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WO2024149237A1 - TGFβ1结合分子、GARP-TGFβ1结合分子及其医药用途 - Google Patents

TGFβ1结合分子、GARP-TGFβ1结合分子及其医药用途 Download PDF

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Publication number
WO2024149237A1
WO2024149237A1 PCT/CN2024/071321 CN2024071321W WO2024149237A1 WO 2024149237 A1 WO2024149237 A1 WO 2024149237A1 CN 2024071321 W CN2024071321 W CN 2024071321W WO 2024149237 A1 WO2024149237 A1 WO 2024149237A1
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WIPO (PCT)
Prior art keywords
tgfβ1
seq
garp
binding molecule
antibody
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PCT/CN2024/071321
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English (en)
French (fr)
Inventor
曹保华
何湘
徐金敬
杜延平
王宇
刘潇
张晓谦
李元元
申晨曦
王雷
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北京拓界生物医药科技有限公司
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Publication of WO2024149237A1 publication Critical patent/WO2024149237A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • the present disclosure relates to the field of biomedicine, and specifically to TGF ⁇ 1 binding molecules, pharmaceutical compositions comprising TGF ⁇ 1 binding molecules, and medical uses, preparation methods, and methods for preventing or treating diseases related to the TGF ⁇ 1 signaling pathway.
  • TGF ⁇ Transforming growth factor ⁇
  • TGF ⁇ 1 Transforming growth factor ⁇
  • TGF ⁇ 2 Transforming growth factor ⁇ 3
  • TGF ⁇ 3 pleiotropic growth factors
  • TGF ⁇ 1 TGF ⁇ 2
  • TGF ⁇ 3 TGF ⁇ 3
  • TGF ⁇ 3 TGF ⁇ 3
  • TGF ⁇ can inhibit tumor growth in the early stage of tumorigenesis, but promote tumor growth in the late stage of tumor progression (Liu S, Ren J, Ten Dijke P. Signal Transduct Target Ther. 2021 Jan 8; 6(1):8.).
  • An important mechanism by which TGF ⁇ promotes late tumor growth is to inhibit immune cells in the tumor microenvironment.
  • TGF ⁇ 1 rather than TGF ⁇ 2 and TGF ⁇ 3, is a key factor in promoting late tumor growth (Constance J Martin, et al. Sci Transl Med. 2020 Mar 25; 12(536):eaay8456.).
  • TGF ⁇ 1 produced by tumor cells, regulatory T cells (Treg) and inhibitory macrophages can directly promote the activity of Treg cells and inhibit effector T cells and antigen-presenting cells, thereby achieving the effect of suppressing immunity.
  • the tumor immune checkpoint inhibitor anti-PD-1 (Programmed Cell Death-1) antibody has achieved great success in the process of tumor immunotherapy, but the overall response rate of patients is only about 20%.
  • the combination of anti-TGF ⁇ 1 antibody and anti-PD-1 antibody in tumor models such as EMT-6, MBT-2, Cloudman S91, CT26 and MC38 can significantly overcome the immune escape of tumors and enhance the efficacy of anti-PD-1 antibodies.
  • pan-TGF ⁇ antibodies targeting mature TGF ⁇ 1 showed dose-dependent in vivo toxicity in experiments. Targeting the pro/latent TGF ⁇ 1 complex inhibits the production of TGF ⁇ 1 from the source, which has better efficacy in animal models than targeting mature TGF ⁇ 1 and has significantly reduced toxicity.
  • TGF ⁇ 1 The production of TGF ⁇ 1 is tightly regulated by a multi-step process.
  • the mature TGF- ⁇ 1 domain at its C-terminus is covalently or non-covalently bound to the domain at the N-terminus called latency associated peptide (LAP). Because LAP prevents mature TGF- ⁇ 1 from binding to its receptor, it is usually inactive.
  • LAP latency associated peptide
  • Such a TGF ⁇ 1 precursor protein can also further form a large latent complex with GARP, LRRC33, LTBP1, LTBP3, etc.
  • GARP-TGF ⁇ 1 is mainly produced by Treg and tumor cells
  • LRRC33-TGF ⁇ 1 is mainly produced by immunosuppressive phagocytes
  • LTBP1-TGF ⁇ 1 and LTBP3-TGF ⁇ 1 are mainly present in the tumor stroma.
  • TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex there are no antibodies targeting TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex on the market worldwide. This field still needs to be able to bind to TGF ⁇ 1 precursor protein or TGF ⁇ 1
  • the complex binds with high affinity and can block its activation, and is used to treat TGF ⁇ -related diseases such as tumors and fibrosis.
  • the present disclosure provides TGF ⁇ 1 binding molecules, GARP-TGF ⁇ 1 binding molecules, pharmaceutical compositions comprising the aforementioned binding molecules, as well as medical uses, preparation methods, and methods for preventing or treating diseases related to the TGF ⁇ signaling pathway, particularly methods for preventing or treating cancer or tumors.
  • the present disclosure provides a TGF ⁇ 1 binding molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:11
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in any one of SEQ ID NO:12, 21-35.
  • the above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat definition scheme.
  • the CDRs may also be defined by other numbering systems, for example, according to the IMGT, Chothia, AbM or Contact numbering systems.
  • exemplary the following provides HCDR1, HCDR2, HCDR3 in VH shown in SEQ ID NO: 11, and LCDR1, LCDR2, LCDR3 in VL shown in SEQ ID NO: 35, defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.
  • the TGF ⁇ 1 binding molecule comprises VH and VL as shown below:
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:13-15, and/or the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:38, 64, 18.
  • amino acid sequence shown in SEQ ID NO: 38 is RASQX 1 ISX 2 YLN, X 1 is selected from S, A, F, G, I, P, Y, V or K, and X 2 is selected from S, D, E, P or H;
  • amino acid sequence shown in SEQ ID NO: 64 is X 3 ASX 4 LX 5 S, wherein X 3 is selected from A, T, S or M, X 4 is selected from S, Y, A, E or G, and X 5 is selected from Q, T, D or E.
  • the amino acid sequence shown in SEQ ID NO:38 is RASQX1ISX2YLN , X1 is selected from F , Y or A, and X2 is selected from D or P; the amino acid sequence shown in SEQ ID NO:64 is X3ASX4LX5S , X3 is selected from A, T or S, X4 is selected from S , Y or E, and X5 is selected from Q, D or E.
  • the TGF ⁇ 1 binding molecule wherein:
  • HCDR1 The amino acid sequence of HCDR1 is shown in SEQ ID NO: 13,
  • HCDR2 The amino acid sequence of HCDR2 is shown in SEQ ID NO: 14,
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 15,
  • amino acid sequence of LCDR1 is shown in any one of SEQ ID Nos: 16, 36, 39, 41-42, 44, 46, 49, 52, 54, 56-59,
  • amino acid sequence of LCDR2 is shown in any one of SEQ ID NO: 17, 37, 40, 43, 45, 47-48, 50-51, 53, 55, and/or,
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:18.
  • the TGF ⁇ 1 binding molecule wherein
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 59, 40, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 36-37, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 39-40, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 41, 40, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 42-43, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 44-45, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 46-47, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 46, 48, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 49-50, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 46, 51, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 52-53, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 54-55, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 56, 53, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 57, 45, 18;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 58, 50, 18; or,
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:13-15
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:16-18.
  • the TGF ⁇ 1 binding molecule comprises VH and VL, wherein:
  • Any HCDR contained in the VH shown has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned HCDRs; and/or, any LCDR contained in the VL shown has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned HCDRs.
  • the amino acid mutations in the HCDR or LCDR are conservative substitutions.
  • the present disclosure provides a TGF ⁇ 1 binding molecule comprising any one or a combination of any several (eg, 2, 3, 4, 5, or 6) of the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3.
  • the TGF ⁇ 1 binding molecule is an anti-TGF ⁇ 1 antibody or an antigen-binding fragment thereof.
  • the anti-TGF ⁇ 1 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment of any of the foregoing.
  • the anti-TGF ⁇ 1 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, or a human antibody or antigen-binding fragment thereof. In some specific embodiments, the anti-TGF ⁇ 1 antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.
  • the anti-TGF ⁇ 1 antibody or antigen-binding fragment thereof is affinity matured.
  • the affinity matured anti-TGF ⁇ 1 antibody or antigen-binding fragment thereof comprises one or more mutations in VH and/or VL.
  • the mutation in VH comprises any one or any combination of the following:
  • the mutations located in VL include mutations at any one or any combination of the following positions: 99, 100, 53, 56, 58, 31, 33, 95, 100.
  • the mutations located in VL include mutations at any one or any combination of the following positions: 24, 28, 31, 32, 50, 53, 55, 92, 93, 94.
  • the positions of the above mutations are numbered according to the Kabat numbering convention.
  • the TGF ⁇ 1 binding molecule comprises VH and VL, wherein,
  • the VH comprises the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence having at least 80% sequence identity thereto; and/or,
  • the VL comprises an amino acid sequence as shown in any one of SEQ ID NO:12, 21-35, or an amino acid sequence having at least 80% sequence identity therewith.
  • the TGF ⁇ 1 binding molecule comprises VH and VL, wherein: the VH has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared with any of the aforementioned VH; and/or the VL has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared with any of the aforementioned VL.
  • the present disclosure provides a TGF ⁇ 1-binding molecule comprising any one or a combination of any two of the aforementioned VH and VL.
  • the TGF ⁇ 1 binding molecules provided by the present disclosure further comprise an immunoglobulin Fc region.
  • the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any of the foregoing.
  • the immunoglobulin Fc region is derived from human IgG4 or a variant thereof.
  • the human IgG4 variant comprises a mutation that reduces or eliminates the Fc effector function.
  • the human IgG4 variant comprises a mutation S228P. The sites of the above mutations are numbered according to the EU numbering rules.
  • the TGF ⁇ 1 binding molecule further comprises a light chain constant region and/or a heavy chain constant region.
  • the light chain constant region is derived from a kappa light chain, a lambda light chain, or a variant of any of the foregoing. In some embodiments, the light chain constant region is derived from a human kappa light chain, a human lambda light chain, or a variant of any of the foregoing.
  • the light chain constant region is derived from a human kappa light chain or a variant thereof.
  • the light chain constant region is derived from a murine kappa light chain or a variant thereof.
  • the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:19, or an amino acid sequence having at least 80% sequence identity thereto.
  • the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any of the foregoing. In some embodiments, the heavy chain constant region is derived from human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any of the foregoing.
  • the heavy chain constant region is derived from human IgG4 or a variant thereof.
  • the human IgG4 variant comprises a mutation that reduces or eliminates Fc effector function.
  • the human IgG4 variant comprises a mutation S228P.
  • the sites of the above mutations are numbered according to EU numbering rules.
  • the heavy chain constant region is derived from murine IgG2 or a variant thereof.
  • the mouse IgG2 variant comprises a mutation that reduces or eliminates the ADCC effect.
  • the mouse IgG2 variant comprises at least one of the following mutations: L234A/L235E/G237A/D327Q/A330S/P331S.
  • the sites of the above mutations are numbered according to the EU numbering convention.
  • the immunoglobulin Fc region is an Fc region with reduced or eliminated effector function, for example, having a mutation that reduces or eliminates the ADCC effect.
  • the mutation that reduces or eliminates the ADCC effect comprises at least one of the following:
  • L234A/L235E/G237A/D327Q/A330S/P331S (IgG2a); N297A or N297Q (IgG1); L234A/L235A (IgG1); V234A/G237A (IgG2); L235A/G237A/E318A (IgG4); H268Q/V309L/A330S/A331S (IgG2); C220S/C226S/C229S/P238S (IgG1); C226S/C229S/E233P/L234V/L235A (IgG1); L234F/L235E/P331S (IgG1); or S267E/L328F (IgG1).
  • the positions of the above mutations are numbered according to EU numbering conventions.
  • the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 binding molecule comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having at least 80% sequence identity thereto; and/or,
  • the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:65, 67-81, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 binding molecule comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 80% sequence identity thereto; and/or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO:83, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 binding molecule comprises a heavy chain and a light chain, wherein: the heavy chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to any of the aforementioned heavy chains; and/or the light chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to any of the aforementioned light chains.
  • amino acid mutations in the heavy chain and/or light chain are not conservative substitutions.
  • the TGF ⁇ 1 binding molecule of the present disclosure comprises any one or a combination of any two of the aforementioned heavy chains and light chains.
  • At least 80% encompasses 80% and above, for example at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any numerical range therebetween.
  • the aforementioned TGF ⁇ 1-binding molecules specifically bind to TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex.
  • the TGF ⁇ 1 in the TGF ⁇ 1 complex is present as a TGF ⁇ 1 precursor protein.
  • the TGF ⁇ 1 complex comprises any of the following:
  • TGF ⁇ 1 or "TGF ⁇ 1 protein” is understood in a broad sense, including the proprotein form (also known as pro-TGF ⁇ 1) or latent form (latent TGF ⁇ 1) of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein, or mature TGF ⁇ 1 after release.
  • proprotein form also known as pro-TGF ⁇ 1
  • latent TGF ⁇ 1 of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein
  • TGF ⁇ 1 or “TGF ⁇ 1 protein” as used in the present disclosure refers to "TGF ⁇ 1 precursor protein.”
  • the TGF ⁇ 1 precursor protein comprises: (i) a mature TGF ⁇ 1 domain, (ii) a potency-associated peptide (LAP).
  • LAP potency-associated peptide
  • the TGF ⁇ 1 precursor protein is pro-TGF ⁇ 1 or latent TGF ⁇ 1.
  • the mature TGF ⁇ 1 domain in pro-TGF ⁇ 1 is covalently bound to latency-associated peptides (LAPs), and the mature TGF ⁇ 1 domain in latent TGF ⁇ 1 is non-covalently bound to latency-associated peptides (LAPs).
  • pro-TGF ⁇ 1 or “latent TGF ⁇ 1” may be used interchangeably.
  • latent TGF ⁇ 1 (also called small latent complex (SLC)) becomes linked to a "presenting molecule" via a disulfide bridge to form a large latent complex (LLC), including, for example, GARP-TGF ⁇ 1 complex.
  • LLC large latent complex
  • TGF ⁇ 1 present in the GARP-TGF ⁇ 1 complex may be in a latent form (latent TGF ⁇ 1) or a precursor form (pro-TGF ⁇ 1).
  • the TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex further comprises a protein fragment, a functional variant, etc. of any of the aforementioned proteins or protein complexes.
  • fragments and protein fragments include, but are not limited to, growth factor domains, N-terminal prodomains, latency-associated peptides (LAPs), LAP-like domains, straight jacket regions, fastener regions, furin cleavage site regions, arm regions, fingers regions, latency loops, alpha 1 helical regions, alpha 2 helical regions, RGD sequence regions, trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, etc.
  • LAPs latency-associated peptides
  • RGD sequence regions trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, etc.
  • variant refers to a protein or protein complex comprising one or more amino acid substitutions, deletions and/or added amino acids and having comparable biological activity.
  • the variant of the TGF ⁇ 1 precursor protein comprises the amino acid sequence shown in SEQ ID NO: The amino acid mutation at position 4 is listed in the natural numbering.
  • the variant of the TGF ⁇ 1 precursor protein comprises a mutation C4S, and the position of the mutation is a position in the natural numbering relative to the amino acid sequence shown in SEQ ID NO:6.
  • the TGF ⁇ 1 complex is a complex formed by TGF ⁇ 1 precursor protein and other types of proteins or their protein fragments and functional variants, for example, a complex formed with LTBP1S, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, etc.
  • the TGF ⁇ 1 precursor protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 complex is a GARP-TGF ⁇ 1 complex, which comprises GARP and TGF ⁇ 1 precursor protein; wherein:
  • GARP comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 5, 8, 100, 103, or an amino acid sequence having at least 80% sequence identity thereto;
  • the TGF ⁇ 1 protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 binding molecule binds to a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex (e.g., LTBP1-TGF ⁇ 1 complex, LTBP3-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex, GARP-TGF ⁇ 1 complex, etc.) with a KD of 10-7 M, 10-8 M, 10-9 M, 10-10 M or less.
  • the TGF ⁇ 1 precursor protein is included in the TGF ⁇ 1 complex.
  • the TGF ⁇ 1 binding molecule does not bind or very weakly binds to the TGF ⁇ 2 protein or TGF ⁇ 2 complex.
  • the anti-TGF ⁇ 1 antibody or its antigen-binding fragment does not specifically bind or very weakly binds to at least one of the following: mature TGF ⁇ 2, TGF ⁇ 2 precursor protein, GARP-TGF ⁇ 2 complex, LTBP-TGF ⁇ 2 complex, etc.
  • the TGF ⁇ 1 binding molecule does not bind or very weakly binds to the TGF ⁇ 3 protein or TGF ⁇ 3 complex.
  • the anti-TGF ⁇ 1 antibody or its antigen-binding fragment does not specifically bind or very weakly binds to at least one of the following: mature TGF ⁇ 3, TGF ⁇ 3 precursor protein, GARP-TGF ⁇ 3 complex, LTBP-TGF ⁇ 3 complex, etc.
  • TGF ⁇ 2 protein should be understood in a broad sense, covering mature TGF ⁇ 2, as well as inactive precursor protein forms such as pro-TGF ⁇ 2 and latent TGF ⁇ 2.
  • Pro-TGF ⁇ 2 and latent TGF ⁇ 2 can be used interchangeably.
  • TGF ⁇ 3 protein should be understood in a broad sense, covering mature TGF ⁇ 3, as well as inactive precursor protein forms such as pro-TGF ⁇ 3 and latent TGF ⁇ 3.
  • Pro-TGF ⁇ 3 and latent TGF ⁇ 3 can be used interchangeably.
  • the TGF ⁇ 1 binding molecules disclosed herein inhibit TGF ⁇ 1 activity.
  • the TGF ⁇ 1 binding molecules bind to TGF ⁇ 1 precursor proteins (original/latent TGF ⁇ 1) or TGF ⁇ 1 complexes (e.g., GARP-TGF ⁇ 1 complexes, LTBP1-TGF ⁇ 1 complexes, LTBP3-TGF ⁇ 1 complexes, LRRC33-TGF ⁇ 1 complexes), thereby selectively inhibiting the activity of TGF ⁇ 1 (e.g., inhibiting the activation of TGF ⁇ 1, and/or inhibiting the release of mature TGF ⁇ 1; and/or, inhibiting TGF ⁇ 1 signal transduction).
  • TGF ⁇ 1 precursor proteins original/latent TGF ⁇ 1
  • TGF ⁇ 1 complexes e.g., GARP-TGF ⁇ 1 complexes, LTBP1-TGF ⁇ 1 complexes, LTBP3-TGF ⁇ 1 complexes, LRRC33-TGF ⁇ 1 complexes
  • TGF ⁇ 2 and/or TGF ⁇ 3 are not selectively inhibited, for example, the activation of TGF ⁇ 1 is selectively inhibited, but the activation of TGF ⁇ 2 and/or TGF ⁇ 3 is not inhibited.
  • the TGF ⁇ 1 in the TGF ⁇ 1 complex exists in the form of TGF ⁇ 1 precursor protein.
  • the TGF ⁇ 1 binding molecules of the present disclosure have improved safety (eg, reduced in vivo toxicity and/or adverse effects).
  • the TGF ⁇ 1 binding molecules of the present disclosure further have at least one of the following properties: inhibiting the immunosuppressive activity of regulatory T (T reg ) cells, inhibiting tumor growth, and inhibiting fibrosis.
  • the TGF ⁇ 1 binding molecule is an anti-TGF ⁇ 1 antibody or an antigen-binding fragment thereof, further Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, double-chain antibody (diabody), three-chain antibody (triabody) and four-chain antibody (tetrabody), tandem di-scFv, tandem tri-scFv), for example, specifically scFv, Fv, Fab or Fab' fragment.
  • an anti-TGF ⁇ 1 antibody or an antigen-binding fragment thereof which binds or competes with the aforementioned TGF ⁇ 1 binding molecule for the same epitope of a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex (e.g., a human or mouse GARP-TGF ⁇ 1 complex, a LTBP-TGF ⁇ 1 complex, a LRRC33-TGF ⁇ 1 complex).
  • a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex e.g., a human or mouse GARP-TGF ⁇ 1 complex, a LTBP-TGF ⁇ 1 complex, a LRRC33-TGF ⁇ 1 complex.
  • the TGF ⁇ 1 in the TGF ⁇ 1 complex exists in the form of a TGF ⁇ 1 precursor protein.
  • an anti-TGF ⁇ 1 antibody or an antigen-binding fragment thereof which blocks the binding of the aforementioned TGF ⁇ 1 binding molecule to a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex (e.g., a human or mouse GARP-TGF ⁇ 1 complex, a LTBP-TGF ⁇ 1 complex, a LRRC33-TGF ⁇ 1 complex).
  • a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex e.g., a human or mouse GARP-TGF ⁇ 1 complex, a LTBP-TGF ⁇ 1 complex, a LRRC33-TGF ⁇ 1 complex.
  • the TGF ⁇ 1 in the TGF ⁇ 1 complex exists in the form of a TGF ⁇ 1 precursor protein.
  • an anti-TGF ⁇ 1 antibody or an antigen-binding fragment thereof is provided, the binding of which to a TGF ⁇ 1 protein or a TGF ⁇ 1 complex (e.g., a human or mouse GARP-TGF ⁇ 1 complex, a LTBP-TGF ⁇ 1 complex, a LRRC33-TGF ⁇ 1 complex) is blocked by the aforementioned TGF ⁇ 1 binding molecule.
  • a TGF ⁇ 1 protein or a TGF ⁇ 1 complex e.g., a human or mouse GARP-TGF ⁇ 1 complex, a LTBP-TGF ⁇ 1 complex, a LRRC33-TGF ⁇ 1 complex
  • the TGF ⁇ 1 in the TGF ⁇ 1 complex exists in the form of a TGF ⁇ 1 precursor protein.
  • the present disclosure provides a GARP-TGF ⁇ 1 binding molecule comprising at least one immunoglobulin single variable domain that binds to a TGF ⁇ 1 complex.
  • the TGF ⁇ 1 complex is a GARP-TGF ⁇ 1 complex.
  • the TGF ⁇ 1 in the GARP-TGF ⁇ 1 complex is a TGF ⁇ 1 precursor protein.
  • the TGF ⁇ 1 precursor protein is selected from pro-TGF ⁇ 1 or latent TGF ⁇ 1.
  • pro-TGF ⁇ 1 and latent TGF ⁇ 1 are used interchangeably. Can be used interchangeably.
  • the present disclosure provides GARP-TGF ⁇ 1 binding molecules comprising at least one immunoglobulin single variable domain that binds to a GARP-TGF ⁇ 1 complex.
  • the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, CDR3, wherein:
  • the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 84;
  • the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 90;
  • the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 91; or,
  • the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 92.
  • CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system. Exemplarily, CDR1, CDR2 and CDR3 defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems are provided below.
  • the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, CDR3, wherein:
  • CDR1 comprises the amino acid sequence shown in SEQ ID NO:85;
  • CDR2 comprises the amino acid sequence shown in SEQ ID NO:86; and/or,
  • CDR2 comprises the amino acid sequence shown in SEQ ID NO:87.
  • the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 shown in SEQ ID NO:85-87.
  • the CDR1 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:85; and/or,
  • the CDR2 contained in the immunoglobulin single variable domain has the same amino acid sequence as shown in SEQ ID NO:86. Compared with the above sequence, the above sequence has 0, 1, 2, 3, 4 or 5 amino acid mutations; and/or,
  • the CDR3 contained in the single variable domain of the immunoglobulin has 0, 1, 2, 3, 4 or 5 amino acid mutations compared with the amino acid sequence shown in SEQ ID NO:87.
  • amino acid mutations in CDR1, CDR2 or CDR3 are conservative substitutions.
  • the GARP-TGF ⁇ 1 binding molecule comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, etc.) of the immunoglobulin single variable domains described above. In some embodiments, the GARP-TGF ⁇ 1 binding molecule comprises two or more immunoglobulin single variable domains, and any two immunoglobulin single variable domains may be the same or different.
  • the GARP-TGF ⁇ 1 binding molecule may comprise any of the aforementioned complete immunoglobulin single variable domains; it may also comprise a functional portion of any of the aforementioned immunoglobulin single variable domains or a variant thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the variant of the functional portion of the immunoglobulin single variable domain may be a polypeptide that retains the serum albumin binding function of CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3 and has at least 80%, at least 90% sequence homology therewith.
  • it may be a polypeptide that retains the TGF ⁇ 1 complex binding function of CDR3 and has a certain sequence homology therewith, for example, a polypeptide that has at least 80%, at least 90% sequence homology with any of the above-mentioned CDR3.
  • the immunoglobulin single variable domain is a VHH.
  • the immunoglobulin single variable domain is a camelid VHH, a fully human VHH, or a humanized VHH.
  • the immunoglobulin single variable domain is a humanized VHH.
  • the human germline template of the humanized VHH is selected from at least one of IGHV3-23 and IGJH4.
  • the humanized VHH comprises FR1, FR2, and FR3 derived from IGHV3-23, and the humanized VHH comprises FR4 derived from IGJH4.
  • the humanized VHH comprises at least one back mutation as shown below: 23T, 29Y, 30C, 37Y, 44E, 45R, 47F, 71Q, 74A, 75R, 78G, 81E, 93K and 94T.
  • the framework region of the humanized VHH comprises at least one back mutation as described above.
  • the immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 84, 90-92, or an amino acid sequence having at least 80% sequence identity thereto.
  • the amino acid sequence of the immunoglobulin single variable domain has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 etc.) amino acid mutations.
  • amino acid mutations in the immunoglobulin single variable domain are conservative substitutions.
  • the GARP-TGF ⁇ 1 binding molecule further comprises an immunoglobulin Fc region.
  • the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3 or IgG4.
  • the immunoglobulin Fc region is derived from IgG2 or IgG4.
  • the immunoglobulin Fc region is derived from human IgG4.
  • the immunoglobulin Fc region comprises a mutation that reduces or eliminates Fc effector function.
  • the immunoglobulin Fc region comprises the mutation: S228P.
  • the positions of the above mutations are numbered according to EU numbering conventions.
  • the immunoglobulin Fc region comprises an amino acid sequence as shown in SEQ ID NO:88 or an amino acid sequence having at least 80% sequence identity thereto.
  • the immunoglobulin Fc region is derived from mouse IgG2.
  • the immunoglobulin Fc region is an Fc region with reduced or eliminated effector function, for example, having a mutation that reduces or eliminates the ADCC effect.
  • the mutation that reduces or eliminates the ADCC effect comprises at least one of the following: L234A/L235E/G237A/D327Q/A330S/P331S (IgG2a); N297A or N297Q (IgG1); L234A/L235A (IgG1); V234A/G237A (IgG2); L235A/G237A/E318A (IgG2a); gG4); H268Q/V309L/A330S/A331S (IgG2); C220S/C226S/C229S/P238S (IgG1); C226S/C229S/E233P/L234V/L235A (IgG1); L234F/L235E
  • the GARP-TGF ⁇ 1 binding molecule is an antibody or an antigen-binding fragment thereof that binds to the GARP-TGF ⁇ 1 complex.
  • the GARP-TGF ⁇ 1 binding molecule comprises an amino acid sequence as shown in any one of SEQ ID NOs: 89, 93-95, 106, or an amino acid sequence having at least 80% or at least 90% sequence identity thereto.
  • the GARP-TGF ⁇ 1 binding molecule comprises one or more (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) amino acid mutations compared to the amino acid sequence shown in any one of SEQ ID NOs: 89, 93-95, 106, and has the functional activity of specifically binding to the GARP-TGF ⁇ 1 complex.
  • the amino acid mutations in the GARP-TGF ⁇ 1 binding molecule are conservative substitutions.
  • At least 80% encompasses 80% and above, for example at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any numerical range therebetween.
  • the aforementioned GARP-TGF ⁇ 1 binding molecule specifically binds to the GARP-TGF ⁇ 1 complex, thereby inhibiting the release of mature TGF ⁇ 1 from the GARP-TGF ⁇ 1 complex, and/or inhibiting TGF ⁇ 1 activity, and/or inhibiting TGF ⁇ 1 signal transduction.
  • the GARP-TGF ⁇ 1 binding molecule does not bind to free mature TGF ⁇ 1.
  • the GARP-TGF ⁇ 1 binding molecule does not bind or very weakly binds to TGF ⁇ 2 protein or TGF ⁇ 2 complex.
  • the TGF ⁇ 1 binding molecule does not bind or very weakly binds to mature TGF ⁇ 2, TGF ⁇ 2 precursor protein, GARP-TGF ⁇ 2 complex, LTBP-TGF ⁇ 2 complex, etc.
  • the GARP-TGF ⁇ 1 binding molecule does not bind or very weakly binds to TGF ⁇ 3 protein or TGF ⁇ 3 complex.
  • the GARP-TGF ⁇ 1 binding molecule does not bind or very weakly binds to mature TGF ⁇ 3, TGF ⁇ 3 precursor protein, GARP-TGF ⁇ 3 complex, LTBP-TGF ⁇ 3 complex, etc.
  • TGF ⁇ 2 protein should be understood in a broad sense, covering mature TGF ⁇ 2, as well as inactive precursor protein forms such as pro-TGF ⁇ 2 and latent TGF ⁇ 2.
  • TGF ⁇ 3 protein should be understood in a broad sense, covering mature TGF ⁇ 3, as well as inactive precursor protein forms such as pro-TGF ⁇ 3 and latent TGF ⁇ 3.
  • the aforementioned GARP-TGF ⁇ 1 binding molecules can selectively inhibit the activity of TGF ⁇ 1, but not the activity of TGF ⁇ 2 and/or TGF ⁇ 3.
  • the GARP-TGF ⁇ 1 binding molecules selectively inhibit the activation of TGF ⁇ 1, but not the activation of TGF ⁇ 2 and/or TGF ⁇ 3.
  • the GARP-TGF ⁇ 1 binding molecules of the present disclosure have improved safety (e.g., reduced in vivo toxicity and/or adverse reactions).
  • mature TGF ⁇ 1, mature TGF ⁇ 2, and/or mature TGF ⁇ 3 are in a free state in the cell.
  • the GARP-TGF ⁇ 1 binding molecule specifically binds to a GARP-TGF ⁇ 1 complex, wherein the GARP-TGF ⁇ 1 complex comprises:
  • Glycoprotein A repeat dominant sequence (GARP);
  • Latency-associated peptide LAP
  • the GARP comprises an amino acid sequence as shown in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 90% sequence identity thereto
  • the mature TGF ⁇ 1 domain comprises an amino acid sequence as shown in SEQ ID NO: 101 or 104, or an amino acid sequence having at least 90% sequence identity thereto
  • the LAP comprises an amino acid sequence as shown in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 90% sequence identity thereto.
  • the GARP-TGF ⁇ 1 binding molecule binds to the GARP-TGF ⁇ 1 complex or a fragment thereof with a KD of 10 "7 M, 10 "8 M, 10 "9 M or less.
  • the GARP-TGF ⁇ 1 binding molecule has the following properties: inhibiting TGF ⁇ 1 activity, inhibiting the immunosuppressive activity of regulatory T cells, and/or inhibiting tumor growth.
  • the GARP-TGF ⁇ 1 binding molecule is an anti-GARP-TGF ⁇ 1 antibody or an antigen-binding fragment thereof; in some specific embodiments, it is a chimeric antibody, a camelid antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.
  • the GARP-TGF ⁇ 1 binding molecule is an anti-GARP-TGF ⁇ 1 antibody or an antigen-binding fragment thereof; in some specific embodiments, the anti-GARP-TGF ⁇ 1 antibody or an antigen-binding fragment thereof includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab') 2 , linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv), for example, scFv, Fv, Fab or Fab' fragments.
  • an anti-GARP-TGF ⁇ 1 antibody or antigen-binding fragment thereof that binds to or competes for binding to the same epitope of a GARP-TGF ⁇ 1 complex (eg, a human or mouse GARP-TGF ⁇ 1 complex) as the aforementioned GARP-TGF ⁇ 1 binding molecule.
  • a GARP-TGF ⁇ 1 complex eg, a human or mouse GARP-TGF ⁇ 1 complex
  • an anti-GARP-TGF ⁇ 1 antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the aforementioned GARP-TGF ⁇ 1 binding molecules to GARP-TGF ⁇ 1 (eg, human or mouse GARP-TGF ⁇ 1 complex).
  • an anti-GARP-TGF ⁇ 1 antibody or an antigen-binding fragment thereof is provided, whose binding to a GARP-TGF ⁇ 1 complex (eg, a human or mouse GARP-TGF ⁇ 1 complex) is blocked by the aforementioned GARP-TGF ⁇ 1 binding molecule.
  • a GARP-TGF ⁇ 1 complex eg, a human or mouse GARP-TGF ⁇ 1 complex
  • the GARP-TGF ⁇ 1 complex comprises (i) GARP, and (ii) TGF ⁇ 1 precursor protein.
  • the GARP-TGF ⁇ 1 complex comprises (i) GARP, and (ii) TGF ⁇ 1 precursor protein.
  • GARP comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 5, 8, 100, 103, or an amino acid sequence having at least 80% sequence identity thereto;
  • the TGF ⁇ 1 protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.
  • the present disclosure provides TGF ⁇ 1 binding molecules.
  • the TGF ⁇ 1 binding molecule specifically binds to a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex.
  • the TGF ⁇ 1 complex comprises a TGF ⁇ 1 precursor protein.
  • TGF ⁇ 1 or “TGF ⁇ 1 protein” is understood in a broad sense, including the proprotein form (also known as proTGF ⁇ 1) or latent form (latent TGF ⁇ 1) of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein, or mature TGF ⁇ 1 after release.
  • proTGF ⁇ 1 or latent TGF ⁇ 1 protein refers to TGF ⁇ 1 precursor protein.
  • the TGF ⁇ 1 precursor protein comprises: (i) a mature TGF ⁇ 1 domain, (ii) a potency-associated peptide (LAP).
  • LAP potency-associated peptide
  • the TGF ⁇ 1 precursor protein is pro-TGF ⁇ 1 or latent TGF ⁇ 1.
  • the mature TGF ⁇ 1 domain in pro-TGF ⁇ 1 is covalently bound to latency-associated peptides (LAPs), and the mature TGF ⁇ 1 domain in latent TGF ⁇ 1 is non-covalently bound to latency-associated peptides (LAPs).
  • pro-TGF ⁇ 1 or “latent TGF ⁇ 1” may be used interchangeably.
  • the mature TGF ⁇ 1 domain comprises an amino acid sequence as shown in SEQ ID NO:101 or 104 or an amino acid sequence having at least 80% sequence identity thereto.
  • LAP comprises an amino acid sequence as shown in SEQ ID NO:102 or 105, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 precursor protein comprises an amino acid sequence as described in any one of SEQ ID NO: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.
  • the TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex further comprises a protein fragment, a functional variant, etc. of any of the aforementioned proteins or protein complexes.
  • the TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex is derived from any vertebrate, including mammals such as primates (eg, humans) and other species (eg, mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.).
  • mammals such as primates (eg, humans) and other species (eg, mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.).
  • fragments and protein fragments include, but are not limited to, growth factor domains, N-terminal prodomains, latency-associated peptides (LAPs), LAP-like domains, straight jacket regions, fastener regions, furin cleavage site regions, arm regions, fingers regions, latency loops, alpha 1 helical regions, alpha 2 helical regions, RGD sequence regions, trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, etc.
  • LAPs latency-associated peptides
  • RGD sequence regions trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, etc.
  • variant refers to a protein or protein complex comprising one or more amino acid substitutions, deletions and/or added amino acids and having comparable biological activity.
  • the variant of the TGF ⁇ 1 precursor protein comprises a mutation in the amino acid at position 4 according to natural counting relative to the amino acid sequence shown in SEQ ID NO: 6.
  • the variant of the TGF ⁇ 1 precursor protein comprises a mutation C4S, wherein the site of the mutation is a site according to natural counting relative to the amino acid sequence shown in SEQ ID NO: 6.
  • the TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex may or may not contain a leader sequence.
  • the leader sequence is a signal peptide sequence.
  • the TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex may or may not contain a tag sequence.
  • the tag sequence is His-tag, AVI-tag, myc-tag, fluorescent tag, etc.
