WO2022002009A1 - 靶向ox40的抗体及其制备方法和应用 - Google Patents

靶向ox40的抗体及其制备方法和应用 Download PDF

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WO2022002009A1
WO2022002009A1 PCT/CN2021/102946 CN2021102946W WO2022002009A1 WO 2022002009 A1 WO2022002009 A1 WO 2022002009A1 CN 2021102946 W CN2021102946 W CN 2021102946W WO 2022002009 A1 WO2022002009 A1 WO 2022002009A1
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seq
amino acid
antibody
cdr1
cdr3
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French (fr)
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石磊
黄冰
王健
贾星星
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和铂医药(上海)有限责任公司
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Priority to CN202180030974.2A priority Critical patent/CN115461371A/zh
Priority to US18/013,707 priority patent/US20230295324A1/en
Publication of WO2022002009A1 publication Critical patent/WO2022002009A1/zh

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Definitions

  • the present application relates to the field of biomedicine, in particular to an OX40-targeting antibody or an antigen-binding fragment thereof, as well as a preparation method and application thereof, and a bispecific comprising the OX40-targeting antibody or its antigen-binding fragment antibody.
  • OX40 also known as CD134, tumor necrosis factor receptor superfamily member 4 (TNFRSF4), is one of the tumor necrosis factor receptor superfamily members (TNFRSF), it is a 50kDa ⁇ 55kDa type I transmembrane glycoprotein, Has an intracytoplasmic part, a transmembrane domain and an extracellular domain. It is involved in enhancing the T cell response triggered by T Cell Receptor (TCR) and is a costimulatory receptor molecule. OX40 is mainly expressed on the surface of activated CD4+ and CD8+ T cells after TCR stimulation.
  • TCR T Cell Receptor
  • OX40 has also been reported to be expressed on human neutrophils (the signaling pathway supports survival) and on murine natural killer (NK) and NK T cells (Baumann R et al., (2004) Eur J Immunol. 34 (8) ): 2268-2275; Croft et al., (2009) Nat Rev Immunol. 9(4): 271-285.).
  • OX40L The only known ligand for OX40 is OX40L (TNFSF4), a type II transmembrane protein containing a conserved tumor necrosis factor (TNF) homology domain that enables trimerization (Bodmer JL et al., (2002) Trends Biochem Sci. 27(1): 19-26). Upon activation, three OX40 molecules can bind to the OX40L trimer, a typical feature of ligand-receptor pairing in TNFRSF (Banner DW et al., (1993) Cell. 73(3):431-445.) .
  • OX40L is induced on human dendritic cells (DC) following exposure to thymic stromal lymphopoietin (Krause P et al., (2009) Blood. 113(11):2451-2460).
  • DC dendritic cells
  • human monocytes, neutrophils, mast cells, lymphoid tissue-inducing cells, smooth muscle cells, endothelial cells and in vitro activated B cells all express OX40L under appropriate conditions (Byun M et al., (2013) J Exp Med. 210(9): 1743-1759; Karulf M et al., (2010) J Immunol. 185(8): 4856-4862).
  • TRAF TNF receptor-associated factor
  • OX40L-Fc fusion protein MEDI6383 OX40L fusion Protein
  • Others are agonistic antibodies, such as the OX40 antibody MEDI6469 (9B12, mouse anti-human OX40 mAb, replaced by MEDI0562 humanized mAb), MEDI0562 (AstraZeneca, Tavolixizumab), Ivuxolimab (Pfizer, PF-04518600), GSK3174998 (GSK), BMS-986178 (BMS), Pogalizumab (MOXRO0916/RG7888), etc.
  • Pogalizumab also known as Vonlerolizumab
  • Vonlerolizumab is a humanized OX40 antibody developed by Roche and is currently in Phase II clinical use for the treatment of solid tumors.
  • anti-OX40 mAbs produce deleterious immunosuppressive side effects by promoting MDSC accumulation and Th2 cytokine production (Gough MJ et al., (2012) Immunology 136:437e47.).
  • agonist mAbs targeting OX40 can confer tumor protection in mice, their role is limited in less immunogenic settings (Kjaergaard J et al., (2000) Cancer Res. 60 (19) ): 5514-5521).
  • VHH Heavy chain antibodies and nanobodies
  • VHH are superior to traditional antibodies in many aspects. They have small molecular weight and can penetrate the blood-brain barrier. Weakness. And heavy chain antibodies or nanobodies are especially suitable for the development of bispecific antibodies, and can solve the problems of light chain mismatch and heterodimerization. In the prior art, however, there are almost no relevant reports on OX40 heavy chain-only antibodies.
  • the technical problem to be solved by the present invention is to provide an antibody targeting OX40 and its preparation method and application, as well as a bispecific antibody developed based on the antibody and its application, in order to overcome the deficiencies of the current antibody targeting OX40.
  • the antibody or antigen-binding fragment thereof of the present invention has the activity of specifically binding to human OX40 and cynomolgus monkey (cyno) OX40.
  • the antibody or antigen-binding fragment thereof of the present invention can promote the greater activation of NF-Kb, thereby stimulating the OX40 signaling pathway, and can activate the OX40 pathway in vitro and induce the function of activating T cells.
  • Existing antibodies are comparable or stronger, while the antibodies or antigen-binding fragments thereof of the present invention are cross-linking dependent on one of the Fc ⁇ receptor members, Fc ⁇ RIIB (CD32B).
  • Fc ⁇ RIIB Fc ⁇ RIIB
  • the antibody of the present invention or its antigen-binding fragment is prepared into a bispecific antibody, the obtained bispecific antibody can bind to human OX40 and the corresponding tumor-associated antigen, and one end of the antibody can recognize the tumor target TAA (TAA) that is specifically expressed on the surface of tumor cells.
  • TAA tumor target TAA
  • PSMA ECAM
  • CLDN18.2, B7H4, PD-L1 tumor target TAA
  • the other end can bind to OX40 molecules on T cells, which can recruit and activate T cells near tumor cells, thereby killing tumor cells.
  • the first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting OX40, which comprises a heavy chain variable region (VH),
  • VH comprises the following complementarity determining regions (CDRs) or mutations thereof: VH CDR1 as shown in the amino acid sequence of SEQ ID NO: 10; VH CDR2 as shown in the amino acid sequence of SEQ ID NO: 44; and/ or, a VH CDR3 as shown in the amino acid sequence of SEQ ID NO:86, SEQ ID NO:84 or SEQ ID NO:89;
  • CDRs complementarity determining regions
  • the mutation is an insertion, deletion or substitution of 3, 2 or 1 amino acid respectively on the basis of the amino acid sequence of VH CDR1, VH CDR2 and VH CDR3 of the VH.
  • amino acid mutation in “insertion, deletion or substitution with 3, 2 or 1 amino acid” refers to the amino acid mutation in the sequence of the variant compared to the original amino acid sequence, including the original amino acid sequence. Amino acid insertions, deletions or substitutions occur on the basis of the sequence.
  • the mutation of CDRs can include mutations of 3, 2 or 1 amino acid, and the same or different numbers of amino acid residues can be optionally selected for mutation among these CDRs, for example, 1 mutation of CDR1 can be performed. 1 amino acid mutation, no amino acid mutation for CDR2 and CDR3.
  • the mutations may include mutations that are currently known to those skilled in the art, for example, during the production or application of antibodies, some mutations that may be performed on antibodies, such as transcription of possible existing, especially CDR regions Post-modification (Potential post-translational modifications, PTMs) site mutation, including antibody aggregation, deamidation (asparagine deamidation, site (NG, NS, NH, etc.), aspartic acid isomerism (DG, DP) sensitive sites, N-glycosylation (N- ⁇ P ⁇ S/T) sensitive sites and oxidation sensitive sites and other related mutations.
  • PTMs Post-modification
  • PTMs post-translational modifications
  • the mutation of the VH CDR1 is that F2, T3, S5, and/or S6 are mutated to L, S, P, I, D and/or C on the amino acid sequence shown in SEQ ID NO: 10, preferably at The amino acid sequence shown in SEQ ID NO: 10 has amino acid substitutions of F2L, T3S/P/I, S5D, and/or S6D/C; its amino acid sequence is, for example, as SEQ ID NO: 13-16, SEQ ID NO: Any of 20-24 are shown.
  • the VH CDR2 is mutated to T, H, L, G, S, N, D, I at the amino acid sequence shown in SEQ ID NO: 44 from S1, R3, G4, G5 and/or S6 to T, H, L, G, S, N, D, I and/or Q, preferably having 3, 2 or 1 of S1T, R3H/L/G/S, G4S, G5N/D, S6N/I/Q/T on the amino acid sequence shown in SEQ ID NO: 44 Amino acid substitution; its amino acid sequence is shown in any of SEQ ID NO: 42-43, SEQ ID NO: 45-50, SEQ ID NO: 54-60, for example.
  • the VH CDR3 is mutated to T2, T5, T6, D9 and/or Y10 on the amino acid sequence shown in SEQ ID NO: 86 to M, I, V, S, W, Y, C, F and/or W, preferably have 2 or 1 amino acid substitutions in T2M/I/V, T5S, T6W/Y, D9C and Y10F/W on the amino acid sequence shown in SEQ ID NO: 86; its amino acid sequence such as As shown in any of SEQ ID NO: 82-83, SEQ ID NO: 85, SEQ ID NO: 87-88, SEQ ID NO: 90, SEQ ID NO: 94-99.
  • F2L generally refers to the mutation of the second amino acid F of the amino acid sequence shown in SEQ ID NO: 10 to L, and other amino acid substitutions such as T3S/P/I, S5D, and/or S6D/C, etc.
  • substitutions such as T3S/P/I, S5D, and/or S6D/C, etc.
  • the OX40-targeting antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises the following complementarity determining regions (CDRs) or mutations thereof: such as SEQ ID NO : 10, VH CDR1 shown in any amino acid sequence of SEQ ID NO: 13-16, SEQ ID NO: 20-24; such as any amino acid sequence of SEQ ID NO: 42-50, SEQ ID NO: 54-60 The VH CDR2 shown; and/or, the VH CDR3 shown in any of the amino acid sequences of SEQ ID NOs: 82-90, SEQ ID NOs: 94-99.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 13, 42 and 82, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 42 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 14, 42 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 43 and 84, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 13, 44 and 82, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 15, 44 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 45 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 46 and 85, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 16, 42 and 82, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 47 and 87, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 16, 44 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 45 and 88, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 48 and 89, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 49 and 90, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 49 and 83, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 50 and 90, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 20, 44 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 21, 44 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 54 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 55 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 56 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 42 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 57 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 58 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 59 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 60 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 94, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 95, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 96, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 97, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 98, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 44 and 99, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 54 and 95, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 54 and 97, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 55 and 95, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 55 and 97, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 56 and 95, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 56 and 97, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 22, 54 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 23, 54 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 24, 54 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 21, 54 and 86, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 10, 54 and 99, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 22, 54 and 99, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 23, 54 and 99, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 24, 54 and 99, respectively.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises a heavy chain variable region (VH), the VH comprises VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of which is These are shown in SEQ ID NOs: 21, 54 and 99, respectively.
  • VH heavy chain variable region
  • the above-mentioned VH also includes a heavy chain variable region framework region (VH FWR); it may for example be selected from germline IGHV3-23 or backmutation thereof. More preferably, the VH FWR is a heavy chain variable region framework region of a human antibody.
  • VH FWR heavy chain variable region framework region
  • the VH comprises any amino acid sequence shown in SEQ ID NO: 142-164 or SEQ ID NO: 168-198 or a mutation thereof; the mutation is that one or more amino acid sequences have occurred on the VH amino acid sequence Deletion, substitution or addition of amino acid residues, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the VH, and maintains or improves the binding of the antibody to OX40; the at least 85
  • the % sequence identity is preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.
  • the amino acid sequences of the CDRs listed above are all shown in accordance with the Chothia definition rules (the sequences in the claims of the present application are also shown in accordance with the Chothia definition rules).
  • the CDRs of antibodies can be defined by a variety of methods in the art, for example, based on the Kabat definition rules for sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • amino acid residues in variable domain sequences can also be determined using the Combined definition rule comprising the Kabat definition and the Chothia definition.
  • the Combined definition rule is to combine the range defined by Kabat and Chothia, and a larger range is taken based on this, see Table a for details.
  • CDRs and “complementarity determining regions” of a given antibody or regions thereof (eg, variable regions) are to be understood to encompass the above-described already described above as described by the present invention. complementarity-determining regions defined by any of the known schemes.
  • Haa-Hbb may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain, from the aa-th position to the bb-th position.
  • H26-H32 may refer to the amino acid sequence from position 26 to position 32 according to the Chothia coding rules, starting from the N-terminus of the antibody heavy chain.
  • the VH contains the CDRs described in Table b below.
  • the OX40-targeting antibody or antigen-binding fragment thereof further comprises the heavy chain constant region Fc domain of a human antibody.
  • the heavy chain constant region Fc domain of the human antibody includes, for example, the heavy chain constant region Fc domain of human IgG1, IgG2, IgG3 or IgG4.
  • the OX40-targeting antibody or antigen-binding fragment thereof comprises a polypeptide chain comprising any of the amino acid sequences shown in SEQ ID NOs: 208-230 or SEQ ID NOs: 234-264 or its mutation.
  • the mutation is the deletion, substitution or addition of one or more amino acid residues in the amino acid sequence, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence, and maintains or improves the Binding of said antibody to OX40; said at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most preferably at least 99% sequence identity.
  • the antibody or antigen-binding fragment thereof targeting OX40 comprises IgG, Fab, Fab', F(ab') 2 , Fv, scFv, HCAb, VH, bispecific antibody, multispecific antibody, single domain
  • an antibody or any other antibody that retains the ability of the antibody to specifically bind to an antigen (which may be part of the ability of the antibody to specifically bind to an antigen), or a monoclonal antibody or a polyclonal antibody prepared from the above-mentioned antibody.
  • the antibody targeting OX40 or an antigen-binding fragment thereof is a blocking antibody.
  • the antibody or antigen-binding fragment thereof targeting OX40 is a weakly blocking or non-blocking antibody.
  • the "Fab fragment” is composed of the CH1 and variable regions of a light chain and a heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc” region contains two heavy chain fragments of the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • Said "Fab fragment” contains a light chain and part of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains, whereby the two heavy chains of the two Fab' fragments can be separated. Interchain disulfide bonds are formed between them to form F(ab') 2 molecules.
  • F(ab') 2 fragment contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide between the two heavy chains key.
  • the F(ab') 2 fragment therefore consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • the term "Fv” means an antibody fragment consisting of the one-armed VL and VH domains of an antibody, but lacking the constant regions.
  • the scFv single chain antibody fragment, single chain antibody
  • the scFv can be a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids .
  • the VL and VH domains form monovalent molecules by pairing of linkers that enable them to be produced as a single polypeptide chain [see, eg, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad . Sci. USA 85: 5879-5883 (1988)].
  • Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linker consists of repeated amino acid sequences of G 4 S, or a variant thereof. For example, a 3 amino acid linker having the sequence (G 4 S) 4, or (G 4 S), but may also be used variants thereof.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitope specificity; having two or more Antibodies with VL and VH regions, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable regions, each single variable region with Different targets or different epitopes of the same target bind; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments covalently or non-covalently linked together Wait.
  • the monoclonal antibody or mAb or Ab refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • the single-domain antibody can be a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
  • the second aspect of the present invention provides a bispecific binding protein, which contains at least two protein functional domains: protein functional domain A and protein functional domain B; the protein functional domain A and the protein functional domain B
  • the protein functional domain B targets different antigens, wherein the protein functional domain B targets OX40, and the protein functional domain A targets non-OX40 antigens; the protein functional domain B is selected from the targets described in the first aspect of the present invention An antibody or antigen-binding fragment thereof to OX40.
  • the protein functional domain A targets PD-L1, B7H4, PSMA, EPCAM or CLDN18.2.
  • the protein functional region A is PSMA antibody or its antigen-binding fragment, EPCAM antibody or its antigen-binding fragment, CLDN18.2 antibody or its antigen-binding fragment, B7H4 antibody or its antigen-binding fragment, PD-L1 antibody or its antigen-binding fragment Antigen-binding fragments.
  • the PD-L1 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, its amino acid sequence is shown as SEQ ID NO:111,119 and 129 respectively, and described VH comprises VH CDR1, VH CDR2 and VH CDR3, its amino acid sequence is shown as SEQ ID NO:10,39 and 79 respectively.
  • VL light chain variable region
  • VH heavy chain variable region
  • the EPCAM antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, Its amino acid sequence is shown as SEQ ID NO: 112, 120 and 130 respectively, and described VH comprises VH CDR1, VH CDR2 and VH CDR3, and its amino acid sequence is shown as SEQ ID NO: 11, 40 and 80 respectively.
  • the PSMA antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, Its amino acid sequence is shown as SEQ ID NO: 113, 121 and 131 respectively, and described VH comprises VH CDR1, VH CDR2 and VH CDR3, and its amino acid sequence is shown as SEQ ID NO: 12, 41 and 81 respectively.
  • the B7H4 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH);
  • VL comprises VL CDR1, VL CDR2 and VL CDR3,
  • SEQ ID NO: 114, 122 and 132 amino acid sequence is shown as SEQ ID NO: 114, 122 and 132 respectively
  • described VH comprises VH CDR1, VH CDR2 and VH CDR3, and its amino acid sequence is shown as SEQ ID NO: 17, 51 and 91 respectively.
  • the CLDN18.2 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH);
  • VL comprises VL CDR1, VL CDR2 and VL CDR3, its amino acid sequence is shown as SEQ ID NO:112,120 and 133 respectively
  • described VH comprises VH CDR1, VH CDR2 and VH CDR3, its amino acid sequence is shown as SEQ ID NO:18,52 and 92 respectively.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B;
  • the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH);
  • the VL comprises VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 111, 119 and 129, respectively, and the VH comprises VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NOs: 10, 39 and 79, respectively;
  • the protein functional domain B comprises a heavy chain variable region (VH), and the VH comprises VH CDR1, VH CDR2 and VH CDR3, which are The amino acid sequences are shown in SEQ ID NOs: 10, 44 and 86, respectively.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B;
  • the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 114, 122 and 132, respectively, the VH comprising VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NOs: 17, 51 and 91, respectively;
  • the protein functional region B comprises a heavy chain variable region (VH), and the VH comprises VH CDR1, VH CDR2 and VH CDR3, which The amino acid sequences are shown in SEQ ID NOs: 10, 44 and 86, respectively.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B;
  • the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 112, 120 and 130, respectively, the VH comprising VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NOs: 11, 40 and 80, respectively;
  • the protein functional domain B comprises a heavy chain variable region (VH), and the VH comprises VH CDR1, VH CDR2 and VH CDR3, which The amino acid sequences are shown in SEQ ID NOs: 10, 44 and 86, respectively.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B;
  • the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 113, 121 and 131, respectively, the VH comprising VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NOs: 12, 41 and 81, respectively;
  • the protein functional domain B comprises a heavy chain variable region (VH), and the VH comprises VH CDR1, VH CDR2 and VH CDR3, which are The amino acid sequences are shown in SEQ ID NOs: 10, 44 and 86, respectively.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B;
  • the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL comprising VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 112, 120 and 133, respectively, the VH comprising VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NOs: 18, 52 and 92, respectively;
  • the protein functional region B comprises a heavy chain variable region (VH), and the VH comprises VH CDR1, VH CDR2 and VH CDR3, which are The amino acid sequences are shown in SEQ ID NOs: 10, 44 and 86, respectively.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B; the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL includes the amino acid sequence shown in SEQ ID NO: 199, the VH includes the amino acid sequence shown in SEQ ID NO: 139; the protein functional region B includes a heavy chain variable region, the VH comprises the amino acid sequence shown in SEQ ID NO:154.
  • VL light chain variable domain
  • VH heavy chain variable region
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B; the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL includes the amino acid sequence shown in SEQ ID NO: 202, the VH includes the amino acid sequence shown in SEQ ID NO: 165; the protein functional region B includes a heavy A chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:154.
  • VL light chain variable domain
  • VH heavy chain variable region
  • VL includes the amino acid sequence shown in SEQ ID NO: 202
  • the VH includes the amino acid sequence shown in SEQ ID NO: 165
  • the protein functional region B includes a heavy A chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:154.
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B; the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL includes the amino acid sequence shown in SEQ ID NO: 200, the VH includes the amino acid sequence shown in SEQ ID NO: 140; the protein functional region B includes a heavy A chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:154.
  • VL light chain variable domain
  • VH heavy chain variable region
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B; the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL includes the amino acid sequence shown in SEQ ID NO: 201, the VH includes the amino acid sequence shown in SEQ ID NO: 141; the protein functional region B includes a heavy A chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:154.
  • VL light chain variable domain
  • VH heavy chain variable region
  • the bispecific binding protein contains at least two protein functional domains: protein functional domain A and protein functional domain B; the protein functional domain A comprises a light chain variable domain (VL) and a heavy chain variable region (VH), the VL includes the amino acid sequence shown in SEQ ID NO: 203, the VH includes the amino acid sequence shown in SEQ ID NO: 166; the protein functional region B includes a heavy A chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:154.
