。
实施例22.MDA-MB-468异种移植小鼠模型中B7-H4×4-1BB双特异性抗体的抗肿瘤药效评估
为了评估B7-H4×4-1BB双特异性抗体的体内抗肿瘤药效。采用6-8周雌性NCG小鼠，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 6MDA-MB-468细胞。当平均肿瘤体积达到120mm 3时对小鼠进行随机分组，每组6只小鼠。分组后接种人5×10 6PBMC细胞，第二天将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、每周给药2次总共给药6次(BIW*3)的方式进行给药，以同型对照IgG为对照组。在初次给药当天及之后第7、12、15、19、22、26和29天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图25所示B7-H4×4-1BB双特异性抗体在MDA-MB-468异种移植小鼠模型中的抗肿瘤药效。如图25-(A)所示，B7-H4×4-1BB双特异性抗体PR003338在18mpk浓度下对小鼠有抑制肿瘤生长的药效，且优于Urelumab在15mpk浓度下的药效。如图25-(B)所示，受试各组小鼠体重变化均在正常范围内。
实施例23.OVCAR3异种移植小鼠模型中B7-H4×4-1BB双特异性抗体的抗肿瘤药效评估
为了评估B7-H4×4-1BB双特异性抗体的体内抗肿瘤药效。采用6-8周雌性NCG小鼠，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 6OVCAR3细胞。当平均肿瘤体积达到120mm 3时对小鼠进行随机分组，每组6只小鼠。分组后接种人5×10 6PBMC细胞，第二天将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、每周给药2次总共给药6次(BIW*3)的方式进行给药，以同型对照IgG为对照组。在初次给药当天及之后第7、 12、15、19、22、26和29天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图26所示B7-H4×4-1BB双特异性抗体PR004282在OVCAR3异种移植小鼠模型中的抗肿瘤药效。如图26-(A)所示，B7-H4×4-1BB双特异性抗体PR004282在18mpk浓度下对小鼠有抑制肿瘤生长的药效。如图26-(B)所示，受试各组小鼠体重变化均在正常范围内。
实施例24.BALB/c-hCD137/CT26-hB7-H4小鼠模型中B7-H4×4-1BB双特异性抗体的抗肿瘤药效和记忆性免疫效应评估
为了评估B7-H4×4-1BB双特异性抗体的体内抗肿瘤药效。采用6-8周雌性BALB/c-hCD137小鼠，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 5CT26-B7-H4细胞。当平均肿瘤体积达到80mm 3时对小鼠进行随机分组，每组6只小鼠。分组后将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、每周给药2次总共给药6次(BIW*3)的方式进行给药，以PBS为空白对照组，Urelumab作为阳性对照组。在初次给药当天及之后持续45天内一周三次对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。给药后45天对肿瘤消除的小鼠的另一侧皮下接种5×10 5CT26/hB7H4细胞，以同龄小鼠作为新接种对照组。一周三次对肿瘤体积和小鼠体重进行测量，第66天处死老鼠。
图27所示B7-H4×4-1BB双特异性抗体PR004282在BALB/c-hCD137/CT26-B7-H4小鼠模型中的抗肿瘤药效和记忆性免疫效果。如图27-(A)-(D)所示，B7-H4×4-1BB双特异性抗体PR004282在1mpk浓度下对小鼠有抑制肿瘤生长的药效，与Urelumab药效相当。如图27-(E)所示，受试各组小鼠体重变化均在正常范围内。如图27--(F)-(I)所示，肿瘤消除后的小鼠新接种同样的肿瘤后与未接种未打药的同龄的小鼠相比，肿瘤不再生长，说明PR004282能够诱导记忆性免疫T细胞的产生从而可以持续长效的杀伤肿瘤。
实施例25.B7-H4×4-1BB双特异性抗体的抗肿瘤药效特异性评估
25.1 JIMT-1异种移植小鼠模型评估B7-H4×4-1BB双特异性抗体的抗肿瘤药效特异性
采用6-8周雌性NCG小鼠，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 6JIMT-1细胞。当平均肿瘤体积达到120mm 3时对小鼠进行随机分组，每组6只小鼠。分组后接种人5×10 6PBMC细胞，第二天将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、 每周给药2次总共给药6次(BIW*3)的方式进行给药，以同型对照IgG为对照组。在初次给药当天及之后第7、12、15、19、22、26和29天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图28–(A)PR004282在JIMT-1异种移植小鼠模型中的抗肿瘤药效。如图所示，PR004282在18mpk浓度下对小鼠无抑制肿瘤生长的药效，而Urelumab在15mpk浓度下则显示出一定的药效，JIMT-1是不表达B7H4的细胞，说明PR004282不能杀伤不表达B7H4的肿瘤细胞。
25.2 BALB/c-hCD137/CT26小鼠模型评估B7-H4×4-1BB双特异性抗体的抗肿瘤药效特异性
