WO2022262749A1 - Protéine de liaison spécifique ciblant pd1 et/ou ox40 - Google Patents

Protéine de liaison spécifique ciblant pd1 et/ou 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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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/zh
Publication of WO2022262749A1 publication Critical patent/WO2022262749A1/fr

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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.

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Abstract

La divulgation concerne un anticorps ciblant PD1, ou un fragment de liaison à l'antigène de celui-ci, et un anticorps bispécifique ciblant PD1 et OX40, ou un fragment de celui-ci. La divulgation concerne en outre une molécule d'acide nucléique codant pour l'anticorps ou un fragment de liaison à l'antigène de celui-ci, l'anticorps ciblant PD1, ou le fragment de liaison à l'antigène de celui-ci, l'anticorps bispécifique ciblant PD1 et OX40, ou un fragment de celui-ci, et l'utilisation de la molécule d'acide nucléique codant pour le traitement, la prévention et/ou le diagnostic d'une maladie. La maladie peut être, par exemple, une maladie immunitaire, une maladie inflammatoire aiguë, une maladie inflammatoire chronique ou une maladie tumorale.
PCT/CN2022/098838 2021-06-17 2022-06-15 Protéine de liaison spécifique ciblant pd1 et/ou ox40 WO2022262749A1 (fr)

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