WO2021093760A1 - 含有TGF-β受体的融合蛋白及其医药用途 - Google Patents

含有TGF-β受体的融合蛋白及其医药用途 Download PDF

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WO2021093760A1
WO2021093760A1 PCT/CN2020/128045 CN2020128045W WO2021093760A1 WO 2021093760 A1 WO2021093760 A1 WO 2021093760A1 CN 2020128045 W CN2020128045 W CN 2020128045W WO 2021093760 A1 WO2021093760 A1 WO 2021093760A1
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seq
sequence
antibody
variable region
cancer
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French (fr)
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孙乐
叶鑫
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure belongs to the field of biotechnology. More specifically, the present disclosure relates to a fusion protein of an anti-PD-1 antibody and a TGF- ⁇ receptor and its application.
  • PD-1 Programmed death receptor 1
  • PD-1 belongs to the CD28 superfamily and is expressed on activated T cells, B cells and myeloid cells.
  • PD-1 is a type I membrane protein of approximately 55 kDa, including an extracellular IgV domain, a transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine inhibitory motif.
  • PD-1 has two ligands, programmed death ligand-1 (PD-L1) and PD-L2. After PD-1 binds to the ligand, it can negatively regulate the activation of T cells, allowing tumor cells to obtain immune escape.
  • PD-L1 is highly expressed in a variety of cancers, and its expression level is also related to the prognosis of patients. Therefore, inhibiting the interaction between PD-1 and PD-L1 can block the negative regulatory pathway and enhance the response of T cells in the immune response. It is a promising treatment in the field of tumor immunotherapy.
  • multiple PD-1 and PD-L1 inhibitors such as Nivolumab of BMS, Pembrolizumab of Merck, Atezolizumab of Roche
  • PD-1 and PD-L1 inhibitors also have the defect of low efficiency, which may be caused by the existence of other immune checkpoints and the complexity of tumor microenvironment.
  • Transforming growth factor- ⁇ (transforming growth factor- ⁇ , TGF- ⁇ ) belongs to the TGF- ⁇ superfamily, and the TGF- ⁇ signaling pathway plays an important role in regulating cell growth and differentiation. After the expression of TGF- ⁇ is released, it will bind to the receptor TGF- ⁇ RII, activate TGF- ⁇ RII, and then form a complex with TGF- ⁇ RI. Subsequently, the complex will phosphorylate downstream Smad2 and Smad3. The phosphorylated Smad2 and Smad3 combine with Smad4 to form a phosphorylated Smad2/3/4 complex that enters the nucleus and regulates downstream gene expression.
  • TGF- ⁇ signaling can promote the epithelial-mesenchymal transition of tumors and initiate tumor metastasis.
  • TGF- ⁇ targeted drugs although the positive effect of inhibiting the TGF- ⁇ signaling pathway on disease progression has been seen, the effect of TGF- ⁇ targeted drugs alone is still not ideal.
  • inhibiting the PD-1/PD-L1 pathway on the basis of TGF- ⁇ that neutralizes the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of suppressing the occurrence and development of tumors.
  • Antibody/TGF- ⁇ receptor fusion proteins have been disclosed, such as WO2006074451A2, WO2009152610A1, WO2011109789A2, WO2013164694A1, WO2014164427A1, WO2015077540A2, WO9309228A1, WO9409815A1, WO2015077540A2, WO2015118175A2 and WO2018205985, but they are not directed against anti-PD-1 antibodies and TGF- ⁇
  • the receptor's fusion protein drug was approved for marketing.
  • the present disclosure provides a fusion protein comprising a targeting portion and a TGF- ⁇ receptor portion, wherein the TGF- ⁇ receptor portion is the extracellular region of TGF- ⁇ RII, and the targeting portion is anti-PD- 1 Antibody or its antigen-binding fragment.
  • the aforementioned fusion protein wherein the extracellular region of TGF- ⁇ RII is an N-terminal truncated form of the extracellular region of TGF- ⁇ RII.
  • the aforementioned fusion protein wherein the N-terminal truncated form of the extracellular region of TGF- ⁇ RII is a deletion of 0 to 26 amino acids at the N-terminus of the extracellular region of TGF- ⁇ RII; preferably the N-terminal deletion 14-26 amino acids; more preferably 14-21 amino acids are deleted; most preferably 14, 19 or 21 amino acids are deleted.
  • the aforementioned fusion protein wherein the N-terminal truncated form of the extracellular region of TGF- ⁇ RII is a deletion of 1 to 26 consecutive amino acids at the N-terminus of the extracellular region of TGF- ⁇ RII; preferably The N-terminal deletion of 14-26 consecutive amino acids; more preferably the deletion of 14-21 consecutive amino acids; most preferably the deletion of 14, 19 or 21 consecutive amino acids.
  • the sequence of the extracellular region of TGF- ⁇ RII is shown in SEQ ID NO: 61.
  • the sequence of the N-terminal truncated form of the extracellular region of TGF- ⁇ RII is shown in SEQ ID NO: 62, 63 or 64.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region contains the sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and
  • the light chain variable region includes the LCDR1 shown in SEQ ID NO: 11, 32, 33, 34, 35, 36 or 37, and LCDR2 shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.
  • the heavy chain variable region contains the sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence such as The HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the light chain variable region comprising sequences such as SEQ ID NO: 34, SEQ ID NO: 12 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in 13.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as described below:
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 4, and the light chain variable region includes the same sequence as SEQ ID NO: :
  • the light chain variable region shown in the sequence 5 has LCDR1, LCDR2 and LCDR3 with the same sequence; or
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region includes the same sequence as SEQ ID NO: :
  • the light chain variable region shown in the 7 sequence has LCDR1, LCDR2 and LCDR3 with the same sequence.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof has an affinity of less than 4 ⁇ 10 -8 M and a KD value of less than 6 ⁇ 10 -9 M with human PD- 1 (or its epitope) binding.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof can effectively block the binding of PD-1 to a ligand (such as PD-L1).
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof binds to human PD-1 (or its epitope) with an affinity of less than 2 ⁇ 10 -9 M. ; And/or bind to PD-1 (or its epitope) of cynomolgus monkeys with an affinity of less than 3 ⁇ 10 -9 M KD.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof binds to human PD-1 (or its epitope) with an affinity of less than 1 ⁇ 10 -9 M. ; And/or bind to PD-1 (or its epitope) of cynomolgus monkeys with an affinity of less than 1 ⁇ 10 -9 M KD.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof can effectively activate T cells to secrete IFN- ⁇ .
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof can effectively inhibit the growth of transplanted tumors in transgenic PD-1 mice (for example, but not limited to colon cancer transplanted tumors, Such as MC38 cell transplantation tumor).
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof can effectively inhibit the growth of transplanted tumors in transgenic PD-1 mice (for example, but not limited to colon cancer transplanted tumors, Such as MC38 cell transplantation tumor), when the dosage is 1mpk, its tumor inhibition rate is less than 46%.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof can effectively inhibit the growth of transplanted tumors in transgenic PD-1 mice (for example, but not limited to colon cancer transplanted tumors, Such as MC38 cell transplantation tumor), when the dosage is 3mpk, its tumor inhibition rate is less than 60%, or less than 76%.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody or a humanized antibody.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof.
  • the body is a back mutation of up to 6 amino acids in the light chain framework region and/or heavy chain framework region of a human antibody;
  • the back mutation is selected from the following:
  • the amino acid back mutation in the heavy chain framework region is selected from one or more of 27Y, 48I, 67T, 69L, 82F and 93T; or
  • the amino acid back mutation in the heavy chain framework region is selected from: 1K and/or 94S; the above-mentioned back mutation position complies with the Kabat numbering rule.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region selected from:
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the FR region contains selected from 27Y, 48I, 67T, 69L, One or more amino acid back mutations in 82F and 93T, and
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 34, SEQ ID NO: 12, and SEQ ID NO: 13, and includes 2G amino acid back mutations in the FR region.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region selected from:
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and contains 1K and/or 94S amino acid back mutations in the FR region, and
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 respectively, and contains one selected from 42G, 44V and 71Y in the FR region Or more amino acid back mutations; the positions of the above amino acid back mutations comply with the Kabat numbering rules.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region selected from:
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the FR region contains selected from 27Y, 48I, 67T, 69L, One or more amino acid back mutations in 82F and 93T, and
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and includes 2G amino acid back mutations in the FR region.
  • the aforementioned fusion protein wherein the heavy chain variable region and light chain variable region of the anti-PD-1 antibody or antigen-binding fragment thereof are selected from any of the following (g) to (k) item:
  • the heavy chain variable region sequence is shown in SEQ ID NO: 4 or has at least 90% sequence identity and the light chain variable region sequence is shown in SEQ ID NO: 5 or has at least 90% sequence identity thereto;
  • the heavy chain variable region sequence is shown in SEQ ID NO: 6 or has at least 90% sequence identity and the light chain variable region sequence is shown in SEQ ID NO: 7 or has at least 90% sequence identity with it ;
  • the heavy chain variable region sequence is shown in SEQ ID NO: 20, 23, 24 or 25 or has at least 90% sequence identity with SEQ ID NO: 20, 23, 24 or 25, and the light chain variable region
  • the sequence is shown in SEQ ID NO: 21, 22, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49, or with SEQ ID NO: 21, 22, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 have at least 90% sequence identity;
  • the heavy chain variable region sequence is shown in SEQ ID NO: 26, 30 or 31 or has at least 90% sequence identity with SEQ ID NO: 26, 30 or 31 respectively, and the light chain variable region sequence is shown in SEQ ID NO: 27, 28, or 29 or have at least 90% sequence identity with SEQ ID NO: 27, 28, or 29;
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region sequence is shown in SEQ ID NO: 20 and the light chain variable region sequence is shown in SEQ ID NO: 40; or
  • the heavy chain variable region sequence is shown in SEQ ID NO: 30 and the light chain variable region sequence is shown in SEQ ID NO: 28; or
  • the heavy chain variable region sequence is shown in SEQ ID NO: 31 and the light chain variable region sequence is shown in SEQ ID NO: 28.
