WO2022262749A1 - Specific binding protein targeting pd1 and/or ox40 - Google Patents

Specific binding protein targeting pd1 and/or ox40 Download PDF

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WO2022262749A1
WO2022262749A1 PCT/CN2022/098838 CN2022098838W WO2022262749A1 WO 2022262749 A1 WO2022262749 A1 WO 2022262749A1 CN 2022098838 W CN2022098838 W CN 2022098838W WO 2022262749 A1 WO2022262749 A1 WO 2022262749A1
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antibody
amino acid
acid sequence
seq
antigen
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PCT/CN2022/098838
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French (fr)
Chinese (zh)
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卢恩杰森
包木胜
戎一平
尹跃翔
罗海山
黄冰
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和铂医药(上海)有限责任公司
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Priority to CN202280037353.1A priority Critical patent/CN117460746A/en
Publication of WO2022262749A1 publication Critical patent/WO2022262749A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present invention relates to the field of biomedicine, more specifically to the field of antibody therapy.
  • the mammalian immune system is a finely balanced system, but it can sometimes be disrupted by diseases such as cancer.
  • Immune checkpoint receptors play an important role in the immune system's response to disease by exerting co-stimulatory or co-inhibitory roles, and the delicate balance of the two determines the efficacy of the immune response.
  • Co-inhibitors inhibit T cell proliferation and induce anti-inflammatory cytokine release. They reduce inflammation and avoid organ/tissue damage from an overactive immune response.
  • co-stimulators promote the development of protective immune responses by promoting T cell clonal expansion, effector differentiation and survival.
  • a well-established approach to cancer immunotherapy can trigger the immune system to recognize and kill tumor cells by targeting these checkpoint receptors with antibodies that block co-inhibitory receptor function or induce co-stimulatory receptor activity (Pardoll, 2012 ).
  • Antibodies that block the activity of co-inhibitory receptors have shown promising clinical activity and are currently approved for the treatment of cancer (Larkin et al., 2015).
  • Antibodies that induce co-stimulatory receptor activity have shown great potential in preclinical model systems (Moran et al., 2013; Schaer et al., 2014), and several agents are currently in clinical trials (Mayes et al., 2018; Melero et al., 2013 ).
  • These antibodies are designed to mimic the ligands of these co-stimulatory receptors and are therefore also known as agonist antibodies.
  • OX40 (also known as CD134, ACT45, TNFRSF4) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is mainly expressed on activated T cells, including CD4+ T cells, CD8+ T cells, type 1 and type 2 T helper cells (Th1 and Th2) and regulatory T (Treg) cells, and expressed on activated natural killer (NK) cells.
  • TNFR tumor necrosis factor receptor
  • APCs antigen-presenting cells
  • OX40L OX40 ligand
  • OX40 stimulation can have direct effects on T cells, promoting their proliferation and survival, or indirect effects by enhancing the production of inflammatory cytokines such as IL2 and IFN ⁇ .
  • OX40 signaling can also regulate the function of Treg cells, although OX40 signaling on Treg cells can abrogate the suppressive activity of Treg cells (Takeda et al., 2004).
  • OX40 has been found to be expressed in tumor-infiltrating T cells from patients with head and neck cancer, melanoma, and colorectal cancer, however, high levels of OX40-positive lymphocytes are associated with better patient survival (Petty et al., 2002; Vetto et al., 1997).
  • Agonist antibodies to OX40 are currently in clinical trials for cancer, most showing good safety and limited clinical activity. The effect of targeting OX40 as a monotherapy is not ideal, and has not always achieved the expected results.
  • PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immune suppression.
  • PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA.
  • Two cell surface glycoprotein ligands of PD-1 have been identified, programmed death-ligand-1 (PD-L1) and programmed death-ligand-2 (PD-L2), which are expressed on antigen-presenting cells and They have been shown to downregulate T cell activation and cytokine secretion by binding PD-1 on many types of human cancer cells.
  • the interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancer cells.
  • CTLA4, PD1/PDL1 and other checkpoint receptor inhibitors enhance the anti-cancer effect of T cells, while co-stimulatory receptors Agonists CD28, 4-1BB, OX40, GITR, CD27 and ICOS promote anti-tumor immunity of T cells.
  • co-administration of co-stimulatory receptor agonists and checkpoint receptor inhibitors has not achieved good clinical results.
  • stimulation of these co-stimulatory receptors with agonists should enhance antitumor immunity in the highly immunosuppressive tumor microenvironment. Extensive mouse data also confirm the therapeutic potential of this class of drugs.
  • the present invention provides a specific binding protein capable of binding to one or more antigens with high affinity and high specificity, the antigen being PD-1 or a fragment thereof, and/or OX40 or a fragment thereof .
  • the present invention also provides nucleic acid molecules encoding the specific binding protein, expression vectors for producing the specific binding protein, host cells and methods for preparing the specific binding protein.
  • the invention also relates to the use of said specific binding protein in the treatment, prevention and/or diagnosis of diseases, such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.
  • the present invention provides a specific antibody or an antigen-binding fragment thereof, which binds to PD-1 or a fragment thereof, comprising a light chain variable region VL and a heavy chain variable region VH
  • the VL comprises CDR1, CDR2 and CDR3, and its amino acid sequences are shown in SEQ ID NO: 39, 46 and 54 respectively
  • the VH comprises CDR1, CDR2 and CDR3, and its amino acid sequences are respectively shown in SEQ ID NO: 8, 18 and 28, or as shown in SEQ ID NO:81, 86 and 91, respectively.
  • 1 to 3 amino acid substitutions may be included in these CDRs.
  • the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54, respectively, and the VH comprises CDR1, CDR2 and CDR3.
  • CDR3, its amino acid sequence is shown in SEQ ID NO:8, 18 and 28 respectively.
  • the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54, respectively, and the VH comprises CDR1, CDR2 and CDR3, Its amino acid sequence is shown in SEQ ID NO:81, 86 and 91 respectively.
  • amino acid mutations may be included in these CDRs, and can maintain the function of the antibody specifically binding to PD-1.
  • the amino acid mutation is amino acid substitution, and the number of amino acid substitutions can be 1-3.
  • the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95% , 97%, 98%, 99% or 100% identical amino acid sequences.
  • the VL of the above-mentioned specific binding protein comprises the amino acid sequence shown in SEQ ID NO:67.
  • the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 62, or has at least 80%, 85%, 88%, 90%, 92%, 95% of it , 97%, 98%, 99% or 100% identical amino acid sequences.
  • the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:62.
  • the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 80%, 85%, 88%, 90%, 92%, 95% of the amino acid sequence thereof , 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 102.
  • the above-mentioned specific antibody or antigen-binding fragment thereof is selected from IgG, Fab, Fab', F(ab') 2 , Fv or scFv. In some embodiments, the above-mentioned specific antibody or antigen-binding fragment thereof is IgG. In some embodiments, the specific antibody or antigen-binding fragment thereof described above includes a heavy chain constant region and/or a light chain constant region. In some embodiments, the heavy chain constant region described above may be derived from human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the light chain constant region may be selected from a kappa chain or a lambda chain. In some embodiments, the above-mentioned specific antibodies or antigen-binding fragments thereof may be prepared polyclonal antibodies or monoclonal antibodies.
  • amino acid substitutions occur at position 234 and/or position 235 of the Fc of the specific antibody or antigen-binding fragment thereof.
  • the above-mentioned specific antibodies or antigen-binding fragments thereof comprise L234A and/or L235A mutations.
  • the above-mentioned specific binding protein comprises two mutations, L234A and L235A.
  • the Fc of the above-mentioned specific antibody is the Fc of human IgG1. In some embodiments, the Fc of the above-mentioned specific antibody is the Fc of human IgG4. In some embodiments, the Fc comprises the L234A and L235A mutations.
  • the aforementioned specific antibodies or antigen-binding fragments thereof include heavy chains and light chains.
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97% thereof %, 98%, 99% or 100% identical amino acid sequences.
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 106 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97% %, 98%, 99% or 100% identical amino acid sequences.
  • the present invention provides a specific binding protein comprising at least two structural domains capable of binding PD-1 or a fragment thereof, and/or OX40 or a fragment thereof.
  • the specific binding protein is a bispecific antibody or an antigen-binding antibody thereof comprising a first domain and a second domain, the first domain binds PD-1 or a fragment thereof, and The second domain binds OX40 or a fragment thereof.
  • the second domain is a VH containing only the heavy chain and no light chain.
  • the first domain is a PD-1 antibody or an antigen-binding fragment thereof.
  • the second domain is an OX40 antibody or antigen-binding fragment thereof.
  • the structure of the first domain and/or the second domain is selected from one of IgG, Fab, Fab', F(ab') 2 , Fv, scFv, VH, or HCAb , preferably, the number of Fab, Fab', F(ab') 2 , Fv, scFv, VH is one or more.
  • the first domain is in the form of an IgG.
  • the IgG heavy chain constant region is a human heavy chain constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region; preferably, the IgG Fc in the first domain is human IgG1 or IgG4 Fc.
  • the human IgG preferably comprises one, two or three mutations among L234A, L235A and P329G, more preferably comprises two mutations of L234A and L235A or comprises three mutations of L234A, L235A and P329G;
  • the Fc of IgG in the first domain is the Fc of human IgG1, more preferably, the Fc comprises L234A, L235A and P329G mutations;
  • the IgG Fc in the first domain is the Fc of human IgG4, and more preferably, the Fc contains L234A, L235A and P329G mutations.
  • the human IgG preferably comprises one, two or three mutations among L234A, L235A and G237A, more preferably comprises two mutations of L234A and L235A or comprises three mutations of L234A, L235A and G237A;
  • the Fc of IgG in the first domain is the Fc of human IgG1, more preferably, the Fc comprises L234A, L235A and G237A mutations;
  • the IgG Fc in the first domain is the Fc of human IgG4, and more preferably, the Fc contains L234A, L235A and G237A mutations.
  • the first domain is a tetravalent structure.
  • the second domain is a VH structure, and the number of VHs is preferably 1, 2, 3 or 4; more preferably, the number of VHs is 2.
  • the first domain and the second domain are linked directly or via a linker peptide L to form a bispecific binding protein.
  • the specific binding protein is a bispecific antibody with IgG_HC-VH tetravalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 - CL 1 -C'; polypeptide chain 1, from the amino terminal to the carboxyl terminal, which comprises N'-VH 1 -CH 1 -h-CH2-CH3-L-VH 2 -C'; wherein, the VL 1 and VH 1 is the VL and VH of the first domain, the VH 2 is the VH of the second domain, the h is the hinge region of the IgG antibody, the CL 1 is the CL of the first domain, and the CH 1 is the CH1 of the first structural domain, the L is a connecting peptide, and CH3 of the polypeptide chain 1 is connected to VH 2 via L.
  • polypeptide chain 2 from the amino terminal to the carboxyl terminal, which contains N'-VL 1 - CL 1 -C
  • the specific binding protein is a bispecific antibody with a tetravalent symmetrical structure of VH-IgG_HC, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which comprises N'-VL 1 - CL 1 -C'; Polypeptide chain 1, from the amino terminal to the carboxyl terminal, which comprises N'-VH 2 -L-VH 1 -CH 1 -h-CH2-CH3-C'; wherein, the VL 1 and VH 1 is the VL and VH of the first domain, the VH 2 is the VH of the second domain, the h is the hinge region, the CL 1 is the CL of the first domain, and the CH1 is the first domain CH 1 of the domain, the L is a connecting peptide, VH 2 of the polypeptide chain 1 is connected to VH 1 via the connecting peptide L.
  • polypeptide chain 2 from the amino terminus to the carboxyl terminus, which comprises N'-VL 1
  • the specific binding protein is a bispecific antibody with a tetravalent symmetrical structure of Fab(CL)-VH-Fc, which comprises two polypeptide chains, polypeptide chain 2, from the amino terminus to the carboxyl terminus, which comprises N '-VH'-CH1'-C'; polypeptide chain 1, from amino terminus to carboxy terminus, comprising N'-VL'-CL'-L-VH2 - h- CH2 - CH3 -C'.
  • VL' and VH' are VL and VH of the first structural domain respectively
  • said VH2 is VH of the second structural domain
  • said h is a hinge region
  • said L is a connecting peptide
  • said CL ' is the CL of the first structural domain
  • the CH1' is the CH1 of the first structural domain
  • the CL' of the polypeptide chain 1 is directly fused with VH 2 , that is, the length of L is 0.
  • the specific binding protein is a bispecific antibody with IgG_HC-VH-VH hexavalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which comprises N'-VL 1 -CL 1 -C'; polypeptide chain 1, from amino terminus to carboxy terminus, comprising N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L 1 -VH 2 -L2-VH 2 -C '.
  • VL 1 and VH 1 are respectively VL and VH of the first structural domain
  • said VH 2 is VH of the second structural domain
  • said h is a hinge region
  • said L1 and L2 are connecting peptides
  • the CL 1 is the CL of the first domain
  • the CH 1 is the CH1 of the first domain
  • the CH3 of the polypeptide chain 1 is connected to the VH 2 through L1
  • the two VH 2 are connected through L2.
  • the specific binding protein is a bispecific antibody with an octavalent symmetrical structure of IgG_HC-VH-VH-VH, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N' - VL1-CL1-C'; polypeptide chain 1, from amino-terminus to carboxy-terminus, comprising N'-VH1-CH1-h-CH2-CH3-L1-VH2-L2-VH2-L3-VH2-C'.
  • said VL1 and VH1 are respectively the VL and VH of the first structural domain
  • said VH2 is the VH of the second structural domain
  • said h is a hinge region
  • said L1, L2 and L3 are connecting peptides
  • said CL1 is the CL of the first structural domain
  • the CH1 is the CH1 of the first structural domain
  • CH3 in the polypeptide chain 1 is connected to the first VH2 from the N-terminal to the C-terminal of the polypeptide chain 1 via the connecting peptide L1
  • the first The VH2 is connected to the second VH2 through the connecting peptide L2
  • the second VH2 is connected to the third VH2 through the connecting peptide L3.
  • the hinge region h is a common hinge region in the immunoglobulin field, usually contains a large amount of proline, is flexible, and forms 2-5 disulfide bonds.
  • said L is a peptide with a length of 0-30 amino acids, and its amino acid sequence is shown in any one of SEQ ID NO: 116-140, see Table 1.
  • CH3 of the second polypeptide chain is directly linked to VH2, ie the length of L is zero.
  • the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), and the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are as SEQ ID NO: As shown in 39, 46 and 54, the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 8, 18 and 28 or shown in SEQ ID NO: 81, 86 and 91 respectively.
  • VL light chain variable region
  • VH heavy chain variable region
  • the first domain comprises VL and VH.
  • the amino acid sequence of the VL is as shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% thereof Or 100% consistency.
  • the amino acid sequence of the VL is as shown in SEQ ID NO:67.
  • the VH has an amino acid sequence such as SEQ ID NO: 62 or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof. %consistency.
  • the amino acid sequence of the VH is as SEQ ID NO:62.
  • the amino acid sequence of the VH is as shown in SEQ ID NO: 102, or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% % or 100% consistency. In a specific embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO: 102.
  • the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, Heavy chains that are 95%, 97%, 98%, 99% or 100% identical.
  • the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain.
  • the first domain comprises a light chain as shown in SEQ ID NO:77, and a heavy chain as shown in SEQ ID NO:71.
  • the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain.
  • said first domain comprises a light chain as shown in SEQ ID NO:77 and a heavy chain as shown in SEQ ID NO:72.
  • the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical heavy chain.
  • said first domain comprises a light chain as shown in SEQ ID NO:77 and a heavy chain as shown in SEQ ID NO:106.
  • the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively.
  • the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 85 and 90, respectively.
  • the second domain comprises a VH having an amino acid sequence as shown in SEQ ID NO: 63, or at least 80%, 85%, 88%, 90%, 92%, 95% therewith. %, 97%, 98%, 99% or 100% agreement.
  • the amino acid sequence of the VH of the second domain is as shown in SEQ ID NO:63.
  • the second domain comprises VH, the amino acid sequence of the VH is as shown in SEQ ID NO: 101, or at least 80%, 85%, 88%, 90%, 92%, 95% therewith %, 97%, 98%, 99% or 100% agreement.
  • the amino acid sequence of the VH of the second domain is shown in SEQ ID NO: 101.
  • the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain. In specific embodiments, said second domain comprises a heavy chain as set forth in SEQ ID NO:73. In some embodiments, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain. In specific embodiments, said second domain comprises a heavy chain as set forth in SEQ ID NO: 105.
  • the bispecific antibody comprises a first domain and a second domain
  • the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • the Said VL comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:39, 46 and 54 respectively
  • said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:8, 18 and 28 respectively
  • the second domain comprises a heavy chain variable region (VH)
  • the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively.
  • the bispecific antibody comprises a first domain and a second domain
  • the first domain comprises VL and VH
  • the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are as follows: Shown in SEQ ID NO:39, 46 and 54, said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:81, 86 and 91 respectively;
  • said second structural domain comprises VH
  • the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively.
  • the bispecific antibody comprises a first domain and a second domain
  • the first domain comprises VL and VH
  • the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are as follows: Shown in SEQ ID NO:39, 46 and 54, said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:81, 86 and 91 respectively;
  • said second structural domain comprises VH
  • the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 85 and 90, respectively.
  • the bispecific antibody comprises a first domain and a second domain
  • the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequences of the VL and VH are shown in SEQ ID NO:67 and SEQ ID NO:62 respectively
  • the second domain comprises VH
  • the amino acid sequence of the VH is shown in SEQ ID NO:63.
  • the bispecific antibody comprises a first domain and a second domain
  • the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequences of the VL and VH are shown in SEQ ID NO:67 and SEQ ID NO:102, respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO:63.
  • the bispecific antibody comprises a first domain and a second domain
  • the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequences of the VL and VH are shown in SEQ ID NO:67 and SEQ ID NO:102, respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO:101.
  • the bispecific binding protein comprises polypeptide chain 1 and polypeptide chain 2, wherein polypeptide chain 1 is a long chain and polypeptide chain 2 is a short chain.
  • the polypeptide chain 1 has SEQ ID NO: 79, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID The amino acid sequence shown in any one of NO:115, or the amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity; the polypeptide chain 2 have the amino acid sequence shown in any one of SEQ ID NO:77, SEQ ID NO:113, or have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical Sexual amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 79; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 109; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 110; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 111; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 112; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 114; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 113 amino acid sequence.
  • the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 115; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
  • the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof in the first aspect of the present invention, the specific binding protein or fragment thereof in the second aspect.
  • the nucleic acid molecule is an mRNA molecule.
  • the present invention provides an expression vector comprising the isolated nucleic acid molecule described in the third aspect of the present invention.
  • the expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a retrovirus vector, a lentivirus vector, a phage vector, an adenovirus vector, an adeno-associated vector or a herpes simplex vector.
  • a retrovirus vector such as a retrovirus vector, a lentivirus vector, a phage vector, an adenovirus vector, an adeno-associated vector or a herpes simplex vector.
  • the expression vector is present in nanoparticles, liposomes, exosomes, microvesicles, or gene guns.
  • the present invention provides a host cell comprising the isolated nucleic acid molecule of the third aspect, or the expression vector of the fourth aspect.
  • the host cell is a conventional host cell in the art, as long as the expression vector of the fourth aspect can stably express the carried nucleic acid molecule as the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody described in the second aspect.
  • sexual antibodies are prokaryotic cells and/or eukaryotic cells
  • the prokaryotic cells are preferably E.coli cells such as TG1, BL21 (expressing single-chain antibodies or Fab antibodies)
  • the eukaryotic cells are preferably HEK293 cells or CHO cells cells (expressing full-length IgG antibodies).
  • the host cell of the present invention can be obtained by transforming the expression vector of the fourth aspect into a host cell.
  • the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect.
  • the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization technology.
  • the method of preparation includes the step of culturing the host cell of the fourth aspect.
  • Harbor HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the second domain of the bispecific antibody of the second aspect.
  • the HCAb transgenic mouse is a transgenic mouse carrying the immune repertoire of human immunoglobulins, capable of producing novel "heavy chain only” antibodies that are half the size of traditional IgG antibodies. It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains.
  • the method for preparing the second domain of the bispecific antibody of the second aspect using HCAb transgenic mice comprises the following steps:
  • the human OX40 antigen is recombinant human OX40-ECD-Fc, specifically, the antigen is a recombinant fusion protein composed of the extracellular region of OX40 linked to Fc;
  • the IgG antibody is an IgG1 antibody or an IgG4 antibody.
  • H2L2 transgenic mice (hereinafter referred to as H2L2 transgenic mice) are used to prepare the specific antibody or antigen-binding fragment thereof of the first aspect or the first domain of the bispecific antibody of the second aspect.
  • the H2L2 transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune library, and the antibody produced by it has a complete human antibody variable domain and a rat constant domain.
  • the method for preparing the first domain of the specific antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect using H2L2 transgenic mice comprises the following steps:
  • the human PD-1 antigen is soluble recombinant human PD-1-hFc, specifically, the antigen is composed of PD-1 linked to Fc recombinant fusion protein;
  • Hybridoma cells were obtained by fusion of splenocytes from immunized H2L2 transgenic mice with myeloma cell lines, and the isolated hybridomas expressed heavy and light chain antibodies with complete human variable domains and rat constant domains molecular;
  • the antibody light chain variable domain sequence (VL) obtained in step (b) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human antibody kappa light chain constant domain sequence to encode the antibody-producing full-length light chain;
  • the antibody heavy chain variable domain sequence (VH) obtained in step (b) is synthesized through genes and cloned into a mammalian cell expression vector encoding a human IgG antibody heavy chain constant domain sequence to encode an IgG antibody full-length heavy chain;
  • step (e) Simultaneously transfect the expression vectors of steps (c) and (d) into mammalian host cells, and use conventional recombinant protein expression and purification techniques to obtain recombinant antibodies with correct pairing and assembly of light and heavy chains.
  • the antibody heavy chain variable domain sequence (VH) obtained in step (b) is gene-synthesized and cloned into a mammalian cell expression vector encoding a human IgG4 antibody heavy chain constant domain sequence to encode the IgG4 antibody produced full-length heavy chain.
  • the antibody heavy chain variable domain sequence (VH) obtained in step (b) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human IgG1 antibody heavy chain constant domain sequence, so as to encode the IgG1 antibody produced full-length heavy chain.
  • the prepared second domain and the prepared first domain are combined into a specific binding protein.
  • the specific binding protein can simultaneously bind two targets, wherein the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, and the second domain can bind OX40 molecules on T cells. After the protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
  • the prepared second domain and the prepared first domain are constructed into a bispecific binding protein.
  • the bispecific binding protein can simultaneously bind two targets, wherein the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, while the second domain can bind OX40 molecules on T cells, and the specificity After the binding protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
  • the bispecific binding protein has a bivalent structure or a tetravalent symmetrical structure. More preferably, the bispecific binding protein has a tetravalent symmetrical structure.
  • the bispecific binding protein has the structure and sequence described in the second aspect.
  • the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof in the first aspect or the bispecific antibody in the second aspect, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes other ingredients as active ingredients, such as other small molecule drugs or antibodies or polypeptides 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 adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the field, preferably including pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the specific binding protein and/or other small molecule drugs or antibodies or polypeptides, and 0.01-99.99% of the pharmaceutical carrier, the percentage being The mass percent of the pharmaceutical composition.
  • the administration route of the pharmaceutical composition can be parenteral, injection or oral administration.
  • the pharmaceutical composition can be prepared in a form suitable for administration, such as solid, semi-solid or liquid form, which can be aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion form.
  • Administration can be intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation, or intrathoracic instillation.
  • the pharmaceutical composition can also be administered in the form of aerosol or spray, such as nasal administration; or, intrathecal, intramedullary or intraventricular administration, and can also be transdermal, transdermal, topical, enteral, intravaginal , sublingual or rectal administration.
  • the pharmaceutical composition can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dose beneficial to the patient.
  • the specific binding protein and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.
  • the specific binding protein is a bispecific binding protein.
  • the present invention provides the antibody or antigen-binding fragment thereof in the first aspect, the bispecific antibody in the second aspect, the isolated nucleic acid molecule in the third aspect, and the pharmaceutical composition in the seventh aspect Application in preparation of medicines for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases.
  • the tumor can be breast cancer, renal cell carcinoma, melanoma, colon cancer, and B cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin's lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignancy, etc.
  • the inflammatory disease may be atopic dermatitis, ulcerative colitis and the like.
  • the immune disease may be graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma and the like.
  • the present invention provides a method for detecting OX40 and PD-1 in a sample, the method comprising using the antibody or antigen-binding fragment described in the first aspect or the bispecific antibody in the second aspect to detect OX40 and PD-1 in the sample OX40 and PD-1 steps.
  • the sample can be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
  • a biological sample for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
  • the method of detecting OX40 and PD-1 in a sample is for non-diagnostic purposes.
  • the present invention also provides a kit, which includes one or more kits, comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the antibody or antigen-binding fragment thereof as described in the second aspect.
  • a kit which includes one or more kits, comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the antibody or antigen-binding fragment thereof as described in the second aspect.
  • the set of kits comprises a first kit comprising the antibody or antigen-binding fragment thereof of the first aspect, or the first domain and the second structure of the second aspect Domain-composed bispecific antibodies.
  • the kit of parts may further include a second kit comprising other therapeutic agents including, but not limited to, chemotherapeutics, radiotherapeutics, immunosuppressive agents and cytotoxic drugs.
  • the above-mentioned first medicine box and the second medicine box can be used at the same time, or the above-mentioned first medicine box can be used first and then the above-mentioned second medicine box, or the above-mentioned second medicine box can be used first and then the above-mentioned first medicine box can be used.
  • a medicine box can be determined according to the actual needs of specific applications.
  • the present invention provides methods for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antibody of the first aspect of the present invention or its The antigen-binding fragment or the bispecific antibody of the second aspect or the pharmaceutical composition of the seventh aspect.
  • the PD1-specific antibody of the present invention can specifically bind to cells expressing human PD-1 and cells expressing cynomolgus monkey PD-1;
  • the first domain of the PD1 ⁇ OX40 bispecific antibody of the present invention can specifically bind to cells expressing human PD-1 and cells expressing cynomolgus PD-1; the second domain can bind to human OX40 protein and cynomolgus Monkey OX40 protein; the second domain showed concentration-dependent enhancement of NF- ⁇ B signaling pathway under CHO-K1/CD32b cross-linking conditions;
  • the PD1 ⁇ OX40 bispecific antibody of the present invention is PD-1 cross-linking dependent; under the assistance of CHO-K1/PD-1 cell cross-linking, it can specifically cause concentration-dependent enhancement of NF- ⁇ b signaling pathway effect. It can specifically induce the promotion of PD-1 cross-linking-dependent OX40-mediated NF- ⁇ b signaling pathway, and the signal strength it causes increases in a positive correlation with its concentration;
  • the PD-1 ⁇ OX40 bispecific antibody of the present invention has an inhibitory effect on the PD-1 signaling pathway; can enhance the secretion of TNF ⁇ and IFN ⁇ ; has an inhibitory effect on the secretion of IL-10 by Treg cells; and can promote cytokine production in T cells. Secretion of Granzyme B.
  • the PD-1 ⁇ OX40 bispecific antibody of the present invention can not only enhance the function and survival of effector T cells, but also inhibit the suppressive function of Treg; compared with individual PD-1 monoclonal antibody and OX40 monoclonal antibody, and The combination of PD-1 mAb and OX40 mAb showed obvious synergistic activity.
  • ABSOR and “approximately” generally mean an acceptable range of error for the measured value given the nature or precision of the measurement. Typically the margin of error is within 20%, typically within 10%, and even more typically within 5% of a given value or range of values.
  • antigen binding molecule or “specific binding protein” refers broadly to a molecule that specifically binds an antigenic determinant.
  • Antigen binding molecules include, for example, antibodies, antibody fragments, and backbone antigen binding proteins.
  • antibody of the present invention encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single chain molecules and antibody fragments as long as they exhibit the desired antigen-binding activity.
  • the term "monoclonal antibody” in the present invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible traces of variant antibodies (e.g., containing naturally occurring mutations or arising during the production of monoclonal antibody preparations). , usually present in small amounts), the individual antibodies comprised by the population of antibodies are identical and/or bind the same epitope. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • multispecific antibody of the present invention is used in its broadest sense to encompass antibodies with polyepitopic specificities.
  • 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 polyepitopic specificity; Antibodies with VL and VH domains, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable domains, each single variable domain binds to Binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently Wait.
  • VH heavy chain variable region
  • VL light chain variable region
  • bispecific binding protein or “bispecific antibody” of the present invention refers to the ability to specifically bind at least two different antigenic determinants, for example each composed of an antibody heavy chain variable domain (VH) and antibody light
  • VH antibody heavy chain variable domain
  • VL chain variable domain
  • Bispecific antibodies can be in a 1+1 format, a 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (comprising a second antigen or epitope).
  • bispecific antibodies comprise two antigen-binding sites, each specific for a different antigenic determinant.
  • valence in the present invention means that an antigen-binding molecule has a specified number of binding domains present.
  • bispecific antibody is at least “bivalent”, and may be “trivalent”, “tetravalent” or “more valent”).
  • the antibodies have two or more binding sites and are bispecific. That is, antibodies can be bispecific even in cases where there are more than two binding sites (ie, the antibody is trivalent or multivalent).
  • full-length antibody and “intact antibody” of the present invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody.
  • “Native antibody” refers to a naturally occurring immunoglobulin molecule.
  • antibodies of the native IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) (also known as the heavy chain constant region).
  • VH variable region
  • CH1 and CH3 constant domains
  • each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain domain). chain constant region).
  • the heavy chain of an antibody can be of one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), which can be further divided into Subtypes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1 ) and ⁇ 2 (IgA2).
  • the light chains of an antibody based on the amino acid sequence of their constant domains, can be of one of two types, kappa light chains and lambda light chains.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed 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 is composed 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.
  • Antibody fragments comprise a portion of an intact antibody.
  • Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv; diabodies, triabodies, tetrabodies, crossed Fab fragments; linear antibodies; single chain antibody molecules (e.g. scFv); Multispecific antibodies formed from antibody fragments and single domain antibodies.
  • an antigen binding domain refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term “antigen-binding domain” refers to a part of an antibody comprising a region that specifically binds and is complementary to a part or all of an antigen. In cases where the antigen molecule is large, the antigen-binding molecule can bind only a specific part of the antigen, called an epitope.
  • An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions).
  • the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • an antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.
  • antigenic determinant in the present invention is synonymous with “antigen” and “epitope”, and refers to a site on a polypeptide macromolecule (such as a stretch of continuous amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids ), the antigen-binding moiety binds to said site, thereby forming an antigen-binding moiety-antigen complex.
  • the antigenic determinant may be present, for example, on the surface of tumor cells, microbially infected cells, other diseased cells, immune cells, free matter in serum and/or in the extracellular matrix (ECM).
  • the proteins used as antigens in the present invention may be proteins in any native form from any vertebrate source, including, for example, primates (e.g., humans) and rodents (e.g., mice and rats) and other mammals.
  • the antigen can also be a human protein, or the antigen can be "full length", unprocessed protein, any form of protein resulting from intracellular processing, or a naturally occurring variant of a protein, such as a splice variant or an allele Variants.
  • Specifically binds means that binding is selective for an antigen and can be distinguished from unwanted or non-specific binding.
  • the ability of an antigen-binding molecule to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques as well as traditional binding assays.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, as measured by SPR.
  • the solution of the molecule bound to the antigen is ⁇ 1 ⁇ M, ⁇ 100nM, ⁇ 10nM, ⁇ 1nM, ⁇ 0.1nM, ⁇ 0.01nM or ⁇ 0.001nM (for example, 10 -7 M or lower, such as 10 -7 M to 10 -13 M, eg 10- 9 M to 10 -13 M).
  • Binding affinity refers to the strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Binding affinity can generally be expressed in terms of a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (k off and k on , respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • high affinity in the present invention means that the Kd of the antibody for the target antigen is 10 -9 M or lower, even 10 -10 M or lower.
  • low affinity in the present invention means that the Kd of the antibody is 10 -8 M or higher.
  • An “affinity matured” antibody is one that has one or more modifications in one or more hypervariable regions (HVRs) that result in greater Affinity improvements.
  • HVRs hypervariable regions
  • single domain antibody and “nanobody” in the present invention have the same meaning, referring to only having the variable region of the heavy chain of an antibody, and constructing a single domain antibody consisting of only one heavy chain variable region, which is fully functional the smallest antigen-binding fragment.
  • HCAb antibody refers to an antibody that lacks the light chain of the antibody and only contains the heavy chain, compared to the double-chain antibody (immunoglobulin), specifically contains the variable structure of the heavy chain domain and Fc constant domain.
  • bispecific antibody comprising a first antigen-binding domain specifically binding to PD1 and a second antigen-binding domain specifically binding to OX40
  • bispecific antibody specifically binding to PD1 and OX40 specifically binding to PD1 and OX40
  • specific to PD1 and OX40 bispecific antigen-binding molecule or "anti-PD1/anti-OX40 antibody” or "PD-1 x OX40 bispecific antibody” are used interchangeably herein, and refer to the ability to bind PD1 and OX40 with sufficient affinity , so that the antibody can be used as a bispecific antibody targeting PD1 and OX40 as a diagnostic and/or therapeutic agent.
  • T effector cells in the present invention refers to T cells with cytolytic activity (for example, CD4+ and CD8+ T cells) and T helper (Th) cells, T effector cells secrete cytokines, and activate and guide other immune cells, Regulatory T cells (Treg cells) are not included.
  • the anti-OX40 antibody of the present invention can activate T effector cells, such as CD4+ and CD8+ T effector cells.
  • Treg cell refers to a special type of CD4+ T cell that can block the response of other T cells.
  • Treg cells are characterized by the expression of CD4, the alpha subunit of the IL-2 receptor (CD25), and the transcription factor FOXP3, and play a crucial role in the induction and maintenance of peripheral self-tolerance against tumor-expressed antigens .
  • PD1 also known as programmed cell death protein 1
  • programmed cell death protein 1 is a type I membrane protein composed of 288 amino acids, which belongs to the immunoglobulin superfamily, and was first disclosed in 1992 (Ishida et al., EMBO J., 11(1992), 3887-3895).
  • PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators and has at least two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273).
  • the protein structure consists of an extracellular IgV domain, followed by a transmembrane region and an intracellular tail.
  • the intracellular tail contains two phosphorylation sites located in the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates T-cell receptor TCR signaling. This is consistent with the binding of SHP-1 phosphatase and SHP-2 phosphatase to the cytoplasmic tail of PD-1 after ligand binding.
  • PD-1 is not expressed on naive T cells, it is upregulated following T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately.
  • PD-1 has a relatively broad expression profile, its most important role may be as a co-inhibitory receptor on T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches mainly block the interaction of PD-1 with its ligands to enhance T cell responses.
  • the terms "programmed death 1" "programmed cell death 1" "protein PD-1” "PD-1” “PD1” “PDCD1” “hPD-1” and “hPD-1” are used interchangeably and include human Variants, isoforms, species homologues of PD-1, and analogs that share at least one common epitope with PD-1.
  • the amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) accession number Q15116.
  • PD1 antibody of the present invention is capable of binding to PD1, especially a PD1 polypeptide expressed on the cell surface, with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD1.
  • PD1 antibodies bind less to irrelevant, non-PD1 proteins than to PD1 as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system. About 10% of the binding capacity.
  • the KD value of the antigen binding protein that binds to human PD1 is ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM or ⁇ 0.001 nM (for example, 10 ⁇ 8 M or lower, eg 10 -13 M to 10 -8 M, eg 10 -13 M to 10 -9 M).