  • the TGF ⁇ 1 complex comprises TGF ⁇ 1 protein and any second protein selected from the following: glycoprotein A repeat dominant sequence (GARP), latent TGF- ⁇ binding protein (Latent TGF- ⁇ -binding proteins, LTBP; for example, LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4), fibrillin (for example, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4), LRRC33 (Leucine-Rich Repeat-Containing Protein 33), or any variant or protein fragment of the foregoing.
  • GARP glycoprotein A repeat dominant sequence
  • LTBP latent TGF- ⁇ binding protein
  • fibrillin for example, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4
  • LRRC33 Leucine-Rich Repeat-Containing Protein 33
  • protein fragment or “fragment” includes, but is not limited to, an extracellular domain, a transmembrane domain, or an intracellular domain, etc.
  • the TGF ⁇ 1 complex includes, but is not limited to, a GARP-TGF ⁇ 1 complex, an LTBP-TGF ⁇ 1 complex (e.g., an LTBP1-TGF ⁇ 1 complex, an LTBP3-TGF ⁇ 1 complex), or an LRRC33-TGF ⁇ 1 complex.
  • TGF ⁇ 1 in the TGF ⁇ 1 complex exists in the form of a TGF ⁇ 1 precursor protein.
  • the GARP-TGF ⁇ 1 complex comprises:
  • Glycoprotein A repeat dominant sequence (GARP);
  • Latency-associated peptide LAP
  • the GARP comprises an amino acid sequence as shown in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 80% sequence identity thereto
  • the TGF ⁇ 1 comprises an amino acid sequence as shown in SEQ ID NO: 101 or 104, or an amino acid sequence having at least 80% sequence identity thereto
  • the LAP comprises an amino acid sequence as shown in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 80% sequence identity thereto.
  • the GARP-TGF ⁇ 1 complex is formed by GARP and TGF ⁇ 1 precursor protein.
  • the GARP-TGF ⁇ 1 complex is formed by the extracellular region of GARP and the TGF ⁇ 1 precursor protein.
  • GARP may be a wild-type GARP of natural origin, or a functional variant of GARP (e.g., comprising one or more modifications, truncations and/or mutations compared to wild-type GARP).
  • GARP may be full-length or a partial domain thereof (e.g., an extracellular domain, a transmembrane domain or an intracellular domain).
  • natural origin refers to any vertebrate source, including mammals such as primates (e.g., humans) and other species (e.g., mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.), etc.
  • GARP may or may not include a leader sequence.
  • the leader sequence is a signal peptide sequence.
  • GARP may include one or more tag sequences, such as His-tag, AVI-tag, myc-tag, fluorescent tag, etc.
  • GARP may not include a tag sequence.
  • the extracellular region of GARP comprises an amino acid sequence as shown in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 80% sequence identity thereto.
  • GARP comprises an amino acid sequence as shown in any one of SEQ ID NO: 2, 4, 5, 8, 100, 103, or an amino acid sequence having at least 80% sequence identity thereto.
  • the protein binding molecule that binds to the TGF ⁇ 1 precursor protein or the TGF ⁇ 1 complex is selected from an antibody or an antigen-binding fragment thereof.
  • the antibody or its antigen-binding fragment includes, but is not limited to, any of the following: linear antibody, single-chain antibody (scFv), single-domain antibody (sdAb), nanobody, peptide antibody peptibody, domain antibody and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv), Fab, Fv, sFv, Fab', F(ab')2.
  • antibodies or antigen-binding fragments thereof that specifically bind to an epitope of a TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex are provided.
  • TGF ⁇ 1 when TGF ⁇ 1 is in the form of a precursor protein or exists in The epitope is available for binding by an antibody or antigen binding fragment thereof when in a complex with GARP, LTBP1, LTBP3 and/or LRRC33.
  • the epitope is available due to a conformational change in TGF ⁇ 1 when in complex with GARP, LTBP and/or LRRC33.
  • the epitope bound by the antibody or antigen binding fragment thereof in TGF ⁇ 1 is unavailable when TGF ⁇ 1 is not in complex with GARP, LTBP and/or LRRC33, or when TGF ⁇ 1 is mature TGF ⁇ 1.
  • the antibody or antigen binding fragment thereof does not prevent TGF ⁇ 1 from binding to integrins.
  • the antibody or antigen binding fragment thereof does not mask the integrin binding site of TGF ⁇ 1.
  • the protein binding molecule does not bind or very weakly binds to the TGF ⁇ 2 protein or TGF ⁇ 2 complex.
  • the protein binding molecule does not specifically bind to at least one of the following: mature TGF ⁇ 2, TGF ⁇ 2 precursor protein, GARP-TGF ⁇ 2 complex, LTBP-TGF ⁇ 2 complex, etc.
  • the protein binding molecule does not bind or very weakly binds to the TGF ⁇ 3 protein or TGF ⁇ 3 complex.
  • the protein binding molecule does not specifically bind to at least one of the following: mature TGF ⁇ 3, TGF ⁇ 3 precursor protein, GARP-TGF ⁇ 3 complex, LTBP-TGF ⁇ 3 complex, etc.
  • TGF ⁇ 2 protein should be understood in a broad sense, covering mature TGF ⁇ 2, as well as inactive precursor protein forms such as pro-TGF ⁇ 2 and latent TGF ⁇ 2.
  • Pro-TGF ⁇ 2 and latent TGF ⁇ 2 can be used interchangeably.
  • TGF ⁇ 3 protein should be understood in a broad sense, covering mature TGF ⁇ 3, as well as inactive precursor protein forms such as pro-TGF ⁇ 3 and latent TGF ⁇ 3.
  • Pro-TGF ⁇ 3 and latent TGF ⁇ 3 can be used interchangeably.
  • mature TGF ⁇ 1, mature TGF ⁇ 2, and/or mature TGF ⁇ 3 are present in a free state in the cell.
  • the protein binding molecule specifically binds to the TGF ⁇ 1 precursor protein or the TGF ⁇ 1 complex (e.g., GARP-TGF ⁇ 1 complex, LTBP1-TGF ⁇ 1 complex, LTBP3-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex, etc.), and can selectively inhibit the activity of TGF ⁇ 1. For example, inhibit the activation of TGF ⁇ 1, and/or inhibit the release of mature TGF ⁇ 1, and/or inhibit TGF ⁇ 1 signal transduction.
  • the TGF ⁇ 1 precursor protein or the TGF ⁇ 1 complex e.g., GARP-TGF ⁇ 1 complex, LTBP1-TGF ⁇ 1 complex, LTBP3-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex, etc.
  • the present disclosure provides polynucleotides encoding any of the aforementioned protein binding molecules. In some embodiments, the present disclosure provides polynucleotides encoding any of the aforementioned TGF ⁇ 1 binding molecules and GARP-TGF ⁇ 1 binding molecules.
  • the present disclosure provides a polynucleotide encoding the TGF ⁇ 1 binding molecule of the first aspect of the present disclosure. In some embodiments, the present disclosure provides a polynucleotide encoding the GARP-TGF ⁇ 1 binding molecule of the second aspect of the present disclosure.
  • the polynucleotides of the present disclosure may be RNA, DNA, or cDNA.
  • the polynucleotides of the present disclosure are isolated polynucleotides.
  • the polynucleotides of the present disclosure may also be in the form of a vector, may be present in a vector and/or may be part of a vector, which may be a eukaryotic vector, a prokaryotic vector, a viral vector, such as a plasmid.
  • the vector may be an expression vector, i.e., a vector that provides for the expression of a binding molecule (e.g., a TGF ⁇ 1 binding molecule) in vitro and/or in vivo (i.e., in a suitable host cell, host organism, and/or expression system).
  • the expression vector generally comprises at least one polynucleotide of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host. Regulatory elements and other elements that are useful or necessary for the expression of the protein binding molecules, TGF ⁇ 1 binding molecules, and GARP-TGF ⁇ 1 binding molecules of the present disclosure are, for example, promoters, enhancers, terminators, integrons, selection markers, leader sequences, and reporter genes.
  • suitable expression control elements e.g., promoters, enhancers, terminators, etc.
  • polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and/or can be isolated from suitable natural sources.
  • the polynucleotides and vectors of the present disclosure can be used to prepare TGF ⁇ 1 binding molecules. In some embodiments, the polynucleotides and vectors of the present disclosure are used to express TGF ⁇ 1 binding molecules in vitro or in vivo, and the TGF ⁇ 1 binding molecules bind to TGF ⁇ 1 precursor protein or TGF ⁇ 1 complex for different purposes such as detection, diagnosis, treatment, and regulation.
  • the polynucleotides and vectors disclosed herein can be used to prepare GARP-TGF ⁇ 1 binding molecules.
  • the polynucleotides and vectors disclosed herein are used to express GARP-TGF ⁇ 1 binding molecules in vitro or in vivo, and the GARP-TGF ⁇ 1 binding molecules bind to the GARP-TGF ⁇ 1 complex for different purposes such as detection, diagnosis, treatment, and regulation.
  • the present disclosure provides host cells expressing one or more protein binding molecules of the present disclosure.
  • the present disclosure provides host cells comprising any of the aforementioned polynucleotides or vectors; or, the host cells express any of the aforementioned TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules.
  • the host cell expresses the TGF ⁇ 1 binding molecule described in the first aspect of the present disclosure. In some embodiments, the host cell expresses the GARP-TGF ⁇ 1 binding molecule described in the second aspect of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • bacterial cells include, for example, cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).
  • Gram-negative bacterial strains e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.
  • Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris), pastoris and Pichia methanolica ) and cells of species of the genus Hansenula .
  • Saccharomyces e.g., Saccharomyces cerevisiae
  • Schizosaccharomyces e.g., Schizosaccharomyces pombe
  • Pichia e.g., Pichia pastoris
  • pastoris e.g., Pichia methanolica
  • mammalian cells such as monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), human glioma cells (LN229 cells), human cervical cancer cells (HeLa cells), ), human breast cancer cells (MCF-7), human prostate cancer cells (PC3), mouse breast cancer cells (EMT-6 cells), mouse breast tumor cells (MMT060562), mouse colon cancer cells (CT26 cells), TRI cells (as described, for example, in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells, Chinese hamster ovary (CHO) cells, myeloma cell lines such as YO,
  • the present disclosure may also use amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.
  • the cells of the present disclosure are incapable of developing into complete plant or animal individuals.
  • the present disclosure provides methods of making the protein binding molecules of the present disclosure.
  • the present disclosure provides a method for preparing the TGF ⁇ 1 binding molecule of the first aspect, comprising the following steps:
  • TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex comprising the TGF ⁇ 1 precursor protein as an antigen protein
  • the method for preparing a TGF ⁇ 1 binding molecule further comprises the following steps:
  • the above preparation method is also a method for screening TGF ⁇ 1 binding molecules that specifically bind to TGF ⁇ 1 precursor protein or a TGF ⁇ 1 complex containing the TGF ⁇ 1 precursor protein.
  • the present disclosure provides a method for preparing the GARP-TGF ⁇ 1 binding molecule of the aforementioned second aspect, comprising the following steps:
  • the method for preparing the GARP-TGF ⁇ 1 binding molecule further comprises the following steps:
  • step b is further:
  • Camels are immunized with the antigen protein, peripheral blood of the immunized camels is collected, nucleic acids are extracted, and phage libraries are constructed using the nucleic acids.
  • the above preparation method is also a method for screening GARP-TGF ⁇ 1 binding molecules that specifically bind to the GARP-TGF ⁇ 1 complex.
  • the present disclosure provides a method for preparing any of the aforementioned TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules, comprising:
  • the TGF ⁇ 1-binding molecules or GARP-TGF ⁇ 1-binding molecules of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.
  • Recombinant immunoglobulin expression vectors can stably transfect cells.
  • Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies.
  • the culture fluid secreting the antibodies can be purified and collected by conventional techniques.
  • the antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange, etc.
  • the obtained product should be immediately frozen, such as at -70°C, or lyophilized.
  • TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules of the present disclosure can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
  • the present disclosure provides a pharmaceutical composition, which contains any one or a combination selected from the following: the protein binding molecules or polynucleotides encoding them as described above.
  • the pharmaceutical composition contains a preventive or therapeutically effective amount of a TGF ⁇ 1 binding molecule or a GARP-TGF ⁇ 1 binding molecule as described in any of the foregoing, or a polynucleotide or vector encoding the TGF ⁇ 1 binding molecule or GARP-TGF ⁇ 1 binding molecule.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants. material, diluent, buffer or excipient.
  • the pharmaceutical composition can be formulated into any dosage form known in the medical art, with the dosage form selected depending on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition further comprises an immune checkpoint inhibitor.
  • immune checkpoint inhibitors include but are not limited to one or more inhibitors of PD-1, PD-L1, and PD-L2.
  • the pharmaceutical composition further comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.
  • the TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules disclosed in the present invention can inhibit the activity of TGF ⁇ 1 and the immunosuppressive activity of regulatory T cells.
  • the combination of TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules and anti-PD-1 antibodies can relieve the tumor immunosuppressive microenvironment.
  • the embodiments disclosed in the present invention found that the combination of TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules and anti-PD-1 antibodies exerts a significantly improved therapeutic effect in tumor treatment.
  • the pharmaceutical composition may contain 0.01 to 99% by weight of the TGF ⁇ 1 binding molecule in a unit dose. In other specific embodiments, the pharmaceutical composition contains 0.1-2000 mg of the TGF ⁇ 1 binding molecule in a unit dose; in some specific embodiments, 1-1000 mg.
  • the pharmaceutical composition may contain 0.01 to 99% by weight of the GARP-TGF ⁇ 1 binding molecule in a unit dose.
  • the amount of the GARP-TGF ⁇ 1 binding molecule in a unit dose of the pharmaceutical composition is 0.1-2000 mg; in some specific embodiments, it is 1-1000 mg.
  • immune checkpoint inhibitors include but are not limited to one or more inhibitors of PD-1, PD-L1, and PD-L2.
  • a TGF ⁇ 1 binding molecule is used in combination with an immune checkpoint inhibitor for treating a disease associated with the TGF ⁇ signaling pathway.
  • a use of a TGF ⁇ 1 binding molecule in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof for treating a disease associated with the TGF ⁇ signaling pathway is provided.
  • a GARP-TGF ⁇ 1 binding molecule is used in combination with an immune checkpoint inhibitor for treating a disease associated with the TGF ⁇ signaling pathway.
  • a GARP-TGF ⁇ 1 binding molecule is used in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof for treating a disease associated with the TGF ⁇ signaling pathway.
  • TGF ⁇ signaling pathway-related disease refers to any disease, disorder and/or condition associated with the expression, activity and/or metabolism of TGF ⁇ family proteins or any disease, disorder and/or condition that may benefit from the regulation of the activity and/or level of one or more TGF ⁇ family proteins.
  • Diseases associated with the TGF ⁇ signaling pathway may include, but are not limited to, tumors or cancers.
  • the disease associated with the TGF ⁇ signaling pathway is cancer.
  • the present disclosure provides a TGF ⁇ 1 binding molecule or a GARP-TGF ⁇ 1 binding molecule in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof for use in treating Use in cancer treatment.
  • TGF ⁇ 1 binding molecules or GARP-TGF ⁇ 1 binding molecules significantly improve the immunosuppressive microenvironment of tumors, and when used in combination with anti-PD-1 antibodies, they exert significantly improved therapeutic effects in tumor treatment.
  • the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, head and neck cancer.
  • Kit (or medicine box)
  • the present disclosure provides a kit or a medicine box, comprising one or more containers, each of which independently contains any one or a combination selected from the following: a protein binding molecule of the present disclosure (e.g., the TGF ⁇ 1 binding molecule or GARP-TGF ⁇ 1 binding molecule), or a polynucleotide or vector encoding the same.
  • a protein binding molecule of the present disclosure e.g., the TGF ⁇ 1 binding molecule or GARP-TGF ⁇ 1 binding molecule
  • a polynucleotide or vector encoding the same e.g., a polynucleotide or vector encoding the same.
  • the present disclosure provides protein-binding molecules, or their encoding polynucleotides, vectors, pharmaceutical compositions, and methods for preventing, treating, and alleviating diseases or symptoms.
  • the present disclosure provides for use of a TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition in at least one of the following:
  • TGF ⁇ 1 activity for example, inhibiting TGF ⁇ 1 activation, inhibiting the release of mature TGF ⁇ 1 from the TGF ⁇ 1 complex, and/or inhibiting TGF ⁇ 1 signal transduction;
  • the present disclosure provides a method for preventing or treating a disease or disorder associated with the TGF ⁇ signaling pathway, comprising administering to a subject a preventively or therapeutically effective amount of a TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition.
  • the present disclosure provides a method for inhibiting TGF ⁇ 1 activity in vitro, comprising: administering a TGF ⁇ 1 antibody or antigen-binding fragment thereof, encoding polynucleotide, vector or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of a TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition is administered.
  • the present disclosure provides a method of inhibiting TGF ⁇ 1 activity in vivo, comprising: administering a TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition to a subject.
  • a TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition is administered to a subject.
  • the subject suffers from a disease or disorder associated with the TGF ⁇ signaling pathway.
  • Diseases associated with the TGF ⁇ signaling pathway refers to diseases related to the expression, activity and/or production of TGF ⁇ family proteins. Any disease, disorder and/or condition related to TGF ⁇ signaling pathway or any disease, disorder and/or condition that may benefit from the regulation of the activity and/or level of one or more TGF ⁇ family proteins. Diseases related to the TGF ⁇ signaling pathway may include, but are not limited to, diseases or conditions related to fibrosis, tumors or cancer.
  • the disease associated with the TGF ⁇ signaling pathway is cancer.
  • the disease or disorder associated with the TGF ⁇ signaling pathway is fibrosis.
  • a method for preventing or treating cancer wherein the components shown in 1) and 2) below are administered in combination in effective amounts for prevention or treatment:
  • anti-TGF ⁇ 1 antibody or antigen-binding fragment thereof or encoding polynucleotide, vector, or pharmaceutical composition
  • the PD-1 signaling pathway inhibitor includes, but is not limited to, one or more inhibitors of PD-1, PD-L1, and PD-L2.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof.
  • the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, head and neck cancer.
  • the TGF ⁇ 1 binding molecules or pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, and the administration may be systemic or local.
  • the dosage regimen can be adjusted to obtain the best intended response (e.g., a therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.
  • the best intended response e.g., a therapeutic or preventive response
  • the present disclosure provides for use of a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition in at least one of the following:
  • TGF ⁇ 1 activity for example, inhibiting TGF ⁇ 1 activation, inhibiting the release of mature TGF ⁇ 1 from the TGF ⁇ 1 complex, and/or inhibiting TGF ⁇ 1 signal transduction;
  • the present disclosure provides a method for preventing or treating a disease or disorder associated with the TGF ⁇ signaling pathway, comprising administering to a subject a preventively or therapeutically effective amount of a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition.
  • the present disclosure provides a method for preventing or treating a disease associated with the TGF ⁇ signaling pathway, comprising administering to a subject a preventively or therapeutically effective amount of a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition; and a preventively or therapeutically effective amount of an immune checkpoint inhibitor.
  • immune checkpoint inhibitors include, but are not limited to, one or more inhibitors of PD-1, PD-L1, and PD-L2.
  • immune checkpoint inhibitors are anti-PD-1 antibodies or their antigen binding partners. Combined fragments.
  • the present disclosure provides a method for inhibiting TGF ⁇ 1 activity in vitro, comprising: administering a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition is administered.
  • the present disclosure provides a method for inhibiting TGF ⁇ 1 activation or inhibiting the release of mature TGF ⁇ 1 from a GARP-TGF ⁇ 1 complex in vitro, comprising: administering a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition is administered.
  • the present disclosure provides a method for inhibiting TGF ⁇ 1 activity in vivo, comprising: administering a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition to a subject.
  • a method for inhibiting TGF ⁇ 1 activity in vivo comprising: administering a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition to a subject.
  • an inhibitory effective amount of a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition is administered to a subject.
  • the present disclosure provides a method for inhibiting TGF ⁇ 1 activation or inhibiting the release of mature TGF ⁇ 1 from the GARP-TGF ⁇ 1 complex in vivo, comprising: administering a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition to a subject.
  • a GARP-TGF ⁇ 1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition is administered to a subject.
  • the subject suffers from a disease or disorder associated with the TGF ⁇ signaling pathway.
  • the disease associated with the TGF ⁇ signaling pathway is cancer.
  • the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, head and neck cancer.
  • the GARP-TGF ⁇ 1 binding molecules or pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, and the administration may be systemic or local.
  • the dosage regimen can be adjusted to obtain the best intended response (e.g., a therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.
  • the best intended response e.g., a therapeutic or preventive response
  • “About”, “approximately” means that the value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation. Alternatively, “about” or “substantially include” can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, each case in which the term “about” is preceding a number or a numerical range also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “substantially include” should be assumed to be within the acceptable error range of the specific value.
  • TGF- ⁇ transforming growth factor- ⁇ family is composed of a class of structurally and functionally related polypeptide growth factor subfamily, which participates in many different biological pathways. In addition to TGF- ⁇ , it also includes activins, inhibins, growth and differentiation factors (GDFs), bone morphogenetic proteins (BMPs), etc. TGF ⁇ has three forms of isoforms: TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3, which are widely expressed in almost all cell types of mammals.
  • Pro-TGF ⁇ 1 separates the homodimeric growth factor domain from its prodomain (also known as latency-associated peptide (LAP)) by cleavage by furin.
  • LAP latency-associated peptide
  • the growth factor and LAP remain non-covalently bound, thereby forming a latent complex that cannot bind to its receptor and induce signal transduction.
  • latent TGF ⁇ 1 also known as a small latent complex (SLC)
  • SLC small latent complex
  • LLC large latent complex
  • presenting molecule two cysteines near the N-terminus of latent TGF ⁇ 1 are connected to appropriately positioned cysteines on the presenting molecule.
  • the identity of the presenting molecule depends on the environment and the cell type that produces latent TGF ⁇ 1.
  • fibroblasts secrete latent TGF ⁇ 1 tethered to TGF ⁇ -binding proteins (LTBPs), which then bind to proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibrillin-1) to link latent TGF ⁇ to the ECM (Robertson et al. Matrix Biol 47:44-53 (2015)).
  • ECM extracellular matrix
  • latent TGF ⁇ 1 is covalently linked to the transmembrane protein GARP, and a protein closely related to GARP, LRRC33, was recently identified as a presenting molecule for TGF ⁇ 1 on the surface of monocytes, macrophages, and microglia (Wang, R. et al., Mol Biol Cell, 2012. 23(6): p. 1129-39 and T.A. Springer, Int. BMP Conference 2016).
  • TGF ⁇ 1 protein should be understood in the broadest sense.
  • the term covers the natural form of TGF ⁇ 1 in nature, naturally occurring variants, and also includes artificially expressed forms, functional variants, etc.
  • TGF ⁇ 1 covers the scope of mature TGF ⁇ 1, TGF ⁇ 1 precursor protein and protein epitopes thereof.
  • TGF ⁇ 1 precursor protein covers pro-TGF ⁇ 1, latent TGF ⁇ 1 and fragments thereof.
  • TGF ⁇ 1 sequences can be obtained from GenBank, UniProt, etc. For example, human pro-TGF ⁇ 1 (Uniprot: P01137), mouse pro-TGF ⁇ 1 (Uniprot: P04202).
  • TGF ⁇ 1 complex refers to a complex formed by TGF ⁇ 1 and a protein molecule including but not limited to GARP, LRRC33, LTBP3 or LTBP1 through a disulfide bridge, also referred to as LTBP1-TGF ⁇ 1 complex, LTBP3-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex or GARP-TGF ⁇ 1 complex.
  • TGF ⁇ 1 exists in the form of a TGF ⁇ 1 precursor protein (e.g., pro/latent TGF ⁇ 1).
  • the term "functional variant” includes but is not limited to homologs, fragments, truncations, mutants, modifications, etc. of wild-type proteins.
  • the functional variant of a protein has improved, reduced or maintained protein activity compared to the wild-type protein.
  • binding molecule encompasses any molecule that can specifically bind to an antigen or antigenic epitope, such as an antibody, an antigen-binding fragment thereof, or a conjugate or fusion protein thereof as defined in the present disclosure.
  • Antibody is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibodies may refer to immunoglobulins, which are tetrapeptide chains consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, respectively.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into ⁇ chains or ⁇ chains according to the differences in the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes three hypervariable regions (CDRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the deterministic depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography.
  • a variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, conformational definitions.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is often used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions. (See, e.g., For example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198).
  • the contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45).
  • the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).
  • Still other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or extended based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding.
  • a CDR may refer to a CDR defined by any method known in the art (including a combination of methods).
  • the correspondence between the various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 4 below.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without loss of the function of the rest of the protein and/or the domain.
  • an “immunoglobulin variable domain” refers to an immunoglobulin domain that is essentially composed of four "framework regions” referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, and “framework region 4" or “FR4", respectively, wherein the framework regions are separated by three “complementarity determining regions” or “CDRs” referred to in the art and hereinafter as “complementarity determining region 1" or “CDR1", “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3".
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • An immunoglobulin variable domain confers specificity to an antigen by having an antigen binding site.
  • Antibody framework (FR) refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain.
  • Immunoglobulin single variable domain is generally used to refer to an immunoglobulin variable domain (which may be a heavy chain or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the required VH/VL interaction between the VH and VL domains of conventional four-chain monoclonal antibodies).
  • immunoglobulin single variable domains include nanobodies (including VHH, humanized VHH and/or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies (such as dAbs TM ) that are VH domains or derived from VH domains, and (single domain) antibodies (such as dAbs TM ) that are VL domains or derived from VL domains.
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody” (i.e., an "antibody lacking a light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain (which is referred to as a "VH domain” in the present disclosure) and the light chain variable domain (which is referred to as a "VL domain” in the present disclosure) present in a conventional tetrapeptide chain structure antibody.
  • the VHH domain specifically binds to an epitope without the need for other antigen binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids that are then humanized, or fully human antibodies obtained by phage display technology screening.
  • the total number of amino acid residues in the VHH domain will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R.
  • the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Other numbering systems or coding conventions include Chothia, IMGT, AbM.
  • Humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable region framework.
  • the strong immune response induced by chimeric antibodies due to carrying a large amount of non-human protein components can be overcome.
  • the fully human antibody variable region can be subjected to minimal reverse mutations to maintain activity.
  • humanization examples include “humanizing” a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody (also referred to as “sequence optimization" in this disclosure, in addition to humanization, “sequence optimization” may also cover other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removing potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.
  • Fully human antibodies or “fully humanized antibodies” include antibodies with variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). "Fully human antibodies” do not include “humanized antibodies”.
  • antigen binding proteins e.g., neutralizing antigen binding proteins or neutralizing antibodies
  • competition means competition between antigen binding proteins, which is determined by the following assay: the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen.
  • the antigen binding protein e.g., antibody or immunologically functional fragment thereof
  • a reference antigen binding protein e.g., ligand or reference antibody
  • RIA solid phase direct or indirect radioimmunoassays
  • EIA solid phase direct or indirect enzyme immunoassays
  • sandwich competition assays see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619
  • solid phase direct label assays solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Manual).
  • solid phase direct labeling RIA with I-125 label see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15
  • solid phase direct biotin-avidin EIA see, e.g., Cheung et al., 1990, Virology 176: 546-552
  • directly labeled RIA Methyl ⁇ -(Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82).
  • the assay involves the use of purified antigen that binds to an unlabeled test antigen binding protein and a labeled reference antigen binding protein (the antigen is on a solid surface or on a cell surface).
  • Antigen binding proteins identified by competition assays include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to an epitope that is sufficiently close to the epitope bound by the reference antigen binding protein that the two epitopes sterically interfere with each other's binding.
  • the competing antigen binding protein when it is present in excess, it will Inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of a reference antigen-binding protein to a common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • GARP Glycoprotein-A Repetitions Predominant
  • GARP is a protein with a single transmembrane structure
  • GARP is also called Leucin Rich Repeat Containing 32 (Leucin Rich Repeat Containing 32, LRRC32), and belongs to the Leucine Rich Repeat family.
  • GARP can be expressed on the cell surface of activated Tregs and form a complex with TGF- ⁇ precursor (such as latent TGF- ⁇ ).
  • GARP sequences can be obtained from GenBank, UniProt, etc. For example, human GARP (LRRC32, Uniprot: Q14392), mouse GARP (LRRC32, Uniprot: G3XA59).
  • GARP should be understood in the broadest sense.
  • the term covers the natural form of GARP in nature, naturally occurring variants, as well as artificially expressed forms, functional variants, etc.
  • GARP covers the scope of the complete protein, the extracellular domain, and its epitopes.
  • GARP-TGF ⁇ 1 complex refers to a protein complex comprising a precursor protein of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein and a glycoprotein-A repeat-based protein (GARP).
  • TGF ⁇ 1 transforming growth factor- ⁇ 1
  • GARP glycoprotein-A repeat-based protein
  • the proprotein form or latent form of the TGF ⁇ 1 protein may be referred to as a "pro/latent TGF ⁇ 1 protein”.
  • the GARP-TGF ⁇ 1 complex comprises a GARP covalently linked to a TGF ⁇ 1 precursor protein (pro/latent TGF ⁇ 1 protein) via one or more disulfide bonds.
  • the GARP-TGF ⁇ 1 complex comprises a GARP non-covalently linked to a TGF ⁇ 1 precursor protein (pro/latent TGF ⁇ 1 protein).
  • the GARP-TGF ⁇ 1 complex is a naturally occurring complex, such as a GARP-TGF ⁇ 1 complex in a cell.
  • LTBP latent transforming growth factor ⁇ binding proteins, which are important components of the extracellular matrix (ECM) and whose main function is related to the regulation of fibrillin and transforming growth factor ⁇ (TGF- ⁇ ).
  • ECM extracellular matrix
  • TGF- ⁇ fibrillin and transforming growth factor ⁇
  • GARP-TGF ⁇ 1 complex refers to a protein complex comprising a precursor protein form or a latent form of the transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein and a glycoprotein-A repeat-based protein (GARP).
  • TGF ⁇ 1 transforming growth factor- ⁇ 1
  • GARP glycoprotein-A repeat-based protein
  • the proprotein form or the latent form of the TGF ⁇ 1 protein may be referred to as a "pro/latent TGF ⁇ 1 protein”.
  • the GARP-TGF ⁇ 1 complex comprises a proprotein or a latent form of the TGF ⁇ 1 protein covalently linked to the TGF ⁇ 1 precursor protein (pro/latent TGF ⁇ 1 protein) via one or more disulfide bonds.
  • the GARP-TGF ⁇ 1 complex comprises GARP non-covalently linked to a TGF ⁇ 1 precursor protein (pro/latent TGF ⁇ 1 protein).
  • the GARP-TGF ⁇ 1 complex is a naturally occurring complex, such as a GARP-TGF ⁇ 1 complex in a cell.
  • LTBP1-TGF ⁇ 1 complex refers to a protein complex comprising a precursor protein of transforming growth factor ⁇ 1 (TGF ⁇ 1) protein and a latent TGF ⁇ binding protein (e.g., LTBP1, LTBP3).
  • TGF ⁇ 1 transforming growth factor ⁇ 1
  • LTBP1-TGF ⁇ 1 complex comprises LTBP1 covalently linked to the TGF ⁇ 1 precursor protein via one or more disulfide bonds.
  • the LTBP1-TGF ⁇ 1 complex comprises LTBP1 non-covalently linked to the TGF ⁇ 1 precursor protein.
  • the LTBP1-TGF ⁇ 1 complex is a naturally occurring complex, such as an LTBP1-TGF ⁇ 1 complex in a cell.