  • the above-mentioned having at least 90% includes having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity.
  • the sequence identity can be through conventional sequence Obtained by comparison software.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises the heavy chain variable region shown in SEQ ID NO: 20 or a variant thereof, and the variant is in SEQ ID NO: 20 has one or more back mutations selected from G27Y, M48I, V67T, I69L, L82F and A93T; and the anti-PD-1 antibody comprises the light chain variable region shown in SEQ ID NO: 21 or Its variants, wherein the variant has an I2G back mutation in SEQ ID NO:21.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises the heavy chain variable region shown in SEQ ID NO: 26 or a variant thereof, and the variant is in SEQ ID NO: 26 has R94S and/or E1K back mutations; and the anti-PD-1 antibody comprises the light chain variable region shown in SEQ ID NO: 27 or a variant thereof, wherein the variant is described in SEQ ID NO: 27 has one or more back mutations selected from K42G, P44V and F71Y.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises the heavy chain variable region shown in SEQ ID NO: 20 or a variant thereof, and the variant is in SEQ ID NO: 20 has one or more back mutations selected from G27Y, M48I, V67T, I69L, L82F and A93T; and the anti-PD-1 antibody comprises the light chain variable region shown in SEQ ID NO: 40 or The variants, wherein the variant has an I2G back mutation in SEQ ID NO: 40.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 20 and a heavy chain variable region as shown in SEQ ID NO: 40 Light chain variable region.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 30 and a heavy chain variable region as shown in SEQ ID NO: 28 Light chain variable region.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 31 and a heavy chain variable region as shown in SEQ ID NO: 28 Light chain variable region.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody or antigen-binding fragment thereof further comprises an antibody heavy chain constant region and a light chain constant region;
  • the heavy chain constant region is selected from human IgG1, IgG2 , IgG3 and IgG4 constant regions and conventional variants thereof, the light chain constant region is selected from human antibody ⁇ and ⁇ chain constant regions and conventional variants thereof;
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises The sequence of the heavy chain constant region shown in SEQ ID NO: 50 or 51 and/or the sequence of the light chain constant region shown in SEQ ID NO: 52.
  • the aforementioned fusion protein wherein the anti-PD-1 antibody comprises a heavy chain and a light chain selected from any one of the following (1)-(n):
  • the heavy chain sequence is shown in SEQ ID NO: 53 or 54 or has at least 85% sequence identity with SEQ ID NO: 53 or 54; and the light chain sequence is shown in SEQ ID NO: 55 or is the same as SEQ ID NO: 55 has at least 85% sequence identity;
  • the heavy chain sequence is shown in SEQ ID NO: 56 or 57 or has at least 85% sequence identity with SEQ ID NO: 56 or 57; and the light chain sequence is shown in SEQ ID NO: 58 or the same as SEQ ID NO: 58 has at least 85% sequence identity; and
  • the heavy chain sequence is shown in SEQ ID NO: 59 or 60 or has at least 85% sequence identity with SEQ ID NO: 59 or 60; and the light chain sequence is shown in SEQ ID NO: 58 or is shown in SEQ ID NO: 58 has at least 85% sequence identity;
  • sequence identity can be obtained by conventional sequence alignment software.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody comprises: a heavy chain of SEQ ID NO: 53 and a light chain of SEQ ID NO: 55.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody comprises: a heavy chain of SEQ ID NO: 56 and a light chain of SEQ ID NO: 58.
  • the aforementioned fusion protein, wherein the anti-PD-1 antibody comprises: a heavy chain of SEQ ID NO: 59 and a light chain of SEQ ID NO: 58.
  • the aforementioned fusion protein wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single-chain antibody (scFv), dimerized V region (diabody) and two Sulfur bond stabilized V region (dsFv).
  • the aforementioned fusion protein wherein the extracellular region of TGF- ⁇ RII can be fused to the carboxyl terminal (C-terminal) of the heavy chain of the anti-PD-1 antibody or antigen-binding fragment thereof via a linker ,
  • the aforementioned fusion protein wherein the linker can be a commonly used linker in the art, including but not limited to (ASTKGP)n, (ASTKGPSVFPLAP)n, (TVAAP)n, (TVAAPSVFIFPP)n or ( G 4 S) nG, where n is 1, 2, 3, 4, 5 or 6, preferably 4 or 5.
  • the aforementioned fusion protein comprises:
  • the first chain comprising the extracellular region of TGF- ⁇ RII fused to the heavy chain of the anti-PD-1 antibody
  • the second chain which contains the light chain of an anti-PD-1 antibody
  • the sequence of the first strand is as shown in SEQ ID NO: 69 or 71 or has at least 85% identity with the amino acid sequence of SEQ ID NO: 69 or 71
  • the sequence of the second strand is as SEQ ID NO: 55 or at least 85% identity with the amino acid sequence shown in SEQ ID NO: 55; or
  • the sequence of the first strand is shown in SEQ ID NO: 70, 72, 73 or 74 or has at least 85% identity with the amino acid sequence shown in SEQ ID NO: 70, 72, 73 or 74,
  • the second strand sequence is shown in SEQ ID NO: 58 or has at least 85% identity with the amino acid sequence shown in SEQ ID NO: 58;
  • sequence identity can be obtained by conventional sequence alignment software.
  • the aforementioned fusion protein comprises two first and second chains with the same sequence, wherein:
  • the first chain comprising the extracellular region of TGF- ⁇ RII fused to the heavy chain of the anti-PD-1 antibody
  • the second chain which contains the light chain of an anti-PD-1 antibody
  • the aforementioned fusion protein comprises two first and second chains with the same sequence, wherein:
  • sequence of the first chain is shown in SEQ ID NO: 71
  • sequence of the second chain is shown in SEQ ID NO: 55; or
  • the sequence of the first chain is shown in SEQ ID NO: 72, and the sequence of the second chain is shown in SEQ ID NO: 58.
  • the aforementioned fusion protein has a KD value of affinity with human PD-1 (or its epitope) less than 1 ⁇ 10 -8 M, less than 5 ⁇ 10 -9 M, and less than 3 ⁇ 10 -9 M or less than 1 ⁇ 10 -9 M; wherein the KD value can be obtained by Biacore detection.
  • the aforementioned fusion protein has a KD value of affinity with human TGF- ⁇ 1 that is less than 1 ⁇ 10 -11 M, less than 5 ⁇ 10 -11 M, less than 7 ⁇ 10 -12 M, or less than 5 ⁇ 10 -12 M; wherein the KD value can be obtained by Biacore detection.
  • the aforementioned fusion protein binds to PD-1 expressed on the cell surface; in some embodiments, its binding EC50 is less than 10 nM, less than 8 nM, less than 5 nM or less than 2 nM, wherein the binding EC50 value can pass the test example 8 Obtained by method detection.
  • the aforementioned fusion protein can block the binding of PD-1 to its ligand (such as PD-L1); in some embodiments, its blocking IC50 value is less than 15nM, less than 13nM, less than 10nM or less than 5nM ;
  • the blocking IC50 value can be obtained by the method of Test Example 9.
  • the aforementioned fusion protein can inhibit the activation of Smad3 induced by TGF- ⁇ 1; in some embodiments, its inhibitory IC50 is less than 5 nM, less than 3 nM, less than 1 nM, less than 0.5 nM or less than 0.2 nM; wherein the blocking IC50 value It can be obtained by the method of Test Example 10.
  • the aforementioned fusion protein can stimulate T lymphocytes to secrete cytokines, especially the secretion of IFN- ⁇ ; in some embodiments, stimulate the secretion of IFN- ⁇ to increase at least 5-fold, at least 8-fold, at least 10 times. Times, at least 11 times or at least 12 times.
  • the aforementioned fusion protein can block the binding of PD-1 to its ligand; in some embodiments, the aforementioned fusion protein has an IC50 value that blocks the binding of PD-1 and PD-L1 to less than 5 nM and less than 2.5. nM or less than 2nM, or less than 1.7nM, or less than 1.4nM, wherein the IC50 value can be obtained through the protocol detection of Test Example 12.
  • the aforementioned fusion protein can block the binding of PD-1 to its ligand; in some embodiments, the aforementioned fusion protein has an IC50 value that blocks the binding of PD-1 and PD-L2 to less than 10 nM and less than 8 nM. Or less than 5 nM, or less than 4.0 nM, or less than 3.5 nM, wherein the IC50 value can be obtained by the protocol detection of Test Example 12.
  • the aforementioned fusion protein can inhibit the growth of transplanted tumors in mice.