  • the corresponding KD value of the binding affinity is determined using human PD1 (PD1-ECD) in a surface plasmon resonance assay to obtain the PD1 binding affinity.
  • Therapeutic strategies with PD-1/PD-L1 antibodies are a standard treatment strategy in several metastatic tumors and have shown their role in early disease stages and adjuvant therapy, especially in melanoma and non-small cell lung cancer.
  • OX40 in the present invention is also called CD134, which is expressed in activated CD4+T cells, CD8+T cells, dendritic cells, neutrophils and Treg cells 1-3 days after activation.
  • the ligand OX40L of OX40 is expressed on the surface of antigen-presenting cells (APC) such as dendritic cells and B cells.
  • APC antigen-presenting cells
  • OX40/OX40L can recruit TRAF molecules in the intracellular region of OX40, and can also activate the canonical NF- ⁇ B1 pathway or non-canonical NF- ⁇ B2 pathway, PI3k/PKB and NFAT pathways, thereby regulating T cell division and survival
  • the genes that promote the transcription of cytokine genes and the expression of cytokine receptors can also promote the differentiation of B cells into plasma cells and the production of antibodies, etc. In other words, it can enhance the activity of effector T cells, NK, and NK-T cells , Relieving the immunosuppressive effect of Treg can not only enhance the specific immune response, but also enhance the innate immune response, thereby enhancing anti-tumor immunity.
  • OX40 antibody of the present invention is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting OX40.
  • an OX40 antibody binds to an irrelevant OX40 protein less than the antibody binds to OX40 as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system about 10% of capacity.
  • RIA radioimmunoassay
  • FACS flow cytometry
  • the antibody that binds OX40 has a concentration of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10 ⁇ 8 M or less, such as 10 ⁇ 13 M to 10 -8 M, eg, 10 -13 M to 10 -9 M) dissociation constant (KD).
  • concentration of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM e.g., 10 ⁇ 8 M or less, such as 10 ⁇ 13 M to 10 -8 M, eg, 10 -13 M to 10 -9 M
  • KD dissociation constant
  • H2L2 transgenic mouse or "Harbour H2L2 mouse” in this application is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces a protein consisting of two heavy chains with a fully human variable region.
  • the antibodies produced by the transgenic mice are affinity matured, fully humanized variable regions, and have excellent solubility.
  • HardbourHCAb mouse (WO2002/085945A2) in this application is a transgenic mouse carrying an immune repertoire of human immunoglobulins capable of producing novel "heavy chain only” antibodies that are only the size of traditional IgG antibodies half. It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains. Due to the absence of light chains, this "heavy chain only” 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.
  • variable region refers to the domain of antibody heavy chain or light chain that participates in the binding of antigen-binding molecule to antigen.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • variable in the present invention means that certain segments of the variable domains generally differ in sequence between antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains.
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, mostly in a ⁇ -sheet configuration, connected by three HVRs that form loops connecting and in some cases forming part of the ⁇ -sheet structure.
  • the HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)).
  • the constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.
  • hypervariable region refers to the region in the variable domain region of an antibody that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops").
  • native four-chain antibodies typically contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).
  • HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)" that have the highest sequence variability and/or are involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53 -55 (H2) and 96-101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987).
  • Exemplary CDRs occur At amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89-97 (L3), amino acid residues 31-35 (H1), amino acid residues 50-65 (H2) , and amino acid residues 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). For comparison, in Table 2 The corresponding amino acid residues of the CDRs defined in the above cited references are listed in.
  • the amino acid sequences of the CDRs listed above are all shown according to the Chothia definition rule (claims of the application is also shown in accordance with the Chothia definition rules).
  • the CDRs of antibodies can be defined by various methods in the art, such as the Kabat definition rules based on sequence variability and the position of the structural loop region The Chothia definition rule (seeing J Mol Biol 273:927-48,1997).In this application, can also use the Combined definition rule that comprises Kabat definition and Chothia definition to determine the amino acid residue in the variable domain sequence.
  • Combined The definition rule is to combine the scope defined by Kabat and Chothia, based on which a larger scope is taken.
  • FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4.
  • HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • the "class" of an antibody refers to the type of constant domain or constant region that the heavy chain of the antibody has.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • a “humanized antibody” comprises amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody comprises at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs Corresponds to FRs of human antibodies.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, eg, a non-human antibody refers to an antibody that has been humanized.
  • a "human antibody” has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • Fc domain or "Fc region” of the present invention is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the IgG Fc region consists of an IgG CH2 domain and an IgG CH3 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • the CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain.
  • the CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fcy receptors (eg, FcyRI, FcyRIIa, FcyRIIb, FcyRIII) and/or complement. It is hypothesized that reducing or eliminating binding to Fc receptor gamma will reduce or eliminate ADCC mediated by antibody molecules. Similarly, reducing or eliminating binding to complement is expected to reduce or eliminate CDC mediated by antibody molecules. Mutations that reduce or eliminate binding of the CH2 domain to one or more Fc ⁇ receptors and/or complement are known in the art (Wang et al., 2018).
  • LALA mutation involves the replacement of the leucine residues at positions 1.3 and 1.2 of the IMGT of the CH2 domain with alanine (L1.3A and L1.2A).
  • CH2 Mutation of the asparagine (N) at position 84.4 of the IMGT position in the domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q) to glycosylate the conserved N-chain Site mutations to generate a-glycosyl antibodies to reduce IgG1 effector function are also known (Wang et al., 2018).
  • complement activation C1q binding
  • ADCC can be achieved through IMGT of the CH2 domain.
  • Mutation of proline at position 114 to alanine or glycine P114A or P114G reduces (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to produce ADCC or CDC with further reduced or no Active antibody molecules.
  • an "antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind a given antigen (eg, PD-1 or OX4).
  • the antigen-binding function of an antibody can be performed by fragments of an intact antibody.
  • antigen-binding portion of an antibody or antigen-binding fragment examples include, but are not limited to, a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; an F(ab) 2 fragment, a fragment comprising two A bivalent fragment of a Fab fragment, the two Fab fragments are linked by a disulfide bridge at the hinge region; an Fd fragment, which consists of the VH and CH1 domains; an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody Composition: a single domain antibody (dAb) fragment consisting of a VH domain or a VL domain; and isolated complementarity determining regions (CDRs).
  • a Fab fragment a monovalent fragment consisting of VL, VH, CL and CH1 domains
  • an F(ab) 2 fragment a fragment comprising two A bivalent fragment of a Fab fragment, the two Fab fragments are linked by a disulfide bridge at
  • a region equivalent to the Fc region of an immunoglobulin includes naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having substitutions, additions or deletions that do not substantially reduce immunoglobulin-mediated effector functions ( Modified variants with capabilities such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, eg, Bowie, J.U. et al., Science 247:1306-10 (1990)).
  • effector function of the present invention is attributable to the Fc region of an antibody and is a biological activity that varies with the antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine Secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (such as B cell receptors), and B cell activation, among others.
  • peptide linker or "connecting peptide” of the present invention refers to a peptide comprising one or more amino acids, usually about 2 to 20 amino acids.
  • the connecting peptide is a connecting peptide known in the art or described herein.
  • fused to refers to segments (eg antigen binding domain and FC domain) linked by peptide bonds either directly or via one or more linking peptides.
  • the invention also relates to amino acid sequence variants of the bispecific antibodies of the invention.
  • Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be made to arrive at a final construct having the desired properties, such as antigen binding activity. Sites for substitution typically include HVRs and frameworks (FRs). See Table 3 for possible amino acid substitutions.
  • polynucleotide or “nucleic acid” or “nucleotide sequence” or “nucleic acid molecule” in the present invention refers to an isolated nucleic acid molecule or construct such as messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA ).
  • mRNA messenger RNA
  • pDNA virus-derived RNA
  • pDNA plasmid DNA
  • a polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNAs)).
  • PNAs peptide nucleic acids
  • nucleic acid molecule refers to any one or more nucleic acid segments, such as DNA or RNA segments, present in a polynucleotide.
  • isolated nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA or RNA, which has been separated from its natural environment.
  • the recombinant polynucleotide encoding the polypeptide contained in the vector is also isolated.
  • isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution.
  • An isolated polynucleotide includes a polynucleotide molecule normally contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, in plus- and minus-strand form, as well as in double-stranded form.
  • Isolated polynucleotides or nucleic acids of the invention further include such molecules produced synthetically.
  • a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, ribosomal binding site, or transcription terminator.
  • expression cassette refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow the transcription of a particular nucleic acid in a target cell.
  • Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
  • the term "vector” or "expression vector” and “expression construct” in the present invention can be used interchangeably, and a specific gene operably linked to it is introduced into a target cell and directs the expression of a DNA molecule.
  • the vector includes a vector that is a self-replicating nucleic acid structure as well as a vector that is incorporated into the genome of a host cell into which it has been introduced.
  • the expression vectors of the present invention comprise expression cassettes. Expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery.
  • the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants/transformants” and “transformed cells”, including primary transformed cells and progeny derived therefrom.
  • the nucleic acid of the progeny may not be identical to that of the parental cell and may contain mutations.
  • a host cell is any type of cell that can be used to produce a bispecific antigen binding molecule of the invention.
  • Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or hybridoma cells, yeast cells, insect cells and plant cells, also cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.
  • cultured mammalian cells such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or hybridoma cells, yeast cells, insect cells and plant cells, also cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.
  • an “effective amount” of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which it is administered.
  • An “effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dosage of administration and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • the bispecific antibodies according to the invention have a synergistic effect.
  • “Synergistic effect” means that the combined effect of two drugs is greater than the sum of their individual effects and statistically different from the control and single drugs.
  • the additive effect of the present invention means that the combined effect of the two drugs is the sum of their individual effects and is statistically different from the control and/or single drug.
  • a “therapeutically effective amount” of a drug refers to that amount, in dosage and administration interval and time, effective to achieve the desired therapeutic or prophylactic effect.
  • a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes or prevents the adverse effects of a disease.
  • “Individual” or “subject” refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human.
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats
  • “Pharmaceutical composition” means a mixture comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure and other chemical components such as physiologically/pharmaceutically acceptable carriers or excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • “Pharmaceutically acceptable carrier” refers to an ingredient of a pharmaceutical composition other than the active ingredient that is nontoxic to the subject.
  • Pharmaceutically acceptable excipients include, but are not limited to, buffers, diluents, stabilizers and/or preservatives.
  • cancer is meant to describe diseases in mammals characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal carcinoma, hepatocellular carcinoma, Gastric cancer (including gastrointestinal and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer cancer, endometrial or cervical cancer, salivary gland cancer, kidney or ureter cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer
  • Treatment refers to administering to a patient an internal or external therapeutic agent, such as a composition comprising any specific binding protein of the present disclosure, a bispecific antibody, an antibody or an antigen-binding fragment thereof or an encoding specific binding protein, bispecific Antibodies, nucleic acid molecules of antibodies or antigen-binding fragments thereof, the patient has one or more diseases or conditions, and the therapeutic agent has a therapeutic effect on these diseases or conditions.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to ameliorate one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.
  • preventing cancer means delaying, inhibiting or preventing the onset of cancer in a mammal in which the onset of carcinogenesis or tumorigenesis has not been proven, but has been identified, for example, by genetic screening or other methods have a susceptibility to cancer.
  • the term also includes treating a mammal with a premalignant condition to halt the progression of the premalignant condition to a malignancy or to cause its regression.
  • liposome is meant to include a variety of unilamellar and multilamellar lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • administration refers to providing the specific binding protein, isolated nucleic acid molecule, expression vector, host cell or pharmaceutical composition of the present invention to a subject in need, for example, by oral administration, injection or local administration. who apply.
  • sequence numbers (SEQ ID NO:) of the light chain, heavy chain and variable region of the antibody of the present application are shown in Table 4.
  • sequence number (SEQ ID NO:) of the framework region of the antibody of the present application is shown in Table 5.
  • Figure 1 shows the PD-1 ⁇ OX40 bispecific antibody IgG_HC-VH structure (structure 1).
  • Figure 2 shows the structure of PD-1 ⁇ OX40 bispecific antibody VH-IgG_HC (structure 2).
  • Figure 3 shows the PD-1 ⁇ OX40 bispecific antibody Fab(CL)-VH-Fc structure (Structure 3).
  • Figure 4 shows the PD-1 ⁇ OX40 bispecific antibody IgG_HC-VH-VH structure (structure 4).
  • Figure 5 shows the PD-1 ⁇ OX40 bispecific antibody IgG_HC-VH-VH-VH structure (Structure 5).
  • Figure 6 shows that PD-1 monoclonal antibody blocks the binding of PD-1 and PD-L1.
  • Figure 7 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to PD-1 on the cell surface as the concentration increases in CHO cells expressing human PD-1 determined by flow cytometry.
  • Figure 8 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to OX40 on the cell surface as the concentration increases in the HEK 293 cells of human OX40 and NFkB promoter-luc determined by flow cytometry .
  • Figure 9 shows that in the HEK 293 cells of human OX40, PD1 and NFkB promoter-luc determined by flow cytometry, the bispecific antibody of the present invention or the monoclonal antibody constituting its structure increase with the concentration on the cell surface combined.
  • Fig. 10 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to the cell surface as the concentration increases in activated human T cells determined by flow cytometry.
  • Figure 11 shows the detection of luciferase reporter gene expression in HEK293 cells expressing human OX40 and NFkB promoter-luc, after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure.
  • Figure 12 shows that CHO cells expressing human CD32b and HEK 293 cells expressing human OX40 and NF ⁇ B promoter-luc are co-cultured, and after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure, the effect on luciferase Detection of reporter gene expression.
  • Figure 13 shows the co-culture of CHO cells expressing human PD-1 and HEK 293 cells expressing human OX40 and NF ⁇ B promoter-luc, after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure, the effect on fluorescence Detection of primease reporter gene expression.
  • Figure 14 shows the co-culture of HEK 293 cells expressing human PD-L1 and OS8 and Jurkat cells expressing human PD-1 and NFAT promoter-luc, adding different concentrations of the bispecific antibody of the present invention or the monoclonal composition of its structure Detection of luciferase reporter gene expression after antibody.
  • Figure 15 shows that in the co-culture system of human peripheral blood mononuclear cells (PBMC) and CHO cells overexpressing human PD-L1, adding PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal composition of its structure Secretion of the cytokine TNF ⁇ following antibody.
  • PBMC peripheral blood mononuclear cells
  • Figure 16 shows that in the co-culture system of human peripheral blood mononuclear cells (PBMC) and CHO cells overexpressing human PD-L1, PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal composition of its structure are added Secretion of the cytokine IFN ⁇ following antibody.
  • PBMC peripheral blood mononuclear cells
  • Fig. 17 shows that the Treg secretion of IL10 was detected by flow cytometry after adding different concentrations of the bispecific antibody of the present invention to the expanded Treg cells in vitro.
  • Figure 18 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of IFN ⁇ .
  • Figure 19 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of Granzyme B.
  • Figure 20 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of IL2.
  • Figure 21 shows the secretion of cytokine IL2 in human peripheral blood mononuclear cells (PBMC) after adding PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure.
  • PBMC peripheral blood mononuclear cells
  • Example 1 The acquisition of anti-OX40 fully human HCAb antibody
  • the Harbor HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces novel "heavy chain only” antibodies the size of traditional IgG antibodies half. It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains. Due to the fact that it does not contain light chains, 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.
  • an immunogenic reagent prepared by mixing 50 ⁇ g of antigenic protein with complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1.
  • each mouse was immunized with an immunogenic reagent prepared by mixing 25 ⁇ g of antigenic protein with Ribi adjuvant (Sigma Adjuvant System, Sigma, #S6322).
  • Immunization scheme 2 the HEK293/OX40 (ChemPartner, Shanghai) stable cell line overexpressing human OX40 was used for immunization.
  • Each mouse was intraperitoneally injected with 2 ⁇ 10 6 cell suspension each time it was immunized.
  • the interval between each booster round is at least two weeks and usually no more than five booster rounds.
  • the immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected.
  • Collect mouse blood dilute the blood 10 times, take 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), and put it on the ELISA plate coated with human OX40-ECD-Fc ELISA was performed to determine the titer of anti-human OX40 in mouse blood, and two concentrations of mouse blood (1:100, 1:1000) were detected by flow cytometry against CHO-K1/hOX40 cells with high expression of OX40 (Chempartner, Shanghai) and atopic reactivity of CHO-K1 blasts.
  • the blank control group (PB) was the serum of mice before immunization.
  • the splenocytes of the mice are taken out to separate B cells, and CD138-positive cells are sorted by BD flow cytometry sorter (BD Biosciences, FACS AriaII Cell Sorter) Plasma cells and human OX40 antigen positive B cell population. Extract B cell RNA, reverse transcribe cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, #18091200), and then amplify human VH gene by PCR with specific primers.
  • BD flow cytometry sorter BD Biosciences, FACS AriaII Cell Sorter
  • PCR forward primer 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' SEQ ID NO: 141
  • PCR reverse primer 5'-AATCCCTGGGCACTGAAGAGACGGTGACC-3' (SEQ ID NO: 142).
  • 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
  • HCAb antibodies Detect the binding of the HCAb-expressing supernatant to the stable cell line CHO-K1/OX40 (CHO-K1/hu OX40, Genscript, #M00561) overexpressing human OX40, and use a positive antibody (porcizumab) as a positive control ,conduct 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) and incubate for 30 minutes in a dark place. After centrifuging the cells, wash the cells with culture medium and adjust the cell density to 1 ⁇ 10 5 cells/mL. Then add 1:1000 diluted Alexa 488, AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-545-098), take 30 ⁇ L of the mixture per well and add it 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 tested for NF-kb function again, thus obtaining 64 proteins that simultaneously bind to CHO-K1/hu OX40 and cynomolgus monkey OX40 A functional fully human OX40 monoclonal antibody with a unique sequence. According to the results of human-monkey binding ability and NF-Kb function test, select the antibody with the highest comprehensive ranking for recombinant expression.
  • the plasmid encoding the HCAb antibody obtained above was transfected into a mammalian host cell (such as human embryonic kidney cell HEK293), and a purified anti-OX40 recombinant heavy chain antibody was obtained by using conventional recombinant protein expression and purification techniques.
  • a mammalian host cell such as human embryonic kidney cell HEK293
  • a purified anti-OX40 recombinant heavy chain antibody was obtained by using conventional recombinant protein expression and purification techniques.
  • HEK293 cells were expanded in FreeStyle TM F17Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to 6 ⁇ 10 5 cells/mL, and cultured in an 8% CO 2 shaker at 37°C for 24 hours with a cell concentration of 1.2 ⁇ 10 6 cells/mL. Prepare 30 mL of cultured cells.
  • 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 MabSelect TM (GE Healthcare Life Science, #71-5020-91AE) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample 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 into PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain the purified antibody Human OX40 HCAb monoclonal antibody solution.
  • Antibody concentration was determined by NanoDrop detection of 280nm absorbance, and the purity of antibody was determined by SEC-HPLC and SDS-PAGE.
  • OX40 antibodies PR002067 and PR002063 were obtained, and the corresponding heavy chain amino acid sequences are shown in SEQ ID NO:73 and SEQ ID NO:105.
  • the applicant produced and prepared the anti-OX40 positive control antibody Pogalizumab (pogalizumab) analog (PR003475), its amino acid sequence is derived from IMGT data, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 74, and the amino acid of the light chain of the antibody See SEQ ID NO:78 for the sequence.
  • the purity and aggregated form of the antibody protein samples obtained above were analyzed using analytical size exclusion chromatography (SEC). Connect the analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 ⁇ m, 7.8 mm ⁇ 30 cm) to a high-pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II), and equilibrate with PBS buffer at room temperature for at least 1 Hour.
  • SEC analytical size exclusion chromatography
  • An appropriate amount of protein sample (at least 10 ⁇ g, the sample concentration adjusted to 1 mg/mL) is filtered through a 0.22 ⁇ m filter membrane and injected into the system, and the HPLC program is set: use pH 7.4 PBS buffer to flow the sample through the chromatography at a flow rate of 1.0 mL/min Column, the maximum time is 20 minutes; the detection wavelength is 280nm.
  • ChemStation software After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample.
  • the purity and hydrophobicity of the antibody protein samples obtained above were analyzed using analytical hydrophobic interaction chromatography (HIC).
  • the analytical chromatographic column TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6mm ⁇ 3.5cm) was connected to a high pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), equilibrated with PBS buffer at room temperature At least 1 hour.
  • the set method consists of a linear gradient from 100% mobile phase A (20mM histidine, 1.8M ammonium sulfate, pH 6.0) to 100% mobile phase B (20mM histidine, pH 6.0) within 16 minutes, with the flow rate set at 0.7mL/min, protein sample concentration 1mg/mL, injection volume 20 ⁇ L, detection wavelength 280nm.
  • ChemStation software uses ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample.
  • DSF Differential Scanning Fluorimetry
  • Table 6 shows the detection results of the physicochemical properties of the OX40 antibodies PR002067 and PR002063.
  • This example is to study the activity of the prepared anti-OX40 HCAb monobody in vitro binding to human and cynomolgus monkey OX40 proteins.
  • Human OX40 protein (Acro biosystem, #OX0-H5224) and cynomolgus monkey OX40 protein (Novoprotein, #CB17) were used to conduct antibody binding experiments at the protein level. Briefly, each well of a 384-well plate (PerkinElmer, #6007509) was coated with 20 ⁇ L of 1 ug/mL human OX40 protein and cynomolgus monkey OX40 protein dissolved in PBS, overnight at 4°C.
  • the OX40 PR002067 antibody and PR002063 antibody of this example can bind to human OX40 and cynomolgus monkey (cyno) OX40 protein, and the detected antibody binding ability is positively correlated with the concentration of the antibody.
  • the EC50 of PR002067 and PR002063 binding to human OX40 protein and cynomolgus monkey OX40 protein is equivalent to that of Tab (Pogalizumab), indicating that the antibody can be more effective at a lower concentration. Sensitively binds human OX40.
  • the binding activity of OX40 antibody to human OX40 protein is shown in Table 7.
  • OX40 antibody The binding activity of OX40 antibody to cynomolgus monkey OX40 protein is shown in Table 8.
  • experimental animals can usually be immunized with PD-1 antigen, and the experimental animals can be mice, rats, rabbits, sheep, camels, etc.
  • the resulting antibody molecules are non-human.
  • these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability.
  • the humanization process of antibodies has its own technical complexity, and molecules after humanization often reduce the affinity for antigens.
  • transgenic technology advances in transgenic technology have made it possible to create genetically engineered mice that carry the human immunoglobulin immune repertoire and have their endogenous murine immune repertoire deleted.
  • the antibody produced by this transgenic mouse has a fully human sequence, so there is no need for further humanization, which greatly improves the efficiency of therapeutic antibody development.
  • Harbour Antibodies BV is a transgenic mouse carrying a human immunoglobulin immune repertoire, which produces antibodies with complete human antibody variable domains and rat constant domains.
  • mice were immunized for multiple rounds with soluble recombinant human PD-1-hFc fusion protein (Shanghai ChemPartner).
  • the antigenic protein is mixed with an immune adjuvant to form an immunogenic reagent, which is then injected subcutaneously through the groin or intraperitoneally.
  • each mouse received a total injection dose of 100 ⁇ L.
  • each mouse was immunized with an immunogenic reagent prepared by mixing 50 ⁇ g of antigenic protein (human PD-1-hFc) with complete Freund’s adjuvant (Sigma, #F5881) at a volume ratio of 1:1.
  • each mouse was immunized with an immunogenic reagent prepared by mixing 25 ⁇ g of antigenic protein with Ribi adjuvant (Sigma Adjuvant System, #S6322).
  • the interval between each booster round is at least two weeks and usually no more than five booster rounds.
  • the immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected.
  • the last booster immunization was performed with a dose of 25 ⁇ g antigenic protein per mouse.
  • mice dilute the blood 10 times, take 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), in the human PD-1-His (Shanghai ChemPartner ) ELISA plate for ELISA detection to determine the titer of anti-human PD-1 in mouse blood, and the anti-PD-1 titer of two concentrations of mouse blood (1:100, 1:1000) was detected by flow cytometry Specific reactivity of expressed CHO-K1/hPD-1 cells (Shanghai ChemPartner) and CHO-K1 blasts.
  • the blank control group (PB) was the serum of mice before immunization.
  • the spleen cells of the mouse are taken out and fused with myeloma cell lines to obtain hybridoma cells; the hybridoma cells are screened and cloned through multiple rounds Afterwards, at least 8 hybridomas expressing anti-PD-1 monoclonal antibody molecules were isolated.
  • the isolated hybridoma cells and their expressed monoclonal antibodies are indicated by the corresponding clone numbers, for example: 4004_10H9A12, 4004_12H9C1 and so on.
  • the isolated hybridomas express antibody molecules with intact human variable domains and heavy and light chains of rat constant domains.
  • the above-mentioned monoclonal antibodies were further identified, and several hybridomas were selected according to their binding ability to human PD-1, binding ability to cynomolgus monkey PD-1, and ability to inhibit the binding of PD-1 and PD-L1.
  • Clones were sequenced.
  • the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence were obtained by conventional hybridoma sequencing means.
  • the sequences of the variable domains of anti-PD-1 monoclonal antibody molecules obtained from immunized Harbor H2L2 mice are human antibody sequences.
  • the CDR sequences are divided by Chothia definition rules.
  • the heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic high-frequency mutation of the germline gene V, D, and J gene fragments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline gene V and J gene fragments of the light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • the positive control antibody Pembrolizumab (Keytruda) analog (PR000150) of the anti-PD-1 of the present application its corresponding amino acid sequence is derived from the IMGT database, the antibody heavy chain sequence is shown in SEQ ID NO: 69, and the antibody light chain sequence As shown in SEQ ID NO:75.
  • the antibody light chain variable domain sequence (VL) obtained from Harbor H2L2 mice was synthesized by gene and cloned into a mammalian cell expression plasmid vector encoding the human antibody kappa light chain constant domain sequence to encode the The full-length light chain of the antibody.
  • the antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG4 antibody heavy chain constant domain sequence (SEQ ID NO: 143) to encode the full IgG4 antibody production long heavy chain.
  • the plasmid encoding the heavy chain of the Harbor H2L2 antibody and the plasmid encoding the light chain of the antibody are simultaneously transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and using conventional recombinant protein expression and purification techniques, the correct paired assembly of the light and heavy chains can be obtained. Purified recombinant antibodies.
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to (6-8) ⁇ 10 5 cells/mL, and cultured in a 8% CO 2 shaker at 37°C for 24 hours, and the cell concentration was 1.2 ⁇ 10 6 cells/mL. Prepare 30 mL of cultured cells. The above-mentioned heavy chain plasmid and light chain plasmid encoding the H2L2 antibody were mixed and dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088) at a ratio of 2:3, and sterilized by filtration with a 0.22 ⁇ m filter membrane.
  • Opti-MEM reduced serum medium Thermo, #31985088
  • a gravity column (Bio-Rad, #7311550) containing MabSelect TM (GE Healthcare Life Science, #71-5020-91AE) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. Then use 0.1M glycine at pH 3.5 to elute the target protein, then adjust to neutrality with Tris-HCl at pH 8.0, and finally use an ultrafiltration tube (Millipore, #UFC901024) to concentrate and replace the medium to PBS buffer to obtain purified antibody solution. Then use NanoDrop (Thermo Scientific TM NanoDrop TM One) to measure the concentration, aliquot and store for future use.
  • PBS pH 7.4
  • Protein samples were analyzed for purity and aggregated form using analytical size exclusion chromatography (SEC). Connect the analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8mm x 30cm) to a high-pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), equilibrate with PBS buffer at room temperature At least 1 hour.
  • SEC analytical size exclusion chromatography
  • Transform the Fc of PR000674 switch the antibody type from IgG4 to IgG1 and introduce LALA (L234A and L235A mutations, according to EU index numbering) mutations to obtain PR001985, and the variable light and heavy chains of the PR001985 antibody are listed in Table 10 Domain amino acid sequence, light chain full-length amino acid sequence, heavy chain (human IgG1) full-length amino acid sequence, and amino acid sequences of CDRs defined according to Chothia definition rules.
  • the potential PTM site of the PR001985 antibody was mutated to obtain a new antibody molecule PR006429 (called the PTM removal variant).
  • Table 10 lists the light and heavy chain variable domain amino acid sequences of the PR006429 antibody, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG1), and the amino acid sequences of CDRs defined according to Chothia's definition rules. All designed PTM-removal variants PR006429 were purified recombinant antibodies according to the method described in Example 2.3, and further verified in subsequent functional experiments.
  • CHO-K1 cell lines (CHO-K1-hPD-1, CHO-K1-cPD-1, derived from GenScript) overexpressing human or cynomolgus monkey PD-1. Briefly, digest CHO-K1-hPD-1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x 106 cells/mL.
  • the binding activity of PD-1 antigen binding protein to CHO-K1 cells overexpressing human PD-1 is shown in Table 12.
  • the binding activity of PD-1 antigen binding protein to CHO-K1 cells overexpressing cynomolgus monkey PD-1 is shown in Table 13.
  • Antigen-binding proteins block the binding of human PD-L1 to CHO-K1 cells overexpressing human PD-1
  • the CHO-K1 cell line (CHO-K1-hPD-1) overexpressing human PD-1 was used to carry out the cell level detection.
  • Human PD-1/human PD-L1 binding blocking experiment Briefly, digest CHO-K1-hPD-1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x 106 cells/mL. Inoculate 100 ⁇ L cells/well in 96-well V-bottom plate (Corning, Cat#3894), then add 100 ⁇ L/well, 2 times the final concentration of 3 times or 5 times the concentration of the antigen-binding protein to be tested, and mix evenly.
  • the highest final concentration of antigen-binding protein was 100nM or 300nM, a total of 8 concentrations, and hIgG was used as a control. Place the cells at 4°C and incubate for 1 hour in the dark. Afterwards, centrifuge at 4°C for 5 minutes, discard the supernatant, then add 50 ⁇ L/well of biotin-labeled human PD-L1 protein (AcroBiosystems, PD1-H82F2) at a concentration of 1 ⁇ g/mL, and incubate at 4°C for 30 minutes in the dark. Add 100 ⁇ L/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant.
  • biotin-labeled human PD-L1 protein AcroBiosystems, PD1-H82F2
  • Figure 6 and Table 14 show that both antibodies PR001985 and PR006429 can block the binding of human PD-L1 to human PD-1 on the cell surface, and the inhibitory ability is equivalent to that of the positive control Keytruda (pembrolizumab).
  • Combining the anti-OX40 antibody prepared in Example 1 and the anti-PD-1 antibody prepared in Example 2 is used to prepare a bispecific antibody, which can bind to two targets at the same time, and one end (the first domain) can recognize the surface of tumor cells Specifically expressed PD-1, while the other end (second domain) can bind to OX40 molecules on T cells.
  • the PD-1 ⁇ OX40 double antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
  • the PD-1 ⁇ OX40 bispecific antibody prepared in this example is IgG1
  • PR003787 has Fc L234A, L235A and P329G mutations (according to EU index numbering)
  • R200538, PR200539, PR200531, PR200536, PR200528 and PR200600 have Fc L234A, L235A and G237A)
  • the molecular structure is shown in Figure 1-5.
  • Bispecific antibodies PR003787, PR200531 and PR200528 with IgG_HC-VH tetravalent symmetrical structure were constructed by using anti-PD-1 H2L2 antibody and anti-OX40 HCAb antibody.
  • the binding protein of IgG_HC-VH tetravalent symmetrical structure contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 -CL 1 - C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L-VH 2 -C'.
  • said VL 1 and VH 1 are respectively the VL and VH of the first structural domain
  • said VH 2 is the VH of the second structural domain
  • said h is the hinge region of an IgG antibody
  • said CL 1 is the first structural domain
  • the CL of the domain, the CH 1 is the CH1 of the first domain
  • the L is the connecting peptide
  • the CH3 of the polypeptide chain 1 is connected to the VH 2 via L.
  • the connecting peptide L in the bispecific antibody PR003787 is H1, and its amino acid sequence is shown in SEQ ID NO: 116 in Table 15.
  • the connecting peptide L in the bispecific antibodies PR200531 and PR200528 is (G2S) 2 , and its sequence is shown in Shown in SEQ ID NO: 117 in Table 15.
  • the anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200538 with VH-IgG_HC tetravalent symmetrical structure.
  • the binding protein of the VH-IgG_HC tetravalent symmetrical structure contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 -CL 1 - C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VH 2 -L-VH 1 -CH 1 -h-CH 2 -CH 3 -C'. .
  • VL 1 and VH 1 are respectively VL and VH of the first structural domain
  • said VH 2 is the VH of the second structural domain
  • said h is the hinge region
  • said CL 1 is the CL of the first structural domain
  • the CH 1 is the CH1 of the first structural domain
  • the VH 2 of the polypeptide chain 1 is connected to VH1 via the linker peptide L
  • the L is the linker peptide (G4S) 3 , its sequence is as shown in SEQ ID NO: 118 in Table 15 shown
  • the anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200539 with a tetravalent symmetrical structure of Fab(CL)-VH-Fc.
  • the binding protein of the Fab(CL)-VH-Fc tetravalent symmetrical structure contains two polypeptide chains: Polypeptide chain 2, also known as the short chain, from the amino terminal to the carboxyl terminal, which contains N'-VH '-CH1'-C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VL'-CL'-L-VH2 - h-CH2-CH3-C'.
  • VL' and VH' are VL and VH of the first structural domain respectively
  • said VH2 is VH of the second structural domain
  • said h is a hinge region
  • said L is a connecting peptide
  • said CL ' is the CL of the first structural domain
  • the CH1' is the CH1 of the first structural domain
  • the CL' of the polypeptide chain 1 in PR200539 is directly fused with VH2, that is, the length of L is 0.
  • the anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200536 with IgG_HC-VH-VH hexavalent symmetrical structure.
  • the binding protein of IgG_HC-VH-VH hexavalent symmetrical structure contains two polypeptide chains: polypeptide chain 2, also known as short chain, from amino terminal to carboxyl terminal, which contains N'-VL 1 -CL 1 -C'; Polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, comprising N'-VH1- CH1 -h- CH2 -CH3-L1-VH2-L2-VH2 - C'.
  • VL 1 and VH 1 are respectively VL and VH of the first structural domain
  • said VH 2 is VH of the second structural domain
  • said h is a hinge region
  • said L1 and L2 are connecting peptides
  • the CL1 is the CL of the first domain
  • the CH1 is the CH1 of the first domain
  • CH3 of polypeptide chain 1 is connected to VH 2 through L1
  • the two VH 2 are connected through L2.
  • the sequences of L1 and L2 are shown in SEQ ID NO: 117 and 118 in Table 15, respectively.
  • the anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002603 were used to construct the bispecific antibody PR200600 with an octavalent symmetrical structure of IgG_HC-VH-VH-VH.
  • the binding protein of the IgG_HC-VH-VH-VH octavalent symmetrical structure contains two polypeptide chains: polypeptide chain 2, also known as the short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 -CL 1 -C'; Polypeptide chain 1, also known as long chain, from amino-terminus to carboxy-terminus, which contains N'-VH1-CH 1 -h-CH 2 -CH3-L1-VH 2 -L 2 -VH 2 -L3-VH 2 -C'.
  • VL 1 and VH 1 are VL and VH of the first structural domain respectively
  • said VH 2 is VH of the second structural domain
  • said h is a hinge region
  • said L1, L2 and L3 are A connecting peptide
  • the CL 1 is the CL of the first domain
  • the CH 1 is the CH 1 of the first domain.