  • VL light chain variable domain
  • VH heavy chain variable region
  • VL includes the amino acid sequence shown in SEQ ID NO: 203
  • the VH includes the amino acid sequence shown in SEQ ID NO: 166
  • the protein functional region B includes a heavy A chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:154.
  • the protein functional domain A and/or the protein functional domain B is in the form of IgG, Fab, Fab', F(ab') 2 , Fv, scFv, VH, or HCAb; wherein, the protein function Domain A and protein functional domain B are not IgG at the same time.
  • the heavy chain constant region of the IgG is a human heavy chain constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region; wherein the human IgG preferably comprises one of L234A, L235A and P329G, Two or three mutations, more preferably mutations comprising L234A and L235A or mutations comprising L234A, L235A and P329G.
  • the number of said Fab, Fab', F(ab') 2 , Fv, scFv, VH is preferably one or more.
  • the protein functional domain B is a single VH structure, and the protein functional domain A is an IgG structure; the protein functional domain B is preferably connected to the C-terminus of the protein functional domain A.
  • the bispecific antibody comprising a first polypeptide chain and a second polypeptide chain, said first polypeptide chain of formula: N'-VL _ A-CL -C ' of the shows, as a second polypeptide chain of formula: N'-VH _A -CH1-h -CH2-CH3-L- VH _B -C shown ';
  • the VH_B is the VH of the protein functional region B
  • the VL_A and VH_A are the VL and VH of the protein functional region A, respectively
  • the h is the hinge region
  • the hinge region can be conventional in the art, usually contains a large amount of proline, and has elasticity.
  • the length of the L is preferably 0 or its amino acid sequence is shown in any of SEQ ID NO.278-295.
  • CH3 of the above-mentioned second polypeptide chain is directly fused to VH_B, that is, the length of L is 0.
  • CH3 of the second polypeptide chain is linked to VH_B via a linker peptide L; L may be the sequence listed in Table 11 in the Examples.
  • the protein functional region B has the structure of HCAb, and the protein functional region A has the structure of Fab; the protein functional region B is preferably connected to the C-terminus of the protein functional region A.
  • the bispecific antibody comprising a first polypeptide chain and a second polypeptide chain, said first polypeptide chain of formula: N'-VH _A -CH1-C ' shown in FIG. a second polypeptide chain of formula: N'-VL _A -CL-L1 -VH _B -L2-CH2-CH3-C ' shown in FIG.
  • the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain
  • the first polypeptide chain is represented by the formula: N'- VL_A- CL-C' a second polypeptide chain of formula: N'-VH _A -CH1-L1 -VH _B -L2-CH2-CH3-C ' shown in FIG.
  • VH_B is the VH of the protein functional region B
  • VL_A and VH_A are the VL and VH of the protein functional region A, respectively
  • the L1 and L2 are connecting peptides.
  • the length of the L1 or L2 is preferably 0 or its amino acid sequence is preferably as shown in any of SEQ ID NO.278-295, or its amino acid sequence is GS, for example, the amino acid sequence of the L1 is as shown in SEQ ID NO.286 As shown, the amino acid sequence of the L2 is shown in SEQ ID NO.285.
  • the VH_B of the second polypeptide chain is linked to CH2 via the connecting peptide L2;
  • L2 can be the hinge region of IgG or a connecting peptide sequence derived from the hinge region;
  • L2 can be the sequence listed in Table 11, preferably a human IgG1 hinge Either the sequence of human IgG1 hinge (C220S) or G5-LH.
  • the CL of the second polypeptide chain is directly fused to VH_B, that is, the length of L1 is 0.
  • the CL of the second polypeptide chain is linked to VH_B via a linking peptide L1; L1 may be the sequence listed in Table 11 in the Examples.
  • the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 265,
  • the two-polypeptide chain includes the amino acid sequence shown in SEQ ID NO:271.
  • the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 268,
  • the two-polypeptide chain includes the amino acid sequence shown in SEQ ID NO:272.
  • the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 273,
  • the two-polypeptide chain includes the amino acid sequence shown in SEQ ID NO:274.
  • the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 266,
  • the two-polypeptide chain includes the amino acid sequence shown in SEQ ID NO:275.
  • the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 267,
  • the two-polypeptide chain includes the amino acid sequence shown in SEQ ID NO:276.
  • the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 269,
  • the two-polypeptide chain includes the amino acid sequence shown in SEQ ID NO:277.
  • the third aspect of the present invention provides a chimeric antigen receptor, which comprises the antibody targeting OX40 or an antigen-binding fragment thereof as described in the first aspect of the present invention or as described in the second aspect of the present invention.
  • the fourth aspect of the present invention provides an immune cell comprising the chimeric antigen receptor according to the third aspect of the present invention.
  • the immune cells are T cells, or NK cells.
  • the fifth aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof targeting OX40 as described in the first aspect of the present invention, or as described in the second aspect of the present invention
  • the bispecific antibody or the chimeric antigen receptor according to the third aspect of the present invention is provided.
  • the preparation method of the nucleic acid is a conventional preparation method in the field, preferably, comprising the following steps: obtaining a nucleic acid molecule encoding the above-mentioned antibody by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned antibody by artificial full sequence synthesis .
  • substitutions, deletions, alterations, insertions or additions can be appropriately introduced into the base sequence encoding the amino acid sequence of the above-mentioned antibody to provide a homolog of a polynucleotide.
  • Homologs of the polynucleotides of the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the antibody sequence within a range that maintains the activity of the antibody.
  • the sixth aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid according to the fifth aspect of the present invention.
  • the recombinant expression vector can be obtained by conventional methods in the art, that is, the nucleic acid molecule described in this application is connected to various expression vectors to construct.
  • the expression vector is any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule.
  • the expression vector comprises a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
  • the seventh aspect of the present invention provides a transformant comprising the isolated nucleic acid according to the fifth aspect of the present invention or the recombinant expression vector according to the sixth aspect of the present invention.
  • the preparation method of the transformant can be a conventional preparation method in the field, for example, by transforming the above-mentioned recombinant expression vector into a host cell.
  • the host cells of the transformants are various conventional host cells in the field, as long as the above-mentioned recombinant expression vectors can be stably self-replicated and the nucleic acid carried by them can be effectively expressed.
  • the host cells are prokaryotic cells and/or eukaryotic cells, the prokaryotic cells are preferably E.coli cells such as TG1, BL21 (expressing single chain antibody or Fab antibody), the eukaryotic cells are preferably HEK293 cells or CHO cells cells (expressing full-length IgG antibodies).
  • the preferred recombinant expression transformants of the present invention can be obtained by transforming the aforementioned recombinant expression plasmids into host cells.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a thermal shock method or an electrotransformation method.
  • the eighth aspect of the present invention provides a preparation method of an antibody targeting OX40 or an antigen-binding fragment thereof, or a bispecific antibody, which comprises culturing the transformant according to the seventh aspect of the present invention, Antibodies or antigen-binding fragments thereof, or bispecific antibodies targeting OX40 are obtained from the culture.
  • the ninth aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate includes an antibody part and a coupling part, and the antibody part comprises the first aspect of the present invention.
  • the OX40-targeting antibody or antigen-binding fragment thereof and/or the bispecific antibody according to the second aspect of the present invention the conjugated moieties include, but are not limited to, detectable labels, drugs, toxins, cytokines, radioactivity Nuclide, enzyme, or a combination thereof, the antibody moiety and the conjugation moiety are coupled via a chemical bond or linker.
  • the tenth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising the antibody targeting OX40 or its antigen-binding fragment as described in the first aspect of the present invention, as described in the second aspect of the present invention
  • the bispecific antibody of the aspect and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes other anti-tumor antibodies as active ingredients.
  • the pharmaceutically acceptable carrier can be a conventional carrier in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and/or the above-mentioned antibody-drug conjugate, and 0.01-99.99% of a pharmaceutically acceptable carrier, the percentage being 0.01-99.99% of the pharmaceutical composition mass percentage.
  • the route of administration of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
  • the pharmaceutical composition is in various conventional dosage forms in the field, preferably in the form of solid, semi-solid or liquid, that is, an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, or a granule. doses, injections or infusions. More preferably via intravascular, subcutaneous, intraperitoneal or intramuscular administration.
  • the pharmaceutical composition can also be administered as an aerosol or coarse spray, ie nasally; alternatively, intrathecally, intramedullary or intraventricularly. More preferably, the pharmaceutical composition may also be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually or rectally.
  • the pharmaceutical composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by physicians according to factors such as the type, age, weight and general disease state of the patient, administration mode and other factors that are beneficial to the patient.
  • the mode of administration can be, for example, by injection or other treatment.
  • the dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition to achieve the desired diagnostic or therapeutic result.
  • the administration regimen may also be a single injection or multiple injections, or adjusted.
  • the dosage level and regimen selected will depend on factors including the activity and stability (i.e., half-life) of the pharmaceutical composition, formulation, route of administration, combination with other drugs or treatments, the disease or disorder to be detected and/or treated, As well as various factors such as the subject's health status and previous medical history to be treated, it is reasonably adjusted.
  • Therapeutically effective doses for the pharmaceutical compositions of the present invention can be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine appropriate ranges and routes of administration. It can then be used to determine useful doses and routes of administration in humans. In general, the determination and adjustment of an effective amount or dosage for administration and the assessment of when and how to make such adjustments are known to those skilled in the art.
  • the above-described OX40-targeting antibodies, the above-described antibody drug conjugates, and/or additional therapeutic or diagnostic agents may each be administered as a single agent within any time frame suitable for performing the desired treatment or diagnosis.
  • the single agents may be administered substantially simultaneously (ie, as a single formulation or within minutes or hours) or in sequential order.
  • the eleventh aspect of the present invention provides an antibody targeting OX40 or an antigen-binding fragment thereof according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, The chimeric antigen receptor according to the third aspect of the present invention, the immune cell according to the fourth aspect of the present invention, the antibody drug conjugate according to the ninth aspect of the present invention and/or the tenth aspect of the present invention
  • Application of the pharmaceutical composition in the preparation of medicaments, kits and/or drug delivery devices for diagnosing, preventing and/or treating tumors; or providing an antibody targeting OX40 as described in the first aspect of the present invention or an antigen-binding fragment thereof, the bispecific antibody according to the second aspect of the present invention, the chimeric antigen receptor according to the third aspect of the present invention, the immune cell according to the fourth aspect of the present invention, the The antibody drug conjugate of the ninth aspect and/or the pharmaceutical composition of the tenth aspect of the present invention are used for diagnosis, prevention and/or treatment of
  • the tumor is a positive tumor related to PSMA, EPCAM, CLDN18.2, B7H4 and/or PD-L1,
  • PSMA positive tumor related to PSMA, EPCAM, CLDN18.2, B7H4 and/or PD-L1
  • the tumor is a positive tumor related to PSMA, EPCAM, CLDN18.2, B7H4 and/or PD-L1
  • PSMA positive tumor related to PSMA, EPCAM, CLDN18.2, B7H4 and/or PD-L1
  • PD-L1 metastatic lesions thereof.
  • the twelfth aspect of the present invention provides a method for detecting OX40 in a sample, which comprises using the OX40-targeting antibody or its antigen-binding fragment as described in the first aspect of the present invention and/or as described in the present invention
  • the bispecific antibody described in the second aspect of the invention is used for detection.
  • the detection method is for non-diagnostic purposes.
  • the thirteenth aspect of the present invention provides a kit, which comprises the OX40-targeting antibody or its antigen-binding fragment as described in the first aspect of the present invention, the antibody or its antigen-binding fragment as described in the second aspect of the present invention Bispecific antibodies, chimeric antigen receptors as described in the third aspect of the present invention, immune cells as described in the fourth aspect of the present invention, antibody drug conjugates as described in the ninth aspect of the present invention and/or as The pharmaceutical composition of the tenth aspect of the present invention, and optionally, instructions.
  • the fourteenth aspect of the present invention provides a drug delivery device, the drug delivery device comprising: (1) for administering the drug according to the tenth aspect of the present invention to a subject in need An infusion module for the pharmaceutical composition, and (2) an optional pharmacodynamic monitoring module.
  • the present invention also provides the OX40-targeting antibody or its antigen-binding fragment according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, and the third aspect of the present invention.
  • the chimeric antigen receptor described in the aspect, the immune cell according to the fourth aspect of the present invention, the antibody drug conjugate according to the ninth aspect of the present invention, and/or the drug according to the tenth aspect of the present invention Use of the composition in the diagnosis, prevention and/or treatment of tumors.
  • the tumor is as described in the eleventh aspect of the present invention.
  • the present invention also provides a kit comprising a kit A and a kit B, wherein the kit A is the OX40-targeting antibody or its antigen as described in the first aspect of the present invention Binding fragments, bispecific antibodies according to the second aspect of the present invention, chimeric antigen receptors according to the third aspect of the present invention, immune cells according to the fourth aspect of the present invention, and ninth aspect of the present invention
  • the antibody drug conjugate, the pharmaceutical composition according to the tenth aspect of the present invention, the kit B is other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies.
  • the medicine box A and the medicine box B can be used at the same time, or the medicine box A can be used first and then the medicine box B can be used, or the medicine box B can be used first and then the medicine box A can be used, which can be determined according to the actual needs of the specific application. .
  • the present invention also provides a method for diagnosing, preventing and/or treating tumors, which comprises administering to a subject in need a therapeutically effective amount of the OX40-targeting agent according to the first aspect of the present invention.
  • 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 light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portion of the variable domain is called the framework (FWR).
  • the variable domains of native heavy and light chains each comprise four FWR 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 in close proximity by the FWR region and together with the CDRs from the other chain form the antigen-binding site of the antibody, the constant region is 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.
  • compositions and methods are intended to mean that the compositions and methods include the stated elements but do not exclude other elements, but also include “consisting of” as the context understands.
  • the HCAb can be produced by a transgenic mouse-Harbour HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) carrying a human immunoglobulin immune repertoire, which contains only the "heavy chain” Human antibody (Heavy Chain Only Antibody), which is half the size of traditional IgG antibodies, usually only has human antibody “heavy chain” variable domains and mouse Fc constant domains.
  • antibody may include immunoglobulins, which are tetrapeptide chain structures formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • immunoglobulins are tetrapeptide chain structures formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains and epsilon chains.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3 and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain variable region described in the present application may further comprise a light chain constant region, and the light chain constant region comprises human kappa, lambda chains or variants thereof.
  • the antibody heavy chain variable region described in the present application may further comprise a heavy chain constant region, and the heavy chain constant region comprises human IgG1, 2, 3, 4 or a variant thereof.
  • variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • CL light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FWR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FWR regions.
  • the order from the amino terminus to the carboxy terminus is: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4.
  • the three CDR regions of the light chain are referred to as VL CDR1, VL CDR2 and VL CDR3; the three CDR regions of the heavy chain are referred to as VH CDR1, VH CDR2 and VH CDR3.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Human antibodies of the present application may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human backbone sequences (ie, "humanized antibodies”) .
  • the term "specific" in reference to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample.
  • an antibody that specifically binds an antigen from one species can also bind that antigen from one or more species.
  • this interspecies cross-reactivity does not in itself alter the classification of antibodies according to specificity.
  • an antibody that specifically binds an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity by itself does not alter the classification of antibodies according to specificity.
  • the terms “specificity” or “specific binding” may be used to refer to the interaction of an antibody, protein or peptide with a second chemical, meaning that the interaction depends on a specific structure on the chemical (eg, antigenic determination of clusters or epitopes); for example, antibodies generally recognize and bind to specific protein structures, not proteins. If the antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A” and the antibody will reduce binding to the antibody The amount of marked A.
  • the term "antigen-binding fragment” refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parental antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the parent binding activity when the activity is expressed on a molar basis. Preferably, the antibody fragment retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% or more of the binding affinity of the parent antibody for the target.
  • antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab') 2 , Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
  • chimeric antigen receptor or "CAR” as used herein refers to an extracellular domain capable of binding an antigen (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region), and a cytoplasmic A polypeptide that signals to a domain (ie, an intracellular signaling domain).
  • the hinge domain can be thought of as part of providing flexibility to the extracellular antigen binding region.
  • Intracellular signaling domains refer to proteins that transmit information into cells to regulate cellular activity by generating second messengers via defined signaling pathways, or proteins that function as effectors by corresponding to such messengers, producing CARs that can promote Signals of immune effector function of cells such as CART cells.
  • Intracellular signaling domains include signaling domains and may also include co-stimulatory intracellular domains derived from co-stimulatory molecules.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position .
  • the percent identity between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matched or homologous. In general, comparisons are made when two sequences are aligned for the greatest percent identity.
  • polypeptide if single chain
  • protein if single chain
  • nucleic acid nucleic acid sequence
  • nucleotide sequence nucleotide sequence
  • polynucleotide sequence nucleotide sequence
  • polynucleotide sequence nucleotide sequence
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
  • host cell refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli, fungal cells such as yeast cells, or fibroblasts, CHO cells, COS cells, NSO cells , HeLa cells, BHK cells, HEK 293 cells or animal cells such as human cells.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • immunode refers to a cell that can elicit an immune response
  • immunoreactive cell and "immune cell” and other grammatical forms thereof can refer to immune cells of any origin.
  • Immunune cells include, for example, white blood cells (leukocytes), lymphocytes (T cells, B cells, natural killer (NK) cells, and bone marrow-derived cells (neutrophils) derived from hematopoietic stem cells (HSCs) produced in the bone marrow , eosinophils, basophils, monocytes, macrophages, dendritic cells).
  • HSCs hematopoietic stem cells
  • the term “immune cells” may also be human or non-human.
  • immune cells may be blood-derived, Such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, and can also be derived from cell lines, such as the use of EBV virus infection to prepare NK cell lines, NK cells and NK92 cells induced and differentiated from embryonic stem cells and iPSCs Department and so on.
  • T cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and differ from other lymphocytes, such as B cells, by the presence of T cell receptors on the cell surface.
  • T cells include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and ⁇ -ST cells.
  • CD4+ cells T helper cells
  • CD8+ cells cytotoxic T cells
  • Tug T regulatory cells
  • ⁇ -ST cells ⁇ -ST cells.
  • Cytotoxic cells include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.
  • NK cells refers to a class of lymphocytes that originate in the bone marrow and play an important role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other stressed cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface.
  • Exemplary degrees of error are generally within 10% thereof and more generally within 5% thereof.
  • the methods and compositions disclosed herein encompass polypeptides and nucleic acids having a specified sequence, a variant sequence, or a sequence substantially identical or similar thereto, eg, at least 85%, 90%, 95%, 99% of the specified sequence. or more of the same sequence.
  • the term "substantially identical" is used in the present invention to refer to a first amino acid sequence.
  • the term "pharmaceutically acceptable carrier” refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient and is well known in the art (see, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining agents, absorption delaying agents Reagents, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate salts and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate) and the like.
  • sugars such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dry whey, albumin or casein
  • degradation products such as lactalbumin hydrolyzate
  • EC 50 refers to the concentration for 50% of maximal effect, ie the concentration that elicits 50% of the maximal effect.
  • cancer As used herein, the terms “cancer”, “cancer”, “cancer patient” are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathology What is the type or aggressiveness stage. Examples include, but are not limited to, solid tumors, hematologic cancers, soft tissue tumors, and metastatic lesions.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the OX40 antibody of the present invention can be a fully human antibody containing only a "heavy chain", the size of the antibody containing only the heavy chain is only half of that of a traditional IgG antibody, and the absence of a light chain allows the antibody to be used as a bispecific antibody, At the same time, the problems of light chain mismatch and heterodimerization can be solved; fully human antibodies can be safely administered to human subjects without eliciting an immunogenic response.
  • the antibody or antigen-binding fragment thereof of the present invention has the activity of specifically binding to human OX40 and cynomolgus monkey (cyno) OX40.
  • the antibody or antigen-binding fragment thereof of the present invention can promote the greater activation of NF-Kb, thereby stimulating the OX40 signaling pathway, and can activate the OX40 pathway in vitro and induce the function of activating T cells.
  • Existing antibodies eg, Pogalizumab
  • the antibodies or antigen-binding fragments thereof of the invention are CD32b cross-linking dependent.
  • the antibody targeting OX40 can bind to human OX40 and cynomolgus monkey OX40 protein, and the binding capacity of the antibody increases in a positive correlation with the concentration of the antibody.
  • the antibody or antigen-binding fragment thereof of the present invention can specifically bind to a cell line overexpressing OX40, but not to other members of the TNF receptor superfamily.
  • the antibody or antigen-binding fragment thereof of the present invention may be a fully human antibody targeting OX40 containing only "heavy chain", and the size of these heavy chain-only antibodies is only traditional IgG Half of the antibody, due to the lack of light chain, can be used in bispecific antibodies, and can solve the problems of light chain mismatch and heterodimerization.
  • the bispecific antibody can bind to human OX40 and the corresponding tumor-related antigen, and the bispecific antibody of the present invention does not affect the binding to tumor cells.
  • the bispecific antibody has one or two or three or more binding sites to OX40, so that the activity of the OX40 terminal can be optimized.
  • one end of the bispecific antibody can recognize and specifically bind to tumor cells, such as EPCAM, PSMA, CLDN18.2, B7H4 or PD-L1, thereby specifically activating the tumor microenvironment of T cells, reducing the toxicity caused by OX40 activation.