  • the tumor inhibition rate of the fusion protein on the transplanted tumor of MC38 is greater than 50%, greater than 70%, greater than 80%, or greater than 85%.
  • the aforementioned fusion protein is a monomer; for example, when the targeting moiety assumes a single-chain structure, the extracellular region of TGF- ⁇ RII is fused to the carboxyl or amino terminus of the single-chain antibody via a linker.
  • the aforementioned fusion protein is a dimer, such as a homodimer or a heterodimer; preferably a homodimer.
  • the present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the aforementioned fusion protein, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1 mg-3000 mg of the aforementioned fusion protein.
  • the present disclosure also provides a nucleic acid molecule that encodes the fusion protein described in any one of the foregoing.
  • the present disclosure also provides a host cell comprising the aforementioned nucleic acid molecule; the host cell is selected from bacteria, yeast and mammalian cells; preferably mammalian cells. More preferably, the mammalian cell is a non-human cell.
  • the present disclosure also provides a method for treating or preventing tumors, the method comprising administering a therapeutically effective amount or a prophylactically effective amount of the aforementioned fusion protein, or the aforementioned pharmaceutical composition, or the aforementioned Nucleic acid molecule; preferably, wherein the tumor is a PD-1 related cancer.
  • the present disclosure also provides a use of the aforementioned fusion protein, or the aforementioned pharmaceutical composition, or the aforementioned nucleic acid molecule in the preparation of a medicament for the treatment or prevention of tumors, preferably, wherein the The tumor is a PD-1 related cancer.
  • the present disclosure also provides the aforementioned fusion protein, or the aforementioned pharmaceutical composition, or the aforementioned nucleic acid molecule for use as a drug, preferably as a drug for the treatment of tumors, and more preferably for the treatment of PD-1 related drugs. Cancer drugs.
  • the aforementioned tumor is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system Cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer , Colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, cartilage Sarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myelodysplastic tumor, squamous cell carcinoma, Ewing's
  • FIG. 1 Schematic diagram of TGF- ⁇ receptor fusion protein structure
  • Figure 2 Test result of anti-PD-1 antibody blocking the binding of PD-1 and its ligand
  • Figure 3 The effect of anti-PD-1 antibody on the secretion of IFN ⁇ from PBMC cells
  • Figure 4 The effect of anti-PD-1 antibody on mouse colon cancer MC38 transplantation tumor
  • Figure 5 The effect of anti-PD-1 antibody on the tumor volume of colon cancer MC38 transplanted in mice with human PD-1 gene;
  • Figure 6 The results of the affinity detection of the fusion protein with CHO-S cells stably transfected with PD-1;
  • Figure 7 In vivo efficacy test of the fusion protein.
  • programmed death 1 means programmed death 1
  • protein PD-1 protein PD-1
  • PD-1 protein PD-1
  • PDCD1 protein PD-1
  • hPD-1 hPD-1
  • P-L1 programmed death ligand-1
  • PD-L2 is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which down-regulates T when it binds to PD-1 Cell activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isotypes, and interspecies homologues of hPD-L1, as well as having at least one common table with hPD-L1 Analogues of bits. The complete hPD-L1 sequence can be found with GenBank accession number Q9NZQ7.
  • cytokine is a general term for proteins that are released by a cell population and act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monocytes, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17 and IL-5.
  • the "immunomodulatory molecules" described in the present disclosure can be used to weaken the immune tolerance of cancer cells.
  • This disclosure uses the extracellular domain of TGF- ⁇ RII (also called the extracellular domain of TGF- ⁇ RII) as the immunomodulatory molecule part of the fusion protein.
  • TGF- ⁇ RII extracellular domain described in this disclosure includes a full-length form and an N-terminal truncated form.
  • the present disclosure adopts a truncated form of the N-terminus of the extracellular domain of TGF- ⁇ RII as the immunomodulatory molecule part of the fusion protein.
  • TGF- ⁇ receptor II refers to a cell surface receptor that can bind ligands (including but not limited to TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3).
  • the TGF- ⁇ RII/TGF- ⁇ complex recruits TGF- ⁇ RI to form a signal transduction complex (Won et al., Cancer Res. 1999; 59: 1273-7).
  • the extracellular domain of the full-length TGF- ⁇ RII is a 136 amino acid residue peptide that starts from the N-terminus outside the cell of TGF- ⁇ RII.
  • Other variants that are about 136 amino acids in length and are derived from humans that have the function of the extracellular domain of TGF- ⁇ RII and can bind to TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 also belong to the TGF- ⁇ RII disclosed herein. The extent of the extracellular domain.
  • N-terminal truncated form of the extracellular domain of TGF- ⁇ RII or “the N-terminal truncated form of the extracellular domain of TGF- ⁇ RII” described in the present disclosure is truncated from the N-terminal of the extracellular domain of TGF- ⁇ RII, That is, the TGF- ⁇ RII extracellular domain obtained after consecutive amino acid deletions from the N-terminus, preferably less than 26 consecutive amino acid deletions from the N-terminus of the TGF- ⁇ RII extracellular domain, preferably 14-26 amino acids A deletion, more preferably a deletion of 14-21 amino acids from the N-terminal, and most preferably a deletion of 19 or 21 consecutive amino acids from the N-terminal.
  • TGF- ⁇ RII extracellular domain and “TGF- ⁇ RII extracellular domain” can be replaced with each other.
  • the "antibody” mentioned in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence 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.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, and humanized antibodies are preferred.
  • murine antibody in the present disclosure refers to a monoclonal antibody against human PD-1 (or its epitope) prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with PD-1 antigen (or its epitope), and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • the murine anti-PD-1 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chain or a variant thereof, or further comprise murine source The heavy chain constant region of IgG1, IgG2, IgG3 or variants thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable region of the antibody of the first species with the constant region of the antibody of the second species, which can reduce the immune response induced by the heterologous antibody.
  • a hybridoma that secretes murine-specific monoclonal antibodies must be established first, and then the variable region genes from the murine hybridoma cells will be cloned, and then the constant region genes of the human antibody will be cloned as needed.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the anti-PD-1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the anti-PD-1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or IgG1, IgG2 or IgG4 variants with amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations) are used.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of CDR sequences from non-human species to the framework of human antibody variable regions, that is, different types of human species
  • the antibody produced in the framework sequence of the antibody It can overcome the heterologous reaction induced by the chimeric antibody carrying a large number of heterologous protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further displayed by yeast and subjected to affinity maturation mutations to the CDRs.
  • the "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refer to the heavy chain constant region that has been disclosed in the prior art and does not change the structure and function of the antibody variable region. Or variants of the constant region of the light chain. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions. The specific substitutions are YTE known in the art.
  • HumanMAb (HuMAb), “human antibody”, “fully human antibody”, and “fully human antibody” can be used interchangeably, and can be antibodies derived from humans or antibodies obtained from a genetically modified organism.
  • the transgenic organism is "engineered” to produce specific human antibodies in response to antigen stimulation and can be produced by any method known in the art.
  • the elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted. To destruction.
  • Transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce human antibody-secreting hybridomas.
  • a human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources.
  • Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.
  • full-length antibody “whole antibody”, “whole antibody” and “whole antibody” are used interchangeably herein and refer to an antibody in a substantially complete form, as distinguished from the antigen-binding fragments defined below.
  • the term specifically refers to an antibody whose heavy chain contains an Fc region.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (or an epitope thereof). It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of the antibody examples include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including A bivalent fragment of two Fab fragments connected by a disulfide bridge in the hinge region, (iii) Fd fragment, usually composed of VH and CH1 domains; (iv) Fv fragment, usually composed of one-arm VH and VL domains of an antibody ; (V) Single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which is composed of VH domain; (vi) Single chain Fv (scFv).
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to connect them through a synthetic linker so that it can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (Known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • scFv single-chain Fv
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single-chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), and the like.
  • Fab is a fragment with a molecular weight of about 50,000 obtained by treating an IgG antibody molecule with an enzyme, and has an antigen-binding activity, in which about half of the N-terminal side of the heavy chain and the entire L chain are bonded together by disulfide bonds.
  • the Fab of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure with an enzyme.
  • the Fab can be produced by inserting the DNA encoding the Fab into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic organism or eukaryotic organism to express the Fab.
  • F(ab')2 is a fragment obtained by enzymatically digesting the downstream part of the disulfide bond in the hinge region of IgG, and has antigen binding activity, and contains two Fab regions connected at the hinge position.
  • the F(ab')2 of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure with an enzyme.
  • the F(ab')2 can be produced by linking Fab' with a thioether bond or a disulfide bond.
  • Fab' is a fragment obtained by cleaving the disulfide bond in the hinge region of F(ab')2, and has antigen binding activity.
  • the Fab' of the present disclosure can be produced by treating F(ab') 2 of the present disclosure with a reducing agent.
  • the Fab' can be produced by inserting the DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof (for example Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present disclosure are described by Alfthan et al.
  • the scFv of the present disclosure can be produced by the following steps, for example: obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure, constructing the DNA encoding the scFv, and inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector Then, the expression vector is introduced into prokaryotes or eukaryotes to express scFv.
  • Diabodies are antibody fragments with bivalent antigen binding activity.
  • the two antigens can be the same or different.
  • the diabody of the present disclosure can be produced by the following steps, for example: obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure, constructing the DNA encoding the scFv so that the amino acid sequence length of the linker is 8 residues or less, The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryotic organism or a eukaryotic organism to express the diabody.