  • CH3 in polypeptide chain 1 is connected to the first VH 2 via connecting peptide L1 (ordered from the N-terminal to the C-terminal of polypeptide chain-1)
  • the first VH 2 is connected to the second VH 2 through connecting peptide L2
  • the first VH 2 is connected to the second VH 2 through the connecting peptide L2.
  • the two VH 2 and the third VH 2 are linked by linker peptide L3, wherein L1 is (G2S)2, L2 is (G4S)3, L3 is (G4S) 2 , and the sequences of L1, L2 and L3 are shown in Table 15 Shown in SEQ117, 118, 119.
  • amino acid sequence of the polypeptide chain of the double antibody molecule obtained in the present invention is shown in Table 16 in the numbering in the sequence listing.
  • Antibody number polypeptide chain 1 polypeptide chain 2 PR003787 79 77 PR200528 109 77 PR200531 110 77 PR200536 111 77 PR200538 112 77 PR200539 114 113 PR200600 115 77
  • the CDR sequence numbers of the antigenic domains of the PD-1 ⁇ OX40 bispecific antibody are shown in Table 17.
  • the 1# sequence number is the antigenic domain that binds to PD-1
  • the 2# sequence number is the antigenic domain that binds to OX40.
  • the molecular structure information of the bispecific antibody of the present invention is shown in Table 18.
  • This example is to study the in vitro binding activity of the PD-1 antibody arm of the PD-1 ⁇ OX40 bispecific antibody to human PD-1.
  • the CHO-K1 cell line (CHO-K1-hu PD-1, Shanghai ChemPartner) overexpressing human PD-1 was used for antibody binding experiments at the cellular level. Briefly, CHO-K1-hu PD-1 cells were digested and resuspended with DMEM complete medium, and the cell density was adjusted to 1 ⁇ 106 cells/mL, respectively.
  • 100 ⁇ L cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 100 ⁇ L/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration. Place the cells at 4°C and incubate for 1 hour in the dark. Afterwards, add 100 ⁇ L/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant.
  • Figure 7 shows the in vitro binding of the PD-1 ⁇ OX40 bispecific antibody on the CHO-K1 cell line overexpressing human PD-1.
  • the PD-1 ⁇ OX40 bispecific antibodies PR003787, PR200538, PR200539 or their monoclonal antibodies PR001985 and PR006429 in this example are all specific to CHO-K1 cells overexpressing human PD-1 sexual union. And the binding ability of PR003787 to human PD-1 is consistent with the binding ability of PD-1 antibody to human PD-1.
  • the PD-1 antibody arm of the PD-1 ⁇ OX40 bispecific antibody PR003787 in this implementation has the same binding ability as the PD-1 monoclonal antibody.
  • the HEK293 cell line (OX40/NF- ⁇ B Reporter-HEK293, BPS BioScience, Cat#60482) with high expression of OX40 was used for cell and Human OX40 binding assay.
  • HEK293 cell suspensions with high expression of OX40 were collected, and the cell density was adjusted to 2 ⁇ 10 6 cells/mL. 50 ⁇ L cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 ⁇ L/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration.
  • FIGS. 8A and 8B show the PD-1 ⁇ OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies constituting their structures in human HEK293/OX40/NF-kb reporter cells determined by flow cytometry PR002067 specifically binds to HEK293 cells overexpressing human OX40, and binds to OX40 on the cell surface as the concentration increases.
  • the OX40 antibody arms of the double-antibody PR003787 and PR200538 have the same or better binding ability as the OX40 monoclonal antibody.
  • This example is to study the activity of PD-1 ⁇ OX40 bispecific antibody binding to OX40 and PD-1 co-expression cells.
  • HEK293 cell line (OX40/NF- ⁇ B Reporter–HEK293, BPS BioScience, Cat#60482) with high expression of OX40 was used to further construct a cell line stably expressing human PD1 (Origene, Cat#PS100092V) with lentivirus, which was screened for resistance to puromycin
  • HEK293 cell lines stably expressing OX40 and PD-1 were obtained, and the obtained cell lines were used for cell binding experiments.
  • HEK293 cell suspensions highly expressing OX40 and PD-1 were collected, and the cell density was adjusted to 2 ⁇ 10 6 cells/mL, respectively.
  • 50 ⁇ L cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 ⁇ L/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration. Place the cells at 4°C and incubate for 2 hours in the dark. Afterwards, add 100 ⁇ L/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant.
  • Figure 9 shows that in human HEK293/OX40/PD1NF-kb reporter cells determined by flow cytometry, the PD-1 ⁇ OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies that make up their structures all interact with HEK293 cells overexpressing human OX40 and PD-1 specifically bind, and bind to cell surface OX40 and PD-1 as the concentration increases.
  • the double-antibody PR003787, PR200538 and PR200539 showed stronger binding ability; compared with the PD1 monoclonal antibody, the double-antibody PR003787, PR200538 and PR200539 showed similar or slightly weaker binding ability.
  • PD-1 ⁇ OX40 bispecific antibody-activated T cells Human leukocyte concentrates (Research blood components LLC) were purchased, and human peripheral blood mononuclear cells (PBMC) were isolated, and T cells were stimulated for 48 hours with anti-CD3 antibody coated on a cell culture plate. The stimulated PBMCs were collected and the cell density was adjusted to 4 ⁇ 10 6 cells/mL. 50 ⁇ L cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 ⁇ L/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration.
  • PBMC peripheral blood mononuclear cells
  • Figure 10 shows that in the activated human T cells determined by flow cytometry, the PD-1 ⁇ OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies that constitute their structures all bind to the activated T cells , and the binding to T cells increases with the concentration.
  • Bi-antibodies PR003787, PR200538 and PR200539 showed stronger binding abilities compared with OX40 mAb and PD1 mAb. It shows that the double antibody of the present invention can specifically bind PD1 and OX40 positive T cells.
  • Example 8 Using a reporter gene cell line to detect the stimulating effect of PD-1 ⁇ OX40 bispecific antibody on OX40 signaling pathway
  • HEK293 reporter cells HEK293/OX40/NF-kb reporter cells, BPS Biosciences, #60482
  • luciferase reporter genes of OX40 and NF-kb response elements were added to each well.
  • ONE-Glo TM luciferase reagent Promega, #E6110
  • the results are shown in Figures 11 to 13.
  • the results in Figure 11 to Figure 13 show that the PD-1 ⁇ OX40 bispecific antibody PR003787 described in this application is PD-1 cross-linking dependent.
  • the results in Figure 11 show that the PD-1 ⁇ OX40 bispecific antibody PR003787 has no direct activation effect on HEK293/OX40/NF-kb reporter cells.
  • the results in Figure 12 show that the OX40 parental monoclonal antibody showed a concentration-dependent effect of enhancing the NF-Kb signaling pathway under CHO-K1/CD32b cross-linking conditions, while the PD-1 ⁇ OX40 bispecific antibody PR003787 did not show NF- Activation of Kb signaling.
  • the results in Figure 13 show that the PD-1 ⁇ OX40 bispecific antibody PR003787, with the help of CHO-K1/PD-1 cell cross-linking, specifically caused a concentration-dependent enhancement of the NF-Kb signaling pathway.
  • the PD-1 ⁇ OX40 bispecific antibody PR003787 described in this application can specifically induce the promotion of PD-1 cross-linking-dependent OX40-mediated NF-Kb signaling pathway, and the signal intensity it causes increases in a positive correlation with its concentration .
  • Example 9 Using a reporter gene cell line to detect the inhibitory effect of PD-1 ⁇ OX40 bispecific antibody (PR003787) on PD-1 signaling pathway
  • HEK293 cells overexpressing PD-L1 and OS8 were plated on a 96-well plate with a cell volume of 1.25 ⁇ 10 4 /well, 100 ⁇ L/well.
  • Add ONE-Glo TM luciferase reagent Promega, #E6110), incubate at room temperature for 15 minutes, and detect the luminescence value with a microplate reader.
  • the PD-1 ⁇ OX40 bispecific antibody (PR003787) and the parent antibody of PD-1 (PR001985) described in this application can both enhance TNF ⁇ compared with the human IgG1 isotype (PR000324) and IFN ⁇ secretion.
  • Example 11 PD-1 ⁇ OX40 bispecific antibody inhibits the production of IL-10 in Tregs cells
  • Treg cells were thawed and cultured for 2 days with the medium containing recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF), and then the Treg cells and mitomycin-treated CHOK1- huPD-L1 cells were co-cultured with CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) for 5 days.
  • IL-2 recombinant human interleukin 2
  • IL-2 human interleukin 2
  • the PD-1 ⁇ OX40 bispecific antibody (PR003787) and the parental antibody of OX40 (PR002067) described in this application have higher effects on the secretion of IL-10 by Treg cells. inhibition.
  • Example 12 Functional difference between PD-1 ⁇ OX40 bispecific antibody (PR003787) and parental monoclonal antibody, combined use (IFN ⁇ , Granzyme B, IL-2 secretion)
  • Treg cells Purchase concentrated human leukocytes (Research blood components LLC), isolate T cells (StemCell, #17951), and cryopreserve. Simultaneously isolate Treg cells (StemCell, Cat#18063), add CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF), during the culture period according to the cell growth status every 2 - Add fresh IL-2-containing culture solution on day 4, and after 12 days of culture, analyze the ratio of Treg by flow cytometry.
  • IL-2 human interleukin 2
  • the activity of the PD-1 ⁇ OX40 bispecific antibody (PR003787) described in this application is superior to that of PD-1 monoclonal antibody (PR001985) and OX40 monoclonal antibody (PR002067), and is superior to The combination of PD-1 mAb (PR001985) and OX40 mAb (PR002067) showed synergistic activity.
  • the PD-1 ⁇ OX40 bispecific antibodies PR003787, PR500531, PR200538, PR200539, PR200536, PR200528 and PR200600 described in this application have stronger activation of T cells Ability to secrete IL-2. And compared with PR003787, PR200538, PR200539, and PR200528 exhibit dose-dependent T cell activation ability, and have stronger T cell activation ability under high concentration conditions.

Abstract

Disclosed are an antibody targeting PD1, or an antigen-binding fragment thereof, and a bispecific antibody targeting PD1 and OX40, or a fragment thereof. Further disclosed are a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, the antibody targeting PD1, or the antigen-binding fragment thereof, the bispecific antibody targeting PD1 and OX40, or the fragment thereof, and the use of the encoding nucleic acid molecule in the treatment, prevention and/or diagnosis of a disease. The disease may be, for example, an immune disease, an acute inflammatory disease, a chronic inflammatory disease or a tumor disease.

Description

靶向PD1和/或OX40的特异性结合蛋白Specific binding proteins targeting PD1 and/or OX40 技术领域technical field
本发明涉及生物医药领域，更具体地涉及抗体治疗领域。The present invention relates to the field of biomedicine, more specifically to the field of antibody therapy.
背景介绍background introduction
哺乳动物的免疫系统是一个精细平衡的系统，但有时会被诸如癌症的疾病破坏。免疫检查点受体通过发挥共刺激或共抑制作用，在免疫系统对疾病的反应中发挥重要作用，而二者的微妙平衡决定了免疫应答的功效。共抑制剂抑制T细胞增殖并诱导抗炎细胞因子释放。它们可以减轻炎症，避免过度免疫反应造成器官/组织损害。另一方面，共刺激剂通过促进T细胞克隆扩增，效应子分化和存活，以促进保护性免疫应答的发展。The mammalian immune system is a finely balanced system, but it can sometimes be disrupted by diseases such as cancer. Immune checkpoint receptors play an important role in the immune system's response to disease by exerting co-stimulatory or co-inhibitory roles, and the delicate balance of the two determines the efficacy of the immune response. Co-inhibitors inhibit T cell proliferation and induce anti-inflammatory cytokine release. They reduce inflammation and avoid organ/tissue damage from an overactive immune response. On the other hand, co-stimulators promote the development of protective immune responses by promoting T cell clonal expansion, effector differentiation and survival.
一种行之有效的癌症免疫治疗方法可以通过阻断共抑制受体功能或诱导共刺激受体活性的抗体靶向这些检查点受体，来触发免疫系统识别并杀死肿瘤细胞(Pardoll,2012)。阻断共抑制受体活性的抗体已显示出良好的临床活性，目前已被批准用于治疗癌症(Larkin等,2015)。诱导共刺激受体活性的抗体已在临床前模型系统中显示出巨大潜力(Moran等,2013；Schaer等,2014)，目前有几种药物正在临床试验中(Mayes等,2018；Melero等,2013)。这些抗体旨在模拟这些共刺激受体的配体，因此也被称为激动剂抗体。A well-established approach to cancer immunotherapy can trigger the immune system to recognize and kill tumor cells by targeting these checkpoint receptors with antibodies that block co-inhibitory receptor function or induce co-stimulatory receptor activity (Pardoll, 2012 ). Antibodies that block the activity of co-inhibitory receptors have shown promising clinical activity and are currently approved for the treatment of cancer (Larkin et al., 2015). Antibodies that induce co-stimulatory receptor activity have shown great potential in preclinical model systems (Moran et al., 2013; Schaer et al., 2014), and several agents are currently in clinical trials (Mayes et al., 2018; Melero et al., 2013 ). These antibodies are designed to mimic the ligands of these co-stimulatory receptors and are therefore also known as agonist antibodies.
OX40(又称CD134，ACT45，TNFRSF4)属于肿瘤坏死因子受体(TNFR)超家族的一员，主要在活化的T细胞上表达，包括CD4+T细胞、CD8+T细胞、1型和2型T辅助细胞(Th1和Th2)和调节性T(Treg)细胞，并在活化的自然杀伤细胞(NK)上表达。在抗原呈递细胞(APC)上表达的OX40及其配体OX40配体(OX40L)的相互作用，增加了T细胞的克隆扩增、分化和存活，并增强了记忆性T细胞的生成(Croft等,2009)。OX40刺激可对T细胞产生直接影响，促进其增殖和存活，或者通过增强炎症细胞因子(例如IL2和IFNγ)的产生而产生间接影响。OX40信号传导也可调节Treg细胞的功能，尽管在Treg细胞上OX40信号传导可以消除Treg细胞的抑制活性(Takeda等,2004)。已经发现OX40在患有头颈部癌、黑色素瘤和结肠直肠癌患者的肿瘤浸润T细胞中表达，但是，高水平的OX40阳性淋巴细胞与患者更高的存活率相关(Petty等,2002；Vetto等,1997)。OX40的激动剂抗体目前正在癌症的临床试验中，大多数显示出良好的安全性和有限的临床活性。以靶向OX40作为单一疗法的效果却并不理想，始终未能达到预期的结果。OX40 (also known as CD134, ACT45, TNFRSF4) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is mainly expressed on activated T cells, including CD4+ T cells, CD8+ T cells, type 1 and type 2 T helper cells (Th1 and Th2) and regulatory T (Treg) cells, and expressed on activated natural killer (NK) cells. The interaction of OX40 expressed on antigen-presenting cells (APCs) and its ligand OX40 ligand (OX40L) increases the clonal expansion, differentiation and survival of T cells and enhances the generation of memory T cells (Croft et al. ,2009). OX40 stimulation can have direct effects on T cells, promoting their proliferation and survival, or indirect effects by enhancing the production of inflammatory cytokines such as IL2 and IFNγ. OX40 signaling can also regulate the function of Treg cells, although OX40 signaling on Treg cells can abrogate the suppressive activity of Treg cells (Takeda et al., 2004). OX40 has been found to be expressed in tumor-infiltrating T cells from patients with head and neck cancer, melanoma, and colorectal cancer, however, high levels of OX40-positive lymphocytes are associated with better patient survival (Petty et al., 2002; Vetto et al., 1997). Agonist antibodies to OX40 are currently in clinical trials for cancer, most showing good safety and limited clinical activity. The effect of targeting OX40 as a monotherapy is not ideal, and has not always achieved the expected results.
PD-1是由活化的T和B细胞表达的关键免疫检查点受体，并介导免疫抑制。PD-1是受体CD28家族的成员，该家族包括CD28、CTLA-4、ICOS、PD-1和BTLA。已鉴定出PD-1的两种细胞表面糖蛋白配体，为程序性死亡配体-1(PD-L1)和程序性死亡配体-2(PD-L2)，它们表达在抗原呈递细胞以及许多种类的人癌症细胞上，已显示它们通过结合PD-1下调T细胞活化和细胞因子分泌。PD-1和PD-L1之间的相互作用导致肿瘤浸润性淋巴细胞减少，T细胞受体介导的增殖降低，和癌细胞的免疫逃避。PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immune suppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA. Two cell surface glycoprotein ligands of PD-1 have been identified, programmed death-ligand-1 (PD-L1) and programmed death-ligand-2 (PD-L2), which are expressed on antigen-presenting cells and They have been shown to downregulate T cell activation and cytokine secretion by binding PD-1 on many types of human cancer cells. The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancer cells.
虽然利用激动剂刺激共刺激受体或者检查点受体抑制剂的抗癌原理是明确的：CTLA4、PD1/PDL1等检查点受体抑制剂是增强T细胞的抗癌作用，而共刺激受体激动剂CD28，4-1BB、OX40、GITR、CD27和ICOS 则是促进T细胞的抗肿瘤免疫力。但在临床上将共刺激受体激动剂和检查点受体抑制剂共同使用并未取得良好的效果。理论上，在高度免疫抑制的肿瘤微环境中，利用激动剂刺激这些共刺激受体应该能够增强抗肿瘤免疫力。大量的小鼠数据也证实了这类药物的治疗潜力。例如，基因泰克将OX40激动剂抗体和PD-L1抗体联合起来，在一种小鼠模型中，90％的结直肠癌肿瘤获得了完全缓解。然而，虽然动物研究数据喜人，但临床试验的数据并不理想。在基因泰克关于OX40激动剂MOXR0916联合PD-L1抗体atezolizumab的Ib期试验中，仅4％(2 of 51)的患者获得了部分缓解，因此，基因泰克关闭了其OX40项目。大多数情况下，化疗药的联合用药可能都会起到协同作用，从而使得肿瘤治疗效果更好，但是免疫疗法的联合用药并不是简单的1+1>2，它可能会与预期的结果相差甚远，亦或是完全相反。在2017年发表的两组小鼠模型数据显示，当同时给予OX40激动剂和PD-1抑制剂时，由于T细胞衰竭或程序性细胞死亡，这两种抗体的作用相互抵消。但是，序贯给药则增强了这些药物的抗肿瘤活性。一种假设是，共刺激必须首先促进肿瘤特异性T细胞达到检查点抑制免疫反应的程度，然后检查点抑制剂才能有效。Although the anti-cancer principle of using agonists to stimulate co-stimulatory receptors or checkpoint receptor inhibitors is clear: CTLA4, PD1/PDL1 and other checkpoint receptor inhibitors enhance the anti-cancer effect of T cells, while co-stimulatory receptors Agonists CD28, 4-1BB, OX40, GITR, CD27 and ICOS promote anti-tumor immunity of T cells. However, co-administration of co-stimulatory receptor agonists and checkpoint receptor inhibitors has not achieved good clinical results. In theory, stimulation of these co-stimulatory receptors with agonists should enhance antitumor immunity in the highly immunosuppressive tumor microenvironment. Extensive mouse data also confirm the therapeutic potential of this class of drugs. For example, Genentech combined an OX40 agonist antibody with a PD-L1 antibody, and in a mouse model, 90% of colorectal cancer tumors achieved complete remission. However, while the data from animal studies is promising, the data from clinical trials is not ideal. In Genentech's phase Ib trial of the OX40 agonist MOXR0916 combined with the PD-L1 antibody atezolizumab, only 4% (2 of 51) of patients achieved a partial response, so Genentech shut down its OX40 program. In most cases, the combination of chemotherapy drugs may have a synergistic effect, so that the tumor treatment effect is better, but the combination of immunotherapy is not a simple 1+1>2, it may be quite different from the expected results far, or quite the opposite. Data from two sets of mouse models published in 2017 showed that when an OX40 agonist and a PD-1 inhibitor are given together, the effects of the two antibodies cancel each other out due to T cell exhaustion or programmed cell death. However, sequential administration enhanced the antitumor activity of these drugs. One hypothesis is that co-stimulation must first promote tumor-specific T cells to the extent that checkpoint-suppressed immune responses exist before checkpoint inhibitors can be effective.
目前尚未有能够将靶向PD1和OX40的抗体共同使用实现有效抗癌的方法，因此发掘能够使两者功能同时发挥的方法，如制备成双特异性抗体，具有重要的临床意义。At present, there is no method that can use antibodies targeting PD1 and OX40 together to achieve effective anti-cancer. Therefore, it is of great clinical significance to discover methods that can enable both functions to function simultaneously, such as preparing bispecific antibodies.
发明内容Contents of the invention
本发明提供了特异性结合蛋白，所述特异性结合蛋白能够以高亲和力和高特异性与一种或多种抗原结合，所述抗原为PD-1或其片段，和/或OX40或其片段。本发明还提供了编码所述特异性结合蛋白的核酸分子，用于产生所述特异性结合蛋白的表达载体，宿主细胞以及用于制备所述特异性结合蛋白的方法。本发明还涉及所述特异性结合蛋白在治疗、预防和/或诊断疾病中的用途，所述疾病例如为免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病。The present invention provides a specific binding protein capable of binding to one or more antigens with high affinity and high specificity, the antigen being PD-1 or a fragment thereof, and/or OX40 or a fragment thereof . The present invention also provides nucleic acid molecules encoding the specific binding protein, expression vectors for producing the specific binding protein, host cells and methods for preparing the specific binding protein. The invention also relates to the use of said specific binding protein in the treatment, prevention and/or diagnosis of diseases, such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.
第一方面，本发明提供了一种特异性抗体或其抗原结合片段，所述抗体或其抗原结合片段结合PD-1或其片段，包含轻链可变区VL和重链可变区VH，其中，所述VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:8、18和28所示，或者分别如SEQ ID NO:81、86和91所示。在一些实施方案中，这些CDR中可以包含1至3个氨基酸取代。In a first aspect, the present invention provides a specific antibody or an antigen-binding fragment thereof, which binds to PD-1 or a fragment thereof, comprising a light chain variable region VL and a heavy chain variable region VH, Wherein, the VL comprises CDR1, CDR2 and CDR3, and its amino acid sequences are shown in SEQ ID NO: 39, 46 and 54 respectively, and the VH comprises CDR1, CDR2 and CDR3, and its amino acid sequences are respectively shown in SEQ ID NO: 8, 18 and 28, or as shown in SEQ ID NO:81, 86 and 91, respectively. In some embodiments, 1 to 3 amino acid substitutions may be included in these CDRs.
在一些实施方案中，上述特异性抗体或其抗原结合片段的VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，且所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:8、18和28所示。在一些实施方案中，上述特异性抗体或其抗原结合片段的VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，且VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:81、86和91所示。在一些实施方案中，这些CDR中可以包含氨基酸突变，并能维持所述抗体的特异性结合PD-1的功能。优选地，所述氨基酸突变为氨基酸替换，所述氨基酸替换的数目可以为1-3个。In some embodiments, the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54, respectively, and the VH comprises CDR1, CDR2 and CDR3. CDR3, its amino acid sequence is shown in SEQ ID NO:8, 18 and 28 respectively. In some embodiments, the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54, respectively, and the VH comprises CDR1, CDR2 and CDR3, Its amino acid sequence is shown in SEQ ID NO:81, 86 and 91 respectively. In some embodiments, amino acid mutations may be included in these CDRs, and can maintain the function of the antibody specifically binding to PD-1. Preferably, the amino acid mutation is amino acid substitution, and the number of amino acid substitutions can be 1-3.
在一些实施方案中，上述特异性抗体或其抗原结合片段的VL包含如SEQ ID NO:67所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。在具体的实施方案中，上述特异性结合蛋白的VL包含如SEQ ID NO:67所示的氨基酸序列。In some embodiments, the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95% , 97%, 98%, 99% or 100% identical amino acid sequences. In a specific embodiment, the VL of the above-mentioned specific binding protein comprises the amino acid sequence shown in SEQ ID NO:67.
在一些实施方案中，上述特异性抗体或其抗原结合片段的VH包含SEQ ID NO:62所示的氨基酸序列，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。在具体的实施方案中，上述特异性抗体或其抗原结合片段的VH包含SEQ ID NO:62所示的氨基酸序列。在一些实施方案中，上述特异性抗体或其抗原结合片段的VH包含SEQ ID NO:102所示的氨基酸序列，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。在一些实施方案中，上述特异性抗体或其抗原结合片段的VH包含SEQ ID NO:102所示的氨基酸序列。In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 62, or has at least 80%, 85%, 88%, 90%, 92%, 95% of it , 97%, 98%, 99% or 100% identical amino acid sequences. In a specific embodiment, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:62. In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 80%, 85%, 88%, 90%, 92%, 95% of the amino acid sequence thereof , 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 102.
在一些实施方案中，上述特异性抗体或其抗原结合片段选自IgG、Fab、Fab’、F(ab’) 2、Fv或scFv。在一些实施方案中，上述特异性抗体或其抗原结合片段为IgG。在一些实施方案中，上述特异性抗体或其抗原结合片段包括重链恒定区和/或轻链恒定区。在一些实施方案中，上述重链恒定区可以来源于人IgG1、人IgG2、人IgG3或人IgG4。在一些实施方案中，所述轻链恒定区可以选自κ链或者λ链。在一些实施方案中，上述特异性抗体或其抗原结合片段可以为制得的多克隆抗体或单克隆抗体。 In some embodiments, the above-mentioned specific antibody or antigen-binding fragment thereof is selected from IgG, Fab, Fab', F(ab') 2 , Fv or scFv. In some embodiments, the above-mentioned specific antibody or antigen-binding fragment thereof is IgG. In some embodiments, the specific antibody or antigen-binding fragment thereof described above includes a heavy chain constant region and/or a light chain constant region. In some embodiments, the heavy chain constant region described above may be derived from human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the light chain constant region may be selected from a kappa chain or a lambda chain. In some embodiments, the above-mentioned specific antibodies or antigen-binding fragments thereof may be prepared polyclonal antibodies or monoclonal antibodies.
在一些实施方案中，上述特异性抗体或其抗原结合片段的Fc在第234位和/或第235位发生氨基酸替换。在一些实施方案中，上述特异性抗体或其抗原结合片段包含L234A和/或L235A突变。在一些实施方案中，上述特异性结合蛋白包含L234A和L235A两个突变。In some embodiments, amino acid substitutions occur at position 234 and/or position 235 of the Fc of the specific antibody or antigen-binding fragment thereof. In some embodiments, the above-mentioned specific antibodies or antigen-binding fragments thereof comprise L234A and/or L235A mutations. In some embodiments, the above-mentioned specific binding protein comprises two mutations, L234A and L235A.
在一些实施方案中，上述特异性抗体的Fc为人IgG1的Fc。在一些实施方案中，上述特异性抗体的Fc为人IgG4的Fc。在一些实施方案中，所述Fc包含L234A和L235A突变。In some embodiments, the Fc of the above-mentioned specific antibody is the Fc of human IgG1. In some embodiments, the Fc of the above-mentioned specific antibody is the Fc of human IgG4. In some embodiments, the Fc comprises the L234A and L235A mutations.
在一些实施方案中，上述特异性抗体或其抗原结合片段包括重链和轻链。在一些实施方案中，所述轻链包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述重链包括如SEQ ID NO：72所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。在一些实施方案中，所述轻链包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述重链包括如SEQ ID NO：106所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。In some embodiments, the aforementioned specific antibodies or antigen-binding fragments thereof include heavy chains and light chains. In some embodiments, the light chain comprises the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97% thereof %, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the light chain comprises the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 106 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97% %, 98%, 99% or 100% identical amino acid sequences.
第二方面，本发明提供了特异性结合蛋白，所述特异性结合蛋白包含至少两个结构域，能够结合PD-1或其片段，和/或OX40或其片段。In a second aspect, the present invention provides a specific binding protein comprising at least two structural domains capable of binding PD-1 or a fragment thereof, and/or OX40 or a fragment thereof.
在一些实施方案中，所述特异性结合蛋白为双特异性抗体或其抗原结合抗体，其包含第一结构域和第二结构域，所述第一结构域结合PD-1或其片段，并且所述第二结构域结合OX40或其片段。在一些实施方案中，所述第二结构域为仅含重链的VH，不含轻链。In some embodiments, the specific binding protein is a bispecific antibody or an antigen-binding antibody thereof comprising a first domain and a second domain, the first domain binds PD-1 or a fragment thereof, and The second domain binds OX40 or a fragment thereof. In some embodiments, the second domain is a VH containing only the heavy chain and no light chain.
在一些实施方案中，所述第一结构域为PD-1抗体或其抗原结合片段。In some embodiments, the first domain is a PD-1 antibody or an antigen-binding fragment thereof.
在一些实施方案中，所述第二结构域为OX40抗体或其抗原结合片段。In some embodiments, the second domain is an OX40 antibody or antigen-binding fragment thereof.
在一些实施方案中，所述第一结构域和/或所述第二结构域的结构选自IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、VH、或HCAb的一种，优选地，所述Fab、Fab’、F(ab’) 2、Fv、scFv、VH的数量为一个或多个。 In some embodiments, the structure of the first domain and/or the second domain is selected from one of IgG, Fab, Fab', F(ab') 2 , Fv, scFv, VH, or HCAb , preferably, the number of Fab, Fab', F(ab') 2 , Fv, scFv, VH is one or more.
在一些实施方案中，所述第一结构域为IgG的形式。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；优选地，所述第一结构域中IgG的Fc为人IgG1 或IgG4的Fc。In some embodiments, the first domain is in the form of an IgG. Preferably, the IgG heavy chain constant region is a human heavy chain constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region; preferably, the IgG Fc in the first domain is human IgG1 or IgG4 Fc.
在一些实施方案中，人IgG优选包含L234A、L235A和P329G中的一个、两个或三个突变，更优选地包含L234A和L235A两个突变或包含L234A、L235A和P329G三个突变；In some embodiments, the human IgG preferably comprises one, two or three mutations among L234A, L235A and P329G, more preferably comprises two mutations of L234A and L235A or comprises three mutations of L234A, L235A and P329G;
优选地，所述第一结构域中IgG的Fc为人IgG1的Fc，更优选地，所述Fc包含L234A、L235A和P329G突变；Preferably, the Fc of IgG in the first domain is the Fc of human IgG1, more preferably, the Fc comprises L234A, L235A and P329G mutations;
优选地，所述第一结构域中IgG的Fc为人IgG4的Fc，更优选地，所述Fc包含L234A和L235A和P329G突变。Preferably, the IgG Fc in the first domain is the Fc of human IgG4, and more preferably, the Fc contains L234A, L235A and P329G mutations.
在一些实施方案中，人IgG优选包含L234A、L235A和G237A中的一个、两个或三个突变，更优选地包含L234A和L235A两个突变或包含L234A、L235A和G237A三个突变；In some embodiments, the human IgG preferably comprises one, two or three mutations among L234A, L235A and G237A, more preferably comprises two mutations of L234A and L235A or comprises three mutations of L234A, L235A and G237A;
优选地，所述第一结构域中IgG的Fc为人IgG1的Fc，更优选地，所述Fc包含L234A、L235A和G237A突变；Preferably, the Fc of IgG in the first domain is the Fc of human IgG1, more preferably, the Fc comprises L234A, L235A and G237A mutations;
优选地，所述第一结构域中IgG的Fc为人IgG4的Fc，更优选地，所述Fc包含L234A和L235A和G237A突变。Preferably, the IgG Fc in the first domain is the Fc of human IgG4, and more preferably, the Fc contains L234A, L235A and G237A mutations.
优选地，所述第一结构域为四价结构。Preferably, the first domain is a tetravalent structure.
优选地，所述第二结构域为VH结构，VH的数量优选为1、2、3或4个；更优选地，VH的数量为2个。Preferably, the second domain is a VH structure, and the number of VHs is preferably 1, 2, 3 or 4; more preferably, the number of VHs is 2.
在一些实施方案中，所述第一结构域与所述第二结构域直接连接或经连接肽L连接形成双特异性结合蛋白。In some embodiments, the first domain and the second domain are linked directly or via a linker peptide L to form a bispecific binding protein.
在一些实施方案中，特异性结合蛋白为IgG_HC-VH四价对称结构的双特异性抗体，其包含两条多肽链：多肽链2，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，从氨基末端到羧基末端，其包含N’-VH 1-CH 1-h-CH2-CH3-L-VH 2-C’；其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为IgG抗体的铰链区，所述CL 1是第一结构域的CL，所述CH 1是第一结构域的CH1，所述L为连接肽，多肽链1的CH3经由L连接到VH 2。 In some embodiments, the specific binding protein is a bispecific antibody with IgG_HC-VH tetravalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 - CL 1 -C'; polypeptide chain 1, from the amino terminal to the carboxyl terminal, which comprises N'-VH 1 -CH 1 -h-CH2-CH3-L-VH 2 -C'; wherein, the VL 1 and VH 1 is the VL and VH of the first domain, the VH 2 is the VH of the second domain, the h is the hinge region of the IgG antibody, the CL 1 is the CL of the first domain, and the CH 1 is the CH1 of the first structural domain, the L is a connecting peptide, and CH3 of the polypeptide chain 1 is connected to VH 2 via L.
在一些实施方案中，特异性结合蛋白为VH-IgG_HC四价对称结构的双特异性抗体，其包含两条多肽链：多肽链2，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，从氨基末端到羧基末端，其包含N’-VH 2-L-VH 1-CH 1-h-CH2-CH3-C’；其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述CL 1是第一结构域的CL，所述CH1是第一结构域的CH 1，所述L为连接肽，多肽链1的VH 2经由连接肽L连接到VH 1。 In some embodiments, the specific binding protein is a bispecific antibody with a tetravalent symmetrical structure of VH-IgG_HC, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which comprises N'-VL 1 - CL 1 -C'; Polypeptide chain 1, from the amino terminal to the carboxyl terminal, which comprises N'-VH 2 -L-VH 1 -CH 1 -h-CH2-CH3-C'; wherein, the VL 1 and VH 1 is the VL and VH of the first domain, the VH 2 is the VH of the second domain, the h is the hinge region, the CL 1 is the CL of the first domain, and the CH1 is the first domain CH 1 of the domain, the L is a connecting peptide, VH 2 of the polypeptide chain 1 is connected to VH 1 via the connecting peptide L.
在一些实施方案中，特异性结合蛋白为Fab(CL)-VH-Fc四价对称结构的双特异性抗体，其包含两条多肽链，多肽链2，从氨基末端到羧基末端，其包含N’-VH’-CH1’-C’；多肽链1，从氨基末端到羧基末端，其包含N’-VL’-CL’-L-VH 2-h-CH 2-CH 3-C’。其中，所述VL’和VH’分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L为连接肽，所述CL’是第一结构域的CL，所述CH1’是第一结构域的CH1，多肽链1的CL’与VH 2直接融合连接，即L的长度为0。 In some embodiments, the specific binding protein is a bispecific antibody with a tetravalent symmetrical structure of Fab(CL)-VH-Fc, which comprises two polypeptide chains, polypeptide chain 2, from the amino terminus to the carboxyl terminus, which comprises N '-VH'-CH1'-C'; polypeptide chain 1, from amino terminus to carboxy terminus, comprising N'-VL'-CL'-L-VH2 - h- CH2 - CH3 -C'. Wherein, said VL' and VH' are VL and VH of the first structural domain respectively, said VH2 is VH of the second structural domain, said h is a hinge region, said L is a connecting peptide, and said CL ' is the CL of the first structural domain, the CH1' is the CH1 of the first structural domain, and the CL' of the polypeptide chain 1 is directly fused with VH 2 , that is, the length of L is 0.