  • Figure 1 shows a graph of the results of HCAb antibodies against OX40 binding to human OX40 protein.
  • Figure 2 shows a graph of the results of HCAb antibodies against OX40 binding to cynomolgus monkey OX40 protein.
  • Figure 3 shows a graph of the results of in vitro binding of anti-OX40 HCAb antibodies to CHO-K1/human OX40 cells.
  • Figure 4 shows a graph of the results of blocking the binding of human OX40 ligand to human OX40 on the cell surface by an anti-OX40 HCAb antibody.
  • Figure 5 shows the results of using reporter gene cell lines to detect the stimulatory effect of OX40 antibody on the OX40 signaling pathway.
  • FIG. 6A shows that most of the OX40 antigen-binding proteins have the function of activating the OX40 pathway and inducing activation of T cells.
  • FIG. 6B shows that OX40 antigen binding protein is CD32b cross-linking dependent.
  • Figure 7 shows that the antibodies of the present application bind specifically to CHO-K1/OX40 cells, but not to other members of the TNF tumor necrosis factor receptor superfamily.
  • Figure 8A shows the structure diagram of the IgG-VH tetravalent symmetrical structure molecule.
  • Figure 8B shows the structure diagram of the Fab-HCAb tetravalent symmetrical structure molecule.
  • Figure 9 shows a graph of the results of binding of a bispecific antibody of OX40 x TAA to human OX40.
  • Figure 10 shows a graph of the results of binding of the bispecific antibody to OX40xTAA to the corresponding human tumor-associated antigen.
  • FIG 11 shows that the bispecific antibodies of OX40 ⁇ TAA both have the ability to activate OX40-mediated T cell pathway under tumor cell cross-linking.
  • Figure 12 shows a graph of the results of the allogeneic mixed lymphocyte reaction of the bispecific antibody to OX40xTAA.
  • the Harbour HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying a human immunoglobulin immune repertoire capable of producing novel "heavy chain-only" antibodies that are only the size of conventional IgG antibodies half. It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains. Due to the lack of light chain, the antibody almost solves the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with traditional antibody platforms.
  • the HarbourHCAb human antibody transgenic mice aged 6-8 weeks were immunized for multiple rounds of Harbour HCAb mice using two groups of immunization protocols. Specifically: immunization protocol 1, immunization with recombinant human OX40-ECD-Fc (ChemPartner, #21127-022) antigen protein. Each mouse received a total injection dose of 100 microliters per immunization, either by subcutaneous inguinal injection or by intraperitoneal injection. In the first round of immunization, each mouse was immunized with 50 micrograms of antigenic protein mixed with complete Freund's adjuvant (Sigma, #F5881) in a 1:1 volume ratio of immunogenic reagents.
  • immunization protocol 1 immunization with recombinant human OX40-ECD-Fc (ChemPartner, #21127-022) antigen protein.
  • Each mouse received a total injection dose of 100 microliters per immunization, either by subcutaneous inguinal injection or by intraperitoneal
  • each mouse received immunization with an immunogenic reagent formulated with 25 micrograms of antigenic protein mixed with Ribi adjuvant (Sigma Adjuvant System, Sigma, #S6322).
  • Immunization protocol 2 immunization with HEK293/OX40 (ChemPartner, Shanghai) stable cell line overexpressing human OX40.
  • Each mouse was injected intraperitoneally with 2 ⁇ 10 6 cell suspension per immunization.
  • the interval between each booster round is at least two weeks, and usually no more than five rounds of booster rounds.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49th and 77th days, the serum antibody titers of mice were detected.
  • a final boost at a dose of 25 micrograms of OX40-ECD-Fc (ChemPartner, #21127-022) antigenic protein per mouse was performed 5 days prior to HCAb mouse splenic B cell isolation.
  • mice The blood of mice was collected, and the blood was diluted 10 times, and 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000) were taken, and the ELISA plates were coated with human OX40-ECD-Fc.
  • Carry out ELISA detection (detection method is the same as Example 2) to determine the titer of anti-human OX40 in mouse blood, and detect 2 concentrations of mouse blood (1:100 by flow cytometry (detection method is the same as Example 3).
  • PB blank control group
  • RNA from B cells was extracted, cDNA was reverse transcribed (SuperScript IV First-Strand synthesis system, Invitrogen, #18091200), and then the human VH gene was amplified by PCR with specific primers.
  • PCR forward primer 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' SEQ ID NO: 255
  • PCR reverse primer 5'- AATCCCTGGGCACTGAAGAGACGGTGACC-3' SEQ ID NO: 256.
  • the amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the heavy chain Fc domain sequence of human IgG1 antibody.
  • the constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain HCAb antibodies.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the supernatant expressing HCAb was tested for binding to the stable cell line CHO-K1/OX40 overexpressing human OX40 (CHO-K1/hu OX40, (Genscript, #M00561), while a positive antibody (pogazumab) was used as a positive compare, carry out Fluorescence cytometry (SPT Labtech Ltd.) screening.
  • the specific steps are: wash CHO-K1/OX40 cells with serum-free F12K medium (Thermo, #21127022), and resuspend them to 1 ⁇ 10 6 /ml with serum-free medium.
  • Draq5 fluorescent probe (Cell Signaling Technology, #4048L) (1 ⁇ l Draq5 to 1 ml CHO-K1/OX40 cells, 1:1000 dilution) was added and incubated for 30 minutes in the dark. After centrifuging the cells, wash the cells with culture medium and adjust the cell density to 1 ⁇ 10 5 cells/ml. Then add Alexa diluted 1:1000 488, AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-545-098), 30 ⁇ l of the mixture per well was added to a 384-well plate (Greiner Bio One, #781091).
  • the remaining sequenced cloned antibody plasmids were transfected into HEK293 cells for expression, and the obtained supernatant was again subjected to the NF-kb functional test, thus obtaining 64 antibodies that simultaneously bind CHO-K1/huOX40 and cynomolgus monkey OX40 proteins.
  • Functional fully human OX40 monoclonal antibody with unique sequence According to the human-monkey binding ability and NF-Kb functional test results, 23 antibodies with the highest comprehensive ranking were selected for recombinant expression.
  • the CDRs of antibodies can be defined by a variety of methods in the art, such as Kabat's definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health). Hospital, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • amino acid residues in variable domain sequences can also be determined using the Combined definition rule comprising the Kabat definition and the Chothia definition.
  • the Combined definition rule is to combine the range defined by Kabat and Chothia, and based on this, a larger range is taken. For details, see Table a in the content of the invention.
  • Table 1 The antibody sequence information obtained by sequencing the above 23 antibodies is shown in Table 1 below (PR002055-PR002077).
  • This example utilizes the method of antibody engineering yeast to display antibody mutation library to improve the binding affinity of HCAb antibody PR002067 to OX40.
  • the CDR sequences of antibody variable domains were analyzed by Chothia's rule. Mutations were randomly introduced into the three CDRs of PR002067 to create a 3CDRs (CDR1, CDR2, CDR3) yeast display mutant library. This affinity maturation sorting is divided into four rounds.
  • the yeast cells with binding ability in the three mutant pools were enriched with MACS, and then expanded and cultured, and after induction, they were used as the yeast cells sorted in the first round of FACS.
  • 0.2nM Bio-huOX40-his (Acro biosystem, #TN4-H82E4) was used to sort out yeast cells with higher binding ability; then the expanded culture was collected, and after induction, the yeast cells were used as the next round of sorting;
  • yeast cells sorted in the fourth round are sent for sequencing, and hot spots are found for random combination.
  • the variant molecules were prepared by conventional recombinant protein expression and purification techniques, and their corresponding sequence numbers were listed in Table 1 (PR005362-PR005392), and their corresponding CDR sequences were listed in Table 1-1 (PR005362-PR005392).
  • the binding ability of the recombinant mutant molecules was determined by FACS and BLI.
  • the plasmid encoding HCAb antibody obtained above is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and the purified anti-OX40 recombinant heavy chain antibody can be obtained by using conventional recombinant protein expression and purification techniques.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the purified anti-OX40 recombinant heavy chain antibody can be obtained by using conventional recombinant protein expression and purification techniques.
  • HEK293 cells were expanded in FreeStyleTM F17 Expression Medium (Thermo, #A1383504). Before the transient transfection started, the cell concentration was adjusted to 6 x 10 5 cells/ml, and incubated for 24 hours at 37°C in an 8% CO 2 shaker at a cell concentration of 1.2 x 10 6 cells/ml.
  • 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.
  • a gravity column (Bio-Rad, #7311550) containing MabSelectTM (GE Healthcare Life Science, #71-5020-91 AE) was equilibrated with PBS (pH 7.4) and washed for 2-5 column volumes. The supernatant sample was passed through the column. Rinse the column with 5-10 column volumes of PBS.
  • the target protein was then eluted with 0.1M glycine at pH 3.5, adjusted to neutrality with Tris-HCl at pH 8.0, and finally concentrated and exchanged to PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain the purified antibody.
  • Human OX40 HCAb mAb solution Antibody concentration was determined by NanoDrop detection with absorbance at 280 nm, and antibody purity was determined by SEC-HPLC and SDS-PAGE.
  • the present application produced and prepared an anti-OX40 positive control antibody Pogalizumab analog, and the corresponding antibody number was PR003475. Its corresponding amino acid sequence is derived from IMGT database, heavy chain SEQ ID NO: 233, light chain SEQ ID NO: 270.
  • the antibody protein samples obtained above were analyzed for purity and aggregate form using analytical size exclusion chromatography (SEC).
  • the analytical column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 ⁇ m, 7.8 mm x 30 cm) was connected to a high pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 Hour.
  • An appropriate amount of protein sample (at least 10 ⁇ g, the sample concentration is adjusted to 1mg/ml) is filtered through a 0.22 ⁇ m filter membrane and injected into the system, and the HPLC program is set: the sample is passed through the chromatograph at a flow rate of 1.0ml/min with pH 7.4 PBS buffer Column, the maximum time is 20 minutes; detection wavelength 280nm.
  • ChemStation software is used to integrate the chromatogram and calculate the relevant data, and generate an analysis report, reporting the retention time of components of different molecular sizes in the sample.
  • the antibody protein samples obtained above were analyzed for purity and hydrophobicity using analytical hydrophobic interaction chromatography (HIC).
  • the analytical column TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6mm x 3.5cm) was connected to a high pressure liquid chromatograph (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), equilibrated with PBS buffer at room temperature At least 1 hour.
  • the method was set up by a linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) in 16 minutes at a flow rate set It was set at 0.7ml/min, the protein sample concentration was 1mg/ml, the injection volume was 20 ⁇ l, and the detection wavelength was 280nm.
  • ChemStation software is used to integrate the chromatogram and calculate the relevant data, and generate an analysis report, reporting the retention time of components of different molecular sizes in the sample.
  • DSF Differential Scanning Fluorimetry
  • the PCR plate was sealed, placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad, model CFX96 PCR System), incubated at 25°C for 5 minutes, and then gradually heated from 25°C to 95°C with a gradient of 0.2°C/0.2 minutes. The temperature was lowered to 25°C at the end of the test. Data analysis was performed using FRET scan mode and using Bio-Rad CFX Maestro software and the Tm of the samples was calculated. The test results are shown in Table 2 below.
  • Example 2 ELISA detects the binding ability of HCAb monoclonal antibody protein level of OX40
  • This example is to study the activity of the anti-OX40 HCAb monoclonal antibody prepared in Example 1 in external binding to human and cynomolgus monkey OX40 protein.
  • Antibody binding experiments at the protein level were performed using human OX40 protein (Acro biosystem, #OX0-H5224) and cynomolgus monkey OX40 protein (Novoprotein, #CB17). Briefly, 384 well plates (PerkinElmer, #6007509) were coated with 20 [mu]l per well of 1 ug/ml human OX40 protein and cynomolgus monkey OX40 protein in PBS overnight at 4 degrees.
  • the OX40 antibodies of this example can all bind to human OX40 and cynomolgus monkey (cyno) OX40 proteins, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration.
  • Example 3 FACS detects the binding ability of HCAb monoclonal antibody to OX40 at the cellular level
  • the purpose of this example is to study the activity of the anti-human OX40 HCAb monoclonal antibody binding to human OX40.
  • Antibody binding experiments at the cellular level were performed using a CHO-K1 stably transfected cell line (CHO-K1/hu OX40) overexpressing human OX40. Briefly, cells CHO-K1/hu OX40 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 of cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), and after centrifugation to remove the supernatant, 100 ⁇ l of 2 times the final concentration of the 3-fold serial dilution of the antibody to be tested was added to each well.
  • 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, and the cells were centrifuged at 500 g and 4° C. for 5 minutes, and the supernatant was discarded.
  • all the OX40 antibodies of the present invention can bind to CHO-K1/hu OX40 cells, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration.
  • the CHO-K1 cell line (CHO-K1/hu OX40) overexpressing human OX40 was used to conduct cell-level human OX40/OX40L Binding blocking experiments. Briefly, digestion of CHO-K1 / huOX40 cells were resuspended in complete medium with F-12K, the cell density was adjusted to 1 ⁇ 10 6 cells / mL.
  • 100 ⁇ l cells/well were inoculated on a 96-well V-bottom plate (Corning, #: 3894), and after centrifugation to remove the supernatant, 100 ⁇ l of 2 times the final concentration of the 3-fold serial dilution of the antigen-binding protein to be tested was added to each well, and mixed evenly.
  • the highest final concentration of binding protein was 100 nM, with a total of 8 serial dilution concentrations, Pogalizumab was used as a positive control, and hIgG1 was used as a negative control.
  • one non-blocking control was the addition of biotin-labeled human OX40L protein and secondary antibody without antibody; the other 100% blocking control was only secondary antibody added.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. After that, centrifuge at 4°C for 5 minutes, discard the supernatant, and then add 50 ⁇ l of 0.1 ⁇ g/ml biotin-labeled human OX40L protein (Acro biosysytem, OXL-H82Q6) to each well except the 100% blocking well, 4 °C, incubate in the dark for 30 minutes.
  • the cells were washed twice by adding 100 ⁇ l/well of pre-cooled PBS, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. 100 ⁇ l of 1:200 fluorescent secondary antibody PE Streptavidin (BD Biosicences, #554061) was added to each well, and incubated at 4° C. for 30 minutes in the dark. The cells were washed twice with 200 ⁇ l/well of pre-cooled PBS, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded.
  • PE Streptavidin BD Biosicences, #554061
  • Figure 4 and Table 6 show that the OX40 antibody (OX40 antigen binding protein) described in this application has a weak ability to block the binding of human OX40 ligand to human OX40 on the cell surface.
  • PR002059 showed a blocking effect comparable to that of the reference antibody Tab (Pogalizumab); the remaining antibodies showed a weaker blocking effect. This indicates that most of the OX40 antigen-binding proteins described in this application are weakly blocking or non-blocking antibodies.
  • Example 5 Using a reporter gene cell line to detect the stimulatory effect of OX40 antibody (antigen-binding protein) on OX40 signaling pathway
  • HEK293 reporter cells HEK293/OX40/NF-kb reporter cells, BPS Biosciences, #60482
  • HEK293/OX40/NF-kb reporter cells BPS Biosciences, #60482
  • NF-kb response elements 40 ⁇ l of 4.5 ⁇ 10 4 HEK293 reporter cells (HEK293/OX40/NF-kb reporter cells, BPS Biosciences, #60482) sustainably expressing the luciferase reporter for OX40 and NF-kb response elements were added per well.
  • HEK293/OX40/NF-kb reporter cells BPS Biosciences, #60482
  • Example 6 Antigen-binding protein can activate OX40 pathway in vitro
  • CHO-K1 ATCC, #CCL-61
  • CHO-K1/CD32b cells were treated with 10 ⁇ g/ml mitomycin (Beijing Zhongsheng Ruitai Technology, 10107409001) for 30 minutes at 37°C. It was then washed 4 times with 10% FBS in F-12K medium. 1.5 ⁇ 10 4 cells of these two treatments were plated in each well of a 96-well flat bottom plate (Corning, #3559), 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).
  • the ELISA detection method refers to the relevant kit operation instructions.
  • the absorbance values at 450 nm and 570 nM were read by a microplate reader (Molecular Devices, model SpectraMax Plus), and the concentration of IFN- ⁇ in the supernatant was calculated by back-calculation from the readings of the standard (OD450-OD570).
  • the software GraphPad Prism 8 was used for data processing and graph analysis. The results are shown in FIG. 6A . Most of the OX40 antigen-binding proteins described in this application have the function of activating the OX40 pathway and inducing activation of T cells.
  • the OX40 antigen-binding protein of the present invention has CD32b cross-linking dependence, and has a greater activation effect on T cells than the reference antibody.
  • the binding kinetics between antigen and antibody was analyzed by Biofilm Interferometry (BLI) technique using OctetRed96e (Fortebio) molecular interaction analyzer. Affinity was determined using an Octet RED96 instrument (Pall Fortiebio) and a ProA avidin sensor (Pall ForteBio, #18-5010) following the detailed procedures and methods provided by the manufacturer.
  • the ProA avidin sensor placed in a column was first equilibrated in the test buffer for 10 minutes, and then the ProA sensor was used to capture 200nM OX40 antibody with a capture height of 0.8nm; the ProA sensor was equilibrated in the buffer for 120s and then mixed with 2-fold gradient dilution Human OX40 protein or cynomolgus monkey OX40 protein (200-6.25nM and OnM for OX40HCAB; 25-1.56nM and OnM for Pogalizumab) binds for 180s and dissociates for 800s (PR002063, PR002065, PR002066 and PR002077 with cynomolgus monkeys) The dissociation time was 400 s); finally, the ProA sensor was immersed in 10 mM glycine-hydrochloric acid pH 1.5 solution for regeneration to elute the protein bound to the sensor.
  • Octet Red 96 records the binding and dissociation signals of OX40 antibody and OX40 protein in real time.
  • Octet Data Analysis software Form 11.0
  • take 0 nM as the reference well deduct the reference signal (reference subtraction)
  • select the "1:1 Global fitting” method for data fitting select the "1:1 Global fitting” method for data fitting, and calculate the antigen-antigen binding Kinetic parameters of protein binding, kon(1/Ms) value, kdis(1/s) value and KD(M) value were obtained.
  • the results are shown in Table 9.
  • OX40 antigen-binding proteins described in this application have a slightly higher KD (M) for binding to human OX40 or cynomolgus monkey OX40 than Pogalizumab, indicating that their binding affinity to OX40 is relatively higher than that of the reference antibody. Weak, possibly related to the structural differences between the OX40HCAb of the present invention and Pogalizumab.
  • OX40 belongs to the TNF tumor necrosis factor receptor superfamily, which consists of a large class of multifunctional receptors that mediate immune and non-immune cell functions. Six receptors have been identified as important immune co-stimulators, including CD40, OX40, 4-BB, CD27, GITR and CD30. Likewise, inducible T cell costimulatory factors (ICOS) are another class of receptors that are important for the function and survival of activated T cells or memory T cells.
  • TNF tumor necrosis factor receptor superfamily which consists of a large class of multifunctional receptors that mediate immune and non-immune cell functions. Six receptors have been identified as important immune co-stimulators, including CD40, OX40, 4-BB, CD27, GITR and CD30.
  • inducible T cell costimulatory factors are another class of receptors that are important for the function and survival of activated T cells or memory T cells.
  • the CHO-K1 cell line overexpressing human OX40 (CHO-K1/hu OX40), the CHO-K1 cell line overexpressing human CD40 (CHO-K1/hu CD40, Beijing Kangyuan Bochuang, #KC-1286), Cellular level of CHO-K1 cell line overexpressing human 4-1BB (CHO-K1/hu4-1BB, Genscript, #M00538) and HEK293 cell line overexpressing human ICOS (HEK293T/ICOS, Genscript, #KC-0210) Antibody binding experiments on .
  • these cells were digested and resuspended in F12K or DMEM complete medium to adjust the cell density to 1 x 106 cells/ml, respectively.
  • 100 ⁇ l cells/well were seeded in 96-well V-bottom plates, centrifuged to remove the supernatant, and 100 ⁇ l/well of 3-fold concentration gradient dilution 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.
  • Anti-OX40 heavy chain antibody and anti-PSMA PR001331, H2L2 antibody, 202010096322.6), anti-OX40 heavy chain antibody and EPCAM (PR001081, H2L2 antibody, 202010114063.5), anti-OX40 heavy chain antibody and CLDN 18.2 (PR002726, H2L2 antibody, 201910941316.3), anti-OX40 heavy chain antibody and B7H4 (PR002408, H2L2 antibody, ), anti-OX40 heavy chain antibody and PD-L1 (PR000265, H2L2 antibody, 201910944996.4) antibody were used to prepare bispecifics Antibodies can bind to two targets at the same time, one end of which can recognize the tumor target TAA specifically expressed on the surface of tumor cells (such as PSMA, EPCAM, CLDN18.2, B7H4, PD-L1), while the other end can bind to T cells on T cells. The OX40 molecule can recruit and activate T cells near tumor cells, thereby killing tumor cells.