  • dsFv can be obtained, for example, by replacing one amino acid residue in VH and VL with a cysteine residue, and a fragment obtained by forming a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present disclosure can be produced through the following steps, for example: obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure, constructing the DNA encoding the dsFv, and inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector Then, the expression vector is introduced into prokaryotes or eukaryotes to express dsFv.
  • amino acid difference or “amino acid mutation” means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes, mutations, or modifications, including the occurrence of one, two, Insertion, deletion, substitution, or modification of 3 or more amino acids.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid residue numbers in VH are roughly 27-38 (CDR1), 56-65 (CDR2) and 105-117 (CDR3)
  • the CDR amino acid residue numbers in VL are roughly 27-38 (CDR1). ), 56-65 (CDR2) and 105-117 (CDR3).
  • epitopes refers to a site on an antigen that is specifically bound by an immunoglobulin (or antibody) (for example, a specific site on a PD-1 molecule).
  • Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10 -8 M, for example, about less than 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD or "Kd” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibodies of the present disclosure bind PD-1 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9 M, for example, as using surface plasmon resonance (SPR)
  • SPR surface plasmon resonance
  • Linker refers to the connecting peptide sequence used to connect protein domains. It usually has a certain degree of flexibility. The use of linker will not lose the original function of the protein domain .
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • mice can be immunized with human PD-1 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequence can be obtained from ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT ImmunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered fusion protein or antibody and antigen-binding fragment thereof of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the first and second strands can be cloned and recombined into an expression vector.
  • the recombinant expression vector can stably transfect cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Positive clones are obtained through screening, and the positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the culture medium from which the fusion protein is secreted can be purified by conventional techniques.
  • a or G Sepharose FF column for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, for example, a composition comprising any one of the fusion proteins of the present disclosure, the subject has one or more disease symptoms, and the therapeutic agent is known It has a therapeutic effect on these symptoms.
  • the therapeutic agent is administered to the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, in order to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired therapeutic effect in the subject. ability.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can evaluate whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. Exemplary conservative substitutions are set out in the table "Exemplary Amino Acid Conservative Substitutions" below.
  • Effective amount refers to the amount of the drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired preventive/therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease. Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the effectiveness of other agents required to treat the disorder. Dosage, enhance the efficacy of another agent, and/or delay the progression of the patient’s disease related to the target antigen of the present disclosure.
  • Exogenous refers to substances that are produced in organisms, cells, or in vitro depending on the situation.
  • Endogenous refers to substances produced in organisms, cells, or in vivo according to circumstances.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position (Or the same). The percentage of homology (homology) between two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • the two sequences are 60% homologous or identical; if there are 95 positions in 100 positions in the two sequences Matches or homology, then the two sequences are 95% homologous or identical.
  • a comparison is made to give the maximum percentage homology or identity.
  • the comparison can be performed by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence.
  • the following references refer to the BLAST algorithm frequently used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol.
  • Other conventional BLAST algorithms provided by NCBI BLAST are also well known to those skilled in the art.
  • the sequence identity in the present disclosure can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the words “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.
  • “Pharmaceutical composition” means a mixture containing one or more of the fusion proteins described herein or their physiologically/pharmaceutically acceptable salts or prodrugs, and other chemical components; the other components are, for example, Physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc.
  • suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g. olive oil), saline, buffers, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol, and sodium chloride.
  • the present disclosure includes agents for treating diseases related to target antigen (eg, PD-1) positive cells, the agents comprising the fusion protein of the present disclosure as an active ingredient.
  • target antigen eg, PD-1
  • the agents comprising the fusion protein of the present disclosure as an active ingredient.
  • the cancer related to PD-1 in the present disclosure there is no restriction on the cancer related to PD-1 in the present disclosure, as long as it is a disease related to PD-1.
  • the therapeutic response induced by the fusion protein of the present disclosure can be achieved by binding PD-1 and then inhibiting PD-1 from Ligand binding, or killing tumor cells. Therefore, when in preparations and preparations suitable for therapeutic applications, the fusion protein of the present disclosure is very useful for such subjects who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, and lung cancer. , Stomach cancer, bowel cancer, kidney cancer, non-small cell lung cancer, bladder cancer, liver cancer, esophageal cancer, etc.
  • the above-mentioned PD-1 related cancers can be diagnosed by detecting or measuring PD-1 expressing cells with anti-PD-1 antibodies or antigen-binding fragments thereof.
  • Suitable immunodetection methods can be used, and immunoprecipitation, fluorescent cell staining, immunotissue staining, and the like are preferably used.
  • a fluorescent antibody staining method using the FMAT8100HTS system can be used.
  • the living body sample used to detect or measure the target antigen for example, PD-1
  • the target antigen for example, PD-1
  • cells expressing the target antigen such as tissue cells.
  • tissue cells Blood, plasma, serum, pancreatic juice, urine, stool, tissue fluid or culture fluid.
  • the disclosure is further described below in conjunction with embodiments, but these embodiments do not limit the scope of the disclosure.
  • the experimental methods that do not specify specific conditions in the examples of this disclosure usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the human PD-1-IgG1Fc fusion protein was designed and synthesized.
  • the N-terminus is 150 amino acids in the extracellular region of human PD-1, and the C-terminus is the Fc section of human IgG1 (hIgG1Fc).
  • Purified by Protein A affinity column, high-purity recombinant PD-1-Fc protein can be obtained, which can be used to detect the binding of anti-PD-1 antibody to antigen.
  • Human PD-1-IgG1Fc (SEQ ID NO: 1; signal peptide + extracellular region + hIgG1Fc):
  • the underlined part is the signal peptide
  • the normal part is the extracellular region of human PD-1
  • the italic part is hIgG1Fc.
  • PD-1 antigen (SEQ ID NO: 3) encoded by transfected cell nucleic acid:
  • Anti-human PD-1 antibodies can be produced by immunizing mice, and can also be obtained by anti-human PD-1 phage mouse immunization library.
  • the method of preparing anti-human PD-1 antibody by immunizing mice is as follows:
  • mice Female, 6-8 weeks old and Balb/c white mice, female, 6-8 weeks old.
  • Feeding environment SPF level. After the mice are purchased, they are reared in a laboratory environment for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 °C; humidity 40-60%. The mice that have adapted to the environment were immunized according to different protocols, with 6-10 mice in each group.
  • the immune antigen can be the purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2), or PD-1 as the antigen (see SEQ ID NO: 3)
  • Transfected Jurkat/CHO-PD-1 cells can be cross-immunized with a single reagent and different immune adjuvants or different types of immunogens.
  • the immune site can be the abdominal cavity or under the skin on the back, or alternate immunization of the two positions.
  • Immune adjuvant Gold Adjuvant (hereinafter referred to as Titermax, purchased from Sigma, product number T2684) and Imject Alum Adjuvant (hereinafter referred to as Alum, purchased from Pierce, product number 77161) are cross-immunized.
  • Titermax 1:1
  • Alum 3:1
  • 25-50 ⁇ g/head first immunization
  • 50 ⁇ g/head boost immunization
  • 1 ⁇ 10 7 One Jurkat/CHO-PD-1 cell/only.
  • intraperitoneal injection of 25-50 ⁇ g/head of emulsified antigen once a week or once every two weeks after the first immunization, Titermax and Alum were used alternately, 5-8 times in total.
  • the fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI), and aliquoted into 96-well cell culture plates (1 ⁇ 10 5 /150 ⁇ l /Well), 37°C, 5% CO 2 incubate, about 10-30 seed plates.
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • Hybridoma cell screening 7-9 days after fusion, according to the cell growth density, carry out the ELISA method to detect the binding of antibody and PD-1, and carry out the PD-1/PDL1 binding blocking ELISA for the detected positive well cells
  • the positive wells are exchanged, and the cells are expanded to a 24-well plate in time according to the cell density.
  • the cell line transferred into the 24-well plate was retested and then subcloned for the first time. Those who are positive in the first subcloning screening will be preserved, and the second or third subcloning will be performed until a single cell clone is obtained. Repeated fusions to obtain hybridoma cells that can block the combination of PD-1 and PDL1.
  • Anti-human PD-1 phage mouse immune library select the spleen of mice with high antibody titers in the serum, and extract total tissue RNA with Trizol (Invitrogen Cat No. 15596-018). CDNA was obtained by reverse transcription using PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara Cat No. 6210A). Design and synthesize the primers to construct the library according to the IMGT database. Through three rounds of PCR reactions, single-chain antibody fragments are obtained.
  • the single-chain antibody fragment and the modified library construction vector pCantab5E were digested with Sfi1 (NEB Cat No.#R0123L), and used after electrophoresis Gel Extraction Kit (Omega Cat No. D2500-02) was used for purification and recovery. Then use T4DNA ligase (NEB Cat No.#M0202L) to ligate at 16°C for 16-18 hours, and then use the above kit for purification and recovery, and finally elution with deionized water. Take 1 ⁇ g of the ligation product and mix it with a piece of electrotransformation competent TG1 (Lucigen Cat No.
  • the phage mouse immune library uses biotinylated human PD-1-ECD-his antigen, after 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen), and finally obtains binding PD-1 and blocking PD-1
  • MACS screening streptomycin magnetic beads, Invitrogen
  • the single clone that binds to PD-L1 was verified by sequencing, and the variable region sequence of the antibody was obtained.
