WO2023019505A1 - 抗TGFβ抗体及其制备方法和应用 - Google Patents

抗TGFβ抗体及其制备方法和应用 Download PDF

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WO2023019505A1
WO2023019505A1 PCT/CN2021/113494 CN2021113494W WO2023019505A1 WO 2023019505 A1 WO2023019505 A1 WO 2023019505A1 CN 2021113494 W CN2021113494 W CN 2021113494W WO 2023019505 A1 WO2023019505 A1 WO 2023019505A1
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seq
cdr3
cdr2
cdr1
amino acid
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PCT/CN2021/113494
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English (en)
French (fr)
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石剑
方丽娟
严永祥
张敬
华珊
周鹏飞
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武汉友芝友生物制药股份有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

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  • the invention relates to the field of medicine, in particular to an antibody to TGF- ⁇ (transforming growth factor beta, transforming growth factor beta) and its preparation method and application.
  • TGF- ⁇ transforming growth factor beta, transforming growth factor beta
  • TGF- ⁇ (transforming growth factor beta, transforming growth factor ⁇ ) is a multifunctional cytokine that plays an important role in regulating cell growth and differentiation.
  • TGF- ⁇ is a disulfide bonded by two subunits with the same or similar structure and a molecular weight of 12.5kDa.
  • At least four subtypes of TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3 and TGF- ⁇ 1 ⁇ 2 are found in mammals.
  • Human TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 genes are located on chromosome 19q3, 1q41 and 14q24, respectively.
  • the homology of human and mouse TGF- ⁇ 1 is as high as 99%.
  • TGF- ⁇ After TGF- ⁇ binds to the receptor, it mediates a series of biological responses through Smad and non-Smad signaling pathways, including promoting cell epithelial-mesenchymal transition, promoting tissue Fibrosis, promoting angiogenesis, promoting tumor immune escape, dual effects of tumor suppressor and tumor promotion, etc. Since the signaling pathway mediated by overactivated TGF- ⁇ and its receptors plays an important pathophysiological role in the occurrence and development of various diseases such as malignant tumors and tissue fibrosis, inhibitors targeting this molecule have been used Blocking this signaling pathway provides a therapeutic option for the control of these diseases.
  • TGF- ⁇ 1 Transforming growth factor ⁇ 1
  • TGF- ⁇ 1 and TGF- ⁇ 3 TGF- ⁇ 1
  • TGF- ⁇ 2 also plays an important role in cell proliferation, differentiation, embryonic development, extracellular matrix formation, bone formation and reconstruction, tumor suppression and metastasis.
  • TGF- ⁇ 2 is pointed out that it can inhibit the growth of IL-2-dependent T cells, and can inhibit the immune surveillance in tumor development, thus promoting tumor growth in an autocrine manner.
  • TGF- ⁇ 2 can affect the viability of killer cells and reduce the expression of IL-2, IL-6, IL-10, IFN- ⁇ and other cytokines.
  • TGF- ⁇ 3 stimulates target cells (fibroblasts, vascular endothelial cells, etc.) to synthesize extracellular matrix and accelerate the process of vascularization during tissue repair, and promotes wound healing and reduces scar formation.
  • target cells fibroblasts, vascular endothelial cells, etc.
  • TGF- ⁇ 3 can promote morphogenesis and play an important role in vertebral formation, limb sprouting, teething, facial bone formation and heart valve formation.
  • TGF- ⁇ 3 regulates bone formation and can affect adult bone regeneration.
  • Drugs targeting TGF- ⁇ have a wide range of indications, including kidney disease, systemic scleroderma, solid tumors, idiopathic pulmonary fibrosis, diabetes, etc., but currently there is no drug targeting TGF- ⁇ on the market.
  • the invention provides a specific antibody against TGF ⁇ . Specifically, the present invention provides the following aspects:
  • An anti-TGF ⁇ antibody comprising, or consisting of, a sequence selected from the group consisting of:
  • B2G8 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 7, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 49, shown in SEQ ID NO: 50 CDR2 and CDR3 shown in SEQ ID NO: 51, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 49, CDR2 shown in SEQ ID NO: 50 and/or SEQ ID NO:
  • the CDR3 shown in 51 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retains an amino acid sequence with TGF ⁇ binding affinity, preferably comprising SEQ ID NO CDR1 shown in: 49, the variant of CDR2 shown in SEQ ID NO: 50 and the CDR3 shown in SEQ ID NO: 51, wherein the variant of CDR2 shown in SEQ ID NO: 50 is in SEQ ID NO :
  • CDR1 shown in SEQ ID NO: 49 CDR2 shown in SEQ ID NO: 50 and CDR3 shown in SEQ ID NO: 51, wherein amino acids 5-8 in CDR1 shown in SEQ ID NO: 49, The 1st-5th amino acid, the 7th-9th amino acid in the CDR2 shown in SEQ ID NO: 50, the 1st amino acid and the 4th-9th amino acid in the CDR3 shown in SEQ ID NO: 51 are selected from amino acid X, so Said amino acid X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val,
  • B1B8 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 4, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 40, shown in SEQ ID NO: 41 CDR2 and CDR3 shown in SEQ ID NO: 42, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 40, CDR2 shown in SEQ ID NO: 41 and/or SEQ ID NO: Compared with CDR3 shown in 42, there are one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retain the amino acid sequence with TGF ⁇ binding affinity; or
  • It comprises CDR1 shown in SEQ ID NO: 40, CDR2 shown in SEQ ID NO: 41 and CDR3 shown in SEQ ID NO: 42, wherein the 3rd amino acid in the CDR1 shown in SEQ ID NO: 40, the 5th amino acid -8 amino acids, amino acids 1, 3 and 5-10 in CDR2 shown in SEQ ID NO: 41, amino acids 4-9 and 11 in CDR3 shown in SEQ ID NO: 42
  • amino acid X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val group,
  • B3F3 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 16, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 76, shown in SEQ ID NO: 77 CDR2 and CDR3 shown in SEQ ID NO: 78, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 76, CDR2 shown in SEQ ID NO: 77 and/or SEQ ID NO: Compared with CDR3 shown in 78, there are one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retain the amino acid sequence with TGF ⁇ binding affinity; or
  • CDR1 shown in SEQ ID NO: 76 CDR2 shown in SEQ ID NO: 77 and CDR3 shown in SEQ ID NO: 78, wherein amino acids 2-3 in CDR1 shown in SEQ ID NO: 76, Amino acids at positions 5-8, amino acids at positions 2-4 and amino acids at position 6 in CDR2 shown in SEQ ID NO: 77, amino acids at positions 1 and 3-8 in CDR3 shown in SEQ ID NO: 78
  • said amino acid X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val group,
  • B2A1 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 1, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 31, shown in SEQ ID NO: 32 CDR2 and CDR3 shown in SEQ ID NO: 33, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32 and/or SEQ ID NO: CDR3 shown in 33 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B1F8 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 5, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 43, shown in SEQ ID NO: 44 CDR2 and CDR3 shown in SEQ ID NO: 45, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 44 and/or SEQ ID NO: CDR3 shown in 45 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B3D6 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 6, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 46, shown in SEQ ID NO: 47 CDR2 and CDR3 shown in SEQ ID NO: 48, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 46, CDR2 shown in SEQ ID NO: 47 and/or SEQ ID NO: CDR3 shown in 48 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B2A9 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 2, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 34, shown in SEQ ID NO: 35 CDR2 and CDR3 shown in SEQ ID NO: 36, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35 and/or SEQ ID NO: CDR3 shown in 36 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B2C2 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 8, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 52, shown in SEQ ID NO: 53 CDR2 and CDR3 shown in SEQ ID NO: 54, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 52, CDR2 shown in SEQ ID NO: 53 and/or SEQ ID NO: CDR3 shown in 54 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • (9) B1B7 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 9, preferably according to Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 55, shown in SEQ ID NO: 56 CDR2 and CDR3 shown in SEQ ID NO: 57, or a variant thereof, said variant being the same as CDR1 shown in SEQ ID NO: 55, CDR2 shown in SEQ ID NO: 56 and/or SEQ ID NO: CDR3 shown in 57 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B1C5 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 10, preferably according to Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 58, shown in SEQ ID NO: 59 CDR2 and CDR3 shown in SEQ ID NO: 60, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 58, CDR2 shown in SEQ ID NO: 59 and/or SEQ ID NO: Compared with the CDR3 shown in 60, there are one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retain the amino acid sequence with TGF ⁇ binding affinity;
  • B2F6, which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 11, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 61, shown in SEQ ID NO: 62 CDR2 and CDR3 shown in SEQ ID NO: 63, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 61, CDR2 shown in SEQ ID NO: 62 and/or SEQ ID NO: CDR3 shown in 63 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B2H1 comprising CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 12, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 64, shown in SEQ ID NO: 65 CDR2 and CDR3 shown in SEQ ID NO: 66, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 64, CDR2 shown in SEQ ID NO: 65 and/or SEQ ID NO: CDR3 shown in 66 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B2A2 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 13, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 67, shown in SEQ ID NO: 68 CDR2 and CDR3 shown in SEQ ID NO: 69, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 67, CDR2 shown in SEQ ID NO: 68 and/or SEQ ID NO: CDR3 shown in 69 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B3D2 comprising CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 14, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 70, shown in SEQ ID NO: 71 CDR2 and CDR3 shown in SEQ ID NO: 72, or a variant thereof, said variant being CDR1 shown in SEQ ID NO: 70, CDR2 shown in SEQ ID NO: 71 and/or SEQ ID NO: CDR3 shown in 72 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B3C5 comprising CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 15, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 73, shown in SEQ ID NO: 74 CDR2 and CDR3 shown in SEQ ID NO: 75, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 73, CDR2 shown in SEQ ID NO: 74 and/or SEQ ID NO: CDR3 shown in 75 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with CDR3 and retains the amino acid sequence with TGF ⁇ binding affinity;
  • B2C9 which comprises CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 3, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 37, shown in SEQ ID NO: 38 CDR2 and CDR3 shown in SEQ ID NO: 39, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38 and/or SEQ ID NO: Compared with CDR3 shown in 39, there are one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retain the amino acid sequence with TGF ⁇ binding affinity; and
  • B3F1 comprising CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO: 17, preferably according to the Kabat and IMGT numbering system, comprising CDR1 shown in SEQ ID NO: 79, shown in SEQ ID NO: 80 CDR2 and CDR3 shown in SEQ ID NO: 81, or a variant thereof that is the same as CDR1 shown in SEQ ID NO: 79, CDR2 shown in SEQ ID NO: 80 and/or SEQ ID NO:
  • the CDR3 shown in 81 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retains the amino acid sequence with binding affinity to TGF ⁇ compared with CDR3.
  • the anti-TGF ⁇ antibody described in item 1 which comprises a sequence selected from SEQ ID NO: 1-17 or a variant thereof, or a sequence selected from SEQ ID NO: 1-17 or a variant thereof composition; wherein the variant is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in the sequence number , 99% homology and retain the sequence of binding affinity with TGF ⁇ , or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9) compared with the amino acid sequence shown in the sequence number or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retain amino acid sequences with TGF ⁇ binding affinity.