  • H2L2 antibody sequence information of the anti-tumor punctuation (eg PSMA, EPCAM, CLDN18.2, B7H4, PD-L1) used in this example is shown in Table 10 below.
  • the TAA ⁇ OX40 bispecific antibody prepared in this example includes a variety of molecular structures:
  • IgG-VH tetravalent symmetrical structure molecule the structure is shown in Figure 8A, it comprises two polypeptide chains: polypeptide chain 1, also called short chain, from the amino terminus to the carboxyl terminus, it 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-L-VH_B.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, that is, 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 11.
  • FIG. 8B shows two polypeptide chains: polypeptide chain 1, also called short chain, from the amino terminus to the carboxyl terminus, it includes VH_A-CH1; Polypeptide chain 2 , also called long chain, from amino terminus to carboxyl terminus, which comprises VL_A-CL-L1-VH_B-L2-CH2-CH3.
  • polypeptide chain 1 also called short chain, from the amino terminus to the carboxyl terminus, it includes VH_A-CH1
  • Polypeptide chain 2 also called long chain, from amino terminus to carboxyl terminus, which comprises VL_A-CL-L1-VH_B-L2-CH2-CH3.
  • the VL_A of antibody A and the VH_B of heavy chain antibody B are fused to the same polypeptide chain, which can avoid the mismatch by-products generated by the association of VL_A and VH_B.
  • VH_B of polypeptide chain 2 is linked to CH2 via connecting peptide L2;
  • L2 can be the hinge region of IgG or a connecting peptide sequence derived from the hinge region; L2 can be the sequence listed in Table 11, preferably human IgG1 hinge or human IgG1 hinge (C220S) Or the sequence of G5-LH.
  • the CL of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • the CL of polypeptide chain 2 is linked to VH_B via a linker peptide L1; L1 may be the sequence listed in Table 11.
  • the bispecific antibody contains the Fc domain of IgG1 with the mutations L234A and L235A or L234A and L235A and P329G (numbering according to the EU index).
  • the information of the bispecific antibody with the tetravalent symmetric structure of IgG-VH constructed in this example is shown in Table 12 below, and the information of the bispecific antibody with the Fab-HCAb quaternary symmetrical structure constructed in this example is shown in Table 13 below. Its physicochemical properties are shown in Table 14.
  • the CDR numbering information of the heavy chain and light chain sequences of the TAA ⁇ OX40 bispecific antibody constructed in this example is shown in Table 15 below, and the polypeptide chain numbering information is shown in Table 16 below.
  • Example 10 FACS detects the binding ability of bispecific antibodies to OX40 cells
  • This example is to study the in vitro binding activity of the OX40 ⁇ TAA bispecific antibody obtained in Example 9 to human OX40.
  • Antibody binding experiments at the cellular level were performed using the CHO-K1 cell line (CHO-K1/hu OX40#) overexpressing human OX40. Briefly, cells CHO-K1/hu OX40 cells were digested, resuspended in F12K complete medium, washed with PBS and adjusted to 1 ⁇ 10 6 cells/ml with PBS, respectively. 100 ⁇ l of cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by the addition of 100 ⁇ l/well of a 3-fold dilution of the antibody to be tested at 2 times the final concentration.
  • 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. Then add 100 ⁇ l/well of 1:1000 diluted fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific, Jackson, #109-545-06), and incubate at 4°C for 30 minutes in the dark. The cells were washed twice with 100 ⁇ l/well of pre-cooled PBS, centrifuged at 500 g and 4° C.
  • fluorescent secondary antibody Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific, Jackson, #109-545-06
  • the OX40 ⁇ TAA bispecific antibody diabodies of the present application can all bind to human OX40, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration. Its binding capacity was comparable to that of the reference antibody Tab (Pogalizumab).
  • Example 11 FACS detects the binding ability of bispecific antibodies to corresponding tumor-associated antigen cells
  • This example is to study the in vitro binding activity of the OX40 ⁇ TAA bispecific antibody obtained in Example 9 to human tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • the cells were digested, resuspended in complete medium, washed with PBS and adjusted to cell density of 1 x 106 cells/ml with PBS, respectively.
  • 100 ⁇ l cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), centrifuged to remove the supernatant, and 100 ⁇ l/well of 3-fold concentration gradient dilution 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 OX40 ⁇ TAA bispecific antibody of the present invention can bind to the corresponding human tumor-associated antigen, and The detected antibody binding capacity increases in a positive correlation with the antibody concentration.
  • the bispecific antibodies exhibited comparable EC50s and maxima compared to the corresponding TAA parental mAbs.
  • This example is to study the activity of OX40 ⁇ TAA bispecific antibody in the presence of target cells to activate T cells by binding to the costimulatory molecule OX40.
  • the target cells are cells expressing specific antigens (such as tumor-specific antigens), such as MDA-MB-231 (ATCC, HTB-26) that highly expresses human PD-L1, or CHO-MB-231 that highly expresses human B7H4 K1-huB7H4 (in-house construction), or HEK293-huPSMA (Beijing Kangyuan, #KC-1005) with high expression of human PSMA, or Capan-2 (ATCC, HTB-80) with high expression of human EPCAM, or high expression of human CLDN18 .2 of NUGC4 (JCRB, JCRB0834). Effector cells are isolated human T cells.
  • specific antigens such as tumor-specific antigens
  • MDA-MB-231 ATCC, HTB-26
  • CHO-MB-231 that highly expresses human B7H4 K1-huB7H4 (in-house construction)
  • HEK293-huPSMA Beijing Kangyuan
  • 0.3 ⁇ g/ml anti-CD3 antibody OKT3 (Thermo, #16-0037-81) was plated in 96-well flat bottom plate (Corning, #3599) at 100 ⁇ l/well.
  • the density of human T cells (separated from human PBMCs using a T cell sorting kit (Miltenyi, #130-096-535)) was adjusted to 2 ⁇ 10 6 cells/ml, and the density of target cells was adjusted to 3 ⁇ 10 5 cells / ml, then each of the two kinds of cell suspension in 50 ⁇ l / well were seeded in 96-well plates.
  • hIgG1 iso (CrownBio, #C0001)
  • hIgG4 iso (CrownBio, #C0045) were used as controls.
  • the 96 well plate was placed 37 °C, 5% CO 2 incubator for 2 days. The supernatant after culturing for 48 hours was collected, and the IL-2 concentration in the supernatant was detected by IL-2 ELISA kit (Thermo, #88-7025-88).
  • IL-2 concentration is calculated by reading the standard value (OD 450 -OD 570) thrust reverser. Data processing and graph analysis were performed using the software GraphPad Prism 8.
  • mixed lymphocyte reaction was used to study the activation effect of PD-L1 ⁇ OX40 bispecific antibody molecule on T cells.
  • CD14 magnetic beads (Meltenyi, #130-050-201) were used to separate monocytes from the first donor PBMC cells (Miaotong Biotechnology); for specific operations, refer to the relevant kit instructions.
  • 50ng/ml recombinant human IL-4 (PeproTech, #200-02-A)
  • 100ng/ml recombinant human GM-CSF (PeproTech, #300-03-A) were added and induced at 37°C for 6 days to obtain Immature dendritic cells (iDC cells).
  • mDC cells mature dendritic cells
  • LPS lipopolysaccharide Lipopolysaccharide
  • mDC cells mature dendritic cells
  • T lymphocytes were isolated from the second donor PBMC cells (Miaotong Bio) by using a T cell isolation kit (Meltenyi, #130-096-535).
  • the obtained T cells and mDC cells were seeded into a 96-well plate at a ratio of 5:1 (1 ⁇ 10 5 /well of T cells and 2 ⁇ 10 4 /well of mDC cells). Then, different concentrations of antibody molecules were added at 50 ⁇ l/well.
  • the final concentration of the antibody can be (10nM, 1nM), or a total of 8 concentrations diluted by 3-fold concentration gradient from the highest final concentration of 50nM, and two replicate wells were loaded; hIgG1 iso (CrownBio, #C0001) or blank wells served as controls. Incubate for 5 days at 37 °C, 5% CO 2 incubator.
  • the fourth step is to collect the supernatants on the 3rd day and the 5th day respectively, and use the IL-2 ELISA kit (Thermo, #88-7025-88) to detect the IL-2 concentration in the supernatant on the 3rd day.
  • ELISA detection method refers to the relevant kit operation instructions.
  • Microplate reader (Molecular Devices, Model SpectraMax Plus) and 570nM absorbance 450nm reading, calculate the concentration of IFN- ⁇ or IL-2 by standard reading (OD 450 -OD 570) thrust reverser. Data processing and graph analysis were performed using the software GraphPad Prism 8.
  • the present invention obtains a type of fully human heavy chain antibody by immunizing Harbour HCAb mice.
  • the antibody of the present invention has the activity of specifically binding to human OX40 and cynomolgus monkey OX40, can promote the greater activation of NF-Kb, thus has a stimulating effect on the OX40 signaling pathway, and can activate the OX40 pathway in vitro and induce activation of T cells Its activating effect is comparable to or stronger than that of existing antibodies (eg Pogalizumab), and the antibody or antigen-binding fragment thereof of the present invention has cross-linking dependence of one of the Fc ⁇ receptor members, Fc ⁇ RIIB (CD32B).
  • the antibody of the present invention contains only "heavy chain” fully human antibodies, and the size of these heavy chain-only antibodies is only half of that of traditional IgG antibodies. Due to the feature of no light chain, the antibody can be used for bispecific Antibodies can also solve the problems of light chain mismatch and heterodimerization.
  • the antibody of the present invention and the anti-tumor target H2L2 antibody are prepared into IgG-VH tetravalent symmetrical structure diabody molecules and Fab-HCAb structure diabody molecules, and the obtained bispecific antibodies can bind to human OX40 and corresponding tumors.
  • tumor target TAA specifically expressed on the surface of tumor cells such as PSMA, EPCAM, CLDN18.2, B7H4, PD-L1
  • the other end can bind to OX40 molecules on T cells to specifically activate T cells in the tumor microenvironment, reduce toxicity caused by OX40 activation, and kill tumor cells.

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Abstract

靶向OX40的抗体或其抗原结合片段，其包含VH，VH包含以下的CDR或其突变：如SEQ ID NO：10的氨基酸序列所示的VH CDR1；如SEQ ID NO：44的氨基酸序列所示的VH CDR2；和如SEQ ID NO：86、SEQ ID NO：84或SEQ ID NO：89的氨基酸序列所示的VH CDR3。还公开了制备方法、应用、及包含其的双抗。抗体或其抗原结合片段能与OX40特异性结合、促进NF-Kb较大激活，且在体外激活OX40通路，并诱导激活T细胞功能。所述双抗可识别肿瘤靶点TAA，并可结合T细胞上的OX40，可以招募并激活肿瘤细胞附近的T细胞，以杀死肿瘤细胞。

Description

靶向OX40的抗体及其制备方法和应用
本申请要求申请日为2020/6/30的中国专利申请202010618134.5的优先权。本申请引用上述中国专利申请的全文。
技术领域
本申请涉及生物医药领域，具体涉及一种靶向OX40的抗体或其抗原结合片段，以及其制备方法和应用，还涉及一种包含所述靶向OX40的抗体或其抗原结合片段的双特异性抗体。
背景技术
OX40，也称为CD134、肿瘤坏死因子受体超家族成员4(TNFRSF4)，是肿瘤坏死因子受体超家族成员(TNFRSF)之一，它是一种50kDa～55kDa的I型跨膜糖蛋白，具有细胞质内部分、跨膜结构域和细胞外区域。它参与增强T细胞受体(T Cell Receptor，TCR)触发的T细胞反应，是共刺激受体分子。TCR刺激后OX40主要表达于激活的CD4+和CD8+T细胞表面，与CD8+T细胞相比，CD4+T在体外和肿瘤部位的表达更高(Fujita T et al.，(2006)Immunol Lett.106(1)：27-33；Montler R et al.，(2016)Clin Transl Immunology.5(4)：e70)。此外，研究表明在人类多种肿瘤中，与常规的CD4+T细胞相比调节型T细胞(Tregs)表达更多的OX40(Timperi E etal.，(2016)OncoImmunology.；5(7)：e1175800；Piconese S et al.，(2014)Hepatology.60(5)：1494-1507.)，因此也存在优先使用去除型抗体(depleting mAb)靶向OX40hi群落的可能性。OX40也被报道表达在人类嗜中性粒细胞(该信号通路支持生存)和鼠类的自然杀伤(NK)和NK T细胞上(Baumann R et al.，(2004)Eur J Immunol.34(8)：2268-2275；Croft et al.，(2009)Nat Rev Immunol.9(4)：271-285.)。
OX40已知的唯一配体是OX40L(TNFSF4)，它是II型跨膜蛋白，含有保守的肿瘤坏死因子(TNF)同源结构域，该结构域可实现三聚化(Bodmer JL et al.，(2002)Trends Biochem Sci.27(1)：19-26)。激活后，三个OX40分子可与OX40L三聚体结合，这是TNFRSF中配体-受体配对的典型特征(Banner DW et al.，(1993)Cell.73(3)：431-445.)。暴露于胸腺基质淋巴细胞生成素后，OX40L在人树突状细胞(DC)上被诱导(Krause P et al.，(2009)Blood.113(11)：2451-2460)。此外，人单核细胞，嗜中性粒细胞，肥大细胞，淋巴组织诱导细胞，平滑肌细胞，内皮细胞和体外活化的B细胞都在适当条件下表达OX40L(Byun M et  al.，(2013)J Exp Med.210(9)：1743-1759；Karulf M et al.，(2010)J Immunol.185(8)：4856-4862)。
对于OX40，与TNFRSF的其他成员相同，跨膜信号传导主要通过TNF受体相关因子(TRAF)家族的成员介导。TRAF是三聚体蛋白，可与配体结合的TNFRSF受体三聚体的细胞质尾部的短基元相互作用(McWhirter SM et al.，(1999)Proc Natl Acad Sci USA.96(15)：8408-8413.；ParkYC et al.，(1999)Nature.398(6727)：533-538)。对于OX40，受体与配体相互作用刺激OX40和TRAF2进入细胞质内，激活下游的PI3K/PKB，NF-κB and NFAT介导的信号通路，从而激活T细胞分裂及存活和细胞因子的产生(Croft et al.，(2009)Nat Rev Immunol.9(4)：271-285.；Watts，(2005)Annu.Rev.Immunol.23，23-68)。因此，CD4+和CD8+T细胞都是OX40定向免疫治疗癌症的潜在靶标。
目前有多种靶向OX40的不同的分子在针对转移性癌症的临床试验中使用，其中一种是OX40L-Fc融合蛋白MEDI6383(OX40L fusion Protein)，目前处于临床一期。另外的则是激动性抗体，例如OX40抗体MEDI6469(9B12，mouse anti-human OX40 mAb，replaced by MEDI0562 humanized mAb)、MEDI0562(AstraZeneca，Tavolixizumab)、Ivuxolimab(Pfizer，PF-04518600)、GSK3174998(GSK)、BMS-986178(BMS)、Pogalizumab(MOXRO0916/RG7888)等。其中Pogalizumab(泊加珠单抗)，也称为Vonlerolizumab，是Roche开发的一种人源化的OX40抗体，目前处于临床二期，用于治疗实体瘤。一些临床前研究表明抗OX40单抗通过促进MDSC的积累和生成Th2细胞因子而产生有害的免疫抑制副作用(Gough MJ et al.，(2012)Immunology 136：437e47.)。另外，尽管靶向OX40的激动剂单抗可以在小鼠中赋予肿瘤保护作用，但在免疫原性较差的环境中其作用有限(Kjaergaard J et al.，(2000)Cancer Res.60(19)：5514-5521)。
仅重链抗体由比利时科学家于1993年在自然杂志中首次报道，重链抗体及纳米抗体(VHH)在诸多方面优于传统抗体，其分子量小，可穿透血脑屏障，对人的免疫原性弱。且重链抗体或纳米抗体特别适用于双特异性抗体的开发，同时能够解决轻链错配和异源二聚化的问题。而现有技术中几乎没有关于OX40仅重链抗体的相关报道。
因此急需研发更加安全有效的靶向OX40的联合治疗策略，例如，增强抗原利用率，增强炎症或抑制免疫抑制信号，以此来应用于多种癌症的治疗。
发明内容
本发明所要解决的技术问题是为了克服目前靶向OX40的抗体的不足，提供了一种靶向OX40的抗体及其制备方法和应用、以及基于所述抗体开发的双特异性抗体及其应用。本发明的抗体或其抗原结合片段具有与人OX40和食蟹猴(cyno)OX40特异性结合 的活性。此外，本发明的抗体或其抗原结合片段能促进NF-Kb较大的激活，从而对OX40信号通路有刺激作用，且能够在体外激活OX40通路，并诱导激活T细胞的功能，其激活作用与现有抗体(例如Pogalizumab)相当或比其更强，同时本发明的抗体或其抗原结合片段具有Fcγ受体成员之一FcγRIIB(CD32B)交联依赖性。将本发明的抗体或其抗原结合片段制备成双特异性抗体时，所得双特异性抗体均能结合人OX40和相应的肿瘤相关抗原，其中一端可以识别肿瘤细胞表面特异表达的肿瘤靶点TAA(例如PSMA、EPCAM、CLDN18.2、B7H4、PD-L1)，而另一端可以结合T细胞上的OX40分子，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。
为了解决上述技术问题，本发明第一方面提供了一种靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，
其中，所述VH包含以下的互补决定区(CDR)或其突变：如SEQ ID NO：10的氨基酸序列所示的VH CDR1；如SEQ ID NO：44的氨基酸序列所示的VH CDR2；和/或，如SEQ ID NO：86、SEQ ID NO：84或SEQ ID NO：89的氨基酸序列所示的VH CDR3；
其中，所述突变为在所述VH的VH CDR1、VH CDR2、VH CDR3的氨基酸序列的基础上分别具有3、2或1个氨基酸的插入、缺失或替换。