  • antigen refers to a molecule used to immunize an immunologically active vertebrate to produce an antibody that recognizes the antigen, or to screen an expression library (e.g., a phage, yeast, or ribosome display library, in particular).
  • antigens are defined more broadly to include target molecules specifically recognized by antibodies, as well as a portion or mimetic of a molecule used in an immunization process for producing antibodies or in a library screening for selecting antibodies.
  • antibodies that bind to the human GARP-TGF ⁇ 1 complex of the present disclosure, as well as truncated variants and other variants of the human GARP-TGF ⁇ 1 complex are referred to as antigens.
  • epitope refers to a site on an antigen that binds to an immunoglobulin or antibody.
  • An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent.
  • An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described in the present disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • a human GARP-TGF ⁇ 1 complex or an epitope thereof is used as an analyte and an antibody is used as a ligand
  • the antibody binds to the predetermined antigen or its epitope with an equilibrium dissociation constant ( KD ) of approximately less than 10-7 M or even less when measured in an instrument by surface plasmon resonance (SPR) technology, and its affinity for binding to the predetermined antigen or its epitope is at least twice its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen.
  • KD equilibrium dissociation constant
  • SPR surface plasmon resonance
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well-known methods and can be performed even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). Calculated.
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432).
  • Other standard assays for assessing the binding ability of an antibody against a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometric analysis, as well as other assays exemplified elsewhere in this disclosure.
  • binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), such as by using a Biacore TM system or KinExA.
  • SPR surface plasmon resonance
  • Binding affinities associated with different molecular interactions can be compared by comparing the KD values of individual antibody/antigen complexes, for example, a comparison of the binding affinities of different antibodies for a given antigen.
  • the specificity of an interaction can be evaluated by determining and comparing the KD value of an interaction of interest (e.g., a specific interaction between an antibody and an antigen) with the KD value of a non-interaction of interest (e.g., a control antibody known not to bind to the antigen).
  • conservative substitution refers to substitution with another amino acid residue having properties similar to the original amino acid residue.
  • lysine, arginine and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains.
  • tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when replacing an amino acid residue in a group showing similar properties as described above, it will not show a specific change in properties.
  • sequence identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two compared sequences are occupied by the same nucleotide or amino acid monomer, for example, if every position of the two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position.
  • the percentage of homology between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is performed when the two sequences are aligned to obtain the maximum percentage of homology.
  • nucleic acid molecule and “polynucleotide” are used interchangeably to refer to DNA molecules and RNA molecules.
  • Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA.
  • a nucleic acid is "operably linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • host cell includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • Host cells include cells transfected and/or transformed in vivo with a polynucleotide of the present disclosure.
  • Cell Cellline
  • cell culture are used interchangeably, and all such designations include their progeny. Also, It is understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations, and mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • an “inhibitory antibody” refers to an antibody that inhibits the release of a mature growth factor or reduces the activity of a growth factor.
  • Inhibitory antibodies include antibodies that target any epitope that reduces the release or activity of a growth factor when bound to such an antibody. Such an epitope may be located on the prodomain of a TGF ⁇ protein (e.g., TGF ⁇ 1), a growth factor, or other epitopes that result in reduced growth factor activity when bound by an antibody.
  • Inhibitory antibodies of the present invention include, but are not limited to, TGF ⁇ 1-inhibitory antibodies.
  • an "effective amount” includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method, 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 subject of the present disclosure may be an animal or a human subject.
  • composition refers to a mixture containing one or more active ingredients described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.
  • pharmaceutically acceptable excipient or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system.
  • examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline.
  • PBS phosphate-buffered saline
  • Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).
  • administer refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, such as for treatment, pharmacokinetics, diagnosis, research, and experimental procedures.
  • Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administer also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell.
  • it refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • treatment means administering a therapeutic agent, such as a fusion protein or insulin analog comprising any of the disclosed compounds, to a subject who has, is suspected of having, or is prone to having one or more diabetes or hyperglycemia-related diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • a therapeutic agent such as a fusion protein or insulin analog comprising any of the disclosed compounds
  • the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in a treated subject or population, by preventing or delaying the onset of symptoms or complications, alleviating symptoms or complications, or eliminating the disease, condition or illness to any clinically measurable extent.
  • the amount of the therapeutic agent that effectively alleviates any specific disease symptom may vary according to a variety of factors, such as the disease state, age and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms.
  • the embodiments of the present disclosure may be ineffective in alleviating the target disease symptoms in a subject, it should alleviate the target disease symptoms in a statistically significant number of subjects according to any statistical test method known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test.
  • the patient to be treated is a mammal, and preferably a human.
  • prevent refers to reducing the risk or incidence, or eliminating or slowing the progression of one or more conditions, symptoms, complications, or disorders.
  • subject refers to mammals, particularly primates, and especially humans.
  • Figure 1 shows the results of detecting the inhibition of the activity of anti-TGF ⁇ 1 antibodies (H27, SL2-2, SL2-9, SL2-12, SL2-19 and SL2-22) on human or mouse TGF ⁇ 1 complexes (human proTGF ⁇ 1, GARP-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex, mouse GARP-TGF ⁇ 1 complex).
  • A is a graph showing the inhibitory effect of antibodies on human proTGF ⁇ 1 activation
  • B is a graph showing the inhibitory effect of antibodies on human GARP-TGF ⁇ 1 complex activation
  • C is a graph showing the inhibitory effect of antibodies on human LRRC33-TGF ⁇ 1 complex activation
  • D is a graph showing the inhibitory effect of antibodies on mouse GARP-TGF ⁇ 1 complex activation.
  • Figure 2 shows the results of an experiment detecting the binding of anti-TGF ⁇ 1 antibodies (SL2-22, Ab6) to HEK293E cells expressing human or mouse original TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3, and TGF ⁇ complexes (GARP-TGF ⁇ 1 complex, GARP-TGF ⁇ 2 complex, GARP-TGF ⁇ 3 complex).
  • Figures 3A-3C show the results of the combination of anti-TGF ⁇ 1 antibody and anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+SL2-22-mIgG2a-FcS) in inhibiting tumor growth in the mouse EMT-6 model.
  • Figure 3A is a graph of mouse tumor volume
  • Figure 3B is a graph of mouse survival rate
  • Figure 3C is a graph of mouse weight.
  • Figures 4A-4C show the results of anti-TGF ⁇ 1 antibody and anti-PD-1 antibody combined with RMP1-14-mIgG2a-FcS+SL2-22-mIgG2a-FcS) inhibiting tumor growth in the mouse CT26 model.
  • Figure 4A is a graph of mouse tumor volume
  • Figure 4B is a graph of mouse survival rate
  • Figure 4C is a graph of mouse weight.
  • Figures 5A-5E show the results of anti-TGF ⁇ 1 antibody (SL2-22-mIgG2a-FcS) inhibiting fibrosis in a mouse idiopathic fibrosis model.
  • Figure 5A is a curve of weight change of mice in the pulmonary fibrosis model
  • Figure 5B is a curve of survival rate of mice in the pulmonary fibrosis model
  • Figure 5C is a curve of lung weight and lung tissue hydroxyproline of mice at the end of the pulmonary fibrosis model experiment.
  • Figure 5D shows the lung sections of mice at the end point of the pulmonary fibrosis model experiment
  • Figure 5E shows the collagen area ratio stained by Masson.
  • Figure 6 shows the inhibition of GARP-TGF ⁇ 1 complex activation by anti-GARP-TGF ⁇ 1 single domain antibodies detected by LN229 experiments.
  • LN229 cells were transfected with human or mouse original TGF ⁇ 1 and human or mouse GARP expression plasmids, and the inhibition of Abbv-151, C19, C19-3, C19-7, and C19-8 on human or mouse GARP-TGF ⁇ 1 complex activation was detected by HepG2CAGA12-luc luciferase reporter cells.
  • Figure 6A is a diagram showing the inhibitory effect of antibodies on human GARP-TGF ⁇ 1 complex activation
  • Figure 6B is a diagram showing the inhibitory effect of antibodies on mouse GARP-TGF ⁇ 1 complex activation.
  • Figure 7 shows the results of the binding experiment of C19-8 with HEK293E cells expressing human or mouse GARP-TGF ⁇ 1 complex.
  • HEK293E cells were transfected with the plasmids in Table 3, and C19-8 and Abbv-151 were added, and then stained and detected to obtain the results.
  • Figures 8A-8C show the results of C19-8 combined with anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+C19-8-mIgG2a-FcS) inhibiting tumor growth in the mouse EMT-6 model.
  • Figure 8A is a graph of mouse tumor volume
  • Figure 8B is a graph of mouse survival rate
  • Figure 8C is a graph of mouse weight.
  • Figures 9A-9C show the results of C19-8 combined with anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+C19-8-mIgG2a-FcS) inhibiting tumor growth in the mouse CT26 model.
  • Figure 9A is a graph of mouse tumor volume
  • Figure 9B is a graph of mouse survival rate
  • Figure 9C is a graph of mouse weight.
  • the italic part is the signal peptide; the underlined part is the latency-associated peptide (LAP); the italic bold part is the TGF ⁇ 1 domain
  • the italic part is the signal peptide; the underlined part is the extracellular domain; the italic bold part is the transmembrane domain; the italic underlined part is the cytoplasmic domain)
  • the italic part is the signal peptide; the underlined part is the potential associated peptide (LAP); the italic bold part is the TGF ⁇ 1 domain)
  • the italic part is the signal peptide; the underlined part is the extracellular region; the italic bold part is the transmembrane region; the italic underlined part is the intracellular region)
  • the human GARP/TGF ⁇ 1 complex for screening and detection was prepared as follows: human GARP-avi-his and human natural TGF ⁇ 1 (shown in SEQ ID NO: 5 and 6, respectively) were co-expressed in ExpiCHO cells (ThermoFisher, A29127) and purified to obtain a protein complex; human TGF ⁇ 1 C4S protein is shown in SEQ ID NO: 7; mouse GARP/TGF ⁇ 1 complex was prepared as follows: mouse GARP-avi-his and mouse natural TGF ⁇ 1 (shown in SEQ ID NO: 8 and 9, respectively) were co-expressed in ExpiCHO cells (ThermoFisher, A29127) and purified to obtain a protein complex; mouse TGF ⁇ 1 C4S protein is shown in SEQ ID NO: 10:
  • the underlined part is the extracellular region of human GARP; the italic part is the (G 4 S) 2 linker; Avi tag; His tag)
  • the underlined part is the mouse GARP extracellular region; the italic part is the (G 4 S) 2 linker; The lower curve is the His tag)
  • Example 2 Screening of anti-TGF ⁇ 1 monoclonal antibodies that specifically bind to human TGF ⁇ 1 complex
  • the human antibody phage library was screened to obtain antibodies with high affinity to the human TGF ⁇ 1 complex.
  • human antibody phage libraries (1: semi-synthetic human Fab library 1 (germline 3-23); 2: semi-synthetic human Fab library 2 (germline 1-69); 3: fully human Fab library; 4: fully human scFV library), all donated by Shanghai Hengrui Medicine Co., Ltd. 10 ⁇ g of biotinylated human GARP-TGF ⁇ 1 complex (containing SEQ ID NO: 5 and 6) protein was combined with 1 mg of Dynabeads M-280 streptavidin (Cat No.
  • the phages that did not bind were washed 10 times with 1 ⁇ PBST (containing 0.05% Tween-20), pH 7.4 solution to remove the unbound phages, and then washed 2 more times with 1 ⁇ PBS, and the phages that specifically bound to human GARP/TGF ⁇ 1 were eluted with 0.5 mL of trypsin (1 mg/mL), and infected with E. coli TG1 in the logarithmic growth phase, 2YT (containing 2% glucose) resistance plates to grow overnight, and the phages were produced and purified from the plates for the next round of screening.
  • 1 ⁇ PBST containing 0.05% Tween-20
  • pH 7.4 solution to remove the unbound phages
  • 1 ⁇ PBS washed 2 more times with 1 ⁇ PBS
  • the phages that specifically bound to human GARP/TGF ⁇ 1 were eluted with 0.5 mL of trypsin (1 mg/mL), and infected with E. coli
  • biotinylated mouse TGF ⁇ 1_C4S (SEQ ID NO: 10) was used as the screening antigen in the second round.
  • biotinylated human GARP-TGF ⁇ 1 complex (containing SEQ ID NO: 5 and 6) was used as the screening antigen in the third round. After three rounds of screening, positive clones were enriched.
  • ELISA plates (Cat No.9018, Corning) were coated with 2 ⁇ g/mL human GARP-TGF ⁇ 1 complex (SEQ ID NO: 5 and 6) and mouse TGF ⁇ 1_C4S (SEQ ID NO: 10) proteins, respectively, and placed at 4°C overnight, washed 3 times with 1 ⁇ PBST, blocked with 2% BSA at 37°C for 1 hour, washed 3 times with PBST, added with phage supernatant diluted with blocking solution, reacted at room temperature for 1 hour, washed 6 times with PBST, added with anti-M13HRP (Cat No.11973-MM05T-H, Sino Biological), reacted at room temperature for 1 hour, washed 3 times with PBST, added with 100 ⁇ L TMB colorimetric substrate, and terminated with 100 ⁇ L 1M sulfuric acid, and read the absorb
  • the clones with OD450 readings greater than 3 times the negative control in the ELISA binding test were sequenced and 43 specific antibody sequences were obtained.
  • the SPR experiment showed that antibodies H5, H14, H16, H17, H19, H20, H21, H23, H24, H27, H28, H30, H32, H34 and H39 could bind to the TGF ⁇ 1 complex.
  • the cell function experiment showed that only H27 had a good inhibitory function and had good binding to human and mouse GARP-TGF ⁇ 1 complexes and human and mouse TGF ⁇ 1_C4S (i.e., TGF ⁇ 1 precursor protein).
  • the sequence of H27 obtained by screening the human antibody phage library in Example 2 was used to construct a complete recombinant antibody: the antibody light chain variable region VL was cloned into the pTT5 expression vector containing the human kappa light chain constant region (SEQ ID NO: 19), and the antibody heavy chain variable region VH was cloned into the pTT5 expression vector containing the human IgG4-S228P (CH1-CH2-CH3) heavy chain constant region (SEQ ID NO: 20).
  • the cloned light chain and heavy chain plasmids were co-transfected into HEK293E cells (donated by Shanghai Hengrui Medicine Co., Ltd.) or ExpiCHO (Cat No. A29127, ThermoFisher) cells.
  • the cell culture supernatant was harvested 5 days (37 degrees, HEK29E3 cells) or 10-12 days (32 degrees, expiCHO cells) after transfection, centrifuged at 4000 rpm for 20 minutes, and the supernatant was filtered using a 0.45 ⁇ M filter.
  • the first step of affinity purification was performed using a MabSelectSure LX column (GE Healthcare).
  • the culture supernatant was passed through a MabSelectSure LX column equilibrated with PBS, washed with PBS, eluted with 0.1M Glycine acidic eluent at pH 3.0, and neutralized with 1M MES (pH 6.0), and finally finely purified using a HiTrap SP HP ion column. After testing, the target antibody was obtained.
  • the affinity of the antibody to human/mouse GARP/TGF ⁇ 1 and human/mouse TGF ⁇ 1_C4S was determined using Biacore T200 (GE Healthcare).
  • a certain amount of the antibody to be tested was affinity captured using a Protein A biosensor chip (Cat No.#29127556, GE), and then a series of gradient dilutions of human or mouse GARP-TGF ⁇ 1 complex TGF ⁇ 1_C4S antigen were passed through the chip surface at a flow rate of 50 ⁇ L/min and a dissociation time of 5 minutes. After each cycle, the chip was regenerated using pH 1.5 glycine-hydrochloric acid (Cat.#BR-1003-54, GE). The reaction buffer was HBS-EP+ buffer solution (Cat No.#BR-1006-69, GE) diluted to 1 x with distilled water (pH 7.4).
  • Biacore T200 was used to detect the reaction signal in real time to obtain the binding and dissociation curves.
  • the data were fitted with the Langmuir 1:1 binding model using BIAevaluation version 4.1 and GE software to obtain the affinity values. The results are shown in Table 6.
  • the H27 antibody molecule was subjected to three-dimensional structural simulation. With reference to the human germline gene mutation hotspots and three-dimensional structural simulation results, some key amino acid residues in the framework region and CDR region were selected to establish four random mutation phage libraries (Table 7). Phage library display technology was used to screen functional antibodies with improved affinity. New amino acid residues obtained from different libraries were combined and verified to obtain functional antibodies with improved affinity and function.
  • the cloned light chain and heavy chain plasmids were paired and co-transfected into HEK293E cells. After 5 days, the cell culture supernatant was collected and centrifuged at 4000 rpm to remove the cells. After purification using MabSelect Sure, affinity measurement was performed according to the method of Example 4.
  • the antibodies with affinity more than 5 times higher than H27 determined by SPR protein interaction were all from the second random mutation library, and the heavy chain of the library antibody was the H27 heavy chain.
  • the light chain variable region sequence is shown in Table 8 (the underlined part is the CDR region, using the Kabat coding rule).
  • the full-length sequence of the light chain and the CDR combination are shown in Tables 9 and 10, respectively.
  • SL2-1, SL2-2, SL2-3, SL2-4, SL2-5, SL2-6, SL2-8, SL2-9, SL2-12, SL2-13, SL2-15, SL2-17, SL2-18, SL2-19, SL2-22 have the following general structure:
  • LCDR1 is RASQX1ISX2YLN (SEQ ID NO: 38 ), X1 is selected from S, A, F, G, I, P, Y, V or K, and X2 is selected from S, D, E, P or H;
  • LCDR2 is X 3 ASX 4 LX 5 S (SEQ ID NO: 64), X 3 is selected from A, T, S or M, X 4 is selected from S, Y, A, E or G, and X 5 is selected from Q, T, D or E.
  • SL2-2, SL2-9, SL2-12, SL2-19 and SL2-22 have the following general structure:
  • LCDR1 is RASQX 1 ISX 2 YLN (SEQ ID NO: 38), wherein X 1 is selected from F, Y or A, and X 2 is selected from D or P;
  • LCDR2 is X 3 ASX 4 LX 5 S (SEQ ID NO: 64), wherein X 3 is selected from A, T or S, X 4 is selected from S, Y or E, and X 5 is selected from Q, D or E.
  • the negative control of this example is HBS-EP, and the positive control is Ab6 (Scholar rock, WO2020014460Al) and Abbv-151 (Abbvie, US10793627B2).
  • Ab6 binds to the GARP-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex, LTBP3-TGF ⁇ 1 complex, and LTBP1-TGF ⁇ 1 complex, and the binding epitope is entirely on TGF ⁇ 1 (Martin et al., 2020); while the binding epitope of Abbv-151 includes GARP and TGF ⁇ 1 in the GARP-TGF ⁇ 1 complex, and does not bind to GARP or TGF ⁇ 1 alone (Streel et al., 2020).
  • the sequences of Ab6 and Abbv-151 are as follows:
  • TGF ⁇ 1 monoclonal antibody inhibits the function of GARP-TGF ⁇ 1 complex in vitro
  • 2 ⁇ 10 6 LN229 cells (Procell, CL-0578) were transferred to T75 culture flasks (Nunc). After 24 hours, 293fectin TM (Invitrogen) transfection reagent was used to transfect human or mouse pro-TGF ⁇ 1 alone, or co-transfect human or mouse pro-TGF ⁇ 1 and GARP or LRRC33 plasmids. After 24 hours, 1 ⁇ 10 4 cells were transferred to white opaque 96-well cell culture plates (PerkinElmer) per well.
  • the culture medium was aspirated, and HepG2CAGA12-luc luciferase reporter cells resuspended in DMEM+0.5% BSA were added, and H27, H27 affinity matured antibody and control antibody Ab6 were added in multiple dilutions.
  • ONE-Glo TM luciferase reagent was added, and after incubation for 5 minutes, the chemiluminescence value was read on a multi-function microplate reader (SpectraMax M5).
  • Prism 9 was used to plot the results using a nonlinear fit and a three-parameter log inhibitor response model.
  • FIGS. 1A and 1B The affinity-matured SL2-2, SL2-9, SL2-12, SL2-19 and SL2-22 have higher inhibitory activities against pro-TGF ⁇ 1 and each complex of TGF ⁇ 1 than the parent H27.
  • SL2-2, SL2-9, SL2-12, SL2-19 and SL2-22 are comparable to the positive control Ab6 in terms of inhibiting the activities of human pro-TGF ⁇ 1, GARP-TGF ⁇ 1 complex and mouse GARP-TGF ⁇ 1 complex, and are superior to Ab6 in terms of inhibiting the activity of human LRRC33-TGF ⁇ 1 complex.
  • HEK293E cells were prepared to 1 ⁇ 10 6 cells/mL, 5 mL was taken and placed in a 50 mL culture tube, and the plasmids in Table 13 were transfected with 293fectin TM reagent. After 48 hours, the cells were washed twice with 1 x PBS, and 1 x 10 5 cells were placed in a 96-well round-bottom cell culture plate (Nunc) per well. 100 ⁇ L of 10nM antibody or control antibody prepared with 1 x PBS was added, and the cells were stained at room temperature for 1 hour. After washing 3 times with 1 ⁇ PBS, 100 ⁇ L of FITC-labeled anti-human Fc antibody was added, and the cells were stained for 30 minutes at room temperature.
  • the negative control of this example is HBS-EP, and the positive controls are Ab6 and Abbv-151.
  • the CM5 sensor chip (Cat. 29-1049-88, GE) was tested for antibody using an amino coupling kit (Cat No. BR-1000-50, GE) and a His capture kit (Cat No. 28-9950-56, GE) according to the kit instructions.
  • the His tag antibody was coupled to make the surface response value reach about 10,000 RU, and then blocked with ethanolamine for later use.
  • Second antibody concentration 100 ⁇ g/ml, flow rate 50 ⁇ L/min, 60 seconds, HBS-EP buffer as control
  • TGF ⁇ 1 monoclonal antibody SL2-22 inhibits tumor growth in the mouse EMT-6 model
  • mice Female, 6 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Mouse breast cancer cells EMT-6 Procell, CL-0573
  • PBS PBS
  • inoculated subcutaneously in the right rib of BALB/c mice at a concentration of 5 ⁇ 10 6 cells/mL and a volume of 0.1 mL/mouse.
  • mice with moderate individual tumor volumes were selected and randomly grouped as shown in Table 15.
  • mIgG2a-FcS is mIgG2a with L234A/L235E/G237A/D327Q/A330S/P331S that removes the ADCC effect.
  • RMP1-14-mIgG2a-FcS is the variable region of the anti-PD1 antibody RMP1-14 (sequences 285 (heavy chain variable region) and 286 (light chain variable region) with the signal peptide removed in WO2018223182A1; WO2018223182A1 is incorporated herein by reference in its entirety) and L234A/L235E/G237A/D327Q/A with ADCC effect removed
  • SL2-22-mIgG2a-FcS is an antibody fused with the variable region of SL2-22 and the mIgG2a heavy chain constant region with L234A/L235E/G237A/D327Q/A330S/P331S/murine kappa light chain constant region with ADCC removed, and the sequence is as follows:
  • the drug was administered on the day of grouping, at the same molar dose, twice a week, for a total of 7 times, by intraperitoneal injection.
  • the molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS and SL2-22-mIgG2a-FcS is the same, about 68 ⁇ mol/kg.
  • TGF ⁇ 1 antibody SL2-22 inhibits tumor growth in the mouse CT26 model
  • mice Female, 6 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Mouse colon cancer cells CT26 National Biomedical Experimental Cell Resource Bank, 1101MOU-PUMC000275
  • the cells were inoculated at a concentration of 3 ⁇ 10 6 cells/mL and a volume of 0.1 mL/mouse.
  • the mice were implanted subcutaneously in the right flank of BALB/c mice.
  • the molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS and SL2-22-mIgG2a-FcS is the same, about 68 ⁇ mol/kg
  • TGF ⁇ 1 monoclonal antibody SL2-22 inhibits fibrosis progression in a mouse idiopathic fibrosis model
  • C57BL/6J mice Male, 8 weeks old, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and were adaptively raised for 1 week. They were numbered and weighed on the day of the experiment. They were numbered and weighed on the day of modeling and randomly divided into groups according to the weight distribution, with 15 mice in each group. The specific groups are shown in Table 17 below.
  • antibody drugs or solvent controls were given by intraperitoneal injection according to body weight.
  • bleomycin was instilled through the trachea using a flat-head needle at a dose of 2U/kg, and each animal was slowly instilled with about 50 ⁇ L. Then, according to the above groups, the drug was administered twice a week for a total of 3 weeks. On the 22nd day of the experiment, bronchoalveolar lavage fluid and lung tissue were collected to analyze the degree of immune cell infiltration, lung tissue hydroxyproline level, and Masson staining analysis.
  • the SL2-22-mIgG2a-Fc administration group significantly improved the overall body weight and survival rate of the animals ( Figures 5A and 5B), and reduced the hydroxyproline level in the lung tissue, which was reduced by 40.44% compared with the modeling group ( Figure 5C); at the same time, it significantly reduced the collagen deposition and fibrosis levels in the lungs after bleomycin modeling, which was reduced by 46.51% compared with the modeling group ( Figures 5D and 5E).
  • a Bactrian camel was immunized with human GARP/TGF ⁇ 1 protein (a protein complex obtained by co-expression and purification of the proteins shown in SEQ ID NO: 5 and 6) as an antigen. After Freund's complete adjuvant and the antigen were mixed in a 1:1 volume, the camel was immunized subcutaneously at multiple points every two weeks. The first immunization dose was 200 ⁇ g protein, followed by four immunization doses of 100 ⁇ g protein/time, for a total of five immunizations, and then the titer was determined using human GARP/TGF ⁇ 1 protein. When the titer was qualified, peripheral blood of the camel was collected, lymphocytes were separated, cells were lysed with Trizol, RNA was extracted, cDNA was obtained by reverse transcription, and a phage library was constructed.
  • human GARP/TGF ⁇ 1 protein a protein complex obtained by co-expression and purification of the proteins shown in SEQ ID NO: 5 and 6
  • 96 monoclonal colonies were selected from the positive clones screened and enriched and packaged into phage single-chain antibodies for phage ELISA test.
  • 2 ⁇ g/mL human GARP-TGF ⁇ 1 complex protein was coated on the ELISA plate, phage supernatant diluted with blocking solution was added, and anti-M13 HRP-labeled antibody was used for detection.
  • the clones with OD450 value/background value>5 in the ELISA binding test results were sequenced to obtain the sequence.
  • the C19-VHH sequence is shown below.
  • SEQ ID NO:84 the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequence are FR sequences, and the underlines are CDR1, CDR2 and CDR3 sequences respectively.
  • the numbering rules of the anti-GARP-TGF ⁇ 1 single domain antibodies provided in the present disclosure are all Kabat.
  • the C19-VHH sequence was fused with the following human IgG4-Fc (CH2-CH3, containing S228P) fragment to obtain C19.
  • the C19 sequence was cloned into the mammalian expression vector pTT5 and transfected into HEK293E or ExpiCHO (ThermoFisher, A29127) cells.
  • the cell culture supernatant was harvested by centrifugation at 4000rpm for 20min 5 days after transfection (HEK293E cells, 37°C) or 10-12 days (ExpiCHO cells, 32°C) and filtered with a 0.45 ⁇ M filter.
  • the first step of affinity purification was performed using a MabSelectSure LX column (GE Healthcare).
  • the culture supernatant was passed through a MabSelectSure LX column equilibrated with PBS, washed with PBS, eluted with 0.1M Glycine acidic eluent at pH 3.0, and neutralized with 1M Tris-HCl (pH 8.0), and finally finely purified with a HiTrap Q HP ion column. After testing, the target antibody was obtained.
  • C19 has good inhibitory function and has good binding to both human and mouse GARP-TGF ⁇ 1 complexes.
  • the heavy chain variable region germline gene IGHV3-23 with high sequence homology to C19 was selected as the template for FR1, FR2, and FR3, and IGJH4 was used as the template for FR4.
  • the CDR of the camel-derived single domain antibody was transplanted into the corresponding human template to form a variable region sequence with the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • a series of back mutations were performed in the framework region.
  • the humanized VHH framework region contains at least one amino acid mutation selected from the following: 23T, 29Y, 30C, 37Y, 44E, 45R, 47F, 71Q, 74A, 75R, 78G, 81E, 93K and 94T.
  • the above amino acid positions are according to the Kabat numbering.
  • the obtained humanized sequence is as follows, the CDR region is underlined, and the coding rule is Kabat coding:
  • the whole antibody sequence of humanized single domain antibody VHH fused with the Fc region of hIgG4 was constructed using the method in Example 2.
  • the obtained humanized whole antibody sequence is as follows:
  • the negative control of this example is HBS-EP (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH7.4).
  • the positive control is Abbv-151 (Abbvie, US10793627B2). Its sequence is as follows:
  • the affinity of C19 and its humanized antibody, positive control antibody Abbv-151 and human GARP-TGF ⁇ 1 complex (formed by the protein complex of SEQ ID NO: 5 and 6) or mouse GARP-TGF ⁇ 1 complex (formed by the protein complex of SEQ ID NO: 8 and 9) was measured by Biacore T200 (GE Healthcare).
  • the antibody to be detected was captured on the chip surface using chip Series S sensor chip Protein A (GE Healthcare, 29127556), and then different concentrations of human or mouse GARP-TGF ⁇ 1 complex were passed on the chip surface.
  • the reaction signal was detected in real time to obtain the binding and dissociation curves, and the binding force constant was obtained by fitting.
  • the solution used in the experiment was HBS-EP solution.
  • the chip was regenerated with pH 1.5 Glycine (GE Healthcare, BR-1003-54) solution.
  • the affinity results of the antibodies are shown in Table 19. The results showed that the affinity of the antibodies C19, C19-3, C19-7 and C19-8 obtained by the present disclosure screening to the human GARP-TGF ⁇ 1 complex was comparable to that of the positive control Abbv-151, except that they also had a certain binding to the mouse GARP-TGF ⁇ 1 complex.
  • 2 x 10 6 LN229 cells (Procell, CL-0578) were transferred to T75 culture flasks (Nunc). After 24 hours, 293fectin TM (Invitrogen) transfection reagent was used to transfect human or mouse pro-TGF ⁇ 1 alone, or co-transfect human and mouse pro-TGF ⁇ 1 and GARP plasmids. After 24 hours, the cells were transferred to white opaque 96-well cell culture plates (PerkinElmer) at 1 ⁇ 10 4 per well.
  • the culture medium was aspirated, and HepG2CAGA12-luc luciferase reporter cells resuspended in DMEM+0.5% BSA were added, and C19, C19 humanized antibody and control antibody Abbv-151 were added in multiple dilutions.
  • ONE-Glo TM luciferase reagent was added, and after incubation for 5 minutes, the chemiluminescence value was read on a multi-function microplate reader (SpectraMax M5).
  • Prism 9 was used to plot the nonlinear fitting and three-parameter log inhibitor response model.
  • C19, C19-3, C19-7 and C19-8 are comparable to the positive control Abbv-151 in inhibiting the activity of the human GARP-TGF ⁇ 1 complex, except that they also inhibit the mouse GARP-TGF ⁇ 1 complex.
  • Example 17 Binding experiment of anti-GARP-TGF ⁇ 1 single domain antibody C19-8 and cells expressing GARP-TGF ⁇ 1
  • HEK293E cells were prepared to 1 ⁇ 10 6 cells/mL, 5 mL was taken and placed in a 50 mL culture tube, and the plasmids in Table 20 were transfected with 293fectin TM reagent. After 48 hours, the cells were washed twice with 1 ⁇ PBS and placed in a 96-well round-bottom cell culture plate (Nunc) at 1 ⁇ 10 5 cells per well. 100 ⁇ L of 10nM antibody or control antibody prepared in 1 ⁇ PBS was added, and the cells were stained at room temperature for 1 hour. After washing 3 times with 1 ⁇ PBS, 100 ⁇ L of FITC-labeled anti-human Fc antibody was added and stained at room temperature for 30 minutes.
  • the cells were washed 3 times with 1 ⁇ PBS. 7-AAD was added and incubated for 5 minutes. After washing 3 times with 1 ⁇ PBS, the cells were detected by flow cytometry. The number of FITC-positive cells in living cells was analyzed by Flowjo, and the percentage of FITC-positive cells in the total number of living cells was calculated and plotted.
  • the negative control of this example is HBS-EP, and the positive control is Ab6 (Scholar rock, WO2020014460A1, the entire text is incorporated into this disclosure) and Abbv-151.
  • Ab6 binds to the GARP-TGF ⁇ 1 complex, LRRC33-TGF ⁇ 1 complex, LTBP1-TGF ⁇ 1 complex, and LTBP3-TGF ⁇ 1 complex, and the binding epitope is entirely on TGF ⁇ 1 (Martin et al., 2020); while the binding epitope of Abbv-151 includes GARP and TGF ⁇ 1 in the GARP-TGF ⁇ 1 complex, and does not bind to GARP or TGF ⁇ 1 alone (Streel et al., 2020).
  • the sequence of Ab6 is as follows:
  • the epitope competition between C19-8 and positive antibodies Abbv-151 and Ab6 and human GARP/TGF ⁇ 1 was determined by Biacore T200 (GE Healthcare).
  • the chip Series S sensor chip CM5 GE Healthcare, Br100530
  • the kit used was the His capture kit (GE Healthcare, 28-9950-56).
  • the antigen protein human GARP-TGF ⁇ 1 complex was captured on the chip surface, and then antibodies of the same concentration (500nM) were sequentially passed on the chip surface, according to the combination of Table 21, 1st antibody is the first antibody to pass through, and 2nd antibody is the second antibody to pass through.
  • the reaction signal was detected in real time using a Biacore instrument to obtain an epitope competition curve.
  • the solution used in the experiment was HBS-EP solution.
  • the chip was regenerated with pH 1.5 Glycine (GE Healthcare, BR-1003-54) solution.
  • the results of the antibody epitope competition assay are shown in Table 22.
  • mice Female, 6 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Mouse breast cancer cells EMT-6 Procell, CL-0573
  • mice with moderate individual tumor volumes were selected and randomly grouped as shown in Table 23.
  • the positive drug M7824 is PD-L1/TGF ⁇ -trap (Merck KGaA, sequence 3 (heavy chain) and sequence 1 (light chain) in WO2018029367A1 (incorporated herein by reference in its entirety));
  • mIgG2a-FcS is mIgG2a Fc with L234A/L235E/G237A/D327Q/A330S/P331S that removes the ADCC effect, which is used as an isotype control here;
  • RMP1-14-mIgG2a-FcS is the variable region of the anti-PD1 antibody RMP1-14 (WO2018223182A1 (incorporated herein by reference in its entirety)).
  • C19-8-mIgG2a-FcS is an antibody in which the variable region of C19-8 is fused with the mIgG2a heavy chain constant region/mouse kappa light chain constant region with L234A/L235E/G237A/D327Q/A330S/P331S and the ADCC effect is removed, and the sequence is as follows:
  • the drug was administered on the day of grouping, at the same molar dose, twice a week, for a total of 7 times, by intraperitoneal injection.
  • the molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS, C19-8-mIgG2a-FcS and M7824 is the same, about 68 ⁇ mol/kg.
  • the molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS and C19-8-mIgG2a-FcS is the same, about 68 ⁇ mol/kg