  • a humanized anti-TGF ⁇ antibody comprising CDR1, CDR2 and CDR3 of the anti-TGF ⁇ antibody described in item 1, preferably comprising a FR region from Germline IGHV3-23*01 of human IgG, preferably further comprising an Fc constant region , the constant region comprises the sequence shown in SEQ ID NO: 18 or the human IgG1 heavy chain Fc constant region (GenBank No.AK303185.1), more preferably, the humanized anti-TGF ⁇ antibody comprises the sequence shown in SEQ ID NO: 20 The humanized sequence or consists of it, further preferably, the serine Ser at position 13 of CDR2 of the humanized sequence shown in SEQ ID NO: 20 is mutated to alanine Ala, most preferably, the humanized anti-TGF ⁇ antibody Comprising or consisting of humanized sequences selected from SEQ ID NO: 21-23.
  • a bivalent or multivalent anti-TGF ⁇ antibody comprising two or more anti-TGF ⁇ antibodies according to any one of items 1-2, or comprising two or more humanized anti-TGF ⁇ antibodies described in item 3, preferably
  • the sequence of the bivalent anti-TGF ⁇ antibody is selected from the group consisting of: (1) the sequence shown by SEQ ID NO: 22, 19, 18, 19 and 22 is in the order from N-terminal to C-terminal (2)
  • the sequence shown by SEQ ID NO: 4, 19, 18, 19 and 4 is connected in sequence from the N-terminal to the C-terminal; (3) It is composed of SEQ ID NO: 23, 19,
  • the sequences shown in 18, 19 and 23 are connected according to the sequence from the N-terminal to the C-terminal;
  • the sequences shown in SEQ ID NO: 21, 19, 18, 19 and 21 are connected according to the sequence from the N-terminal to the C-terminal (5) the sequence shown by SEQ ID NO: 7, 19, 18, 19 and 7 is formed by connecting the sequence from N-terminal to C-terminal; and
  • a multispecific antibody comprising the anti-TGF beta antibody described in any one of items 1-2 or the humanized anti-TGF beta antibody described in item 3, preferably a bispecific antibody comprising an anti-TGF beta antibody targeting the first A first antibody against an antigen TGF ⁇ and a second antibody against a second antigen, wherein the first antibody is selected from the anti-TGF ⁇ antibody described in any one of items 1-2 or the humanized anti-TGF ⁇ antibody described in item 3 , the second antigen is selected from immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines, or combinations thereof; more preferably selected from: PD-L1, PD-1, VEGFA, IL-10, IL- 10R, BCMA, VEGF, TGF- ⁇ , CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2,
  • a vector comprising the polynucleotide according to item 6.
  • a host cell comprising the vector according to item 7.
  • a conjugate comprising the anti-TGF ⁇ antibody of any one of items 1-2, the humanized anti-TGF ⁇ antibody of item 3, the bivalent or multivalent anti-TGF ⁇ antibody of item 4, or item 5
  • the coupling part is radioactive isotope, fluorescent substance, chemiluminescent substance, colored substance, chemotherapeutic agent, biotoxin, polyethylene glycol or enzyme.
  • a kit comprising the anti-TGF ⁇ antibody described in any one of items 1-2, the humanized anti-TGF ⁇ antibody described in item 3, the bivalent or multivalent anti-TGF ⁇ antibody described in item 4, or the anti-TGF ⁇ antibody described in item 5.
  • the multispecific antibody described above or the conjugate described in item 9; preferably, the kit also includes a second antibody that specifically recognizes the anti-TGF ⁇ antibody described in any one of items 1-2, or the conjugate described in item 3.
  • the kit comprises (1) the anti-TGF ⁇ antibody described in any one of items 1-2, the humanized anti-TGF ⁇ antibody described in item 3, the bivalent or multivalent anti-TGF ⁇ antibody described in item 4, and the anti-TGF ⁇ antibody described in item 5
  • the multispecific antibody or the conjugate described in item 9 and (2) antibodies against other antigens or antigen-binding fragments thereof, and/or cytotoxic agents, and/or chemotherapeutic agents, and optionally, user's Guide.
  • a pharmaceutical composition comprising the anti-TGF ⁇ antibody of any one of items 1-2, the humanized anti-TGF ⁇ antibody of item 3, the bivalent or multivalent anti-TGF ⁇ antibody of item 4, or item 5
  • the multispecific antibody or the conjugate described in item 9; optionally, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition is Forms suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.
  • the anti-TGF ⁇ antibody described in any one of items 1-2, the humanized anti-TGF ⁇ antibody described in item 3, the bivalent or multivalent anti-TGF ⁇ antibody described in item 4, or the multispecific antibody described in item 5 The antibody or the conjugate described in Item 9 is used as a drug for treating and/or preventing tumors (for example, treating solid tumors), diabetes, systemic scleroderma, kidney disease, idiopathic pulmonary fibrosis and/or multiple fibrosis use in .
  • the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen.
  • the known antibody heavy and light chain variable region sequences there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia, and AbM numbering systems.
  • each application of the definition to the CDRs of an antibody or variant thereof will be within the scope of the terms as defined and used herein.
  • the variable region amino acid sequence of the antibody one skilled in the art can generally determine the specific CDRs without reliance on any experimental data other than the sequence itself.
  • Antibodies of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) having substitutions in one or more amino acid residues or (iii) a mature polypeptide fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence to form The polypeptide (such as a leader sequence or a secretory sequence or a sequence or protein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • a “conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g.
  • aspartic acid glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • side chains of ⁇ -branches for example, threonine, valine, isoleucine amino acids
  • aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine
  • nonessential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family.
  • a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or composition of side chain families.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between these two amino acids.
  • the conservative substitution is preferably a substitution wherein one amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small Aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e ) Aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala by Gly or by Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly replaced by Ala or replaced by Pro; His replaced by Asn or replaced by Gln; Ile replaced by Leu or replaced by Val; Leu replaced by Ile or replaced by Val; Lys replaced by Arg, replaced by Gln or replaced by Glu; Met Leu, Tyr, or Ile; Phe, Met, Leu, or Tyr; Ser, Thr; Thr, Ser; Trp, Tyr; Tyr, Trp; and/or Phe Replace with Val, replace with Ile or replace with Leu.
  • the antibodies of the invention can bind to a therapeutic agent (such as a chemotherapeutic drug, such as cisplatin, carboplatin), a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.
  • a therapeutic agent such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisp
  • the anti-TGF ⁇ antibody of the present invention has excellent biological activity and specificity, still maintains high affinity with TGF ⁇ , significantly improves the expression level, and has good stability of the antibody, especially in acidic environment and heat treatment conditions.
  • Figure 7 The biological activity of bispecific antibodies to inhibit the proliferation of HUVEC cells induced by VEGFA.
  • TGF ⁇ 1 (Z03411), TGF ⁇ 2 (Z03429) and TGF ⁇ 3 (Z03430) proteins used for immunization were purchased from GenScript. Two alpacas (Alpaca) were selected for antigen immunization, the first time was immunized with TGF ⁇ 3 1mg, and the next three times were immunized with TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 mixed in equal proportions (each 0.5mg). After the 4 times of immunization, lymphocytes from 100ml peripheral blood of alpaca were extracted, total RNA was extracted with RNAiso Plus reagent (TAKARA, 9109), and the extracted RNA was reverse transcribed into cDNA using PrimeScript II kit (TAKARA, 6210A).
  • TTKARA RNAiso Plus reagent
  • the vector pComb3XSS purchased from Apak Biotechnology, China
  • the ligation product was electrotransformed into Escherichia coli competent cell TG1, and the ligation product of each alpaca was transformed by electroporation 10 times.
  • the size of the library was calculated by gradient dilution plating, and the sizes of the phage libraries of the two alpacas were: 1.21 ⁇ 10 9 and 1 ⁇ 10 9 .
  • 96 clones were randomly selected from the titer plate for identification, and the results showed that the insertion rate was 100%.
  • the phages specifically bound to TGF ⁇ were eluted with glycine-hydrochloric acid (200 mM), and infected with Escherichia coli TG1 in logarithmic phase growth, and the phages were produced and purified for the next round of screening. After the same screening process was repeated for 3 rounds, the obtained phages were infected with Escherichia coli TG1 and plated, and single clones were picked from the plate and sequenced. According to the sequence comparison results, the protein sequences of each clone were analyzed, and the clones with different CDR1, CDR2, and CDR3 sequences were regarded as different antibody strains, and finally 17 different antibody strains were obtained.
  • the TG1 host bacteria of the obtained 17 heavy chain single domain antibodies were inoculated with a single colony and cultured overnight.
  • the overnight strain was transferred and amplified the next day, induced with 0.5mM IPTG, and cultured overnight at 37°C on a shaking table.
  • the next day centrifuge to collect the supernatant for detection.
  • the heavy chain variable region (SEQ ID NO: 1-17) corresponding to the coding DNA sequence cloned by PCR was connected with the coding DNA corresponding to the human IgG1 heavy chain constant region (GenBank No. AK303185.1) to construct the heavy chain chain antibody to obtain the expression plasmid of anti-TGF ⁇ single-domain heavy chain antibody, and the vector generally uses pcDNA3.1(-) (purchased from Invitrogen) or other eukaryotic expression vectors.
  • the endotoxin-free large-scale extraction plasmid kit (Qiagen, product number 12391) was used for plasmid extraction, and the specific operation was performed according to the instructions provided by the manufacturer.
  • CHO-S cells were cultured in CD CHO medium (Gibco, Cat. No. 10743-029) according to the instructions provided by the manufacturer, and placed in a 37°C, 5% CO2 cell incubator for cultivation. After the cells were prepared, the cells containing the heavy chain Sequence plasmids were co-transfected into CHO-S cells to express anti-TGF ⁇ antibodies. On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% 2 ⁇ EFC+ (Gibco, Cat. No.
  • A2503105 was supplemented every day. After 14 days of culture, the expression supernatant was harvested by centrifugation at 800 ⁇ g. And filter with 0.22 ⁇ m membrane filter. The anti-TGF ⁇ antibody in the culture supernatant was purified by protein A affinity chromatography and cation exchange chromatography. Purified antibody concentrations were determined by UV absorbance at 280 nm and corresponding extinction coefficients for each protein. Antibody purity and homogeneity were assessed by SDS-PAGE and SE-HPLC. Or use ion exchange and Superdex 200 SEC for secondary purification to prepare high-purity antibody samples for later use.
  • ELISA detection method was used to evaluate the affinity of 17 heavy chain single domain antibodies.
  • Example 5 Antibodies inhibit the biological activity of TGF- ⁇ 1/2/3
  • Antibody molecules affect the proliferation of TF-1 cells by inhibiting TGF- ⁇ 1/2/3, and use chemiluminescence to detect the number of cells.
  • TF-1 cells ATCC
  • a negative control (only TGF- ⁇ , no antibody) and a blank control group (only TF-1 cells, no TGF- ⁇ and antibody) were set up. Place in an incubator for 120 h, then add 80 ⁇ L of Cell Counting-Lite 2.0 Luminescent Cell Viability Assay (purchased from Vazyme, DD1101) reagent to each well for 10-15 min, and detect the absorbance at OD450 nm with a LUX multi-functional microplate reader.