  • ProteinG is the first choice for affinity chromatography.
  • the cultured hybridoma is centrifuged to take the supernatant, and 10-15% of the volume of 1M Tris-HCl (pH 8.0-8.5) is added according to the volume of the supernatant. Clear pH.
  • the ProteinG column uses 6M guanidine hydrochloride to wash 3-5 times the column volume, and then uses pure water to wash 3-5 times the column volume; use a buffer system such as 1 ⁇ PBS (pH7.4) as an equilibration buffer to equilibrate the column for 3-5 Times the column volume; the cell supernatant is combined with low flow rate loading, and the flow rate is controlled so that the retention time is about 1 min or longer; the column is washed with 1 ⁇ PBS (pH 7.4) 3-5 times the column volume until the UV absorption falls to Baseline: Use 0.1M acetic acid/sodium acetate (pH3.0) buffer for sample elution, collect elution peaks based on UV detection, and use 1M Tris-HCl (pH8.0) to quickly adjust the pH of the eluted product to 5-6.
  • a buffer system such as 1 ⁇ PBS (pH7.4) as an equilibration buffer to equilibrate the column for 3-5 Times the
  • the eluted product can be replaced by a method well known to those skilled in the art (for example, using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion to replace it with the required buffer system, Or use a molecular exclusion column to remove the polymer components in the eluted product to improve the purity of the sample).
  • a method well known to those skilled in the art for example, using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion to replace it with the required buffer system, Or use a molecular exclusion column to remove the polymer components in the eluted product to improve the purity of the sample).
  • the cell culture supernatant expressing the antigen protein or antibody is centrifuged at a high speed to collect the supernatant.
  • the Protein A affinity column was washed with 6M guanidine hydrochloride by 3-5 times the column volume, and then washed with pure water for 3-5 times the column volume.
  • Use a buffer system such as 1 ⁇ PBS (pH 7.4) as an equilibration buffer to equilibrate the chromatography column by 3-5 times the column volume.
  • the cell supernatant is loaded and bound at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer.
  • the column is washed with 1 ⁇ PBS (pH 7.4) by 3-5 times the column volume until the UV absorption falls back to the baseline .
  • the sample was eluted with 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer, the elution peaks were collected according to UV detection, and the eluted product was quickly adjusted to pH 5-6 with 1M Tris-HCl (pH8.0).
  • the eluted product can be replaced by a solution well known to those skilled in the art (ibid.).
  • the anti-human PD-1 murine antibody obtained by the aforementioned method was subjected to antigen binding experiments, and two clones with good activity were screened: M23 and M33.
  • the single cell clones were expanded and cultured, RNA was extracted, and the degeneracy of mouse-Ig was used.
  • the primers are subjected to reverse transcription amplification (RT-PCR) to obtain the variable region sequence of the antibody.
  • the murine antibody variable region sequence is connected with the human antibody constant region sequence, the chimeric antibody of the murine monoclonal antibody is cloned and recombinantly expressed, and the in vitro activity experiment is performed to confirm that the obtained monoclonal antibody variable region sequence is correct.
  • variable region sequences of murine antibodies M23 and M33 were determined as follows:
  • the heavy chain variable region of the murine antibody M23 (SEQ ID NO: 4):
  • the light chain variable region of the murine antibody M23 (SEQ ID NO: 5):
  • the heavy chain variable region of the murine antibody M33 (SEQ ID NO: 6)
  • the underline is the CDR sequence determined by the Kabat numbering system, followed by FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antibody CDR sequences in the table are determined according to the Kabat numbering system.
  • the heavy and light chain variable region germline genes with high sequence identity with the M23 and M33 light and heavy chain sequences were selected as templates, and these two The CDRs of the murine antibody were transplanted into the corresponding human antibody templates, and the corresponding humanized antibodies were constructed respectively.
  • the humanized light chain templates of the murine antibody M23 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01.
  • the sequence of the variable region after humanization is as follows ( The underline is the CDR sequence):
  • Hu23VL-CDR transplantation (SEQ ID NO: 21)
  • Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence; the amino acid residues are determined and annotated by the Kabat numbering system, such as I2G means that the 2nd position I of the Kabat numbering is mutated back to G according to the Kabat numbering system.
  • the light/heavy chain variable region sequence of the humanized antibody of M23 is as follows:
  • variable region back mutations are combined into different humanized antibody variable regions, as shown in the table below.
  • “Hu23-1” in the table means that the variable region of the light chain of the antibody is Hu23VL1, and the variable region of the heavy chain is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu23-1) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; if not explicitly stated in this disclosure, a full-length antibody is formed.
  • the variable region of the light chain and the constant region of the Kappa chain shown in SEQ ID NO: 52 are connected to form an antibody light chain
  • the variable region of the heavy chain is connected to the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 50 or 51. Linked to form an antibody heavy chain.
  • the humanized light chain templates of the murine antibody M33 are IGKV1-39*01 and IGKJ4*01, the humanized heavy chain templates are IGHV3-7 and IGHJ6*01, and the humanized variable region sequences are as follows:
  • Grafted represents the insertion of mouse antibody CDR into human germline FR region sequence. Amino acid residues are determined and annotated by the Kabat numbering system.
  • F71Y means that the F 71 of Kabat numbering is mutated back to Y according to the Kabat numbering system.
  • sequences of the light chain variable region and heavy chain variable region of the humanized antibody of the murine antibody M33 are as follows:
  • “Hu33-6" in the table means that the variable region of the light chain of the antibody is Hu33VL2 and the variable region of the heavy chain is Hu33VH3, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu33-6) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; if not explicitly stated in this disclosure, a full-length antibody is formed.
  • the variable region of the light chain and the constant region of the Kappa chain shown in SEQ ID NO: 52 are connected to form an antibody light chain
  • the variable region of the heavy chain is connected to the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 50 or 51. Linked to form an antibody heavy chain.
  • Hu23LCDR1 (N28Q) represents the LCDR1 mutation sequence of the variable region after the mutation of the 28th N of the Hu23VL1 or Hu23VL2 light chain variable region of Hu23 humanized antibody to Q.
  • Hu23LCDR1 (G29A) represents the light chain variable region of Hu23
  • the LCDR1 mutation sequence of the variable region after the 29th G of the chain variable region Hu23VL1 or Hu23VL2 is mutated to A (the amino acid position and CDR are determined by the Kabat numbering system), and others can be deduced by analogy.
  • the sequence of the light chain variable region of the Hu23 humanized antibody after LCDR1 mutation is as follows:
  • Hu23-11 in the table means that the variable region of the light chain of the antibody is Hu23VL1 (N28T) and the variable region of the heavy chain is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu23-11) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; if not explicitly stated in this disclosure, a full-length antibody is formed.
  • the variable region of the light chain and the constant region of the Kappa chain shown in SEQ ID NO: 52 are connected to form an antibody light chain
  • the variable region of the heavy chain is connected to the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 50 or 51. Linked to form an antibody heavy chain.
  • IgG4-AA represents F234A (corresponding to the 114th position of the sequence SEQ ID NO: 50), L235A (corresponding to the 115th position of the sequence SEQ ID NO: 50) mutation and S228P (corresponding to the 108th position of the sequence SEQ ID NO: 50) ), IgG4-AA can be obtained by simple point mutation in the form of IgG4 antibody.
  • the last amino acid K of the IgG4 constant region is mutated to A, and the mutation will not affect the activity of the antibody.
  • An exemplary light chain constant region is the constant region of the Kappa chain.
  • the IgG4-AA heavy chain constant region variant sequence is as follows (SEQ ID NO: 50):
  • the sequence of the IgG4-AA heavy chain constant region is as follows (SEQ ID NO: 51):
  • the sequence of the constant region of the Kappa chain is as follows (SEQ ID NO: 52):
  • the constructed IgG4AA format full-length antibody sequence is exemplified as follows:
  • Hu23-11(A) antibody heavy chain (SEQ ID NO: 53):
  • Hu23-11 antibody heavy chain (SEQ ID NO: 54):
  • Hu23-11(A)/Hu23-11 antibody light chain (SEQ ID NO: 55):
  • Hu33-5(A) antibody heavy chain (SEQ ID NO: 56):
  • Hu33-5 antibody heavy chain (SEQ ID NO: 57):
  • Hu33-5(A)/Hu33-5 antibody light chain (SEQ ID NO: 58):
  • Hu33-6(A) antibody heavy chain (SEQ ID NO: 59):
  • Hu33-6 antibody heavy chain (SEQ ID NO: 60):
  • Hu33-6(A)/Hu33-6 antibody light chain (SEQ ID NO: 58):
  • the extracellular domain of TGF- ⁇ RII (also called the extracellular domain of TGF- ⁇ RII) is used as the immunomodulatory molecule part of the fusion protein, and the PD-1 antibody is used as the targeting part of the fusion protein to form PD-1 antibody/TGF- ⁇ RII cells Fusion protein of the outer domain (PD-1/TGF- ⁇ trap).
  • the structure and function of the fusion protein containing the truncated form of the extracellular domain of TGF- ⁇ RII is relatively stable, especially after the 19 amino acid truncation at the N-terminus of the extracellular domain of TGF- ⁇ RII, the fusion protein has Higher expression and stable structure.