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Abstract

涉及TGFβ1结合分子、GARP-TGFβ1结合分子及其医药用途。具体涉及TGFβ1结合分子、GARP-TGFβ1结合分子、包含其的药物组合物、其制备方法及医药用途。还涉及使用所述的TGFβ1结合分子、GARP-TGFβ1结合分子和包含其的药物组合物治疗和/或预防疾病(例如肿瘤或癌症)的方法。

Description

TGFβ1结合分子、GARP-TGFβ1结合分子及其医药用途
本公开要求2023年01月09日提交的中国专利申请202310027965.9、以及2023年01月09日提交的中国专利申请202310037692.6的优先权，前述专利申请的全部内容通过引用并入本公开。
技术领域
本公开涉及生物医药领域，具体涉及TGFβ1结合分子、包含TGFβ1结合分子的药物组合物，及医药用途、制备方法，预防或治疗治疗与TGFβ1信号通路相关的疾病的方法。
背景技术
TGFβ(Transforming growth factorβ)是一类多效的生长因子，包括TGFβ1、TGFβ2和TGFβ3。它们调节许多重要的生理过程，如细胞生长、分化、增殖、凋亡和基质产生。研究发现，TGFβ在肿瘤发生的早期能够抑制肿瘤的生长，但是在肿瘤进展的晚期却促进肿瘤生长(Liu S,Ren J,Ten Dijke P.Signal Transduct Target Ther.2021 Jan 8；6(1):8.)。TGFβ促进晚期肿瘤生长的一个重要机理是抑制肿瘤微环境的免疫细胞。其中，TGFβ1，而非TGFβ2和TGFβ3，是促进晚期肿瘤生长的关键因子(Constance J Martin,et al.Sci Transl Med.2020Mar 25；12(536):eaay8456.)。肿瘤细胞、调控T细胞(Treg)和抑制性的吞噬细胞等产生的TGFβ1能够直接促进Treg细胞的活性并抑制效应T细胞和抗原呈递细胞，从而达到抑制免疫的效果。
目前，肿瘤免疫检查点抑制剂抗PD-1(Programmed Cell Death-1)抗体在肿瘤的免疫治疗过程中取得了巨大的成功，但是患者的总应答率只有20％左右。抗TGFβ1抗体与抗PD-1抗体联用在EMT-6、MBT-2、Cloudman S91、CT26和MC38等肿瘤模型上能够显著克服肿瘤的免疫逃避，增强抗PD-1抗体的药效。然而，靶向成熟TGFβ1的pan-TGFβ抗体在实验中呈现出剂量依赖的体内毒性。靶向原/潜伏TGFβ1复合物从源头上抑制TGFβ1的产生，这比靶向成熟的TGFβ1在动物模型上具有更好的药效，并具有显著减少的毒性。
TGFβ1的产生受到多步过程的严密调控。TGFβ1前体蛋白中，其C末端的成熟TGF-β1结构域与N末端的称作潜伏相关肽(Latency Associated Peptide，LAP)的结构域保持共价或非共价结合。因为LAP阻止成熟TGF-β1与其受体结合，所以通常是无活性的。这样的TGFβ1前体蛋白还可以进一步与GARP、LRRC33、LTBP1、LTBP3等形成大的潜伏复合物。在肿瘤微环境中，GARP-TGFβ1主要由Treg和肿瘤细胞产生，LRRC33-TGFβ1主要由免疫抑制的吞噬细胞产生，而LTBP1-TGFβ1、LTBP3-TGFβ1主要存在于肿瘤基质中。目前，全球范围内尚未有针对TGFβ1前体蛋白或TGFβ1复合物的抗体上市。本领域仍需要能够与TGFβ1前体蛋白或TGFβ1 复合物高亲和力结合并能阻断其激活，用于对肿瘤、纤维化等TGFβ相关疾病的治疗。
发明内容
本公开提供TGFβ1结合分子，GARP-TGFβ1结合分子，包含前述结合分子的药物组合物，及医药用途、制备方法，预防或治疗治疗与TGFβ信号通路相关的疾病的方法，特别是预防或治疗癌症或肿瘤的方法。
TGFβ1结合分子
第一方面，本公开提供TGFβ1结合分子，包含重链可变区(VH)和轻链可变区(VL)。
在一些实施方案中，所述VH包含SEQ ID NO:11所示氨基酸序列中的HCDR1、HCDR2和HCDR3，和/或，所述VL包含SEQ ID NO:12、21-35中任一项所示氨基酸序列中的LCDR1、LCDR2和LCDR3。
上述的CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的。在一些具体的实施方案中，所述CDR是根据Kabat定义方案定义的。
在一些实施方案中，所述CDR还可以由其他编号系统定义，例如，根据IMGT、Chothia、AbM或Contact编号系统定义。示例性的，以下提供根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的SEQ ID NO:11所示VH中的HCDR1、HCDR2、HCDR3，以及SEQ ID NO：35所示VL中的LCDR1、LCDR2、LCDR3。
表1.SEQ ID NO:11所示VH中的HCDR1、HCDR2、HCDR3
表2.SEQ ID NO：35所示VL中的LCDR1、LCDR2、LCDR3