为了评估B7-H4×4-1BB双特异性抗体的体内抗肿瘤药效的特异性。采用6-8周雌性BALB/c-hCD137小鼠，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 5CT26野生型细胞。当平均肿瘤体积达到80mm 3时对小鼠进行随机分组，每组6只小鼠。分组后将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、每周给药2次总共给药6次(BIW*3)的方式进行给药，以PBS为空白对照组。在初次给药当天及之后第4、7、11、14和18天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图28-(B)：B7-H4×4-1BB双特异性抗体PR004282在BALB/c-hCD137/CT26小鼠模型中没有显示出对小鼠较强的抑制肿瘤生长的药效，而Urelumab显示出很强的药效。由于CT26是不表达B7H4的细胞，说明PR004282不能杀伤不表达B7H4的细胞。另一方面，根据实施例24中的结果，在CT26细胞上过表达人B7H4以后，该CT26-hB7-H4肿瘤细胞的生长会受到PR004282的抑制。结合两个实验可以推论出，PR004282对肿瘤细胞的杀伤是B7H4表达依赖的。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (25)

  1. 一种双特异性抗体，其包含靶向B7-H4的抗原结合结构域和靶向4-1BB的抗原结合结构域。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述的靶向B7-H4的抗原结合结构域含有HCDR1、HCDR2和HCDR3，所述的HCDR1含有如SEQ ID NO.16或者23所示的氨基酸序列，所述的HCDR2含有如SEQ ID NO.46、59或者60所示的氨基酸序列，所述的HCDR3含有如SEQ ID NO.98或者84所示的氨基酸序列；
    较佳地，所述的靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:46所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3；序列如SEQ ID NO:23所示的HCDR1、序列如SEQ ID NO:59所示的HCDR2以及序列如SEQ ID NO:98所示的HCDR3；或者，序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:60所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3；优选包含序列如SEQ ID NO:142所示、如SEQ ID NO:159所示或者如SEQ ID NO:160所示的VH；更优选包含序列如SEQ ID NO:174所示、如SEQ ID NO:191所示或者如SEQ ID NO:192所示的重链；
    更佳地，所述的靶向B7-H4的抗原结合结构域还含有LCDR1、LCDR2和LCDR3，所述的LCDR1的氨基酸序列如SEQ ID NO.112或者113所示，所述的LCDR2的氨基酸序列如SEQ ID NO.118所示，所述的LCDR3的氨基酸序列如SEQ ID NO.131～133中任一个所示；
    进一步更佳地，所述的靶向B7-H4的抗原结合结构域还含有序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:131所示的LCDR3；序列如SEQ ID NO:113所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:132所示的LCDR3；或者序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:133所示的LCDR3；优选还含有序列如SEQ ID NO:166～169任一个所示的VL；更优选还含有序列如SEQ ID NO:198～201任一个所示的轻链。
  3. 如权利要求2所述的双特异性抗体，其特征在于，所述的靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:46所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3，序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:131所示的LCDR3；序列 如SEQ ID NO:23所示的HCDR1、序列如SEQ ID NO:59所示的HCDR2以及序列如SEQ ID NO:98所示的HCDR3，序列如SEQ ID NO:113所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:132所示的LCDR3；或者，序列如SEQ ID NO:16所示的HCDR1、序列如SEQ ID NO:60所示的HCDR2以及序列如SEQ ID NO:84所示的HCDR3，序列如SEQ ID NO:112所示的LCDR1、序列如SEQ ID NO:118所示的LCDR2以及序列如SEQ ID NO:133所示的LCDR3；
    较佳地，所述的靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:142所示的VH和序列如SEQ ID NO:166所示的VL；序列如SEQ ID NO:159所示的VH和序列如SEQ ID NO:167所示的VL；序列如SEQ ID NO:160所示的VH和序列如SEQ ID NO:168所示的VL；或者序列如SEQ ID NO:159所示的VH和序列如SEQ ID NO:169所示的VL；
    更佳地，所述靶向B7-H4的抗原结合结构域含有序列如SEQ ID NO:174所示的重链和序列如SEQ ID NO:198所示的轻链；序列如SEQ ID NO:191所示的重链和序列如SEQ ID NO:199所示的轻链；序列如SEQ ID NO:192所示的重链和序列如SEQ ID NO:200所示的轻链；或者，序列如SEQ ID NO:191所示的重链和序列如SEQ ID NO:201所示的轻链。