  • Non-limiting example sequences of the TGF- ⁇ RII extracellular domain and its truncated form in the present disclosure are as follows:
  • TGF- ⁇ RII extracellular domain sequence has a 19 amino acid truncation at the N-terminus: ECD(20-136)
  • the TGF- ⁇ RII extracellular domain sequence has a 21 amino acid truncation at the N-terminus: ECD(22-136)
  • the TGF- ⁇ RII extracellular domain sequence has a 14 amino acid truncation at the N-terminus: ECD(15-136)
  • the C-terminal amino acid of the heavy chain of the anti-PD-1 antibody of the present disclosure is connected to the extracellular region of TGF- ⁇ RII of different lengths through a conventional linker, together with the light chain, is conventionally expressed through the 293 expression system to obtain PD-1 /TGF- ⁇ trap fusion protein
  • common linkers include but are not limited to:
  • n is an integer from 1 to 6, preferably 4 or 5.
  • Table 10 The structure of the fusion protein of the present disclosure is shown in Figure 1.
  • Table 10 PD-1/TGF- ⁇ trap fusion protein
  • Fusion Proteins Sequence description N-terminal consecutive amino acid deletions Fusion protein 1 Hu23-11(A)-(G 4 S) 4 G-ECD(1-136) Not missing Fusion Protein 2 Hu33-5(A)-(G 4 S) 4 G-ECD(1-136) Not missing
  • Hu23-11(A), Hu33-5(A) and Hu33-6(A) are the anti-PD-1 antibodies described in this disclosure.
  • ECD(n-136) is the extracellular domain of TGF- ⁇ RII Full length or truncated form
  • n is the starting number of amino acids after truncation of the extracellular region of TGF- ⁇ RII.
  • the first chain of fusion protein 1 ie, heavy chain of anti-Hu23-11 (A) antibody-(G 4 S) 4 G-ECD (1-136)):
  • the second chain of fusion protein 1 (same as Hu23-11 antibody light chain sequence):
  • the first chain of fusion protein 2 ie, heavy chain of anti-Hu33-5 (A) antibody-(G 4 S) 4 G-ECD (1-136)):
  • the second chain of fusion protein 2 (same as Hu33-5 antibody light chain sequence):
  • the first chain of fusion protein 3 (ie, heavy chain of anti-Hu23-11 (A) antibody-(G 4 S) 4 G-ECD (20-136)):
  • the second chain of fusion protein 3 (same as Hu23-11 antibody light chain sequence):
  • the first chain of fusion protein 4 (ie, heavy chain of anti-Hu33-5 (A) antibody-(G 4 S) 4 G-ECD (20-136)):
  • the second chain of fusion protein 4 (same as Hu33-5 antibody light chain sequence):
  • the first chain of fusion protein 5 ie, heavy chain of anti-Hu33-6 (A) antibody-(G 4 S) 4 G-ECD (1-136)):
  • the second chain of fusion protein 5 (same as Hu33-6 antibody light chain sequence):
  • the first chain of fusion protein 6 ie, heavy chain of anti-Hu33-6 (A) antibody-(G 4 S) 4 G-ECD (20-136):
  • the second chain of fusion protein 6 (same as Hu33-6 antibody light chain sequence):
  • the underlined part is the heavy chain of the PD-1 antibody, the italic is the linker, and the dotted line is the extracellular region of TGF- ⁇ RII.
  • the nucleotide sequence encoding the anti-PD-1 antibody, the nucleotide sequence encoding the extracellular region of TGF- ⁇ RII, and the nucleotide sequence of the adaptor protein fragment ((G 4 S) 4 G) n were obtained by conventional technical means in the field .
  • the C-terminal nucleotides of the heavy chain of the anti-PD-1 antibody were connected to the N-terminal nucleotides of the extracellular region of TGF- ⁇ RII of different lengths through a linker protein, and cloned into the Phr-BsmbI vector.
  • the recombinant PD-1/TGF- ⁇ trap was expressed in 293 cells and other engineered cells, and was purified in Example 5.
  • the purified protein can be used in the following experiments.
  • the chromatographic medium is Protein A that interacts with Fc or derivative fillers, such as GE's MabSelect SuRe.
  • the equilibration buffer is 1 ⁇ PBS (137mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na 2 HPO 4 , 2mmol/L KH 2 PO 4 , pH7.4). After equilibrating 5 times the column volume, place the cells on In combination with clearing and loading, the flow rate is controlled so that the retention time of the sample on the column is ⁇ 1min.
  • the eluted sample after neutralization was ultrafiltration and concentrated and then subjected to size exclusion chromatography, the buffer was 1 ⁇ PBS, the chromatography column was XK26/60 Superdex200GE), the flow rate was controlled at 4mL/min, and the sample volume was less than 5mL.
  • the collected protein was identified by SEC-HPLC with a purity of greater than 95%, and was identified as correct by LC-MS and then divided into equipment to obtain the PD-1/TGF- ⁇ trap fusion protein.
  • Test Example 1 ELISA experiment in which anti-PD-1 antibody blocks the binding of PD-1 to its ligand in vitro
  • PD-L1 on the surface of tumor cells binds to PD-1 on the surface of T cells, thereby inhibiting the proliferation of T cells.
  • Anti-PD-1 antibodies can block the PD-L1/PD-1 signaling pathway by binding to PD-1, thereby stimulating the proliferation of T cells.
  • the binding experiment of blocking PD-1/PD-L1 is used to detect the blocking activity of anti-PD-1 antibody on the signal pathway.
  • the extracellular domain and Fc fused PD-1 protein (PD-1-Fc, sequence see SEQ ID NO:1) was coated in a 96-well plate, and the anti-PD-1 antibodies to be tested (including Antibodies: Hu23-11, Hu33-6, positive control antibody: H005-1 (refer to the H005-1 antibody in WO2015085847), perform the incubation reaction; later add biotin-labeled PD-L1/PD-L2 to incubate the reaction. After washing the plate, the binding amount of biotin-labeled PD-L1/PD-L2 was detected, and the IC 50 value of the blocking of the binding of the ligand PD-L1/PD-L2 by the anti-PD-1 antibody was calculated.
  • the anti-PD-1 antibodies to be tested including Antibodies: Hu23-11, Hu33-6, positive control antibody: H005-1 (refer to the H005-1 antibody in WO2015085847)
  • pH 9.6CB buffer 1.59g Na 2 CO 3 and 2.93g NaHCO 3 dissolved in 1L distilled water
  • the exemplary anti-PD-1 antibodies Hu23-11 and Hu33-6 of the present disclosure can effectively block the binding of PD-1 and PD-L1/PD-L2, and their blocking activity is similar to that of the positive control antibody H005-1.
  • Test Example 2 Blocking test of exemplary antibodies and ligands
  • Count Jurkat/PD-1 cells Jurkat cells stably transfected with PD-1
  • plant CHOK1/PD-L1 cells in a certain proportion in a cell culture plate (90 ⁇ L/well) while adding 10 ⁇ L/well to the diluted antibody (Antibody: Hu23-11 and Hu33-6, positive control antibody: H005-1), negative control IgG4 protein, antibody gradient dilution concentration is 0.3mg/mL, 3mg/mL, 30mg/mL, placed at 37°C, 5% Incubate in a CO 2 incubator for 5 hours. Take out the cell culture plate and place it at room temperature for 5 minutes, then add 50 ⁇ l Bio-Glo TM Reagent to each well, incubate at room temperature for 5 minutes, and read the plate.
  • the experimental results are shown in Figure 2.
  • Biacore T200 instrument detects PD-1 antibody and antigen PD-1 reaction signals in real time to obtain binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with 10mM glycine-HCl pH1.5 buffer.
  • the experimental buffer system is 1 ⁇ HBS-EP buffer solution (Cat#BR-1001-88, GE). After the experiment, the GE Biacore T200 Evaluation version 3.0 software was used to fit the data with the (1:1) Langmuir model to obtain the affinity value. The results are shown in Table 12.
  • PBMC peripheral blood mononuclear cells
  • serially diluted antibody samples including the antibodies of the present disclosure: Hu23-11 and Hu33-6, positive control antibody H005-1, and negative control IgG4 protein, and the antibody's serial dilution concentrations are 0.3mg/mL, 3mg/mL, 30mg /mL), diluted with PBS (B320, Shanghai Yuanpei Biotechnology Co., Ltd.), 10 ⁇ L per well.
  • PBS B320, Shanghai Yuanpei Biotechnology Co., Ltd.
  • test results are shown in Figure 3.
  • the results show that the anti-PD-1 antibodies Hu23-11 and Hu33-6 of the present disclosure can effectively activate the secretion of IFN- ⁇ , and their ability to activate IFN- ⁇ secretion is similar to the positive H005-1 control.
  • Test Example 5 The effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38
  • mice Inoculate 5 ⁇ 10 5 cells/mouse/100 ⁇ L of MC38 cells into 90 hPD-1TG mice (Biocytometer) subcutaneously on the right ribs. After 10 days, remove the animals with too large and small tumors, and the average tumor volume is about 120mm. 3 The mice were randomly divided into 5 groups: blank control (PBS), positive control H005-1 3mpk, Hu23-11 1mpk, Hu23-11 3mpk, Hu33-6 3mpk, with 8 mice in each group. From Day 0 (day 0), the antibodies of each group were injected intraperitoneally three times a week. After the first week of administration, the tumor was found to be significantly inhibited.