在一些实施方案中，所述TGFβ1结合分子，包含如下所示VH和VL：
所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，和/或，所述VL包含SEQ ID NO:38、64、18所示的LCDR1、LCDR2和LCDR3。
其中，SEQ ID NO:38所示氨基酸序列为RASQX1ISX2YLN，X1选自S、A、F、G、I、P、Y、V或K，X2选自S、D、E、P或H；
SEQ ID NO:64所示氨基酸序列为X3ASX4LX5S，X3选自A、T、S或M，X4选自S、Y、A、E或G，X5选自Q、T、D或E。
在一些实施方案中，SEQ ID NO:38所示氨基酸序列为RASQX1ISX2YLN，X1选自F、Y或A，X2选自D或P；SEQ ID NO:64所示氨基酸序列为X3ASX4LX5S，X3选自A、T或S，X4选自S、Y或E，X5选自Q、D或E。
在一些实施方案中，所述TGFβ1结合分子，其中：
所述HCDR1的氨基酸序列如SEQ ID NO:13所示，
所述HCDR2的氨基酸序列如SEQ ID NO:14所示，
所述HCDR3的氨基酸序列如SEQ ID NO:15所示，
所述LCDR1的氨基酸序列如SEQ ID NO:16、36、39、41-42、44、46、49、52、54、56-59任一项所示，
所述LCDR2的氨基酸序列如SEQ ID NO:17、37、40、43、45、47-48、50-51、53、55任一项所示，和/或，
所述LCDR3的氨基酸序列如SEQ ID NO:18所示。
在一些实施方案中，所述TGFβ1结合分子，其中，
a)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:59、40、18所示的LCDR1、LCDR2和LCDR3；
b)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:36-37、18所示的LCDR1、LCDR2和LCDR3；
c)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:39-40、18所示的LCDR1、LCDR2和LCDR3；
d)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:41、40、18所示的LCDR1、LCDR2和LCDR3；
e)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:42-43、18所示的LCDR1、LCDR2和LCDR3；
f)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:44-45、18所示的LCDR1、LCDR2和LCDR3；
g)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述 VL包含SEQ ID NO:46-47、18所示的LCDR1、LCDR2和LCDR3；
h)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:46、48、18所示的LCDR1、LCDR2和LCDR3；
j)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:49-50、18所示的LCDR1、LCDR2和LCDR3；
k)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:46、51、18所示的LCDR1、LCDR2和LCDR3；
l)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:52-53、18所示的LCDR1、LCDR2和LCDR3；
m)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:54-55、18所示的LCDR1、LCDR2和LCDR3；
n)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:56、53、18所示的LCDR1、LCDR2和LCDR3；
o)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:57、45、18所示的LCDR1、LCDR2和LCDR3；
p)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:58、50、18所示的LCDR1、LCDR2和LCDR3；或，
q)所述VH包含SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO:16-18所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述TGFβ1结合分子，包含VH和VL，其中：
所示VH中包含的任一HCDR与前述任一种HCDR相比，具有0、1、2、3、4或5个氨基酸突变；和/或，所示VL中包含的任一LCDR与前述任一种HCDR相比，具有0、1、2、3、4或5个氨基酸突变。
在一些具体的实施方案中，HCDR或LCDR中的氨基酸突变为保守性取代。
在一些实施方案中，本公开提供的TGFβ1结合分子，其包含前述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3中的任意一个或任意几个(例如，2、3、4、5或6)的组合。
在一些实施方案中，所述TGFβ1结合分子为抗TGFβ1抗体或其抗原结合片段。
在一些实施方案中，所述抗TGFβ1抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、人源抗体或前述任意一种的抗原结合片段。
在一些具体的实施方案中，抗TGFβ1抗体或其抗原结合片段为人源化抗体或其抗原结合片段，或者为人源抗体或其抗原结合片段。在一些具体的实施方案中，抗TGFβ1抗体或其抗原结合片段为人源抗体或其抗原结合片段。
在一些具体的实施方案中，抗TGFβ1抗体或其抗原结合片段是经亲和力成熟改造的。经亲和力成熟改造的抗TGFβ1抗体或其抗原结合片段包含位于VH和/或VL的一个或多个突变。示例性地，位于VH的突变包含如下任意一个或任意组 合的位置处的突变：99、100、53、56、58、31、33、95、100。示例性地，位于VL的突变包含如下任意一个或任意组合的位置处的突变：24、28、31、32、50、53、55、92、93、94。上述突变的位置根据Kabat编号规则编号。
在一些实施方案中，所述TGFβ1结合分子包含VH和VL，其中，
所述VH包含如SEQ ID NO:11所示氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列；和/或，
所述VL包含如SEQ ID NO:12、21-35中任一项所示氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，所述TGFβ1结合分子，包含VH和VL，其中：所示VH中与前述任一种VH相比，具有0、1、2、3、4、5、6、7、8、9或10个氨基酸突变；和/或，所示VL与前述任一种VL相比，具有0、1、2、3、4、5、6、7、8、9或10个氨基酸突变。
在一些实施方案中，本公开提供的TGFβ1结合分子，其包含前述VH、VL中的任意一个或任意2个的组合。
在一些实施方案中，本公开提供的TGFβ1结合分子，其进一步包含免疫球蛋白Fc区。在一些实施方案中，所述免疫球蛋白Fc区来源于IgG1、IgG2、IgG3、IgG4或前述任一种的变体。在一些实施方案中，所述免疫球蛋白Fc区来源于人IgG4或其变体。在一些实施方案中，人IgG4变体中包含降低或消除Fc效应功能的突变。示例性地，人IgG4变体中包含突变S228P。上述突变的位点根据EU编号规则编号。
在一些实施方案中，所述TGFβ1结合分子，其进一步包含轻链恒定区和/或重链恒定区。
在一些实施方案中，轻链恒定区来源于κ轻链、λ轻链或前述任一种的变体。在一些实施方案中，轻链恒定区来源于人κ轻链、人λ轻链或前述任一种的变体。
在一些具体的实施方案中，轻链恒定区来源于人κ轻链或其变体。
在一些具体的实施方案中，轻链恒定区来源于鼠κ轻链或其变体。
在一些具体的实施方案中，轻链恒定区包含如SEQ ID NO:19所示氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，重链恒定区来源于IgG1、IgG2、IgG3、IgG4或前述任一种的变体。在一些实施方案中，重链恒定区来源于人IgG1、人IgG2、人IgG3、人IgG4或前述任一种的变体。
在一些具体的实施方案中，重链恒定区来源于人IgG4或其变体。
在一些具体的实施方案中，人IgG4变体中包含降低或消除Fc效应功能的突变。示例性地，人IgG4变体中包含突变S228P。上述突变的位点根据EU编号规则编号。
在一些具体的实施方案中，重链恒定区来源于鼠IgG2或其变体。
在一些具体的实施方案中，鼠IgG2变体中包含降低或消除ADCC效应的突变。示例性地，鼠IgG2变体中包含至少一种如下所示突变：L234A/L235E/G237A/D327Q/A330S/P331S。上述突变的位点根据EU编号规则编号。
在一些实施方案中，免疫球蛋白Fc区是效应器功能降低或消去除的Fc区，例如，具有降低或去除ADCC效应的突变。示例性地，降低或消除ADCC效应的突变包含如下至少一种：
L234A/L235E/G237A/D327Q/A330S/P331S(IgG2a)；N297A或N297Q(IgG1)；L234A/L235A(IgG1)；V234A/G237A(IgG2)；L235A/G237A/E318A(IgG4)；H268Q/V309L/A330S/A331S(IgG2)；C220S/C226S/C229S/P238S(IgG1)；C226S/C229S/E233P/L234V/L235A(IgG1)；L234F/L235E/P331S(IgG1)；或S267E/L328F(IgG1)。上述突变的位置根据EU编号规则编号。
在一些具体的实施方案中，重链恒定区包含如SEQ ID NO:20所示氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，所述TGFβ1结合分子，其包含重链和轻链，其中：
所述重链包含如SEQ ID NO:66所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列；和/或，
所述轻链包含如SEQ ID NO:65、67-81中任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，所述TGFβ1结合分子，其包含重链和轻链，其中：
所述重链包含如SEQ ID NO:82所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列；和/或，
所述轻链包含如SEQ ID NO:83所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，所述TGFβ1结合分子，包含重链和轻链，其中：所示重链与前述任一种重链相比，具有0、1、2、3、4、5、6、7、8、9或10个氨基酸突变；和/或，所示轻链与前述任一种轻链相比，具有0、1、2、3、4、5、6、7、8、9或10个氨基酸突变。
在一些具体的实施方案中，重链和/或轻链上的氨基酸突变未保守性取代。
在一些实施方案中，本公开的TGFβ1结合分子，其包含前述重链、轻链中的任意一个或任意2个的组合。
在本公开的上下文中，“至少80％”涵盖80％及以上，例如至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％，以及任意两者之间的数值范围。
在一些实施方案中，前述TGFβ1结合分子与TGFβ1前体蛋白或TGFβ1复合物特异性结合。
在一些实施方案中，TGFβ1复合物中的TGFβ1以TGFβ1前体蛋白存在。
在一些实施方案中，TGFβ1复合物包含如下任一种：
(2)LTBP1-TGFβ1复合物，
(3)LTBP3-TGFβ1复合物，
(4)LRRC33-TGFβ1复合物，
(5)GARP-TGFβ1复合物。
如本公开所使用，“TGFβ1”或“TGFβ1蛋白”以广泛的含义理解，包含转化生长因子-β1(TGFβ1)蛋白的原蛋白形式(又称，原TGFβ1(pro-TGFβ1))或潜伏形式(潜伏TGFβ1(latent TGFβ1))，或经释放后的成熟TGFβ1。
在一些实施方案中，本公开中所使用的“TGFβ1”或“TGFβ1蛋白”是指“TGFβ1前体蛋白”。
在一些实施方案中，TGFβ1前体蛋白包含：(i)成熟TGFβ1结构域，(ii)潜能相关肽(LAP)。
示例性地，TGFβ1前体蛋白是原TGFβ1或潜伏TGFβ1。原TGFβ1中成熟TGFβ1结构域与潜能相关肽(latency-associated peptides，LAPs)共价结合，潜伏TGFβ1中成熟TGFβ1结构域与潜能相关肽(latency-associated peptides，LAPs)非共价结合。
在本公开中，“原TGFβ1”或“潜伏TGFβ1”可以互换地使用。
在翻译过程中，潜伏TGFβ1(也称为小潜伏复合物(SLC))变成通过二硫桥与“呈递分子”连接，从而形成大潜伏复合物(LLC)，包括，例如，与GARP形成GARP-TGFβ1复合物。在GARP-TGFβ1复合物中存在的TGFβ1可以为潜伏形式(潜伏TGFβ1)或前体形式(原TGFβ1)。
在一些实施方案中，所述TGFβ1前体蛋白或TGFβ1复合物还包含前述任意蛋白或蛋白复合物的蛋白片段、功能变体等。
示例性地，“片段”、“蛋白片段”包括但不限于：生长因子结构域(growth factor domains)、N-末端前结构域(N-terminal prodomain)、潜伏相关肽(latency-associated peptides，LAPs)、LAP样结构域(LAP-like domains)、直夹克区域(straight jacket regions)、紧固件区域(fastener regions)、弗林蛋白酶切位点(furin cleavage site regions)、手臂区域(arm regions)、手指区域(fingers regions)、潜伏环(latency loops)、α1螺旋区域(alpha 1helical regions)、α2螺旋区域(alpha 2helical regions)、RGD序列区(RGD sequence regions)、触发环区(trigger loop regions)、胞外结构域、跨膜结构域、胞内结构域等。
示例性地，“变体”、“功能变体”是指包含一个或几个氨基酸的取代、缺失和/或添加的氨基酸，且具有相当的生物活性的蛋白或蛋白复合物。
示例性地，TGFβ1前体蛋白的变体包含相对于SEQ ID NO：6所示氨基酸序 列按自然计数第4位氨基酸突变。在一些实施方案中，TGFβ1前体蛋白的变体包含突变C4S，所述突变的位点是相对于SEQ ID NO:6所示氨基酸序列按自然计数的位点。
在一些实施方案中，所述TGFβ1复合物是由TGFβ1前体蛋白与其他种类的蛋白或其蛋白片段、功能变体形成的复合物，例如，与LTBP1S、LTBP4、fibrillin-1、fibrillin-2、fibrillin-3、fibrillin-4等形成的复合物。
在一些实施方案中，TGFβ1前体蛋白包含如SEQ ID NO:1、3、6-7、9-10任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
示例性地，TGFβ1复合物是GARP-TGFβ1复合物，其包含GARP和TGFβ1前体蛋白；其中：
GARP包含如SEQ ID NO:2、4、5、8、100、103任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列；
和/或，TGFβ1蛋白包含如SEQ ID NO:1、3、6-7、9-10任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，TGFβ1结合分子是抗TGFβ1抗体或其抗原结合片段，其与TGFβ1前体蛋白或TGFβ1复合物的抗原表位特异性结合。在一些实施方式中，抗TGFβ1抗体或其抗原结合片段不阻止TGFβ1与整联蛋白结合。例如，在一些实施方式中，抗TGFβ1抗体或其抗原结合片段不掩蔽TGFβ1的整联蛋白结合位点。
在一些实施方案中，TGFβ1结合分子以10-7M、10-8M、10-9M、10-10M或更低的KD结合TGFβ1前体蛋白或TGFβ1复合物(例如，LTBP1-TGFβ1复合物、LTBP3-TGFβ1复合物、LRRC33-TGFβ1复合物、GARP-TGFβ1复合物等)。在一些实施方案中，TGFβ1复合物中包含所述TGFβ1前体蛋白。
在一些实施方式中，TGFβ1结合分子与TGFβ2蛋白或TGFβ2复合物不结合或极微弱结合。示例性地，抗TGFβ1抗体或其抗原结合片段不与如下至少一种特异性结合或极弱地结合：成熟TGFβ2、TGFβ2前体蛋白、GARP-TGFβ2复合物、LTBP-TGFβ2复合物等。
在一些实施方式中，TGFβ1结合分子与TGFβ3蛋白或TGFβ3复合物不结合或极微弱结合。示例性地，抗TGFβ1抗体或其抗原结合片段不与如下至少一种特异性结合或极弱地结合：成熟TGFβ3、TGFβ3前体蛋白、GARP-TGFβ3复合物、LTBP-TGFβ3复合物等。
在本公开中，“TGFβ2蛋白”应作广义理解，其范围覆盖成熟TGFβ2，以及pro-TGFβ2、latent TGFβ2等非活性的前体蛋白形式。“pro-TGFβ2”与“latent TGFβ2”可以互换地使用。
在本公开中，“TGFβ3蛋白”应作广义理解，其范围覆盖成熟TGFβ3，以及pro-TGFβ3、latent TGFβ3等非活性的前体蛋白形式。“pro-TGFβ3”与“latent TGFβ3”可以互换地使用。
在一些实施方案中，本公开的TGFβ1结合分子抑制TGFβ1活性。例如，TGFβ1结合分子与TGFβ1前体蛋白(原/潜伏TGFβ1)或TGFβ1复合物(例如，GARP-TGFβ1复合物，LTBP1-TGFβ1复合物，LTBP3-TGFβ1复合物，LRRC33-TGFβ1复合物)结合，进而选择性抑制TGFβ1的活性(例如，抑制TGFβ1的激活，和/或抑制成熟TGFβ1的释放；和/或，抑制TGFβ1信号转导)。并且，选择性不抑制TGFβ2和/或TGFβ3的活性，例如，选择性抑制TGFβ1的激活，但不抑制TGFβ2和/或TGFβ3的激活。其中，TGFβ1复合物中的TGFβ1以TGFβ1前体蛋白形式存在。
在一些实施方案中，本公开的TGFβ1结合分子具有改善的安全性(例如，降低的体内毒性和/或不良反应)。
在一些实施方案中，本公开的TGFβ1结合分子还具有至少一种如下所示特性：抑制调节性T(Treg)细胞的免疫抑制活性，抑制肿瘤生长，抑制纤维化。
在一些实施方案中，TGFβ1结合分子是抗TGFβ1抗体或其抗原结合片段，进一步为Fab、Fv、sFv、Fab’、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、双链抗体(diabody)、三链抗体(triabody)和四链抗体(tetrabody)、串联二-scFv、串联三-scFv)，例如具体为scFv、Fv、Fab或Fab’片段。
在一些实施方案中，提供抗TGFβ1抗体或其抗原结合片段，其与前述TGFβ1结合分子结合或竞争结合相同的TGFβ1前体蛋白或TGFβ1复合物(例如人或鼠GARP-TGFβ1复合物、LTBP-TGFβ1复合物、LRRC33-TGFβ1复合物)表位。在一些实施方案中，TGFβ1复合物中的TGFβ1以TGFβ1前体蛋白形式存在。
在一些实施方案中，提供抗TGFβ1抗体或其抗原结合片段，其阻断前述TGFβ1结合分子与TGFβ1前体蛋白或TGFβ1复合物(例如人或鼠GARP-TGFβ1复合物、LTBP-TGFβ1复合物、LRRC33-TGFβ1复合物)的结合。在一些实施方案中，TGFβ1复合物中的TGFβ1以TGFβ1前体蛋白形式存在。
在一些实施方案中，提供抗TGFβ1抗体或其抗原结合片段，其与TGFβ1蛋白或TGFβ1复合物(例如人或鼠GARP-TGFβ1复合物、LTBP-TGFβ1复合物、LRRC33-TGFβ1复合物)的结合被前述TGFβ1结合分子阻断。在一些实施方案中，TGFβ1复合物中的TGFβ1以TGFβ1前体蛋白形式存在。
GARP-TGFβ1结合分子
第二方面，本公开提供GARP-TGFβ1结合分子，其包含至少一个与TGFβ1复合物结合的免疫球蛋白单一可变结构域。
在一些实施方案中，所述TGFβ1复合物是GARP-TGFβ1复合物。在一些实施方案中，所述GARP-TGFβ1复合物中TGFβ1为TGFβ1前体蛋白。示例性地，TGFβ1前体蛋白选自原TGFβ1或潜伏TGFβ1。在本公开中，“原TGFβ1”与“潜伏TGFβ1” 可以互换地使用。
在一些实施方案中，本公开提供GARP-TGFβ1结合分子，其包含至少一个与GARP-TGFβ1复合物结合的免疫球蛋白单一可变结构域。
在一些实施方案中，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2、CDR3，其中：
a)所述免疫球蛋白单一可变结构域包含SEQ ID NO：84所示氨基酸序列中的CDR1、CDR2和CDR3；
b)所述免疫球蛋白单一可变结构域包含SEQ ID NO：90所示氨基酸序列中的CDR1、CDR2和CDR3；
c)所述免疫球蛋白单一可变结构域包含SEQ ID NO：91所示氨基酸序列中的CDR1、CDR2和CDR3；或，
d)所述免疫球蛋白单一可变结构域包含SEQ ID NO：92所示氨基酸序列中的CDR1、CDR2和CDR3。
上述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的。在一些具体的实施方案中，所述CDR是根据Kabat编号系统定义的。示例性地，以下提供Kabat、IMGT、Chothia、AbM或Contact编号系统定义的CDR1、CDR2和CDR3。
表3.SEQ ID NO:84所示序列中的CDR1、CDR2和CDR3
在一些实施方案中，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2、CDR3，其中：
CDR1包含如SEQ ID NO:85所示的氨基酸序列；
CDR2包含如SEQ ID NO:86所示的氨基酸序列；和/或，
CDR2包含如SEQ ID NO:87所示的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域包含SEQ ID NO:85-87所示的CDR1、CDR2和CDR3。
在一些实施方案中，所述免疫球蛋白单一可变结构域包含的CDR1与SEQ ID NO:85所示氨基酸序列相比，具有0、1、2、3、4或5个氨基酸突变；和/或，
所述免疫球蛋白单一可变结构域包含的CDR2与SEQ ID NO:86所示氨基酸序 列相比，具有0、1、2、3、4或5个氨基酸突变；和/或，
所述免疫球蛋白单一可变结构域包含的CDR3与SEQ ID NO:87所示氨基酸序列相比，具有0、1、2、3、4或5个氨基酸突变。
在一些具体的实施方案中，CDR1、CDR2或CDR3中的氨基酸突变为保守性取代。
在一些实施方案中，GARP-TGFβ1结合分子包含前述任意1个或多个(例如，1、2、3、4、5、6、7、8、9、10、20、30、50等等)的免疫球蛋白单一可变结构域。在一些实施方案中，GARP-TGFβ1结合分子包含2个或以上的免疫球蛋白单一可变结构域，且其中任意两个免疫球蛋白单一可变结构域可以是相同的或不同的。
在一些实施方案中，GARP-TGFβ1结合分子可以包含前述任一的完整的免疫球蛋白单一可变结构域；也可以包含前述任一的免疫球蛋白单一可变结构域的功能部分或其变体，例如CDR3、CDR3-FR4、CDR2-FR3-CDR3、CDR2-FR3-CDR3-FR4、FR2-CDR2-FR3-CDR3-FR4、CDR1-FR2-CDR2-FR3-CDR3-FR4、FR1-CDR1-FR2-CDR2-FR3-CDR3。
在一些实施方案中，所述免疫球蛋白单一可变结构域的功能部分的变体可以是保留了CDR3、CDR3-FR4、CDR2-FR3-CDR3、CDR2-FR3-CDR3-FR4、FR2-CDR2-FR3-CDR3-FR4、CDR1-FR2-CDR2-FR3-CDR3-FR4、或FR1-CDR1-FR2-CDR2-FR3-CDR3的血清白蛋白结合功能且与之具有至少80％、至少90％序列同源性的多肽，例如可以是保留了CDR3的TGFβ1复合物结合功能并与之具有一定序列同源性的多肽，例如与上述任一CDR3具有至少80％、至少90％序列同源性的多肽。
在一些实施方案中，所述免疫球蛋白单一可变结构域为VHH。示例性地，免疫球蛋白单一可变结构域为骆驼源VHH、全人VHH或人源化VHH。
在一些具体的实施方案中，免疫球蛋白单一可变结构域为人源化VHH。
在一些实施方案中，人源化VHH的人种系模板选自IGHV3-23和IGJH4的至少一种。在一些具体的方案中，人源化VHH包含来源于IGHV3-23的FR1、FR2、FR3，人源化VHH包含来源于IGJH4的FR4。
在一些实施方案中，人源化VHH包含至少一个如下所示的回复突变：23T、29Y、30C、37Y、44E、45R、47F、71Q、74A、75R、78G、81E、93K和94T。在一些具体的实施方案中，人源化VHH的框架区包含至少一个上述的回复突变。
上述的回复突变的位点依据Kabat编号规则编号。
在一些实施方案中，所述免疫球蛋白单一可变结构域包含如SEQ ID NO：84、90-92中任一项所示的氨基酸序列或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域包含的氨基酸序列与SEQ ID NO：84、90-92中任一项所示的序列相比，具有1个或多个(例如2、3、4、 5、6、7、8、9或10个等)的氨基酸突变。
在一些具体的实施方案中，免疫球蛋白单一可变结构域的氨基酸突变为保守性取代。
在一些实施方案中，GARP-TGFβ1结合分子进一步包含免疫球蛋白Fc区。示例性地，免疫球蛋白Fc区来源于IgG1、IgG2、IgG3或IgG4。在一些实施方案中，免疫球蛋白Fc区来源于IgG2或IgG4。在一些具体的实施方案中，免疫球蛋白Fc区来源于人IgG4。
在一些实施方案中，免疫球蛋白Fc区包含降低或消除Fc效应功能的突变。示例性地，免疫球蛋白Fc区包含突变：S228P。上述突变的位置根据EU编号规则编号。
在一些具体实施方案中，免疫球蛋白Fc区包含如SEQ ID NO:88所示的氨基酸序列或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，免疫球蛋白Fc区来源于小鼠IgG2。
在一些实施方案中，免疫球蛋白Fc区是效应器功能降低或消去除的Fc区，例如，具有降低或去除ADCC效应的突变。示例性地，降低或消除ADCC效应的突变包含如下至少一种：L234A/L235E/G237A/D327Q/A330S/P331S(IgG2a)；N297A或N297Q(IgG1)；L234A/L235A(IgG1)；V234A/G237A(IgG2)；L235A/G237A/E318A(IgG4)；H268Q/V309L/A330S/A331S(IgG2)；C220S/C226S/C229S/P238S(IgG1)；C226S/C229S/E233P/L234V/L235A(IgG1)；L234F/L235E/P331S(IgG1)；或S267E/L328F(IgG1)。上述突变的位置根据EU编号规则编号。
在一些实施方案中，所述GARP-TGFβ1结合分子是与GARP-TGFβ1复合物结合的抗体或其抗原结合片段。
在一些实施方案中，GARP-TGFβ1结合分子包含如SEQ ID NO：89、93-95、106任一项所示的氨基酸序列，或与之具有至少80％、至少90％序列同一性的氨基酸序列。
在一些实施方案中，GARP-TGFβ1结合分子包含与SEQ ID NO：89、93-95、106任一项所示的氨基酸序列相比，具有一个多个(例如0、1、2、3、4、5、6、7、8、9或10个等)氨基酸突变，且具有与GARP-TGFβ1复合物特异性结合的功能活性。
在一些具体的实施方案中，GARP-TGFβ1结合分子中的氨基酸突变为保守性取代。
在本公开的上下文中，“至少80％”涵盖80％及以上，例如至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％，以及任意两者之间的数值范围。
在一些实施方案中，前述GARP-TGFβ1结合分子与GARP-TGFβ1复合物特异性结合，从而抑制成熟TGFβ1从GARP-TGFβ1复合物中的释放、和/或抑制TGFβ1活性、和/或抑制TGFβ1信号转导。
在一些实施方案中，GARP-TGFβ1结合分子不结合游离的成熟TGFβ1。
在一些实施方案中，GARP-TGFβ1结合分子不结合或极弱结合TGFβ2蛋白或TGFβ2复合物。例如，TGFβ1结合分子不结合或极弱结合成熟TGFβ2、TGFβ2前体蛋白、GARP-TGFβ2复合物、LTBP-TGFβ2复合物等。
在一些实施方案中，GARP-TGFβ1结合分子不结合或极弱结合TGFβ3蛋白或TGFβ3复合物。例如，GARP-TGFβ1结合分子不结合或极弱结合成熟TGFβ3、TGFβ3前体蛋白、GARP-TGFβ3复合物、LTBP-TGFβ3复合物等。
在本公开中，“TGFβ2蛋白”应作广义理解，其范围覆盖成熟TGFβ2，以及pro-TGFβ2、latent TGFβ2等非活性的前体蛋白形式。“TGFβ3蛋白”应作广义理解，其范围覆盖成熟TGFβ3，以及pro-TGFβ3、latent TGFβ3等非活性的前体蛋白形式。
在一些实施方案中，前述GARP-TGFβ1结合分子能够选择性抑制TGFβ1活性，但不抑制TGFβ2和/或TGFβ3的活性。例如，GARP-TGFβ1结合分子选择性抑制TGFβ1的激活，但不抑制TGFβ2和/或TGFβ3的激活。因而，本公开的GARP-TGFβ1结合分子具有改善的安全性(例如，降低的体内毒性和/或不良反应)。
在本开的一些实施方案中，成熟TGFβ1、成熟TGFβ2、和/或成熟TGFβ3在细胞中呈现游离状态。
在一些实施方案中，GARP-TGFβ1结合分子与GARP-TGFβ1复合物特异性结合。其中，GARP-TGFβ1复合物包含：
a)糖蛋白A重复主导序列(GARP)；
b)成熟TGFβ1结构域；以及，
c)潜能相关肽(LAP)。
在一些实施方案中，所述GARP包含如SEQ ID NO:100或103所示的氨基酸序列或与之具有至少90％序列同一性的氨基酸序列，所述成熟TGFβ1结构域包含如SEQ ID NO:101或104所示的氨基酸序列或与之具有至少90％序列同一性的氨基酸序列，和/或，所述LAP包含如SEQ ID NO:102或105所示的氨基酸序列或与之具有至少90％序列同一性的氨基酸序列。
在一些实施方案中，GARP-TGFβ1结合分子以10-7M、10-8M、10-9M或更低的KD结合GARP-TGFβ1复合物或其片段。
在一些实施方案中，GARP-TGFβ1结合分子，其具有如下所示特性：抑制TGFβ1活性，抑制调节性T细胞的免疫抑制活性，和/或抑制肿瘤生长。
在一些实施方案中，GARP-TGFβ1结合分子为抗GARP-TGFβ1抗体或其抗原结合片段；在一些具体实施方案中，为嵌合抗体、骆驼抗体、人源化抗体、全人抗体或其抗原结合片段。
在一些实施方案中，GARP-TGFβ1结合分子为抗GARP-TGFβ1抗体或其抗原结合片段；在一些具体实施方案中，抗GARP-TGFβ1抗体或其抗原结合片段包括但不限于：Fab、Fv、sFv、Fab’、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如为scFv、Fv、Fab或Fab’片段。
在一些实施方案中，提供抗GARP-TGFβ1抗体或其抗原结合片段，其与前述GARP-TGFβ1结合分子结合或竞争结合相同的GARP-TGFβ1复合物(例如人或鼠GARP-TGFβ1复合物)表位。
在一些实施方案中，提供抗GARP-TGFβ1抗体或其抗原结合片段，其阻断前述GARP-TGFβ1结合分子与GARP-TGFβ1(例如人或鼠GARP-TGFβ1复合物)的结合。
在一些实施方案中，提供抗GARP-TGFβ1抗体或其抗原结合片段，其与GARP-TGFβ1复合物(例如人或鼠GARP-TGFβ1复合物)的结合被前述GARP-TGFβ1结合分子阻断。
在一些实施方案中，所述GARP-TGFβ1复合物包含(i)GARP，和(ii)TGFβ1前体蛋白。示例性地，所述GARP-TGFβ1复合物中：
GARP包含如SEQ ID NO:2、4、5、8、100、103任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列；
和/或，TGFβ1蛋白包含如SEQ ID NO:1、3、6-7、9-10任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
蛋白结合分子
在一些实施方案中，本公开提供TGFβ1结合分子。
在一些实施方案中，所述TGFβ1结合分子特异性结合TGFβ1前体蛋白或TGFβ1复合物。在一些实施方案中，所述TGFβ1复合物包含TGFβ1前体蛋白。
如本公开所使用，“TGFβ1”或“TGFβ1蛋白”以广泛的含义理解，包含转化生长因子-β1(TGFβ1)蛋白的原蛋白形式(又称，原TGFβ1)或潜伏形式(潜伏TGFβ1)，或经释放后的成熟TGFβ1。在一些实施方案中，TGFβ1或TGFβ1蛋白是指TGFβ1前体蛋白。
在一些实施方案中，TGFβ1前体蛋白包含：(i)成熟TGFβ1结构域，(ii)潜能相关肽(LAP)。
示例性地，TGFβ1前体蛋白是原TGFβ1或潜伏TGFβ1。原TGFβ1中成熟TGFβ1结构域与潜能相关肽(latency-associated peptides，LAPs)共价结合，潜伏TGFβ1中成熟TGFβ1结构域与潜能相关肽(latency-associated peptides，LAPs)非共价结合。
在本公开中，“原TGFβ1”或“潜伏TGFβ1”可以互换地使用。
示例性地，成熟TGFβ1结构域包含如SEQ ID NO:101或104所示的氨基酸序列或与之具有至少80％序列同一性的氨基酸序列。
示例性地，LAP包含如SEQ ID NO:102或105所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
示例性地，TGFβ1前体蛋白包含如SEQ ID NO:1、3、6-7、9-10任一项所述的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，所述TGFβ1前体蛋白或TGFβ1复合物还包含前述任意蛋白或蛋白复合物的蛋白片段、功能变体等。
在一些实施方案中，所述TGFβ1前体蛋白或TGFβ1复合物来源于任意的脊椎动物，包括哺乳动物如灵长类(例如人)和其他物种(例如，小鼠、大鼠、豚鼠、兔子、狗、猪、羊等)等。
示例性地，“片段”、“蛋白片段”包括但不限于：生长因子结构域(growth factor domains)、N-末端前结构域(N-terminal prodomain)、潜伏相关肽(latency-associated peptides，LAPs)、LAP样结构域(LAP-like domains)、直夹克区域(straight jacket regions)、紧固件区域(fastener regions)、弗林蛋白酶切位点(furin cleavage site regions)、手臂区域(arm regions)、手指区域(fingers regions)、潜伏环(latency loops)、α1螺旋区域(alpha 1helical regions)、α2螺旋区域(alpha 2helical regions)、RGD序列区(RGD sequence regions)、触发环区(trigger loop regions)、胞外结构域、跨膜结构域、胞内结构域等。
示例性地，“变体”、“功能变体”是指包含一个或几个氨基酸的取代、缺失和/或添加的氨基酸，且具有相当的生物活性的蛋白或蛋白复合物。
示例性地，TGFβ1前体蛋白的变体包含相对于SEQ ID NO：6所示氨基酸序列按自然计数第4位氨基酸突变。在一些实施方案中，TGFβ1前体蛋白的变体包含突变C4S，所述突变的位点是相对于SEQ ID NO:6所示氨基酸序列按自然计数的位点。
在一些实施方案中，所述TGFβ1前体蛋白或TGFβ1复合物可以包含或不包含前导序列。示例性地，前导序列为信号肽序列。在一些实施方案中，所述TGFβ1前体蛋白或TGFβ1复合物可以包含或不包含标签序列。示例性地，标签序列为His-tag、AVI-tag、myc-tag、荧光标签等。
在一些实施方案中，所述TGFβ1复合物包含TGFβ1蛋白，以及选自如下所示的任意的第二蛋白：糖蛋白A重复主导序列(GARP)、潜伏TGF-β结合蛋白(Latent TGF-β-binding proteins，LTBP；例如LTBP1、LTBP1S、LTBP2、LTBP3、LTBP4)、原纤蛋白(例如，fibrillin-1、fibrillin-2、fibrillin-3、fibrillin-4)、LRRC33(Leucine-Rich Repeat-Containing Protein 33)、或前述任意一种的变体或蛋白片段等。
示例性地，“蛋白片段”或“片段”包括但不限于胞外结构域、跨膜结构域或胞内结构域等。
在一些实施方案中，所述TGFβ1复合物包括但不限于：GARP-TGFβ1复合物、LTBP-TGFβ1复合物(例如，LTBP1-TGFβ1复合物、LTBP3-TGFβ1复合物)、或LRRC33-TGFβ1复合物。在一些实施方案中，所述TGFβ1复合物中TGFβ1以TGFβ1前体蛋白形式存在。
示例性地，GARP-TGFβ1复合物包括：
a)糖蛋白A重复主导序列(GARP)；
b)成熟TGFβ1结构域；以及，
c)潜能相关肽(LAP)。
在一些实施方案中，所述GARP包含如SEQ ID NO:100或103所示的氨基酸序列或与之具有至少80％序列同一性的氨基酸序列，所述TGFβ1包含如SEQ ID NO:101或104所示的氨基酸序列或与之具有至少80％序列同一性的氨基酸序列，和/或，所述LAP包含如SEQ ID NO:102或105所示的氨基酸序列或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，GARP-TGFβ1复合物由GARP与TGFβ1前体蛋白形成。
在一些具体的实施方案中，GARP-TGFβ1复合物由GARP的胞外区与TGFβ1前体蛋白形成。
本公开中“GARP”可以是天然来源的野生型GARP，也可以是GARP的功能变体(例如，与野生型GARP相比，包含一个或多个修饰、截短和/或突变)。在一些实施方案中，GARP可以是全长或其部分结构域(例如，胞外结构域、跨膜结构域或胞内结构域)。例如，天然来源指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和其他物种(例如，小鼠、大鼠、豚鼠、兔子、狗、猪、羊等)等。
在一些实施方案中，GARP可以包含或不包含前导序列。示例性地，前导序列为信号肽序列。在一些实施方案中，GARP可以包含一个或多个标签序列，例如His-tag、AVI-tag、myc-tag、荧光标签等。在一些实施方案中，GARP可以不包含标签序列。
示例性地，GARP的胞外区包含如SEQ ID NO:100或103所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
示例性地，GARP包含如SEQ ID NO:2、4、5、8、100、103任一项所示的氨基酸序列，或与之具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，与TGFβ1前体蛋白或TGFβ1复合物结合的蛋白结合分子选自抗体或其抗原结合片段。示例性地，抗体或其抗原结合片段包括但不限于如下任意一种：线性抗体、单链抗体(scFv)、单域抗体(sdAb)、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)、Fab、Fv、sFv、Fab’、F(ab’)2。