在一些实施方案中，特异性结合蛋白为IgG_HC-VH-VH六价对称结构的双特异性抗体，其包含两条多肽 链：多肽链2，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，从氨基末端到羧基末端，其包含N’-VH 1-CH 1-h-CH 2-CH 3-L 1-VH 2-L2-VH 2-C’。其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L1和L2为连接肽，所述CL 1是第一结构域的CL，所述CH 1是第一结构域的CH1，多肽链1的CH3经由L1连接到VH 2，两个VH 2之间通过L2连接。 In some embodiments, the specific binding protein is a bispecific antibody with IgG_HC-VH-VH hexavalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which comprises N'-VL 1 -CL 1 -C'; polypeptide chain 1, from amino terminus to carboxy terminus, comprising N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L 1 -VH 2 -L2-VH 2 -C '. Wherein, said VL 1 and VH 1 are respectively VL and VH of the first structural domain, said VH 2 is VH of the second structural domain, said h is a hinge region, and said L1 and L2 are connecting peptides, so The CL 1 is the CL of the first domain, the CH 1 is the CH1 of the first domain, the CH3 of the polypeptide chain 1 is connected to the VH 2 through L1, and the two VH 2 are connected through L2.
在一些实施方案中，特异性结合蛋白为IgG_HC-VH-VH-VH八价对称结构的双特异性抗体，其包含两条多肽链：多肽链2，从氨基末端到羧基末端，其包含N’-VL1-CL1-C’；多肽链1，从氨基末端到羧基末端，其包含N’-VH1-CH1-h-CH2-CH3-L1-VH2-L2-VH2-L3-VH2-C’。其中，所述VL1和VH1分别为第一结构域的VL和VH，所述VH2为第二结构域的VH，所述h为铰链区，所述的L1、L2和L3为连接肽，所述CL1是第一结构域的CL，所述CH1是第一结构域的CH1，多肽链1中的CH3经由连接肽L1连接到自多肽链1的N端至C端的第一个VH2，第一个VH2与第二个VH2通过连接肽L2连接，第二个VH2与第三个VH2通过连接肽L3连接。In some embodiments, the specific binding protein is a bispecific antibody with an octavalent symmetrical structure of IgG_HC-VH-VH-VH, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N' - VL1-CL1-C'; polypeptide chain 1, from amino-terminus to carboxy-terminus, comprising N'-VH1-CH1-h-CH2-CH3-L1-VH2-L2-VH2-L3-VH2-C'. Wherein, said VL1 and VH1 are respectively the VL and VH of the first structural domain, said VH2 is the VH of the second structural domain, said h is a hinge region, said L1, L2 and L3 are connecting peptides, said CL1 is the CL of the first structural domain, the CH1 is the CH1 of the first structural domain, CH3 in the polypeptide chain 1 is connected to the first VH2 from the N-terminal to the C-terminal of the polypeptide chain 1 via the connecting peptide L1, the first The VH2 is connected to the second VH2 through the connecting peptide L2, and the second VH2 is connected to the third VH2 through the connecting peptide L3.
其中，所述的铰链区h为免疫球蛋白领域常见的铰链区，通常含大量脯氨酸，具有挠性，形成2-5个二硫键。Wherein, the hinge region h is a common hinge region in the immunoglobulin field, usually contains a large amount of proline, is flexible, and forms 2-5 disulfide bonds.
优选地，所述L为长度为0-30个氨基酸长度的肽，其氨基酸序列如SEQ ID NO：116-140任一所示所示，见表1。Preferably, said L is a peptide with a length of 0-30 amino acids, and its amino acid sequence is shown in any one of SEQ ID NO: 116-140, see Table 1.
在一些实施方案中，所述第二多肽链的CH3与VH 2直接连接，即L的长度为0。 In some embodiments, CH3 of the second polypeptide chain is directly linked to VH2, ie the length of L is zero.
表1.连接肽序列Table 1. Linker peptide sequences
Figure PCTCN2022098838-appb-000001
Figure PCTCN2022098838-appb-000001
Figure PCTCN2022098838-appb-000002
Figure PCTCN2022098838-appb-000002
在一些实施方案中，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:8、18和28所示或者分别如SEQ ID NO:81、86和91所示。In some embodiments, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), and the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are as SEQ ID NO: As shown in 39, 46 and 54, the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 8, 18 and 28 or shown in SEQ ID NO: 81, 86 and 91 respectively.
在一些实施方案中，所述第一结构域包含VL和VH。在一些实施方案中，所述VL的氨基酸序列如SEQ ID NO:67所示或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。在具体的实施方案中，所述VL的氨基酸序列如SEQ ID NO:67所示。在一些实施方案中，所述VH的氨基酸序列如SEQ ID NO:62或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。在具体的实施方案中，所述VH的氨基酸序列如SEQ ID NO:62。在一些实施方案中，所述VH的氨基酸序列如SEQ ID NO:102所示，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。在具体的实施方案中，所述VH的氨基酸序列如SEQ ID NO:102所示。In some embodiments, the first domain comprises VL and VH. In some embodiments, the amino acid sequence of the VL is as shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% thereof Or 100% consistency. In a specific embodiment, the amino acid sequence of the VL is as shown in SEQ ID NO:67. In some embodiments, the VH has an amino acid sequence such as SEQ ID NO: 62 or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof. %consistency. In specific embodiments, the amino acid sequence of the VH is as SEQ ID NO:62. In some embodiments, the amino acid sequence of the VH is as shown in SEQ ID NO: 102, or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% % or 100% consistency. In a specific embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO: 102.
在一些实施方案中，所述第一结构域包含与SEQ ID NO:77所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的轻链，和与SEQ ID NO:71、SEQ ID NO:72或SEQ ID NO:106所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, Heavy chains that are 95%, 97%, 98%, 99% or 100% identical.
在一些实施方案中，所述第一结构域包含与SEQ ID NO:77所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的轻链，和与SEQ ID NO:71所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。在具体的实施方案中， 所述第一结构域包含如SEQ ID NO:77所示的轻链，和如SEQ ID NO:71所示的重链。In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain. In specific embodiments, the first domain comprises a light chain as shown in SEQ ID NO:77, and a heavy chain as shown in SEQ ID NO:71.
在一些实施方案中，所述第一结构域包含与SEQ ID NO:77所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的轻链，和与SEQ ID NO:72所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。在具体的实施方案中，所述第一结构域包含如SEQ ID NO:77所示的轻链和如SEQ ID NO:72所示的重链。In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain. In specific embodiments, said first domain comprises a light chain as shown in SEQ ID NO:77 and a heavy chain as shown in SEQ ID NO:72.
在一些实施方案中，所述第一结构域包含与SEQ ID NO:77所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的轻链，和与SEQ ID NO:106所示的氨基酸具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。在具体的实施方案中，所述第一结构域包含如SEQ ID NO:77所示的轻链和如SEQ ID NO:106所示的重链。In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% of the amino acids shown in SEQ ID NO:77 A light chain of % or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical heavy chain. In specific embodiments, said first domain comprises a light chain as shown in SEQ ID NO:77 and a heavy chain as shown in SEQ ID NO:106.
在一些实施方案中，所述第二结构域包含重链可变区(VH)，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:9、19和29所示。在一些实施方案中，所述第二结构域包含重链可变区(VH)，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:9、85和90所示。In some embodiments, the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively. In some embodiments, the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 85 and 90, respectively.
在一些实施方案中，所述第二结构域包含VH，所述VH的氨基酸序列如SEQ ID NO:63所示，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。在具体的实施方案中，所述第二结构域的VH的氨基酸序列如SEQ ID NO:63所示。在一些实施方案中，所述第二结构域包含VH，所述VH的氨基酸序列如SEQ ID NO:101所示，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。在具体的实施方案中，所述第二结构域的VH的氨基酸序列如SEQ ID NO:101所示。In some embodiments, the second domain comprises a VH having an amino acid sequence as shown in SEQ ID NO: 63, or at least 80%, 85%, 88%, 90%, 92%, 95% therewith. %, 97%, 98%, 99% or 100% agreement. In a specific embodiment, the amino acid sequence of the VH of the second domain is as shown in SEQ ID NO:63. In some embodiments, the second domain comprises VH, the amino acid sequence of the VH is as shown in SEQ ID NO: 101, or at least 80%, 85%, 88%, 90%, 92%, 95% therewith %, 97%, 98%, 99% or 100% agreement. In a specific embodiment, the amino acid sequence of the VH of the second domain is shown in SEQ ID NO: 101.
在一些实施方案中，所述第二结构域包含与SEQ ID NO:73所示的氨基酸序列具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。在具体的实施方案中，所述第二结构域包含如SEQ ID NO:73所示的重链。在一些实施方案中，所述第二结构域包含与SEQ ID NO:105所示的氨基酸序列具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。在具体的实施方案中，所述第二结构域包含如SEQ ID NO:105所示的重链。In some embodiments, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain. In specific embodiments, said second domain comprises a heavy chain as set forth in SEQ ID NO:73. In some embodiments, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain. In specific embodiments, said second domain comprises a heavy chain as set forth in SEQ ID NO: 105.
在具体的实施方案中，所述双特异性抗体包含第一结构域和第二结构域，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:8、18和28所示；并且，所述第二结构域包含重链可变区(VH)，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:9、19和29所示。In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the Said VL comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:39, 46 and 54 respectively, and said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:8, 18 and 28 respectively and, the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively.
在具体的实施方案中，所述双特异性抗体包含第一结构域和第二结构域，所述第一结构域包含VL和VH，所述VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:81、86和91所示；并且，所述第二结构域包含VH，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:9、19和29所示。In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises VL and VH, the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are as follows: Shown in SEQ ID NO:39, 46 and 54, said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:81, 86 and 91 respectively; And, said second structural domain comprises VH, The VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively.
在具体的实施方案中，所述双特异性抗体包含第一结构域和第二结构域，所述第一结构域包含VL和VH，所述VL包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:39、46和54所示，所述VH包含 CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:81、86和91所示；并且，所述第二结构域包含VH，所述VH包含CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID NO:9、85和90所示。In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises VL and VH, the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are as follows: Shown in SEQ ID NO:39, 46 and 54, said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequence is shown in SEQ ID NO:81, 86 and 91 respectively; And, said second structural domain comprises VH, The VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 85 and 90, respectively.
在具体的实施方案中，所述双特异性抗体包含第一结构域和第二结构域，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL和VH的氨基酸序列分别如SEQ ID NO:67和SEQ ID NO:62所示；并且，所述第二结构域包含VH，所述VH的氨基酸序列如SEQ ID NO:63所示。In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the The amino acid sequences of the VL and VH are shown in SEQ ID NO:67 and SEQ ID NO:62 respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO:63.
在具体的实施方案中，所述双特异性抗体包含第一结构域和第二结构域，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL和VH的氨基酸序列分别如SEQ ID NO:67和SEQ ID NO:102所示；并且，所述第二结构域包含VH，所述VH的氨基酸序列如SEQ ID NO:63所示。In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the The amino acid sequences of the VL and VH are shown in SEQ ID NO:67 and SEQ ID NO:102, respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO:63.
在具体的实施方案中，所述双特异性抗体包含第一结构域和第二结构域，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL和VH的氨基酸序列分别如SEQ ID NO:67和SEQ ID NO:102所示；并且，所述第二结构域包含VH，所述VH的氨基酸序列如SEQ ID NO:101所示。In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the The amino acid sequences of the VL and VH are shown in SEQ ID NO:67 and SEQ ID NO:102, respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO:101.
在一些实施方案中，所述双特异性结合蛋白包含多肽链1和多肽链2，所述多肽链1为长链，所述多肽链2为短链。In some embodiments, the bispecific binding protein comprises polypeptide chain 1 and polypeptide chain 2, wherein polypeptide chain 1 is a long chain and polypeptide chain 2 is a short chain.
在一些实施方案中，所述多肽链1具有SEQ ID NO:79、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:115中任一项所示的氨基酸序列，或与具有至少80％、85％、90％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述多肽链2具有SEQ ID NO:77、SEQ ID NO:113中任一所示的氨基酸序列，或与具有至少80％、85％、90％、95％、97％、98％、99％或100％一致性的氨基酸序列。In some embodiments, the polypeptide chain 1 has SEQ ID NO: 79, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID The amino acid sequence shown in any one of NO:115, or the amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity; the polypeptide chain 2 have the amino acid sequence shown in any one of SEQ ID NO:77, SEQ ID NO:113, or have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical Sexual amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：79所示的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 79; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：109所示的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 109; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：110所示的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 110; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：111所示的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 111; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：112所示的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 112; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：114所示的氨基酸序列；多肽链2包括如SEQ ID NO：113所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 114; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 113 amino acid sequence.
在具体的实施方案中，所述双特异性抗体包含两条多肽链：其中，多肽链1包括如SEQ ID NO：115所示的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列。In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 115; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 amino acid sequence.
第三方面，本发明提供了分离的核酸分子，其编码本发明第一方面的抗体或其抗原结合片段、第二方面所述的特异性结合蛋白或其片段。In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof in the first aspect of the present invention, the specific binding protein or fragment thereof in the second aspect.
在一些实施方案中，所述核酸分子是mRNA分子。In some embodiments, the nucleic acid molecule is an mRNA molecule.
第四方面，本发明提供了表达载体，其包含本发明第三方面所述的分离的核酸分子。In a fourth aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule described in the third aspect of the present invention.
所述表达载体可以是真核细胞表达载体和/或原核细胞表达载体，例如为逆转录病毒载体、慢病毒载体、噬菌体载体、腺病毒载体、腺相关载体或单纯疱疹载体。The expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a retrovirus vector, a lentivirus vector, a phage vector, an adenovirus vector, an adeno-associated vector or a herpes simplex vector.
在一些实施方案中，所述表达载体存在于纳米颗粒、脂质体、外来体、微泡或基因枪中。In some embodiments, the expression vector is present in nanoparticles, liposomes, exosomes, microvesicles, or gene guns.
第五方面，本发明提供了宿主细胞，所述宿主细胞包含第三方面的分离的核酸分子，或第四方面的表达载体。In a fifth aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the third aspect, or the expression vector of the fourth aspect.
所述宿主细胞为本领域常规的宿主细胞，只要能使第四方面的表达载体稳定地将所携带的核酸分子表达为第一方面的抗体或其抗原结合片段或第二方面所述的双特异性抗体。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)。将第四方面的表达载体转化至宿主细胞中，即可得本发明的宿主细胞。其中所述转化方法为本领域的常规转化方法，较佳地为化学转化法，热激法或电转法。The host cell is a conventional host cell in the art, as long as the expression vector of the fourth aspect can stably express the carried nucleic acid molecule as the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody described in the second aspect. Sexual antibodies. Preferably, 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 antibodies or Fab antibodies), and the eukaryotic cells are preferably HEK293 cells or CHO cells cells (expressing full-length IgG antibodies). The host cell of the present invention can be obtained by transforming the expression vector of the fourth aspect into a host cell. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.
第六方面，本发明提供了第一方面的抗体或其抗原结合片段或第二方面的双特异性抗体的制备方法。In a sixth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect.
在一些实施方案中，利用杂交瘤技术或者本领域的其他常规技术制备，例如人源化技术等制备第一方面的抗体或其抗原结合片段或第二方面的双特异性抗体。In some embodiments, the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization technology.
在一些实施方案中，所述制备方法包括培养第四方面的宿主细胞的步骤。In some embodiments, the method of preparation includes the step of culturing the host cell of the fourth aspect.
在一些实施方案中，利用Harbour HCAb转基因小鼠(以下简称HCAb转基因小鼠)制备第二方面的双特异性抗体的第二结构域。In some embodiments, Harbor HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the second domain of the bispecific antibody of the second aspect.
所述HCAb转基因小鼠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。The HCAb transgenic mouse is a transgenic mouse carrying the immune repertoire of human immunoglobulins, capable of producing novel "heavy chain only" antibodies that are half the size of traditional IgG antibodies. It produces antibodies with only human antibody "heavy chain" variable domains and mouse Fc constant domains.
优选地，所述利用HCAb转基因小鼠制备第二方面的双特异性抗体的第二结构域的方法包括如下步骤：Preferably, the method for preparing the second domain of the bispecific antibody of the second aspect using HCAb transgenic mice comprises the following steps:
(a)利用人OX40免疫HCAb转基因小鼠，更优选地，所述人OX40抗原为重组人OX40-ECD-Fc，具体地，所述抗原为OX40的胞外区连接Fc构成的重组融合蛋白；(a) using human OX40 to immunize HCAb transgenic mice, more preferably, the human OX40 antigen is recombinant human OX40-ECD-Fc, specifically, the antigen is a recombinant fusion protein composed of the extracellular region of OX40 linked to Fc;
(b)利用免疫后的HCAb转基因小鼠脾B细胞的RNA反转录后的cDNA，以及特异性引物扩增人VH基因；(b) Utilize the cDNA after the RNA reverse transcription of the splenic B cells of the HCAb transgenic mouse after immunization, and specific primers to amplify the human VH gene;
(c)将扩增的VH基因构建到编码人IgG抗体重链Fc结构域序列的哺乳动物细胞表达载体中；和(c) constructing the amplified VH gene into a mammalian cell expression vector encoding the human IgG antibody heavy chain Fc domain sequence; and
(d)纯化步骤(c)获得的全人源OX40单抗。(d) Purifying the fully human OX40 monoclonal antibody obtained in step (c).
优选地，所述IgG抗体为IgG1抗体或IgG4抗体。Preferably, the IgG antibody is an IgG1 antibody or an IgG4 antibody.
在一些实施方案中，利用Harbour H2L2转基因小鼠(以下简称H2L2转基因小鼠)制备第一方面的特异性抗体或其抗原结合片段或者第二方面的双特异性抗体的第一结构域。In some embodiments, Harbor H2L2 transgenic mice (hereinafter referred to as H2L2 transgenic mice) are used to prepare the specific antibody or antigen-binding fragment thereof of the first aspect or the first domain of the bispecific antibody of the second aspect.
所述H2L2转基因小鼠是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。The H2L2 transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune library, and the antibody produced by it has a complete human antibody variable domain and a rat constant domain.
优选地，所述利用H2L2转基因小鼠制备第一方面的特异性抗体或其抗原结合片段或者第二方面的双特 异性抗体的的第一结构域的方法包括如下步骤：Preferably, the method for preparing the first domain of the specific antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect using H2L2 transgenic mice comprises the following steps:
(a)利用人PD-1免疫H2L2转基因小鼠，更优选地，所述人PD-1抗原为可溶性的重组人PD-1-hFc，具体地，所述抗原为PD-1连接Fc构成的重组融合蛋白；(a) Using human PD-1 to immunize H2L2 transgenic mice, more preferably, the human PD-1 antigen is soluble recombinant human PD-1-hFc, specifically, the antigen is composed of PD-1 linked to Fc recombinant fusion protein;
(b)免疫后的H2L2转基因小鼠脾细胞与骨髓瘤细胞系融合得到杂交瘤细胞，分离的杂交瘤表达具有完整的人可变结构域和大鼠恒定结构域的重链和轻链的抗体分子；(b) Hybridoma cells were obtained by fusion of splenocytes from immunized H2L2 transgenic mice with myeloma cell lines, and the isolated hybridomas expressed heavy and light chain antibodies with complete human variable domains and rat constant domains molecular;
(c)将步骤(b)获得的抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体κ轻链恒定结构域序列的哺乳动物细胞表达载体中，以编码产生抗体的全长轻链；(c) The antibody light chain variable domain sequence (VL) obtained in step (b) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human antibody kappa light chain constant domain sequence to encode the antibody-producing full-length light chain;
(d)将步骤(b)获得的抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG抗体重链恒定结构域序列的哺乳动物细胞表达载体中，以编码产生IgG抗体的全长重链；(d) The antibody heavy chain variable domain sequence (VH) obtained in step (b) is synthesized through genes and cloned into a mammalian cell expression vector encoding a human IgG antibody heavy chain constant domain sequence to encode an IgG antibody full-length heavy chain;
(e)将步骤(c)和(d)的表达载体同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到轻重链正确配对组装的重组抗体。(e) Simultaneously transfect the expression vectors of steps (c) and (d) into mammalian host cells, and use conventional recombinant protein expression and purification techniques to obtain recombinant antibodies with correct pairing and assembly of light and heavy chains.
优选地，将步骤(b)获得的抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG4抗体重链恒定结构域序列的哺乳动物细胞表达载体中，以编码产生IgG4抗体的全长重链。Preferably, the antibody heavy chain variable domain sequence (VH) obtained in step (b) is gene-synthesized and cloned into a mammalian cell expression vector encoding a human IgG4 antibody heavy chain constant domain sequence to encode the IgG4 antibody produced full-length heavy chain.
选地，将步骤(b)获得的抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达载体中，以编码产生IgG1抗体的全长重链。Optionally, the antibody heavy chain variable domain sequence (VH) obtained in step (b) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human IgG1 antibody heavy chain constant domain sequence, so as to encode the IgG1 antibody produced full-length heavy chain.
在一些实施方案中，将所制备的第二结构域和所制备的第一结构域组合成特异性结合蛋白。所述特异性结合蛋白可以同时结合两个靶点，其中第一结构域可以识别肿瘤细胞表面特异表达的PD-1，而第二结构域可以结合T细胞上的OX40分子，所述特异性结合蛋白结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，从而杀死肿瘤细胞。In some embodiments, the prepared second domain and the prepared first domain are combined into a specific binding protein. The specific binding protein can simultaneously bind two targets, wherein the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, and the second domain can bind OX40 molecules on T cells. After the protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
在一些实施方案中，将所制备的第二结构域和所制备的第一结构域构建成双特异性结合蛋白。所述双特异性结合蛋白可以同时结合两个靶点，其中第一结构域可以识别肿瘤细胞表面特异表达的PD-1，而第二结构域可以结合T细胞上的OX40分子，所述特异性结合蛋白结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，从而杀死肿瘤细胞。In some embodiments, the prepared second domain and the prepared first domain are constructed into a bispecific binding protein. The bispecific binding protein can simultaneously bind two targets, wherein the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, while the second domain can bind OX40 molecules on T cells, and the specificity After the binding protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
优选地，所述双特异性结合蛋白为二价结构或为四价对称结构。更优选地，所述双特异性结合蛋白为四价对称结构。Preferably, the bispecific binding protein has a bivalent structure or a tetravalent symmetrical structure. More preferably, the bispecific binding protein has a tetravalent symmetrical structure.
优选地，所述双特异性结合蛋白具有第二方面中所述的结构和序列。Preferably, the bispecific binding protein has the structure and sequence described in the second aspect.
第七方面，本发明提供了一种药物组合物，所述药物组合物包含第一方面所述的抗体或其抗原结合片段或第二方面所述的双特异性抗体，以及药学上可接受的载体。In the seventh aspect, the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof in the first aspect or the bispecific antibody in the second aspect, and a pharmaceutically acceptable carrier.
在一些实施方案中，所述药物组合物还包括其他的成分作为活性成分，例如其他的小分子药物或者抗体或多肽作为活性成分。In some embodiments, the pharmaceutical composition further includes other ingredients as active ingredients, such as other small molecule drugs or antibodies or polypeptides as active ingredients.
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％的所述特异性结合蛋白和/或其他的小分子药物或者抗体或多肽，以及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。The pharmaceutically acceptable carrier can be a conventional carrier in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, preferably including pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the specific binding protein and/or other small molecule drugs or antibodies or polypeptides, and 0.01-99.99% of the pharmaceutical carrier, the percentage being The mass percent of the pharmaceutical composition.
所述的药物组合物的给药途径可以是经肠胃外、注射或口服给药。所述药物组合物可以制备成适于给药的形式，例如固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，粉末、片剂、胶囊、颗粒、注射剂或输注剂的形式。可以经血管内、皮下、腹膜内、肌内、吸入、鼻内、气道滴注或胸腔内滴注施用。所述药物组合物还可以气雾剂或喷雾剂的形式施用，例如经鼻施用；或者，鞘内、髓内或心室内施用，还可以经透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。所述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。The administration route of the pharmaceutical composition can be parenteral, injection or oral administration. The pharmaceutical composition can be prepared in a form suitable for administration, such as solid, semi-solid or liquid form, which can be aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion form. Administration can be intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation, or intrathoracic instillation. The pharmaceutical composition can also be administered in the form of aerosol or spray, such as nasal administration; or, intrathecal, intramedullary or intraventricular administration, and can also be transdermal, transdermal, topical, enteral, intravaginal , sublingual or rectal administration. The pharmaceutical composition can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dose beneficial to the patient.
在一些实施方案中，所述药物组合物中的特异性结合蛋白与其他的活性成分可以同时给药或者按顺序给药。In some embodiments, the specific binding protein and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.
在一些实施方案中，所述特异性结合蛋白是双特异性结合蛋白。In some embodiments, the specific binding protein is a bispecific binding protein.
第八方面，本发明提供了第一方面所述的抗体或其抗原结合片段、第二方面所述的双特异性抗体、第三方面所述的分离的核酸分子、第七方面的药物组合物在制备用于预防、治疗和/或诊断免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病的药物中的应用。In the eighth aspect, the present invention provides the antibody or antigen-binding fragment thereof in the first aspect, the bispecific antibody in the second aspect, the isolated nucleic acid molecule in the third aspect, and the pharmaceutical composition in the seventh aspect Application in preparation of medicines for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases.
所述肿瘤可以为乳腺癌、肾细胞癌、黑色素瘤、结肠癌，以及B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期或转移性恶性肿瘤等。The tumor can be breast cancer, renal cell carcinoma, melanoma, colon cancer, and B cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin's lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignancy, etc.
所述炎性疾病可以为特应性皮炎、溃疡性结肠炎等。The inflammatory disease may be atopic dermatitis, ulcerative colitis and the like.
所述免疫性疾病可以为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮、哮喘等。The immune disease may be graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma and the like.
第九方面，本发明提供了用于检测样品中的OX40和PD-1的方法，所述方法包括用第一方面所述的抗体或抗原结合片段或第二方面的双特异性抗体检测样品中的OX40和PD-1的步骤。In the ninth aspect, the present invention provides a method for detecting OX40 and PD-1 in a sample, the method comprising using the antibody or antigen-binding fragment described in the first aspect or the bispecific antibody in the second aspect to detect OX40 and PD-1 in the sample OX40 and PD-1 steps.
所述样品可以是生物学样品，例如，全血，红血细胞浓缩物，血小板浓缩物，白细胞浓缩物，组织，骨髓吸出物，血浆，血清，脑脊液，粪便，尿液，培养的细胞，唾液，口腔分泌物，和鼻腔分泌物等生物学样品。The sample can be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
在一些实施方案中，所述检测样品中的OX40和PD-1的方法出于非诊断的目的。In some embodiments, the method of detecting OX40 and PD-1 in a sample is for non-diagnostic purposes.
第十方面，本发明还提供了一种套装药盒，该套装药盒包括一个或多个药盒，包含如本发明第一方面所述的抗体或其抗原结合片段或第二方面所述的双特异性抗体或如本发明第七方面所述的药物组合物。In the tenth aspect, the present invention also provides a kit, which includes one or more kits, comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the antibody or antigen-binding fragment thereof as described in the second aspect. The bispecific antibody or the pharmaceutical composition according to the seventh aspect of the present invention.
在一些实施方案中，所述套装药盒包括第一药盒，所述第一药盒包括第一方面的抗体或其抗原结合片段，或者第二方面所述的第一结构域和第二结构域组成的双特异性抗体。In some embodiments, the set of kits comprises a first kit comprising the antibody or antigen-binding fragment thereof of the first aspect, or the first domain and the second structure of the second aspect Domain-composed bispecific antibodies.
在一些实施方案中，所述套装药盒可以进一步包括第二药盒，所述第二药盒包括其他治疗剂，所述其他治疗剂包括，但不限于化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。In some embodiments, the kit of parts may further include a second kit comprising other therapeutic agents including, but not limited to, chemotherapeutics, radiotherapeutics, immunosuppressive agents and cytotoxic drugs.
在一些实施方案中，上述第一药盒和第二药盒可以同时使用，也可以先使用上述第一药盒再使用上述第二药盒，还可以先使用上述第二药盒再使用上述第一药盒，可以根据具体应用时的实际需求而定。In some embodiments, the above-mentioned first medicine box and the second medicine box can be used at the same time, or the above-mentioned first medicine box can be used first and then the above-mentioned second medicine box, or the above-mentioned second medicine box can be used first and then the above-mentioned first medicine box can be used. A medicine box can be determined according to the actual needs of specific applications.
第十方面，本发明提供了预防、治疗和/或诊断免疫性疾病、急性和慢性炎性疾病以及肿瘤疾病的方法，其包括向受试者施用治疗有效量的本发明第一方面抗体或其抗原结合片段或第二方面所述的双特异性抗体或 第七方面所述的药物组合物。In a tenth aspect, the present invention provides methods for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antibody of the first aspect of the present invention or its The antigen-binding fragment or the bispecific antibody of the second aspect or the pharmaceutical composition of the seventh aspect.
本发明的特异性结合蛋白具有如下技术效果：The specific binding protein of the present invention has the following technical effects:
1，本发明的PD1的特异性抗体能够特异性结合表达人PD-1的细胞和表达食蟹猴PD-1的细胞；1. The PD1-specific antibody of the present invention can specifically bind to cells expressing human PD-1 and cells expressing cynomolgus monkey PD-1;
2，本发明的PD1×OX40双特异性抗体的第一结构域能够特异性结合表达人PD-1的细胞和表达食蟹猴PD-1的细胞；第二结构域能够结合人OX40蛋白和食蟹猴OX40蛋白；第二结构域在CHO-K1/CD32b交联条件下显示出浓度依赖的增强NF-κb信号通路的作用；2. The first domain of the PD1×OX40 bispecific antibody of the present invention can specifically bind to cells expressing human PD-1 and cells expressing cynomolgus PD-1; the second domain can bind to human OX40 protein and cynomolgus Monkey OX40 protein; the second domain showed concentration-dependent enhancement of NF-κB signaling pathway under CHO-K1/CD32b cross-linking conditions;
3，本发明的PD1×OX40双特异性抗体是PD-1交联依赖型；在CHO-K1/PD-1细胞交联辅助作用下，特异性的引起浓度依赖的增强NF-κb信号通路的作用。能够特异地引起PD-1交联依赖的OX40介导的NF-κb信号通路的促进作用，而且其引起的信号强度与其浓度成正相关关系递增；3. The PD1×OX40 bispecific antibody of the present invention is PD-1 cross-linking dependent; under the assistance of CHO-K1/PD-1 cell cross-linking, it can specifically cause concentration-dependent enhancement of NF-κb signaling pathway effect. It can specifically induce the promotion of PD-1 cross-linking-dependent OX40-mediated NF-κb signaling pathway, and the signal strength it causes increases in a positive correlation with its concentration;
4，本发明的PD-1×OX40双特异性抗体对PD-1信号通路有抑制作用；能够增强TNFα和IFNγ的分泌；对Treg细胞分泌IL-10有抑制作用；能够促进T细胞中细胞因子Granzyme B的分泌。4. The PD-1×OX40 bispecific antibody of the present invention has an inhibitory effect on the PD-1 signaling pathway; can enhance the secretion of TNFα and IFNγ; has an inhibitory effect on the secretion of IL-10 by Treg cells; and can promote cytokine production in T cells. Secretion of Granzyme B.
5，本发明的PD-1×OX40双特异性抗体不仅能够增强效应T细胞的功能和存活，而且还能抑制Treg的抑制性功能；相对于单独的PD-1单抗和OX40单抗，以及PD-1单抗和OX40单抗的组合显示出明显的协同活性。5. The PD-1×OX40 bispecific antibody of the present invention can not only enhance the function and survival of effector T cells, but also inhibit the suppressive function of Treg; compared with individual PD-1 monoclonal antibody and OX40 monoclonal antibody, and The combination of PD-1 mAb and OX40 mAb showed obvious synergistic activity.
定义definition
除非另有定义，否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的，将应用以下定义，并且在适当时，以单数形式使用的术语也将包括复数形式，反之亦然。Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa.
“约”和“大约”通常意指鉴于测量的性质或精度，所测量值的可接受误差范围。通常误差范围在所给出的值或值范围的20％范围内，一般在10％的范围内，甚至更一般在5％的范围内。"About" and "approximately" generally mean an acceptable range of error for the measured value given the nature or precision of the measurement. Typically the margin of error is within 20%, typically within 10%, and even more typically within 5% of a given value or range of values.
术语“抗原结合分子”或“特异性结合蛋白”泛指特异性结合抗原决定簇的分子。抗原结合分子包括例如抗体、抗体片段和骨架抗原结合蛋白。The term "antigen binding molecule" or "specific binding protein" refers broadly to a molecule that specifically binds an antigenic determinant. Antigen binding molecules include, for example, antibodies, antibody fragments, and backbone antigen binding proteins.
本发明的术语“抗体”涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、单特异性和多特异性抗体(例如，双特异性抗体或三特异性抗体)，单链分子以及抗体片段，只要表现出所需的抗原结合活性即可。The term "antibody" of the present invention encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single chain molecules and antibody fragments as long as they exhibit the desired antigen-binding activity.
本发明的术语“单克隆抗体”是指从基本上同质的抗体群获得的抗体，即，除了可能微量存在的变异抗体(例如含有天然存在的突变或在单克隆抗体制剂的生产过程中产生的，通常以少量存在)之外，所述抗体群所包含的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同抗原决定簇(表位)的不同抗体的多克隆抗体制剂不同，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单个决定簇。The term "monoclonal antibody" in the present invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible traces of variant antibodies (e.g., containing naturally occurring mutations or arising during the production of monoclonal antibody preparations). , usually present in small amounts), the individual antibodies comprised by the population of antibodies are identical and/or bind the same epitope. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
本发明的术语“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。The term "multispecific antibody" of the present invention is used in its broadest sense to encompass antibodies with polyepitopic specificities. 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 polyepitopic specificity; Antibodies with VL and VH domains, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable domains, each single variable domain binds to Binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently Wait.
本发明的术语“双特异性结合蛋白”或“双特异性抗体”是指能够特异性结合至少两种不同的抗原决定簇，例如各自由一对抗体重链可变结构域(VH)和抗体轻链可变结构域(VL)形成的两个结合位点与不同抗原或同一抗原上的不同表位结合。双特异性抗体可以是1+1形式，2+1形式(包含第一抗原或表位的两个结合位点和第二抗原或表位的一个结合位点)或2+2形式(包含第一抗原或表位的两个结合位点和第二抗原或表位的两个结合位点)。通常，双特异性抗体包含两个抗原结合位点，每个抗原结合位点特异性针对不同的抗原决定簇。The term "bispecific binding protein" or "bispecific antibody" of the present invention refers to the ability to specifically bind at least two different antigenic determinants, for example each composed of an antibody heavy chain variable domain (VH) and antibody light The two binding sites formed by the chain variable domain (VL) bind different antigens or different epitopes on the same antigen. Bispecific antibodies can be in a 1+1 format, a 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (comprising a second antigen or epitope). two binding sites for one antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies comprise two antigen-binding sites, each specific for a different antigenic determinant.