  • PBS blank control
  • H005-1 3mpk positive control
  • Hu23-11 1mpk Hu23-11 3mpk
  • Hu33-6 3mpk Hu33-6 3mpk
  • the administration frequency was adjusted to once a week for a total of 5 administrations.
  • the tumor volume and animal weight were monitored twice a week and the data was recorded. When the tumor volume exceeds 2000 mm 3 or most tumors appear ulcerated or lose weight by 20%, the tumor-bearing animals are euthanized as the experimental end point.
  • Tumor volume (TV) 1/2 ⁇ L long ⁇ L short 2
  • Tumor growth rate (T/C%) (TT 0 )/(CC 0 ) ⁇ 100%
  • TGI%) 1-T/C%
  • T and T 0 respectively represent the tumor volume at the end of the experiment and the beginning of the experiment in the antibody administration group
  • C and C 0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the blank control group, respectively.
  • the test results are shown in Table 13 and Figure 4, and the test results show that compared with the blank control, the antibodies of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 transplanted tumors.
  • the dosing frequency is 3 times a week
  • the results show that the anti-tumor rate of the antibody of the present disclosure is significantly better than that of the positive control antibody H005-1; after that, the dosing frequency is reduced to once a week.
  • the drug was administered twice (day 21)
  • the efficacy of the antibodies of the present disclosure gradually widened, and showed a dose-dependence.
  • the tumor-bearing mice can tolerate the anti-PD-1 antibody well, and their weight rises steadily during the whole administration process, and no obvious drug-induced weight loss and other symptoms occur.
  • Test Example 6 The effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38
  • the transgenic PD-1 mice were derived from purchased transgenic PD-1 mice (ISIS INNOVATION LIMITED, University Offices, Wellington Square, Oxford OX1 2JD, England) and cultivated in Cephrim Biosciences, Inc. of the fifth generation of mice.
  • MC38 cells were inoculated into hPD-1 transgenic mice (male and female) at the rate of 5 ⁇ 10 5 cells/100 ⁇ l per subcutaneously on the posterior right rib of hPD-1 transgenic mice. When the average tumor volume of the mice reached between 80-100 mm 3 , the body weight was removed.
  • the tumor-bearing mice were randomly divided into 5 groups (8 in each group) according to the tumor size: negative control hIgG 30mpk, H005-1 10mpk, H005-1 30mpk, Hu33-6 10mpk, Hu33 -6 30mpk.
  • the group administration date is set as D0 (day 0).
  • each drug was administered intraperitoneally for a period of 22 days, once every two days, for a total of 11 times.
  • the tumor volume was measured twice a week, the weight was weighed, and the data was recorded.
  • the animal body weight and tumor volume of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and graphed with Graphpad Prism 5 and Excel software, and statistical analysis was performed using Student’s T test.
  • Tumor growth rate T/C% (TT 0 )/(CC 0 ) ⁇ 100%
  • T and T 0 respectively represent the tumor volume at the end of the experiment and the beginning of the experiment in the antibody administration group
  • C and C 0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the blank control group, respectively.
  • the test results are shown in Table 14 and attached Figure 5.
  • the test results show that compared with the control group, the antibodies of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 transplanted tumors.
  • Hu33-6 30mpk has the highest tumor inhibition rate.
  • the tumor inhibition rate was 80.4% when measured on the 20th day.
  • the efficacy of Hu33-6 10mpk is better than the positive control H005-1-10mpk.
  • the unit of the average tumor volume of each group in the table is mm 3 ;
  • Test Example 7 In vitro binding and kinetic experiment of PD-1/TGF- ⁇ trap fusion protein detected by Biacore
  • the affinity of the test molecule with human TGF- ⁇ 1 or human PD-1 protein is determined by Biacore T200 (GE). The experimental process is described as follows:
  • Biosensor chip to affinity capture PD-1/TGF- ⁇ trap, and then flow high concentration of antigen 1 (human PD1 (Sino Biological, Cat.#10377-H08H) or human TGF- ⁇ 1 ( Acro, Cat.1G1-H4212#)) 120s, saturate the site on the antibody against antigen 1, then load antigen 2 (human TGF- ⁇ 1 or human PD1), and use Biacore T200 instrument to detect the reaction signal in real time to obtain binding and dissociation curve. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with glycine-hydrochloric acid (pH 1.5, GE). The buffer solution used in the experiment is HBS-EP buffer (GE). The data obtained in the experiment was fitted with the (1:1) Langmuir model using BIAevaluation 4.1 software (GE), and the affinity values shown in Table 15 were obtained.
  • antigen 1 human PD1 (Sino Biological, Cat.#10377-H
  • Test Example 8 In vitro cell binding experiment of PD-1/TGF- ⁇ trap
  • Fusion protein or antibody to be detected is a Fusion protein or antibody to be detected:
  • Negative control C25 HIV antibody (see patent US6114143 for preparation)
  • Test Example 9 In vitro detection of PD-1/PD-L1 pathway blocking experiment
  • Anti-PD-1 antibodies Hu23-11, Hu33-5;
  • CHO/PD-L1 cells (CS187108, Promega), digest and resuspend the cells with F-12 Nutrient Mixture (Gibco, 22400-089) complete medium, adjust the cell density to 4 ⁇ 10 5 with complete medium according to the cell count results /mL, transfer the cell suspension to the sample tank, use a multichannel pipette to add 100 ⁇ L/well to a 96-well plate, place it in a 37°C, 5% CO 2 incubator and incubate for 20 to 24 hours; the next day Prepare Jurkat/PD-1 (CS187102, Promega) cell suspension, resuspend the cells in analytical medium according to the cell count results, and adjust the cell density to 1.25 ⁇ 10 6 /mL; culture the cells with CHO/PD-L1 cells Take out the plate from the incubator, use a multichannel pipette to take out 95 ⁇ L of culture medium from each well, add 410 ⁇ L/well of serially diluted fusion protein and PD-1 antibody, and then transfer the Jurkat/
  • the Bio-Glo TM reagent (Promega, G7940) was taken out and the temperature returned to room temperature. Take out the cell culture plate and place it at room temperature for 5 to 10 minutes, then add 450 ⁇ L of Bio-Glo TM reagent to each well, incubate in a safe cabinet for 5 to 10 minutes, and read the chemiluminescence signal value with a multifunctional microplate reader.
  • the fusion protein 3 and fusion protein 4 of the present disclosure can effectively block the binding of Jurkat cells expressing PD-1 molecules with CHO/PD-L1 cells.
  • Antibody IC50(nM) Maximum blocking multiple C25 / / Fusion Protein 3 12.32 3.56 Hu23-11 6.00 3.6 Fusion protein 4 3.03 3.63 Hu33-5 1.82 3.63
  • the maximum blocking multiple is the multiple that is enhanced compared to the blank fluorescence.
  • HepG2 cells expressed the Smad3 binding element (SBE) with a luciferase reporter gene to study the inhibitory effect of PD-1/TGF- ⁇ trap on TGF- ⁇ 1-induced Smad3 activation.
  • SBE Smad3 binding element
  • the PD-1/TGF- ⁇ was evaluated based on the IC50. In vitro activity of ⁇ trap.
  • Test sample Fusion protein 3, Fusion protein 4, negative control (C25).
  • HepG2 cells (ATCC, HB-8065 TM ) were cultured in MEM complete medium (GE, SH30243.01) containing 10% FBS, and passaged every 3 days. On the first day of the experiment, a 96-well plate (Corning, 3903) was seeded at a density of 25,000 cells per well, and cultured at 37°C and 5% CO 2 for 24 hours. On the second day, the medium in the cell culture plate was discarded, and 100 ng of 3TP-Lux plasmid (Prutin Biotechnology (Beijing) Co., Ltd., catalog number 11767) was transfected per well. The cells were cultured at 37°C and 5% CO 2 for 24 hours.
  • the complete medium in the 96-well plate was discarded, and 80 ⁇ L of incomplete medium (MEM+0.5% FBS) was added to each well.
  • 10 ⁇ L of human TGF- ⁇ 1 (R&D, 240-B-010) solution prepared with incomplete medium the final concentration is 2ng/mL and 10 ⁇ L of the sample to be tested, the final concentration of the sample to be tested is 500, 50 , 5, 0.5, 0.05, 0.005, 0.0005, 0.00005, 0.000005 and 0 nM, with human TGF- ⁇ 1 solvent as a control, the cells were cultured at 37°C and 5% CO 2 for 18 hours.
  • Test Example 11 In vitro detection of tuberculin (TB) to stimulate PBMC to release IFN ⁇
  • PBMC peripheral blood mononuclear cells
  • TB tuberculin
  • Test sample 1C25; 2PD-1 antibody; 3fusion protein 3; 4fusion protein 4.
  • the cell culture plate was placed in a 37°C, 5% CO 2 incubator and incubated for 3 days. Take out the cell culture plate, centrifuge (4000rpm, 10min) to take the supernatant from each well, after 10 times dilution, use ELISA method (human IFN- ⁇ detection kit, Xinbosheng, EHC102g.96) to detect the level of IFN- ⁇ . Refer to the reagent instructions for specific operations. The results are shown in Table 19.
  • fusion protein 3 and fusion protein 4 can dose-dependently enhance the secretion of cytokine IFN- ⁇ by activated T lymphocytes, and have a stronger activating effect than anti-PD-1 antibodies.