本申请中，在类似“具有3、2或1个氨基酸的插入、缺失或替换”中“氨基酸突变”是指相较于原氨基酸序列而言，变体的序列存在氨基酸的突变，包括在原氨基酸序列的基础上发生氨基酸的插入、缺失或替换。示例性的解释是对CDR的突变可以包含3个、2个或1个氨基酸的突变，这些CDR之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如可以是对CDR1进行1个氨基酸的突变，对CDR2和CDR3不进行氨基酸突变。
本申请中，所述突变可以包括目前如本领域技术人员公知的突变，例如在抗体的生产或者应用过程中，可能会对抗体进行的一些突变，例如对可能存在的，特别是CDR区的转录后修饰(Potential post-translational modifications，PTMs)的位点进行突变，包括抗体的聚集、脱酰胺基敏感(asparagine deamidation，位点(NG，NS，NH等)、天冬氨酸异构(DG，DP)敏感位点、N糖基化(N-{P}S/T)敏感位点及氧化敏感位点等相关突变。
优选地，VH CDR1的突变为在如SEQ ID NO：10所示的氨基酸序列上的F2、T3、S5、和/或S6突变为L、S、P、I、D和/或C，优选在如SEQ ID NO：10所示的氨基酸序列上具有F2L、T3S/P/I、S5D、和/或S6D/C的氨基酸替换；其氨基酸序列例如如SEQ ID NO：13-16、SEQ ID NO：20-24任一所示。
优选地，VH CDR2的突变为在如SEQ ID NO：44所示的氨基酸序列上的S1、R3、 G4、G5和/或S6突变为T、H、L、G、S、N、D、I和/或Q，优选在如SEQ ID NO：44所示的氨基酸序列上具有S1T、R3H/L/G/S、G4S、G5N/D、S6N/I/Q/T的3、2或1个氨基酸替换；其氨基酸序列例如如SEQ ID NO：42-43、SEQ ID NO：45-50、SEQ ID NO：54-60任一所示。
优选地，VH CDR3的突变为在如SEQ ID NO：86所示的氨基酸序列上的T2、T5、T6、D9和/或Y10突变为M、I、V、S、W、Y、C、F和/或W，优选在如SEQ ID NO：86所示的氨基酸序列上具有T2M/I/V、T5S、T6W/Y、D9C和Y10F/W中的2或1个氨基酸替换；其氨基酸序列例如如SEQ ID NO：82-83、SEQ ID NO：85、SEQ ID NO：87-88、SEQ ID NO：90、SEQ ID NO：94-99任一所示。
以上所述的F2L一般是指如SEQ ID NO：10所示的氨基酸序列的第二位氨基酸F突变为L，其他的氨基酸替换例如T3S/P/I、S5D、和/或S6D/C等等以此类推，本领域人员都应当理解其含义。
优选地，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，其中，所述VH包含以下的互补决定区(CDR)或其突变：如SEQ ID NO：10、SEQ ID NO：13-16、SEQ ID NO：20-24的任一氨基酸序列所示的VH CDR1；如SEQ ID NO：42-50、SEQ ID NO：54-60的任一氨基酸序列所示的VH CDR2；和/或，如SEQ ID NO：82-90、SEQ ID NO：94-99的任一氨基酸序列所示的VH CDR3。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：13、42和82所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、42和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：14、42和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、43和84所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如 SEQ ID NO：13、44和82所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：15、44和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、45和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、46和85所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：16、42和82所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、47和87所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：16、44和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、45和88所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、48和89所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、49和90所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、49和83所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、50和90所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：20、44和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：21、44和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、55和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、56和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、42和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、57和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可 变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、58和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、59和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、60和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和94所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和95所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和96所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和97所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和98所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和99所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和95所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如 SEQ ID NO：10、54和97所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、55和95所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、55和97所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、56和95所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、56和97所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：22、54和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：23、54和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：24、54和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：21、54和86所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和99所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：22、54和99所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：23、54和99所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：24、54和99所示。
在某一较佳实施例中，所述的靶向OX40的抗体或其抗原结合片段，其包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：21、54和99所示。
优选地，上述的VH还包括重链可变区框架区(VH FWR)；其例如可以选自种系IGHV3-23或其回复突变。更选地，所述VH FWR为人抗体的重链可变区框架区。
优选地，所述VH包括如SEQ ID NO：142-164或SEQ ID NO：168一198所示的任一氨基酸序列或其突变；所述突变为所述VH的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VH的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与OX40的结合；所述至少85％序列同一性优选为至少90％序列同一性，更优选为至少95％序列同一性，最优选为至少99％序列同一性。
在本申请中，上述所列CDR的氨基酸序列均是按照Chothia定义规则所示出的(本申请的权利要求中也是按照Chothia定义规则所示出的序列)。但是，本领域人员公知，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JMol Biol 273：927-48，1997)。在本申请中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见表a。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表a 本申请抗体CDR定义方法(可参见http：//bioinf.org.uk/abs/)
  Kabat Chothia Combined
VHCDR1 H31--H35 H26--H32 H26-H35
VHCDR2 H50--H65 H52--H56 H50-H65
VHCDR3 H95--H102 H95--H102 H95-H102
其中，Haa-Hbb可以指从抗体重链的N端开始，第aa位至第bb位的氨基酸序列。例如，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。本领域技术人员应当知晓的是，在用Chothia编码CDR时，有些位置会有插入位点的情况。
例如，按照Chothia编号规则定义，所述VH包含下表b所述的CDR。
表b
抗体编号 HCDR1 HCDR2 HCDR3
PR002055 13 42 82
PR002056 10 42 83
PR002057 14 42 83
PR002058 10 43 84
PR002059 13 44 82
PR002060 15 44 83
PR002061 10 44 83
PR002062 10 45 83
PR002063 10 42 83
PR002064 10 45 83
PR002065 10 46 85
PR002066 10 44 83
PR002067 10 44 86
PR002068 16 42 82
PR002069 10 47 87
PR002070 16 44 83
PR002071 10 42 83
PR002072 10 45 88
PR002073 10 43 84
PR002074 10 48 89
PR002075 10 49 90
PR002076 10 49 83
PR002077 10 50 90
PR005362 20 44 86
PR005363 21 44 86
PR005364 10 54 86
PR005365 10 55 86
PR005366 10 56 86
PR005367 10 42 86
PR005368 10 57 86
PR005369 10 58 86
PR005370 10 59 86
PR005371 10 60 86
PR005372 10 44 94
PR005373 10 44 95
PR005374 10 44 96
PR005375 10 44 97
PR005376 10 44 98
PR005377 10 44 99
PR005378 10 54 95
PR005379 10 54 97
PR005380 10 55 95
PR005381 10 55 97
PR005382 10 56 95
PR005383 10 56 97
PR005384 22 54 86
PR005385 23 54 86
PR005386 24 54 86
PR005387 21 54 86
PR005388 10 54 99
PR005389 22 54 99
PR005390 23 54 99
PR005391 24 54 99
PR005392 21 54 99
优选地，所述靶向OX40的抗体或其抗原结合片段还包含人抗体的重链恒定区Fc结构域。其中，所述人抗体的重链恒定区Fc结构域例如包括人IgG1、IgG2、IgG3或IgG4的重链恒定区Fc结构域。
优选地，所述靶向OX40的抗体或其抗原结合片段包含一个多肽链，所述多肽链包含如SEQ ID NO：208-230或SEQ ID NO：234-264所示的任一氨基酸序列或其突变。所述突变为所述氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与OX40的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
优选地，所述靶向OX40的抗体或其抗原结合片段包括IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、HCAb、VH、双特异性抗体、多特异性抗体、单域抗体或者其他任何保留抗体特异性结合抗原的能力(可以是保留抗体的特异性结合抗原的部分能力)的抗体，或者由上述抗体制得的单克隆抗体或多克隆抗体。
在某一较佳实施例中，所述靶向OX40的抗体或其抗原结合片段为阻断性抗体。
在某一较佳实施例中，所述靶向OX40的抗体或其抗原结合片段为弱阻断或者非阻断性抗体。
本申请中，所述的“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc”区含有抗体的CH2和CH3结构域的 两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。所述的“Fab片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’) 2分子。所述的“F(ab’) 2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’) 2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。所述的术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段，但缺少恒定区。
本申请中，所述的scFv(single chain antibody fragment，单链抗体)可为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见，例如，Bird等人，Science 242：423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883(1988)]。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的G 4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G 4S) 4或(G 4S) 3接头，但也可使用其变体。
本申请中，术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
本申请中，所述的单克隆抗体或mAb或Ab，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。
本申请中，所述的单域抗体可为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
为了解决上述技术问题，本发明第二方面提供了一种双特异性的结合蛋白，其含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原，其中所述蛋白功能区B靶向OX40，所述蛋白功能区A靶向非OX40抗原；所述蛋白功能区B选自如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段。
优选地，所述蛋白功能区A靶向PD-L1、B7H4、PSMA、EPCAM或CLDN18.2。
优选地，所述蛋白功能区A为PSMA抗体或其抗原结合片段、EPCAM抗体或其抗 原结合片段、CLDN18.2抗体或其抗原结合片段、B7H4抗体或其抗原结合片段、PD-L1抗体或其抗原结合片段。
在某一较佳实施例中，所述PD-L1抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：111、119和129所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、39和79所示。
在某一较佳实施例中，所述EPCAM抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和130所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：11、40和80所示。
在某一较佳实施例中，所述PSMA抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：113、121和131所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：12、41和81所示。
在某一较佳实施例中，所述B7H4抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)；所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：114、122和132所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：17、51和91所示。
在某一较佳实施例中，所述CLDN18.2抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)；所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和133所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：18、52和92所示。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)；所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：111、119和129所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、39和79所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)， 所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：114、122和132所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：17、51和91所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和130所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：11、40和80所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：113、121和131所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：12、41和81所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和133所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：18、52和92所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：199所示的氨基酸序列，所述VH包括如SEQ ID NO：139所示的氨基酸序列；所述蛋白功能区B包含重链可变区，所述VH包括如SEQ ID NO：154所示的氨基酸序列。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：202所示的氨基酸序列，所述VH包括如SEQ ID NO：165所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：200所示的氨基酸序列，所述VH包括如SEQ ID NO：140所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：201所示的氨基酸序列，所述VH包括如SEQ ID NO：141所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列。
在某一较佳实施例中，所述双特异性的结合蛋白含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：203所示的氨基酸序列，所述VH包括如SEQ ID NO：166所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列。
优选地，所述蛋白功能区A和/或所述蛋白功能区B为IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、VH、或者HCAb的形式；其中，所述蛋白功能区A和蛋白功能区B不同时为IgG。
更优选地，所述IgG的重链恒定区为人重链恒定区，更优选为人IgG1、人IgG2、人IgG3或人IgG4重链恒定区；其中人IgG优选包含L234A、L235A和P329G中的一个、两个或三个突变，更优选包含L234A和L235A的突变或包含L234A、L235A和P329G的突变。
更优选地，所述Fab、Fab’、F(ab’) 2、Fv、scFv、VH的数量优选为一个或多个。
优选地，所述蛋白功能区B为单个VH的结构，所述蛋白功能区A为IgG的结构；所述蛋白功能区B优选连接在所述蛋白功能区A的C末端。
在某一较佳实施例中，所述双特异性抗体包含第一多肽链和第二多肽链，所述第一多肽链如式：N’-VL _A-CL-C’所示，所述第二多肽链如式：N’-VH _A-CH1-h-CH2-CH3-L- VH _B-C’所示；
其中，所述的VH _B为所述蛋白功能区B的VH，所述的VL _A和VH _A分别为所述蛋白功能区A的VL和VH，所述的h为铰链区，所述的L为连接肽。其中，所述的铰链区为本领域常规即可，通常含大量脯氨酸，具有弹性。
优选地，所述L的长度优选为0或其氨基酸序列如SEQ ID NO.278-295任一所示所示。在一个实施方案中，上述第二多肽链的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，第二多肽链的CH3经由连接肽L联结到VH_B；L可以是实施例中的表11中所列序列。
优选地，所述蛋白功能区B为HCAb的结构，所述蛋白功能区A为Fab的结构；所述蛋白功能区B优选连接在所述蛋白功能区A的C末端。
在某一较佳实施例中，所述双特异性抗体包含第一多肽链和第二多肽链，所述第一多肽链如式：N’-VH _A-CH1-C’所示，所述第二多肽链如式：N’-VL _A-CL-L1-VH _B-L2-CH2-CH3-C’所示。
在某一较佳实施例中，所述双特异性抗体包含第一多肽链和第二多肽链，所述第一多肽链如式：N’-VL _A-CL-C’所示，所述第二多肽链如式：N’-VH _A-CH1-L1-VH _B-L2-CH2-CH3-C’所示。
其中，上述的VH _B为所述蛋白功能区B的VH，所述的VL _A和VH _A分别为所述蛋白功能区A的VL和VH，所述的L1和L2为连接肽。
其中，所述L1或L2的长度优选为0或其氨基酸序列优选如SEQ ID NO.278-295任一所示、或其氨基酸序列为GS，例如所述L1的氨基酸序列如SEQ ID NO.286所示，所述L2的氨基酸序列如SEQ ID NO.285所示。其中，所述的第二多肽链的VH_B经由连接肽L2联结到CH2；L2可以是IgG的铰链区或者铰链区衍生的连接肽序列；L2可以是表11中所列序列，优选为人IgG1铰链或者人IgG1铰链(C220S)或者G5-LH的序列。在一个实施方案中，第二多肽链的CL与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，第二多肽链的CL经由连接肽L1联结到VH_B；L1可以是实施例中的表11中所列序列。
在某一较佳实施例中，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，第一多肽链包括如SEQ ID NO：265所示的氨基酸序列，第二多肽链包括如SEQ ID NO：271所示的氨基酸序列。
在某一较佳实施例中，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，第一多肽链包括如SEQ ID NO：268所示的氨基酸序列，第二多肽链包括如SEQ ID NO： 272所示的氨基酸序列。
在某一较佳实施例中，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，第一多肽链包括如SEQ ID NO：273所示的氨基酸序列，第二多肽链包括如SEQ ID NO：274所示的氨基酸序列。
在某一较佳实施例中，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，第一多肽链包括如SEQ ID NO：266所示的氨基酸序列，第二多肽链包括如SEQ ID NO：275所示的氨基酸序列。
在某一较佳实施例中，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，第一多肽链包括如SEQ ID NO：267所示的氨基酸序列，第二多肽链包括如SEQ ID NO：276所示的氨基酸序列。
在某一较佳实施例中，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，第一多肽链包括如SEQ ID NO：269所示的氨基酸序列，第二多肽链包括如SEQ ID NO：277所示的氨基酸序列。
为了解决上述技术问题，本发明第三方面提供了一种嵌合抗原受体，其包含如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段或如本发明第二方面所述的双特异性抗体。
为了解决上述技术问题，本发明第四方面提供了一种免疫细胞，其包含如本发明第三方面所述的嵌合抗原受体。优选地，所述免疫细胞为T细胞，或NK细胞。
为了解决上述技术问题，本发明第五方面提供了一种分离的核酸，其编码如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、或如本发明第二方面所述的双特异性抗体或如本发明第三方面所述的嵌合抗原受体。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述抗体的核酸分子，或者通过人工全序列合成的方法得到编码上述抗体的核酸分子。
本领域技术人员知晓，编码上述抗体的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该抗体序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
为了解决上述技术问题，本发明第六方面提供了一种重组表达载体，其包含如本发明第五方面所述的分离的核酸。
所述重组表达载体可通过本领域常规方法获得，即：将本申请所述的核酸分子连接 于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。
优选地，所述表达载体包含真核细胞表达载体和/或原核细胞表达载体。
为了解决上述技术问题，本发明第七方面提供了一种转化体，其包含如本发明第五方面所述的分离的核酸或如本发明第六方面所述的重组表达载体。
所述转化体的制备方法可为本领域常规的制备方法，例如为：将上述重组表达载体转化至宿主细胞中制得。所述转化体的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。优选地，所述宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21(表达单链抗体或Fab抗体)，所述真核细胞优选HEK293细胞或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
为了解决上述技术问题，本发明第八方面提供了一种靶向OX40的抗体或其抗原结合片段、或双特异性抗体的制备方法，其包含培养如本发明第七方面所述的转化体，从培养物中获得靶向OX40的抗体或其抗原结合片段、或双特异性抗体。
为了解决上述技术问题，本发明第九方面提供了一种抗体药物偶联物，所述的抗体药物偶联物包括抗体部分和偶联部分，所述抗体部分包含如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段和/或如本发明第二方面所述的双特异性抗体，所述偶联部分包括但不限于可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合，所述抗体部分和偶联部分通过化学键或接头进行偶联。
为了解决上述技术问题，本发明第十方面提供了一种药物组合物，所述药物组合物包含如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体，以及药学上可接受的载体。
优选地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分。