本发明的术语“价”表示抗原结合分子存在指定数目的结合结构域。因此，术语“二价”“四价”和“六价”分别表示抗原结合分子中存在两个结合结构域、四个结合结构域和六个结合结构域。所述双特异性抗体至少是“二价的”，并且可以是“三价的”“四价的”或“更多价的”)。在一些情况下，所述抗体具有两个或更多个结合位点并且是双特异性的。也就是说，即使在存在多于两个结合位点(即抗体是三价的或多价的)的情况下，抗体也可以是双特异性的。The term "valence" in the present invention means that an antigen-binding molecule has a specified number of binding domains present. Thus, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two binding domains, four binding domains and six binding domains, respectively, in the antigen-binding molecule. The bispecific antibody is at least "bivalent", and may be "trivalent", "tetravalent" or "more valent"). In some instances, the antibodies have two or more binding sites and are bispecific. That is, antibodies can be bispecific even in cases where there are more than two binding sites (ie, the antibody is trivalent or multivalent).
本发明的术语“全长抗体”和“完整抗体”在本文中可互换使用，指代与天然抗体结构基本上相似的抗体。“天然抗体”是指天然存在的免疫球蛋白分子。例如，天然IgG类抗体是约150,000道尔顿的异四聚体糖蛋白，由二硫键键合的两条轻链和两条重链组成。从N末端到C末端，每条重链具有可变区(VH)(也称为可变重链结构域或重链可变结构域)和三个恒定结构域(CH1、CH2和CH3)(也称为重链恒定区)。从N末端到C末端，每条轻链具有可变区(VL)(也称为可变轻链结构域或轻链可变结构域)和轻链恒定结构域(CL)(也称为轻链恒定区)。抗体的重链可以是五种类型中的一种，所述五种类型为α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，还可以进一步分为亚型，例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。抗体的轻链基于其恒定结构域的氨基酸序列，可以是两种类型中的一种，所述两种类型为κ轻链和λ轻链。The terms "full-length antibody" and "intact antibody" of the present invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody. "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, antibodies of the native IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) ( Also known as the heavy chain constant region). From N-terminus to C-terminus, each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain domain). chain constant region). The heavy chain of an antibody can be of one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), which can be further divided into Subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1 ) and α2 (IgA2). The light chains of an antibody, based on the amino acid sequence of their constant domains, can be of one of two types, kappa light chains and lambda light chains.
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。Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids. Each heavy chain is composed 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 is composed of a light chain variable region (VL) and a light chain constant region (CL). The 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.
“抗体片段”包含完整抗体的一部分。抗体片段的示例包括但不限于Fab、Fab’、F(ab’) 2和Fv；双体抗体、三体抗体、四体抗体、交叉Fab片段；线性抗体；单链抗体分子(例如scFv)；由抗体片段和单结构域抗体形成的多特异性抗体。 "Antibody fragments" comprise a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv; diabodies, triabodies, tetrabodies, crossed Fab fragments; linear antibodies; single chain antibody molecules (e.g. scFv); Multispecific antibodies formed from antibody fragments and single domain antibodies.
本发明的术语“抗原结合结构域”或“抗原结合位点”是指抗原结合分子中特异性结合抗原决定簇的部分。更具体地，术语“抗原结合结构域”是指抗体的一部分，所述部分包含与抗原的一部分或全部特异性结合并互补的区域。在抗原分子很大的情况下，抗原结合分子可以仅结合抗原的特定部分，该部分称为表位。抗原结合结构域可以由例如一个或多个可变结构域(也称为可变区)提供。优选地，抗原结合结构域包含抗体轻链可变区(VL)和抗体重链可变区(VH)。在一个方面，抗原结合结构域能够结合其抗原并阻断或部分阻断所述抗原的功能。The term "antigen-binding domain" or "antigen-binding site" in the present invention refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a part of an antibody comprising a region that specifically binds and is complementary to a part or all of an antigen. In cases where the antigen molecule is large, the antigen-binding molecule can bind only a specific part of the antigen, called an epitope. An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, an antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.
本发明的术语“抗原决定簇”与“抗原”和“表位”同义，并且是指多肽大分子上的位点(例如一段连续的氨基酸或由非连续氨基酸的不同区域组成的构象构型)，抗原结合部分与所述位点结合，从而形成抗原结合 部分-抗原复合物。抗原决定簇可以存在于例如肿瘤细胞的表面、微生物感染细胞的表面、其他患病细胞的表面、免疫细胞的表面、血清中游离物和/或细胞外基质(ECM)中。除非另有说明，本发明中用作抗原的蛋白可以是任何脊椎动物来源的任何天然形式的蛋白质，所述脊椎动物来源包括诸如灵长类动物(例如人类)和啮齿类动物(例如小鼠和大鼠)等哺乳动物。抗原也可以是人蛋白质，或者抗原为“全长”、未加工的蛋白质，以及由细胞内加工而产生的任何形式的蛋白质，或者为天然存在的蛋白质变体，例如剪接变体或等位基因变体。The term "antigenic determinant" in the present invention is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (such as a stretch of continuous amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids ), the antigen-binding moiety binds to said site, thereby forming an antigen-binding moiety-antigen complex. The antigenic determinant may be present, for example, on the surface of tumor cells, microbially infected cells, other diseased cells, immune cells, free matter in serum and/or in the extracellular matrix (ECM). Unless otherwise stated, the proteins used as antigens in the present invention may be proteins in any native form from any vertebrate source, including, for example, primates (e.g., humans) and rodents (e.g., mice and rats) and other mammals. The antigen can also be a human protein, or the antigen can be "full length", unprocessed protein, any form of protein resulting from intracellular processing, or a naturally occurring variant of a protein, such as a splice variant or an allele Variants.
“特异性结合”是指对于抗原具有结合选择性，并且可以与不需要的或非特异性的结合区分开来。抗原结合分子与特定抗原结合的能力可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术(例如表面等离子体共振(SPR)技术以及传统的结合测定进行测量。在一个实施例中，例如通过SPR所测得的，抗原结合分子与不相关蛋白的结合程度小于所述抗原结合分子与抗原的结合程度的约10％。在某些实施例中，与抗原结合的分子的解离常数(Kd)为≤1μM、≤100nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10 -7M或更低，例如10 -7M至10 -13M，例如10- 9M至10 -13M)。 "Specifically binds" means that binding is selective for an antigen and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques as well as traditional binding assays. In one embodiment In certain embodiments, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, as measured by SPR. In certain embodiments, the solution of the molecule bound to the antigen The separation constant (Kd) is ≤1μM, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM or ≤0.001nM (for example, 10 -7 M or lower, such as 10 -7 M to 10 -13 M, eg 10- 9 M to 10 -13 M).
“亲和力”或“结合亲和力”是指分子(例如抗体)的单个结合位点与其结合配体(例如抗原)之间的非共价相互作用的强度。结合亲和力通常可以用解离常数(Kd)表示，解离常数(Kd)是解离速率常数与缔合速率常数(分别为k off和k on)的比率。因此，等效亲和力可以包括不同的速率常数，只要速率常数的比率保持相同即可。亲和力可以通过本领域已知的常规方法测量，例如表面等离子体共振(SPR)。 "Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Binding affinity can generally be expressed in terms of a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (k off and k on , respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).
本发明的术语“高亲和力”是指抗体对靶抗原的Kd为10 -9M或更低，甚至10 -10M或更低。本发明的术语“低亲和力”是指抗体的Kd为10 -8M或更高。 The term "high affinity" in the present invention means that the Kd of the antibody for the target antigen is 10 -9 M or lower, even 10 -10 M or lower. The term "low affinity" in the present invention means that the Kd of the antibody is 10 -8 M or higher.
“亲和力成熟的”的抗体是指在一个或多个高变区(HVR)中具有一个或多个修饰的抗体，与不具有此类修饰的亲本抗体相比，此类修饰导致了抗体对抗原亲和力的改善。An "affinity matured" antibody is one that has one or more modifications in one or more hypervariable regions (HVRs) that result in greater Affinity improvements.
本发明的术语“单域抗体”和“纳米抗体”具有相同的含义，指仅具有抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。The terms "single domain antibody" and "nanobody" in the present invention have the same meaning, referring to only having the variable region of the heavy chain of an antibody, and constructing a single domain antibody consisting of only one heavy chain variable region, which is fully functional the smallest antigen-binding fragment.
本发明的术语“重链抗体”也称为HCAb抗体，是指相对双链抗体(免疫球蛋白)来说，缺失抗体轻链，只包含重链的抗体，具体来说包含重链可变结构域和Fc恒定结构域。The term "heavy chain antibody" of the present invention is also called HCAb antibody, which refers to an antibody that lacks the light chain of the antibody and only contains the heavy chain, compared to the double-chain antibody (immunoglobulin), specifically contains the variable structure of the heavy chain domain and Fc constant domain.
术语“包含特异性结合PD1的第一抗原结合结构域和特异性结合OX40的第二抗原结合结构域的双特异性抗体”“特异性结合PD1和OX40的双特异性抗体”“特异于PD1和OX40的双特异性抗原结合分子”或“抗PD1/抗OX40抗体”或“PD-1×OX40双特异性抗体”在本文中可互换使用，并且是指能够以足够的亲和力结合PD1和OX40，使得该抗体可用作靶向PD1和OX40的诊断和/或治疗剂的双特异性抗体。The term "bispecific antibody comprising a first antigen-binding domain specifically binding to PD1 and a second antigen-binding domain specifically binding to OX40", "bispecific antibody specifically binding to PD1 and OX40", "specific to PD1 and OX40 bispecific antigen-binding molecule" or "anti-PD1/anti-OX40 antibody" or "PD-1 x OX40 bispecific antibody" are used interchangeably herein, and refer to the ability to bind PD1 and OX40 with sufficient affinity , so that the antibody can be used as a bispecific antibody targeting PD1 and OX40 as a diagnostic and/or therapeutic agent.
本发明的术语“T效应细胞”指具有细胞溶解活性的T细胞(例如，CD4+及CD8+T细胞)以及T辅助(Th)细胞，T效应细胞分泌细胞因子、且激活并引导其他免疫细胞，但不包括调控性T细胞(Treg细胞)。本发明的所述抗OX40抗体可激活T效应细胞，例如CD4+及CD8+T效应细胞。The term "T effector cells" in the present invention refers to T cells with cytolytic activity (for example, CD4+ and CD8+ T cells) and T helper (Th) cells, T effector cells secrete cytokines, and activate and guide other immune cells, Regulatory T cells (Treg cells) are not included. The anti-OX40 antibody of the present invention can activate T effector cells, such as CD4+ and CD8+ T effector cells.
本发明的术语“调节性T细胞”或“Treg细胞”是指特殊类型的CD4+T细胞，能阻断其它T细胞的应答。Treg细胞特征在于表达CD4，IL-2受体的α亚基(CD25)和转录因子FOXP3，并且在诱导和维持外周自体耐受中发挥至关重要的作用，所述耐受针对肿瘤表达的抗原。The term "regulatory T cell" or "Treg cell" in the present invention refers to a special type of CD4+ T cell that can block the response of other T cells. Treg cells are characterized by the expression of CD4, the alpha subunit of the IL-2 receptor (CD25), and the transcription factor FOXP3, and play a crucial role in the induction and maintenance of peripheral self-tolerance against tumor-expressed antigens .
本发明的术语“PD1”，也称为程序性细胞死亡蛋白1，是一种由288个氨基酸组成的I型膜蛋白，是免 疫球蛋白超家族，最早公开于1992年(Ishida等人,EMBO J.,11(1992),3887-3895)。PD-1是扩展的CD28/CTLA-4家族T细胞调节因子的成员，并具有至少两个配体PD-L1(B7-H1、CD274)和PD-L2(B7-DC、CD273)。蛋白质结构包括细胞外IgV结构域，然后是跨膜区和细胞内尾部。细胞内尾部含有位于免疫受体酪氨酸基的抑制基序和免疫受体酪氨酸基的开关基序中的两个磷酸化位点，表明PD-1负调节T细胞受体TCR信号。这与配体结合后SHP-1磷酸酶和SHP-2磷酸酶与PD-1的细胞质尾部的结合一致。虽然PD-1不在原初T细胞上表达，但在T细胞受体(TCR)介导的激活后上调，并且在激活的和耗竭的T细胞上都被观察到(Agata等人，Int.Immunology 8(1996),765-772)。这些耗竭的T细胞具有功能障碍的表型并且不能适当地作出反应。尽管PD-1具有相对较宽的表达谱，但其最重要的作用可能是作为T细胞上的共抑制受体(Chinai等人，Trends in Pharmacological Sciences 36(2015),587-595)。因此，目前的治疗方法主要是阻断PD-1与其配体的相互作用以增强T细胞应答。术语“程序性死亡1”“程序性细胞死亡1”“蛋白质PD-1”“PD-1”“PD1”“PDCD1”“hPD-1”和“hPD-I”可互换使用，并且包括人PD-1的变体、同种型、物种同源物，以及与PD-1具有至少一个共同表位的类似物。人PD1的氨基酸序列显示于UniProt(www.uniprot.org)登录号Q15116中。The term "PD1" of the present invention, also known as programmed cell death protein 1, is a type I membrane protein composed of 288 amino acids, which belongs to the immunoglobulin superfamily, and was first disclosed in 1992 (Ishida et al., EMBO J., 11(1992), 3887-3895). PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators and has at least two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein structure consists of an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates T-cell receptor TCR signaling. This is consistent with the binding of SHP-1 phosphatase and SHP-2 phosphatase to the cytoplasmic tail of PD-1 after ligand binding. Although PD-1 is not expressed on naive T cells, it is upregulated following T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately. Although PD-1 has a relatively broad expression profile, its most important role may be as a co-inhibitory receptor on T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches mainly block the interaction of PD-1 with its ligands to enhance T cell responses. The terms "programmed death 1" "programmed cell death 1" "protein PD-1" "PD-1" "PD1" "PDCD1" "hPD-1" and "hPD-1" are used interchangeably and include human Variants, isoforms, species homologues of PD-1, and analogs that share at least one common epitope with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) accession number Q15116.
本发明的术语“PD1抗体”能够结合PD1，尤其是在细胞表面上表达的PD1多肽，并具有足够的亲和力以使得所述抗体可用作靶向PD1的诊断和/或治疗剂。通常情况下，经放射免疫测定(RIA)或流式细胞术(FACS)或使用生物传感器系统通过表面等离子体共振测定，PD1抗体与不相关的非PD1蛋白的结合能力小于所述抗体与PD1的结合能力的约10％。在某些实施方案中，结合人PD1的抗原结合蛋白的KD值≤1μM、≤100nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10 -8M或更低，例如10 -13M至10 -8M，例如10 -13M至10 -9M)。在一些实施方案中，在表面等离子体共振测定中，使用人PD1(PD1-ECD)测定结合亲和力的相应KD值，以获得PD1结合亲和力。PD-1/PD-L1抗体的治疗策略是几种转移性肿瘤中的标准治疗策略，并已显示出它们在早期疾病阶段和辅助治疗中的作用，尤其是黑色素瘤和非小细胞肺癌。 The term "PD1 antibody" of the present invention is capable of binding to PD1, especially a PD1 polypeptide expressed on the cell surface, with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD1. Typically, PD1 antibodies bind less to irrelevant, non-PD1 proteins than to PD1 as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system. About 10% of the binding capacity. In certain embodiments, the KD value of the antigen binding protein that binds to human PD1 is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (for example, 10 −8 M or lower, eg 10 -13 M to 10 -8 M, eg 10 -13 M to 10 -9 M). In some embodiments, the corresponding KD value of the binding affinity is determined using human PD1 (PD1-ECD) in a surface plasmon resonance assay to obtain the PD1 binding affinity. Therapeutic strategies with PD-1/PD-L1 antibodies are a standard treatment strategy in several metastatic tumors and have shown their role in early disease stages and adjuvant therapy, especially in melanoma and non-small cell lung cancer.
本发明的术语“OX40”又称CD134，表达于激活后1-3天的活化CD4+T细胞，以及CD8+T细胞、树突状细胞、中性粒细胞和Treg细胞等。OX40的配体OX40L表达于树突状细胞、B细胞等抗原呈递细胞(APC)的表面。OX40/OX40L的相互作用能够在OX40的胞内区域内招募TRAF分子，也可激活经典的NF-κB1途径或非经典的NF-κB2途径、PI3k/PKB和NFAT途径，进而调控T细胞分裂和存活的基因，以及促进细胞因子基因的转录以及细胞因子受体的表达，还能促进B细胞分化成浆细胞，促进抗体产生等，换句话说，可增强效应T细胞、NK、NK-T细胞活性，解除Treg的免疫抑制作用，既可增强特异性免疫反应，也可增强固有免疫反应，从而增强抗肿瘤免疫。The term "OX40" in the present invention is also called CD134, which is expressed in activated CD4+T cells, CD8+T cells, dendritic cells, neutrophils and Treg cells 1-3 days after activation. The ligand OX40L of OX40 is expressed on the surface of antigen-presenting cells (APC) such as dendritic cells and B cells. The interaction of OX40/OX40L can recruit TRAF molecules in the intracellular region of OX40, and can also activate the canonical NF-κB1 pathway or non-canonical NF-κB2 pathway, PI3k/PKB and NFAT pathways, thereby regulating T cell division and survival The genes that promote the transcription of cytokine genes and the expression of cytokine receptors can also promote the differentiation of B cells into plasma cells and the production of antibodies, etc. In other words, it can enhance the activity of effector T cells, NK, and NK-T cells , Relieving the immunosuppressive effect of Treg can not only enhance the specific immune response, but also enhance the innate immune response, thereby enhancing anti-tumor immunity.
本发明的术语“OX40抗体”能够结合OX40，并具有足够的亲和力以使得所述抗体可用作靶向OX40的诊断和/或治疗剂。通常情况下，经放射免疫测定(RIA)或流式细胞术(FACS)或使用生物传感器系统通过表面等离子体共振测定，OX40抗体与不相关的OX40蛋白的结合能力小于所述抗体与OX40的结合能力的约10％。在某些实施方案中，结合OX40的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M或更低，例如10 -13M到10 -8M，例如10 -13M到10 -9M)的解离常数(KD)。 The term "OX40 antibody" of the present invention is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting OX40. Typically, an OX40 antibody binds to an irrelevant OX40 protein less than the antibody binds to OX40 as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system about 10% of capacity. In certain embodiments, the antibody that binds OX40 has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 −8 M or less, such as 10 −13 M to 10 -8 M, eg, 10 -13 M to 10 -9 M) dissociation constant (KD).
本申请中的术语“H2L2转基因小鼠”或“Harbour H2L2小鼠”是一种携带人免疫球蛋白免疫库的转基因小鼠，所述小鼠产生具有完全人可变区的由两条重链和两条轻链(H2L2)组成的传统四聚体抗体，具有完整 的人抗体可变结构域和大鼠恒定结构域。由所述转基因小鼠所产生的抗体亲和力成熟，可变区完全人源化，并且具有优异的溶解性。The term "H2L2 transgenic mouse" or "Harbour H2L2 mouse" in this application is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces a protein consisting of two heavy chains with a fully human variable region. Traditional tetrameric antibody consisting of two light chains (H2L2) with fully human antibody variable domains and rat constant domains. The antibodies produced by the transgenic mice are affinity matured, fully humanized variable regions, and have excellent solubility.
本申请中的术语“HarbourHCAb小鼠”(WO2002/085945A2)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的恒定结构域。由于不含轻链这一特点，该仅“重链”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。The term "HarbourHCAb mouse" (WO2002/085945A2) in this application is a transgenic mouse carrying an immune repertoire of human immunoglobulins capable of producing novel "heavy chain only" antibodies that are only the size of traditional IgG antibodies half. It produces antibodies with only human antibody "heavy chain" variable domains and mouse Fc constant domains. Due to the absence of light chains, this "heavy chain only" 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.
本发明的术语“可变区”或“可变结构域”是指参与抗原结合分子与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域(分别为VH和VL)通常具有相似的结构，其中每个结构域包含四个保守框架区(FR)和三个高变区(HVR)。单个VH或VL结构域可足以赋予抗原结合特异性。The term "variable region" or "variable domain" in the present invention refers to the domain of antibody heavy chain or light chain that participates in the binding of antigen-binding molecule to antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.
本发明的术语“可变”是指可变域的某些区段在抗体之间在序列上普遍不同。V结构域介导抗原结合并限定特定抗体对于其特定抗原的特异性。然而，可变性在整个可变域并非均匀分布的。相反，集中于轻链与重链可变域内三个称为高变区(HVR)的区段中。可变域的更高度保守部分被称作框架区(FR)。天然重链与轻链的可变域各自包含四个FR区，大部分采用β-折叠构型，由三个HVR连接，其形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的HVR通过FR区紧密保持在一起，并且与其它链的HVR一起促成抗体的抗原结合位点的形成(参见Kabat等,Sequences of Immunological Interest,第5版,National Institute of Health,Bethesda,MD(1991))。恒定域不直接参与抗体与抗原的结合，具有其他效应功能，例如参与抗体的抗体依赖性细胞毒性。The term "variable" in the present invention means that certain segments of the variable domains generally differ in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β-sheet configuration, connected by three HVRs that form loops connecting and in some cases forming part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.
本发明的术语“高变区”或“HVR”是指在抗体可变结构域区域中序列上高变和/或形成结构上限定的环(“高变环”)的区域。通常，天然四链抗体包含六个HVR：三个存在于VH中(H1、H2、H3)，三个存在于VL中(L1、L2、L3)。HVR通常包含来自高变环和/或来自“互补决定区(CDR)”的氨基酸残基，来自“互补决定区(CDR)”的氨基酸残基具有最高的序列可变性和/或参与抗原识别。示例性CDR(LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3)发生在氨基酸残基26-32(L1)、50-52(L2)、91-96(L3)、26-32(H1)、53-55(H2)和96-101(H3)处(Chothia等人,J.Mol.Biol.196:901-917(1987)。示例性CDR(LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3)发生在氨基酸残基24-34(L1)、氨基酸残基50-56(L2)、氨基酸残基89-97(L3)、氨基酸残基31-35(H1)、氨基酸残基50-65(H2)，以及氨基酸残基95-102(H3)处(Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991)。作为比较，在表2中列出了包含上文引用的参考文献中所定义的CDR的相应氨基酸残基。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定义规则和基于结构环区域位置的Chothia定义规则(参见J Mol Biol 273:927-48,1997)。在本申请中，还可以使用包含Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列中的氨基酸残基。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。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”应理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。The term "hypervariable region" or "HVR" according to the present invention refers to the region in the variable domain region of an antibody that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, native four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)" that have the highest sequence variability and/or are involved in antigen recognition. Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53 -55 (H2) and 96-101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) occur At amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89-97 (L3), amino acid residues 31-35 (H1), amino acid residues 50-65 (H2) , and amino acid residues 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). For comparison, in Table 2 The corresponding amino acid residues of the CDRs defined in the above cited references are listed in. In this application, the amino acid sequences of the CDRs listed above are all shown according to the Chothia definition rule (claims of the application is also shown in accordance with the Chothia definition rules). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined by various methods in the art, such as the Kabat definition rules based on sequence variability and the position of the structural loop region The Chothia definition rule (seeing J Mol Biol 273:927-48,1997).In this application, can also use the Combined definition rule that comprises Kabat definition and Chothia definition to determine the amino acid residue in the variable domain sequence. Wherein Combined The definition rule is to combine the scope defined by Kabat and Chothia, based on which a larger scope is taken. It will be understood by those skilled in the art that unless otherwise specified, the term given antibody or its region (such as can "CDR" and "complementarity-determining region" of variable region) should be understood as encompassing a complementarity-determining region as defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present invention is based on the Chothia definition The sequence shown by the rules, but the amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.
表2.各CDR区的氨基酸编号系统(可参见http://bioinf.org.uk/abs/)Table 2. Amino acid numbering system for each CDR region (see http://bioinf.org.uk/abs/)
CDR\编号体系CDR\numbering system KabatKabat ChothiaChothia CombinedCombined
LCDR1LCDR1 24-3424-34 26-3226-32 24-3424-34
LCDR2LCDR2 50-5650-56 50-5250-52 50-5650-56
LCDR3LCDR3 89-9789-97 91-9691-96 89-9789-97
HCDR1HCDR1 31-3531-35 26-3226-32 26-3526-35
HCDR2HCDR2 50-6550-65 52-5852-58 50-6550-65
HCR3HCR3 95-10295-102 95-10295-102 95-10295-102
“框架”或“FR”是指除高变区(HVR)残基之外的可变结构域残基。可变结构域的FR通常由以下四个FR结构域组成：FR1、FR2、FR3和FR4。因此，HVR和FR序列通常在VH(或VL)中以如下序列出现：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
抗体的“类别”是指抗体的重链所具有的恒定结构域或恒定区的类型。存在五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些类别中的若干可以进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。The "class" of an antibody refers to the type of constant domain or constant region that the heavy chain of the antibody has. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
“人源化抗体”包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体包含至少一个，通常两个可变结构域，其中所有或基本上所有HVR(例如CDR)对应于非人抗体的HVR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。“人源化形式”的抗体，例如非人抗体，是指已经进行了人源化的抗体。A "humanized antibody" comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs Corresponds to FRs of human antibodies. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has been humanized.
“人抗体”具有的氨基酸序列与由人或人细胞产生的或来源于利用人抗体库或其他人抗体编码序列的非人来源的抗体的氨基酸序列相对应。人抗体的该定义特别地排除了包含非人抗原结合残基的人源化抗体。A "human antibody" has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
本发明的术语“Fc结构域”或“Fc区”用于定义含有恒定区的至少一部分的抗体重链C末端区域。该术语包括天然序列Fc区和变体Fc区。IgG Fc区包含IgG CH2结构域和IgG CH3结构域。本文的CH2结构域可以是天然序列CH2结构域或变体CH2结构域。本文的CH3结构域可以是天然序列CH3结构域或变体CH3结构域。CH2结构域可包含一种或多种减少或消除CH2结构域结合一种或多种Fcγ受体(例如FcγRI、FcγRIIa、FcγRIIb、FcγRIII)和/或补体的突变。推测减少或消除与Fc受体γ的结合将减少或消除抗体分子介导的ADCC。类似地，减少或消除与补体的结合预期将减少或消除抗体分子介导的CDC。减少或消除CH2结构域与一种或多种Fcγ受体和/或补体结合的突变是本领域已知的(Wang等，2018)。这些突变包括所谓的LALA突变”，所述突变涉及将CH2域的IMGT位置的1.3和1.2处的亮氨酸残基替换为丙氨酸(L1.3A和L1.2A)。或者，通过将CH2结构域中IMGT位置的84.4位的天冬酰胺(N)突变为丙氨酸、甘氨酸或谷氨酰胺(N84.4A、N84.4G或N84.4Q)，从而将保守的N-链糖基化位点突变产生a-糖基抗体，以降低IgG1效应子功能也是已知的(Wang等，2018)。作为另一选择，已知补体激活(C1q结合)和ADCC可通过将CH2结构域的IMGT位置第114位的脯氨酸突变为丙氨酸或甘氨酸(P114A或P114G)而降低(Idusogie等，2000；Klein等，2016)。这些突 变可以被组合，以产生具有进一步降低或没有ADCC或CDC活性的抗体分子。The term "Fc domain" or "Fc region" of the present invention is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region consists of an IgG CH2 domain and an IgG CH3 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain. The CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fcy receptors (eg, FcyRI, FcyRIIa, FcyRIIb, FcyRIII) and/or complement. It is hypothesized that reducing or eliminating binding to Fc receptor gamma will reduce or eliminate ADCC mediated by antibody molecules. Similarly, reducing or eliminating binding to complement is expected to reduce or eliminate CDC mediated by antibody molecules. Mutations that reduce or eliminate binding of the CH2 domain to one or more Fcγ receptors and/or complement are known in the art (Wang et al., 2018). These mutations include the so-called "LALA mutation" which involves the replacement of the leucine residues at positions 1.3 and 1.2 of the IMGT of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, the CH2 Mutation of the asparagine (N) at position 84.4 of the IMGT position in the domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q) to glycosylate the conserved N-chain Site mutations to generate a-glycosyl antibodies to reduce IgG1 effector function are also known (Wang et al., 2018). Alternatively, it is known that complement activation (C1q binding) and ADCC can be achieved through IMGT of the CH2 domain. Mutation of proline at position 114 to alanine or glycine (P114A or P114G) reduces (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to produce ADCC or CDC with further reduced or no Active antibody molecules.
抗体的“抗原结合部分”或“抗原结合片段”是指保留与给定抗原(例如PD-1或OX4)特异性结合的能力的完整抗体的一或多个片段。抗体的抗原结合功能可由完整抗体的片段执行。术语抗体的抗原结合部分或抗原结合片段的实例包括，但不限于，Fab片段，一种由VL、VH、CL及CH1结构域组成的单价片段；F(ab) 2片段，一种包含两个Fab片段的二价片段，该两个Fab片段经位于铰链区的二硫桥键连接；Fd片段，其由VH及CH1结构域组成；Fv片段，其由抗体的单臂的VL及VH结构域组成；单域抗体(dAb)片段，其由VH结构域或VL结构域组成；及分离的互补决定区(CDR)。 An "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind a given antigen (eg, PD-1 or OX4). The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of the term antigen-binding portion of an antibody or antigen-binding fragment include, but are not limited to, a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; an F(ab) 2 fragment, a fragment comprising two A bivalent fragment of a Fab fragment, the two Fab fragments are linked by a disulfide bridge at the hinge region; an Fd fragment, which consists of the VH and CH1 domains; an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody Composition: a single domain antibody (dAb) fragment consisting of a VH domain or a VL domain; and isolated complementarity determining regions (CDRs).
“与免疫球蛋白的Fc区等同的区域”包括免疫球蛋白Fc区的天然存在等位基因的变体，以及具有取代、添加或缺失但基本上不降低免疫球蛋白介导的效应子功能(诸如抗体依赖性细胞毒性)的能力的修饰变体。例如，可以使一个或多个氨基酸从免疫球蛋白的Fc区的N末端或C末端缺失，而基本上不丧失生物学功能。可以根据本领域中已知的一般规则来选择此类变体，以便对活性具有最小的影响(参见例如Bowie,J.U.等人，Science 247:1306-10(1990))。"A region equivalent to the Fc region of an immunoglobulin" includes naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having substitutions, additions or deletions that do not substantially reduce immunoglobulin-mediated effector functions ( Modified variants with capabilities such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, eg, Bowie, J.U. et al., Science 247:1306-10 (1990)).
本发明的术语“效应子功能”可归因于抗体的Fc区，是随着抗体同种型的变化而变化的生物活性。抗体效应子功能的示例包括：C1q结合和补体依赖性细胞毒性(CDC)、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞的抗原摄取、下调细胞表面受体(例如B细胞受体)，以及B细胞激活等。The term "effector function" of the present invention is attributable to the Fc region of an antibody and is a biological activity that varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine Secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (such as B cell receptors), and B cell activation, among others.
本发明的术语“肽接头”或者“连接肽”是指包含一个或多个氨基酸，通常为约2至20个氨基酸的肽。连接肽是本领域中已知的连接肽或在本文中描述的连接肽。The term "peptide linker" or "connecting peptide" of the present invention refers to a peptide comprising one or more amino acids, usually about 2 to 20 amino acids. The connecting peptide is a connecting peptide known in the art or described herein.
本发明的术语“融合至”“融合连接”或“连接至”是指区段(例如抗原结合结构域和FC结构域)或者直接地或经由一个或多个连接肽而通过肽键连接。The terms "fused to", "fusion-linked" or "linked to" of the present invention refer to segments (eg antigen binding domain and FC domain) linked by peptide bonds either directly or via one or more linking peptides.
本发明还涉及本发明的双特异性抗体的氨基酸序列变体。双特异性抗体的氨基酸序列变体可以通过向编码分子的核苷酸序列中引入适当的修饰或通过肽合成来制备。这类修饰包括例如抗体氨基酸序列中残基的缺失、插入和/或取代。可以进行缺失、插入和取代的任何组合获得最终的构建体，最终构建体具有所需的特性，例如抗原结合活性。用于取代的位点通常包括HVR和框架(FR)。参见表3中氨基酸的可能取代。The invention also relates to amino acid sequence variants of the bispecific antibodies of the invention. Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be made to arrive at a final construct having the desired properties, such as antigen binding activity. Sites for substitution typically include HVRs and frameworks (FRs). See Table 3 for possible amino acid substitutions.
表3.保守氨基酸取代Table 3. Conservative Amino Acid Substitutions
氨基酸残基amino acid residue 保守取代conservative substitution 优选保守取代Preferred conservative substitution
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Asp；Lys；ArgGln; His; Asp; Lys; Arg GlnGln
Asp(D)Asp(D) Glu；AsnGlu;Asn GluGlu
Cys(C)Cys(C) Ser；AlaSer; Ala SerSer
Gln(Q)Gln(Q) Asn；GluAsn; Glu AsnAsn
Glu(E)Glu(E) Asp；GlnAsp; Gln AspAsp
Gly(G)Gly(G) AlaAla AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile (I) Leu；Val；Met；Ala；Phe；NleLeu; Val; Met; Ala; Phe; Nle LeuLeu
Leu(L)Leu(L) Nle；Ile；Val；Met；Ala；PheNle; Ile; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr TyrTyr
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) Val；SerVal; SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；Ala；NleIle; Leu; Met; Phe; Ala; Nle LeuLeu
本发明的术语“多核苷酸”或“核酸”或“核苷酸序列”或“核酸分子”指分离的核酸分子或构建物，例如信使RNA(mRNA)，病毒衍生的RNA或质粒DNA(pDNA)。多核苷酸可包含常规的磷酸二酯键或非常规键(例如酰胺键,诸如在肽核酸(PNA)中找到的)。术语“核酸分子”指多核苷酸中存在的任一个或多个核酸区段，例如DNA或RNA片段。The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" or "nucleic acid molecule" in the present invention refers to an isolated nucleic acid molecule or construct such as messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA ). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA segments, present in a polynucleotide.
本发明的术语“分离的”核酸分子或多核苷酸是指已经与其天然环境分隔开的核酸分子，DNA或RNA。在本发明中，载体中包含的编码多肽的重组多核苷酸也是分离的。分离的多核苷酸的其他实例包括在异源宿主细胞中的重组多核苷酸或溶液中纯化的多核苷酸。分离的多核苷酸包括通常是含有该多核苷酸分子的细胞中所含有的多核苷酸分子,但是该多核苷酸分子存在于染色体外或与其天然染色体位置不同的染色体位置。分离的RNA分子包括本发明的体内或体外RNA转录物，为正和负链形式，以及双链形式。本发明的分离的多核苷酸或核酸进一步包括合成生成的此类分子。另外，多核苷酸或核酸可以为或可以包括调节元件,诸如启动子,核糖体结合位点,或转录终止子。The term "isolated" nucleic acid molecule or polynucleotide according to the present invention refers to a nucleic acid molecule, DNA or RNA, which has been separated from its natural environment. In the present invention, the recombinant polynucleotide encoding the polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. An isolated polynucleotide includes a polynucleotide molecule normally contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, in plus- and minus-strand form, as well as in double-stranded form. Isolated polynucleotides or nucleic acids of the invention further include such molecules produced synthetically. Additionally, a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, ribosomal binding site, or transcription terminator.