在一些实施方案中，提供特异性地结合TGFβ1前体蛋白或TGFβ1复合物的表位的抗体或其抗原结合片段。在一些实施方案中，当TGFβ1为前体蛋白形式或存 在于与GARP、LTBP1、LTBP3和/或LRRC33复合物中时，该表位可用于被抗体或其抗原结合片段结合。在一些实施方式中，表位由于在与GARP、LTBP和/或LRRC33复合时TGFβ1的构象变化而可用。在一些实施方式中，TGFβ1中抗体或其抗原结合片段结合的表位在TGFβ1未与GARP、LTBP和/或LRRC33复合时，或TGFβ1是成熟TGFβ1时是不可用的。在一些实施方式中，抗体或其抗原结合片段不阻止TGFβ1与整联蛋白结合。例如，在一些实施方式中，抗体或其抗原结合片段不掩蔽TGFβ1的整联蛋白结合位点。
在一些实施方式中，蛋白结合分子与TGFβ2蛋白或TGFβ2复合物不结合或极弱结合。示例性地，蛋白结合分子不与如下至少一种特异性结合：成熟TGFβ2、TGFβ2前体蛋白、GARP-TGFβ2复合物、LTBP-TGFβ2复合物等。
在一些实施方式中，蛋白结合分子与TGFβ3蛋白或TGFβ3复合物不结合或极弱结合。示例性地，蛋白结合分子不与如下至少一种特异性结合：成熟TGFβ3、TGFβ3前体蛋白、GARP-TGFβ3复合物、LTBP-TGFβ3复合物等。
在本公开中，“TGFβ2蛋白”应作广义理解，其范围覆盖成熟TGFβ2，以及pro-TGFβ2、latent TGFβ2等非活性的前体蛋白形式。“pro-TGFβ2”与“latent TGFβ2”可以互换地使用。
在本公开中，“TGFβ3蛋白”应作广义理解，其范围覆盖成熟TGFβ3，以及pro-TGFβ3、latent TGFβ3等非活性的前体蛋白形式。“pro-TGFβ3”与“latent TGFβ3”可以互换地使用。
在一些实施方案中，成熟TGFβ1、成熟TGFβ2、和/或成熟TGFβ3在细胞中呈现游离状态。
在一些实施方案中，蛋白结合分子特异性结合TGFβ1前体蛋白或TGFβ1复合物(例如，GARP-TGFβ1复合物，LTBP1-TGFβ1复合物，LTBP3-TGFβ1复合物，LRRC33-TGFβ1复合物等)，可以选择性抑制TGFβ1的活性。例如，抑制TGFβ1的激活，和/或抑制成熟TGFβ1的释放，和/或抑制TGFβ1信号转导。
多核苷酸
本公开提供编码前述任一种蛋白结合分子的多核苷酸。在一些实施方案中，本公开提供编码前述任一种TGFβ1结合分子、GARP-TGFβ1结合分子的多核苷酸。
在一些实施方案中，本公开提供编码本公开第一方面所述TGFβ1结合分子的多核苷酸。在一些实施方案中，本公开提供编码本公开第二方面所述GARP-TGFβ1结合分子的多核苷酸。
在一些实施方案中，本公开的多核苷酸可为RNA、DNA或cDNA。
在一些实施方案中，本公开的多核苷酸是分离的多核苷酸。
在一些实施方案中，本公开的多核苷酸是也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体可以是真核载体、原核载体、病毒载体，例如质 粒、粘端质粒、YAC或病毒载体等。载体可尤其为表达载体，即可提供结合分子(例如，TGFβ1结合分子)在体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的多核苷酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的蛋白结合分子、TGFβ1结合分子、GARP-TGFβ1结合分子表达有用或必需的调控元件及其他元件例如为启动子、增强子、终止子、整合子、选择标记物、前导序列、报告基因。
本公开的多核苷酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在一些实施方案中，本公开中的多核苷酸及载体可用于制备TGFβ1结合分子。在一些实施方案中，本公开中的多核苷酸及载体用于在体外或体内表达TGFβ1结合分子，TGFβ1结合分子结合TGFβ1前体蛋白或TGFβ1复合物，用于检测、诊断、治疗、调节等不同目的。
在一些实施方案中，本公开中的多核苷酸及载体可用于制备GARP-TGFβ1结合分子。在一些实施方案中，本公开中的多核苷酸及载体用于在体外或体内表达GARP-TGFβ1结合分子，GARP-TGFβ1结合分子结合GARP-TGFβ1复合物，用于检测、诊断、治疗、调节等不同目的。
宿主细胞
本公开提供表达一种或多种本公开的蛋白结合分子的宿主细胞。在一些实施方案中，本公开提供的宿主细胞其包含前述任一种的多核苷酸或载体；或者，宿主细胞表达前述任一种的TGFβ1结合分子或GARP-TGFβ1结合分子。
在一些实施方案中，宿主细胞表达本公开第一方面所述的TGFβ1结合分子。在一些实施方案中，宿主细胞表达本公开第二方面所述的GARP-TGFβ1结合分子。
在一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
示例性地，细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
示例性地，真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia  pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
示例性地，哺乳动物细胞例如猴肾CV1系(COS-7)、人胚胎肾系(293或293T细胞)、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、犬肾细胞(MDCK)、buffalo大鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(HepG2)、人胶质瘤细胞(LN229细胞)、人宫颈癌细胞(HeLa细胞)、人乳腺癌细胞(MCF-7)、人前列腺癌细胞(PC3)，小鼠乳腺癌细胞(EMT-6细胞)、小鼠乳房肿瘤细胞(MMT060562)、小鼠结肠癌细胞(CT26细胞)，TRI细胞(如例如记载于Mather等，Annals N.Y.Acad Sci383，44-68(1982))、MRC5细胞和FS4细胞、中国仓鼠卵巢(CHO)细胞、骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0。
本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本公开的细胞不能发育成完整的植物或动物个体。
生产或制备方法
本公开提供制备本公开的蛋白结合分子的方法。
在一些实施方案中，本公开提供制备前述第一方面所述的TGFβ1结合分子的方法，其包括如下步骤：
a.形成TGFβ1前体蛋白或包含所述TGFβ1前体蛋白的TGFβ1复合物，作为抗原蛋白；
b.构建人源抗体噬菌体库；
c.利用所述噬菌体文库筛选与所述抗原蛋白特异性结合的TGFβ1结合分子。
在一些实施方案中，TGFβ1结合分子的制备方法还包括如下步骤：
d.对所述TGFβ1结合分子进行亲和力成熟改造。
上述的制备方法也是与TGFβ1前体蛋白或包含所述TGFβ1前体蛋白的TGFβ1复合物特异性结合的TGFβ1结合分子的筛选方法。
在一些实施方案中，本公开提供制备前述第二方面所述GARP-TGFβ1结合分子的方法，其包括如下步骤：
a.形成包含人GARP和人TGFβ1前体蛋白的复合物，作为抗原蛋白；
b.利用所述抗原蛋白免疫动物，构建噬菌体文库；
c.利用所述噬菌体文库筛选GARP-TGFβ1结合分子，所述GARP-TGFβ1结合分子包含与GARP-TGFβ1复合物结合的免疫球蛋白单一可变结构域。
在一些实施方案中，GARP-TGFβ1结合分子的制备方法还包括如下步骤：
d.对所述GARP-TGFβ1结合分子进行人源化改造。
在一些具体的实施方案中，步骤b进一步为：
利用所述抗原蛋白免疫骆驼，采集免疫后的骆驼外周血，提取核酸，利用所述核酸构建噬菌体文库。
上述的制备方法也是与GARP-TGFβ1复合物特异性结合的GARP-TGFβ1结合分子的筛选方法。
在另外一些实施方案中，本公开提供制备前述任一种TGFβ1结合分子或GARP-TGFβ1结合分子的方法，包括：
-在允许表达本公开的TGFβ1结合分子或GARP-TGFβ1结合分子的条件下培养本公开的宿主细胞；及
-从培养物回收由所述宿主细胞表达的目的蛋白；及
-任选的，包括进一步纯化和/或修饰本公开的目的蛋白。
本公开的TGFβ1结合分子或GARP-TGFβ1结合分子可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生蛋白或多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的结合分子或抗体等目的蛋白的分离及纯化技术为本领域技术人员所公知。生产和纯化抗体的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。本公开工程化的抗体也可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
然而，本公开的TGFβ1结合分子或GARP-TGFβ1结合分子也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
药物组合物
本公开提供药物组合物，其含有选自以下的任一项或其组合：如上所述的蛋白结合分子或其编码多核苷酸。在一些实施方案中，药物组合物中包含预防或治疗有效量的如前述任一种所述的TGFβ1结合分子或GARP-TGFβ1结合分子，或编码所述TGFβ1结合分子或GARP-TGFβ1结合分子的多核苷酸、载体。
在一些实施方案中，药物组合物中进一步包含一种或多种药学上可接受的辅 料、稀释剂、缓冲剂或赋形剂。
在一些实施方案中，药物组合物可以配制成医学领域已知的任何剂型，剂型选择取决于预期的给药方式和治疗用途。
在一些实施方案中，药物组合物进一步包含免疫检查点抑制剂。例如，免疫检查点抑制剂包括但不限于PD-1、PD-L1、PD-L2中的一种或多种抑制剂。在一些实施方案中，药物组合物中进一步包含抗PD-1抗体或其抗原结合片段。
本公开中的TGFβ1结合分子或GARP-TGFβ1结合分子能够抑制TGFβ1活性、抑制调节性T细胞的免疫抑制活性。TGFβ1结合分子或GARP-TGFβ1结合分子与抗PD-1抗体联用可以解除肿瘤免疫抑制微环境，本公开实施例发现，TGFβ1结合分子或GARP-TGFβ1结合分子与抗PD-1抗体联用后在肿瘤治疗中发挥显著改善的治疗效果。
在一些实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的TGFβ1结合分子。另一些具体实施方案中，药物组合物单位剂量中含TGFβ1结合分子的量为0.1-2000mg；一些具体实施方案中为1-1000mg。
在一些实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的GARP-TGFβ1结合分子。另一些具体实施方案中，药物组合物单位剂量中含GARP-TGFβ1结合分子的量为0.1-2000mg；一些具体实施方案中为1-1000mg。
联合用药
本公开提供任一种蛋白结合分子或其编码多核苷酸，联合免疫检查点抑制剂在用于治疗与TGFβ信号通路相关的疾病中的用途。示例性地，免疫检查点抑制剂包括但不限于PD-1、PD-L1、PD-L2中的一种或多种抑制剂。
在一些实施方案中，提供TGFβ1结合分子联合免疫检查点抑制剂在用于治疗与TGFβ信号通路相关的疾病中的用途。
在一些实施方案中，提供TGFβ1结合分子联合抗PD-1抗体或其抗原结合片段在用于治疗与TGFβ信号通路相关的疾病中的用途。
在一些实施方案中，提供GARP-TGFβ1结合分子联合免疫检查点抑制剂在用于治疗与TGFβ信号通路相关的疾病中的用途。
在一些实施方案中，提供GARP-TGFβ1结合分子联合抗PD-1抗体或其抗原结合片段在用于治疗与TGFβ信号通路相关的疾病中的用途。
术语“TGFβ信号通路相关的疾病”是指与TGFβ家族蛋白的表达、活性和/或代谢相关的任何疾病、障碍和/或病症或者可能受益于一种或多种TGFβ家族蛋白的活性和/或水平的调节的任何疾病、障碍和/或病症。TGFβ信号通路相关的疾病可以包括，但不限于肿瘤或癌症。
在一些实施方案中，与TGFβ信号通路相关的疾病为癌症。本公开提供TGFβ1结合分子或GARP-TGFβ1结合分子联合抗PD-1抗体或其抗原结合片段在用于治 疗癌症中的用途。
TGFβ1结合分子或GARP-TGFβ1结合分子显著改善肿瘤的免疫抑制微环境，其与抗PD-1抗体联用后在肿瘤治疗中发挥显著改善的治疗效果。
在一些实施方案中，癌症选自肺癌、肠癌、肾癌、膀胱癌、肝癌、胃癌、乳腺癌、结肠癌、宫颈癌、前列腺癌、头颈癌。
试剂盒(或药盒)
本公开提供试剂盒或药盒，包含一个或多个容器，其各自独立地包含选自以下的任一项或其组合：本公开的蛋白结合分子(例如，所述的TGFβ1结合分子或GARP-TGFβ1结合分子)，或其编码多核苷酸、载体。
预防、治疗疾病的方法和制药用途
本公开提供蛋白结合分子，或其编码多核苷酸、载体、药物组合物的医药用途，和预防、治疗、缓解疾病或症状的方法。
在一些实施方案中，本公开提供TGFβ1结合分子、编码多核苷酸、载体或药物组合物在如下至少一种中的用途：
(1)用于抑制TGFβ1活性；例如，抑制TGFβ1激活，抑制成熟TGFβ1从TGFβ1复合物中的释放，和/或抑制TGFβ1信号转导；
(2)制备用于抑制TGFβ1活性的药物；
(3)用于抑制调节性T细胞的免疫抑制活性；
(4)制备用于抑制调节性T细胞的免疫抑制活性的药物；
(5)用于预防或治疗与TGFβ信号通路相关的疾病或病症。
在一些实施方案中，本公开提供预防或治疗与TGFβ信号通路相关的疾病或病症的方法，包括向受试者施用预防或治疗有效量的TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些实施方案中，本公开提供在体外抑制TGFβ1活性的方法，其包括：在体外施用TGFβ1抗体或其抗原结合片段、编码多核苷酸、载体或药物组合物。在一些具体的实施方案中，施用抑制有效量的TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些实施方案中，本公开提供在体内抑制TGFβ1活性的方法，其包括：向受试者施用TGFβ1结合分子、编码多核苷酸、载体或药物组合物。在一些具体的实施方案中，向受试者施用抑制有效量的TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些具体的实施方案中，所述受试者患有与TGFβ信号通路相关的疾病或病症。
术语“TGFβ信号通路相关的疾病”是指与TGFβ家族蛋白的表达、活性和/或代 谢相关的任何疾病、障碍和/或病症或者可能受益于一种或多种TGFβ家族蛋白的活性和/或水平的调节的任何疾病、障碍和/或病症。TGFβ信号通路相关的疾病可以包括，但不限于纤维化相关的疾病或病症、肿瘤或癌症。
在一些实施方案中，所述与TGFβ信号通路相关的疾病为癌症。
在一些实施方案中，所述与TGFβ信号通路相关的疾病或病症为纤维化。
在一些实施方案中，提供预防或治疗癌症的方法，联合施用预防或治疗有效量的如下1)和2)所示的组分：
1)抗TGFβ1抗体或其抗原结合片段，或编码多核苷酸、载体，或药物组合物；
2)免疫检查点抑制剂。
示例性地，所述PD-1信号通路抑制剂包括但不限于PD-1、PD-L1、PD-L2中的一种或多种抑制剂。在一些实施方案中，所述免疫检查点抑制剂为抗PD-1抗体或其抗原结合片段。
在一些实施方案中，所述癌症选自肺癌、肠癌、肾癌、膀胱癌、肝癌、胃癌、乳腺癌、结肠癌、宫颈癌、前列腺癌、头颈癌。
在一些实施方案中，本公开的TGFβ1结合分子或药物组合物可以通过本领域已知的任何合适的方法来施用，施用可以为全身性或局部的。
在一些实施方案中，可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
在一些实施方案中，本公开提供GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物在如下至少一种中的用途：
(1)用于抑制TGFβ1活性；例如，抑制TGFβ1激活，抑制成熟TGFβ1从TGFβ1复合物中的释放，和/或抑制TGFβ1信号转导；
(2)制备用于抑制TGFβ1活性的药物；
(3)用于抑制调节性T细胞的免疫抑制活性；
(4)制备用于抑制调节性T细胞的免疫抑制活性的药物；
(5)用于预防或治疗与TGFβ信号通路相关的疾病或病症。
在一些实施方案中，本公开提供预防或治疗与TGFβ信号通路相关的疾病或病症的方法，包括向受试者施用预防或治疗有效量的GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些实施方案中，本公开提供预防或治疗与TGFβ信号通路相关的疾病的方法，包括向受试者施用预防或治疗有效量的GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物；以及，预防或治疗有效量的免疫检查点抑制剂。
示例性地，免疫检查点抑制剂包括但不限于PD-1、PD-L1、PD-L2中的一种或多种抑制剂。在一些实施方案中，免疫检查点抑制剂为抗PD-1抗体或其抗原结 合片段。
在一些实施方案中，本公开提供在体外抑制TGFβ1活性的方法，其包括：在体外施用GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。在一些具体的实施方案中，施用抑制有效量的GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些实施方案中，本公开提供在体外抑制TGFβ1激活或抑制成熟TGFβ1从GARP-TGFβ1复合物中释放的方法，其包括：在体外施用GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。在一些具体的实施方案中，施用抑制有效量的GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些实施方案中，本公开提供在体内抑制TGFβ1活性的方法，其包括：向受试者施用GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。在一些具体的实施方案中，向受试者施用抑制有效量的GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些实施方案中，本公开提供在体内抑制TGFβ1激活或抑制成熟TGFβ1从GARP-TGFβ1复合物中释放的方法，其包括：向受试者施用GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。在一些具体的实施方案中，向受试者施用抑制有效量的GARP-TGFβ1结合分子、编码多核苷酸、载体或药物组合物。
在一些具体的实施方案中，所述受试者患有与TGFβ信号通路相关的疾病或病症。
在一些实施方案中，与TGFβ信号通路相关的疾病为癌症。
在一些实施方案中，所述癌症选自肺癌、肠癌、肾癌、膀胱癌、肝癌、胃癌、乳腺癌、结肠癌、宫颈癌、前列腺癌、头颈癌。
在一些实施方案中，本公开的GARP-TGFβ1结合分子或药物组合物可以通过本领域已知的任何合适的方法来施用，施用可以为全身性或局部的。
在一些实施方案中，可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
定义
为了更容易理解本公开，以下具体定义了某些技术和科学。除非在本公开中另有明确定义，本公开使用的所有其它技术和科学都具有本公开所属领域的一般技术人员通常理解的含义。
除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围，例如1％至15％之间、在1％至10％之间、在1％至5％之间、在0.5％至5％之间、在0.5％至1％之间变化，本公开中，数字或数值范围之前有术语“约”的每种情况也包括给定数的实施方案。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“转化生长因子β(transforming growth factor-β)”家族是由一类结构、功能相关的多肽生长因子亚家族组成，参与许多不同的生物学途径。除TGF-β外，还包括激活素、抑制素、生长和分化因子(GDFs)、骨形态发生蛋白(BMP)等。TGFβ具有3种形式的异构体：TGFβ1、TGFβ2和TGFβ3，在哺乳动物的几乎所有细胞类型中被广泛表达。
与其它细胞因子不同，TGFβ超家族成员不作为活性生长因子分泌，而是作为由N-末端前结构域和C-末端生长因子结构域组成的二聚体前体蛋白分泌。原TGFβ1通过弗林蛋白酶的切割将同型二聚的生长因子结构域与其前结构域(也称为潜伏相关肽(LAP))分离。但是，生长因子和LAP保持非共价结合，从而形成潜伏复合物，其不能与其受体结合和诱导信号传导。在翻译过程中，潜伏TGFβ1(也称为小潜伏复合物(SLC))变成通过二硫桥与“呈递分子”连接，从而形成大潜伏复合物(LLC)。这些分子允许原TGFβ1存在于特定细胞或组织情境(contexts)中。靠近潜伏TGFβ1的N-末端的两个半胱氨酸连接于呈递分子上适当定位的半胱氨酸。呈递分子的身份取决于环境和产生潜伏TGFβ1的细胞类型。例如，成纤维细胞分泌系着于TGFβ-结合蛋白(LTBPs)的潜伏TGFβ1，其然后与细胞外基质(ECM)中的蛋白质(即，纤连蛋白、原纤维蛋白-1)结合以将潜伏TGFβ连接于ECM(Robertson等Matrix Biol 47:44-53(2015))。在激活的调节性T细胞的表面上，潜伏TGFβ1共价连接于跨膜蛋白GARP，且最近与GARP，LRRC33密切相关的蛋白质鉴定为在单核细胞、巨噬细胞和小胶质细胞的表面上TGFβ1的呈递分子(Wang,R.等,Mol Biol Cell,2012.23(6):p.1129-39和T.A.Springer,Int.BMP Conference 2016)。
在本公开的范畴内，TGFβ1蛋白应做最广泛的理解。该术语涵盖TGFβ1在自然界中的天然形式、天然存在的变体，也包括人工表达的形式、功能变体等。当上下文没有专门说明时，TGFβ1涵盖成熟TGFβ1、TGFβ1前体蛋白及其蛋白表位的范畴。TGFβ1前体蛋白涵盖原TGFβ1、潜伏TGFβ1及其片段。TGFβ1序列可获取于GenBank、UniProt等。例如，人原TGFβ1(Uniprot:P01137)、鼠原TGFβ1(Uniprot:P04202)。
在本公开的上下文中，“原TGFβ1”与“潜伏TGFβ1”可以互换地使用。
如本文所用，术语“TGFβ1复合物”是指TGFβ1与包括但不限于GARP、LRRC33、LTBP3或LTBP1的蛋白分子通过二硫桥连接形成的复合物，也称为LTBP1-TGFβ1复合物、LTBP3-TGFβ1复合物、LRRC33-TGFβ1复合物或GARP-TGFβ1复合物。在所述TGFβ1复合物中，TGFβ1以TGFβ1前体蛋白(例如，原/潜伏TGFβ1)的形式存在。
术语“功能变体”包括但不限于野生型蛋白的同系物、片段、截短体、突变体、修饰物等，蛋白的功能变体与野生型蛋白相比，具有提高的、降低的或维持的蛋白活性。
术语“结合分子”涵盖任何能够特异性结合抗原或抗原表位的分子，例如本公开所定义的抗体、其抗原结合片段或其缀合物、融合蛋白。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。
Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例 如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表4中所示。
表4.CDR编号系统之间的关系
多肽或蛋白的“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个功能性质，且在许多情况下可添加、移除或转移至其它蛋白而不损失蛋白的其余部分和/或结构域的功能。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“抗体框架(FR)”，是指可变结构域的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。
“免疫球蛋白单一可变结构域”通常用于指可以在不与其他可变结构域相互作用的情况下(例如在没有如常规四链单克隆抗体的VH和VL结构域之间所需要的VH/VL相互作用的情况下)，形成功能性抗原结合位点的免疫球蛋白可变结构域(其可以是重链或轻链结构域，包括VH、VHH或VL结构域)。“免疫球蛋白单一可变结构域”的实例包括纳米抗体(包括VHH、人源化VHH和/或骆驼化VH，例如骆驼化人VH)、IgNAR、结构域、作为VH结构域或衍生自VH结构域的(单结构域)抗体(诸如dAbsTM)和作为VL结构域或衍生自VL结构域的(单结构域)抗体(诸如dAbsTM)。基于和/或衍生自重链可变结构域(诸如VH或VHH结构域)的免疫球蛋白单一可变结构域通常是优选的。免疫球蛋白单一可变结构域的一个具体实例为如下文定义的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段、VHH抗体、纳米抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规四肽链结构抗体中的重链可变结构域(其在本公开中称为“VH结构域”)以及轻链可变结构域(其在本公开中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规四肽链结构抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”、“Nanobody”以及““Nanobody结构域””可互换使用。“VHH结构域”包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的全人源抗体。VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本公开所述的目的。获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。其它的编号系统或编码规则包括Chothia、IMGT、AbM。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。“人源化”的例子包括可将源自骆驼科的VHH结构域通过以人常规四肽链结构抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而“人源化”(本公开中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其它修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列。此外，为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“全人抗体”或“全人源抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的全人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。“全人抗体”不包括“人源化抗体”。
当“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与充分接近参考抗原结合蛋白结合的表位所邻近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。通常当竞争的抗原结合蛋白过量存在时，其将 抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合抗原蛋白上的相同表位的抗体。
术语“糖蛋白A重复主导序列(Glycoprotein-A Repetitions Predominant，GARP)”是具有单次跨膜结构的蛋白质，“GARP”还被称为富含亮氨酸重复序列32(LeucinRichRepeatContaining32，LRRC32)，并且属于富含亮氨酸重复序列家族。GARP可以在活化的Treg的细胞表面上表达，并且与TGF-β前体(例如latent TGF-β)形成复合物。GARP序列可获取于GenBank、UniProt等。例如，人GARP(LRRC32,Uniprot:Q14392)，鼠GARP(LRRC32,Uniprot:G3XA59)。
在本公开的范畴内，GARP应做最广泛的理解。该术语涵盖GARP在自然界中的天然形式、天然存在的变体，也包括人工表达的形式、功能变体等。当上下文没有专门说明时，涉及抗原-抗体相互作用的情形下，GARP涵盖完整蛋白、胞外结构域及其表位的范畴。
术语“GARP-TGFβ1复合物”“GARP/TGFβ1蛋白”“GARP/TGFβ1”是指包含转化生长因子-β1(TGFβ1)蛋白的前体蛋白和糖蛋白-A重复序列为主蛋白(GARP)的蛋白质复合物。在一些实施方式中，TGFβ1蛋白的原蛋白形式或潜伏形式可以称为“原/潜伏TGFβ1蛋白”。在一些实施方式中，GARP-TGFβ1复合物包含通过一个或多个二硫键与TGFβ1前体蛋白(原/潜伏TGFβ1蛋白)共价连接的GARP。在其它实施方式中，GARP-TGFβ1复合物包含与TGFβ1前体蛋白(原/潜伏TGFβ1蛋白)非共价连接的GARP。在一些实施方式中，GARP-TGFβ1复合物是天然发生的复合物，例如细胞中的GARP-TGFβ1复合物。
术语“LTBP”是指潜在转化生长因子β结合蛋白(latent transforming growth factorβbinding proteins)，LTBP是细胞外基质(ECM)的重要组成成分，其主要功能与调节原纤蛋白、转化生长因子β(TGF-β)相关。在哺乳动物中，存在四种已知的LTBPs，LTBP1-4，其各自具有多个剪接变体(Robertson,I.B.等,Matrix Biol,2015.47:p.44-53)。
术语“GARP-TGFβ1复合物”“GARP/TGFβ1蛋白”“GARP/TGFβ1”是指包含转化生长因子-β1(TGFβ1)蛋白的前体蛋白形式或潜伏形式和糖蛋白-A重复序列为主蛋白(GARP)的蛋白质复合物。在一些实施方式中，TGFβ1蛋白的原蛋白形式或潜伏形式可以称为“原/潜伏TGFβ1蛋白”。在一些实施方式中，GARP-TGFβ1复合物包含通过一个或多个二硫键与TGFβ1前体蛋白(原/潜伏TGFβ1蛋白)共价连 接的GARP。在其它实施方式中，GARP-TGFβ1复合物包含与TGFβ1前体蛋白(原/潜伏TGFβ1蛋白)非共价连接的GARP。在一些实施方式中，GARP-TGFβ1复合物是天然发生的复合物，例如细胞中的GARP-TGFβ1复合物。
术语“LTBP1-TGFβ1复合物”是指包含转化生长因子β1(TGFβ1)蛋白的前体蛋白和潜伏TGFβ结合蛋白(例如，LTBP1、LTBP3)的蛋白质复合物。在一些实施方式中，LTBP-TGFβ1复合物包含通过一个或多个二硫键与TGFβ1前体蛋白共价连接的LTBP1。在其它实施方式中，LTBP1-TGFβ1复合物包含与TGFβ1前体蛋白非共价连接的LTBP1。在一些实施方式中，LTBP1-TGFβ1复合物是天然发生的复合物，例如细胞中的LTBP1-TGFβ1复合物。
术语“抗原”指用于免疫接种免疫活性的脊椎动物的分子，以产生识别抗原的抗体，或筛选表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)。在本公开中，抗原被更广义地定义，包括由抗体特异性识别的靶分子，以及包括用于产生抗体的免疫接种过程或用于选择抗体的文库筛选中使用的分子的一部分或模拟物。例如，对于本公开的与人GARP-TGFβ1复合物结合的抗体，以及人GARP-TGFβ1复合物的截短变体和其它变体均被称为抗原。
术语“表位”是指抗原上与免疫球蛋白或抗体结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本公开所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。例如，当使用人GARP-TGFβ1复合物或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10-7M或甚至更小的平衡解离常数(KD)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。“识别抗原的抗体”在本公开中可以与“特异性结合的抗体”互换使用。
“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。KD通过方程KD＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且可通过方法例如Caceci等人(1984，Byte 9：340-362)中所述的那些甚至对于复杂混合物进行 计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定KD。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开其它地方例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用BiacoreTM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的KD值来比较与不同分子相互作用相关的结合亲和力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的KD值与非目的相互作用(例如已知不结合抗原的对照抗体)的KD值进行评价。
术语“保守性取代”、“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
术语“同源性”、“同一性”或“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
术语“核酸分子”与“多核苷酸”可互换使用，其是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“宿主细胞”包括各个细胞或细胞培养物，其可为或已是用于掺入多核苷酸插入片段的载体的受体。宿主细胞包括单个宿主细胞的子代，并且由于天然、偶然或有意的突变，子代可不一定与原始亲本细胞完全相同(在形态学或基因组DNA互补体中)。宿主细胞包括用本公开的多核苷酸在体内转染和/或转化的细胞。“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还 应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。例如，“抑制抗体”是指抑制成熟生长因子释放或降低生长因子活性的抗体。抑制抗体包括靶向在与这样的抗体结合时降低生长因子释放或活性的任何表位的抗体。这样的表位可以位于TGFβ蛋白(例如，TGFβ1)的前结构域、生长因子或在被抗体结合时导致降低的生长因子活性的其它表位上。本发明的抑制抗体包括，但不限于TGFβ1-抑制抗体。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。本公开的受试者可以是动物或人类受试者。
术语“药物组合物”表示含有一种或多种本文所述活性成分或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学可接受的辅料”或“药学可接受的赋形剂”包括当与活性成分组合时，允许该成分保留生物学活性并且不与受试者的免疫系统反应的任何材料。例子包括但不限于任何标准药物载体，例如磷酸盐缓冲盐水溶液、水、乳剂如油/水乳剂、和各种类型的润湿剂。在一些实施例中，用于气雾剂或肠胃外施用的稀释剂是磷酸盐缓冲盐水(PBS)或生理(0.9％)盐水。包含此类载体的组合物通过众所周知的常规方法配制(参见例如Remington′s Pharmaceutical Sciences，第18版，A.Gennaro，编辑，Mack PublishingCo.，Easton，PA，1990；以及R Remington，The Science and Practice of Pharmacy第20版Mack Publishing，2000)。
“癌症”、“癌性”、“增殖性病症”和“肿瘤”在本公开中提到时并不互相排斥。
术语“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触，例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
术语“治疗”意指给予受试者治疗剂，诸如包含本公开的任一种融合蛋白或胰岛素类似物为治疗剂，所述受试者已经患有、疑似患有、倾向于患有一种或多种糖尿病或高血糖相关疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。 通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，是通过以预防或延迟症状或并发症的发作，减轻症状或并发症，或者消除疾病、病况或病症到任何临床能测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。待治疗的患者是哺乳动物，并且优选地为人类。
术语“预防”是指降低一种或多种病况、症状、并发症或病症的风险或发病率，或者消除或减缓一种或多种病况、症状、并发症或病症的进展。
术语“受试者”、“患者”意指哺乳动物，尤其灵长类动物，尤其是人。
附图说明
图1示出了检测抗TGFβ1抗体(H27、SL2-2、SL2-9、SL2-12、SL2-19及SL2-22)对人或小鼠TGFβ1复合物(人proTGFβ1、GARP-TGFβ1复合物、LRRC33-TGFβ1复合物，鼠GARP-TGFβ1复合物)的活性抑制结果图。图1中：A为抗体对人proTGFβ1激活的抑制效果图，B为抗体对人GARP-TGFβ1复合物激活的抑制效果图，C为抗体对人LRRC33-TGFβ1复合物激活的抑制效果图，D为抗体对鼠GARP-TGFβ1复合物激活的抑制效果图。
图2示出了检测抗TGFβ1抗体(SL2-22、Ab6)与表达人或小鼠的原TGFβ1、TGFβ2、TGFβ3、及TGFβ复合物(GARP-TGFβ1复合物、GARP-TGFβ2复合物、GARP-TGFβ3复合物)的HEK293E细胞结合实验的结果图。
图3A-3C示出了抗TGFβ1抗体与抗PD-1抗体联合(RMP1-14-mIgG2a-FcS+SL2-22-mIgG2a-FcS)在小鼠EMT-6模型中抑制肿瘤生长的结果图。图3A为小鼠肿瘤体积曲线图，图3B为小鼠生存率曲线图，图3C为小鼠体重曲线图。
图4A-4C示出了抗TGFβ1抗体与抗PD-1抗体联合RMP1-14-mIgG2a-FcS+SL2-22-mIgG2a-FcS)在小鼠CT26模型中抑制肿瘤生长结果图。图4A为小鼠肿瘤体积曲线图，图4B为小鼠生存率曲线图，图4C为小鼠体重曲线图。
图5A-5E示出了抗TGFβ1抗体(SL2-22-mIgG2a-FcS)在小鼠特发性纤维化模型中抑制纤维化结果图。图5A为肺纤维化模型小鼠体重变化曲线，图5B为肺纤维化模型小鼠存活率曲线，图5C为肺纤维化模型实验终点小鼠肺重及肺组织羟脯 氨酸含量，图5D为肺纤维化模型实验终点小鼠肺切片，图5E为Masson染色胶原面积比。
图6示出了通过LN229实验检测抗GARP-TGFβ1单域抗体对GARP-TGFβ1复合物激活的抑制。LN229细胞转染人或鼠原TGFβ1和人或鼠GARP表达质粒，通过HepG2CAGA12-luc荧光素酶报道细胞，检测Abbv-151、C19、C19-3、C19-7、C19-8对人或鼠GARP-TGFβ1复合物激活的抑制。图6A为抗体对人GARP-TGFβ1复合物激活的抑制效果图，图6B为抗体对鼠GARP-TGFβ1复合物激活的抑制效果图。
图7示出了C19-8与表达人或小鼠的GARP-TGFβ1复合物的HEK293E细胞结合实验的结果图。HEK293E细胞转染表3中质粒，加入C19-8和Abbv-151，分别染色，检测，获得结果。
图8A-8C示出了C19-8与抗PD-1抗体联合(RMP1-14-mIgG2a-FcS+C19-8-mIgG2a-FcS)在小鼠EMT-6模型中抑制肿瘤生长结果图。图8A为小鼠肿瘤体积曲线图，图8B为小鼠生存率曲线图，图8C为小鼠体重曲线图。
图9A-9C示出了C19-8与抗PD-1抗体联合(RMP1-14-mIgG2a-FcS+C19-8-mIgG2a-FcS)在小鼠CT26模型中抑制肿瘤生长结果图。图9A为小鼠肿瘤体积曲线图，图9B为小鼠生存率曲线图，图9C为小鼠体重曲线图。
具体实施方式
实施例
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗原及检测用蛋白的制备
以人原TGFβ1(Uniprot:P01137)、人GARP(LRRC32,Uniprot:Q14392)、鼠原TGFβ1(Uniprot:P04202)和鼠GARP(LRRC32,Uniprot:G3XA59)为模板，设计筛选和检测用相关蛋白的氨基酸序列。
>人原TGFβ1全长氨基酸序列：