  • GraphPad Prism6.0 software was used to analyze the data and compare the inhibitory effect of antibodies on the biological activity of TGF- ⁇ on TF-1 cells.
  • B2G8, B3F3 and B1B8 have better inhibitory activity on TGF ⁇ 1 than other candidate antibodies, and also have biological activity inhibitory effect on TGF- ⁇ 2/3.
  • antibodies B1B8, B2G8 and B3F3 have high affinity and strong biological inhibitory activity for TGF- ⁇ 1, and have cross-inhibitory activity for TGF- ⁇ 2/3.
  • Example 6 Construction and preparation of humanized heavy chain single domain antibody and bivalent heavy chain antibody
  • the main method of humanizing B2G8 is human IgG Germline IGHV3-23*01 as a template through CDR (complementarity determining region, complementarity determining region) transplantation, and the alpaca CDR sequence is grafted to the human heavy chain sequence , and carry out the amino acid back mutation of the framework region to obtain the humanized sequence shown in SEQ ID NO: 20-23.
  • the amino acid S marked by the box in the CDR2 of the humanized sequence shown in SEQ ID NO: 21-23 is mutated into amino acid A.
  • the plasmid preparation of the antibody is to construct the FcG2D heavy chain antibody by linking the gene-synthesized monoclonal antibody heavy chain variable region coding DNA sequence with the modified Fc coding DNA to obtain the anti-TGF ⁇ antibody expression plasmid.
  • the vector is generally pcDNA3.1( -) (purchased from Invitrogen) or other eukaryotic expression vectors.
  • the sequence information of the parental antibody (FcG2D-B2G8) and the humanized heavy chain antibody (FcG2D-huG89, FcG2D-huG891, FcG2D-huG892 and FcG2D-huG893) is shown in Table 4, and other heavy chain single domain antibodies were also designed ( Humanized antibodies including B1B8-Fc, B3F3-Fc, etc.).
  • bivalent heavy chain antibodies huG892-Fc-huG892 and B1B8-Fc-B1B8 were constructed, the specific sequences of which are shown in Table 4, and other bivalent heavy chain antibodies (including huG891-Fc-huG891, huG893-Fc- huG893, B2G8-Fc-B2G8, B3F3-Fc-B3F3, etc.).
  • the endotoxin-free large-scale extraction plasmid kit (Qiagen, product number 12391) was used for plasmid extraction, and the specific operation was performed according to the instructions provided by the manufacturer.
  • CHO-S cells were cultured in CD CHO medium (Gibco, Cat. No. 10743-029) according to the instructions provided by the manufacturer, and placed in a 37°C, 5% CO2 cell incubator for cultivation. After the cells were prepared, the cells containing the heavy chain The sequenced plasmids were co-transfected into CHO-S cells to express antibodies. On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% 2 ⁇ EFC+ (Gibco, Cat. No.
  • A2503105 was supplemented every day. After 14 days of culture, the expression supernatant was harvested by centrifugation at 800 ⁇ g. And filter with 0.22 ⁇ m membrane filter. The antibody in the culture supernatant was purified by protein A affinity chromatography and cation exchange chromatography. Purified antibody concentrations were determined by UV absorbance at 280 nm and corresponding extinction coefficients for each protein. Antibody purity and homogeneity were assessed by SDS-PAGE and SE-HPLC. Or use ion exchange and Superdex 200 SEC for secondary purification to prepare high-purity antibody samples for later use.
  • the antibodies in Table 4 were evaluated according to the following acid stability and thermal stability evaluation methods.
  • antibody molecules are subjected to protein A affinity chromatography, in the acid elution step (using citrate buffer at pH 3.5), the eluted antibody solution is not neutralized, and after being kept in the buffer for a period of time, At 30 minutes, a sample was added to 1/10 volume of 1M Tris-HCl (pH 8.0) for neutralization, and the HPLC-SEC detection of the sample was carried out.
  • the humanized antibody molecule did not show aggregation or degradation after being treated at pH 3.5 for 30 minutes, and the change in purity was less than 4%, indicating that it can maintain stability in an acidic environment.
  • the HPLC-SEC detection of the sample was carried out after 14 days of incubation in a 40°C incubator, no aggregation or degradation occurred, and the change in purity was less than 4%, indicating that it can maintain stability in an environment of 40°C, as shown in Table 6 , acid stability of other humanized antibodies and bivalent heavy chain antibodies (including huB1B8-Fc, huB3F3-Fc, huG891-Fc-huG891, huG893-Fc-huG893, B2G8-Fc-B2G8, B3F3-Fc-B3F3, etc.) Similar results were obtained from the thermal and thermal stability experiments.
  • Antibody molecules affect the proliferation of TF-1 cells by inhibiting TGF- ⁇ , and the number of cells is detected by chemiluminescence.
  • TF-1 cells ATCC
  • 1640 medium containing 10% FBS and 2ng/mL rhGM-CSF were inoculated into 96-well culture plates at 6000/well, and TGF- ⁇ 1 with a concentration of 16ng/mL was added to each well protein, and then add the antibody samples to be tested in serial dilutions, and successively dilute by 4 times, a total of 8 concentration gradients.
  • Antibodies obtained similar results.
  • huB1B8-Fc, huB3F3-Fc, huG891-Fc-huG891, huG893-Fc-huG893, B2G8-Fc-B2G8, B3F3-Fc-B3F3, etc. obtained similar results.
  • bispecific antibody amino acid sequence shown in Table 7 the reverse translation of the DNA code and the synthesis of the DNA fragment (Jinkarui) were performed, constructed into the expression vector pcDNA3.1, and transiently transfected into 293 or CHO cells for expression. The supernatant was harvested for protein purification to obtain bispecific antibodies with a purity of not less than 95%.
  • transient transfection and protein purification methods please refer to the previous examples.
  • the structure of the bispecific antibody is: comprising a light chain and a heavy chain, wherein the light-heavy chain pairs and forms an interchain disulfide bond, and two heavy chains pair and forms an interchain disulfide bond, wherein the heavy chain is (VH )-(CH1)-(hinge region)-(Fc)-(connecting peptide)-(VHH), light chain is (VL)-(light chain constant region), VH and VL are heavy chain and light chain of anti-VEGF antibody Chain variable region, VHH is the heavy chain variable region of anti-TGF- ⁇ single domain antibody.
  • the two prepared bispecific antibodies G6-Fc-huG892 and Beva-Fc-huG892 were subjected to thermal acceleration stability investigation, and the samples were detected by HPLC-SEC after being incubated in a 40°C incubator for 14 days, and there was no aggregation or degradation phenomenon , the change in purity is less than 4%, indicating that it can maintain stability in an environment of 40°C.
  • the results of the investigation are shown in Table 8. The results showed that the stability of the two bispecific antibodies was good.
  • G6-Fc-huG892 can bind human or mouse VEGF and human or mouse TGF- ⁇
  • Beva-Fc-huG892 can bind human VEGF and human or mouse TGF- ⁇ .
  • CT26 mouse colon cancer cell line, purchased from ATCC
  • EMT-6 mouse breast cancer cell line, purchased from ATCC
  • mice BALB/c wild-type mice, female, purchased from Beijing Weitong Lihua;
  • Inoculation method Cultivate CT26 cells and EMT-6 cells, collect and adjust the cell concentration, and subcutaneously inoculate 1*10 6 cells per mouse (0.1ml per mouse) on the back. When the tumor grows to 100-200mm, it will be administered in 3 groups, 6 rats in each group, and two groups for each type of cell;
  • Test drug G6-Fc-huG892.
  • Negative control normal saline
  • Administration method intraperitoneal administration was performed when the tumor grew to 100-200 mm 3 , and the administration dose was 10 mg/kg, and intraperitoneal administration was performed, starting on day 0, 3 times a week for 2 weeks.
  • Tumor volume The length and width of the tumor were measured 3 times a week. A group will be terminated when the tumor volume approaches 2000mm 3 or a single mouse tumor volume reaches 3000mm 3 .
  • Beva-Fc-huG892 was administered intraperitoneally to monitor the tumor size and evaluate the antitumor efficacy of the antibody.
  • A549 human lung cancer cell line, purchased from ATCC
  • mice balb/c-nude nude mice, female, purchased from Beijing Weitong Lihua;
  • Inoculation method culture A549 cells, collect and adjust the cell concentration, and subcutaneously inoculate on the back of 5 ⁇ 10 6 cells (0.1ml/body). When the tumor grows to 100-200mm, 3 groups are administered, 6 rats in each group, 2 groups in total;
  • Test drug Beva-Fc-huG892.
  • Negative control normal saline
  • Dosing method intraperitoneal administration was performed when the tumor grew to 100-200 mm 3 , the bispecific antibody was administered at a dose of 10 mg/kg, and intraperitoneal administration was performed separately, starting on day 0, 3 times a week, 2 week.
  • Tumor volume The length and width of the tumor were measured 3 times a week. A group will be terminated when the tumor volume approaches 2000mm 3 or a single mouse tumor volume reaches 3000mm 3 .
  • Antibody molecules affect the proliferation of HUVEC cells by inhibiting VEGF, and the number of cells is detected by chemiluminescence method and the inhibition rate is calculated.
  • HUVEC cells purchased from AllCells
  • HUVEC cells cultured in the complete medium of endothelial cells were inoculated into 96-well culture plates at 5000 cells/well, and cultivated overnight; the medium was discarded the next day, and 200 ng/mL VEGFA protein ( (purchased from GeneScript, Z02689), and then add the antibody samples to be tested Beva-Fc-hG892 and G6-Fc-hG892 in serial dilution, the highest concentration is 300 ⁇ g/ml, and then three-fold dilutions in sequence, and a total of 9-11 concentration gradients are set up.
  • Antibody name EC50( ⁇ g/mL) G6-Fc-hG892 2.090 Beva-Fc-hG892 2.602
  • Antibody molecules affect the proliferation of TF-1 cells by inhibiting TGF- ⁇ , and the number of cells is detected by chemiluminescence.
  • TF-1 cells ATCC
  • 1640 medium containing 10% FBS and 2ng/mL rhGM-CSF were inoculated into 96-well culture plates at 1000 cells/well, and TGF- ⁇ 1 at a concentration of 1ng/mL was added to each well (GenScript, Z03411) or TGF- ⁇ 2 (GenScript, Z03429) or TGF- ⁇ 3 (GenScript, Z03430) protein, and then add the antibody samples to be tested Beva-Fc-hG892 and G6-Fc-hG892 in serial dilution, the highest concentration is 100nM, successively 5-fold dilution, a total of 9-11 concentration gradients were set up, and 3 replicate wells.