本发明的术语“表达盒”指重组或合成生成的多核苷酸，具有允许特定核酸在靶细胞中转录的一系列核酸元件。重组表达盒可导入质粒，染色体，线粒体DNA，质体DNA，病毒或核酸片段。典型地，在其它序列以外，表达载体的重组表达盒部分包括要转录的核酸序列和启动子。在某些实施方案中，本发明的表达盒包含编码本发明的双特异性抗原结合分子或其片段的多核苷酸序列。The term "expression cassette" according to the present invention refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow the transcription of a particular nucleic acid in a target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
本发明的术语“载体”或“表达载体”与“表达构建物”可以互换使用，将与其可操作连接的特定基因导入靶细胞并指导表达的DNA分子。所述载体包括作为自身复制性核酸结构的载体以及并入其已经导入的宿主细胞的基因组的载体。本发明的表达载体包含表达盒。表达载体可以进行大量稳定mRNA的转录。一旦表达载体在靶细胞内，则由细胞转录和/或翻译机制生成由该基因编码的核糖核酸分子或蛋白质。在一个实施方案中，本发明的表达载体包括含有编码本发明的双特异性抗原结合分子或其片段的多核苷酸序列的表达盒。The term "vector" or "expression vector" and "expression construct" in the present invention can be used interchangeably, and a specific gene operably linked to it is introduced into a target cell and directs the expression of a DNA molecule. The vector includes a vector that is a self-replicating nucleic acid structure as well as a vector that is incorporated into the genome of a host cell into which it has been introduced. The expression vectors of the present invention comprise expression cassettes. Expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
术语“宿主细胞”“宿主细胞系”和“宿主细胞培养物”可互换使用，是指其中已经导入外源核酸的细胞，还包括这类细胞的后代。宿主细胞包括“转化体/转化子”和“转化细胞”，包括原代转化细胞以及由其衍生 的后代。后代的核酸与亲本细胞可以不完全一致，可含有突变。宿主细胞是可用于生成本发明的双特异性抗原结合分子的任何类型的细胞。宿主细胞包括培养的细胞，例如培养的哺乳动物细胞，诸如CHO细胞，HEK293细胞，BHK细胞，NS0细胞，SP2/0细胞，YO骨髓瘤细胞，P3X63小鼠骨髓瘤细胞，PER细胞，PER.C6细胞或杂交瘤细胞，酵母细胞，昆虫细胞和植物细胞，还包括转基因动物，转基因植物或培养的植物或动物组织内所包含的细胞。The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants/transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom. The nucleic acid of the progeny may not be identical to that of the parental cell and may contain mutations. A host cell is any type of cell that can be used to produce a bispecific antigen binding molecule of the invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or hybridoma cells, yeast cells, insect cells and plant cells, also cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.
药物的“有效量”指在接受其施用的细胞或组织中导致生理变化所必需的量。“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用的严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which it is administered. An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dosage of administration and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
依照本发明的双特异性抗体具有协同效应。“协同效应”是指两种药物的组合效果大于它们单独的效果之和且在统计学上不同于对照和单一药物。本发明的叠加效应是指两种药物的组合效果是它们单独的效果之和且在统计学上不同于对照和/或单一药物。The bispecific antibodies according to the invention have a synergistic effect. "Synergistic effect" means that the combined effect of two drugs is greater than the sum of their individual effects and statistically different from the control and single drugs. The additive effect of the present invention means that the combined effect of the two drugs is the sum of their individual effects and is statistically different from the control and/or single drug.
药物(例如药用组合物)的“治疗有效量”指在剂量和给药间隔和时间上有效实现想要的治疗或预防效果必需的量。例如，治疗有效量的药物消除、减轻/减少、延迟、最小化或预防疾病的不利影响。A "therapeutically effective amount" of a drug (eg, a pharmaceutical composition) refers to that amount, in dosage and administration interval and time, effective to achieve the desired therapeutic or prophylactic effect. For example, a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes or prevents the adverse effects of a disease.
“个体”或“受试者”是指哺乳动物。哺乳动物包括但不限于驯养的动物(例如牛，绵羊，猫，犬和马)，灵长类动物(例如人和非人灵长类，诸如猴)，家兔和啮齿类动物(例如小鼠和大鼠)。具体地，个体或受试者是人。"Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human.
“药物组合物”表示含有一种或多种本公开的抗体或其抗原结合片段与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体或赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means a mixture comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure and other chemical components such as physiologically/pharmaceutically acceptable carriers or excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
“药学可接受的载体”指药用组合物中除了活性组分以外，对受试者无毒的组分。药学可接受赋形剂包括但不限于缓冲剂，稀释剂，稳定剂和/或防腐剂。"Pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition other than the active ingredient that is nontoxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, diluents, stabilizers and/or preservatives.
术语“癌症”是指描述哺乳动物中以细胞生长不受调节为特征的疾病。癌症的例子包括但不限于肿瘤，淋巴瘤、母细胞瘤、肉瘤和白血病或淋巴样恶性肿瘤。癌症更具体的实例包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、腺癌和肺的鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠基质癌)、骨癌、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、尿道癌、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫颈癌、唾液腺癌、肾癌或输尿管癌、前列腺癌、阴道癌、外阴癌、甲状腺癌、肛门癌、阴茎癌、黑素瘤、胆管癌、中枢神经系统(CNS)肿瘤、脊椎轴肿瘤、脑干胶质瘤、多形性成胶质细胞瘤、星形细胞瘤、神经鞘瘤、室管膜瘤、成髓细胞瘤、脑膜瘤、鳞状细胞癌、垂体腺瘤和尤文氏肉瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端黑素瘤、结节性黑素瘤、多发性骨髓瘤和B细胞淋巴瘤、慢性淋巴细胞白血病(CLL)、急性成淋巴细胞性白血病(ALL)、毛细胞性白血病、慢性成髓细胞性白血病和移植后淋巴增殖性疾病(PTLD)、以及与瘢痣病(phakomatoses)、水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖、脑瘤和脑癌、以及头或颈癌和相关的转移癌。The term "cancer" is meant to describe diseases in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal carcinoma, hepatocellular carcinoma, Gastric cancer (including gastrointestinal and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer cancer, endometrial or cervical cancer, salivary gland cancer, kidney or ureter cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, bile duct cancer, central nervous system (CNS ) tumors, spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, myeloblastomas, meningiomas, squamous cell carcinomas, pituitary Adenoma and Ewing's sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplantation lymphoproliferative disorder (PTLD), as well as those associated with phakomatoses, edema (such as with brain tumor-related) and abnormal vascular proliferation associated with Meigs' syndrome, brain tumors and brain cancers, and head or neck cancer and related metastases.
“治疗”是指给予患者内用或外用治疗剂，例如包含本公开的任一种特异性结合蛋白，双特异性抗体， 抗体或其抗原结合片段的组合物或编码特异性结合蛋白，双特异性抗体，抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病或症状，所述治疗剂对这些疾病或症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。"Treatment" refers to administering to a patient an internal or external therapeutic agent, such as a composition comprising any specific binding protein of the present disclosure, a bispecific antibody, an antibody or an antigen-binding fragment thereof or an encoding specific binding protein, bispecific Antibodies, nucleic acid molecules of antibodies or antigen-binding fragments thereof, the patient has one or more diseases or conditions, and the therapeutic agent has a therapeutic effect on these diseases or conditions. Generally, a therapeutic agent is administered in a patient or population to be treated in an amount effective to ameliorate one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.
术语“预防癌症”是指在哺乳动物中延迟、抑制或防止癌症发作，所述哺乳动物中癌发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定其具有癌症易感性。该术语还包括治疗具有癌变前病症的哺乳动物以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。The term "preventing cancer" means delaying, inhibiting or preventing the onset of cancer in a mammal in which the onset of carcinogenesis or tumorigenesis has not been proven, but has been identified, for example, by genetic screening or other methods have a susceptibility to cancer. The term also includes treating a mammal with a premalignant condition to halt the progression of the premalignant condition to a malignancy or to cause its regression.
术语“脂质体”是指包括通过产生封闭的脂质双层或聚集体形成的多种单层和多层脂质载体。脂质体可表征为具有磷脂双层膜和内部水性介质的囊泡结构。多层脂质体具有由水性介质分开的多个脂质层。它们在磷脂悬浮于过量的水溶液中时自发形成。The term "liposome" is meant to include a variety of unilamellar and multilamellar lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
术语“施用”是指向有需要的受试者提供本发明的特异性结合蛋白、分离的核酸分子、表达载体、宿主细胞或药物组合物，例如通过口服、注射、局部给药的方式向受试者施用。The term "administration" refers to providing the specific binding protein, isolated nucleic acid molecule, expression vector, host cell or pharmaceutical composition of the present invention to a subject in need, for example, by oral administration, injection or local administration. who apply.
本申请的抗体的轻链、重链及可变区的序列编号(SEQ ID NO:)见表4。The sequence numbers (SEQ ID NO:) of the light chain, heavy chain and variable region of the antibody of the present application are shown in Table 4.
表4.本申请的抗体的轻链、重链及可变区的序列编号(SEQ ID NO:)Table 4. The sequence numbering (SEQ ID NO:) of the light chain of the antibody of the present application, heavy chain and variable region
Figure PCTCN2022098838-appb-000003
Figure PCTCN2022098838-appb-000003
本申请的抗体的框架区的序列编号(SEQ ID NO:)见表5。The sequence number (SEQ ID NO:) of the framework region of the antibody of the present application is shown in Table 5.
表5.本申请的抗体的框架区的序列编号(SEQ ID NO:)Table 5. Sequence numbering (SEQ ID NO:) of the framework region of the antibody of the present application
Figure PCTCN2022098838-appb-000004
Figure PCTCN2022098838-appb-000004
Figure PCTCN2022098838-appb-000005
Figure PCTCN2022098838-appb-000005
附图说明Description of drawings
图1示出PD-1×OX40双特异性抗体IgG_HC-VH结构(结构1)。Figure 1 shows the PD-1×OX40 bispecific antibody IgG_HC-VH structure (structure 1).
图2示出PD-1×OX40双特异性抗体VH-IgG_HC结构(结构2)。Figure 2 shows the structure of PD-1×OX40 bispecific antibody VH-IgG_HC (structure 2).
图3示出PD-1×OX40双特异性抗体Fab(CL)-VH-Fc结构(结构3)。Figure 3 shows the PD-1×OX40 bispecific antibody Fab(CL)-VH-Fc structure (Structure 3).
图4示出PD-1×OX40双特异性抗体IgG_HC-VH-VH结构(结构4)。Figure 4 shows the PD-1×OX40 bispecific antibody IgG_HC-VH-VH structure (structure 4).
图5示出PD-1×OX40双特异性抗体IgG_HC-VH-VH-VH结构(结构5)。Figure 5 shows the PD-1×OX40 bispecific antibody IgG_HC-VH-VH-VH structure (Structure 5).
图6示出PD-1单克隆抗体阻断PD-1与PD-L1结合。Figure 6 shows that PD-1 monoclonal antibody blocks the binding of PD-1 and PD-L1.
图7示出通过流式细胞术测定的在表达人PD-1的CHO细胞中，本发明的双特异抗体或组成其结构的单克隆抗体随着浓度增加对细胞表面PD-1的结合。Figure 7 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to PD-1 on the cell surface as the concentration increases in CHO cells expressing human PD-1 determined by flow cytometry.
图8示出通过流式细胞术测定的在人OX40和NFkB启动子-luc的HEK 293细胞中，本发明的双特异抗体或组成其结构的单克隆抗体随着浓度增加对细胞表面OX40的结合。Figure 8 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to OX40 on the cell surface as the concentration increases in the HEK 293 cells of human OX40 and NFkB promoter-luc determined by flow cytometry .
图9示出通过流式细胞术测定的在人OX40、PD1和NFkB启动子-luc的HEK 293细胞中，本发明的双特异抗体或组成其结构的单克隆抗体随着浓度增加对细胞表面的结合。Figure 9 shows that in the HEK 293 cells of human OX40, PD1 and NFkB promoter-luc determined by flow cytometry, the bispecific antibody of the present invention or the monoclonal antibody constituting its structure increase with the concentration on the cell surface combined.
图10示出通过流式细胞术测定的在激活的人T细胞中，本发明的双特异抗体或组成其结构的单克隆抗体随着浓度增加对细胞表面的结合。Fig. 10 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to the cell surface as the concentration increases in activated human T cells determined by flow cytometry.
图11示出在表达人OX40和NFkB启动子-luc的HEK293细胞中，加入不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后对荧光素酶报告基因表达的检测。Figure 11 shows the detection of luciferase reporter gene expression in HEK293 cells expressing human OX40 and NFkB promoter-luc, after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure.
图12示出表达人CD32b的CHO细胞和表达人OX40和NFκB启动子-luc的HEK 293细胞共培养，加入不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后对荧光素酶报告基因表达的检测。Figure 12 shows that CHO cells expressing human CD32b and HEK 293 cells expressing human OX40 and NFκB promoter-luc are co-cultured, and after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure, the effect on luciferase Detection of reporter gene expression.
图13示出表达人PD-1的CHO细胞和表达人OX40和NFκB启动子-luc的HEK 293细胞共培养，加入不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后对荧光素酶报告基因表达的检测。Figure 13 shows the co-culture of CHO cells expressing human PD-1 and HEK 293 cells expressing human OX40 and NFκB promoter-luc, after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure, the effect on fluorescence Detection of primease reporter gene expression.
图14示出表达人PD-L1和OS8的HEK 293细胞和表达人PD-1和NFAT启动子-luc的Jurkat细胞共培养，加入不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后对荧光素酶报告基因表达的检测。Figure 14 shows the co-culture of HEK 293 cells expressing human PD-L1 and OS8 and Jurkat cells expressing human PD-1 and NFAT promoter-luc, adding different concentrations of the bispecific antibody of the present invention or the monoclonal composition of its structure Detection of luciferase reporter gene expression after antibody.
图15示出在人外周血单个核细胞(PBMC)与过表达人PD-L1的CHO细胞共培养体系里，加入PHA-L和不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后细胞因子TNFα的分泌。Figure 15 shows that in the co-culture system of human peripheral blood mononuclear cells (PBMC) and CHO cells overexpressing human PD-L1, adding PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal composition of its structure Secretion of the cytokine TNFα following antibody.
图16示出在人外周血单个核细胞(PBMC)与过表达人PD-L1的CHO细胞共培养体系里，加入PHA-L和不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后细胞因子IFNγ的分泌。Figure 16 shows that in the co-culture system of human peripheral blood mononuclear cells (PBMC) and CHO cells overexpressing human PD-L1, PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal composition of its structure are added Secretion of the cytokine IFNγ following antibody.
图17示出在体外培养扩增后的Treg细胞中加入不同浓度的本发明的双特异抗体后用流式细胞术检测Treg的IL10分泌。Fig. 17 shows that the Treg secretion of IL10 was detected by flow cytometry after adding different concentrations of the bispecific antibody of the present invention to the expanded Treg cells in vitro.
图18示出在分离的人T细胞、体外扩增的Treg细胞和体外诱导分离的DC细胞共培养体系中，加入不 同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后细胞因子IFNγ的分泌。Figure 18 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of IFNγ.
图19示出在分离的人T细胞、体外扩增的Treg细胞和体外诱导分离的DC细胞共培养体系中，加入不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后细胞因子Granzyme B的分泌。Figure 19 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of Granzyme B.
图20示出在分离的人T细胞、体外扩增的Treg细胞和体外诱导分离的DC细胞共培养体系中，加入不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后细胞因子IL2的分泌。Figure 20 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of IL2.
图21示出在人外周血单个核细胞(PBMC)里，加入PHA-L和不同浓度的本发明的双特异抗体或组成其结构的单克隆抗体后细胞因子IL2的分泌。Figure 21 shows the secretion of cytokine IL2 in human peripheral blood mononuclear cells (PBMC) after adding PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure.
具体实施方式detailed description
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.和Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。The examples shown below are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of the specification or claims in any way. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods are useful for this Those of ordinary skill in the field are well known and described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, 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Example 1 The acquisition of anti-OX40 fully human HCAb antibody
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。The Harbor HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces novel "heavy chain only" antibodies the size of traditional IgG antibodies half. It produces antibodies with only human antibody "heavy chain" variable domains and mouse Fc constant domains. Due to the fact that it does not contain light chains, 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.
1.1免疫HCAb小鼠1.1 Immunization of HCAb mice
6～8周龄的上述Harbour HCAb人源抗体转基因小鼠利用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100μL。在首轮免疫中，每只小鼠用50μg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25μg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天、第14天、第28天、第42天、第56天、第70天；并且在第49天、第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25μgOX40-ECD-Fc(ChemPartner,#21127-022)抗原蛋白的剂量进行最后一次增强免疫。 The above-mentioned Harbor HCAb human antibody transgenic mice aged 6 to 8 weeks were immunized with Harbor HCAb mice for multiple rounds using two immunization schemes. Specifically: immunization scheme 1, immunization with recombinant human OX40-ECD-Fc (ChemPartner, #21127-022) antigen protein. The total injected dose per mouse received by subcutaneous inguinal injection or by intraperitoneal injection was 100 [mu]L per immunization. In the first round of immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 50 μg of antigenic protein with complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of booster immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 25 μg of antigenic protein with Ribi adjuvant (Sigma Adjuvant System, Sigma, #S6322). Immunization scheme 2, the HEK293/OX40 (ChemPartner, Shanghai) stable cell line overexpressing human OX40 was used for immunization. Each mouse was intraperitoneally injected with 2×10 6 cell suspension each time it was immunized. The interval between each booster round is at least two weeks and usually no more than five booster rounds. The immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected. Five days before HCAb mouse splenic B cell isolation, the last booster immunization was performed with a dose of 25 μg OX40-ECD-Fc (ChemPartner, #21127-022) antigen protein per mouse.
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检测来确定小鼠血液中抗人OX40的滴度，并经流式细胞术检测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)为免疫前小鼠的血清。Collect mouse blood, dilute the blood 10 times, take 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), and put it on the ELISA plate coated with human OX40-ECD-Fc ELISA was performed to determine the titer of anti-human OX40 in mouse blood, and two concentrations of mouse blood (1:100, 1:1000) were detected by flow cytometry against CHO-K1/hOX40 cells with high expression of OX40 (Chempartner, Shanghai) and atopic reactivity of CHO-K1 blasts. The blank control group (PB) was the serum of mice before immunization.
1.2获得抗OX40的HCAb抗体序列1.2 Obtain the anti-OX40 HCAb antibody sequence
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:141)，PCR反向引物5’-AATCCCTGGGCACTGAAGAGACGGTGACC-3’(SEQ ID NO:142)。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。When the OX40-specific antibody titer in the serum of the above-mentioned mice reaches a certain level, the splenocytes of the mice are taken out to separate B cells, and CD138-positive cells are sorted by BD flow cytometry sorter (BD Biosciences, FACS AriaII Cell Sorter) Plasma cells and human OX40 antigen positive B cell population. Extract B cell RNA, reverse transcribe cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, #18091200), and then amplify human VH gene by PCR with specific primers. PCR forward primer 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' (SEQ ID NO: 141), PCR reverse primer 5'-AATCCCTGGGCACTGAAGAGACGGTGACC-3' (SEQ ID NO: 142). 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.
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 PCTCN2022098838-appb-000006
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 PCTCN2022098838-appb-000007
488,AffiniPure Goat Anti-Human IgG,FcγFragment Specific二抗(Jackson ImmunoResearchLaboratories 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荧光流式仪器上读取荧光值。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/hu OX40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抗体进行重组表达。 The constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain HCAb antibodies. Detect the binding of the HCAb-expressing supernatant to the stable cell line CHO-K1/OX40 (CHO-K1/hu OX40, Genscript, #M00561) overexpressing human OX40, and use a positive antibody (porcizumab) as a positive control ,conduct
Figure PCTCN2022098838-appb-000006
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. Add Draq5 fluorescent probe (Cell Signaling Technology, #4048L) (1 μL Draq5 to 1 mL CHO-K1/OX40 cells, 1:1000 dilution) and incubate for 30 minutes in a dark place. After centrifuging the cells, wash the cells with culture medium and adjust the cell density to 1×10 5 cells/mL. Then add 1:1000 diluted Alexa
Figure PCTCN2022098838-appb-000007
488, AffiniPure Goat Anti-Human IgG, FcγFragment Specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-545-098), take 30 μL of the mixture per well and add it to a 384-well plate (Greiner Bio One, #781091). Add 10 μL positive control or supernatant expressing HCAB to the 384-well plate and incubate for 2 hours. Read fluorescence values on a mirrorball fluorescence flow instrument. The positive clone antibody was further tested by ELISA with human OX40 protein (Acrobiosystem, #OX0-H5224) and cynomolgus monkey OX40 protein (Novoprotein, #CB17) to verify the cross-binding activity. At the same time, the binding activity to CHO-K1/hu OX40# cells was further detected by FACS. The nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence of the cloned antibody were obtained by conventional sequencing means. After removing the repetitive sequence, the remaining sequenced cloned antibody plasmids were transfected into HEK293 cells for expression, and the obtained supernatant was tested for NF-kb function again, thus obtaining 64 proteins that simultaneously bind to CHO-K1/hu OX40 and cynomolgus monkey OX40 A functional fully human OX40 monoclonal antibody with a unique sequence. According to the results of human-monkey binding ability and NF-Kb function test, select the antibody with the highest comprehensive ranking for recombinant expression.
1.3制备抗OX40全人重组抗体1.3 Preparation of anti-OX40 fully human recombinant antibody
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，得到纯化的抗OX40重组重链抗体。具体说来，将HEK293细胞在FreeStyle TMF17Expression 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,#71-5020-91AE)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。 The plasmid encoding the HCAb antibody obtained above was transfected into a mammalian host cell (such as human embryonic kidney cell HEK293), and a purified anti-OX40 recombinant heavy chain antibody was obtained by using conventional recombinant protein expression and purification techniques. Specifically, HEK293 cells were expanded in FreeStyle ™ F17Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to 6×10 5 cells/mL, and cultured in an 8% CO 2 shaker at 37°C for 24 hours with a cell concentration of 1.2×10 6 cells/mL. Prepare 30 mL of cultured cells. Dissolve 30 μg of the plasmid encoding the HCAb heavy chain in 1.5 mL of Opti-MEM serum-free medium (Thermo, #31985088), and then dissolve 1.5 mL of Opti-MEM into 1 mg/mL PEI (Polysciences, Inc. , #23966-2) 120 μL, let stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop into the plasmid PEI mixed solution while shaking the culture bottle, and incubate at 37°C for 5 days in an 8% CO2 shaker. After 5 days, the cell viability was observed. 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 MabSelect ™ (GE Healthcare Life Science, #71-5020-91AE) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample 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 into PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain the purified antibody Human OX40 HCAb monoclonal antibody solution. Antibody concentration was determined by NanoDrop detection of 280nm absorbance, and the purity of antibody was determined by SEC-HPLC and SDS-PAGE.
获得OX40抗体PR002067和PR002063，其对应的重链氨基酸序列参见SEQ ID NO:73和SEQ ID NO:105。同时本申请生产制备了抗OX40的阳性对照抗体Pogalizumab(泊加珠单抗)类似物(PR003475)，其氨基酸序列来源于IMGT数据，抗体重链氨基酸序列参见SEQ ID NO:74，抗体轻链氨基酸序列参见SEQ ID NO:78。OX40 antibodies PR002067 and PR002063 were obtained, and the corresponding heavy chain amino acid sequences are shown in SEQ ID NO:73 and SEQ ID NO:105. At the same time, the applicant produced and prepared the anti-OX40 positive control antibody Pogalizumab (pogalizumab) analog (PR003475), its amino acid sequence is derived from IMGT data, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 74, and the amino acid of the light chain of the antibody See SEQ ID NO:78 for the sequence.
1.4利用HPLC-SEC分析蛋白纯度和多聚体1.4 Analysis of protein purity and polymers by HPLC-SEC
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×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。The purity and aggregated form of the antibody protein samples obtained above were analyzed using analytical size exclusion chromatography (SEC). Connect the analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 μm, 7.8 mm × 30 cm) to a high-pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II), and equilibrate with PBS buffer at room temperature for at least 1 Hour. An appropriate amount of protein sample (at least 10 μg, the sample concentration adjusted to 1 mg/mL) is filtered through a 0.22 μm filter membrane and injected into the system, and the HPLC program is set: use pH 7.4 PBS buffer to flow the sample through the chromatography at a flow rate of 1.0 mL/min Column, the maximum time is 20 minutes; the detection wavelength is 280nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample.
1.5利用HPLC-HIC分析蛋白纯度和疏水性1.5 Analysis of protein purity and hydrophobicity by HPLC-HIC
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×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，pH 6.0)至100％流动相B(20mM组氨酸，pH 6.0)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。The purity and hydrophobicity of the antibody protein samples obtained above were analyzed using analytical hydrophobic interaction chromatography (HIC). The analytical chromatographic column TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6mm × 3.5cm) was connected to a high pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), equilibrated with PBS buffer at room temperature At least 1 hour. The set method consists of a linear gradient from 100% mobile phase A (20mM histidine, 1.8M ammonium sulfate, pH 6.0) to 100% mobile phase B (20mM histidine, pH 6.0) within 16 minutes, with the flow rate set at 0.7mL/min, protein sample concentration 1mg/mL, injection volume 20μL, detection wavelength 280nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample.
1.6利用DSF测定蛋白分子的热稳定性1.6 Using DSF to measure the thermal stability of protein molecules
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。其使用实时荧光定量PCR仪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的热变性温度(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(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。Differential Scanning Fluorimetry (DSF) is a commonly used high-throughput method for measuring protein thermal stability. It uses a real-time fluorescent quantitative PCR instrument to reflect the denaturation process of the protein by monitoring the change of the fluorescence intensity of the dye combined with the unfolded protein molecule, thereby reflecting the thermal stability of the protein molecule. In this example, the DSF method is used to determine the thermal denaturation temperature (Tm) of protein molecules. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700/W), followed by 2 μL of 100× diluted dye SYPROTM (Invitrogen, #2008138), followed by buffer to a final volume of 40 μL per well. Seal the PCR plate and place it in a real-time fluorescent quantitative PCR instrument (Bio-Rad, model CFX96 PCR System), incubate at 25°C for 5 minutes, and then gradually increase the temperature from 25°C to 95°C with a gradient of 0.2°C/0.2 minutes. At the end of the test the temperature was lowered to 25°C. Data analysis was performed using FRET scan mode and Bio-Rad CFX Maestro software was used to calculate the Tm of the samples.
对OX40抗体PR002067和PR002063的理化性质的检测结果如表6所示。Table 6 shows the detection results of the physicochemical properties of the OX40 antibodies PR002067 and PR002063.
表6.OX40抗体的理化性质Table 6. Physicochemical properties of OX40 antibody
Figure PCTCN2022098838-appb-000008
Figure PCTCN2022098838-appb-000008
1.7 ELISA检测OX40的HCAb单抗蛋白水平的结合能力1.7 ELISA detection of binding ability of OX40 HCAb monoclonal antibody protein level
本实施例是为了研究制得的抗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℃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℃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℃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。 This example is to study the activity of the prepared anti-OX40 HCAb monobody in vitro binding to human and cynomolgus monkey OX40 proteins. Human OX40 protein (Acro biosystem, #OX0-H5224) and cynomolgus monkey OX40 protein (Novoprotein, #CB17) were used to conduct antibody binding experiments at the protein level. Briefly, each well of a 384-well plate (PerkinElmer, #6007509) was coated with 20 μL of 1 ug/mL human OX40 protein and cynomolgus monkey OX40 protein dissolved in PBS, overnight at 4°C. The next day, wash the 384-well plate three times with PBS containing 0.05% Tween (MEDICAGO, #09-9410-100), and block with PBS containing 2% milk (Bio-Rad, #170-6404) at 37°C for 1 hour . The initial concentration of the OX40 antibody to be tested and the positive antibody (Pogalizumab) was 10 nM, and a 4-fold serial dilution was made. The blocked 384-well plate was washed three times with PBST, 10 μL of PBS or 10 μL of 4-fold serially diluted antibody and positive control (Pogalizumab) were added to the plate, and incubated at room temperature for 1 hour. After washing three times, 20 μL of goat anti-human Fc horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc., #109-035-098) was added to each well, and incubated at 37° C. for 40 minutes. Wash three times, add 20 μL TMB (Sera Care, #5120-0077) to each well, and incubate at room temperature for 5-15 minutes. 20 μL of stop solution (BBI life sciences, #E661006-0200) was added to each well, and the OD 450-650 value was read using a plate reader (Molecular Devices, model SpectraMax Plus). The value was analyzed and graphed with Graphad 8.0.
本实施例的OX40 PR002067抗体和PR002063抗体能结合人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(泊加珠单抗，罗氏)相比，PR002067、PR002063与人OX40蛋白和食蟹猴OX40蛋白结合的EC50与Tab(Pogalizumab)相当，说明该抗体能以较低的浓度更灵敏地结合人OX40。The OX40 PR002067 antibody and PR002063 antibody of this example can bind to human OX40 and cynomolgus monkey (cyno) OX40 protein, and the detected antibody binding ability is positively correlated with the concentration of the antibody. Compared with the reference antibody Tab (Pogalizumab (Pogalizumab, Roche), the EC50 of PR002067 and PR002063 binding to human OX40 protein and cynomolgus monkey OX40 protein is equivalent to that of Tab (Pogalizumab), indicating that the antibody can be more effective at a lower concentration. Sensitively binds human OX40.
OX40抗体与人OX40蛋白的结合活性见表7。The binding activity of OX40 antibody to human OX40 protein is shown in Table 7.
表7.OX40抗体与人OX40蛋白的结合活性Table 7. Binding activity of OX40 antibody to human OX40 protein
Figure PCTCN2022098838-appb-000009
Figure PCTCN2022098838-appb-000009
Figure PCTCN2022098838-appb-000010
Figure PCTCN2022098838-appb-000010
OX40抗体与食蟹猴OX40蛋白的结合活性见表8。The binding activity of OX40 antibody to cynomolgus monkey OX40 protein is shown in Table 8.
表8.OX40抗体与食蟹猴OX40蛋白的结合活性Table 8. Binding activity of OX40 antibody to cynomolgus monkey OX40 protein
抗体Antibody EC50(pM)EC50(pM) OD最大值ODmax
PR002067PR002067 232232 2.1632.163
PR002063PR002063 275.8275.8 2.3182.318
PogalizumabPogalizumab 194.3194.3 2.2262.226
实施例2抗PD-1H2L2抗体的制备Example 2 Preparation of anti-PD-1H2L2 antibody
为制备针对PD-1特异性结合的抗体分子，通常可以利用PD-1抗原对实验动物进行免疫，该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常，其得到的抗体分子是非人源的。在获得非人源抗体后，需要对这些分子利用抗体工程技术进行人源化改造，以降低免疫原性并提高成药性。然而，抗体的人源化过程有其技术复杂性，经过人源化改造的分子往往会降低对抗原的亲和力。另一方面，转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。In order to prepare antibody molecules specifically binding to PD-1, experimental animals can usually be immunized with PD-1 antigen, and the experimental animals can be mice, rats, rabbits, sheep, camels, etc. Typically, the resulting antibody molecules are non-human. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability. However, the humanization process of antibodies has its own technical complexity, and molecules after humanization often reduce the affinity for antigens. On the other hand, advances in transgenic technology have made it possible to create genetically engineered mice that carry the human immunoglobulin immune repertoire and have their endogenous murine immune repertoire deleted. The antibody produced by this transgenic mouse has a fully human sequence, so there is no need for further humanization, which greatly improves the efficiency of therapeutic antibody development.
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人抗体可变结构域和大鼠恒定结构域。Harbor H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune repertoire, which produces antibodies with complete human antibody variable domains and rat constant domains.
2.1 PD-1免疫H2L2小鼠2.1 PD-1 immune H2L2 mice
用可溶的重组人PD-1-hFc融合蛋白(Shanghai ChemPartner)对Harbour H2L2小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100μL。在首轮免疫中，每只小鼠接受用50μg抗原蛋白(人PD-1-hFc)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的免疫。在随后的每轮增强免疫中，每只小鼠接受用25μg抗原蛋白与Ribi佐剂(Sigma Adjuvant System,#S6322)混合配制的免疫原试剂的免疫。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天、第14天、第28天、第42天、第56天、第70天；并且在第49天、第77天，检测小鼠血清抗体滴度。在进行细胞融合前3天，以每只小鼠25μg抗原蛋白的剂量进行最后一次增强免疫。Harbor H2L2 mice were immunized for multiple rounds with soluble recombinant human PD-1-hFc fusion protein (Shanghai ChemPartner). The antigenic protein is mixed with an immune adjuvant to form an immunogenic reagent, which is then injected subcutaneously through the groin or intraperitoneally. In each round of immunization, each mouse received a total injection dose of 100 μL. In the first round of immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 50 μg of antigenic protein (human PD-1-hFc) with complete Freund’s adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of booster immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 25 μg of antigenic protein with Ribi adjuvant (Sigma Adjuvant System, #S6322). The interval between each booster round is at least two weeks and usually no more than five booster rounds. The immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected. Three days before cell fusion, the last booster immunization was performed with a dose of 25 μg antigenic protein per mouse.
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PD-1-His(Shanghai ChemPartner)的ELISA板进行ELISA检测来确定小鼠血液中抗人PD-1的滴度，并经流式细胞术检测2个浓度的小鼠血液(1:100、1:1000)对PD-1高表达的CHO-K1/hPD-1细胞(Shanghai ChemPartner)和CHO-K1母细胞的特异反应性。空白对照组(PB)为免疫前小鼠的血清。Collect mouse blood, dilute the blood 10 times, take 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), in the human PD-1-His (Shanghai ChemPartner ) ELISA plate for ELISA detection to determine the titer of anti-human PD-1 in mouse blood, and the anti-PD-1 titer of two concentrations of mouse blood (1:100, 1:1000) was detected by flow cytometry Specific reactivity of expressed CHO-K1/hPD-1 cells (Shanghai ChemPartner) and CHO-K1 blasts. The blank control group (PB) was the serum of mice before immunization.
2.2获得杂交瘤单克隆和抗体序列2.2 Obtaining hybridoma monoclonal and antibody sequences
当检测H2L2小鼠血清中PD-1特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，分离出至少8个表达抗-PD-1的单克隆抗体分子的杂交瘤。分离的杂交瘤细胞及其表达的单克隆抗体都使用对应的克隆号来表示，例如：4004_10H9A12，4004_12H9C1等等。分离的杂交瘤表达具有完整的人可变结构域和大鼠恒定结构域的重链和轻链的抗体分子。对上述单克隆抗体进行进一步的鉴定，根据其对人PD-1的结合能力、食蟹猴PD-1的结合能力、抑制PD-1与PD-L1结合能力等参数，选出若干个杂交瘤克隆进行测序。利用常规的杂交瘤测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中，从免疫的Harbour H2L2小鼠得到的抗PD-1单克隆抗体分子可变结构域的序列是人源抗体序列。在本申请中，所述CDR序列通过Chothia定义规则划分。When the PD-1-specific antibody titer in the H2L2 mouse serum reaches a certain level, the spleen cells of the mouse are taken out and fused with myeloma cell lines to obtain hybridoma cells; the hybridoma cells are screened and cloned through multiple rounds Afterwards, at least 8 hybridomas expressing anti-PD-1 monoclonal antibody molecules were isolated. The isolated hybridoma cells and their expressed monoclonal antibodies are indicated by the corresponding clone numbers, for example: 4004_10H9A12, 4004_12H9C1 and so on. The isolated hybridomas express antibody molecules with intact human variable domains and heavy and light chains of rat constant domains. The above-mentioned monoclonal antibodies were further identified, and several hybridomas were selected according to their binding ability to human PD-1, binding ability to cynomolgus monkey PD-1, and ability to inhibit the binding of PD-1 and PD-L1. Clones were sequenced. The nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence were obtained by conventional hybridoma sequencing means. In this example, the sequences of the variable domains of anti-PD-1 monoclonal antibody molecules obtained from immunized Harbor H2L2 mice are human antibody sequences. In the present application, the CDR sequences are divided by Chothia definition rules.
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。The heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic high-frequency mutation of the germline gene V, D, and J gene fragments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline gene V and J gene fragments of the light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is high. Using some algorithms, such as IMGT/DomainGapAlign (http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) or NCBI/IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/) can From the sequence of the variable domain of the antibody, the possible germline gene fragments in the event of gene rearrangement were deduced.