所述的药学上可接受的载体可为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，优选地包括药学上可接受的赋形剂、填充剂或稀释剂等。更优选地，所述的药物组合物包括0.01～99.99％的上述蛋白质和/或上述的抗体药物偶联物，和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明所述的药物组合物的给药途径优选地为肠胃外施用、注射给药或口服给药。 所述注射给药优选地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，即可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。更优选地为经由血管内、皮下、腹膜内或肌内施用。较佳地，所述药物组合物还可以作为气雾剂或粗喷雾剂施用，即经鼻施用；或者，鞘内、髓内或心室内施用。更优选地，所述的药物组合物还可以透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。本发明的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
本发明所述的药物组合物的给药剂量水平可以根据达到所需诊断或治疗结果的组合物量而调整。施用方案也可以为单次注射或多次注射，或进行调整。所选择的剂量水平和方案依赖于包括所述药物组合物的活性和稳定性(即，半衰期)、制剂、施用途径、与其他药物或治疗的组合、待检测和/或治疗的疾病或病症、以及待治疗的受试者的健康状况和先前医疗史等各种因素而进行合理地调整。
对于本发明的所述药物组合物的治疗有效剂量可以最初在细胞培养实验或动物模型例如啮齿类动物、兔、犬、猪和/或灵长类动物中进行估计。动物模型也可以用于测定合适的施用浓度范围和途径。随后可以用于确定在人中施用的有用剂量和途径。一般地，施用有效量或剂量的确定和调整以及何时和如何进行此类调整的评估为本领域技术人员已知。
对于组合疗法，上述靶向OX40的抗体、上述抗体药物偶联物和/或另外的治疗或诊断剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行使用。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。
关于制剂、剂量、施用方案和可测量的治疗结果的另外指导，参见Berkow等人(2000)The Merck Manual of Medical Information(Merck医学信息手册)和Merck&Co.Inc.，Whitehouse Station，New Jersey；Ebadi(1998)CRC Desk Reference of Clinical Pharmacology(临床药理学手册)等著作。
为了解决上述技术问题，本发明第十一方面提供了一种如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体、如本发明第三方面所述的嵌合抗原受体、如本发明第四方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物和/或如本发明第十方面所述的药物组合物在制备诊断、预防和/或 治疗肿瘤的药物、试剂盒和/或给药装置中的应用；或提供了一种如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体、如本发明第三方面所述的嵌合抗原受体、如本发明第四方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物和/或如本发明第十方面所述的药物组合物，其用于诊断、预防和/或治疗肿瘤。
优选地，当使用所述双特异性抗体用于制备诊断、预防和/或治疗肿瘤的药物时，所述肿瘤为PSMA、EPCAM、CLDN18.2、B7H4和/或PD-L1相关的阳性肿瘤，例如为乳腺癌、胰腺癌、胃癌和/或前列腺癌等，或其转移性病灶。
为了解决上述技术问题，本发明第十二方面提供了一种检测样品中OX40的方法，其包括使用如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段和/或如本发明第二方面所述的双特异性抗体进行检测。
优选地，所述检测方法为非诊断目的。
为了解决上述技术问题，本发明第十三方面提供了一种试剂盒，其包括如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体、如本发明第三方面所述的嵌合抗原受体、如本发明第四方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物和/或如本发明第十方面所述的药物组合物，及任选地，说明书。
为了解决上述技术问题，本发明第十四方面提供了一种给药装置，所述的给药装置包含：(1)用于对有需要的受试者施用如本发明第十方面所述的药物组合物的输注模块，以及(2)任选的药效监控模块。
为解决上述技术问题，本发明还提供了如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体、如本发明第三方面所述的嵌合抗原受体、如本发明第四方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、和/或如本发明第十方面所述的药物组合物在诊断、预防和/或治疗肿瘤中的应用。优选地，所述肿瘤如本发明第十一方面所述。
为解决上述技术问题，本发明还提供一种套装药盒，其包含药盒A和药盒B，所述的药盒A为如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体、如本发明第三方面所述的嵌合抗原受体、如本发明第四方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、如本发明第十方面所述的药物组合物，所述的药盒B为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。所述的药盒A和药盒B可以同时使用，也可以先使用药盒A再使用药盒B，还可 以先使用药盒B再使用药盒A，可以根据具体应用时的实际需求而定。
为解决上述技术问题，本发明还提供一种诊断、预防和/或治疗肿瘤的方法，其包括向有需要的受试者施用治疗有效量的如本发明第一方面所述的靶向OX40的抗体或其抗原结合片段、如本发明第二方面所述的双特异性抗体、如本发明第三方面所述的嵌合抗原受体、如本发明第四方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、和/或如本发明第十方面所述的药物组合物。
在本申请中，除非另有说明，否则本申请中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本申请中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
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，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FWR)。天然重链和轻链的可变结构域各自包含四个FWR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FWR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。
本申请所用氨基酸三字母代码和单字母代码如本领域技术人员知晓，或J.Biol.Chem，243，p3558(1968)中所述。
如本文使用的，术语“包括”或“包含”旨在表示组合物和方法包括所述的元素但不排除其他元素，但根据上下文的理解，也包括“由……组成”的情况。
本申请中，所述的HCAb可以是由一种携带人免疫球蛋白免疫库的转基因小鼠一Harbour HCAb小鼠(Harbour Antibodies BV，WO 2002/085945 A3)产生，仅含有“重链”的全人源抗体(Heavy Chain Only Antibody)，该抗体的大小只有传统IgG抗体的一半，其通常仅具有人的抗体“重链”可变结构域和小鼠Fc恒定结构域。
本申请所述的术语“抗体”可以是包括免疫球蛋白，其是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、 α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本申请中，本申请所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源的κ、λ链或其变体。在本申请中，本申请所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源的IgG1、2、3、4或其变体。
在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FWR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FWR区组成，从氨基端到羧基端依次排列的顺序为：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4。轻链的3个CDR区指VL CDR1、VL CDR2和VL CDR3；重链的3个CDR区指VH CDR1、VH CDR2和VH CDR3。
术语“人源抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本申请的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
如本申请所用，关于抗体的术语“特异性”意指识别特异性抗原但基本上不识别或结合样品中的其他分子的抗体。例如，特异性结合来自一个物种的抗原的抗体也可以结合来自一个或更多个物种的该抗原。但是，这种种间交叉反应性本身不改变抗体根据特异性的分类。在另一个实例中，特异性结合抗原的抗体也可以结合该抗原的不同等位基因形式。然而，这种交叉反应性本身不改变抗体根据特异性的分类。在一些情况下，术语“特异性”或“特异性结合”可用于指抗体、蛋白质或肽与第二化学物质的相互作用，意味着该相互作用取决于化学物质上特定结构(例如，抗原决定簇或表位)的存在；例如，抗体一般识别并结合特定的蛋白质结构，而不是蛋白质。如果抗体对表位“A”具有特异性， 则在含有经标记的“A”和抗体的反应中，含有表位A的分子(或游离的，未标记的A)的存在将减少结合于抗体的标记的A的量。
本申请中，术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少50％、60％、70％、80％、90％、95％、99％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab’、F(ab’) 2、Fv片段、线性抗体(1inear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson(2005)Nat.Biotechnol.23：1126-1136中。
本文中使用的术语“嵌合抗原受体”或“CAR”指：包含能够结合抗原的胞外域(胞外结合结构域)、铰链结构域、跨膜结构域(跨膜区)和使胞质信号传到结构域的多肽(即胞内信号域)。铰链结构域可以被认为是用于向细胞外抗原结合区提供柔性的一部分。胞内信号域指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，产生可以促进CAR的细胞(例如CART细胞)的免疫效应子功能的信号。胞内信号域包含信号传导结构域，还可以包括源自共刺激分子的共刺激胞内结构域。
“同一性”、“突变”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同一性百分率时进行比较。
术语“多肽”、“肽”和“蛋白质”(如果为单链)在本申请中互换地使用。术语“核酸”、“核酸序列”，“核苷酸序列”或“多核苷酸序列”和“多核苷酸”互换使用。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
本申请使用的表述“细胞”、“细胞系”可互换使用，并且所有这类名称都包括后代。术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌等原核细胞，如酵母细胞等的真菌细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK 293细胞或人细胞等的动物细胞。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“免疫细胞”指可以引发免疫应答的细胞，“免疫细胞”及其语法上的其他形式可以指任何来源的免疫细胞。“免疫细胞”包括例如衍生自在骨髓中产生的造血干细胞(HSC)的白血细胞(白细胞)、淋巴细胞(T细胞、B细胞、自然杀伤(NK)细胞和骨髓来源的细胞(嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、单核细胞、巨噬细胞、树突状细胞)。术语“免疫细胞”也可以是人或非人的。例如，免疫细胞可以是来自血液的，如自体的T细胞、异体T细胞、自体NK细胞、异体NK细胞，也可以来源自细胞系，如利用EBV病毒感染来制备NK细胞系，从胚胎干细胞和iPSC诱导分化来的NK细胞以及NK92细胞系等。
如本申请使用的，术语“T细胞”是指在胸腺中成熟的一类淋巴细胞。T细胞在细胞介导的免疫中起重要作用，并且与与其他淋巴细胞(例如B细胞)的不同点在于细胞表面上存在T细胞受体。“T细胞”包括表达CD3的所有类型的免疫细胞，包括T辅助细胞(CD4+细胞)、细胞毒性T细胞(CD8+细胞)、自然杀伤T细胞、T调节细胞(Treg)和γ-ST细胞。“细胞毒性细胞”包括CD8+T细胞、自然杀伤(NK)细胞和嗜中性粒细胞，这些细胞能够介导细胞毒性反应。如本文使用的，术语“NK细胞”是指起源于骨髓并且在先天免疫系统中起重要作用的一类淋巴细胞。NK细胞提供针对病毒感染的细胞、肿瘤细胞或其他应激细胞的快速免疫反应，即使是细胞表面上不存在抗体和主要组织相容性复合体。
“任选”、“任一”、“任意”或“任一项”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。本发明所用，“一个”和“一种”在本发明中用来指的一个或多于一个的语法对象。除非内容明确提示，否则术语“或”在本发明中用来意指术语“和/或”并且与之互换使用。“约”和“大约”应当通常意指鉴于测量的性质或精度，所测量的量的可接受误差程度。示例性误差程度一般在其10％范围内和更一般 在其5％范围内。本发明公开的方法和组合物涵盖这样的多肽和核酸，它们具有指定的序列，变异序列或与其基本上相同或相似的序列，例如，与序列指定至少85％、90％、95％、99％或更多相同的序列。在氨基酸序列的情况下，术语“基本上相同”在本发明中用来指第一氨基酸序列。
如发明所用，术语“药学上可接受的载体”是指在药理学和/或生理学上与受试者和活性成分相容的载体，是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，即能引起50％最大效应的浓度。
如本发明所用，术语“癌”、“癌症”、“癌症病人”意在包括全部类型的癌性生长物或致瘤过程、转移性组织或恶性转化的细胞、组织或器官，无论组织病理学类型或侵袭力阶段是什么。例子包括但不限于实体瘤、血液学癌、软组织肿瘤和转移性病灶。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
1、本发明OX40抗体可为仅含“重链”的全人源抗体，仅含重链的抗体的大小只有传统IgG抗体的一半，不含轻链使得该抗体可以用于双特异性抗体，同时能够解决轻链错配和异源二聚化的问题；全人源抗体能够安全地施用给人受试者，而不引发免疫原性反 应。
2、本发明的抗体或其抗原结合片段具有与人OX40和食蟹猴(cyno)OX40特异性结合的活性。此外，本发明的抗体或其抗原结合片段能促进NF-Kb较大的激活，从而对OX40信号通路有刺激作用，且能够在体外激活OX40通路，并诱导激活T细胞的功能，其激活作用与现有抗体(例如Pogalizumab)相当或比其更强，同时本发明的抗体或其抗原结合片段具有CD32b交联依赖性。在本发明某一较佳实施例中，所述靶向OX40的抗体能够结合人OX40和食蟹猴OX40蛋白，且抗体的结合能力与抗体浓度成正相关关系递增。在本发明某一较佳实施例中，本发明的抗体或其抗原结合片段能够特异性地结合过表达OX40的细胞系，而不结合TNF肿瘤坏死因子受体超家族的其他成员。在本发明某一较佳实施例中，本发明的抗体或其抗原结合片段可以为靶向OX40的仅含“重链”的全人源抗体，这些仅含重链的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，同时能够解决轻链错配和异源二聚化的问题。
3、将本发明的抗体或其抗原结合片段制备成双特异性抗体时，双特异性抗体均能结合人OX40和相应的肿瘤相关抗原，且本发明的双抗不影响与肿瘤细胞结合。在本发明某一较佳实施例中，所述双特异性抗体具有一个或两个或三个或更多个与OX40结合的位点，从而能够优化OX40端的活性。在本发明某一较佳实施例中，所述双特异性抗体的一端能识别并特异性结合肿瘤细胞，例如EPCAM、PSMA、CLDN18.2、B7H4或PD-L1，从而特异性激活肿瘤微环境的T细胞，降低OX40激活引起的毒性。
附图说明
图1显示了抗OX40的HCAb抗体结合人OX40蛋白的结果图。
图2显示了抗OX40的HCAb抗体结合食蟹猴OX40蛋白的结果图。
图3显示了抗OX40的HCAb抗体体外结合CHO-K1/人OX40细胞的结果图。
图4显示了抗OX40的HCAb抗体阻断人OX40配体与细胞表面的人OX40结合的结果图。
图5显示了利用报告基因细胞系检测OX40抗体对OX40信号通路的刺激作用的结果图。
图6A显示了大部分OX40抗原结合蛋白均具有激活OX40通路，并诱导激活T细胞的功能。
图6B显示了OX40抗原结合蛋白具有CD32b交联依赖性。
图7显示了本申请的抗体特异性地结合CHO-K1/OX40细胞，而不结合TNF肿瘤坏死因子受体超家族的其他成员。
图8A显示了IgG-VH四价对称结构分子的结构图。
图8B显示了Fab-HCAb四价对称结构分子的结构图。
图9显示了OX40×TAA的双特异性抗体结合人OX40的结果图。
图10显示了OX40×TAA的双特异性抗体结合相应的人的肿瘤相关抗原的结果图。
图11显示了OX40×TAA的双特异性抗体在肿瘤细胞交联下均具有激活OX40介导的T细胞通路。
图12显示了OX40×TAA的双特异性抗体的异体混合淋巴细胞反应的结果图。
具体实施方式
下面显示的实施例意在说明本发明的具体实施方案，并且不意在以任何方式限制本说明书或权利要求书的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook，J.，Fritsch，E.F.andManiais，T.(1989)MolecuLar Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1 抗OX40全人源HCAb抗体的获得
Harbour HCAb小鼠(Harbour Antibodies BV，WO 2002/085945 A3)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生全新的仅“重链”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。由于不含轻链的这一特点，该抗体几乎解决了轻链错配和异源二聚化的问题，使得这一技术平台能够开发出传统抗体平台难以实现的产品。
实施例1.1 免疫HCAb小鼠
6～8周龄的上述HarbourHCAb人源抗体转基因小鼠采用了2组免疫方案对Harbour HCAb小鼠进行多轮免疫。具体为：免疫方案1，用重组的人OX40-ECD-Fc(ChemPartner，#21127-022)抗原蛋白进行免疫。每只小鼠每次免疫时通过皮下经腹股沟注射或通过腹腔注射接受的总注射剂量是100微升。在首轮免疫中，每只小鼠用50微克抗原蛋白与完全弗氏佐剂(Sigma，#F5881)以体积比1∶1混合配制的免疫原试剂进行免疫。在随后的每轮增强免疫中，每只小鼠接受用25微克抗原蛋白与Ribi佐剂(Sigma Adjuvant System，Sigma，#S6322)混合配制的免疫原试剂的免疫。免疫方案2，用过表达人OX40的 HEK293/OX40(ChemPartner，Shanghai)稳定细胞系进行免疫。每只小鼠每次免疫时腹腔注射2×10 6细胞悬液。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行HCAb小鼠脾B细胞分离前5天，以每只小鼠25微克OX40-ECD-Fc(ChemPartner，#21127-022)抗原蛋白的剂量进行最后一次增强免疫。
采集小鼠血液，对血液进行10倍稀释，取5个浓度(1∶100、1∶1000、1∶10000、1∶100000、1∶1000000)，在包被有人OX40-ECD-Fc的ELISA板进行ELISA检测(检测方法同实施例2)来确定小鼠血液中抗人OX40的滴度，并经流式细胞术(检测方法同实施例3)检测2个浓度的小鼠血液(1∶100、1∶1000)对OX40高表达的CHO-K1/hOX40细胞(Chempartner，Shanghai)和CHO-K1母细胞的特异反应性。空白对照组(PB)为免疫前老鼠的血清。
实施例1.2 获得抗OX40的HCAb抗体序列
当检测上述小鼠的血清中OX40特异的抗体滴度达到一定的水平后，从小鼠的脾细胞取出分离B细胞，用BD流式分选仪(BD Biosciences，FACS AriaII Cell Sorter)分选CD138阳性的浆细胞和人OX40抗原阳性的B细胞群。提取B细胞的RNA，反转录cDNA(SuperScript IV First-Strand synthesis system，Invitrogen，#18091200)，然后用特异性的引物PCR扩增人VH基因。PCR正向引物5’-GGTGTCCAGTGTSAGGTGCAGCTG-3’(SEQ ID NO：255)，PCR反向引物5’-AATCCCTGGGCACTGAAGAGACGGTGACC-3’(SEQ ID NO：256)。将扩增的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列的哺乳动物细胞表达质粒pCAG载体中。
构建好的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达获得HCAb的抗体。检测表达HCAb的上清与过表达人OX40的稳定细胞系CHO-K1/OX40(CHO-K1/hu OX40，(Genscript，#M00561)的结合，同时用阳性抗体(泊加珠单抗)作为阳性对照，进行
Figure PCTCN2021102946-appb-000001
荧光细胞仪(SPT Labtech Ltd.)筛选。具体步骤是：用无血清的F12K培养基(Thermo，#21127022)洗涤CHO-K1/OX40细胞，将其用无血清的培养基重悬至1×10 6/ml。加入Draq5荧光探针(Cell Signaling Technology，#4048L)(1μl Draq5至1ml CHO-K1/OX40细胞中，1∶1000稀释)，在避光处孵育30分钟。离心细胞后用培养基洗涤细胞，调整细胞密度至1×10 5细胞/ml。再加入1∶1000稀释后的Alexa
Figure PCTCN2021102946-appb-000002
488，AffiniPure Goat Anti-Human IgG，Fcγ Fragment Specific二抗(Jackson ImmunoResearch Laboratories Inc.，#109-545-098)，取每孔30μl的该混合物加入384孔板(Greiner Bio One，#781091)。再在384孔板中加入10μl阳性对照或者表达HCAB的上清，孵育2小时。在Mirrorball 仪器上读取荧光值。阳性克隆抗体进一步与人OX40蛋白(Acrobiosystem，#OX0-H5224)和食蟹猴OX40蛋白(Novoprotein，#CB17)进行ELISA检测来验证交叉结合活性。同时进一步通过FACS检测与CHO-K1/hu OX40#细胞的结合活性。利用常规的测序手段获得克隆抗体的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。去除重复序列后将余下的测序后的克隆抗体质粒转染至HEK293细胞进行表达，获得的上清再次进行NF-kb功能试验，这样得到64个同时结合CHO-K1/huOX40和食蟹猴OX40蛋白的功能性的具有独特序列的全人源OX40单克隆抗体。根据人猴结合能力及NF-Kb功能试验结果，选择综合排名靠前的23个抗体进行重组表达。
本领域人员公知，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JMol Biol 273：927-48，1997)。在本申请中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见发明内容部分的表a。上述23个抗体经测序后所得的抗体序列信息如下表1(PR002055-PR002077)所示。
实施例1.3 优化HCAb抗体序列提高结合OX40的亲和力
本实施例利用抗体工程酵母展示抗体突变库的方法来提高HCAb抗体PR002067结合OX40的亲和力。在本实施例中抗体可变结构域的CDR序列通过Chothia规则进行分析。对PR002067的三个CDRs随机引入突变建立3CDRs(CDR1，CDR2，CDR3)酵母展示突变库。此亲和力成熟的分选分为四轮。
第一轮，用MACS富集3个突变库中有结合能力的酵母细胞，然后扩大培养，诱导后作为FACS第一轮分选的酵母细胞。第二轮，用0.2nM Bio-huOX40-his(Acro biosystem，#TN4-H82E4)分选出结合力更高的酵母细胞；然后收集扩大培养，诱导后作为下一轮分选的酵母细胞；第三轮，降低分选浓度，用0.02nM Bio-huOX40-his分选出结合力更高的酵母细胞；然后收集扩大培养，诱导后作为下一轮分选的酵母细胞；第四轮，继续降低分选浓度，用0.006nM Bio-huOX40-his分选出结合力更高的酵母细胞。最后，将第四轮分选到的酵母细胞送测序，找到热点进行随机组合。然后利用常规的重组蛋白表达和纯化技术制备变体分子，其对应的序列编号列于表1(PR005362-PR005392)，其对应的CDR序列列于表1-1(PR005362-PR005392)。最后利用FACS和BLI等方法对重组突变分子进行结合能力的测定。
表1 抗OX40抗体的序列编号表
抗体编号 重链 VH FWR1 HCDR1 FWR2 HCDR2 FWR3 HCDR3 FWR4
PR002055 208 142 4 13 28 42 64 82 102
PR002056 209 143 4 10 29 42 65 83 102
PR002057 210 144 4 14 29 42 66 83 102
PR002058 211 145 4 10 29 43 66 84 102
PR002059 212 146 5 13 30 44 67 82 102
PR002060 213 147 4 15 29 44 68 83 102
PR002061 214 148 4 10 29 44 64 83 102
PR002062 215 149 4 10 31 45 69 83 102
PR002063 216 150 4 10 32 42 69 83 103
PR002064 217 151 6 10 31 45 69 83 102
PR002065 218 152 1 10 29 46 70 85 102
PR002066 219 153 7 10 29 44 67 83 104
PR002067 220 154 4 10 29 44 71 86 102
PR002068 221 155 4 16 33 42 65 82 104
PR002069 222 156 4 10 30 47 67 87 102
PR002070 223 157 7 16 29 44 72 83 102
PR002071 224 158 1 10 29 42 65 83 102
PR002072 225 159 4 10 34 45 73 88 105
PR002073 226 160 1 10 29 43 66 84 102
PR002074 227 161 4 10 35 48 74 89 102
PR002075 228 162 8 10 29 49 64 90 103
PR002076 229 163 4 10 29 49 64 83 103
PR002077 230 164 4 10 29 50 67 90 102
PR005362 234 168 4 20 29 44 71 86 102
PR005363 235 169 4 21 29 44 71 86 102
PR005364 236 170 4 10 29 54 78 86 102
PR005365 237 171 4 10 29 55 78 86 102
PR005366 238 172 4 10 29 56 78 86 102
PR005367 239 173 4 10 29 42 78 86 102
PR005368 240 174 4 10 29 57 78 86 102
PR005369 241 175 4 10 29 58 78 86 102
PR005370 242 176 4 10 29 59 78 86 102
PR005371 243 177 4 10 29 60 78 86 102
PR005372 244 178 4 10 29 44 71 94 102
PR005373 245 179 4 10 29 44 71 95 102
PR005374 246 180 4 10 29 44 71 96 102
PR005375 247 181 4 10 29 44 71 97 102
PR005376 248 182 4 10 29 44 71 98 102
PR005377 249 183 4 10 29 44 71 99 102
PR005378 250 184 4 10 29 54 78 95 102
PR005379 251 185 4 10 29 54 78 97 102
PR005380 252 186 4 10 29 55 78 95 102
PR005381 253 187 4 10 29 55 78 97 102
PR005382 254 188 4 10 29 56 78 95 102
PR005383 255 189 4 10 29 56 78 97 102
PR005384 256 190 4 22 29 54 78 86 102
PR005385 257 191 4 23 29 54 78 86 102
PR005386 258 192 4 24 29 54 78 86 102
PR005387 259 193 4 21 29 54 78 86 102
PR005388 260 194 4 10 29 54 78 99 102
PR005389 261 195 4 22 29 54 78 99 102
PR005390 262 196 4 23 29 54 78 99 102
PR005391 263 197 4 24 29 54 78 99 102
PR005392 264 198 4 21 29 54 78 99 102
表1-1 抗OX40抗体的CDR序列表
抗体编号 HCDR1 HCDR2 HCDR3
PR002055 GLTFSSY SGGGGS GMTGSTDVDY
PR002056 GFTFSSY SGGGGS GMTGTTDVDY
PR002057 GFIFSSY SGGGGS GMTGTTDVDY
PR002058 GFTFSSY SGGSGS GVTGTDFDF
PR002059 GLTFSSY SGRGGS GMTGSTDVDY
PR002060 GFTFSDY SGRGGS GMTGTTDVDY
PR002061 GFTFSSY SGRGGS GMTGTTDVDY
PR002062 GFTFSSY SGSGGS GMTGTTDVDY
PR002063 GFTFSSY SGGGGS GMTGTTDVDY
PR002064 GFTFSSY SGSGGS GMTGTTDVDY
PR002065 GFTFSSY SGRGDI GMTGSTDVDF
PR002066 GFTFSSY SGRGGS GMTGTTDVDY