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Abstract

本发明涉及抗TGFβ抗体，例如B2G8，其包含SEQ ID NO：7所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和SEQ ID NO：51所示的CDR3，及所述抗TGFβ抗体的应用。

Description

抗TGFβ抗体及其制备方法和应用 技术领域
本发明涉及医药领域，具体地涉及TGF-β(transforming growth factor beta，转化生长因子β)的抗体及其制备方法和应用。
背景技术
TGF-β(transforming growth factor beta，转化生长因子β)是一种多功能的细胞因子，在调节细胞的生长和分化中起重要作用。TGF-β是由两个结构相同或相近的、分子量为12.5kDa的亚单位借二硫键连接的双体。在哺乳动物至少发现有TGF-β1、TGF-β2、TGF-β3、TGF-β1β2四个亚型。人TGF-β1、TGF-β2和TGF-β3的基因分别定位于染色体19q3、1q41和14q24。人和小鼠TGF-β1的同源性高达99％，TGF-β与受体结合后通过Smad与非Smad信号通路介导一系列的生物学反应，包括促细胞上皮-间质转化，促组织纤维化，促血管生成，促肿瘤的免疫逃逸，抑癌和促癌双重作用等。由于过度活化的TGF-β及其受体所介导的信号通路在多种疾病如恶性肿瘤及组织纤维化的发生、发展中具有重要的病理生理学作用，所以针对该分子的抑制剂已被用于阻断该信号通路，从而为控制这些疾病提供了治疗方案。
转化生长因子β1(TGF-β1)广泛参与体内各种病理生理过程，与炎症、创伤、器官纤维化等多种疾病的发生、发展关系密切，尤其是在肿瘤的发生、发展中的调节作用。跟TGF-β1以及TGF-β3一样，TGF-β2在细胞的增殖、分化、胚胎发育、胞外基质形成、骨的形成和重建以及肿瘤的抑制和转移扩散等方面也起着重要的作用。TGF-β2被指出可以抑制IL-2依赖的T细胞的生长，并且能抑制肿瘤发展中的免疫监视，因而以一种自分泌的方式促进肿瘤生长。TGF-β2可以影响杀伤细胞的活力和降低IL-2，IL-6，IL-10，IFN-γ等细胞因子的表达。TGF-β3在组织修复过程中刺激靶细胞(成纤维细胞、血管内皮细胞等)细胞外基质合成和加快血管化进程，且促进创面愈合和减轻瘢痕形成。TGF-β3在哺乳动物胚胎发育过程中，能够促进形态发生、并对脊椎形成、肢端发芽、出牙、面骨形成及心脏瓣膜的形成起重要作用。TGF-β3可调控骨形成，可影响成年骨再生。
针对TGF-β的药物适应症比较广泛，包括肾病，系统性硬皮病，实体瘤，特发性肺纤维化，糖尿病等，但目前没有针对TGF-β的药物上市。
发明内容
本发明提供一种针对TGFβ的特异性抗体。具体地，本发明提供以下几个方面：
1.抗TGFβ抗体，其包含选自以下各项组成的组的序列，或由其组成：
(1)B2G8，其包含SEQ ID NO：7所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和SEQ ID NO：51所示的CDR3，或其变体，所述变体为与 SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和/或SEQ ID NO：51所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列，优选地包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2的变体和SEQ ID NO：51所示的CDR3，其中所述SEQ ID NO：50所示的CDR2的变体为在SEQ ID NO：50所示氨基酸序列的第13位氨基酸置换为丙氨酸；或者
其包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和SEQ ID NO：51所示的CDR3，其中SEQ ID NO：49所示的CDR1中第5-8位氨基酸、SEQ ID NO：50所示的CDR2中第1-5位氨基酸、第7-9位氨基酸、SEQ ID NO：51所示的CDR3第1位氨基酸、第4-9位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
(2)B1B8，其包含SEQ ID NO：4所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2和SEQ ID NO：42所示的CDR3，或其变体，所述变体为与SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2和/或SEQ ID NO：42所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；或者
其包含SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2和SEQ ID NO：42所示的CDR3，其中SEQ ID NO：40所示的CDR1中第3位氨基酸、第5-8位氨基酸、SEQ ID NO：41所示的CDR2中第1位、第3位和第5-10位氨基酸、SEQ ID NO：42所示的CDR3第4-9位和第11位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
(3)B3F3，其包含SEQ ID NO：16所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：76所示的CDR1，SEQ ID NO：77所示的CDR2和SEQ ID NO：78所示的CDR3，或其变体，所述变体为与SEQ ID NO：76所示的CDR1，SEQ ID NO：77所示的CDR2和/或SEQ ID NO：78所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；或者
其包含SEQ ID NO：76所示的CDR1，SEQ ID NO：77所示的CDR2和SEQ ID NO：78所示的CDR3，其中SEQ ID NO：76所示的CDR1中第2-3位氨基酸、第5-8位氨基酸、SEQ ID NO：77所示的CDR2中第2-4位氨基酸、第6位氨基酸、SEQ ID NO：78所示的CDR3第1位氨基酸、第3-8位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
(4)B2A1，其包含SEQ ID NO：1所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：31所示的CDR1，SEQ ID  NO：32所示的CDR2和SEQ ID NO：33所示的CDR3，或其变体，所述变体为与SEQ ID NO：31所示的CDR1，SEQ ID NO：32所示的CDR2和/或SEQ ID NO：33所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(5)B1F8，其包含SEQ ID NO：5所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：43所示的CDR1，SEQ ID NO：44所示的CDR2和SEQ ID NO：45所示的CDR3，或其变体，所述变体为与SEQ ID NO：43所示的CDR1，SEQ ID NO：44所示的CDR2和/或SEQ ID NO：45所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(6)B3D6，其包含SEQ ID NO：6所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：46所示的CDR1，SEQ ID NO：47所示的CDR2和SEQ ID NO：48所示的CDR3，或其变体，所述变体为与SEQ ID NO：46所示的CDR1，SEQ ID NO：47所示的CDR2和/或SEQ ID NO：48所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(7)B2A9，其包含SEQ ID NO：2所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：34所示的CDR1，SEQ ID NO：35所示的CDR2和SEQ ID NO：36所示的CDR3，或其变体，所述变体为与SEQ ID NO：34所示的CDR1，SEQ ID NO：35所示的CDR2和/或SEQ ID NO：36所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(8)B2C2，其包含SEQ ID NO：8所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：52所示的CDR1，SEQ ID NO：53所示的CDR2和SEQ ID NO：54所示的CDR3，或其变体，所述变体为与SEQ ID NO：52所示的CDR1，SEQ ID NO：53所示的CDR2和/或SEQ ID NO：54所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(9)B1B7，其包含SEQ ID NO：9所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：55所示的CDR1，SEQ ID NO：56所示的CDR2和SEQ ID NO：57所示的CDR3，或其变体，所述变体为与SEQ ID NO：55所示的CDR1，SEQ ID NO：56所示的CDR2和/或SEQ ID NO：57所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(10)B1C5，其包含SEQ ID NO：10所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：58所示的CDR1，SEQ ID NO：59所示的CDR2和SEQ ID NO：60所示的CDR3，或其变体，所述变体为与SEQ ID NO：58所示的CDR1，SEQ ID NO：59所示的CDR2和/或SEQ ID NO：60 所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(11)B2F6，其包含SEQ ID NO：11所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：61所示的CDR1，SEQ ID NO：62所示的CDR2和SEQ ID NO：63所示的CDR3，或其变体，所述变体为与SEQ ID NO：61所示的CDR1，SEQ ID NO：62所示的CDR2和/或SEQ ID NO：63所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(12)B2H1，其包含SEQ ID NO：12所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：64所示的CDR1，SEQ ID NO：65所示的CDR2和SEQ ID NO：66所示的CDR3，或其变体，所述变体为与SEQ ID NO：64所示的CDR1，SEQ ID NO：65所示的CDR2和/或SEQ ID NO：66所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(13)B2A2，其包含SEQ ID NO：13所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：67所示的CDR1，SEQ ID NO：68所示的CDR2和SEQ ID NO：69所示的CDR3，或其变体，所述变体为与SEQ ID NO：67所示的CDR1，SEQ ID NO：68所示的CDR2和/或SEQ ID NO：69所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(14)B3D2，其包含SEQ ID NO：14所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：70所示的CDR1，SEQ ID NO：71所示的CDR2和SEQ ID NO：72所示的CDR3，或其变体，所述变体为与SEQ ID NO：70所示的CDR1，SEQ ID NO：71所示的CDR2和/或SEQ ID NO：72所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(15)B3C5，其包含SEQ ID NO：15所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：73所示的CDR1，SEQ ID NO：74所示的CDR2和SEQ ID NO：75所示的CDR3，或其变体，所述变体为与SEQ ID NO：73所示的CDR1，SEQ ID NO：74所示的CDR2和/或SEQ ID NO：75所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