上述获得的抗体的胚系基因分析如表9所示，抗体序列编号如表10所示。The germline gene analysis of the antibodies obtained above is shown in Table 9, and the sequence numbers of the antibodies are shown in Table 10.
同时，本申请的抗PD-1的阳性对照抗体Pembrolizumab(Keytruda)类似物(PR000150)，其相应的氨基酸序列来源于IMGT数据库，抗体重链序列如SEQ ID NO:69所示，抗体轻链序列如SEQ ID NO:75所示。At the same time, the positive control antibody Pembrolizumab (Keytruda) analog (PR000150) of the anti-PD-1 of the present application, its corresponding amino acid sequence is derived from the IMGT database, the antibody heavy chain sequence is shown in SEQ ID NO: 69, and the antibody light chain sequence As shown in SEQ ID NO:75.
表9.抗PD-1H2L2序列胚系基因分析Table 9. Anti-PD-1H2L2 sequence germline gene analysis
Figure PCTCN2022098838-appb-000011
Figure PCTCN2022098838-appb-000011
表10.抗PD-1H2L2抗体的序列编号(SEQ ID NO:)Table 10. Sequence numbering (SEQ ID NO:) of anti-PD-1H2L2 antibody
Figure PCTCN2022098838-appb-000012
Figure PCTCN2022098838-appb-000012
2.3重组全人源抗体的制备2.3 Preparation of recombinant fully human antibody
上述实施例2.1和2.2中得到编码抗体分子的轻链、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻链、重链可变结构域序列和相应的人的抗体轻链、重链恒定结构域序列进行融合表达，得到重组抗体分子。After obtaining the light chain and heavy chain variable domain sequences encoding antibody molecules in the above-mentioned Examples 2.1 and 2.2, conventional recombinant DNA technology can be used to combine the light chain and heavy chain variable domain sequences with the corresponding human antibody light Chain and heavy chain constant domain sequences are fused and expressed to obtain recombinant antibody molecules.
在本实施例中Harbour H2L2小鼠得到的抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体κ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG4抗体重链恒定结构域序列(SEQ ID NO:143)的哺乳动物细胞表达质粒载体中，以编码产生IgG4抗体的全长重链。In this example, the antibody light chain variable domain sequence (VL) obtained from Harbor H2L2 mice was synthesized by gene and cloned into a mammalian cell expression plasmid vector encoding the human antibody kappa light chain constant domain sequence to encode the The full-length light chain of the antibody. The antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG4 antibody heavy chain constant domain sequence (SEQ ID NO: 143) to encode the full IgG4 antibody production long heavy chain.
将编码Harbour H2L2抗体重链的质粒和编码抗体轻链的质粒同时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，可以得到具有轻重链正确配对组装的纯化的重组抗体。The plasmid encoding the heavy chain of the Harbor H2L2 antibody and the plasmid encoding the light chain of the antibody are simultaneously transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and using conventional recombinant protein expression and purification techniques, the correct paired assembly of the light and heavy chains can be obtained. Purified recombinant antibodies.
具体说来，将HEK293细胞在FreeStyle TMF17 Expression Medium培养基(Thermo，#A1383504)扩培。瞬时转染开始之前，调节细胞浓度至(6～8)×10 5细胞/mL，于37℃ 8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/mL。准备30mL培养的细胞。将上述编码H2L2抗体的重链质粒和轻链质粒以2:3的比例混合溶解于1.5mL Opti-MEM减血清培养基(Thermo，#31985088)，并用0.22μm滤膜过滤除菌。再取1.5mL Opti-MEM溶入1mg/mL PEI(Polysciences 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，#71-5020-91AE)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，然后用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，得到纯化的抗体溶液。然后用NanoDrop(Thermo Scientific TMNanoDrop TMOne)测定浓度，分装、存储备用。 Specifically, HEK293 cells were expanded in FreeStyle ™ F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to (6-8)×10 5 cells/mL, and cultured in a 8% CO 2 shaker at 37°C for 24 hours, and the cell concentration was 1.2×10 6 cells/mL. Prepare 30 mL of cultured cells. The above-mentioned heavy chain plasmid and light chain plasmid encoding the H2L2 antibody were mixed and dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088) at a ratio of 2:3, and sterilized by filtration with a 0.22 μm filter membrane. Then take 1.5mL Opti-MEM and dissolve it into 120μL of 1mg/mL PEI (Polysciences Inc, #23966-2) and let stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop into the plasmid PEI mixed solution while shaking the culture bottle, and incubate for 5 days in a shaker at 37°C with 8% CO 2 . After 5 days, the cell viability was observed. The culture was collected, centrifuged at 3300 g 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 MabSelect ™ (GE Healthcare Life Science, #71-5020-91AE) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. Then use 0.1M glycine at pH 3.5 to elute the target protein, then adjust to neutrality with Tris-HCl at pH 8.0, and finally use an ultrafiltration tube (Millipore, #UFC901024) to concentrate and replace the medium to PBS buffer to obtain purified antibody solution. Then use NanoDrop (Thermo Scientific TM NanoDrop TM One) to measure the concentration, aliquot and store for future use.
2.4利用HPLC-SEC分析蛋白纯度和多聚体形式2.4 Analysis of protein purity and multimeric form by HPLC-SEC
使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience,#08541,5μm,7.8mm 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PBS(pH 7.4)缓冲液将样品以1.0mL/min的流速流过色谱柱，最长时间为25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。PD-1抗体的产量和纯度分析结果见表11。Protein samples were analyzed for purity and aggregated form using analytical size exclusion chromatography (SEC). Connect the analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5μm, 7.8mm x 30cm) to a high-pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), equilibrate with PBS buffer at room temperature At least 1 hour. An appropriate amount of protein sample (at least 10 μg, the sample concentration adjusted to 1 mg/mL) was filtered with a 0.22 μm filter membrane and injected into the system, and the HPLC program was set: the sample was flowed at a flow rate of 1.0 mL/min with PBS (pH 7.4) buffer Through the chromatographic column, the maximum time is 25 minutes; the detection wavelength is 280nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample. The yield and purity analysis results of PD-1 antibody are shown in Table 11.
表11.PD-1抗体的产量和纯度分析Table 11. Yield and purity analysis of PD-1 antibody
重组抗体recombinant antibody 分子类型molecular type 表达体系和体积Expression system and volume 第一步纯化后产量Yield after the first step of purification HPLC-SEC纯HPLC-SEC pure
 the  the  the (mg/L)(mg/L) 度(％)Spend(%)
PR000674PR000674 H2L2H2L2 HEK293-F(30mL)HEK293-F (30mL) 119.0119.0 99.5399.53
2.5改变H2L2抗体Fc序列2.5 Changing the Fc sequence of H2L2 antibody
对PR000674的Fc进行改造，将抗体类型由IgG4转换成IgG1并引入LALA(L234A和L235A突变,根据EU索引编号)突变，获得了PR001985，表10中列出了PR001985抗体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG1)全长氨基酸序列，和根据Chothia定义规则定义的CDR的氨基酸序列。Transform the Fc of PR000674, switch the antibody type from IgG4 to IgG1 and introduce LALA (L234A and L235A mutations, according to EU index numbering) mutations to obtain PR001985, and the variable light and heavy chains of the PR001985 antibody are listed in Table 10 Domain amino acid sequence, light chain full-length amino acid sequence, heavy chain (human IgG1) full-length amino acid sequence, and amino acid sequences of CDRs defined according to Chothia definition rules.
2.6抗体的序列优化2.6 Sequence optimization of antibodies
本实施例中对PR001985抗体的潜在PTM位点进行氨基酸突变得到新的抗体分子PR006429(称为PTM removal变体)。表10中列出了PR006429抗体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG1)全长氨基酸序列，和根据Chothia定义规则定义的CDR的氨基酸序列。所有设计出来的PTM-removal变体PR006429按照实施例2.3中描述的方法得到纯化的重组抗体，并在后续的功能实验中进一步验证。In this example, the potential PTM site of the PR001985 antibody was mutated to obtain a new antibody molecule PR006429 (called the PTM removal variant). Table 10 lists the light and heavy chain variable domain amino acid sequences of the PR006429 antibody, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG1), and the amino acid sequences of CDRs defined according to Chothia's definition rules. All designed PTM-removal variants PR006429 were purified recombinant antibodies according to the method described in Example 2.3, and further verified in subsequent functional experiments.
2.7抗原结合蛋白与过表达人/食蟹猴PD-1的细胞的结合(FACS)2.7 Binding of antigen-binding proteins to cells overexpressing human/cynomolgus PD-1 (FACS)
为了研究PD-1抗原结合蛋白体外结合人/食蟹猴PD-1的活性。采用过表达人或食蟹猴PD-1的CHO-K1细胞株(CHO-K1-hPD-1，CHO-K1-cPD-1，来源于GenScript)进行细胞水平的结合实验。简言之，消化CHO-K1-hPD-1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,Cat#3894)，随后加入100μL/孔，2倍于终浓度的5倍浓度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或300nM，共8至11个浓度，hIgG作为对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(山羊抗人IgG(H+L)第二抗体(Alexa
Figure PCTCN2022098838-appb-000013
488conjugate，Invitrogen，Cat#A11013,1:1000))，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，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值。 In order to study the activity of PD-1 antigen binding protein binding to human/cynomolgus monkey PD-1 in vitro. Cell-level binding experiments were performed using CHO-K1 cell lines (CHO-K1-hPD-1, CHO-K1-cPD-1, derived from GenScript) overexpressing human or cynomolgus monkey PD-1. Briefly, digest CHO-K1-hPD-1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x 106 cells/mL. Inoculate 100 μL cells/well in 96-well V-bottom plate (Corning, Cat#3894), then add 100 μL/well, 5-fold dilutions of the antigen-binding protein to be tested, which is 2 times the final concentration, and mix well, in which the antigen-binding protein The highest final concentration was 100nM or 300nM, 8 to 11 concentrations in total, and hIgG was used as a control. Place the cells at 4°C and incubate for 1 hour in the dark. Then, add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well fluorescent secondary antibody (goat anti-human IgG (H+L) secondary antibody (Alexa
Figure PCTCN2022098838-appb-000013
488conjugate, Invitrogen, Cat#A11013, 1:1000)), incubated at 4°C for 30 minutes in the dark. Wash the cells twice with 200 μL/well of pre-cooled PBS, centrifuge at 500 g, 4 °C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well of pre-cooled PBS, and use BD FACS CANTOII to read the fluorescence signal value.
结果显示，本申请所述PD-1抗原结合蛋白PR000674能结合过表达人PD-1的CHO-K1细胞以及过表达食蟹猴PD-1的CHO-K1细胞。The results show that the PD-1 antigen binding protein PR000674 described in this application can bind to CHO-K1 cells overexpressing human PD-1 and CHO-K1 cells overexpressing cynomolgus monkey PD-1.
PD-1抗原结合蛋白与过表达人PD-1的CHO-K1细胞的结合活性见表12。The binding activity of PD-1 antigen binding protein to CHO-K1 cells overexpressing human PD-1 is shown in Table 12.
表12.PD-1抗原结合蛋白与过表达人PD-1的CHO-K1细胞的结合活性Table 12. Binding activity of PD-1 antigen binding proteins to CHO-K1 cells overexpressing human PD-1
抗体Antibody EC50(nM)EC50(nM) MFI最大值MFImaximum
PR000674PR000674 1.9391.939 23470182347018
PembrolizumabPembrolizumab 1.6341.634 23821662382166
PD-1抗原结合蛋白与过表达食蟹猴PD-1的CHO-K1细胞的结合活性见表13。The binding activity of PD-1 antigen binding protein to CHO-K1 cells overexpressing cynomolgus monkey PD-1 is shown in Table 13.
表13.PD-1抗原结合蛋白与过表达食蟹猴PD-1的CHO-K1细胞的结合活性Table 13. Binding activity of PD-1 antigen binding proteins to CHO-K1 cells overexpressing cynomolgus monkey PD-1
抗体Antibody EC50(nM)EC50(nM) MFI最大值MFImaximum
PR000674PR000674 2.3492.349 4116241162
PembrolizumabPembrolizumab 0.9980.998 1909119091
2.8抗原结合蛋白阻断人PD-L1与过表达人PD-1的CHO-K1细胞的结合2.8 Antigen-binding proteins block the binding of human PD-L1 to CHO-K1 cells overexpressing human PD-1
为了研究人PD-1结合蛋白体外阻断人PD-1与人PD-L1结合的活性，采用过表达人PD-1的CHO-K1细胞株(CHO-K1-hPD-1)进行细胞水平的人PD-1/人PD-L1结合阻断实验。简言之，消化CHO-K1-hPD-1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,Cat#3894)，随后加入100μL/孔，2倍于终浓度的3倍或5倍浓度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或300nM，共8个浓度，hIgG作为对照。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，随后加入50μL/孔，1μg/mL浓度的生物素标记的人PD-L1蛋白(AcroBiosystems,PD1-H82F2),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。加入100μL/孔荧光二抗(PE Streptavidin,BD,Cat#554061，1:200)，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，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值，计算IC50。 In order to study the activity of human PD-1 binding protein in blocking the binding of human PD-1 and human PD-L1 in vitro, the CHO-K1 cell line (CHO-K1-hPD-1) overexpressing human PD-1 was used to carry out the cell level detection. Human PD-1/human PD-L1 binding blocking experiment. Briefly, digest CHO-K1-hPD-1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x 106 cells/mL. Inoculate 100 μL cells/well in 96-well V-bottom plate (Corning, Cat#3894), then add 100 μL/well, 2 times the final concentration of 3 times or 5 times the concentration of the antigen-binding protein to be tested, and mix evenly. The highest final concentration of antigen-binding protein was 100nM or 300nM, a total of 8 concentrations, and hIgG was used as a control. Place the cells at 4°C and incubate for 1 hour in the dark. Afterwards, centrifuge at 4°C for 5 minutes, discard the supernatant, then add 50 μL/well of biotin-labeled human PD-L1 protein (AcroBiosystems, PD1-H82F2) at a concentration of 1 μg/mL, and incubate at 4°C for 30 minutes in the dark. Add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL/well fluorescent secondary antibody (PE Streptavidin, BD, Cat#554061, 1:200), and incubate at 4°C for 30 minutes in the dark. Wash the cells twice with 200 μL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well pre-cooled PBS, use BD FACS CANTOII to read the fluorescence signal value, and calculate IC50.
结果如图6和表14所示。图6和表14显示抗体PR001985、PR006429均能阻断人PD-L1与细胞表面的人PD-1结合，且抑制能力与阳性对照Keytruda(pembrolizumab)相当。The results are shown in Figure 6 and Table 14. Figure 6 and Table 14 show that both antibodies PR001985 and PR006429 can block the binding of human PD-L1 to human PD-1 on the cell surface, and the inhibitory ability is equivalent to that of the positive control Keytruda (pembrolizumab).
表14PD-1抗原结合蛋白阻断PD-1与PD-L1结合Table 14 PD-1 antigen-binding proteins block the combination of PD-1 and PD-L1
AbsAbs KeytrudaKeytruda PR001985PR001985 PR006429PR006429
最大值RULMaximum RUL 48314831 47854785 49444944
EC50EC50 2.2672.267 2.042.04 2.2162.216
实施例3抗PD-1 H2L2×OX40双特异性抗体的制备Example 3 Preparation of anti-PD-1 H2L2×OX40 bispecific antibody
将实施例1制备的抗OX40抗体和实施例2制备的抗PD-1抗体组合用于制备双特异性抗体，可以同时结合两个靶点，其中一端(第一结构域)可以识别肿瘤细胞表面特异表达的PD-1，而另一端(第二结构域)可以结合T细胞上的OX40分子。当PD-1×OX40双抗分子结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，从而杀死肿瘤细胞。Combining the anti-OX40 antibody prepared in Example 1 and the anti-PD-1 antibody prepared in Example 2 is used to prepare a bispecific antibody, which can bind to two targets at the same time, and one end (the first domain) can recognize the surface of tumor cells Specifically expressed PD-1, while the other end (second domain) can bind to OX40 molecules on T cells. When the PD-1×OX40 double antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
本实施例制备的PD-1×OX40双特异性抗体为IgG1，PR003787具有Fc L234A和L235A和P329G突变(根据EU索引编号)，R200538、PR200539、PR200531、PR200536、PR200528以及PR200600具有Fc L234A,L235A和G237A)，分子结构参见图1-5。The PD-1×OX40 bispecific antibody prepared in this example is IgG1, PR003787 has Fc L234A, L235A and P329G mutations (according to EU index numbering), R200538, PR200539, PR200531, PR200536, PR200528 and PR200600 have Fc L234A, L235A and G237A), the molecular structure is shown in Figure 1-5.
3.1构建IgG_HC-VH四价对称结构的双特异性抗体(结构1)3.1 Construction of a bispecific antibody with IgG_HC-VH tetravalent symmetrical structure (structure 1)
利用抗PD-1的H2L2抗体和抗OX40的HCAb抗体构建IgG_HC-VH四价对称结构的双特异性抗体PR003787、PR200531、PR200528。IgG_HC-VH四价对称结构(参见图1所示)的结合蛋白包含两条多肽链：多肽链2，也称短链，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，也称长链，从氨基末端到 羧基末端，其包含N’-VH 1-CH 1-h-CH 2-CH 3-L-VH 2-C’。其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为IgG抗体的铰链区，所述CL 1是第一结构域的CL，所述CH 1是第一结构域的CH1，所述L为连接肽，多肽链1的CH3经由L连接到VH 2。双特异性抗体PR003787中的连接肽L为H1，其氨基酸序列如表15中的SEQ ID NO:116所示，双特异性抗体PR200531和PR200528中的连接肽L为(G2S) 2，其序列如表15中的SEQ ID NO:117所示。 Bispecific antibodies PR003787, PR200531 and PR200528 with IgG_HC-VH tetravalent symmetrical structure were constructed by using anti-PD-1 H2L2 antibody and anti-OX40 HCAb antibody. The binding protein of IgG_HC-VH tetravalent symmetrical structure (see Figure 1) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 -CL 1 - C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L-VH 2 -C'. Wherein, said VL 1 and VH 1 are respectively the VL and VH of the first structural domain, said VH 2 is the VH of the second structural domain, said h is the hinge region of an IgG antibody, and said CL 1 is the first structural domain The CL of the domain, the CH 1 is the CH1 of the first domain, the L is the connecting peptide, and the CH3 of the polypeptide chain 1 is connected to the VH 2 via L. The connecting peptide L in the bispecific antibody PR003787 is H1, and its amino acid sequence is shown in SEQ ID NO: 116 in Table 15. The connecting peptide L in the bispecific antibodies PR200531 and PR200528 is (G2S) 2 , and its sequence is shown in Shown in SEQ ID NO: 117 in Table 15.
3.2构建VH-IgG_HC四价对称结构的双特异性抗体(结构2)3.2 Construction of bispecific antibody with VH-IgG_HC tetravalent symmetrical structure (structure 2)
利用抗PD-1的H2L2抗体PR006429和抗OX40的HCAb抗体PR002607构建VH-IgG_HC四价对称结构的双特异性抗体PR200538。VH-IgG_HC四价对称结构(参见图2所示)的结合蛋白包含两条多肽链：多肽链2，也称短链，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，也称长链，从氨基末端到羧基末端，其包含N’-VH 2-L-VH 1-CH 1-h-CH 2-CH 3-C’。。其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述CL 1是第一结构域的CL，所述CH 1是第一结构域的CH1，多肽链1的VH 2经由连接肽L连接到VH1，所述L为连接肽(G4S) 3，其序列如表15中的SEQ ID NO:118所示 The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200538 with VH-IgG_HC tetravalent symmetrical structure. The binding protein of the VH-IgG_HC tetravalent symmetrical structure (see Figure 2) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 -CL 1 - C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VH 2 -L-VH 1 -CH 1 -h-CH 2 -CH 3 -C'. . Wherein, said VL 1 and VH 1 are respectively VL and VH of the first structural domain, said VH 2 is the VH of the second structural domain, said h is the hinge region, and said CL 1 is the CL of the first structural domain , the CH 1 is the CH1 of the first structural domain, and the VH 2 of the polypeptide chain 1 is connected to VH1 via the linker peptide L, and the L is the linker peptide (G4S) 3 , its sequence is as shown in SEQ ID NO: 118 in Table 15 shown
3.3构建Fab(CL)-VH-Fc四价对称结构的双特异性抗体(结构3)3.3 Construction of bispecific antibody with Fab(CL)-VH-Fc tetravalent symmetrical structure (structure 3)
利用抗PD-1的H2L2抗体PR006429和抗OX40的HCAb抗体PR002607构建Fab(CL)-VH-Fc四价对称结构的双特异性抗体PR200539。Fab(CL)-VH-Fc四价对称结构(参见图3所示)的结合蛋白包含两条多肽链：多肽链2，也称短链，从氨基末端到羧基末端，其包含N’-VH’-CH1’-C’；多肽链1，也称长链，从氨基末端到羧基末端，其包含N’-VL’-CL’-L-VH 2-h-CH2-CH3-C’。其中，所述VL’和VH’分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L为连接肽，所述CL’是第一结构域的CL，所述CH1’是第一结构域的CH1，PR200539中多肽链1的CL’与VH2直接融合连接，即L的长度为0。 The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200539 with a tetravalent symmetrical structure of Fab(CL)-VH-Fc. The binding protein of the Fab(CL)-VH-Fc tetravalent symmetrical structure (see Figure 3) contains two polypeptide chains: Polypeptide chain 2, also known as the short chain, from the amino terminal to the carboxyl terminal, which contains N'-VH '-CH1'-C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VL'-CL'-L-VH2 - h-CH2-CH3-C'. Wherein, said VL' and VH' are VL and VH of the first structural domain respectively, said VH2 is VH of the second structural domain, said h is a hinge region, said L is a connecting peptide, and said CL ' is the CL of the first structural domain, the CH1' is the CH1 of the first structural domain, and the CL' of the polypeptide chain 1 in PR200539 is directly fused with VH2, that is, the length of L is 0.
3.4构建IgG_HC-VH-VH六价对称结构的双特异性抗体(结构4)3.4 Construction of bispecific antibody with IgG_HC-VH-VH hexavalent symmetrical structure (structure 4)
利用抗PD-1的H2L2抗体PR006429和抗OX40的HCAb抗体PR002607构建IgG_HC-VH-VH六价对称结构的双特异性抗体PR200536。IgG_HC-VH-VH六价对称结构(参见图4所示)的结合蛋白包含两条多肽链：多肽链2，也称短链，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，也称长链，从氨基末端到羧基末端，其包含N’-VH1-CH 1-h-CH2-CH3-L1-VH 2-L2-VH 2-C’。其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L1和L2为连接肽，所述CL1是第一结构域的CL，所述CH 1是第一结构域的CH1。多肽链1的CH3经由L1连接到VH 2，两个VH 2之间通过L2连接，L1和L2序列分别如表15中的SEQ ID NO:117、118所示。 The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200536 with IgG_HC-VH-VH hexavalent symmetrical structure. The binding protein of IgG_HC-VH-VH hexavalent symmetrical structure (see Figure 4) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from amino terminal to carboxyl terminal, which contains N'-VL 1 -CL 1 -C'; Polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, comprising N'-VH1- CH1 -h- CH2 -CH3-L1-VH2-L2-VH2 - C'. Wherein, said VL 1 and VH 1 are respectively VL and VH of the first structural domain, said VH 2 is VH of the second structural domain, said h is a hinge region, and said L1 and L2 are connecting peptides, so The CL1 is the CL of the first domain, and the CH1 is the CH1 of the first domain. CH3 of polypeptide chain 1 is connected to VH 2 through L1, and the two VH 2 are connected through L2. The sequences of L1 and L2 are shown in SEQ ID NO: 117 and 118 in Table 15, respectively.
3.5构建IgG_HC-VH-VH-VH八价对称结构的双特异性抗体(结构5)3.5 Construction of bispecific antibody with IgG_HC-VH-VH-VH octavalent symmetrical structure (structure 5)
利用抗PD-1的H2L2抗体PR006429和抗OX40的HCAb抗体PR002603构建IgG_HC-VH-VH-VH八价对称结构的双特异性抗体PR200600。IgG_HC-VH-VH-VH八价对称结构(参见图5所示)的结合蛋白包含两条多肽链：多肽链2，也称短链，从氨基末端到羧基末端，其包含N’-VL 1-CL 1-C’；多肽链1，也称长链，从氨基末端到羧基末端，其包含N’-VH1-CH 1-h-CH 2-CH3-L1-VH 2-L 2-VH 2-L3-VH 2-C’。其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L1、L 2和L 3为连接肽，所述CL 1是第一结构域的CL，所述CH 1是第一结构域的CH 1。多肽链1中的CH3经由连接肽L1连接到第一个VH 2(从多肽链-1的N端至C端排序)，第一个VH 2与第二个VH 2通过连接肽L2连接，第二个VH 2与第三个VH 2通过连接肽L3连接，其中L1为(G2S)2，L2为(G4S)3，L3为(G4S) 2，L1、L2和L3的序列分别如表15中的SEQ117、118、119所示。 The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002603 were used to construct the bispecific antibody PR200600 with an octavalent symmetrical structure of IgG_HC-VH-VH-VH. The binding protein of the IgG_HC-VH-VH-VH octavalent symmetrical structure (see Figure 5) contains two polypeptide chains: polypeptide chain 2, also known as the short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL 1 -CL 1 -C'; Polypeptide chain 1, also known as long chain, from amino-terminus to carboxy-terminus, which contains N'-VH1-CH 1 -h-CH 2 -CH3-L1-VH 2 -L 2 -VH 2 -L3-VH 2 -C'. Wherein, said VL 1 and VH 1 are VL and VH of the first structural domain respectively, said VH 2 is VH of the second structural domain, said h is a hinge region, and said L1, L2 and L3 are A connecting peptide, the CL 1 is the CL of the first domain, the CH 1 is the CH 1 of the first domain. CH3 in polypeptide chain 1 is connected to the first VH 2 via connecting peptide L1 (ordered from the N-terminal to the C-terminal of polypeptide chain-1), the first VH 2 is connected to the second VH 2 through connecting peptide L2, and the first VH 2 is connected to the second VH 2 through the connecting peptide L2. The two VH 2 and the third VH 2 are linked by linker peptide L3, wherein L1 is (G2S)2, L2 is (G4S)3, L3 is (G4S) 2 , and the sequences of L1, L2 and L3 are shown in Table 15 Shown in SEQ117, 118, 119.
表15.连接肽序列表Table 15. List of connecting peptide sequences
Figure PCTCN2022098838-appb-000014
Figure PCTCN2022098838-appb-000014
Figure PCTCN2022098838-appb-000015
Figure PCTCN2022098838-appb-000015
本发明所得双抗分子的多肽链的氨基酸序列在序列表中的编号中如表16所示。The amino acid sequence of the polypeptide chain of the double antibody molecule obtained in the present invention is shown in Table 16 in the numbering in the sequence listing.
表16.本发明所得双抗分子的序列编号(SEQ ID NO:)Table 16. Sequence number (SEQ ID NO:) of the obtained double antibody molecule of the present invention
抗体编号Antibody number 多肽链1polypeptide chain 1 多肽链2 polypeptide chain 2
PR003787PR003787 7979 7777
PR200528PR200528 109109 7777
PR200531PR200531 110110 7777
PR200536PR200536 111111 7777
PR200538PR200538 112112 7777
PR200539PR200539 114114 113113
PR200600PR200600 115115 7777
PD-1×OX40双特异性抗体的抗原结构域的CDR序列编号如表17所示，表17中1#序号为结合PD-1的抗原结构域，2#序号为结合OX40的抗原结构域。The CDR sequence numbers of the antigenic domains of the PD-1×OX40 bispecific antibody are shown in Table 17. In Table 17, the 1# sequence number is the antigenic domain that binds to PD-1, and the 2# sequence number is the antigenic domain that binds to OX40.
表17.双抗的抗原结合结构域的CDR序列编号(SEQ ID NO:)Table 17. The CDR sequence number (SEQ ID NO:) of the antigen-binding domain of the double antibody
Figure PCTCN2022098838-appb-000016
Figure PCTCN2022098838-appb-000016
本发明的双特异性抗体分子结构信息见表18。The molecular structure information of the bispecific antibody of the present invention is shown in Table 18.
表18.具有IgG-VH四价对称结构的PD-1×OX40双抗分子Table 18. PD-1×OX40 double antibody molecule with IgG-VH tetravalent symmetrical structure
Figure PCTCN2022098838-appb-000017
Figure PCTCN2022098838-appb-000017
Figure PCTCN2022098838-appb-000018
Figure PCTCN2022098838-appb-000018
实施例4Example 4
FACS检测PD-1×OX40双特异性抗体在过表达人PD-1的CHO-K1细胞株上的体外结合FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on CHO-K1 cell line overexpressing human PD-1
本实施例为了研究PD-1×OX40双特异性抗体PD-1抗体臂体外结合人PD-1的活性。采用过表达人PD-1的CHO-K1细胞株(CHO-K1-hu PD-1,Shanghai ChemPartner)进行细胞水平上的抗体结合实验。简言之，消化CHO-K1-hu PD-1细胞，并用DMEM完全培养基重悬，将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入100μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,Cat#:109-545-06,1：500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD Accuri C6 Plus流式细胞仪读取荧光发光信号值。 This example is to study the in vitro binding activity of the PD-1 antibody arm of the PD-1×OX40 bispecific antibody to human PD-1. The CHO-K1 cell line (CHO-K1-hu PD-1, Shanghai ChemPartner) overexpressing human PD-1 was used for antibody binding experiments at the cellular level. Briefly, CHO-K1-hu PD-1 cells were digested and resuspended with DMEM complete medium, and the cell density was adjusted to 1× 106 cells/mL, respectively. 100 μL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 100 μL/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration. Place the cells at 4°C and incubate for 1 hour in the dark. Afterwards, add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL/well fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific, Jackson, Cat#:109-545-06, 1:500 dilution), and incubate at 4°C for 30 minutes in the dark. Wash the cells twice with 100 μL/well of pre-cooled PBS, centrifuge at 500 g, 4 °C for 5 minutes, and discard the supernatant. Finally, the cells were resuspended in 200 μL/well of pre-cooled PBS, and the fluorescence signal value was read using a BD Accuri C6 Plus flow cytometer.
图7示出PD-1×OX40双特异性抗体在过表达人PD-1的CHO-K1细胞株上的体外结合。Figure 7 shows the in vitro binding of the PD-1×OX40 bispecific antibody on the CHO-K1 cell line overexpressing human PD-1.
如图7A和7B所示，本实例中的PD-1×OX40双特异性抗体PR003787、PR200538、PR200539或其组成的单克隆抗体PR001985、PR006429均与过表达人PD-1的CHO-K1细胞特异性结合。且PR003787与人PD-1的结合能力与PD-1抗体和人PD-1结合能力一致。本实施中的PD-1×OX40双特异抗体PR003787中PD-1抗体臂具有和PD-1单克隆抗体同样的结合能力。As shown in Figures 7A and 7B, the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 or their monoclonal antibodies PR001985 and PR006429 in this example are all specific to CHO-K1 cells overexpressing human PD-1 sexual union. And the binding ability of PR003787 to human PD-1 is consistent with the binding ability of PD-1 antibody to human PD-1. The PD-1 antibody arm of the PD-1×OX40 bispecific antibody PR003787 in this implementation has the same binding ability as the PD-1 monoclonal antibody.
实施例5Example 5
FACS检测PD-1×OX40双特异性抗体在过表达OX40的HEK293细胞系上的体外结合FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on HEK293 cell line overexpressing OX40
本实施例为了研究PD-1×OX40双特异性抗体OX40臂结合人OX40的活性，采用高表达OX40的HEK293细胞系(OX40/NF-κB Reporter–HEK293，BPS BioScience,Cat#60482)进行细胞与人OX40结合实验。简言之，收集高表达OX40的HEK293细胞悬液，将细胞密度分别调整为2×10 6细胞/mL。以50μL细胞/孔接 种于96孔V底板(Corning,Cat#:3894)，随后加入50μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch,Cat#:109-605-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷FACS重悬细胞，使用BD Accuri C6 Plus Flowcytometer读取荧光发光信号值。 In this example, in order to study the activity of the OX40 arm of the PD-1×OX40 bispecific antibody binding to human OX40, the HEK293 cell line (OX40/NF-κB Reporter-HEK293, BPS BioScience, Cat#60482) with high expression of OX40 was used for cell and Human OX40 binding assay. In brief, HEK293 cell suspensions with high expression of OX40 were collected, and the cell density was adjusted to 2×10 6 cells/mL. 50 μL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 μL/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration. Place the cells at 4°C and incubate for 2 hours in the dark. Afterwards, add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific, Jackson ImmunoResearch, Cat#:109-605-098, diluted 1:1000), and incubate at 4°C for 1 hour in the dark . Wash the cells twice with 100 μL/well of pre-cooled PBS, centrifuge at 500 g, 4 °C for 5 minutes, and discard the supernatant. Finally, pre-cool FACS to resuspend the cells at 200 μL/well, and use the BD Accuri C6 Plus Flowcytometer to read the fluorescence signal value.
图8A和8B示出通过流式细胞术测定的在人HEK293/OX40/NF-kb报告细胞中，本发明的PD-1×OX40双特异抗体PR003787、PR200538、PR200539或组成其结构的单克隆抗体PR002067均与过表达人OX40的HEK293细胞特异性结合，且随着浓度增加对细胞表面OX40的结合。而且双抗PR003787、PR200538的OX40抗体臂具有和OX40单克隆抗体同样或更优的结合能力。8A and 8B show the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies constituting their structures in human HEK293/OX40/NF-kb reporter cells determined by flow cytometry PR002067 specifically binds to HEK293 cells overexpressing human OX40, and binds to OX40 on the cell surface as the concentration increases. Moreover, the OX40 antibody arms of the double-antibody PR003787 and PR200538 have the same or better binding ability as the OX40 monoclonal antibody.
实施例6Example 6
FACS检测PD-1×OX40双特异性抗体在过表达OX40和PD-1的HEK293细胞系上的体外结合FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on HEK293 cell line overexpressing OX40 and PD-1
本实施例为了研究PD-1×OX40双特异性抗体结合OX40和PD-1共表达细胞的活性。采用高表达OX40的HEK293细胞系(OX40/NF-κB Reporter–HEK293，BPS BioScience,Cat#60482)，进一步用慢病毒构建稳定表达人PD1细胞系(Origene,Cat#PS100092V)，经过puromycin抗性筛选后，得到稳定表达OX40和PD-1的HEK293细胞系，得到的细胞株用来进行细胞结合实验。简言之，收集高表达OX40和PD-1的HEK293细胞悬液，将细胞密度分别调整为2×10 6细胞/mL。以50μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入50μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch,Cat#:109-605-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷FACS重悬细胞，使用BD Accuri C6 Plus Flowcytometer读取荧光发光信号值。 This example is to study the activity of PD-1×OX40 bispecific antibody binding to OX40 and PD-1 co-expression cells. HEK293 cell line (OX40/NF-κB Reporter–HEK293, BPS BioScience, Cat#60482) with high expression of OX40 was used to further construct a cell line stably expressing human PD1 (Origene, Cat#PS100092V) with lentivirus, which was screened for resistance to puromycin Finally, HEK293 cell lines stably expressing OX40 and PD-1 were obtained, and the obtained cell lines were used for cell binding experiments. In brief, HEK293 cell suspensions highly expressing OX40 and PD-1 were collected, and the cell density was adjusted to 2×10 6 cells/mL, respectively. 50 μL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 μL/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration. Place the cells at 4°C and incubate for 2 hours in the dark. Afterwards, add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific, Jackson ImmunoResearch, Cat#:109-605-098, diluted 1:1000), and incubate at 4°C for 1 hour in the dark . Wash the cells twice with 100 μL/well of pre-cooled PBS, centrifuge at 500 g, 4 °C for 5 minutes, and discard the supernatant. Finally, pre-cool FACS to resuspend the cells at 200 μL/well, and use the BD Accuri C6 Plus Flowcytometer to read the fluorescence signal value.