PR002067 GFTFSSY SGRGGS GTTGTTDVDY
PR002068 GFSFSSY SGGGGS GMTGSTDVDY
PR002069 GFTFSSY SGGGGN GVTGTTDVDY
PR002070 GFSFSSY SGRGGS GMTGTTDVDY
PR002071 GFTFSSY SGGGGS GMTGTTDVDY
PR002072 GFTFSSY SGSGGS GMTGTTDVDF
PR002073 GFTFSSY SGGSGS GVTGTDFDF
PR002074 GFTFSSY SGGGNN GWELPLLEN
PR002075 GFTFSSY SGSGGN GITGTTDVDY
PR002076 GFTFSSY SGSGGN GMTGTTDVDY
PR002077 GFTFSSY SGRGNI GITGTTDVDY
PR005362 GLPFDCY SGRGGS GTTGTTDVDY
PR005363 GLPFDSY SGRGGS GTTGTTDVDY
PR005364 GFTFSSY SGRGGQ GTTGTTDVDY
PR005365 GFTFSSY SGLGGQ GTTGTTDVDY
PR005366 GFTFSSY SGRSGQ GTTGTTDVDY
PR005367 GFTFSSY SGGGGS GTTGTTDVDY
PR005368 GFTFSSY SGLGGS GTTGTTDVDY
PR005369 GFTFSSY TGRGGQ GTTGTTDVDY
PR005370 GFTFSSY SGHGGT GTTGTTDVDY
PR005371 GFTFSSY SGRGGT GTTGTTDVDY
PR005372 GFTFSSY SGRGGS GTTGSWDVCY
PR005373 GFTFSSY SGRGGS GTTGTWDVDW
PR005374 GFTFSSY SGRGGS GTTGSWDVDW
PR005375 GFTFSSY SGRGGS GTTGSYDVDW
PR005376 GFTFSSY SGRGGS GTTGSTDVDW
PR005377 GFTFSSY SGRGGS GTTGTWDVDY
PR005378 GFTFSSY SGRGGQ GTTGTWDVDW
PR005379 GFTFSSY SGRGGQ GTTGSYDVDW
PR005380 GFTFSSY SGLGGQ GTTGTWDVDW
PR005381 GFTFSSY SGLGGQ GTTGSYDVDW
PR005382 GFTFSSY SGRSGQ GTTGTWDVDW
PR005383 GFTFSSY SGRSGQ GTTGSYDVDW
PR005384 GLPFSSY SGRGGQ GTTGTTDVDY
PR005385 GLTFDSY SGRGGQ GTTGTTDVDY
PR005386 GFPFDSY SGRGGQ GTTGTTDVDY
PR005387 GLPFDSY SGRGGQ GTTGTTDVDY
PR005388 GFTFSSY SGRGGQ GTTGTWDVDY
PR005389 GLPFSSY SGRGGQ GTTGTWDVDY
PR005390 GLTFDSY SGRGGQ GTTGTWDVDY
PR005391 GFPFDSY SGRGGQ GTTGTWDVDY
PR005392 GLPFDSY SGRGGQ GTTGTWDVDY
实施例1.4 制备抗OX40全人重组抗体
将上述所得的编码HCAb抗体的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到纯化的抗OX40重组重链抗体。具体说来，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo，#A1383504)扩培。瞬时转染开始之前，调节细胞浓度至6×10 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，将上述编码HCAb重链的质粒30μg质粒溶解于1.5ml Opti-MEM无血清培养基(Thermo，#31985088)，再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences，Inc，#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)平衡含有MabSelect TM(GE Healthcare Life Science，#71-5020-91 AE)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冲洗柱子。再用pH3.5的0.1M甘氨酸洗脱目的蛋白，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，得到纯化的抗人OX40的HCAb单抗溶液。抗体浓度用NanoDrop检测280nm吸光度测定，抗体的纯度用SEC-HPLC和SDS-PAGE测定。
同时，本申请生产制备了抗OX40的阳性对照抗体Pogalizumab类似物，对应的抗体编号为PR003475。其相应的氨基酸序列来源于IMGT数据库，重链SEQ ID NO：233，轻链SEQ ID NO：270。
实施例1.5 利用HPLC-SEC分析蛋白纯度和多聚体
使用分析型分子尺寸排阻层析色谱法(SEC)来分析上述所得抗体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 x 30cm)连接到高压液相色谱仪(HPLC)(型号Agilent Technologies，Agilent 1260 Infinity 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，样品浓度调整到1mg/ml)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用pH 7.4 PBS缓冲液将样品以1.0ml/min的流速流过色谱柱，最长时间为20分钟；检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据，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。
实施例1.6 利用HPLC-HIC分析蛋白纯度和疏水性
使用分析型疏水相互作用层析色谱法(HIC)来分析上述所得抗体蛋白样品的纯度和疏水性。将分析型色谱柱TSKge1 Buty1-NPR(Tosoh Bioscience，14947，4.6mm x 3.5cm)连接到高压液相色谱仪(HPLC)(型号：Agilent Technologies，Agilent 1260 Infinity II)，用PBS缓冲液室温下平衡至少1小时。设定方法由16分钟内从100％流动相A(20mM组氨酸，1.8M硫酸铵，pH6.0)至100％流动相B(20mM组氨酸，pH6.0)的线性梯度，流速设定为0.7ml/min，蛋白样品浓度1mg/ml，进样体积20μl，检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据，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。
实施例1.7 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用来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，型号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。检测结果如下表2所示。
表2.OX40抗体的理化性质
Figure PCTCN2021102946-appb-000003
实施例2 ELISA检测OX40的HCAb单抗蛋白水平的结合能力
本实施例是为了研究实施例1制得的抗OX40的HCAb单抗体外结合人和食蟹猴OX40蛋白的活性。采用人OX40蛋白(Acro biosystem，#OX0-H5224)和食蟹猴OX40蛋白(Novoprotein，#CB17)进行蛋白水平上的抗体结合实验。简言之，在384孔板(PerkinElmer，#6007509)每孔包被20μl的1ug/ml溶于PBS的人OX40蛋白和食蟹猴OX40蛋白，4度过夜。第二天用含0.05％吐温的PBS(MEDICAGO，#09-9410-100)洗涤384孔板三次，用含2％牛奶(Bio-Rad，#170-6404)的PBS在37度封闭1小时。待测的OX40抗体和阳性抗体(Pogalizumab)起始浓度为10nM，做4倍梯度稀释。封闭后的384孔板用PBST洗涤三次，在板内加入10μl PBS或10μl 4倍梯度稀释的抗体和阳性对照(Pogalizumab)，室温孵育1小时。洗涤三次，每孔加入20μl羊抗人Fc辣根过氧化物酶(Jackson ImmunoResearch Laboratories Inc.，#109-035-098)，37度孵育40分钟。洗涤三次，每孔加入20μl TMB(Sera Care，#5120-0077)，室温孵育5-15分钟。每孔加入20μl终止液(BBI life sciences，#E661006-0200)，使用读板机(Molecular Devices，型号SpectraMax Plus)读取OD 450-650值。用Graphad 8.0分析该值并做图。
如图1和图2和表3及表4所示，本实施例的OX40抗体均能结合人OX40和食蟹猴(cyno)OX40蛋白，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(Pogalizumab)相比，其PR002055，PR002056，PR002058，PR002059，PR002065，PR002069，PR002070，PR002074和PR002076与人OX40蛋白和食蟹猴蛋白结合的EC50与Tab(Pogalizumab(泊加珠单抗)，传统的IgG型抗体，其重轻链序列分别如SEQ ID NO：233和SEQ ID NO：270所示)相当或者更低，说明这些抗体均能以较低的浓度更灵敏得结合人OX40，其中以PR002055最佳，其EC50均小于50pM。
表3.OX40抗体与人OX40蛋白的结合活性
抗体 EC50(pM) OD最大值
PR002055 26.84 1.412
PR002056 56.97 1.713
PR002057 124.1 1.76
PR002058 62.92 1.935
PR002059 69.52 2.003
PR002060 82.81 2.094
PR002061 134.4 1.963
PR002062 103.1 1.939
PR002063 100.9 2.281
PR002064 81.43 2.28
PR002065 63.88 2.474
PR002066 118.1 2.47
PR002067 80.6 2.378
PR002068 91.97 2.418
PR002069 50.97 2.236
PR002070 72.89 2.405
PR002071 50.16 2.452
PR002072 57.72 2.471
PR002073 85.02 2.38
PR002074 51.15 2.349
PR002075 61.84 2.484
PR002076 64.97 2.8
PR002077 145.6 2.665
Pogalizumab 82.59 2.134
表4.OX40抗体与食蟹猴OX40蛋白的结合活性
抗体 EC50(pM) OD最大值
PR002055 45.22 1.743
PR002056 68.19 1.685
PR002057 132.8 1.764
PR002058 59.74 1.761
PR002059 77.35 1.904
PR002060 93.8 1.94
PR002061 151.8 1.872
PR002062 125.6 1.815
PR002063 275.8 2.318
PR002064 217.9 2.079
PR002065 103.5 1.918
PR002066 369.7 2.444
PR002067 232 2.163
PR002068 253.1 2.381
PR002069 129 2.238
PR002070 131.9 2.513
PR002071 201.5 2.927
PR002072 216.7 2.968
PR002073 145.7 2.552
PR002074 140.4 2.341
PR002075 414.9 3.072
PR002076 115.6 2.053
PR002077 440.9 2.729
Pogalizumab 194.3 2.226
实施例3 FACS检测OX40的HCAb单抗在细胞水平的结合能力
本实施例是为了研究抗人OX40的HCAb单抗体外结合人OX40的活性。采用过表达人OX40的CHO-K1稳转细胞株(CHO-K1/hu OX40)进行细胞水平上的抗体结合实 验。简言之，消化细胞CHO-K1/hu OX40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)，离心去上清后每孔加入100μl 2倍于终浓度的3倍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1∶1000稀释的荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG，Fcγ Fragment Specific，Jackson，#109-545-06)，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。
如图3和表5所示，本发明的OX40抗体均能结合CHO-K1/hu OX40细胞，且检测到的抗体结合能力与抗体浓度成正相关关系递增。
表5.OX40抗体与CHO-K1/hu OX40的结合活性
抗体 EC50(nM) MFI最大值
PR002055 3.18 2244
PR002056 3.12 2928
PR002057 12.81 1668
PR002058 5.79 3559
PR002059 1.35 3628
PR002060 8.95 1857
PR002061 10.95 1738
PR002062 37.89 1902
PR002063 9 2244
PR002064 22.44 1573
PR002065 3.82 2865
PR002066 4.67 3012
PR002067 5.25 3012
PR002068 10.08 2129
PR002069 4.43 2992
PR002070 4.39 2532
PR002071 4 3059
PR002072 5.64 1405
PR002073 6.55 3258
PR002074 1.27 3541
PR002075 n.d. 501
PR002076 24.59 1380
PR002077 7.22 3982
Pogalizumab 0.34 4516
实施例4 抗原结合蛋白阻断OX40与OX40配体结合
为了研究人OX40结合蛋白体外阻断人OX40与人OX40配体(OX40L)结合的能力，采用过表达人OX40的CHO-K1细胞株(CHO-K1/hu OX40)进行细胞水平的人OX40/OX40L结合阻断实验。简言之，消化CHO-K1/huOX40细胞，并用F-12K完全培养基重悬，将细胞密度调整为1×10 6细胞/mL。以100μl细胞/孔接种于96孔V底板(Corning，#：3894)，离心去上清后每孔加入100μl 2倍于终浓度的3倍梯度稀释的待测抗原结合蛋白，混合均匀，其中抗原结合蛋白最高终浓度为100nM，共8个梯度稀释浓度，Pogalizumab作为阳性对照，而hIgG1作为阴性对照。同时另设两个对照，一个无阻断对照是不加抗体只加生物素标记的人OX40L蛋白和二抗；另一个100％阻断对照是只加二抗。将细胞放置于4℃，避光孵育1小时。之后，4℃下离心5分钟，弃上清，随后除100％阻断孔外其他孔每孔加入50μl 0.1μg/ml浓度的生物素标记的人OX40L蛋白(Acro biosysytem，OXL-H82Q6)，4℃，避光孵育30分钟。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。每孔加入100μl 1∶200荧光二抗PE Streptavidin(BD Biosicences，#554061)，4℃，避光孵育30分钟。用200μl/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，每孔加200μl预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值，计算IC50，抑制率％＝{无阻断对照孔MFI平均值-OX40抗体的MFI值-}/{(无阻断对照孔MFI平均值)-100％阻断对照孔的MFI平均值)}*100。
结果如图4和表6所示。图4和表6显示本申请所述OX40抗体(OX40抗原结合蛋白)阻断人OX40配体与细胞表面的人OX40结合的能力较弱。其中PR002059显示出与参照抗体Tab(Pogalizumab)相当的阻断效果；余下抗体则显示较弱的阻断效果。说明本申请所述OX40抗原结合蛋白大部分为弱阻断或者非阻断性抗体。
表6 抗原结合蛋白阻断OX40配体与OX40结合
抗体 IC50(nM) 最大抑制率(％)
PR002055 59.94 19
PR002056 43.71 34
PR002057 n.d. 12
PR002058 98.1 38
PR002059 15.94 79
PR002060 n.d. 12
PR002061 n.d. 16
PR002062 n.d. 13
PR002063 n.d. 17
PR002064 n.d. 20
PR002065 49.58 44
PR002066 67.61 58
PR002067 166.2 64
PR002068 23.06 25
PR002069 50.86 53
PR002070 67.57 46
PR002071 51.73 50
PR002072 n.d. 14
PR002073 75.21 24
PR002074 43.13 81
PR002075 n.d. 0
PR002076 n.d. 0
PR002077 41.1 34
Pogalizumab 5.575 99
实施例5 利用报告基因细胞系检测OX40抗体(抗原结合蛋白)对OX40信号通路的刺激作用
每孔铺100μl的1.5×10 4将表达CD32b的CHO-K1细胞(CHO-K1/CD32b)(Genscript，#M00587)或CHO-K1(ATCC，#CCL-61)到96孔板(Perkin Elmer，#6005225)，37℃在5％CO 2培养箱内孵育过夜。第二天96孔板去除上清后每孔加入40μl的2倍的待测抗原结合蛋白稀释液，起始终浓度为200nM，5倍梯度稀释，hlgG1为阴性对照组。每孔加入40μl 4.5×10 4的可持续表达OX40和NF-kb反应元件的荧光素酶报告基因的HEK293报告细胞(HEK293/OX40/NF-kb报告细胞，BPS Biosciences，#60482)。37℃在5％CO 2培养箱内培养6小时。之后加入ONE-Glo TM荧光素酶试剂(Promega，#E6110)，室温孵育5分钟，酶标仪检测发光值。
结果如图5和下表7所示。图5和表7的结果显示本申请所述所有OX40抗原结合蛋白均是CD32b交联依赖型。在CHO-K1/CD32b交联下，本申请所述大部分OX40抗原结合蛋白对OX40介导的NF-Kb信号通路的促进作用与其浓度成正相关关系递增。与参照抗体Tab(Pogalizumab)相比，所有的OX40抗体的最大发光值都比参照抗体高，说明这些抗体能促进NF-Kb较大的激活。其中以PR002059最优，其与参照抗体的EC50相当，但最大发光值比参照抗体更高。
表7 抗原结合蛋白对OX40信号通路的刺激作用
抗体 EC50(nM) 最大发光信号值
PR002055 4.66 140624
PR002056 4.89 153884
PR002057 14.35 172165
PR002058 4.29 135546
PR002059 1.83 116834
PR002060 15.1 132365
PR002061 23.2 155668
PR002062 25.22 148937
PR002063 8.64 149686
PR002064 30.34 147258
PR002065 8.55 149211
PR002066 5.45 146566
PR002067 5.37 136921
PR002068 22.95 138931
PR002069 6.27 99697
PR002070 7.6 129161
PR002071 7.85 143752
PR002072 14.21 99625
PR002073 6.39 120867
PR002074 3.08 84796
PR002075 135.9 140219
PR002076 67.03 140541
PR002077 3.51 113373
Pogalizumab 0.91 18147
hIgG1 iso / 2530
实施例6 抗原结合蛋白能在体外激活OX40通路
使用10μg/ml的丝裂霉素(北京中生瑞泰科技，10107409001)处理CHO-K1(ATCC，#CCL-61)或者是CHO-K1/CD32b细胞，30分钟37℃。然后用10％FBS的F-12K培养液，洗涤4次。在96孔平底板(Corning，#3559)内每孔分别铺1.5×10 4个这两种处理过的细胞，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。去掉前一天96孔板的培养液，加入纯化的T细胞，每孔1×10 5个。然后加入相应浓度的OX40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℃5％CO 2箱培养箱培养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。ELISA检测方法参照相关试剂盒操作说明。酶标仪(Molecular Devices，型号SpectraMax Plus)读取450nm和570nM吸光度值，通过标准品读值(OD450-OD570)反推计算上清中IFN-γ浓度。应用软件GraphPad Prism 8进行数据处理和作图分析。结果如图6A所示本申请所述大部分OX40抗原结合蛋白均具有激活OX40通路，并诱导激活T细胞的功能。且其激活作用都比参照抗体Tab(Pogalizumab)更强。如图6B和表8所示，本发明中的OX40抗原结合蛋白具有CD32b交联依赖性，且对T细胞的激活效应比参照抗体大。
表8 OX40抗体对T细胞的激活效应
抗体 EC50(nM) 最大IFN-γ释放值
PR002066 0.03102 1873
PR002067 0.08634 2003
Pogalizumab 0.003736 1142
实施例7 利用BLI方法检测OX40抗体对重组OX40蛋白的结合亲和力和解离常数
通过生物膜干涉(BLI)技术，使用OctetRed96e(Fortebio)分子相互作用分析仪来进行抗原抗体之间的结合动力学分析。按照制造商提供的详细操作和方法，使用Octet RED96仪器(Pall Fortiebio)和ProA亲和素传感器(Pall ForteBio，#18-5010)测定亲和力。
先将置于一列的ProA亲和素传感器在测试缓冲液中平衡10分钟，然后用ProA传感器捕获200nM的OX40抗体，捕获高度0.8nm；ProA传感器在缓冲液中平衡120s后与2倍梯度稀释的人OX40蛋白或食蟹猴OX40蛋白(OX40HCAB用浓度为200-6.25nM和0nM；Pogalizumab则用25-1.56nM和0nM)结合180s，解离800s(PR002063，PR002065，PR002066和PR002077与食蟹猴的解离时间为400s)；最后将ProA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。Octet Red 96实时记录OX40抗体与OX40蛋白的结合和分离信号。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∶1 Global fitting”方法进行数据拟合，计算出抗原与抗原结合蛋白结合的动力学参数，得到kon(1/Ms)值、kdis(1/s)值和KD(M)值。结果如表9所示，本申请所述大部分OX40抗原结合蛋白结合人OX40或食蟹猴OX40的KD(M)都略高于Pogalizumab，表明与参照抗体相比其对OX40的结合亲和力相对较弱，可能是与本发明的OX40HCAb和Pogalizumab的结构差异相关。
表9.抗体与人OX40蛋白和食蟹猴OX40蛋白的结合亲和力
Figure PCTCN2021102946-appb-000004
Figure PCTCN2021102946-appb-000005
实施例8 抗原结合蛋白的特异性结合OX40
OX40属于TNF肿瘤坏死因子受体超家族，该家族是由一大类多功能的受体组成，这些受体具有介导免疫和非免疫细胞功能。已鉴定出6种受体是发挥重要作用的免疫共同刺激者，包括CD40，OX40，4-BB，CD27，GITR和CD30。同样，诱导型T细胞共刺激因子(ICOS)是另一类对激活的T细胞或记忆型T细胞的功能及存活有重要作用的受体。
本实施例是通过流式检测TNF肿瘤坏死因子受体超家族的3个受体和ICOS来研究抗人OX40的HCAb单抗体外结合的特异性。采用过表达人OX40的CHO-K1细胞株(CHO-K1/hu OX40)、过表达人CD40的CHO-K1细胞株(CHO-K1/hu CD40，北京康源博创，#KC-1286)、过表达人4-1BB的CHO-K1细胞株(CHO-K1/hu4-1BB，Genscript，#M00538)和过表达人ICOS的HEK293细胞株(HEK293T/ICOS，Genscript，#KC-0210)进行细胞水平上的抗体结合实验。简言之，消化这些细胞，并用F12K或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，离心去上清后加入100μl/孔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1∶1000稀释后的荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG，Fcγ Fragment Specific，Jackson#109-545-06)，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NovoCyte流式细胞仪(ACEA Biosciences)读取荧光发光信号值。
结果如图7所示，本申请所述的PR002067特异性地结合CHO-K1/OX40细胞，而不结合TNF肿瘤坏死因子受体超家族的其他成员。
实施例9 OX40和肿瘤靶点的双特异性抗体的结构和设计
自实施例1到实施例8所选的抗OX40重链抗体和抗PSMA(PR001331，H2L2抗体，202010096322.6)、抗OX40重链抗体和EPCAM(PR001081，H2L2抗体，202010114063.5)、抗OX40重链抗体和CLDN 18.2(PR002726，H2L2抗体，201910941316.3)、抗OX40重链抗体和B7H4(PR002408，H2L2抗体，)、抗OX40重链抗体和PD-L1(PR000265，H2L2抗体，201910944996.4)抗体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肿瘤靶点TAA(例如PSMA、EPCAM、CLDN18.2、B7H4、PD-L1)，而另一端可以结合T细胞上的OX40分子，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。
本实施例所用到的抗肿瘤标点(例如PSMA、EPCAM、CLDN18.2、B7H4、PD-L1)的H2L2抗体序列的信息如下表10所示。
表10 抗肿瘤靶点的H2L2抗体序列表
Figure PCTCN2021102946-appb-000006
本实施例制备的TAA×OX40双特异性抗体包括多种分子结构：
1)IgG-VH四价对称结构分子，结构如图8A所示，其包含两条多肽链：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。
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11中所列序列。
2)Fab-HCAb四价对称结构分子，结构如图8B所示，其包含两条多肽链：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。在该结构中，抗体A的VL_A和重链抗体B的VH_B融合在同一条多肽链上，这样可以避免VL_A和VH_B的缔合产生的错配副产物。
多肽链2的VH_B经由连接肽L2联结到CH2；L2可以是IgG的铰链区或者铰链区衍生的连接肽序列；L2可以是表11中所列序列，优选为人IgG1铰链或者人IgG1铰链(C220S)或者G5-LH的序列。
在一个实施方案中，多肽链2的CL与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CL经由连接肽L1联结到VH_B；L1可以是表11中所列序列。
表11 连接肽
Figure PCTCN2021102946-appb-000007
双特异性抗体含有IgG1的Fc结构域，其具有突变L234A和L235A或者L234A和L235A和P329G(根据EU索引编号)。
本实施例所构建IgG-VH四价对称结构的双特异性抗体的信息如下表12所示，本实施例所构建Fab-HCAb四价对称结构的双特异性抗体的信息如下表13所示，其物理化学性质如表14所示。
表12 IgG-VH四价对称结构的双特异性抗体
Figure PCTCN2021102946-appb-000008
Figure PCTCN2021102946-appb-000009
表13 Fab-HCAb四价对称结构的双特异性抗体
Figure PCTCN2021102946-appb-000010
表14 双特异性抗体的表达和物理化学性质
Figure PCTCN2021102946-appb-000011
本实施例所构建TAA×OX40双特异性抗体的重链轻链序列的CDR编号信息如下表15所示，多肽链编号信息如下表16所示。
表15 TAA×OX40双特异性抗体的序列CDR编号表
Figure PCTCN2021102946-appb-000012
表16 TAA×OX40双特异性抗体的多肽链编号表
Figure PCTCN2021102946-appb-000013
实施例10 FACS检测双特异性抗体与OX40细胞的结合能力
本实施例是为了研究实施例9中所得的OX40×TAA双特异性抗体体外结合人OX40的活性。采用过表达人OX40的CHO-K1细胞株(CHO-K1/hu OX40#)进行细胞水平上的抗体结合实验。简言之，消化细胞CHO-K1/hu OX40细胞，并用F12K完全培养基重悬，PBS洗涤后用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1∶1000稀释后的荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG，Fcγ Fragment Specific，Jackson，#109-545-06)，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NovoCyte流式细胞仪(ACEA Biosciences)读取荧光发光信号值。
如图9和表17所示，本申请的OX40×TAA的双特异性抗体双抗均能结合人OX40，且检测到的抗体结合能力与抗体浓度成正相关关系递增。其结合能力与参照抗体Tab(Pogalizumab)相当。
表17 OX40×TAA的双特异性抗体结合人OX40细胞
抗体 EC50(nM) MFI最大值
PR003789 3.977 287338
PR004276 2.799 281894
PR004277 3.458 274396
PR004283 1.809 204612