(16)B2C9，其包含SEQ ID NO：3所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：37所示的CDR1，SEQ ID NO：38所示的CDR2和SEQ ID NO：39所示的CDR3，或其变体，所述变体为与SEQ ID NO：37所示的CDR1，SEQ ID NO：38所示的CDR2和/或SEQ ID NO：39所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；和
(17)B3F1，其包含SEQ ID NO：17所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：79所示的CDR1，SEQ ID NO：80所示的CDR2和SEQ ID NO：81所示的CDR3，或其变体，所述变体为与SEQ ID NO：79所示的CDR1，SEQ ID NO：80所示的CDR2和/或SEQ ID NO：81所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列。
2.项目1所述的抗TGFβ抗体，其包含选自SEQ ID NO：1-17所示的序列或其变体，或由选自SEQ ID NO：1-17所示的序列或其变体组成；其中所述变体为与序列编号所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性并保留与TGFβ结合亲和力的序列，或与序列编号所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列。
3.人源化抗TGFβ抗体，其包含项目1所述的抗TGFβ抗体的CDR1，CDR2和CDR3，优选地包含来自人IgG的Germline IGHV3-23*01的FR区，优选地还包含Fc恒定区，所述恒定区包含SEQ ID NO：18所示序列或人IgG1重链Fc恒定区(GenBank No.AK303185.1)，更优选地，人源化抗TGFβ抗体包含SEQ ID NO：20所示的人源化序列或由其组成，进一步优选地，在SEQ ID NO：20所示人源化序列的CDR2第13位的丝氨酸Ser突变为丙氨酸Ala，最优选地，人源化抗TGFβ抗体包含选自SEQ ID NO：21-23所示的人源化序列或由其组成。
4.二价或多价抗TGFβ抗体，其包含2个以上的项目1-2任一项所述的抗TGFβ抗体，或包含2个以上的项目3所述的人源化抗TGFβ抗体，优选地，所述二价抗TGFβ抗体的序列选自以下各项组成的组：(1)由SEQ ID NO：22，19，18，19和22所示的序列按照由N端至C端首尾顺序连接而成；(2)由SEQ ID NO：4，19，18，19和4所示的序列按照由N端至C端首尾顺序连接而成；(3)由SEQ ID NO：23，19，18，19和23所示的序列按照由N端至C端首尾顺序连接而成；(4)由SEQ ID NO：21，19，18，19和21所示的序列按照由N端至C端首尾顺序连接而成；(5)由SEQ ID NO：7，19，18，19和7所示的序列按照由N端至C端首尾顺序连接而成；和(6)由SEQ ID NO：16，19，18，19和16所示的序列按照由N端至C端首尾顺序连接而成。
5.多特异性抗体，其包含项目1-2任一项所述的抗TGFβ抗体或项目3所述的人源化抗TGFβ抗体，优选双特异性抗体，所述双特异性抗体包含针对第一抗原TGFβ的第一抗体和针对第二抗原的第二抗体，其中所述第一抗体选自项目1-2任一项所述的抗TGFβ抗体或项目3所述的人源化抗TGFβ抗体，第二抗原选自免疫细胞表面抗原、肿瘤抗原、病毒、细菌、内毒素、细胞因子、或其组合；更优选地选自：PD-L1，PD-1，VEGFA，IL-10，IL-10R，BCMA，VEGF，TGF-β，CTLA-4，LAG-3，TIGIT，CEA，CD38，SLAMF7，B7-H3，Her2，EpCAM，CD19，CD20，CD30，CD33，CD47，CD52，CD133，EGFR，GD2，GD3，GM2，RANKL， CD3，和/或CD16a，优选第二抗原为VEGF，更优选第二抗体选自抗VEGF抗体，其包含(i)SEQ ID NO：82所示的HCDR1，SEQ ID NO：83所示的HCDR2，SEQ ID NO：84所示的HCDR3，SEQ ID NO：85所示的LCDR1，SEQ ID NO：86所示的LCDR2和SEQ ID NO：87所示的LCDR3，或(ii)SEQ ID NO：88所示的HCDR1，SEQ ID NO：89所示的HCDR2，SEQ ID NO：90所示的HCDR3，SEQ ID NO：91所示的LCDR1，SEQ ID NO：92所示的LCDR2和SEQ ID NO：93所示的LCDR3；更优选地，抗VEGF抗体包含(a)SEQ ID NO：24所示的重链可变区或与SEQ ID NO：24所示的重链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，和SEQ ID NO：27所示的轻链可变区或与SEQ ID NO：27所示的轻链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，或(b)SEQ ID NO：29所示的重链可变区或与SEQ ID NO：29所示的重链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，和SEQ ID NO：30所示的轻链可变区或与SEQ ID NO：30所示的轻链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列；最优选地，抗VEGF抗体进一步包含SEQ ID NO：25所示的CH1，SEQ ID NO：26所示的铰链区，SEQ ID NO：18所示的Fc恒定区，和SEQ ID NO：28所示的轻链恒定区。
6.多核苷酸，其编码项目1-2任一项所述的抗TGFβ抗体或项目3所述的人源化抗TGFβ抗体，或项目4所述的二价或多价抗TGFβ抗体，或项目5所述的多特异性抗体。
7.载体，其包含项目6所述的多核苷酸。
8.宿主细胞，其包含项目7所述的载体。
9.偶联物，其包含项目1-2任一项所述的抗TGFβ抗体、项目3所述的人源化抗TGFβ抗体、项目4所述的二价或多价抗TGFβ抗体或项目5所述的多特异性抗体，以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)、可检测的标记、药物、毒素、细胞因子、酶、或其组合；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、化疗剂、生物毒素、聚乙二醇或酶。
10.试剂盒，其包括项目1-2任一项所述的抗TGFβ抗体、项目3所述的人源化抗TGFβ抗体、项目4所述的二价或多价抗TGFβ抗体、项目5所述的多特异性抗体或项目9所述的偶联物；优选地，所述试剂盒还包括第二抗体，其特异性识别项目1-2任一项所述的抗TGFβ抗体、项目3所述的人源化抗TGFβ抗体、项目4所述的二价或多价抗TGFβ抗体、项目5所述的多特异性抗体或项目9所述的偶联物；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测VEGF在样品中的存在或其水平；或者
所述试剂盒包含(1)项目1-2任一项所述的抗TGFβ抗体、项目3所述的人源 化抗TGFβ抗体、项目4所述的二价或多价抗TGFβ抗体、项目5所述的多特异性抗体或项目9所述的偶联物，和(2)针对其它抗原的抗体或其抗原结合片段，和/或细胞毒性剂，和/或化疗药，和任选地，使用说明书。
11.药物组合物，其包含项目1-2任一项所述的抗TGFβ抗体、项目3所述的人源化抗TGFβ抗体、项目4所述的二价或多价抗TGFβ抗体、项目5所述的多特异性抗体或项目9所述的偶联物；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂；优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
12.项目1-2任一项所述的抗TGFβ抗体、项目3所述的人源化抗TGFβ抗体、项目4所述的二价或多价抗TGFβ抗体、项目5所述的多特异性抗体或项目9所述的偶联物用于治疗和/或预防肿瘤(例如治疗实体瘤)、糖尿病、系统性硬皮病、肾病、特发性肺纤维化和/或多发性纤维化的药物中的用途。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
在本发明中涉及的术语具备本领域技术人员理解的常规含义。在本技术领域内使用和/或可接受的情况下，一个术语有两个或两个以上定义时，本文使用的术语的定义用于包括所有的含义。
本领域普通技术人员可以理解，抗体的CDR区负责抗体对抗原的结合特异性。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定特定CDR，而不依赖于该序列自身之外的任何实验数据。
本发明的抗体可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
“保守氨基酸替换”是其中氨基酸残基被具有类似侧链的氨基酸残基替换。具有类似侧链的氨基酸残基家族已在本领域中定义，其包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，β-支链的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽的非必需氨基酸残基优选被 来自相同侧链家族的其他氨基酸残基替换。在另一些实施方案中，一串氨基酸可被结构上类似的氨基酸串替换，后者在顺序上和/或侧链家族的组成上不同。
在下表中提供了保守性氨基酸替换的非限制性实例，其中相似性得分为0或更高表示在这两个氨基酸之间有保守替换。
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
在一些实施方案中，所述保守替换优选地是这样的替换，即，其中下列组(a)-(e)内的一个氨基酸被同组内的另一氨基酸残基替换：(a)小的脂肪族、非极性或弱极性的残基：Ala，Ser，Thr，Pro和Gly；(b)极性、带负电荷的残基及其(不带电荷的)酰胺：Asp，Asn，Glu和Gln；(c)极性、带正电荷的残基：His，Arg和Lys；(d)大的脂肪族、非极性残基：Met，Leu，Ile，Val和Cys；以及(e)芳族残基：Phe，Tyr和Trp。
特别优选的保守替换如下：Ala替换成Gly或替换成Ser；Arg替换成Lys；Asn替换成Gln或替换成His；Asp替换成Glu；Cys替换成Ser；Gln替换成Asn；Glu替换成Asp；Gly替换成Ala或替换成Pro；His替换成Asn或替换成Gln；Ile替换成Leu或替换成Val；Leu替换成Ile或替换成Val；Lys替换成Arg，替换成Gln或替换成Glu；Met替换成Leu，替换成Tyr或替换成I le；Phe替换成Met，替换成Leu或替换成Tyr；Ser替换成Thr；Thr替换成Ser；Trp替换成Tyr；Tyr替换成Trp；和/或Phe替换成Val，替换成Ile或替换成Leu。
在一些实施方案中，本发明所述抗体可以结合治疗剂(如化疗药，如顺铂、卡铂)、药物前体、肽、蛋白、酶、病毒、脂质、生物反应调节剂、药剂或PEG。本发明抗体可以连接至或融合至治疗剂上，该治疗剂可包括可检测标记物，如放射性标记物、免疫调节剂、激素、酶、寡核苷酸、光活性治疗剂或诊断剂、细胞毒性剂，其可为药物或毒素，超声增强剂，非放射性标记物，它们的组合和其他这类本领域已知的成分。
与现有技术相比，本发明的有益效果为：
本发明的抗TGFβ抗体具有优异的生物活性和特异性，仍然保持与TGFβ的高亲和力，表达水平显著提高，抗体稳定性好，尤其在酸性环境和热处理条件下仍保持良好稳定性。
附图说明
图1.抗体抑制TGF-β1的生物学活性。
图2.抗体抑制TGF-β2的生物学活性。
图3.抗体抑制TGF-β3的生物学活性。
图4.人源化单域抗体抑制TGF-β1的生物学活性。
图5.双特异性抗体G6-Fc-huG892对于小鼠皮下瘤CT-26和EMT-6的体内药效。
图6.双特异性抗体Beva-Fc-huG892对于小鼠异种移植瘤A549的体内药效。
图7.双特异性抗体抑制VEGFA诱导的HUVEC细胞增殖的生物学活性。
图8.双特异性抗体抑制TGFβ1/2/3的生物学活性。
具体实施方式
下面通过实施例详细描述本发明。本领域的普通技术人员可以理解，下述实施例仅是用于举例说明的目的。本发明的精神和范围由权利要求所限定。下述实施例中所用方法如无特别说明均为常规方法，所用的试剂如无特别说明均为可商购的试剂。
实施例1：针对TGFβ的重链单域抗体的筛选
1.1重链单域抗体噬菌体文库的构建：
免疫用的TGFβ1(Z03411)、TGFβ2(Z03429)和TGFβ3(Z03430)蛋白购自金斯瑞。选取两只羊驼(Alpaca)进行抗原免疫，第一次用TGFβ3 1mg免疫，接下来的三次用TGFβ1、TGFβ2和TGFβ3等比例混合后免疫(各0.5mg)。4次免疫结束后，提取羊驼100ml外周血的淋巴细胞，用RNAiso Plus试剂(TAKARA，9109)提取总RNA，使用PrimeScript II试剂盒(TAKARA，6210A)将提取的RNA反转录成cDNA。用PCR扩增的方法先扩增750bp长的重链抗体可变区和恒定区CH2的核酸片段，然后以上一步中回收的750bp长的核酸片段为模板扩增目的片段，即重链抗体可变区片段。将载体pComb3XSS(购自阿帕克生物科技，中国)与目的片段分别用SfiI进行酶切，50℃过夜酶切，回收目的片段，按照连接摩尔比例载体∶片段＝1∶3的比例连接。将连接产物电转化至大肠杆菌感受态细胞TG1中，每一只羊驼的连接产物进行10次电击转化。通过梯度稀释铺板，计算库容的大小，两只羊驼的噬菌体文库大小分别为：1.21×10 9和1×10 9。随机从测滴度平板上挑取96个克隆进行鉴定，结果表明插入率为100％。