  4. 如权利要求2所述的双特异性抗体，其特征在于，所述的靶向B7-H4的抗原结合结构域为单个VH、串联VH、ScFv、Fab或者IgG的形式；所述的串联VH较佳地为2个以上VH串联，如2个、3个或者4个；当为IgG形式时，其含有的恒定区较佳地来自含有突变L234A、L235A和P329G的人IgG1或者含有突变L234A和L235A的人IgG1。
  5. 如权利要求1～4任一项所述的双特异性抗体，其特征在于，所述的靶向4-1BB的抗原结合结构域含有HCDR1、HCDR2和HCDR3，所述的HCDR1的序列如SEQ ID NO:19或其变体1、或者SEQ ID NO:14所示，所述的HCDR2的序列如SEQ ID NO:49或其变体2、SEQ ID NO:51或者SEQ ID NO:43所示，所述的HCDR3的序列如SEQ ID NO:86或其变体3、SEQ ID NO:96、SEQ ID NO:89或者SEQ ID NO:81所示；其中：
    所述的变体1的突变包括T3I、S6N/R以及Y7F中的一个或多个；较佳地，所述变体1的序列优选如序列表中SEQ ID NO:17～18以及SEQ ID NO:20～22中任一个所示；
    所述变体2的突变包括S1N/D、G2S/A、S3D/G、G5D/F/S/V以及S6T/N/D中的一个或多个；所述变体2的序列优选如序列表中SEQ ID NO:47～48、SEQ ID NO:50、SEQ ID NO:52～58以及SEQ ID NO:61中的任一个所示；
    所述的变体3的突变包括G2R/D/A/K、S3A/T、S4G/N/A/T/H、E5T/V/M/G、T6A、 D7G/S、H9Y/S、Y10H、Y11F、N12G/D以及V13I/M/T中的一个或多个；所述变体3的氨基酸序列优选如序列表中SEQ ID NO:85、SEQ ID NO:87～88以及SEQ ID NO:90～95中的任一个所示；
    较佳地，所述的靶向4-1BB的抗原结合结构域含有：序列分别如SEQ ID NO:17、47和85所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、49和87所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:19、50和88所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:20、51和89所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、52和90所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、49和90所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:21、53和91所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:21、54和92所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:19、55和93所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、49和86所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:19、49和94所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:22、56和86所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、57和95所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:19、58和96所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:18、61和95所示的HCDR1、HCDR2和HCDR3；
    或者，序列分别如SEQ ID NO:14、43和81所示的HCDR1、HCDR2和HCDR3；
    更佳地，所述的靶向4-1BB的抗原结合结构域含有一个或者多个序列如SEQ ID NO:143～157、139、284或者161所示的重链可变区；
    进一步更佳地，所述的靶向4-1BB的抗原结合结构域包含序列如SEQ ID NO:175～189、193、285或者171所示的重链。
  6. 如权利要求5所述的双特异性抗体，其特征在于，所述的靶向4-1BB的抗原结合结构域还包含序列分别如SEQ ID NO:109、118和128所示的LCDR1、LCDR2和LCDR3；较佳地，包含序列如SEQ ID NO:163所示的轻链可变区；更佳地，包含序列如SEQ ID NO:195所示的轻链。
  7. 如权利要求5所述的双特异性抗体，其特征在于，所述的靶向4-1BB的抗原结合结构域为单个VH或者串联VH的形式、HCAb的形式或者ScFv的形式；所述的串联VH 较佳地为2个以上VH串联，如2个、3个或者4个。
  8. 如权利要求1～7任一项所述的双特异性抗体，其特征在于，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:109、118和128所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:14、43和81所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:109、118和128所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:14、43和81所示的HCDR1、HCDR2和HCDR3；