(注释：斜体部分为信号肽(Signal peptide)；下划线部分为潜能相关肽(Latency-associated peptide；LAP)；斜体加粗部分为TGFβ1结构域
>成熟的人TGFβ1(mature transforming growth factor beta-1)：
>人LAP：
>人GARP全长氨基酸序列：
(注释：斜体部分为信号肽；下划线部分为胞外区(Extracellular domain)；斜体加粗部分为跨膜区(Transmembrane domain)；斜体下划线部分为胞内区(Cytoplasmic domain))
>人GARP胞外区：

>小鼠原TGFβ1全长氨基酸序列：
(注释：斜体部分为信号肽；下划线部分为潜能相关肽(LAP)；斜体加粗部分为TGFβ1结构域)
>成熟的小鼠TGFβ1：
>小鼠LAP：
>小鼠GARP全长氨基酸序列：

(注释：斜体部分为信号肽；下划线部分为胞外区；斜体加粗部分为跨膜区；斜体下划线部分为胞内区)
>小鼠GARP胞外区：
筛选和检测用人GARP/TGFβ1复合物如下制备：将人GARP-avi-his与人天然TGFβ1(分别如SEQ ID NO:5和6所示)在ExpiCHO细胞(ThermoFisher,A29127)中共表达、纯化获得蛋白复合物；人TGFβ1 C4S蛋白如SEQ ID NO:7所示；小鼠GARP/TGFβ1复合物如下制备：将小鼠GARP-avi-his与小鼠天然TGFβ1(分别如SEQ ID NO:8和9所示)在ExpiCHO细胞(ThermoFisher,A29127)中共表达、纯化获得蛋白复合物；小鼠TGFβ1 C4S蛋白如SEQ ID NO:10所示：
>人GARP-avi-his氨基酸序列：

(注释：下划线部分为人GARP胞外区；斜体部分为(G4S)2接头；为Avi tag；为His标签)
>人TGFβ1-天然氨基酸序列：
>人TGFβ1 C4S氨基酸序列：
(注释：为His标签；斜体部分为(G4S)2接头；Avi标签；下 划线部为人TGFβ1全长蛋白，其中第四位的氨基酸C突变为S)
>小鼠GARP-avi-his氨基酸序列：

(注释：下划线部分为小鼠GARP胞外区；斜体部分为(G4S)2接头；为Avi tag；下曲线部分为His标签)
>小鼠TGFβ1-天然氨基酸序列：
>小鼠TGFβ1 C4S氨基酸序列：
(注释：为His标签；斜体部分为(G4S)2接头；为Avi标签；下划线部分为小鼠TGFβ1全长蛋白，其中第四位的氨基酸C突变为S)
以上序列均可以经本领域的常规方法表达、纯化、分离获得。
实施例2.特异性结合人TGFβ1复合物的抗TGFβ1单克隆抗体的筛选
通过人源抗体噬菌体库的筛选来获得与人TGFβ1复合物具有高亲和力的抗体。人源抗体噬菌体库一共四个(1：半合成人Fab库1(种系3-23)；2：半合成人Fab库2(种系1-69)；3：全人源Fab库；4：全人源scFV库)，均为上海恒瑞医药有限公司赠送。使用10μg生物素化的人GARP-TGFβ1复合物(包含SEQ ID NO：5和6)蛋白结合1mg Dynabeads M-280链霉亲和素(Cat No.11206D,Invitrogen)，室温反应半小时，1×PBS洗3遍，之后加入2％的脱脂牛奶室温封闭1小时，同时使用2％的脱脂牛奶和1mg Dynabeads M-280链霉亲和素对人源抗体噬菌体展 示文库进行封闭和消耗(depletion)，将处理后的噬菌体文库加入到结合了抗原的珠中，室温作用1小时。使用1×PBST(含0.05％Tween-20)，pH 7.4溶液洗10遍，以去除不结合的噬菌体，1×PBS再洗2遍后用0.5mL的胰蛋白酶(1mg/mL)将与人GARP/TGFβ1特异性结合的噬菌体洗脱，并感染处于对数生长期的大肠杆菌TG1，2YT(含2％葡萄糖)抗性平板生长过夜，从平板刮菌生产并纯化噬菌体用于下一轮筛选。
相同筛选过程重复2轮，为获得与小鼠TGFβ1复合物有交叉结合的抗体，第二轮使用生物素化的小鼠TGFβ1_C4S(SEQ ID NO：10)作为筛选抗原。第三轮使用生物素化的人GARP-TGFβ1复合物(包含SEQ ID NO：5和6)作为筛选抗原。三轮筛选过后阳性的克隆被富集。
从筛选富集的克隆中挑取6×92个单克隆包装成单克隆噬菌体，用于噬菌体ELISA测试。ELISA板(Cat No.9018,Corning)分别包被2μg/mL的人GARP-TGFβ1复合物(SEQ ID NO：5和6)和小鼠TGFβ1_C4S(SEQ ID NO：10)蛋白，4℃放置过夜，1×PBST洗3遍，用2％BSA 37℃封闭1个小时，PBST洗3遍后，加入封闭液稀释的噬菌体上清，室温反应1小时，PBST洗6遍，加入抗M13HRP(Cat No.11973-MM05T-H,义翘神州)，室温反应1小时，PBST洗3遍，加入100μL TMB显色底物，并用100μL 1M硫酸终止反应，用SpectraMax M5酶标仪读取450nm处吸收值。将ELISA结合测试中OD450读值大于3倍阴性对照的克隆进行测序，分析得到43个特异性抗体序列。通过SPR实验显示抗体H5、H14、H16、H17、H19、H20、H21、H23、H24、H27、H28、H30、H32、H34和H39能够结合TGFβ1复合物。但是细胞功能实验显示，只有H27有较好的抑制功能，并且和人和小鼠GARP-TGFβ1复合物以及人和小鼠TGFβ1_C4S(也即，TGFβ1前体蛋白)都有很好的结合。
以下示出其中的H27序列。
>H27 VH
>H27 VL
以上序列SEQ ID NO：11和12中，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线分别为CDR1、CDR2和CDR3序列。本公开提供的人源抗体的编号规则均为Kabat，表5是CDR序列。
表5.H27抗体的CDR序列
实施例3.完整单克隆抗体的构建与表达纯化
将实施例2中通过人源抗体噬菌体文库筛选得到的H27的序列构建完整重组抗体：将抗体的轻链可变区VL克隆进含有人源kappa轻链恒定区(SEQ ID NO：19)的pTT5表达载体，将抗体的重链可变区VH克隆进含有人IgG4-S228P(CH1-CH2-CH3)重链恒定区(SEQ ID NO：20)的pTT5表达载体。
>人kappa轻链恒定区：
>人IgG4-S228P(CH1-CH2-CH3)重链恒定区：
H27完整抗体序列如下：
>H27轻链:
>H27重链:

将克隆所得轻链和重链质粒配对共转染HEK293E细胞(上海恒瑞医药有限公司赠送)或者ExpiCHO(Cat No.A29127,ThermoFisher)细胞，转染后5天(37度，HEK29E3细胞)或者10-12天(32度，expiCHO细胞)收获细胞培养上清，4000转离心20分钟，上清使用0.45μM滤器过滤。接着用MabSelectSure LX柱(GE Healthcare)进行第一步亲和纯化。将培养液上清流过PBS平衡的MabSelectSure LX柱，经PBS洗涤后，用pH 3.0的0.1M Glycine酸性洗脱液洗脱目的蛋白后用1M MES(pH 6.0)中和，最后用HiTrap SP HP离子柱精细纯化。经检测，获得目的抗体。
实施例4.TGFβ1单克隆抗体的亲和力测定
用Biacore T200(GE Healthcare)测定抗体与人/小鼠GARP/TGFβ1以及人/小鼠TGFβ1_C4S的亲和力。
使用Protein A生物传感芯片(Cat No.#29127556,GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一系列梯度稀释的人或小鼠GARP-TGFβ1复合物TGFβ1_C4S抗原，流速50μL/min，解离时间5分钟，每个循环结束后使用pH 1.5甘氨酸-盐酸(Cat.#BR-1003-54,GE)对芯片进行再生，反应缓冲液为用蒸馏水稀释至1 x的HBS-EP+缓冲溶液(Cat No.#BR-1006-69，GE)pH 7.4)。
利用Biacore T200实时检测反应信号从而获得结合和解离曲线，得到的数据用BIAevaluation version 4.1，GE软件以Langmuir 1:1结合模型进行拟合，得出亲和力数值，结果见表6。
表6.H27的亲和力数据
实施例5.TGFβ1单克隆抗体H27的亲和力成熟
将H27抗体分子进行三维结构模拟。参考人种系基因突变热点和三维结构模拟结果，选定框架区和CDR区部分关键氨基酸残基，建立4个随机突变噬菌体文库(表7)。利用噬菌体文库展示技术，筛选得到亲和力有所提高的功能性抗体。对不同文库获得新氨基酸残基进行组合与验证，获得亲和力与功能均有提高的功能性抗体。
表7.H27亲和力成熟文库设计
注：“/”为不含突变残基
对4个随机突变噬菌体文库进行筛选，利用生物素化的人GARP-TGFβ1(包含SEQ ID NO：5和6)作为筛选抗原，经过3-4轮筛选，对所获克隆进行序列分析并结合噬菌体ELISA结果，挑选62个抗体序列进行克隆构建，将抗体的轻链可变区VL克隆进含有人源kappa轻链恒定区(SEQ ID NO：19)的pTT5表达载体，将抗体重链可变区VH克隆进含有人hIgG4-S228P(CH1-CH2-CH3)重链恒定区(SEQ ID NO：20)的pTT5表达载体。
将克隆所得轻链重链质粒配对共转染HEK293E细胞，5天后收集细胞培养上清，4000转离心去除细胞，用MabSelect Sure进行纯化后参照实施例4的方法进行亲和力测定。
通过SPR蛋白相互作用确定亲和力比H27提高5倍以上的抗体均来自第二个随机突变库，该库抗体重链为H27重链。其轻链可变区序列如表8所示(下划线部分为CDR区，采用Kabat编码规则)。另外，其轻链全长序列和CDR组合分别如表9和10所示。
表8.TGFβ1单克隆抗体H27的亲和力成熟后的轻链可变区序列

表9.TGFβ1单克隆抗体H27的亲和力成熟后的轻链序列


表10.TGFβ1单克隆抗体H27的亲和力成熟后的轻链CDR序列

H27、SL2-1、SL2-2、SL2-3、SL2-4、SL2-5、SL2-6、SL2-8、SL2-9、SL2-12、SL2-13、SL2-15、SL2-17、SL2-18、SL2-19、SL2-22具有如下通式结构：
LCDR1为RASQX1ISX2YLN(SEQ ID NO:38)，X1选自S、A、F、G、I、P、Y、V或K，X2选自S、D、E、P或H；
LCDR2为X3ASX4LX5S(SEQ ID NO:64)，X3选自A、T、S或M，X4选自S、Y、A、E或G，X5选自Q、T、D或E。
SL2-2、SL2-9、SL2-12、SL2-19、SL2-22具有如下通式结构：
LCDR1为RASQX1ISX2YLN(SEQ ID NO:38)，其中X1选自F、Y或A，X2选自D或P；
LCDR2为X3ASX4LX5S(SEQ ID NO:64)，其中X3选自A、T或S，X4选自S、Y或E，X5选自Q、D或E。
实施例6.TGFβ1亲和力成熟单克隆抗体的亲和力测定
本实施例的阴性对照为HBS-EP，阳性对照为Ab6(Scholar rock,WO2020014460Al)和Abbv-151(Abbvie，US10793627B2)。Ab6与GARP-TGFβ1复合物、LRRC33-TGFβ1复合物、LTBP3-TGFβ1复合物和LTBP1-TGFβ1复合物都结合，结合表位完全在TGFβ1上(Martin等，2020)；而Abbv-151的结合表位包括GARP-TGFβ1复合物中的GARP和TGFβ1，不与单独的GARP或TGFβ1结合(Streel等，2020)。Ab6和Abbv-151的序列如下：
>Ab6抗体重链序列：
>Ab6抗体轻链序列：
>Abbv-151抗体重链序列：
>Abbv-151抗体轻链：
用Biacore T200(GE Healthcare)按照实施例4的方法测定几个亲和力成熟单克隆抗体与人和小鼠GARP-TGFβ1复合物的亲和力。亲和力测定结果参见表11和12。
表11.H27亲和力成熟抗体的亲和力数据

表12.SL2-22的亲和力数据
实施例7.TGFβ1单克隆抗体体外抑制GARP-TGFβ1复合物的功能
将2×106个LN229细胞(Procell,CL-0578)传至T75培养瓶(Nunc)中，24h后利用293fectinTM(Invitrogen)转染试剂单转染人或小鼠的原TGFβ1、或共转染人或小鼠的原TGFβ1和GARP或LRRC33质粒。24h后，将细胞按每孔1×104个传至白色不透明96孔细胞培养板(PerkinElmer)中。24h后，吸出培养基，加入重悬于DMEM+0.5％BSA中的HepG2CAGA12-luc荧光素酶报道细胞，同时加入倍比稀释的H27、H27亲和力成熟抗体以及对照抗体Ab6。混合培养20h后，加入ONE-GloTM萤光素酶试剂，孵育5min后，在多功能酶标仪(SpectraMax M5)上读取化学发光值。溶媒处理组的荧光素酶活性值标准化到1，相对活性＝抗体处理组荧光素酶÷溶媒对照组。使用Prism 9通过非线性拟合、三参数对数抑制剂对反应模型作图。
结果如图1A和图1B显示，亲和力成熟后的SL2-2、SL2-9、SL2-12、SL2-19及SL2-22抑制原TGFβ1及TGFβ1各个复合物的活性均高于母本H27。SL2-2、SL2-9、SL2-12、SL2-19及SL2-22在抑制人原TGFβ1、GARP-TGFβ1复合物和鼠GARP-TGFβ1复合物活性上与阳性对照Ab6相当，在抑制人LRRC33-TGFβ1复合物活性上优于Ab6。
实施例8.TGFβ1单克隆抗体SL2-22与表达不同TGFβ复合物的细胞结合实验
将HEK293E细胞配制成1×106个/mL，取5mL放入50mL培养管中，用293fectinTM试剂转染表13中质粒。48h后，将细胞用1 x PBS洗2次，按每孔1 x 105个放入96孔圆底细胞培养板(Nunc)中。加入100μL 1 x PBS配制的10nM抗体或对照抗体，室温染色1h，1×PBS洗3次后，加入FITC标记的抗人Fc抗体100μL，室温染色30min。染色结束，1 x PBS洗3次。加入7-AAD孵育5min， 1 x PBS洗3次后，流式细胞仪检测。通过Flowjo分析活细胞中FITC阳性的细胞数，计算FITC阳性的细胞数占活细胞总数的百分比，并作图。
表13.质粒结构
结果如图2所示，SL2-22和Ab6均与人和鼠TGFβ1(图2中分别对应TGFβ1和mTGFβ1)、GARP-TGFβ1复合物(图2中分别对应TGFβ1+GARP，mTGFβ1+mGARP)结合，并且不结合TGFβ2、GARP-TGFβ2复合物(图2中对应为TGFβ2+GARP)，不同的是Ab6与人TGFβ3、GARP-TGFβ3复合物(图2中对应TGFβ3+GARP)也有一定结合，而SL2-22只特异结合TGFβ1复合物。
实施例9.TGFβ1抗体的表位竞争研究
本实施例的阴性对照为HBS-EP，阳性对照为Ab6和Abbv-151。
用Biacore T200(GE Healthcare)研究抗人TGFβ1抗体SL2-22与阳性对照抗体与人GARP-TGFβ1复合物的表位竞争。
使用氨基偶联试剂盒(Cat No.BR-1000-50,GE)和His捕获试剂盒(Cat No.28-9950-56,GE)根据试剂盒说明书对CM5传感芯片(Cat.29-1049-88,GE)进行抗 His标签抗体的偶联,使表面响应值达到约10000RU，之后用乙醇胺封闭后待用。
表位竞争研究步骤(样本使用双模式)如下(抗体两两循环配对)：
1)抗原捕获：人GARP-TGFβ1复合物
2)第一个抗体：浓度100μg/ml,流速50μL/min,60秒，HBS-EP缓冲液做对照
3)第二个抗体：浓度100μg/ml,流速50μL/min,60秒，HBS-EP缓冲液做对照
4)对传感图进行分析，SL2-22与Ab6对人GARP-TGFβ1复合物的结合表位有部分竞争，SL2-22与Abbv151对人GARP-TGFβ1复合物的结合表位完全不竞争(表14)。
表14.SL2-22与阳性抗体Ab6、Abbv151表位竞争的测定
(注：“×”表示完全不竞争)
实施例10.TGFβ1单克隆抗体SL2-22在小鼠EMT-6模型中抑制肿瘤生长
为检测SL2-22体内肿瘤抑制活性，将BALB/c小鼠(雌性，6周龄，购买于北京维通利华实验动物技术有限公司)适应性饲养1周，实验当天编号称重。收集处于对数生长期的小鼠乳腺癌细胞EMT-6(Procell，CL-0573)，用PBS重悬，以5×106个/mL浓度，0.1mL/只体积接种于BALB/c小鼠的右侧肋部皮下。接种后第3d，当平均肿瘤体积达到约60mm3时，挑选个体肿瘤体积适中的小鼠，按照表15所示进行随机分组。mIgG2a-FcS为去除ADCC效应的带有L234A/L235E/G237A/D327Q/A330S/P331S的mIgG2a Fc，作为同种型对照；RMP1-14-mIgG2a-FcS为抗PD1抗体RMP1-14的可变区(WO2018223182A1中除去信号肽的序列285(重链可变区)和286(轻链可变区)；WO2018223182A1通过引用全文并入本公开)与去除ADCC效应的带有L234A/L235E/G237A/D327Q/A330S/P331S的mIgG2a重链恒定区/鼠kappa轻链恒定区融合在一起的抗体；SL2-22-mIgG2a-FcS为SL2-22的可变区与去除ADCC效应的带有L234A/L235E/G237A/D327Q/A330S/P331S的mIgG2a重链恒定区/鼠kappa轻链恒定区融合在一起的抗体，序列如下：
>SL2-22-mIgG2a-FcS抗体重链：