1.2针对TGFβ1的重链单域抗体淘选：
用TGFβ1蛋白10μg/孔包被平板，4℃过夜。第二天用1％BSA室温封闭2小时后，加入100μl噬菌体(2×10 9pfu/孔，来自1.1所构建的重链单域抗体噬菌文库)，37℃孵育1小时。之后用PBST(PBS中含有0.05％吐温20)洗5遍，以洗掉不结合的噬菌体。最后用甘氨酸-盐酸(200mM)将与TGFβ特异性结合的噬菌体洗脱下来，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮 的筛选。相同筛选过程重复3轮后，将获得的噬菌体感染大肠杆菌TG1并铺板，从平板上挑取单克隆并测序。根据序列比对结果分析各个克隆的蛋白序列，把CDR1、CDR2、CDR3序列不相同的克隆视为不同抗体株，最终共获得17株不同的抗体。
表1.重链单域抗体可变区氨基酸序列信息(Numbering Scheme IMGT和Kabat)
Figure PCTCN2021113494-appb-000001
Figure PCTCN2021113494-appb-000002
Figure PCTCN2021113494-appb-000003
实施例2：针对TGFβ的重链单域抗体的初步评价鉴定
2.1重链单域抗体在宿主菌大肠杆菌中表达：
将所获得17株重链单域抗体的TG1宿主菌挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，0.5mM IPTG诱导，37℃摇床培养过夜。第二天，离心收集上清检测。
2.2ELISA检测17个重链单域抗体亲和力：
将TGFβ1蛋白溶液和BSA分别以2μl/孔包被酶标板，4度过夜；弃上清，每孔加入300μl封闭液(含3％BSA的PBS)，37度封闭2小时；将离心收集的上清以每孔200μl加入到酶标板孔中，室温下静置孵育2小时，弃上清；用200μl/孔PBST(PBS含0.1％Tween20)洗涤3次；加入稀释好的HRP标记的抗VHH二抗(Genscript，A01861)，二抗以1∶10000稀释后使用，稀释液为1％BSA的PBS，体积为100μl/孔，室温孵育1小时；用200μl/孔PBST洗涤5次；加TMB显色液(BD，55214)100μl/孔，37度显色8分钟。加2M HCl终止液100μl/孔，终止液加入后，在30分钟内进行酶标仪450nm读数。结果见下表2。
表2.重链单域抗体与TGFβ1抗原结合的OD值
Figure PCTCN2021113494-appb-000004
Figure PCTCN2021113494-appb-000005
实施例3：抗体的质粒制备、表达与纯化
通过将PCR克隆的单抗重链可变区(SEQ ID NO：1～17)对应的编码DNA序列与人IgG1重链恒定区(GenBank No.AK303185.1)对应的编码DNA连接，来构建重链抗体，以获得抗TGFβ的单域重链抗体表达质粒，载体一般用pcDNA3.1(-)(购自Invitrogen)或其他真核表达载体。所述的重链抗体蛋白序列的可变区与恒定区之间使用连接肽相连，所述连接肽为(GGGGS)n，n＝1、2或者3。
利用无内毒素大量提取质粒试剂盒(Qiagen，货号12391)进行质粒提取，具体操作按照厂商提供的说明书进行。CHO-S细胞培养根据厂商提供的说明书在CD CHO培养基(Gibco，货号10743-029)中，置于37℃，5％CO 2细胞培养箱中进行培养，准备好细胞后，将含有重链序列的质粒一起共转染到CHO-S细胞中，以表达抗TGFβ的抗体。在转染后的第二天，培养温度下调到32℃，并每天补加3.5％2×EFC+(Gibco，货号A2503105)，培养14天后，800×g离心收获表达上清。并用0.22μm滤膜过滤。通过蛋白A亲和层析和阳离子交换层析纯化得到培养上清液中的抗TGFβ抗体。通过在280nm处的UV吸光度以及每种蛋白对应的消光系数测定纯化的抗体浓度。通过SDS-PAGE和SE-HPLC评估抗体的纯度和均质性。或者使用离子交换和Superdex 200的SEC进行二次纯化制备高纯度抗体样品备用。
实施例4：抗体结合TGF-β的亲和力
本实施例使用ELISA检测方法评估17个重链单域抗体亲和力。
将人TGF-β1/2/3蛋白溶液分别以2μl/孔包被酶标板，4℃过夜；弃上清，每孔加入300μl封闭液(含3％BSA的PBS)，37℃封闭2小时；梯度稀释抗体，稀释液为1％BSA的PBS。比如抗体稀释的初始浓度为500nM，10倍稀释，稀释成8个浓度梯度。将稀释好的抗体以每孔200μl加入到酶标板孔中，室温下静置孵育2小时，弃上清；用200μl/孔PBST(PBS含0.1％Tween20)洗涤3次；加入稀释好的HRP标记的抗人Fc二抗(SIGMA，A8667)，二抗以1∶20000稀释后使用，稀释液为1％BSA的PBS，体积为100μl/孔，室温孵育1小时；用200μl/孔PBST洗涤5次；加TMB显色液(BD，55214)100μl/孔，37℃显色8分钟。加2M HCl终止液100μl/孔，终止液加入后，在30分钟内进行酶标仪450nm读 数。用GraphPad Prism6.0软件分析数据，进行亲和力的拟合，得到EC50值，结果见表3。
表3.重链单域抗体对TGFβ蛋白的亲和力
Figure PCTCN2021113494-appb-000006
实施例5：抗体抑制TGF-β1/2/3的生物学活性
抗体分子通过抑制TGF-β1/2/3而影响TF-1细胞的增殖，使用化学发光法检测细胞的数量。将在含10％FBS、2ng/mL rhGM-CSF(R&D，货号7954-GM-050/CF)的1640培养基中培养的TF-1细胞(ATCC)以1000个/孔接种到96孔培养板，每孔加入浓度1ng/mL的TGF-β1/2/3蛋白，再分别加入梯度稀释的待测抗体样品，最高浓度为100nM，依次20倍稀释，共3个浓度梯度。同时设置阴性对照(只加TGF-β，不加抗体)及空白对照组(仅TF-1细胞，TGF-β及抗体均不加)。放入培养箱培养120h，再每孔加入80μL Cell Counting-Lite 2.0 Luminescent Cell Viability Assay(购自Vazyme，DD1101)试剂反应10-15min，用LUX多功能酶标仪检测OD450nm处的吸光度。用GraphPad Prism6.0软件分析数据，比较抗体对TGF-β在TF-1细胞上生物学活性的抑制作用。
结果见图1、图2和图3。B2G8、B3F3和B1B8对TGFβ1抑制活性优于其它候选抗体，同时对TGF-β2/3也有生物学活性抑制作用。结合实施例2抗体结合TGF-β的亲和力的结果，抗体B1B8，B2G8和B3F3对TGF-β1具有高亲和力和强生物学抑制活性，同时对TGF-β2/3具有交叉抑制活性。
实施例6：人源化重链单域抗体及二价重链抗体构建制备
将B2G8进行人源化改造，主要实施方法是人IgG的Germline IGHV3-23*01 为模板通过CDR(complementarity determining region，互补决定区)移植，将羊驼的CDR序列移植到人的重链序列上，并进行框架区的氨基酸回复突变，获得序列如SEQ ID NO：20-23所示的人源化序列。其中SEQ ID NO：21-23所示的人源化序列的CDR2中方框标记的氨基酸S突变为氨基酸A。抗体的质粒制备通过将基因合成的单抗重链可变区编码DNA序列与改造后的Fc编码DNA连接，来构建FcG2D重链抗体，以获得抗TGFβ抗体表达质粒，载体一般用pcDNA3.1(-)(购自Invitrogen)或其他真核表达载体。母本抗体(FcG2D-B2G8)和人源化重链抗体(FcG2D-huG89、FcG2D-huG891、FcG2D-huG892和FcG2D-huG893)的序列信息如表4所示，同样设计其它重链单域抗体(包括B 1B8-Fc、B3F3-Fc等)的人源化抗体。此外，还构建了二价重链抗体huG892-Fc-huG892和B1B8-Fc-B1B8，具体序列如表4所示，同样设计其它二价重链抗体(包括huG891-Fc-huG891、huG893-Fc-huG893、B2G8-Fc-B2G8、B3F3-Fc-B3F3等)的序列。
表4.母本抗体和人源化重链抗体序列信息(Numbering Scheme IMGT)
Figure PCTCN2021113494-appb-000007
Figure PCTCN2021113494-appb-000008
Figure PCTCN2021113494-appb-000009
利用无内毒素大量提取质粒试剂盒(Qiagen，货号12391)进行质粒提取，具体操作按照厂商提供的说明书进行。CHO-S细胞培养根据厂商提供的说明书在CD CHO培养基(Gibco，货号10743-029)中，置于37℃，5％CO 2细胞培养箱中进行培养，准备好细胞后，将含有重链序列的质粒一起共转染到CHO-S细胞中表达抗体。在转染后的第二天，培养温度下调到32℃，并每天补加3.5％2×EFC+(Gibco，货号A2503105)，培养14天后，800×g离心收获表达上清。并用0.22μm滤膜过滤。通过蛋白A亲和层析和阳离子交换层析纯化得到培养上清液中的抗体。通过在280nm处的UV吸光度以及每种蛋白对应的消光系数测定纯化的抗体浓度。通过SDS-PAGE和SE-HPLC评估抗体的纯度和均质性。或者使用离子交换和Superdex 200的SEC进行二次纯化制备高纯度抗体样品备用。
实施例7：抗体的酸稳定性和热稳定性评估
按照以下酸稳定性和热稳定性评估方法对表4中抗体进行评估。抗体分子进行protein A亲和层析时，在酸洗脱步骤中(使用pH3.5的柠檬酸缓冲液)，洗脱下来的抗体溶液不进行中和，在该缓冲液中保持一段时间后，在第30min取样加入1/10体积的1M Tris-HCl(pH 8.0)进行中和，并进行该样品的HPLC-SEC检测。如表5所示，人源化抗体分子在经过pH 3.5处理30min后未出现聚集或降解现象，纯度变化小于4％，说明其在酸性环境中能保持稳定性。同时，在经40℃培养箱孵育14天后进行该样品的HPLC-SEC检测，未出现聚集或降解现象，纯度变化小于4％，说明其在40℃环境中能保持稳定性，如表6所示，其它人源化抗体和二价重链抗体(包括huB1B8-Fc、huB3F3-Fc、huG891-Fc-huG891、huG893-Fc-huG893、B2G8-Fc-B2G8、B3F3-Fc-B3F3等)的酸稳定性和热稳定性实验获得了类似的结果。
表5.抗体酸稳定性评估结果
Figure PCTCN2021113494-appb-000010
Figure PCTCN2021113494-appb-000011
表6.抗体热稳定性评估结果
Figure PCTCN2021113494-appb-000012
结果显示，人源化分子FcG2D-hu89，FcG2D-hu891，FcG2D-hu892，FcG2D-hu893，huG892-Fc-huG892，和B1B8-Fc-B1B8均具有良好的酸稳定性和热稳定性。且通过proteinA的纯化后，抗体的纯度均大于96％，显示出良好的表达稳定性。
实施例8：抑制TGF-β生物学活性
抗体分子通过抑制TGF-β而影响TF-1细胞的增殖，使用化学发光法检测细胞的数量。将在含10％FBS、2ng/mL rhGM-CSF的1640培养基中培养的TF-1细胞(ATCC)以6000个/孔接种到96孔培养板，每孔加入浓度16ng/mL的TGF-β1蛋白，再分别加入梯度稀释的待测抗体样品，依次4倍稀释，共8个浓度梯度。放入培养箱培养120小时，再每孔加入80μL Cell Counting-Lite 2.0 Luminescent Cell Viability Assay(购自Vazyme，DD1101)试剂反应10-15min，用LUX多功能酶标仪检测读取化学发光值。用GraphPad Prism6.0软件分析数据，以抗体终浓度为横坐标，化学发光值(实验孔读值-阴性对照孔读值)为纵坐标，将数据进行四参数非线性回归，拟合量效曲线，计算供试品和参比品的半效浓度(EC50)。比较抗体对TGF-β1在TF-1细胞上生物学活性的抑制作用。
结果见图4。FcG2D-huG89、FcG2D-huG891和FcG2D-huG892的EC50值 与母本FcG2D-B2G8相差一倍以内，表明这3个人源化分子具有较强的抑制活性。同样的方法对huG892-Fc-huG892和B1B8-Fc-B1B8进行抑制TGF-β1活性评估，EC50值分别为0.424nM和0.813nM，都具有较强的活性，其它人源化抗体和二价重链抗体(包括huB1B8-Fc、huB3F3-Fc、huG891-Fc-huG891、huG893-Fc-huG893、B2G8-Fc-B2G8、B3F3-Fc-B3F3等)获得了类似的结果。
实施例9：构建双特异性抗体及应用
9.1双特异性抗体构建
按表7所示的双特异性抗体氨基酸序列，进行DNA编码的逆向翻译和DNA片段的合成(金开瑞)，构建至表达载体pcDNA3.1中，瞬时转染至293或者CHO细胞中表达。收获上清进行蛋白纯化，获得纯度不低于95％的双特异性抗体。具体载体构建，瞬时转染和蛋白纯化方法参加前述的各实施例。所述双特异性抗体的结构为：包含轻链和重链，其中轻-重链配对并形成链间二硫键，两个重链配对并形成链间二硫键，其中重链为(VH)-(CH1)-(铰链区)-(Fc)-(连接肽)-(VHH)，轻链为(VL)-(轻链恒定区)，VH和VL为抗VEGF抗体的重链和轻链可变区，VHH为抗TGF-β单域抗体的重链可变区。