PR004284 4.955 287445
PR004285 3.548 252325
Pogalizumab 1.153 317098
hIgG1 ～70.97 6902
实施例11 FACS检测双特异性抗体与相应的肿瘤相关抗原细胞的结合能力
本实施例是为了研究实施例9中所得的OX40×TAA双特异性抗体体外结合人的肿瘤相关抗原(TAA)的活性。采用高表达人B7H4的SK-BR-3(中科院细胞库，#TCHu225)，高表达人PD-L1的MDA-MB-231(ATCC，HTB-26)，高表达人PSMA的LNCAP(南京科佰，#CBP60346)，或者高表达人CLDN18.2的NUGC-4(ExPASy，#CVCL_3082)，高表达人EPCAM的Capan-2(ATCC，#HTB-80)进行细胞水平上的抗体结合实验。简言之，消化这些细胞，并用完全培养基重悬，PBS洗涤后用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)，离心去上清后加入100μl/孔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1∶1000稀释后的荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG，Fcγ Fragment Specific，Jackson，#109-545-06)，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NovoCyte流式细胞仪(ACEA Biosciences)读取荧光发光信号值。
如图10和表18-1、表18-2、18-3、18-4、18-5所示，本发明的OX40×TAA双特异性抗体均能结合相应的人的肿瘤相关抗原，且检测到的抗体结合能力与抗体浓度成正相关关系递增。与相对应的TAA亲本单抗相比，双特异性抗体表现出与之相当的EC50和最大值。
表18-1 OX40×PD-L1双特异性抗体结合相应的MDA-MB-231细胞
抗体 EC50(nM) 荧光强度中位值最大值
PR003789 1.040 128390
PR000265 0.6050 141594
表18-2 OX40×B7H4双特异性抗体结合相应的SK-BR-3细胞
抗体 EC50(nM) 荧光强度中位值最大值
PR004276 1.139 354678
PR004277 1.506 362253
PR002408 1.813 373359
表18-3 OX40×EpCAM双特异性抗体结合相应的Capan-2细胞
抗体 EC50(nM) 荧光强度中位值最大值
PR004283 3.518 1544198
PR001081 2.584 1642796
表18-4 OX40×Claudin18.2的双特异性抗体结合相应的NUGC-4细胞
抗体 EC50(nM) 荧光强度中位值最大值
PR004285 7.205 740185
PR002726 4.982 745505
表18-5 OX40×PSMA双特异性抗体结合相应的LNCAP细胞
抗体 EC50(nM) 荧光强度中位值最大值
PR004284 6.701 160857
PR001331 2.578 142270
PR000327 3.025 126604
实施例12 双特异性抗体体外激活OX40通路
本实施例是为了研究OX40×TAA双特异性抗体在靶细胞的存下，通过结合共刺激分子OX40来激活T细胞的活性。
本实施例中，靶细胞是表达特定抗原(例如肿瘤特异性抗原)的细胞，例如高表达人PD-L1的MDA-MB-231(ATCC，HTB-26)，或者高表达人B7H4的CHO-K1-huB7H4(内 部构建)，或者高表达人PSMA的HEK293-huPSMA(北京康源，#KC-1005)，或者高表达人EPCAM的Capan-2(ATCC，HTB-80)，或者高表达人CLDN18.2的NUGC4(JCRB，JCRB0834)。效应细胞是分离的人T细胞。
具体的，首先以100μl/孔将0.3μg/ml抗CD3抗体OKT3(Thermo，#16-0037-81)包板于96孔平底板(Corning，#3599)。接着，将人T细胞(从人PBMC中用T细胞分选试剂盒(Miltenyi，#130-096-535)分离得到)的密度调整为2×10 6细胞/ml，将靶细胞的密度调整为3×10 5细胞/ml，随后把两种细胞悬液各以50μl/孔接种于96孔板。然后，以100μl/孔加入不同浓度的抗体分子，两个复孔加样；hIgG1 iso(CrownBio，#C0001)和hIgG4 iso(CrownBio，#C0045)作为对照。将96孔板置于37℃，5％CO 2培养箱中孵育2天。收集培养48小时后的上清液，用IL-2 ELISA试剂盒(Thermo，#88-7025-88)检测上清中的IL-2浓度，ELISA检测方法参照相关试剂盒操作说明。酶标仪(Molecular Devices，型号SpectraMax Plus)读取450nm和570nM吸光度值，通过标准品读值(OD 450-OD 570)反推计算IL-2浓度。应用软件GraphPad Prism 8进行数据处理和作图分析。
结果如图11所示(因存在个体差异单独PBMC供体可能得不到有效结果，故一般一次实验至少选择两个供体平行进行，实施例中分别标号为供体1和供体2)，可以看出本申请所述的OX40/TAA双特异性抗体在肿瘤细胞交联下均具有激活XO40介导的T细胞通路，说明OX40双特异性抗体在肿瘤靶细胞存在下能特异性激活T细胞。
实施例13 异体混合淋巴细胞反应(Heterologous Mixed Lymphocyte Reaction Assay，MLR)
本实施例是利用混合淋巴细胞反应(MLR)来研究PD-L1×OX40的双特异性抗体分子对T细胞的激活作用。
第一步，利用CD14磁珠(Meltenyi，#130-050-201)从第一供体PBMC细胞(妙通生物)中分离单核细胞(monocytes)；具体操作参照相关试剂盒说明书。然后加入50ng/ml重组人源IL-4(PeproTech，#200-02-A)和100ng/ml重组人源GM-CSF(PeproTech，#300-03-A)，于37℃诱导6天后，获得未成熟的树突状细胞(iDC细胞)。继续加入1μg/ml的脂多糖Lipopolysaccharide(LPS，Sigma，#L6529)，诱导24小时后，获得成熟的树突状细胞(mDC细胞)。第二步，利用T细胞分离试剂盒(Meltenyi，#130-096-535)从第二供体PBMC细胞(妙通生物)中分离得到T淋巴细胞。第三步，将获得的T细胞和mDC细胞按5∶1比例接种至96孔板(1×10 5/孔的T细胞和2×10 4/孔的mDC细胞)。随后以50μl/孔加入不同浓度的抗体分子，抗体终浓度可以是(10nM，1nM)，或者是从最高终浓度为50nM按照3倍浓度梯度稀释的共8个浓度，两个复孔加样；hIgG1 iso(CrownBio， #C0001)或者空白孔作为对照。于37℃，5％CO 2培养箱孵育5天。第四步，分别收集第3天和第5天的上清液，用IL-2 ELISA试剂盒(Thermo，#88-7025-88)检测第3天的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo，#88-7316-77)检测第5天的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。酶标仪(Molecular Devices，型号SpectraMax Plus)读取450nm和570nM吸光度值，通过标准品读值(OD 450-OD 570)反推计算IL-2或IFN-γ浓度。应用软件GraphPad Prism 8进行数据处理和作图分析。
结果如图12和表19-1和表19-2中所示，在两次独立的MLR实验中(不同的供体配对)，抗PD-L1单抗PR000265有较明显的激活作用，但是PD-L1×OX40的双特异性抗体分子PR003789可以进一步提高T细胞的功能。
表19-1 PD-L1×OX40双特异性抗体MLR诱导的细胞因子释放(供体1)
Figure PCTCN2021102946-appb-000014
表19-2 PD-L1×OX40双特异性抗体MLR诱导的细胞因子释放(供体2)
Figure PCTCN2021102946-appb-000015
小结
本发明为了克服目前靶向OX40的抗体的不足，通过免疫Harbour HCAb小鼠获得了一类全人源重链抗体。本发明的抗体具有与人OX40和食蟹猴OX40特异性结合的活性，能促进NF-Kb较大的激活，从而对OX40信号通路有刺激作用，且能够在体外激活OX40通路，并诱导激活T细胞的功能，其激活作用与现有抗体(例如Pogalizumab)相当或比其更强，同时本发明的抗体或其抗原结合片段具有Fcγ受体成员之一FcγRIIB(CD32B)交联依赖性。
本发明的抗体仅含“重链”的全人源抗体，这些仅含重链的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，同时能够解决轻链错配和异源二聚化的问题。将本发明的抗体与抗肿瘤靶点的H2L2抗体制备成IgG-VH四价对称结构的双抗分子和Fab-HCAb结构的双抗分子，所得双特异性抗体均能结合人OX40和相应的肿瘤相关抗原，其中一端可以识别肿瘤细胞表面特异表达的肿瘤靶点TAA(例如PSMA、EPCAM、CLDN18.2、B7H4、PD-L1)，而另一端可以结合T细胞上的OX40分子，从而特异性激活肿瘤微环境的T细胞，降低OX40激活引起的毒 性，并杀死肿瘤细胞。
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. 一种靶向OX40的抗体或其抗原结合片段，其特征在于，其包含重链可变区(VH)，
    其中，所述VH包含以下的互补决定区(CDR)或其突变：如SEQ ID NO：10的氨基酸序列所示的VH CDR1；如SEQ ID NO：44的氨基酸序列所示的VH CDR2；和，如SEQ ID NO：86、SEQ ID NO：84或SEQ ID NO：89的氨基酸序列所示的VH CDR3；
    其中，所述突变为在所述VH的VH CDR1、VH CDR2、VH CDR3的氨基酸序列的基础上分别具有3、2或1个氨基酸的插入、缺失或替换。
  2. 如权利要求1所述的靶向OX40的抗体或其抗原结合片段，其特征在于，VH CDR1的突变为在如SEQ ID NO：10所示的氨基酸序列上具有F2L、T3S/P/I、S5D、和/或S6D/C的氨基酸替换；其氨基酸序列例如如SEQ ID NO：13-16、SEQ ID NO：20-24任一所示；和/或，
    VH CDR2的突变为在如SEQ ID NO：44所示的氨基酸序列上具有S1T、R3H/L/G/S、G4S、G5N/D、S6N/I/Q/T的3、2或1个氨基酸替换；其氨基酸序列例如如SEQ ID NO：42-43、SEQ ID NO：45-50、SEQ ID NO：54-60任一所示；和/或，
    VH CDR3的突变为在如SEQ ID NO：86所示的氨基酸序列上具有T2M/I/V、T5S、T6W/Y、D9C和Y10F/W中的2或1个氨基酸替换；其氨基酸序列例如如SEQ ID NO：82-83、SEQ ID NO：85、SEQ ID NO：87-88、SEQ ID NO：90、SEQ ID NO：94-99任一所示。
  3. 如权利要求1或2所述的靶向OX40的抗体或其抗原结合片段，其特征在于，所述的靶向OX40的抗体或其抗原结合片段包含重链可变区(VH)，其中，所述VH包含以下的互补决定区(CDR)或其突变：如SEQ ID NO：10、SEQ ID NO：13-16、SEQ ID NO：20-24的任一氨基酸序列所示的VH CDR1；如SEQ ID NO：42-50、SEQ ID NO：54-60的任一氨基酸序列所示的VH CDR2；和，如SEQ ID NO：82-90、SEQ ID NO：94-99的任一氨基酸序列所示的VH CDR3；
    优选地，所述靶向OX40的抗体或其抗原结合片段包含重链可变区(VH)，其中，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：13、42和82所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、42和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO： 14、42和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、43和84所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：13、44和82所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：15、44和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、45和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、46和85所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：16、42和82所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、47和87所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：16、44和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、45和88所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、48和89所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、49和90所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、49和83所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、50和90所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：20、44和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：21、44和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、55和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、56和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、42和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、57和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、58和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、59和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、60和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和94所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和95所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和96所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和97所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和98所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO： 10、44和99所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和95所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和97所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、55和95所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、55和97所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、56和95所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、56和97所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：22、54和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：23、54和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：24、54和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：21、54和86所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、54和99所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：22、54和99所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：23、54和99所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：24、54和99所示；或，
    所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：21、54和99所示；
    优选地，所述VH包括如SEQ ID NO：142-164或SEQ ID NO：168-198所示的任一氨基酸序列；
    更优选地，所述靶向OX40的抗体或其抗原结合片段还包含人抗体的重链恒定区Fc结构域；所述人抗体的重链恒定区Fc结构域包括人IgG1、IgG2、IgG3或IgG4的重链恒定区Fc结构域；
    进一步更优选地，所述靶向OX40的抗体或其抗原结合片段包含一个多肽链，所述多肽链包含如SEQ ID NO：208-230或SEQ ID NO：234-264所示的任一氨基酸序列。
  4. 如权利要求1-3中任一项所述的靶向OX40的抗体或其抗原结合片段，其中所述靶向OX40的抗体或其抗原结合片段包括IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、HCAb、VH、双特异性抗体、多特异性抗体、单域抗体或者其他任何保留抗体特异性结合抗原的部分能力的抗体，或者由上述抗体制得的单克隆抗体或多克隆抗体。
  5. 一种双特异性的结合蛋白，其特征在于，其含有至少两个蛋白功能区：蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原，其中所述蛋白功能区B靶向OX40，所述蛋白功能区A靶向非OX40抗原；所述蛋白功能区B选自如权利要求1-4任一项所述的靶向OX40的抗体或其抗原结合片段。
  6. 如权利要求5所述的双特异性结合蛋白，其特征在于，所述蛋白功能区A靶向PD-L1、B7H4、PSMA、EPCAM或CLDN18.2。
  7. 如权利要求6所述的双特异性结合蛋白，其中，所述蛋白功能区A为PD-L1抗体或其抗原结合片段、B7H4抗体或其抗原结合片段、PSMA抗体或其抗原结合片段、EPCAM抗体或其抗原结合片段或CLDN18.2抗体或其抗原结合片段；
    优选地：
    所述PD-L1抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：111、119和129所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、39和79所示；或，
    所述EPCAM抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和130所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：11、40和80所示；或，
    所述PSMA抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：113、 121和131所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：12、41和81所示或，
    所述B7H4抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)；所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：114、122和132所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：17、51和91所示；或，
    所述CLDN18.2抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)；所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和133所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：18、52和92所示。
  8. 如权利要求5-7任一项所述的结合蛋白，其特征在于，所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)；所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：111、119和129所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、39和79所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：114、122和132所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：17、51和91所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和130所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：11、40和80所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：113、121和131所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：12、41和81所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含VL CDR1、VL CDR2和VL CDR3，其氨基酸序列分别如SEQ ID NO：112、120和133所示，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：18、52和92所示；所述蛋白功能区B包含重链可变区(VH)，所述VH包含VH CDR1、VH CDR2和VH CDR3，其氨基酸序列分别如SEQ ID NO：10、44和86所示。
  9. 如权利要求5-8任一项所述的结合蛋白，其特征在于，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：199所示的氨基酸序列，所述VH包括如SEQ ID NO：139所示的氨基酸序列；所述蛋白功能区B包含重链可变区，所述VH包括如SEQ ID NO：154所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：202所示的氨基酸序列，所述VH包括如SEQ ID NO：165所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：200所示的氨基酸序列，所述VH包括如SEQ ID NO：140所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：201所示的氨基酸序列，所述VH包括如SEQ ID NO：141所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：203所示的氨基酸序列，所述VH包括如SEQ ID NO：166所示的氨基酸序列；所述蛋白功能区B包含重链可变区(VH)，所述VH包括如SEQ ID NO：154所示的氨基酸序列。
  10. 如权利要求5-9任一项所述的双特异性结合蛋白，其特征在于，所述蛋白功能区A和/或所述蛋白功能区B为IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、VH、或者HCAb的形式；其中，所述蛋白功能区A和蛋白功能区B不同时为IgG；
    优选地，所述Fab、Fab’、F(ab’) 2、Fv、scFv、VH的数量为一个或多个。
  11. 如权利要求5-10任一项所述的双特异性结合蛋白，其特征在于，所述蛋白功能 区B为单个VH的结构，所述蛋白功能区A为IgG的结构；所述蛋白功能区B优选连接在所述蛋白功能区A的C末端；
    优选地，所述双特异性抗体包含第一多肽链和第二多肽链，所述第一多肽链如式：N’-VL_ A-CL-C’所示，所述第二多肽链如式：N’-VH_ A-CH1-h-CH2-CH3-L-VH_ B-C’所示；
    其中，所述的VH_ B为所述蛋白功能区B的VH，所述的VL_ A和VH_ A分别为所述蛋白功能区A的VL和VH，所述的h为铰链区，所述的L为连接肽；
    更优选地，所述L的长度优选为0或其氨基酸序列如SEQ ID NO.278-295任一所示、或其氨基酸序列为GS。
  12. 如权利要求5-10任一项所述的双特异性结合蛋白，其特征在于，所述蛋白功能区B为HCAb的结构，所述蛋白功能区A为Fab的结构；所述蛋白功能区B优选连接在所述蛋白功能区A的C末端；
    优选地：
    所述双特异性抗体包含第一多肽链和第二多肽链，所述第一多肽链如式：N’-VH_ A-CH1-C’所示，所述第二多肽链如式：N’-VL_ A-CL-L1-VH_ B-L2-CH2-CH3-C’所示；
    或者，所述双特异性抗体包含第一多肽链和第二多肽链，所述第一多肽链如式：N’-VL_ A-CL-C’所示，所述第二多肽链如式：N’-VH_ A-CH1-L1-VH_ B-L2-CH2-CH3-C’所示；
    其中，所述的VH_ B为所述蛋白功能区B的VH，所述的VL_ A和VH_ A分别为所述蛋白功能区A的VL和VH，所述的L1和L2为连接肽；
    更优选地，所述L1或L2的长度优选为0或其氨基酸序列如SEQ ID NO.278-295任一所示、或其氨基酸序列为GS，例如所述L1的氨基酸序列如SEQ ID NO.286所示，所述L2的氨基酸序列如SEQ ID NO.285所示。
  13. 如权利要求5-12任一项所述的双特异性结合蛋白，其特征在于，所述双特异性结合蛋白包含第一多肽链和第二多肽链，其中，
    第一多肽链包括如SEQ ID NO：265所示的氨基酸序列，第二多肽链包括如SEQ ID NO：271所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO：268所示的氨基酸序列，第二多肽链包括如SEQ ID NO：272所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO：273所示的氨基酸序列，第二多肽链包括如SEQ ID NO：274所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO：266所示的氨基酸序列，第二多肽链包括如SEQ ID NO：275所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO：267所示的氨基酸序列，第二多肽链包括如SEQ ID NO：276所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO：269所示的氨基酸序列，第二多肽链包括如SEQ ID NO：277所示的氨基酸序列。
  14. 一种嵌合抗原受体，其特征在于，其包含如权利要求1-4中任一项所述的靶向OX40的抗体或其抗原结合片段或如权利要求5-13中任一项所述的双特异性抗体。
  15. 一种免疫细胞，其特征在于，其包含如权利要求14所述的嵌合抗原受体。
  16. 一种分离的核酸，其编码如权利要求1-4中任一项所述的靶向OX40的抗体或其抗原结合片段或如权利要求5-13中任一项所述的双特异性抗体或权利要求14所述的嵌合抗原受体。
  17. 一种重组表达载体，其包含如权利要求16所述的分离的核酸；优选地，所述表达载体包含真核细胞表达载体和/或原核细胞表达载体。
  18. 一种转化体，其包含如权利要求16所述的分离的核酸或如权利要求17所述的重组表达载体；
    优选地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
  19. 一种靶向OX40的抗体或其抗原结合片段、或双特异性抗体的制备方法，其包含培养如权利要求18中所述的转化体，从培养物中获得靶向OX40的抗体或其抗原结合片段、或双特异性抗体。
  20. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物包括抗体部
    分和偶联部分，所述抗体部分包含权利要求1-4任意一项所述的靶向OX40的抗体或其抗原结合片段和/或如权利要求中5-13任一项所述的双特异性抗体，所述偶联部分包括但不限于可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合，所述抗体部分和偶联部分通过化学键或接头进行偶联。
  21. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-4中任一项所述的靶向OX40的抗体或其抗原结合片段、如权利要求中5-13任一项所述的双特异性抗体，以及药学上可接受的载体；
    优选地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分。
  22. 一种如权利要求1-4中任一项所述的靶向OX40的抗体或其抗原结合片段、如权利要求5-13中任一项所述的双特异性抗体、如权利要求14所述的嵌合抗原受体、如权利要求15所述的免疫细胞、如权利要求20所述的抗体药物偶联物和/或如权利要求21所 述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物、试剂盒和/或给药装置中的应用。
  23. 一种检测样品中OX40的方法，其包括使用如权利要求1-4中任一项所述的靶向OX40的抗体或其抗原结合片段和/或如权利要求中5-13任一项所述的双特异性抗体进行检测；优选地，所述检测方法为非诊断目的。
  24. 试剂盒，其特征在于，所述的试剂盒包括权利要求1-4中任一项所述的靶向OX40的抗体或其抗原结合片段、如权利要求5-13中任一项所述的双特异性抗体、如权利要求14所述的嵌合抗原受体、如权利要求15所述的免疫细胞、如权利要求20所述的抗体药物偶联物和/或如权利要求21所述的药物组合物，及任选地，说明书。
  25. 给药装置，其特征在于，所述的给药装置包含：(1)用于对有需要的受试者施用权利要求21所述的药物组合物的输注模块，以及(2)任选的药效监控模块。
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