>SL2-22-mIgG2a-FcS抗体轻链：
分组当天开始给药，按照相同摩尔剂量，给药频率为一周两次，共给药7次，给药方式为腹腔注射。每周测量2次小鼠体重以及肿瘤体积，肿瘤体积计算公式为TV＝L长×L短 2/2。各组肿瘤体积用平均值±标准差表示，使用Two-way ANOVA进行统计分析，并计算肿瘤抑制率(％TGI)，计算公式为％TGI＝[1-(T-T0)/(C-C0)]×100％。
结果如图3A、3B、表15所示，SL2-22-mIgG2a-FcS与抗PD-1抗体RMP1-14-mIgG2a-FcS联用相比于RMP1-14-mIgG2a-FcS单用组显著抑制小鼠EMT-6肿瘤生长(p＝0.0127,Two-way ANOVA)，并延长动物生存期(p＝0.0323,Log-rank(Mantel-Cox)test)。在治疗过程中，小鼠耐受良好，没有出现明显的体重下降(图3C)。
表15.SL2-22在EMT-6模型上的给药方案、抑瘤效果和中位生存期
注：mIgG2a-FcS、RMP1-14-mIgG2a-FcS和SL2-22-mIgG2a-FcS给药摩尔浓度相同，约为68μmol/kg。
实施例11.TGFβ1抗体SL2-22在小鼠CT26模型中抑制肿瘤生长
为检测SL2-22体内肿瘤抑制活性，将BALB/c小鼠(雌性，6周龄，购买于北京维通利华实验动物技术有限公司)适应性饲养1周，实验当天编号称重。收集处于对数生长期的小鼠结肠癌细胞CT26(国家生物医学实验细胞资源库，1101MOU-PUMC000275)，用PBS重悬，以3×106个/mL浓度，0.1mL/只体积接 种于BALB/c小鼠的右侧肋部皮下。接种后第8-9d，当平均肿瘤体积达到约60mm3时，挑选个体肿瘤体积适中的小鼠，按照表16所示进行随机分组。分组当天开始给药，按照相同摩尔剂量，给药频率为一周两次，共给药6次，给药方式为腹腔注射。每周测量2次小鼠体重以及肿瘤体积，肿瘤体积计算公式为TV＝L长×L短 2/2。各组肿瘤体积用平均值±标准差表示，并计算肿瘤抑制率(％TGI)，计算公式为％TGI＝[1-(T-T0)/(C-C0)]×100％。
结果如图4A、4B、表16所示，SL2-22-mIgG2a-FcS与抗PD-1抗体RMP1-14-mIgG2a-FcS联用能够抑制小鼠CT26肿瘤生长，并延长动物生存期。在治疗过程中，小鼠耐受良好，没有出现明显的体重下降(图4C)。
表16.SL2-22在CT26模型上的给药方案、抑瘤效果和中位生存期
注：mIgG2a-FcS、RMP1-14-mIgG2a-FcS和SL2-22-mIgG2a-FcS给药摩尔浓度相同，约为68μmol/kg
实施例12.TGFβ1单克隆抗体SL2-22在小鼠特发性纤维化模型中抑制纤维化进程
为检测TGFβ1抗体SL2-22体内抑制纤维化进程，使用C57BL/6J小鼠，雄性，8周龄，购买于北京维通利华实验动物技术有限公司，适应性饲养1周，实验当天编号称重。造模当天编号称重，按照体重分布随机分组，每组15只，具体分组见下表17。实验第1天按照体重，腹腔注射给予抗体药物或溶媒对照，2小时后使用平头针经气管滴注博莱霉素，以2U/kg的剂量，每只动物缓慢滴注约50μL。然后按照上述组别，每周给药2次，共给药3周。实验第22天分别采集支气管肺泡灌洗液和肺组织，分析免疫细胞浸润程度、肺组织羟脯氨酸水平，以及Masson染色分析。
表17.实验分组及给药情况
SL2-22-mIgG2a-Fc给药组显著改善动物整体体重和存活率(图5A和5B)，降低肺组织羟脯氨酸水平，相比造模组降低40.44％(图5C)；同时显著降低博莱霉素造模后的肺内胶原沉积和纤维化水平，相比造模组降低46.51％(图5D和5E)。
实施例13.抗人和小鼠GARP-TGFβ1的单域抗体(VHH)的筛选和制备
1.骆驼免疫和文库构建
用人GARP/TGFβ1蛋白(由SEQ ID NO：5和6所示的蛋白共表达、纯化获得的蛋白复合物)作为抗原免疫一头双峰驼。将弗氏完全佐剂与抗原以1：1体积混合后，每两周对骆驼进行皮下多点免疫。第一次免疫剂量为200μg蛋白，随后四次免疫剂量为100μg蛋白/次，共五次免疫，然后用人GARP/TGFβ1蛋白测定效价。效价合格时，采集骆驼外周血，分离淋巴细胞，Trizol裂解细胞，抽提RNA，逆转录得到cDNA，构建噬菌体文库。
2.单域抗体(VHH)的筛选
用20μg的生物素化的人GARP/TGFβ1蛋白结合100μL DynabeadsTM M-280链霉亲和素，37℃放置1h后用2％脱脂奶室温封闭1h，加入前述噬菌体文库，在室温下作用1h。用PBST(含有0.05％Tween-20的PBS)洗9遍，去除不结合的噬菌体。用1mg/mL的胰蛋白酶将与人GARP/TGFβ1蛋白特异性结合的噬菌体洗脱，并感染处于对数期生长的大肠杆菌TG1，产生并纯化获得第一轮阳性噬菌体。基于第一轮筛到的阳性噬菌体，在第二轮筛选中获得与小鼠GARP-TGFbβ1复合物蛋白也具有高亲和力的单域抗体(VHH)。
从筛选富集的阳性克隆中挑取96个单克隆菌落包装成噬菌体单链抗体，用于噬菌体ELISA测试。ELISA板上分别包被2μg/mL的人GARP-TGFβ1复合物蛋白，加入封闭液稀释的噬菌体上清，用抗M13HRP标记的抗体检测。对ELISA结合测试结果中OD450值/背景值>5的克隆测序，获得序列。以下示出其中的C19-VHH序列。
>C19-VHH
SEQ ID NO:84中，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线分别为CDR1、CDR2和CDR3序列。本公开提供的抗GARP-TGFβ1单域抗体的编号规则均为Kabat。
表18.C19 VHH的CDR序列
3.完整抗体的制备
将C19-VHH序列与如下人IgG4-Fc(CH2-CH3，含有S228P)片段融合，获得C19。
>hIgG4-Fc(S228P)
>C19
将C19序列克隆进哺乳动物表达载体pTT5，转染HEK293E或ExpiCHO(ThermoFisher，A29127)细胞，转染后5天(HEK293E细胞，37℃)或10-12天(ExpiCHO细胞，32℃)于4000rpm离心20min收获细胞培养上清，并用0.45μM滤器过滤。接着用MabSelectSure LX柱(GE Healthcare)进行第一步亲和纯化。将培养液上清流过PBS平衡的MabSelectSure LX柱，经PBS洗涤后，用pH 3.0的0.1M Glycine酸性洗脱液洗脱目的蛋白后用1M Tris-HCl(pH 8.0)中和，最后用HiTrap Q HP离子柱精细纯化。经检测，获得目的抗体。
通过SPR和细胞功能实验发现C19有较好的抑制功能，并且和人和小鼠GARP-TGFβ1复合物都有很好的结合。
实施例14.抗GARP-TGFβ1单域抗体的人源化改造
通过对选定的特异性TGFβ1单域抗体C19进行三维结构同源建模，结合与V-base人种系序列数据库和IMGT人类抗体重链可变区种系基因数据库进行比对的结果，挑选与C19序列同源性高的重链可变区种系基因IGHV3-23作为FR1、FR2、FR3的模板，用IGJH4作为FR4的模板，将骆驼来源单域抗体的CDR移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。为保留人源化后单域抗体原有活性，在框架区进行一系列回复突变。人源化的VHH框架区包含选自以下的至少一种氨基酸突变：23T、29Y、30C、37Y、44E、45R、47F、71Q、74A、75R、78G、81E、93K和94T。上述氨基酸位置根据Kabat编码。
获得的人源化序列如下，CDR区用下划线表示，编码规则为Kabat编码：
>C19-hu3
>C19-hu7
>C19-hu8
使用实施例2中的方法构建人源化单域抗体VHH与hIgG4的Fc区融合的全抗体序列。获得的人源化完整抗体序列如下：
>C19-3
>C19-7
>C19-8

实施例15.抗GARP-TGFβ1单域抗体与GARP-TGFβ1复合物的亲和力测定
本实施例的阴性对照为HBS-EP(10mM HEPES，150mM NaCl，3mM EDTA，0.005％P20，pH7.4)。阳性对照为Abbv-151(Abbvie，US10793627B2)。其序列如下：
>Abbv-151抗体重链：
>Abbv-151抗体轻链：
通过Biacore T200(GE Healthcare)仪器测定C19及其人源化抗体、阳性对照抗体Abbv-151与人GARP-TGFβ1复合物(由SEQ ID NO：5和6所示序列的蛋白复合形成)或小鼠GARP-TGFβ1复合物(由SEQ ID NO：8和9所示序列的蛋白复合形成)的亲和力。使用芯片Series S sensor chip Protein A(GE Healthcare，29127556)将待检测抗体捕获至芯片表面，然后于芯片表面流过不同浓度的人或小鼠GARP-TGFβ1复合物，实时检测反应信号从而获得结合解离曲线，通过拟合获得结合力常数。实验使用溶液为HBS-EP溶液。每个实验循环结束时，用pH 1.5Glycine(GE Healthcare，BR-1003-54)溶液将芯片再生。抗体的亲和力结果如表19所示。结果显示，本公开筛选获得的抗体C19、C19-3、C19-7和C19-8与人GARP-TGFβ1复合物的亲和力与阳性对照Abbv-151相当，不同的是它们与小鼠GARP-TGFβ1复合物也有一定结合。
表19.C19和C19人源化抗体与人或小鼠GARP/TGFβ1的亲和力测定

(注：“/”表示没有结合)
实施例16.抗GARP/TGFβ1单域抗体体外抑制GARP/TGFβ1功能
将2 x 106个LN229细胞(Procell,CL-0578)传至T75培养瓶(Nunc)中，24h后利用293fectinTM(Invitrogen)转染试剂单转染人或小鼠的原TGFβ1、或共转染人和小鼠的原TGFβ1和GARP质粒。24h后，将细胞按每孔1×104个传至白色不透明96孔细胞培养板(PerkinElmer)中。24h后，吸出培养基，加入重悬于DMEM+0.5％BSA中的HepG2CAGA12-luc荧光素酶报道细胞，同时加入倍比稀释的C19、C19人源化抗体以及对照抗体Abbv-151。混合培养20h后，加入ONE-GloTM萤光素酶试剂，孵育5min后，在多功能酶标仪(SpectraMax M5)上读取化学发光值。溶媒处理组的荧光素酶活性值标准化到1，相对活性＝抗体处理组荧光素酶÷溶媒对照组。使用Prism 9通过非线性拟合、三参数对数抑制剂对反应模型作图。
结果如图6A和图6B显示，C19、C19-3、C19-7和C19-8在抑制人GARP-TGFβ1复合物活性上与阳性对照Abbv-151相当，不同的是它们也抑制小鼠GARP-TGFβ1复合物。
实施例17.抗GARP-TGFβ1单域抗体C19-8与表达GARP-TGFβ1的细胞结合实验
将HEK293E细胞配制成1×106个/mL，取5mL放入50mL培养管中，用293fectinTM试剂转染表20中质粒。48h后，将细胞用1×PBS洗2次，按每孔1×105个放入96孔圆底细胞培养板(Nunc)中。加入100μL 1×PBS配制的10nM抗体或对照抗体，室温染色1h，1×PBS洗3次后，加入FITC标记的抗人Fc抗体100μL，室温染色30min。染色结束，1×PBS洗3次。加入7-AAD孵育5min，1×PBS洗3次后，流式细胞仪检测。通过Flowjo分析活细胞中FITC阳性的细胞数，计算FITC阳性的细胞数占活细胞总数的百分比，并作图。
表20.质粒结构

结果如图7所示，C19-8和Abbv-151均与人GARP-TGFβ1复合物(对应图7中TGFβ1+GARP)结合，不同的是C19-8与小鼠GARP-TGFβ1复合物(对应图7中mTGFβ1+mGARP)也有一定结合。另外，C19-8和Abbv-151均不与TGFβ2复合物(TGFβ2+GARP)和TGFβ3复合物(TGFβ3+GARP)结合。
实施例18.抗GARP/TGFβ1单域抗体C19-8与Ab6和Abbv-151的表位竞争研究
本实施例的阴性对照为HBS-EP，阳性对照为Ab6(Scholar rock,WO2020014460Al，全文引入本公开)和Abbv-151。Ab6与GARP-TGFβ1复合物、LRRC33-TGFβ1复合物、LTBP1-TGFβ1复合物、LTBP3-TGFβ1复合物都结合，结合表位完全在TGFβ1上(Martin等，2020)；而Abbv-151的结合表位包括GARP-TGFβ1复合物中的GARP和TGFβ1，不与单独的GARP或TGFβ1结合(Streel等，2020)。Ab6的序列如下：
>Ab6抗体重链：

>Ab6抗体轻链：
通过Biacore T200(GE Healthcare)仪器测定C19-8及阳性抗体Abbv-151、Ab6与人GARP/TGFβ1的表位竞争。使用芯片Series S sensor chip CM5(GE Healthacare，Br100530)偶联抗组氨酸标签抗体，所用试剂盒为His捕获试剂盒(GE Healthcare，28-9950-56)。将抗原蛋白人GARP-TGFβ1复合物捕获至芯片表面，然后于芯片表面依次流过相同浓度(500nM)的抗体，按表21组合进行，1st抗体为第一个流过的抗体，2nd抗体为第二个流过的抗体。
表21.抗体组合
利用Biacore仪器实时检测反应信号从而获得表位竞争曲线。实验使用溶液为 HBS-EP溶液。每个实验循环结束时，用pH 1.5Glycine(GE Healthcare，BR-1003-54)溶液将芯片再生。抗体的表位竞争测定结果如表22所示。
结果显示，C19-8与Ab6完全竞争，与Abbv-151完全不竞争。综合前述的体外抑制和细胞结合实验表明，C19-8是一个与Abbv-151一样只抑制GARP-TGFβ1复合物，但结合表位不同的抗体。
表22.C19-8与阳性抗体Abbv-151、Ab6表位竞争的测定
(注：“√”表示完全竞争；“×”表示完全不竞争)
实施例19.抗GARP-TGFβ1单域抗体C19-8在小鼠EMT-6模型中抑瘤生长
为检测C19-8体内肿瘤抑制活性，将BALB/c小鼠(雌性，6周龄，购买于北京维通利华实验动物技术有限公司)适应性饲养1周，实验当天编号称重。收集处于对数生长期的小鼠乳腺癌细胞EMT-6(Procell，CL-0573)，用PBS重悬，以5×106个/mL浓度，0.1mL/只体积接种于BALB/c小鼠的右侧肋部皮下。接种后第3d，当平均肿瘤体积达到约60mm3时，挑选个体肿瘤体积适中的小鼠，按照表23所示进行随机分组。
其中，阳性药M7824为PD-L1/TGFβ-trap(Merck KGaA，WO2018029367A1(通过引用全文并入本公开)中的序列3(重链)和序列1(轻链))；mIgG2a-FcS为去除ADCC效应的带有L234A/L235E/G237A/D327Q/A330S/P331S的mIgG2a Fc，这里作为同种型对照；RMP1-14-mIgG2a-FcS为anti-PD1抗体RMP1-14的可变区(WO2018223182A1(通过引用全文并入本公开)中除去信号肽的序列285(重链可变区)和286(轻链可变区))与去除ADCC效应的带有L234A/L235E/G237A/D327Q/A330S/P331S的mIgG2a重链恒定区/鼠kappa轻链恒定区融合在一起的抗体；C19-8-mIgG2a-FcS为C19-8的可变区与去除ADCC效应的带有L234A/L235E/G237A/D327Q/A330S/P331S的mIgG2a重链Fc融合在一起的抗体，序列如下：
>C19-8-mIgG2a-FcS：

分组当天开始给药，按照相同摩尔剂量，给药频率为一周两次，共给药7次，给药方式为腹腔注射。每周测量2次小鼠体重以及肿瘤体积，肿瘤体积计算公式为TV＝L长×L短 2/2。各组肿瘤体积用平均值±标准差表示，使用Two-way ANOVA进行统计分析，并计算肿瘤抑制率(％TGI)，计算公式为％TGI＝[1-(T-T0)/(C-C0)]×100％。
结果如图8A、8B示，C19-8-mIgG2a-FcS与抗PD-1抗体RMP1-14-mIgG2a-FcS联用相比于RMP1-14-mIgG2a-FcS单用组显著抑制小鼠EMT-6肿瘤生长(p＝0.0017,Two-way ANOVA)，并延长动物生存期(p＝0.0157,Log-rank(Mantel-Cox)test)。在治疗过程中，小鼠耐受良好，没有出现明显的体重下降(图8C)。
表23.C19-8在EMT-6模型上的给药方案、抑瘤效果和中位生存期
注：mIgG2a-FcS、RMP1-14-mIgG2a-FcS、C19-8-mIgG2a-FcS和M7824给药摩尔浓度相同，约为68μmol/kg。
实施例9.抗GARP-TGFβ1单域抗体C19-8在小鼠CT26模型中抑瘤生长
为检测C19-8体内肿瘤抑制活性，将BALB/c小鼠(雌性，6周龄，北京维通利华实验动物技术有限公司)适应性饲养1周，实验当天编号称重。收集处于对数生长期的小鼠结肠癌细胞CT26(国家生物医学实验细胞资源库，1101MOU-PUMC000275)，用PBS重悬，以3×106个/mL浓度，0.1mL/只体积接种于BALB/c小鼠的右侧肋部皮下。接种后第8-9d，当平均肿瘤体积达到约60mm3时，挑选个体肿瘤体积适中的小鼠，按照表24所示进行随机分组。分组当天开始给药，按照相同摩尔剂量，给药频率为一周两次，共给药6次，给药方式为腹腔注射。每周测量2次小鼠体重以及肿瘤体积，肿瘤体积计算公式为TV＝L长×L短 2/2。各组肿瘤体积用平均值±标准差表示，并计算肿瘤抑制率(％TGI)，计算公式为％TGI＝[1-(T-T0)/(C-C0)]×100％。
结果如图9A、9B所示，C19-8-mIgG2a-FcS与抗PD-1抗体RMP1-14-mIgG2a-FcS联用能够抑制小鼠CT26肿瘤生长，并延长动物生存期。在治疗过程中，小鼠耐受良好，没有出现明显的体重下降(图9C)。
表24.C19-8在CT26模型上的给药方案、抑瘤效果和中位生存期
注：mIgG2a-FcS、RMP1-14-mIgG2a-FcS和C19-8-mIgG2a-FcS给药摩尔浓度相同，约为68μmol/kg
虽然以上描述了本公开的具体实施方案，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本公开的原理和实质的前提下，可以对这些实施方案做出多种变更或修改。因此，本公开的保护范围由所附权利要求书限定。

Claims (25)

  1. TGFβ1结合分子，其包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述VH包含SEQ ID NO:11所示氨基酸序列中的HCDR1、HCDR2和HCDR3，和所述VL包含SEQ ID NO:12、21-35中任一项所示氨基酸序列中的LCDR1、LCDR2和LCDR3；所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
    优选地，所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，和，所述VL包含分别如SEQ ID NO:38、64和18所示的LCDR1、LCDR2和LCDR3；
    优选地，所述TGFβ1结合分子包含：
    如SEQ ID NO:13所示的HCDR1，
    如SEQ ID NO:14所示的HCDR2，
    如SEQ ID NO:15所示的HCDR3，
    如SEQ ID NO:16、36、39、41-42、44、46、49、52、54和56-59中任一项所示的LCDR1，
    如SEQ ID NO:17、37、40、43、45、47-48、50-51、53和55中任一项所示的LCDR2，和
    如SEQ ID NO:18所示的LCDR3。
  2. 根据权利要求1所述的TGFβ1结合分子，其中：
    a)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:59、40和18所示的LCDR1、LCDR2和LCDR3；
    b)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:36-37和18所示的LCDR1、LCDR2和LCDR3；
    c)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:39-40和18所示的LCDR1、LCDR2和LCDR3；
    d)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:41、40和18所示的LCDR1、LCDR2和LCDR3；
    e)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:42-43和18所示的LCDR1、LCDR2和LCDR3；
    f)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:44-45和18所示的LCDR1、LCDR2和LCDR3；
    g)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:46-47和18所示的LCDR1、LCDR2和LCDR3；
    h)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3， 所述VL包含分别如SEQ ID NO:46、48和18所示的LCDR1、LCDR2和LCDR3；
    j)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:49-50和18所示的LCDR1、LCDR2和LCDR3；
    k)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:46、51和18所示的LCDR1、LCDR2和LCDR3；
    l)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:52-53和18所示的LCDR1、LCDR2和LCDR3；
    m)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:54-55和18所示的LCDR1、LCDR2和LCDR3；
    n)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:56、53和18所示的LCDR1、LCDR2和LCDR3；
    o)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:57、45和18所示的LCDR1、LCDR2和LCDR3；
    p)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:58、50和18所示的LCDR1、LCDR2和LCDR3；或，
    q)所述VH包含分别如SEQ ID NO:13-15所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO:16-18所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1-2中任一项所述的TGFβ1结合分子，其中：所述VH包含如SEQ ID NO:11所示氨基酸序列，或与之具有至少90％序列同一性的氨基酸序列；
    和，
    所述VL包含如SEQ ID NO:12和21-35中任一项所示氨基酸序列，或与之具有至少90％序列同一性的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的TGFβ1结合分子，其进一步包含免疫球蛋白Fc区；优选地，所述免疫球蛋白Fc区来源于IgG1、IgG2、IgG3、IgG4或前述任一种的变体；更优选地，所述免疫球蛋白Fc区来源于人IgG4或其变体，所述变体包含突变228P。
  5. 根据权利要求1-4中任一项所述的TGFβ1结合分子，其包含重链和轻链，其中：
    所述重链包含如SEQ ID NO:66所示，或与之具有至少90％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:65、67-81中任一项所示，或与之具有至少90％序列同一性的氨基酸序列；或，
    所述重链包含如SEQ ID NO:82所示，或与之具有至少90％序列同一性的氨基 酸序列；和所述轻链包含如SEQ ID NO:83所示，或与之具有至少90％序列同一性的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的TGFβ1结合分子，其是抗TGFβ1抗体或其抗原结合片段；
    优选地，所述抗TGFβ1抗体为鼠源抗体、嵌合抗体、人源化抗体或人抗体，优选为人源化抗体，更优选为经亲和力成熟改造的人源化抗体；
    优选地，所述抗原结合片段选自scFv、dsFv、(dsFv)2、dsFv-dsFv’、Fv片段、Fab、Fab’或F(ab’)2。
  7. 根据权利要求1-6任一项所述的TGFβ1结合分子，其具有至少一种如下所示的特性：
    (1)结合TGFβ1前体蛋白和/或结合TGFβ1复合物，
    (2)不结合TGFβ2或TGFβ2复合物，
    (3)不结合TGFβ3或TGFβ3复合物，
    (4)抑制TGFβ1活性，
    (5)抑制调节性T(Treg)细胞的免疫抑制活性，
    (6)抑制肿瘤生长，
    (7)抑制纤维化；
    优选地，所述抑制TGFβ1活性包括：抑制TGFβ1激活，抑制成熟TGFβ1从TGFβ1复合物中释放，和/或抑制TGFβ1信号转导；
    优选地，所述TGFβ1复合物中的TGFβ1以TGFβ1前体蛋白存在，所述TGFβ1前体蛋白优选包含成熟TGFβ1结构域和潜能相关肽(LAP)；
    优选地，所述TGFβ1复合物选自LTBP1-TGFβ1复合物、LTBP3-TGFβ1复合物、LRRC33-TGFβ1复合物和/或GARP-TGFβ1复合物。
  8. GARP-TGFβ1结合分子，其包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含SEQ ID NO:84和90-92中任一所示氨基酸序列中的CDR1、CDR2和CDR3，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
    优选地，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:85、86和87所示的氨基酸序列。
  9. 根据权利要求8所述的GARP-TGFβ1结合分子，其中，所述免疫球蛋白单一可变结构域是为人源化、亲和力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的；
    优选地，人源化改造过程使用的人种系模板选自IGHV3-23和/或IGJH4。
  10. 根据权利要求8-9中任一项所述的GARP-TGFβ1结合分子，其中，所述免疫球蛋白单一可变结构域包含如SEQ ID NO:84和90-92中任一项所示，或与之具有至少90％序列同一性的氨基酸序列；优选地，所述免疫球蛋白单一可变结构域是VHH。
  11. 根据权利要求8-10中任一项所述的GARP-TGFβ1结合分子，其进一步包含免疫球蛋白Fc区；优选地，所述Fc区来源于IgG1、IgG2、IgG3、IgG4或前述任一种的变体；更优选来源于人IgG4或其变体，所述变体包含突变228P。
  12. 根据权利要求11所述的GARP-TGFβ1结合分子，其包含如SEQ ID NO:89、93-95和106中任一项所示的氨基酸序列，或与之具有至少90％序列同一性的氨基酸序列。
  13. 根据权利要求8-12中任一项所述的GARP-TGFβ1结合分子，其是与GARP-TGFβ1复合物结合的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段选自线性抗体、单链抗体、纳米抗体、肽抗体、结构域抗体、多特异性抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段为骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
  14. GARP-TGFβ1结合分子，其包含与GARP-TGFβ1复合物结合的免疫球蛋白单一可变结构域，所述GARP-TGFβ1复合物包含：
    TGFβ1前体蛋白和糖蛋白A重复主导序列(GARP)；所述TGFβ1前体蛋白包含：成熟TGFβ1结构域以及潜能相关肽(LAP)；
    优选地，所述GARP包含如SEQ ID NO:100或103所示的氨基酸序列或与之具有至少90％序列同一性的氨基酸序列，所述成熟TGFβ1结构域包含如SEQ ID NO:101或104所示的氨基酸序列或与之具有至少90％序列同一性的氨基酸序列，和/或，所述LAP包含如SEQ ID NO:102或105所示的氨基酸序列或与之具有至少90％序列同一性的氨基酸序列。
  15. 根据权利要求8-14任一项所述的GARP-TGFβ1结合分子，其具有至少一种如下所示特性：
    (1)结合GARP-TGFβ1复合物；
    (2)不结合TGFβ2蛋白或TGFβ2复合物；
    (3)不结合TGFβ3蛋白或TGFβ3复合物；
    (4)不结合游离的成熟TGFβ1；
    (5)抑制TGFβ1活性；
    (6)抑制调节性T(Treg)细胞的免疫抑制活性；
    (7)抑制肿瘤生长；
    优选地，所述抑制TGFβ1活性包括：抑制TGFβ1激活，抑制成熟TGFβ1从GARP-TGFβ1复合物中释放，和/或抑制TGFβ1信号转导；
    优选地，所述GARP-TGFβ1复合物包含(i)GARP，和(ii)TGFβ1前体蛋白，所述TGFβ1前体蛋白优选包含成熟TGFβ1结构域和潜能相关肽(LAP)。
  16. 多核苷酸，其编码权利要求1-7任一项所述的TGFβ1结合分子，或权利要求8-15任一项所述的GARP-TGFβ1结合分子。
  17. 载体，其含有权利要求16所述的多核苷酸。
  18. 宿主细胞，其包含权利要求16所述的多核苷酸，或权利要求17所述的载体；
    优选地，所述宿主细胞为细菌、酵母或哺乳动物细胞；更优选地，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞或人胚肾293细胞。
  19. 药物组合物，其含有权利要求1-7任一项所述的TGFβ1结合分子，权利要求8-15任一项所述的GARP-TGFβ1结合分子，权利要求16所述的多核苷酸，或权利要求17所述的载体；
    优选地，所述药物组合物进一步包含一种或多种可药用的赋形剂、稀释剂或辅料；
    优选地，所述药物组合物进一步包含免疫检查点抑制剂；
    优选地，所述药物组合物进一步包含抗PD-1抗体或其抗原结合片段。
  20. 一种制备权利要求1-7任一项所述的TGFβ1结合分子的方法，其中，所述方法包括：
    在宿主细胞中表达权利要求1-7任一项所述的TGFβ1结合分子，以及，
    从所述宿主细胞中分离所述TGFβ1结合分子；
    可选地，所述方法进一步包含纯化所述TGFβ1结合分子的步骤。
  21. 一种制备权利要求8-15任一项所述的GARP-TGFβ1结合分子的方法，其中，所述方法包括：
    在宿主细胞中表达权利要求8-15任一项所述的GARP-TGFβ1结合分子，以及，
    从所述宿主细胞中分离所述GARP-TGFβ1结合分子；
    可选地，所述方法进一步包含纯化所述GARP-TGFβ1结合分子的步骤。
  22. 权利要求1-7任一项所述的TGFβ1结合分子、权利要求8-15任一项所述的GARP-TGFβ1结合分子、权利要求16所述的多核苷酸、权利要求17所述的载体，或权利要求19所述的药物组合物在制备与TGFβ信号通路相关的疾病或症状的药物中的用途；
    优选地，所述与TGFβ信号通路相关的疾病选自癌症或纤维化；
    优选地，所述癌症选自肺癌、肠癌、肾癌、膀胱癌、肝癌、胃癌、乳腺癌、结肠癌、宫颈癌、前列腺癌和头颈癌。
  23. 一种预防或治疗与TGFβ信号通路相关的疾病或病症的方法，其包括向受试者施用预防或治疗有效量的权利要求1-7任一项所述的TGFβ1结合分子，权利要求8-15任一项所述的GARP-TGFβ1结合分子，权利要求16所述的多核苷酸，权利要求17所述的载体，或权利要求19所述的药物组合物；
    或者，
    所述方法包括向受试者施用预防或治疗有效量的权利要求1-7任一项所述的TGFβ1结合分子，或权利要求8-15任一项所述的GARP-TGFβ1结合分子；以及，预防或治疗有效量的免疫检查点抑制剂；
    优选地，所述免疫检查点抑制剂为抗PD-1抗体或其抗原结合片段；
    优选地，所述与TGFβ信号通路相关的疾病或病症选自癌症或纤维化；优选地，所述癌症选自肺癌、肠癌、肾癌、膀胱癌、肝癌、胃癌、乳腺癌、结肠癌、宫颈癌、前列腺癌和头颈癌。
  24. 一种在体外或受试者体内抑制TGFβ1活性的方法，其包括：
    在体外或体内施用权利要求1-7任一项所述的TGFβ1结合分子，权利要求8-15任一项所述的GARP-TGFβ1结合分子，权利要求16所述的多核苷酸，权利要求17所述的载体，或权利要求19所述的药物组合物。
  25. TGFβ1结合分子或GARP-TGFβ1结合分子联合免疫检查点抑制剂在制备治疗癌症的药物中的用途；其中，所述TGFβ1结合分子如权利要求1-7任一项所定义，所述GARP-TGFβ1结合分子如权利要求8-15任一项所定义
    优选地，所述免疫检查点抑制剂为抗PD-1抗体或其抗原结合片段；
    优选地，所述癌症选自肺癌、肠癌、肾癌、膀胱癌、肝癌、胃癌、乳腺癌、结肠癌、宫颈癌、前列腺癌和头颈癌。
PCT/CN2024/071321 2023-01-09 2024-01-09 TGFβ1结合分子、GARP-TGFβ1结合分子及其医药用途 WO2024149237A1 (zh)

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