表7：双特异性抗体氨基酸序列(Numbering scheme IMGT)
Figure PCTCN2021113494-appb-000013
Figure PCTCN2021113494-appb-000014
9.2双特异性抗体的稳定性考察
将制备的两种双特异性抗体G6-Fc-huG892和Beva-Fc-huG892进行热加速稳定考察，在经40℃培养箱孵育14天后进行该样品的HPLC-SEC检测，未出现聚集或降解现象，纯度变化小于4％，说明其在40℃环境中能保持稳定性，考察结果见表8。结果表明两种双特异性抗体的稳定性均良好。
表8.双特异性抗体的40℃热加速稳定性考察
双特异性抗体 第0天浓度 第0天纯度 第14天浓度 第14天纯度
G6-Fc-huG892 6.3mg/mL 99.45％ 6.3mg/mL 98.92％
Beva-Fc-huG892 4.8mg/mL 99.42％ 4.8mg/mL 99.26％
9.3双特异性抗体的体内抗肿瘤药效评估
G6-Fc-huG892能结合人或鼠的VEGF以及人或鼠的TGF-β，Beva-Fc-huG892能结合人的VEGF以及人或鼠的TGF-β。
①BALB/c野生型小鼠同系皮下移植瘤肿瘤模型
在野生型小鼠的CT26和EMT-6皮下瘤模型中，通过腹腔给药G6-Fc-huG892和监测肿瘤大小评估抗体的抑瘤药效。
a).实验材料：
细胞：CT26(小鼠结肠癌细胞株，购自ATCC)，EMT-6(小鼠乳腺癌细胞株，购自ATCC)；
小鼠：BALB/c野生型小鼠，雌性，购自北京维通利华；
接种方式：培养CT26细胞和EMT-6细胞，收集并调整细胞浓度，分别以1*10 6个/只(0.1ml/只)背部皮下接种。肿瘤生长至100-200mm 3分组给药，每组6只，每种细胞各两组；
受试药：G6-Fc-huG892。
阴性对照：生理盐水；
给药方式：肿瘤生长至100-200mm 3进行腹腔给药，给药剂量为10mg/kg，，进行腹腔给药，开始给药为第0天，每周3次，给药2周。
肿瘤体积：每周3次对肿瘤长宽进行测量。一组瘤体积接近2000mm 3或单只小鼠肿瘤体积达3000mm 3时将结束该组。
b).实验结果
G6-Fc-huG892体内药效实验结果显示于图5。在CT-26皮下瘤模型和EMT-6皮下瘤模型中，G6-Fc-huG892均有显著的抑瘤效果。
②balb/c-nude裸鼠异种皮下移植瘤肿瘤模型
在balb/c-nude裸鼠的人肺癌A549皮下瘤模型中，通过腹腔给药Beva-Fc-huG892监测肿瘤大小评估抗体的抑瘤药效。
a).实验材料：
细胞：A549(人肺癌细胞株，购自ATCC)；
小鼠：balb/c-nude裸鼠，雌性，购自北京维通利华；
接种方式：培养A549细胞，收集并调整细胞浓度，以5×10 6个/只(0.1ml/只)背部皮下接种。肿瘤生长至100-200mm 3分组给药，每组6只，共2组；
受试药：Beva-Fc-huG892。
阴性对照：生理盐水；
给药方式：肿瘤生长至100-200mm 3进行腹腔给药，双特异性抗体给药剂量为10mg/kg，分别进行腹腔给药，开始给药为第0天，每周3次，给药2周。
肿瘤体积：每周3次对肿瘤长宽进行测量。一组瘤体积接近2000mm 3或单 只小鼠肿瘤体积达3000mm 3时将结束该组。
b).实验结果
Beva-Fc-huG892体内药效实验结果显示于图6。在A549皮下瘤模型中，Beva-Fc-huG892有显著的抑瘤效果。
③双特异性抗体的抗VEGF的体外生物学活性评估
抗体分子通过抑制VEGF而影响HUVEC细胞的增殖，使用化学发光法检测细胞的数量并计算抑制率。将在内皮细胞完全培养基中培养的HUVEC细胞(购自AllCells)以5000个/孔接种到96孔培养板，培养过夜；第二天弃掉培养基，每孔加入的200ng/mL VEGFA蛋白(购自GeneScript，Z02689)，再分别加入梯度稀释的待测抗体样品Beva-Fc-hG892和G6-Fc-hG892，最高浓度为300μg/ml，依次3倍稀释，共设置9-11个浓度梯度。放入培养箱培养3天，第3天在每孔加入等体积的Cell Counting-Lite 2.0 Luminescent Cell Viability Assay(购自Vazyme，DD1101)试剂100μL反应10-15min，用LUX多功能酶标仪检测OD450nm处的吸光度。用GraphPad Prism6.0软件分析数据，计算抗体抑制VEGFA诱导的HUVEC细胞增殖作用的半数效应浓度(EC50)。
结果见图7和表9。两个双抗体分子对VEGF的抑制活性一致。
表9.抗体抑制VEGF的生物学活性的半数效应浓度(EC50)
抗体名称 EC50(μg/mL)
G6-Fc-hG892 2.090
Beva-Fc-hG892 2.602
④双特异性抗体的抗TGF-β1/2/3的体外生物学活性评估
抗体分子通过抑制TGF-β而影响TF-1细胞的增殖，使用化学发光法检测细胞的数量。将在含10％FBS、2ng/mL rhGM-CSF的1640培养基中培养的TF-1细胞(ATCC)以1000个/孔接种到96孔培养板，每孔加入浓度1ng/mL的TGF-β1(GenScript，Z03411)或TGF-β2(GenScript，Z03429)或TGF-β3(GenScript，Z03430)蛋白，再分别加入梯度稀释的待测抗体样品Beva-Fc-hG892和G6-Fc-hG892，最高浓度为100nM，依次5倍稀释，共设置9-11个浓度梯度，3个复孔。放入培养箱培养120h，再每孔加入80μL Cell Counting-Lite 2.0 Luminescent Cell Viability Assay(购自Vazyme，DD1101)试剂反应10-15min，用LUX多功能酶标仪检测OD450nm处的吸光度。用GraphPad Prism6.0软件分析数据，计算抗体对TGF-β诱导的TF-1细胞增殖抑制的阻断作用的EC50值。
结果见图8和表10。两个双抗体分子对TGF-β1/2/3的抑制活性一致。
表10.抗体抑制TGF-β的生物学活性的半数效应浓度(EC50)
Figure PCTCN2021113494-appb-000015
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本 领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 抗TGFβ抗体，其包含选自以下各项组成的组的序列，或由其组成：
    (1)B2G8，其包含SEQ ID NO：7所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和SEQ ID NO：51所示的CDR3，或其变体，所述变体为与SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和/或SEQ ID NO：51所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；优选地包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2的变体和SEQ ID NO：51所示的CDR3，其中所述SEQ ID NO：50所示的CDR2的变体为在SEQ ID NO：50所示氨基酸序列的第13位氨基酸置换为丙氨酸，或者
    其包含SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2和SEQ ID NO：51所示的CDR3，其中SEQ ID NO：49所示的CDR1中第5-8位氨基酸、SEQ ID NO：50所示的CDR2中第1-5位氨基酸、第7-9位氨基酸、SEQ ID NO：51所示的CDR3第1位氨基酸、第4-9位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
    (2)B1B8，其包含SEQ ID NO：4所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2和SEQ ID NO：42所示的CDR3，或其变体，所述变体为与SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2和/或SEQ ID NO：42所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；或者
    其包含SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2和SEQ ID NO：42所示的CDR3，其中SEQ ID NO：40所示的CDR1中第3位氨基酸、第5-8位氨基酸、SEQ ID NO：41所示的CDR2中第1位、第3位和第5-10位氨基酸、SEQ ID NO：42所示的CDR3第4-9位和第11位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
    (3)B3F3，其包含SEQ ID NO：16所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：76所示的CDR1，SEQ ID NO：77所示的CDR2和SEQ ID NO：78所示的CDR3，或其变体，所述变体为与SEQ ID NO：76所示的CDR1，SEQ ID NO：77所示的CDR2和/或SEQ ID NO：78所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；或者
    其包含SEQ ID NO：76所示的CDR1，SEQ ID NO：77所示的CDR2和SEQ ID NO：78所示的CDR3，其中SEQ ID NO：76所示的CDR1中第2-3位氨基酸、第 5-8位氨基酸、SEQ ID NO：77所示的CDR2中第2-4位氨基酸、第6位氨基酸、SEQ ID NO：78所示的CDR3第1位氨基酸、第3-8位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
    (4)B2A1，其包含SEQ ID NO：1所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：31所示的CDR1，SEQ ID NO：32所示的CDR2和SEQ ID NO：33所示的CDR3，或其变体，所述变体为与SEQ ID NO：31所示的CDR1，SEQ ID NO：32所示的CDR2和/或SEQ ID NO：33所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (5)B1F8，其包含SEQ ID NO：5所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：43所示的CDR1，SEQ ID NO：44所示的CDR2和SEQ ID NO：45所示的CDR3，或其变体，所述变体为与SEQ ID NO：43所示的CDR1，SEQ ID NO：44所示的CDR2和/或SEQ ID NO：45所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (6)B3D6，其包含SEQ ID NO：6所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：46所示的CDR1，SEQ ID NO：47所示的CDR2和SEQ ID NO：48所示的CDR3，或其变体，所述变体为与SEQ ID NO：46所示的CDR1，SEQ ID NO：47所示的CDR2和/或SEQ ID NO：48所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (7)B2A9，其包含SEQ ID NO：2所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：34所示的CDR1，SEQ ID NO：35所示的CDR2和SEQ ID NO：36所示的CDR3，或其变体，所述变体为与SEQ ID NO：34所示的CDR1，SEQ ID NO：35所示的CDR2和/或SEQ ID NO：36所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (8)B2C2，其包含SEQ ID NO：8所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：52所示的CDR1，SEQ ID NO：53所示的CDR2和SEQ ID NO：54所示的CDR3，或其变体，所述变体为与SEQ ID NO：52所示的CDR1，SEQ ID NO：53所示的CDR2和/或SEQ ID NO：54所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (9)B1B7，其包含SEQ ID NO：9所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：55所示的CDR1，SEQ ID