图9示出通过流式细胞术测定的在人HEK293/OX40/PD1NF-kb报告细胞中，本发明的PD-1×OX40双特异抗体PR003787、PR200538、PR200539或组成其结构的单克隆抗体均与过表达人OX40和PD-1的HEK293细胞特异性结合，且随着浓度增加对细胞表面OX40和PD-1的结合。和OX40单克隆抗体相比，双抗PR003787、PR200538和PR200539显示更强的结合能力；和PD1单克隆抗体相比，双抗PR003787、PR200538和PR200539显示出类似或稍弱的结合能力。Figure 9 shows that in human HEK293/OX40/PD1NF-kb reporter cells determined by flow cytometry, the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies that make up their structures all interact with HEK293 cells overexpressing human OX40 and PD-1 specifically bind, and bind to cell surface OX40 and PD-1 as the concentration increases. Compared with the OX40 monoclonal antibody, the double-antibody PR003787, PR200538 and PR200539 showed stronger binding ability; compared with the PD1 monoclonal antibody, the double-antibody PR003787, PR200538 and PR200539 showed similar or slightly weaker binding ability.
实施例7Example 7
FACS检测PD-1×OX40双特异性抗体在激活T细胞上的体外结合FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on activated T cells
由于过表达细胞不能真正反映生理条件下靶点的表达情况，本实施例研究PD-1×OX40双特异性抗体结合激活的T细胞的活性。购买人浓缩白细胞(Research blood components LLC)，分离人的外周血单核细胞(PBMC)，用包被到细胞培养板上的抗CD3抗体刺激T细胞48小时。收集刺激后的PBMC，将细胞密度分别调整为4×10 6细胞/mL。以50μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入50μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Goat anti-Human IgG Fc Secondary Antibody,PE,eBioscience TM，Cat#:12-4998-82，1：250稀释)和APC标记的抗人CD3抗体(BioLegend,Cat#317318)，4℃，避光孵育1小时。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷FACS重悬细胞，使用BD BD LSRFortessa Flowcytometer读取荧光发光信号 值。 Since the overexpression cells cannot truly reflect the expression of the target under physiological conditions, this example studies the activity of PD-1×OX40 bispecific antibody-activated T cells. Human leukocyte concentrates (Research blood components LLC) were purchased, and human peripheral blood mononuclear cells (PBMC) were isolated, and T cells were stimulated for 48 hours with anti-CD3 antibody coated on a cell culture plate. The stimulated PBMCs were collected and the cell density was adjusted to 4×10 6 cells/mL. 50 μL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 μL/well, 5-fold dilutions of the antibody to be tested was added at 2 times the final concentration. Place the cells at 4°C and incubate for 2 hours in the dark. Afterwards, add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL/well fluorescent secondary antibody (Goat anti-Human IgG Fc Secondary Antibody, PE, eBioscience TM , Cat#:12-4998-82, 1:250 dilution) and APC-labeled anti-human CD3 antibody (BioLegend, Cat# 317318), incubated at 4°C for 1 hour in the dark. Wash the cells twice with 100 μL/well of pre-cooled PBS, centrifuge at 500 g, 4 °C for 5 minutes, and discard the supernatant. Finally, pre-cool FACS to resuspend the cells at 200 μL/well, and use the BD BD LSRFortessa Flowcytometer to read the fluorescence signal value.
图10示出通过流式细胞术测定的在激活的人T细胞中，本发明的PD-1×OX40双特异抗体PR003787、PR200538、PR200539或组成其结构的单克隆抗体均与激活的T细胞结合，且随着浓度增加对T细胞的结合。与OX40单克隆抗体和PD1单克隆抗体相比，双抗PR003787、PR200538和PR200539显示更强的结合能力。表明本发明的双抗能够特异性的结合PD1和OX40阳性的T细胞。Figure 10 shows that in the activated human T cells determined by flow cytometry, the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies that constitute their structures all bind to the activated T cells , and the binding to T cells increases with the concentration. Bi-antibodies PR003787, PR200538 and PR200539 showed stronger binding abilities compared with OX40 mAb and PD1 mAb. It shows that the double antibody of the present invention can specifically bind PD1 and OX40 positive T cells.
实施例8利用报告基因细胞系检测PD-1×OX40双特异性抗体对OX40信号通路的刺激作用Example 8 Using a reporter gene cell line to detect the stimulating effect of PD-1×OX40 bispecific antibody on OX40 signaling pathway
每孔铺100μL的培养液，或者1.5×10 4表达CD32b的CHO-K1细胞(CHO-K1/CD32b)(BPS BioScience,#79511)或表达人PD1的CHO细胞(CHO-K1/PD-1)到96孔板(Perkin Elmer，#6005225)，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，4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15分钟，酶标仪检测发光值。 Spread 100 μL of culture medium per well, or 1.5×10 4 CHO-K1 cells expressing CD32b (CHO-K1/CD32b) (BPS BioScience, #79511) or CHO cells expressing human PD1 (CHO-K1/PD-1) To a 96-well plate (Perkin Elmer, #6005225), add 40 μL of 2-fold dilution of the antigen-binding protein to be tested to each well, the initial concentration is 200 nM, and 4-fold serial dilution, hlgG1 is the negative control group. 40 μL of 4.5×10 4 HEK293 reporter cells (HEK293/OX40/NF-kb reporter cells, BPS Biosciences, #60482) continuously expressing luciferase reporter genes of OX40 and NF-kb response elements were added to each well. Incubate for 6 hours at 37°C in a 5% CO 2 incubator. Then add ONE-Glo TM luciferase reagent (Promega, #E6110), incubate at room temperature for 15 minutes, and detect the luminescence value with a microplate reader.
结果如图11至图13所示。图11至图13的结果显示本申请所述PD-1×OX40双特异性抗体PR003787是PD-1交联依赖型。图11结果显示PD-1×OX40双特异性抗体PR003787对HEK293/OX40/NF-kb报告细胞没有直接的激活作用。图12结果显示，OX40亲本单克隆抗体在CHO-K1/CD32b交联条件下显示出浓度依赖的增强NF-Kb信号通路的作用，而PD-1×OX40双特异性抗体PR003787未显示出NF-Kb信号激活作用。图13结果显示PD-1×OX40双特异性抗体PR003787在CHO-K1/PD-1细胞交联辅助作用下，特异性地引起浓度依赖的增强NF-Kb信号通路的作用。The results are shown in Figures 11 to 13. The results in Figure 11 to Figure 13 show that the PD-1×OX40 bispecific antibody PR003787 described in this application is PD-1 cross-linking dependent. The results in Figure 11 show that the PD-1×OX40 bispecific antibody PR003787 has no direct activation effect on HEK293/OX40/NF-kb reporter cells. The results in Figure 12 show that the OX40 parental monoclonal antibody showed a concentration-dependent effect of enhancing the NF-Kb signaling pathway under CHO-K1/CD32b cross-linking conditions, while the PD-1×OX40 bispecific antibody PR003787 did not show NF- Activation of Kb signaling. The results in Figure 13 show that the PD-1×OX40 bispecific antibody PR003787, with the help of CHO-K1/PD-1 cell cross-linking, specifically caused a concentration-dependent enhancement of the NF-Kb signaling pathway.
本申请所述PD-1×OX40双特异性抗体PR003787能够特异地引起PD-1交联依赖的OX40介导的NF-Kb信号通路的促进作用，而且其引起的信号强度与其浓度成正相关关系递增。The PD-1×OX40 bispecific antibody PR003787 described in this application can specifically induce the promotion of PD-1 cross-linking-dependent OX40-mediated NF-Kb signaling pathway, and the signal intensity it causes increases in a positive correlation with its concentration .
实施例9利用报告基因细胞系检测PD-1×OX40双特异性抗体(PR003787)对PD-1信号通路的抑制作用Example 9 Using a reporter gene cell line to detect the inhibitory effect of PD-1×OX40 bispecific antibody (PR003787) on PD-1 signaling pathway
将过表达PD-L1和OS8(CD3单链抗体跨膜蛋白)的HEK293细胞，铺到96孔板上，细胞量为1.25×10 4/孔，100μL/孔。加入50μL/孔的待测抗原结合蛋白稀释液，起始浓度为100nM，4倍浓度稀释，hlgG1为对照组。加入5×10 4/孔的可持续表达PD-1和NFAT-荧光素酶报告基因的Jurkat报告细胞(Shanghai ChemPartner构建)50μL/孔。37℃在5％CO 2环境下培养6小时。加入ONE-Glo TM荧光素酶试剂(Promega,#E6110)，室温孵育15分钟，酶标仪检测发光值。 HEK293 cells overexpressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were plated on a 96-well plate with a cell volume of 1.25×10 4 /well, 100 μL/well. Add 50 μL/well of the antigen-binding protein dilution to be tested, the initial concentration is 100 nM, and the concentration is diluted 4 times, and hlgG1 is used as the control group. Add 5×10 4 /well of Jurkat reporter cells (built by Shanghai ChemPartner) that can sustainably express PD-1 and NFAT-luciferase reporter genes, 50 μL/well. Incubate at 37°C for 6 hours in a 5% CO2 environment. Add ONE-Glo TM luciferase reagent (Promega, #E6110), incubate at room temperature for 15 minutes, and detect the luminescence value with a microplate reader.
结果如图14所示。显示本申请所述PD-1×OX40双特异性抗体(PR003787)及PD-1的亲本抗体对PD-1信号通路均有抑制作用。The result is shown in Figure 14. It shows that the PD-1×OX40 bispecific antibody (PR003787) described in this application and the parent antibody of PD-1 both have inhibitory effect on the PD-1 signaling pathway.
实施例10 PD-1×OX40双特异性抗体对T细胞的活化功能检测Example 10 Detection of the activation function of PD-1×OX40 bispecific antibody on T cells
购买人浓缩白细胞(Research blood components LLC)，分离人的外周血单核细胞(PBMC)，然后将人PBMC细胞和丝裂霉素处理的CHOK1-huPD-L1细胞共培养，加入2.5ug/mL的PHA-L(eBioscience,#00-4977-03)刺激3天。收集上清，通过MSD(Meso Scale Discovery,U-Plex K15067M-2)检测肿瘤坏死因子TNFα和IFNγ的分泌。Purchase human concentrated leukocytes (Research blood components LLC), separate human peripheral blood mononuclear cells (PBMC), then co-culture human PBMC cells and mitomycin-treated CHOK1-huPD-L1 cells, add 2.5ug/mL PHA-L (eBioscience, #00-4977-03) stimulation for 3 days. The supernatant was collected, and the secretion of tumor necrosis factors TNFα and IFNγ was detected by MSD (Meso Scale Discovery, U-Plex K15067M-2).
如图15和图16所示，本申请所述PD-1×OX40双特异性抗体(PR003787)及PD-1的亲本抗体(PR001985)与人IgG1同种型(PR000324)相比均能增强TNFα和IFNγ的分泌。As shown in Figure 15 and Figure 16, the PD-1×OX40 bispecific antibody (PR003787) and the parent antibody of PD-1 (PR001985) described in this application can both enhance TNFα compared with the human IgG1 isotype (PR000324) and IFNγ secretion.
实施例11 PD-1×OX40双特异性抗体抑制Tregs细胞中IL-10的生产Example 11 PD-1×OX40 bispecific antibody inhibits the production of IL-10 in Tregs cells
购买人浓缩白细胞(Research blood components LLC),分离Treg细胞(StemCell,Cat#18063)，加入CD3/CD28磁珠和重组人源白介素2(IL-2)(R&D,#202-IL-010/CF)，培养期间根据细胞生长状态每2-4天补充加入新鲜的含有IL-2的培养液，培养12天后，通过流式细胞仪分析Treg的比例，冷冻保存体外扩增的Treg细胞。解冻保存的Treg细胞，用含有重组人源白介素2(IL-2)(R&D,#202-IL-010/CF)的培养液培养2天，然后将Treg细胞和丝裂霉素处理的CHOK1-huPD-L1细胞共培养，加入CD3/CD28磁珠和重组人源白介素2(IL-2)培养5天。收集细胞，用BD Accuri C6 Plus流式细胞仪检测FoxP3和白介素IL-10分泌细胞的比例。Purchase concentrated human leukocytes (Research blood components LLC), separate Treg cells (StemCell, Cat#18063), add CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF ), according to the state of cell growth, add fresh culture medium containing IL-2 every 2-4 days, after 12 days of culture, analyze the ratio of Treg by flow cytometry, and cryopreserve the Treg cells expanded in vitro. The preserved Treg cells were thawed and cultured for 2 days with the medium containing recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF), and then the Treg cells and mitomycin-treated CHOK1- huPD-L1 cells were co-cultured with CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) for 5 days. Cells were collected, and the ratio of FoxP3 and interleukin IL-10 secreting cells was detected by BD Accuri C6 Plus flow cytometer.
如图17所示，本申请所述PD-1×OX40双特异性抗体(PR003787)及OX40的亲本抗体(PR002067)与人IgG1同种型(PR000324)相比对Treg细胞分泌IL-10均有抑制作用。As shown in Figure 17, compared with the human IgG1 isotype (PR000324), the PD-1×OX40 bispecific antibody (PR003787) and the parental antibody of OX40 (PR002067) described in this application have higher effects on the secretion of IL-10 by Treg cells. inhibition.
实施例12 PD-1×OX40双特异性抗体(PR003787)与亲本单抗、联合使用的功能差异(IFNγ，Granzyme B，IL-2 secretion)Example 12 Functional difference between PD-1×OX40 bispecific antibody (PR003787) and parental monoclonal antibody, combined use (IFNγ, Granzyme B, IL-2 secretion)
购买人浓缩白细胞(Research blood components LLC)，分离T细胞(StemCell，#17951)，冷冻保存。同时分离Treg细胞(StemCell,Cat#18063)，加入CD3/CD28磁珠和重组人源白介素2(IL-2)(R&D,#202-IL-010/CF)，培养期间根据细胞生长状态每2-4天补充加入新鲜的含有IL-2的培养液，培养12天后，通过流式细胞仪分析Treg的比例。Purchase concentrated human leukocytes (Research blood components LLC), isolate T cells (StemCell, #17951), and cryopreserve. Simultaneously isolate Treg cells (StemCell, Cat#18063), add CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF), during the culture period according to the cell growth status every 2 - Add fresh IL-2-containing culture solution on day 4, and after 12 days of culture, analyze the ratio of Treg by flow cytometry.
购买第二供体人浓缩白细胞(Research blood components LLC)，分离monocytes细胞(StemCell，#19359)，加入重组人源白介素4(IL-4)(R&D，#204-GMP)和人源GM-CSF(R&D，#215-GM/CF)诱导6天后，获得未成熟的人CD14+树突状细胞(iDC细胞)。Purchase the second donor human enriched leukocytes (Research blood components LLC), separate monocytes cells (StemCell, #19359), add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) 6 days after induction, immature human CD14+ dendritic cells (iDC cells) were obtained.
将第一供体的5×10 4个T细胞、5×10 4个Treg细胞及第二供体的2×10 4个iDC细胞按照2.5:2.5:1的比例接种至96孔板，加入2nM或10nM本申请所述的PD-1×OX40双特异性抗体(PR003787)，亲本单抗或PD-1和OX40两个亲本联合单抗，共培养4-5天。收集100μL培养上清，通过MSD(Meso Scale Discovery,U-Plex K15067M-2)检测IL-2，Granzyme B和IFNγ的分泌。 Inoculate 5×10 4 T cells, 5×10 4 Treg cells from the first donor and 2×10 4 iDC cells from the second donor into a 96-well plate at a ratio of 2.5:2.5:1, and add 2nM Or 10nM PD-1×OX40 bispecific antibody (PR003787) described in this application, parental monoclonal antibody or two parental monoclonal antibodies of PD-1 and OX40, and co-culture for 4-5 days. 100 μL of the culture supernatant was collected, and the secretion of IL-2, Granzyme B and IFNγ was detected by MSD (Meso Scale Discovery, U-Plex K15067M-2).
如图18、图19和图20所示，本申请所述PD-1×OX40双特异性抗体(PR003787)的活性优于PD-1单抗(PR001985)和OX40单抗(PR002067)，并优于PD-1单抗(PR001985)和OX40单抗(PR002067)的组合，显示了协同活性。As shown in Figure 18, Figure 19 and Figure 20, the activity of the PD-1×OX40 bispecific antibody (PR003787) described in this application is superior to that of PD-1 monoclonal antibody (PR001985) and OX40 monoclonal antibody (PR002067), and is superior to The combination of PD-1 mAb (PR001985) and OX40 mAb (PR002067) showed synergistic activity.
实施例13 PHA检测PD-1×OX40双特异性抗体的T细胞激活作用Example 13 PHA detection of T cell activation of PD-1×OX40 bispecific antibody
购买人浓缩白细胞(Research blood components LLC)，分离人的外周血单核细胞(PBMC)，然后将人PBMC细胞中加入2.5ug/mL的PHA-L(eBioscience,#00-4977-03)刺激3天。收集上清，通过MSD(Meso Scale Discovery,U-Plex K15067M-2)检测肿瘤坏死因子IL-2的分泌。Purchase concentrated human leukocytes (Research blood components LLC), separate human peripheral blood mononuclear cells (PBMC), and then add 2.5ug/mL PHA-L (eBioscience, #00-4977-03) to human PBMC cells to stimulate 3 sky. The supernatant was collected, and the secretion of tumor necrosis factor IL-2 was detected by MSD (Meso Scale Discovery, U-Plex K15067M-2).
如图21A和图21B所示，与PD1单克隆抗体相比，本申请所述PD-1×OX40双特异性抗体PR003787、PR500531、PR200538、PR200539、PR200536、PR200528和PR200600具有更强的激活T细胞分泌IL-2的能力。且与PR003787相比，PR200538、PR200539、PR200528呈现剂量依赖的T细胞的激活能力，在高浓度条件下具有更强的T细胞活化能力。As shown in Figure 21A and Figure 21B, compared with the PD1 monoclonal antibody, the PD-1×OX40 bispecific antibodies PR003787, PR500531, PR200538, PR200539, PR200536, PR200528 and PR200600 described in this application have stronger activation of T cells Ability to secrete IL-2. And compared with PR003787, PR200538, PR200539, and PR200528 exhibit dose-dependent T cell activation ability, and have stronger T cell activation ability under high concentration conditions.

Claims (23)

  1. 一种靶向PD-或其片段的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包括轻链可变区VL和重链可变区VH，所述VL和所述VH分别包括三个CDR，其中：A PD-targeting antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH, the VL and the VH includes three CDRs respectively, among which:
    所述VL包含氨基酸序列分别由SEQ ID NO:39、46和54所示的LCDR1、LCDR2和LCDR3；所述VH包含氨基酸序列分别由SEQ ID NO:8、18和28所示HCDR1、HCDR2和HCDR3；或The VL comprises amino acid sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:39, 46 and 54; the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:8, 18 and 28 respectively ;or
    所述VL包含氨基酸序列分别由SEQ ID NO:39、46和54所示的LCDR1、LCDR2和LCDR3；所述VH包含氨基酸序列分别由SEQ ID NO:81、86和91所示HCDR1、HCDR2和HCDR3；The VL comprises amino acid sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:39, 46 and 54 respectively; the VH comprises amino acid sequences HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:81, 86 and 91 respectively ;
    优选地，所述CDR包含1至3个氨基酸取代。Preferably, the CDRs comprise 1 to 3 amino acid substitutions.
  2. 如权利要求1所述的抗体或其抗原结合片段，其特征在于，The antibody or antigen-binding fragment thereof according to claim 1, wherein,
    所述VL可变区包括如SEQ ID NO:67所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述VH包括如SEQ ID NO:62所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，The VL variable region comprises an amino acid sequence as shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof % identical amino acid sequence; the VH comprises the amino acid sequence shown in SEQ ID NO: 62 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequences; or,
    所述VL可变区包括如SEQ ID NO:67所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述VH包括如SEQ ID NO:102所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。The VL variable region comprises an amino acid sequence as shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof % identical amino acid sequence; the VH comprises the amino acid sequence shown in SEQ ID NO: 102 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, Amino acid sequences with 99% or 100% identity.
  3. 如权利要求1或2所述的抗体或其抗原结合片段，其特征在于，所述抗体还包括重链恒定区和/或轻链恒定区；优选地，所述抗体的重链恒定区选自hIgG1、hIgG2、hIgG3或hIgG4，所述轻链恒定区选自κ链或者λ链；更优选地，所述抗体的Fc的第234位和/或第235位发生氨基酸替换，优选所述氨基酸替换为L234A和/或L235A。The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region of the antibody is selected from hIgG1, hIgG2, hIgG3 or hIgG4, the light chain constant region is selected from κ chain or λ chain; more preferably, the 234th and/or 235th position of Fc of the antibody undergoes an amino acid substitution, preferably the amino acid substitution for L234A and/or L235A.
  4. 如权利要求1-3任一所述的抗体或其抗原结合片段，其特征在于，所述抗体选自全长抗体、Fab、Fab’、F(ab’)2、Fv或scFv。The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody is selected from full-length antibodies, Fab, Fab', F(ab')2, Fv or scFv.
  5. 如权利要求1-4所述的抗体，其特征在于，其包括重链和轻链，其中，The antibody of claims 1-4, comprising a heavy chain and a light chain, wherein,
    所述轻链包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述重链包括如SEQ ID NO：72所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，The light chain comprises an amino acid sequence as shown in SEQ ID NO: 77 or is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical A specific amino acid sequence; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 72 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99 % or 100% identity amino acid sequences; or,
    所述轻链包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；所述重链包括如SEQ ID NO：106所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。The light chain comprises an amino acid sequence as shown in SEQ ID NO: 77 or is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical A specific amino acid sequence; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 106 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, % or 100% identity amino acid sequences.
  6. 一种双特异性抗体或其抗原结合片段，其特征在于，包括：A bispecific antibody or an antigen-binding fragment thereof, characterized by comprising:
    第一结构域，其结合PD-1或其片段，所述第一结构域为如权利要求1-5任一项所述的靶向PD-1的抗体；和A first domain that binds to PD-1 or a fragment thereof, the first domain being the antibody targeting PD-1 according to any one of claims 1-5; and
    第二结构域，其结合OX40或其片段，优选地，所述第二结构域为VH结构。The second domain, which binds to OX40 or a fragment thereof, preferably, the second domain is a VH structure.
  7. 如权利要求6所述的双特异性抗体或其抗原结合片段，其特征在于，所述双特性抗体或其抗原结合片 段包括两条多肽链，其中：The bispecific antibody or its antigen-binding fragment according to claim 6, wherein the bispecific antibody or its antigen-binding fragment comprises two polypeptide chains, wherein:
    多肽链2具有N’-VL 1-CL 1-C’所示的结构，多肽链1具有N’-VH 1-CH 1-h-CH 2-CH 3-L-VH 2-C’所示的结构；或 Polypeptide chain 2 has the structure shown by N'-VL 1 -CL 1 -C', and polypeptide chain 1 has the structure shown by N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L-VH 2 -C'structure; or
    多肽链2具有N’-VL 1-CL 1-C’所示的结构，多肽链1具有N’-VH 2-L-VH 1-CH 1-h-CH 2-CH 3-C’所示的结构；或 Polypeptide chain 2 has the structure shown by N'-VL 1 -CL 1 -C', and polypeptide chain 1 has the structure shown by N'-VH 2 -L-VH 1 -CH 1 -h-CH 2 -CH 3 -C'structure; or
    多肽链2具有N’-VH’-CH 1’-C’所示的结构，多肽链1具有N’-VL’-CL’-L-VH 2-h-CH 2-CH 3-C’所示的结构；或 Polypeptide chain 2 has the structure shown by N'-VH'-CH 1 '-C', and polypeptide chain 1 has the structure shown by N'-VL'-CL'-L-VH 2 -h-CH 2 -CH 3 -C' the structure shown; or
    多肽链2具有N’-VL 1-CL 1-C’所示的结构，多肽链1具有N’-VH 1-CH 1-h-CH 2-CH 3-L 1-VH 2-L 2-VH 2-C’所示的结构；或 Polypeptide chain 2 has the structure shown by N'-VL 1 -CL 1 -C', and polypeptide chain 1 has the structure shown by N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L 1 -VH 2 -L 2 - The structure shown in VH 2 -C'; or
    多肽链2具有N’-VL 1-CL 1-C’所示的结构，多肽链1具有N’-VH 1-CH 1-h-CH 2-CH 3-L 1-VH 2-L 2-VH 2-L 3-VH 2-C’所示的结构， Polypeptide chain 2 has the structure shown by N'-VL 1 -CL 1 -C', and polypeptide chain 1 has the structure shown by N'-VH 1 -CH 1 -h-CH 2 -CH 3 -L 1 -VH 2 -L 2 - The structure shown in VH 2 -L 3 -VH 2 -C',
    其中，所述VL 1、VH 1、VL’和VH’分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L、L 1、L 2和L 3为连接肽，所述CL 1和CL’是第一结构域的CL，所述CH 1和CH 1’是第一结构域的CH 1； Wherein, said VL 1 , VH 1 , VL' and VH' are respectively VL and VH of the first domain, said VH 2 is VH of the second domain, said h is a hinge region, said L, L 1 , L 2 and L 3 are connecting peptides, the CL 1 and CL' are the CL of the first domain, and the CH 1 and CH 1 ' are the CH 1 of the first domain;
    优选地，所述连接肽为长度为0-30个氨基酸的肽，优选地，其氨基酸序列如SEQ ID NO:116-140任一所示；Preferably, the connecting peptide is a peptide with a length of 0-30 amino acids, preferably, its amino acid sequence is as shown in any one of SEQ ID NO: 116-140;
    优选地，所述多肽链1和所述多肽链2形成四价对称结构、六价对称结构或八价对称结构。Preferably, the polypeptide chain 1 and the polypeptide chain 2 form a tetravalent symmetrical structure, a hexavalent symmetrical structure or an octavalent symmetrical structure.
  8. 如权利要求6或7所述的双特异性抗体或其抗原结合片段，其特征在于，所述第二结构域包含重链可变区，其中，所述重链可变区包含氨基酸序列分别由SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29所示的HCDR1、HCDR2、HCDR3；或所述重链可变区包含氨基酸序列分别由SEQ ID NO:9、SEQ ID NO:85、SEQ ID NO:90所示的HCDR1、HCDR2、HCDR3；The bispecific antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the second domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence composed of HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29; or the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 85 , HCDR1, HCDR2, HCDR3 shown in SEQ ID NO:90;
    优选地，所述第二结构域包含与如SEQ ID NO:63或101所示的氨基酸序列具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链可变区。Preferably, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain variable regions.
  9. 如权利要求8所述的双特异性抗体或其抗原结合片段，其特征在于，Fc为人IgG1结构，优选地，所述Fc包含L234A、L235A和P329G，或所述Fc包含L234A、L235A和G237A。The bispecific antibody or antigen-binding fragment thereof according to claim 8, wherein the Fc is a human IgG1 structure, preferably, the Fc comprises L234A, L235A and P329G, or the Fc comprises L234A, L235A and G237A.
  10. 如权利要求7所述的双特异性抗体或其抗原结合片段，其特征在于，所述双特异性抗体或其抗原结合片段包括：The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the bispecific antibody or antigen-binding fragment thereof comprises:
    多肽链1包括如SEQ ID NO：79所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 79 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; or,
    多肽链1包括如SEQ ID NO：109所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列； 或，Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 109 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; or,
    多肽链1包括如SEQ ID NO：110所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 110 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; or,
    多肽链1包括如SEQ ID NO：111所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 111 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; or,
    多肽链1包括如SEQ ID NO：112所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 112 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; or,
    多肽链1包括如SEQ ID NO：114所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：113所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；或，Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 114 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 113 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; or,
    多肽链1包括如SEQ ID NO：115所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；多肽链2包括如SEQ ID NO：77所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 115 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity therewith The amino acid sequence; Polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence.
  11. 分离的核酸分子，其编码权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6-10任一项所述的双特异性抗体或其抗原结合片段，An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5 or the bispecific antibody or antigen-binding fragment thereof of any one of claims 6-10,
    优选地，所述核酸分子是mRNA分子。Preferably, said nucleic acid molecule is an mRNA molecule.
  12. 表达载体，其包含权利要求11的分离核酸分子，所述表达载体为真核细胞表达载体或原核细胞表达载体，优选为逆转录病毒载体、慢病毒载体、噬菌体载体、腺病毒载体、腺相关载体或单纯疱疹载体。Expression vector, it comprises the isolated nucleic acid molecule of claim 11, and described expression vector is eukaryotic cell expression vector or prokaryotic cell expression vector, is preferably retroviral vector, lentiviral vector, phage vector, adenoviral vector, adeno-associated vector or herpes simplex carrier.
  13. 根据权利要求12的表达载体，其中，所述表达载体存在于纳米颗粒、脂质体或基因枪中。The expression vector according to claim 12, wherein said expression vector is present in nanoparticles, liposomes or gene guns.
  14. 宿主细胞，其包含权利要求11的分离的核酸分子，或权利要求12或13的表达载体，所述宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选为E.coli细胞，所述真核细胞优选为HEK293细胞或CHO细胞。Host cell, it comprises the isolated nucleic acid molecule of claim 11, or the expression vector of claim 12 or 13, and described host cell is prokaryotic cell and/or eukaryotic cell, and described prokaryotic cell is preferably E.coli cell, so The eukaryotic cells are preferably HEK293 cells or CHO cells.
  15. 权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求6-9任一项所述的双特异性抗体或其抗原结合片段的制备方法，包括在使得权利要求1-5任一项所述的特异性抗体或其抗原结合片段，或权利要求6-10任一项所述的双特异性抗体或其抗原结合片段表达的情况下，培养权利要求14所述的宿主细胞。The preparation method of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 6-9, comprising making claims 1- In the case of expressing the specific antibody or antigen-binding fragment thereof according to any one of 5, or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 6-10, culturing the host according to claim 14 cell.
  16. 药物组合物，其包含权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求6-10任一项所述的双特异性抗体或其抗原结合片段，以及药学上可接受的载体；优选地，所述的药物组合物还包含第二治疗剂，所述的第二治疗剂包括化学治疗剂、放射治疗剂、免疫抑制剂、细胞毒性药物。A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 6-10, and a pharmaceutically acceptable Acceptable carrier; preferably, the pharmaceutical composition further includes a second therapeutic agent, and the second therapeutic agent includes a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressant, and a cytotoxic drug.
  17. 根据权利要求16的药物组合物，其中，所述药物组合物为固体、半固体或液体的形式；The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition is in the form of solid, semi-solid or liquid;
    优选地，所述药物组合物为水溶液、非水溶液或混悬液，粉末、片剂、胶囊、颗粒、注射剂或输注剂的形式。Preferably, the pharmaceutical composition is in the form of aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion.
  18. 权利要求1-5任一项所述的抗体或其抗原结合片段，权利要求6-10任一项所述的双特异性抗体或其抗原结合片段，权利要求11的分离的核酸分子，权利要求12或13的表达载体，权利要求14的宿主细胞，或权利要求16或17的药物组合物在制备用于预防、治疗和/或诊断肿瘤疾病，急性和慢性炎性疾病和/或免疫性疾病的药物中的应用。The antibody or antigen-binding fragment thereof according to any one of claims 1-5, the bispecific antibody or antigen-binding fragment thereof according to any one of claims 6-10, the isolated nucleic acid molecule of claim 11, or the isolated nucleic acid molecule of claim 11 The expression vector of 12 or 13, the host cell of claim 14, or the pharmaceutical composition of claim 16 or 17 are used in the preparation for prevention, treatment and/or diagnosis of tumor diseases, acute and chronic inflammatory diseases and/or immune diseases application in medicines.
  19. 根据权利要求18的应用，其中，所述肿瘤选自乳腺癌、肾细胞癌、黑色素瘤、结肠癌，B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期或转移性恶性肿瘤中的一种或多种；The use according to claim 18, wherein said tumor is selected from the group consisting of breast cancer, renal cell carcinoma, melanoma, colon cancer, B-cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urinary Road skin cancer, liver cancer, esophageal cancer, gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, One or more of Hodgkin's lymphoma, squamous cell carcinoma of the skin, locally advanced or metastatic malignancies;
    所述炎性疾病为特应性皮炎或溃疡性结肠炎，The inflammatory disease is atopic dermatitis or ulcerative colitis,
    所述免疫性疾病为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮或哮喘。The immune disease is graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus or asthma.
  20. 用于检测样品中的OX40和/或PD-1的方法，所述方法包括利用权利要求1-5任一项的抗体或其抗原结合片段，或权利要求6-10任一项所述的双特异性抗体或其抗原结合片段，检测样品中的OX40和/或PD-1的步骤，所述样品为全血，红血细胞浓缩物，血小板浓缩物，白细胞浓缩物，组织，骨髓吸出物，血浆，血清，脑脊液，粪便，尿液，培养的细胞，唾液，口腔分泌物和/或鼻腔分泌物；优选地，所述检测方法为非诊断目的。A method for detecting OX40 and/or PD-1 in a sample, the method comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the bismuth according to any one of claims 6-10 Specific antibodies or antigen-binding fragments thereof, the step of detecting OX40 and/or PD-1 in samples such as whole blood, red blood cell concentrates, platelet concentrates, white blood cell concentrates, tissues, bone marrow aspirate, plasma , serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral and/or nasal secretions; preferably, said detection method is for non-diagnostic purposes.
  21. 套装药盒，其包括一个或多个药盒，所述一个或多个药盒包括权利要求1-5任一项的抗体或其抗原结合片段，权利要求6-10任一项所述的双特异性抗体或其抗原结合片段，或权利要求16或17的药物组合物。A set of kits comprising one or more kits, said one or more kits comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the bismuth of any one of claims 6-10 A specific antibody or an antigen-binding fragment thereof, or the pharmaceutical composition of claim 16 or 17.
  22. 权利要求21的套装药盒，其中所述套装药盒包括第一药盒，且所述第一药盒包括权利要求6-10任一项所述的双特异性抗体或其抗原结合片段；The set of kits according to claim 21, wherein the set of kits comprises a first kit, and the first kit includes the bispecific antibody or antigen-binding fragment thereof according to any one of claims 6-10;
    可选地，所述套装药盒进一步包括第二药盒，且所述第二药盒包括选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物中的至少一种治疗剂。Optionally, the set of kits further includes a second kit, and the second kit includes at least one therapeutic agent selected from chemotherapeutic agents, radiotherapeutic agents, immunosuppressants and cytotoxic drugs.
  23. 预防、治疗和/或诊断免疫性疾病、急性和慢性炎性疾病以及肿瘤疾病的方法，其包括向受试者施用治疗有效量的权利要求1-5任一项的抗体或其抗原结合片段，权利要求6-10任一项所述的双特异性抗体或其抗原结合片段，权利要求11的分离的核酸分子，权利要求12或13的表达载体，权利要求14的宿主细胞，或权利要求16或17的药物组合物。A method for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, The bispecific antibody or antigen-binding fragment thereof according to any one of claims 6-10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12 or 13, the host cell of claim 14, or claim 16 or 17 pharmaceutical compositions.
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