    所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:17、47和85所示的HCDR1、HCDR2和HCDR3；
    所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和87所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、50和88所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如 SEQ ID NO:20、51和89所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、52和90所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:21、53和91所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:21、54和92所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、55和93所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和86所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、49和94所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:22、56和86所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和131所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、46和84所示 的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、57和95所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:17、47和85所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和86所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、49和94所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:112、118和133所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:16、60和84所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、61和95所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、48和86所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:19、58和96所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118 和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、57和95所示的HCDR1、HCDR2和HCDR3；
    或者，所述的靶向B7-H4的抗原结合结构域包含序列分别如SEQ ID NO:113、118和132所示的LCDR1、LCDR2和LCDR3和序列分别如SEQ ID NO:23、59和98所示的HCDR1、HCDR2和HCDR3；且所述的靶向4-1BB的抗原结合结构域包含序列分别如SEQ ID NO:18、49和90所示的HCDR1、HCDR2和HCDR3。
  9. 如权利要求1～8任一项所述的双特异性抗体，其特征在于，所述靶向B7-H4的抗原结构域为IgG的形式，所述靶向4-1BB的抗原结合结构域为单个VH的形式；
    或者，所述靶向B7-H4的抗原结构域为IgG的形式，所述靶向4-1BB的抗原结合结构域为ScFv的形式；
    或者，所述靶向B7-H4的抗原结构域为IgG的形式，所述靶向4-1BB的抗原结合结构域为2个或者3个VH串联的形式；
    或者，所述靶向B7-H4的抗原结构域为Fab的形式，所述靶向4-1BB的抗原结合结构域为HCAb的形式或者为HCAb的形式和VH的形式(HCAb-VH形式)；
    或者，所述靶向B7-H4的抗原结构域为Fab的形式，所述靶向4-1BB的抗原结合结构域为VH的形式，所述VH的形式优选单个VH、2个或者3个VH串联的形式；
    较佳地，所述的双特异性抗体为如下形式：
    (1)IgG的C端连接有ScFv，所述ScFv的VH连接于所述C端；较佳地，该形式的双特异性抗体的多肽链1如式：VLB7-H4-CL所示，多肽链2如式：VHB7-H4-CH1-铰链-CH2-CH3-接头-VH4-1BB-接头-VL4-1BB所示；
    (2)IgG的C端连接有VH或者串联VH；较佳地，该形式的双特异性抗体的多肽链1如式：VLB7-H4-CL所示，多肽链2如式：VHB7-H4-CH1-铰链-CH2-CH3-接头-(VH4-1BB)n所示；
    (3)HCAb的N端连接有Fab；较佳地，该形式的双特异性抗体的多肽链1如式：VHB7-H4-CH1所示，多肽链2如式：VLB7-H4-CL-接头-VH4-1BB-接头-CH2-CH3所示；
    (4)HCAb的N端连接有Fab，所述Fab的C端连接有VH；较佳地，该形式的双特异性抗体的多肽链1如式：VHB7-H4-CH1所示，多肽链2如式：VLB7-H4-CL-接头-VH4-1BB-接头-CH2-CH3-VH4-1BB所示；