NO：56所示的CDR2和SEQ ID NO：57所示的CDR3，或其变体，所述变体为与SEQ ID NO：55所示的CDR1，SEQ ID NO：56所示的CDR2和/或SEQ ID NO：57 所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (10)B1C5，其包含SEQ ID NO：10所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：58所示的CDR1，SEQ ID NO：59所示的CDR2和SEQ ID NO：60所示的CDR3，或其变体，所述变体为与SEQ ID NO：58所示的CDR1，SEQ ID NO：59所示的CDR2和/或SEQ ID NO：60所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (11)B2F6，其包含SEQ ID NO：11所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：61所示的CDR1，SEQ ID NO：62所示的CDR2和SEQ ID NO：63所示的CDR3，或其变体，所述变体为与SEQ ID NO：61所示的CDR1，SEQ ID NO：62所示的CDR2和/或SEQ ID NO：63所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (12)B2H1，其包含SEQ ID NO：12所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：64所示的CDR1，SEQ ID NO：65所示的CDR2和SEQ ID NO：66所示的CDR3，或其变体，所述变体为与SEQ ID NO：64所示的CDR1，SEQ ID NO：65所示的CDR2和/或SEQ ID NO：66所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (13)B2A2，其包含SEQ ID NO：13所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：67所示的CDR1，SEQ ID NO：68所示的CDR2和SEQ ID NO：69所示的CDR3，或其变体，所述变体为与SEQ ID NO：67所示的CDR1，SEQ ID NO：68所示的CDR2和/或SEQ ID NO：69所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (14)B3D2，其包含SEQ ID NO：14所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：70所示的CDR1，SEQ ID NO：71所示的CDR2和SEQ ID NO：72所示的CDR3，或其变体，所述变体为与SEQ ID NO：70所示的CDR1，SEQ ID NO：71所示的CDR2和/或SEQ ID NO：72所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (15)B3C5，其包含SEQ ID NO：15所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：73所示的CDR1，SEQ ID NO：74所示的CDR2和SEQ ID NO：75所示的CDR3，或其变体，所述变体为与SEQ ID NO：73所示的CDR1，SEQ ID NO：74所示的CDR2和/或SEQ ID NO：75所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；
    (16)B2C9，其包含SEQ ID NO：3所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：37所示的CDR1，SEQ ID NO：38所示的CDR2和SEQ ID NO：39所示的CDR3，或其变体，所述变体为与SEQ ID NO：37所示的CDR1，SEQ ID NO：38所示的CDR2和/或SEQ ID NO：39所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列；和
    (17)B3F1，其包含SEQ ID NO：17所示序列中包含的CDR1，CDR2和CDR3，优选按照Kabat和IMGT编号系统，包含SEQ ID NO：79所示的CDR1，SEQ ID NO：80所示的CDR2和SEQ ID NO：81所示的CDR3，或其变体，所述变体为与SEQ ID NO：79所示的CDR1，SEQ ID NO：80所示的CDR2和/或SEQ ID NO：81所示的CDR3相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列。
  2. 权利要求1所述的抗TGFβ抗体，其包含选自SEQ ID NO：1-17所示的序列或其变体，或由选自SEQ ID NO：1-17所示的序列或其变体组成；其中所述变体为与序列编号所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性并保留与TGFβ结合亲和力的序列，或与序列编号所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)并保留与TGFβ结合亲和力的氨基酸序列。
  3. 人源化抗TGFβ抗体，其包含权利要求1所述的抗TGFβ抗体的CDR1，CDR2和CDR3，优选地包含来自人IgG的Germline IGHV3-23*01的FR区，优选地还包含Fc恒定区，所述恒定区包含SEQ ID NO：18所示序列或人IgG1重链Fc恒定区(GenBankNo.AK303185.1)，更优选地，人源化抗TGFβ抗体包含SEQ ID NO：20所示的人源化序列或由其组成，进一步优选地，在SEQ ID NO：20所示人源化序列的CDR2第13位的丝氨酸Ser突变为丙氨酸Ala，最优选地，人源化抗TGFβ抗体包含选自SEQ ID NO：21-23所示的人源化序列或由其组成。
  4. 二价或多价抗TGFβ抗体，其包含2个以上的权利要求1-2任一项所述的抗TGFβ抗体，或包含2个以上的权利要求3所述的人源化抗TGFβ抗体，优选地，所述二价抗TGFβ抗体的序列选自以下各项组成的组：(1)由SEQ ID NO：22，19，18，19和22所示的序列按照由N端至C端首尾顺序连接而成；(2)由SEQ ID NO：4，19，18，19和4所示的序列按照由N端至C端首尾顺序连接而成；(3)由SEQ ID NO：23，19，18，19和23所示的序列按照由N端至C端首尾顺序连接而成；(4)由SEQ ID NO：21，19，18，19和21所示的序列按照由N端至C端首尾顺序连接而成；(5)由SEQ ID NO：7，19，18，19和7所示的序列按照由N端至C端首尾顺序连接而成；和(6)由SEQ ID NO：16，19，18，19和16所示的序列按照由N端至C端首尾顺序连接而成。
  5. 多特异性抗体，其包含权利要求1-2任一项所述的抗TGFβ抗体或权利要求3所述的人源化抗TGFβ抗体，优选双特异性抗体，所述双特异性抗体包含针 对第一抗原TGFβ的第一抗体和针对第二抗原的第二抗体，其中所述第一抗体选自权利要求1-2任一项所述的抗TGFβ抗体或权利要求3所述的人源化抗TGFβ抗体，第二抗原选自免疫细胞表面抗原、肿瘤抗原、病毒、细菌、内毒素、细胞因子、或其组合；更优选地选自：PD-L1，PD-1，VEGFA，IL-10，IL-10R，BCMA，VEGF，TGF-β，CTLA-4，LAG-3，TIGIT，CEA，CD38，SLAMF7，B7-H3，Her2，EpCAM，CD19，CD20，CD30，CD33，CD47，CD52，CD133，EGFR，GD2，GD3，GM2，RANKL，CD3，和/或CD16a，优选第二抗原为VEGF，更优选第二抗体选自抗VEGF抗体，其包含(i)SEQ ID NO：82所示的HCDR1，SEQ ID NO：83所示的HCDR2，SEQ ID NO：84所示的HCDR3，SEQ ID NO：85所示的LCDR1，SEQ ID NO：86所示的LCDR2和SEQ ID NO：87所示的LCDR3，或(ii)SEQ ID NO：88所示的HCDR1，SEQ ID NO：89所示的HCDR2，SEQ ID NO：90所示的HCDR3，SEQ ID NO：91所示的LCDR1，SEQ ID NO：92所示的LCDR2和SEQ ID NO：93所示的LCDR3；更优选地，抗VEGF抗体包含(a)SEQ ID NO：24所示的重链可变区或与SEQ ID NO：24所示的重链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，和SEQ ID NO：27所示的轻链可变区或与SEQ ID NO：27所示的轻链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，或(b)SEQ ID NO：29所示的重链可变区或与SEQ ID NO：29所示的重链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，和SEQ ID NO：30所示的轻链可变区或与SEQ ID NO：30所示的轻链可变区具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列；最优选地，抗VEGF抗体进一步包含SEQ ID NO：25所示的CH1，SEQ ID NO：26所示的铰链区，SEQ ID NO：18所示的Fc恒定区，和SEQ ID NO：28所示的轻链恒定区。
  6. 多核苷酸，其编码权利要求1-2任一项所述的抗TGFβ抗体或权利要求3所述的人源化抗TGFβ抗体，或权利要求4所述的二价或多价抗TGFβ抗体，或权利要求5所述的多特异性抗体。
  7. 载体，其包含权利要求6所述的多核苷酸。
  8. 宿主细胞，其包含权利要求7所述的载体。
  9. 偶联物，其包含权利要求1-2任一项所述的抗TGFβ抗体、权利要求3所述的人源化抗TGFβ抗体、权利要求4所述的二价或多价抗TGFβ抗体或权利要求5所述的多特异性抗体，以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)、可检测的标记、药物、毒素、细胞因子、酶、或其组合；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、化疗剂、生物毒素、聚乙二醇或酶。
  10. 试剂盒，其包括权利要求1-2任一项所述的抗TGFβ抗体、权利要求3所述的人源化抗TGFβ抗体、权利要求4所述的二价或多价抗TGFβ抗体、权利要求5所述的多特异性抗体或权利要求9所述的偶联物；优选地，所述试剂盒还包 括第二抗体，其特异性识别权利要求1-2任一项所述的抗TGFβ抗体、权利要求3所述的人源化抗TGFβ抗体、权利要求4所述的二价或多价抗TGFβ抗体、权利要求5所述的多特异性抗体或权利要求9所述的偶联物；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测VEGF在样品中的存在或其水平；或者
    所述试剂盒包含(1)权利要求1-2任一项所述的抗TGFβ抗体、权利要求3所述的人源化抗TGFβ抗体、权利要求4所述的二价或多价抗TGFβ抗体、权利要求5所述的多特异性抗体或权利要求9所述的偶联物，和(2)针对其它抗原的抗体或其抗原结合片段，和/或细胞毒性剂，和/或化疗药，和任选地，使用说明书。
  11. 药物组合物，其包含权利要求1-2任一项所述的抗TGFβ抗体、权利要求3所述的人源化抗TGFβ抗体、权利要求4所述的二价或多价抗TGFβ抗体、权利要求5所述的多特异性抗体或权利要求9所述的偶联物；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂；优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
  12. 权利要求1-2任一项所述的抗TGFβ抗体、权利要求3所述的人源化抗TGFβ抗体、权利要求4所述的二价或多价抗TGFβ抗体、权利要求5所述的多特异性抗体或权利要求9所述的偶联物用于治疗和/或预防肿瘤(例如治疗实体瘤)、糖尿病、系统性硬皮病、肾病、特发性肺纤维化和/或多发性纤维化的药物中的用途。
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CN111440238A (zh) * 2020-03-27 2020-07-24 国典(北京)医药科技有限公司 抗人转化生长因子β1的纳米抗体及其制备方法和应用

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CN111440239A (zh) * 2020-03-27 2020-07-24 中国医学科学院基础医学研究所 抗人转化生长因子β1的纳米抗体B3及其制备方法和应用
CN111440238A (zh) * 2020-03-27 2020-07-24 国典(北京)医药科技有限公司 抗人转化生长因子β1的纳米抗体及其制备方法和应用

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