    或者(5)Fc的2个CH2的N端分别连接有Fab，以及VH或者串联VH；较佳地，该形式的双特异性抗体含三条多肽链：
    多肽链1如式：VLB7-H4-CL所示，多肽链2如式：VHB7-H4-CH1-铰链-CH2-CH3所示，多肽链3如式：VH4-1BB-接头-CH2-CH3或者(VH4-1BB)n-接头-CH2-CH3所示。
  10. 如权利要求9所述的双特异性抗体，其特征在于，所述的接头选自由SEQ ID NO:241～261、282以及288～289构成的群组；其中：
    形式(1)的接头优选含有如SEQ ID NO:245所示的序列；
    形式(2)的接头优选含有如SEQ ID NO:243、245～247以及SEQ ID NO:288～289任一所示的序列；
    形式(3)的接头优选含有如SEQ ID NO:250所示的序列；
    形式(4)的接头优选含有如SEQ ID NO:282所示的序列；
    形式(5)的接头优选含有如SEQ ID NO:245所示的序列。
  11. 如权利要求10所述的双特异性抗体，其特征在于，所述多肽链1的氨基酸序列如SEQ ID NO:198所示，且所述多肽链2的氨基酸序列如SEQ ID NO:202～215中任一个所示；
    或者，所述多肽链1的氨基酸序列如SEQ ID NO:201所示，且所述多肽链2的氨基酸序列如SEQ ID NO:217、230、238、240、226、262或者239所示；
    或者，所述多肽链1的氨基酸序列如SEQ ID NO:200所示，且所述多肽链2的氨基酸序列如SEQ ID NO:218～225、235～237、263～268、274～281、286以及287中任一个所示；
    或者，所述多肽链1的氨基酸序列如SEQ ID NO:201所示，所述多肽链2的氨基酸序列如SEQ ID NO:227所示，且所述多肽链3的氨基酸序列如SEQ ID NO:228、229、231或者232所示；
    或者，所述多肽链1的氨基酸序列如SEQ ID NO:233所示，且所述多肽链2的氨基酸序列如SEQ ID NO:234、269～273中的任一个所示。
  12. 一种分离的核酸，其编码如权利要求1～11任一项所述的双特异性抗体。
  13. 一种包含如权利要求12所述的分离的核酸的表达载体。
  14. 一种宿主细胞，其包含根据权利要求13所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞。
  15. 一种双特异性抗体的制备方法，其包含培养如权利要求14所述的宿主细胞，从培养物中获得所述双特异性抗体。
  16. 一种药物组合物，其包含如权利要求1～11任一项所述的双特异性抗体。
  17. 如权利要求1～11任一项所述的双特异性抗体或者如权利要求16所述的药物组合物在制备防治4-1BB和/或B7-H4相关的疾病的药物中的应用；
    所述的疾病优选癌症，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤或者结直肠癌；较佳地，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌或胆管癌；更佳地，所述的癌症优选乳腺癌。
  18. 一种嵌合抗原受体，其包含如权利要求1～11任一项所述的双特异性抗体。
  19. 一种抗体药物偶联物，其包含细胞毒性剂，以及如权利要求1～11任一项所述的双特异性抗体；优选地，所述细胞毒性剂为MMAF或MMAE。
  20. 试剂盒，其包括如权利要求1～11任一项所述的双特异性抗体、如权利要求18所述的嵌合抗原受体、如权利要求19中所述的抗体药物偶联物和/或如权利要求16所述的药物组合物；
    优选地，所述试剂盒还包括(i)施用抗体或其抗原结合片段或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
  21. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A含有如权利要求1～11任一项所述的双特异性抗体、如权利要求18所述的嵌合抗原受体、如权利要求19所述的抗体药物偶联物和/或如权利要求16所述的药物组合物；
    所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  22. 一种诊断、治疗和/或预防4-1BB和/或B7-H4介导的疾病或病症的方法，所述方法包括向有需要的患者施用治疗有效量的如权利要求1～11任一项所述的双特异性抗体、如权利要求18所述的嵌合抗原受体、如权利要求19所述的抗体药物偶联物或如权利要求16所述的药物组合物，或者使用如权利要求21所述的套装药盒治疗有需要的患者。
  23. 如权利要求22所述的方法，其特征在于，所述的疾病或病症为肿瘤，优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤或者结直肠癌；较佳地，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌或胆管癌；更佳地，所述的癌症优选乳腺癌。
  24. 一种免疫检测或者测定4-1BB或B7H4的方法，其包括使用如权利要求1～11任 一项所述的双特异性抗体、如权利要求18所述的嵌合抗原受体、如权利要求19所述的抗体药物偶联物或如权利要求16所述的药物组合物；优选地，所述检测为非诊断和/或治疗目的的。
  25. 一种联合疗法，其包括分别向有需要的患者施用如权利要求1～11任一项所述的双特异性抗体、如权利要求18所述的嵌合抗原受体、如权利要求19所述的抗体药物偶联物或如权利要求16所述的药物组合物，和第二治疗剂；所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
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