  • Test example 12 PD-1/PD-L1, PD-1/PD-L2 blocking experiment
  • Dilute PD-1-IgG1Fc with PBS to 0.5 ⁇ g/mL add 100 ⁇ L/well to a 96-well plate, and incubate overnight at 4°C.
  • the PBS in the 96-well plate was discarded, and the plate was washed 3 times with PBST (pH 7.4 PBS containing 0.05% tween20), 100 ⁇ L/well PBS/3% BSA was added, and the plate was incubated for 1 hour at room temperature for blocking. Discard the blocking solution, wash the plate with PBST 3 times, add 100 ⁇ L of fusion protein or control diluted with PBS/3% BSA to an appropriate concentration, and pre-incubate at 4°C for 1 hour.
  • PBST pH 7.4 PBS containing 0.05% tween20
  • Test Example 13 In vivo efficacy test of fusion protein
  • Human PD1 transgenic C57BL/6J mice were purchased from Biocytometer Co., Ltd. The animals were bred adaptively in the laboratory environment for 7 days with 12/12 hours light/dark cycle adjustment, temperature 23 ⁇ 1°C, humidity 40-50%, animals were given standard sterilized rat feed, and they were free to eat and drink.
  • the human PD1 transgenic C57BL/6J was subcutaneously inoculated with MC38 cells (1 ⁇ 10 5 cells/unit, purchased from Nanjing Yinhe) in the right rib. Eight days later, they were divided into 4 groups, 10 cells/group, and the average tumor volume in each group was 59.08mm 3 .
  • Intraperitoneal injection of equimolar amounts of C25 (2.48mpk), Hu23-11 (0.83mpk) and fusion protein 3 (3mpk and 1mpk) was administered 4 times, 7 days, 9 days, 11 days and 14 days after tumor inoculation Administration.
  • the tumor volume and weight were measured twice a week, and the data was recorded.
  • Use Excel 2003 statistical software the average value is calculated by avg; the SD value is calculated by STDEV; the SEM value is calculated by STDEV/SQRT; the difference P value between groups is calculated by TTEST.
  • V tumor volume
  • Relative tumor growth rate T/C(%) (TT 0 )/(CC 0 ) ⁇ 100%
  • TGI (%) 1-T/C (%).
  • T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 , C 0 are the tumor volumes at the beginning of the experiment.

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Abstract

含有TGF-β受体的融合蛋白及其医药用途。具体地，包含所述PD-1抗体靶向部分和TGF-βRII胞外区的双功能融合蛋白，以及包含所述含有TGF-β受体融合蛋白的药物组合物，以及其作为抗癌药物的用途。

Description

含有TGF-β受体的融合蛋白及其医药用途
本申请要求2019年11月12日提交的中国专利申请的优先权(申请号201911098550.0)，其内容通过引用并入本文。
技术领域
本披露属于生物技术领域，更具体地，本披露涉及抗PD-1抗体与TGF-β受体的融合蛋白及其应用。
背景技术
这里的陈述仅是提供与本披露有关的背景信息，而不必然地构成现有技术。
程序性死亡受体1(programmed death 1，PD-1)属于CD28超家族，表达于活化的T细胞，B细胞和髓系细胞。PD-1是约55kDa的I型膜蛋白，包括细胞外IgV结构域、跨膜域和含有免疫受体酪氨酸抑制基序的胞内域。目前，已发现PD-1有两个配体，程序性死亡配体-1(programmed death ligand 1，PD-L1)和PD-L2。PD-1与配体结合后，可以负调控T细胞的活化，使肿瘤细胞获得免疫逃逸。
PD-L1在多种癌症中有高表达，其表达水平也与患者预后相关。因此，抑制PD-1与PD-L1的相互作用可以阻断负调控通路，增强T细胞在免疫应答中的反应，是肿瘤免疫治疗领域很有潜力的治疗方式。目前，多个PD-1和PD-L1抑制剂(如BMS的Nivolumab，Merck的Pembrolizumab，Roche的Atezolizumab)已经被证实临床有效并批准上市。但是，PD-1和PD-L1抑制剂也存在着有效率不高的缺陷，主要原因可能是其他免疫检查点的存在和肿瘤微坏境的复杂性所导致。
转化生长因子-β(transforming growth factor-β，TGF-β)属于TGF-β超家族，TGF-β信号通路在调节细胞生长和分化中扮演着重要的角色。TGF-β表达释放后，会与受体TGF-βRII结合，激活TGF-βRII，再与TGF-βRI形成复合体。随后，该复合体会磷酸化下游的Smad2和Smad3。被磷酸化的Smad2和Smad3再与Smad4结合，形成磷酸化的Smad2/3/4复合体进入细胞核，调控下游基因表达。研究表明，TGF-β信号能促进肿瘤的上皮间质转化过程，启动肿瘤转移。在目前的TGF-β靶向药物的临床研究中，虽然看到了抑制TGF-β信号通路对疾病进展的积极作用，但单用TGF-β靶向药物的效果却依然不够理想。
因此，在中和肿瘤微环境的TGF-β基础上抑制PD-1/PD-L1通路，可以使T细胞恢复活性，增强免疫应答，更有效地提高抑治肿瘤发生和发展的效果。
目前已有抗体/TGF-β受体融合蛋白公开，如WO2006074451A2、WO2009152610A1、WO2011109789A2、WO2013164694A1、WO2014164427A1、WO2015077540A2、WO9309228A1、WO9409815A1、WO2015077540A2、WO2015118175A2和WO2018205985，但并没有针对抗PD-1抗体和TGF-β受体的 融合蛋白药物获批上市。
在实际生产和临床应用上，仍需进一步开发具有更优性能的产品。
发明内容
本披露提供一种融合蛋白，其包含靶向部分和TGF-β受体部分，其中，所述的TGF-β受体部分为TGF-βRII胞外区，所述的靶向部分为抗PD-1抗体或其抗原结合片段。
在一些实施方案中，前述的融合蛋白，其中所述的TGF-βRII胞外区为TGF-βRII胞外区的N端截短形式。
在一些实施方案中，前述的融合蛋白，其中所述的TGF-βRII胞外区的N端截短形式为在TGF-βRII胞外区的N端缺失0至26个的氨基酸；优选N端缺失14-26个氨基酸；更优选缺失14-21个氨基酸；最优选缺失14、19或21个氨基酸。
在一些具体的实施方案中，前述的融合蛋白，其中所述的TGF-βRII胞外区的N端截短形式为在TGF-βRII胞外区的N端缺失1至26个连续的氨基酸；优选N端缺失14-26个连续的氨基酸；更优选缺失14-21个连续的氨基酸；最优选缺失14、19或21个连续的氨基酸。
在一些实施方案中，前述的融合蛋白，其中所述的TGF-βRII胞外区的序列如SEQ ID NO：61所示。
在一些实施方案中，前述的融合蛋白，其中所述的TGF-βRII胞外区的的N端截短形式序列如SEQ ID NO：62、63或64所示。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中
a)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和
所述轻链可变区包含序列如SEQ ID NO：11、32、33、34、35、36或37所示的LCDR1和分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3；或
b)所述重链可变区包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和
所述轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：34、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含如下所述的重链可变区和轻链可变区：
c)所述重链可变区包含与如SEQ ID NO：4序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含与如SEQ ID NO：5序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；或
d)所述重链可变区包含与如SEQ ID NO：6序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含与如SEQ ID NO：7序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段以小于4×10 -8M、小于6×10 -9M的KD值的亲和力与人PD-1(或其表位)结合。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段能够有效阻断PD-1与配体(如PD-L1)的结合。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段以小于2×10 -9M的KD值的亲和力与人PD-1(或其表位)结合；和/或以小于3×10 -9M的KD值的亲和力与食蟹猴的PD-1(或其表位)结合。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段以小于1×10 -9M的KD值的亲和力与人PD-1(或其表位)结合；和/或以小于1×10 -9M的KD值的亲和力与食蟹猴的PD-1(或其表位)结合。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段能有效激活T细胞分泌IFN-γ。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段能有效抑制转基因PD-1小鼠中移植瘤的生长(例如，但不限于结肠癌移植瘤，如MC38细胞移植瘤)。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段能有效抑制转基因PD-1小鼠中移植瘤的生长(例如，但不限于结肠癌移植瘤，如MC38细胞移植瘤)，当给药量为1mpk时，其抑瘤率小于46％。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段能有效抑制转基因PD-1小鼠中移植瘤的生长(例如，但不限于结肠癌移植瘤，如MC38细胞移植瘤)，当给药量为3mpk时，其抑瘤率小于60％，或小于76％。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段是鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，前述的融合蛋白，其中所述抗PD-1抗体为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多6个氨基酸的回复突变；
优选地，所述的回复突变选自如下：
e)轻链框架区中的氨基酸回复突变，如2G，和/或
重链框架区中的氨基酸回复突变，其选自：27Y、48I、67T、69L、82F和93T中的一个或更多个；或
f)轻链框架区中的氨基酸回复突变，选自：42G、44V和71Y中的一个或更多个，和/或
重链框架区中的氨基酸回复突变，其选自：1K和/或94S；上述回复突变位置符合Kabat编号规则。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体包含选自以下的重链可变区和轻链可变区：
重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，且在FR区包含选自27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变，和
轻链可变区包含序列分别如SEQ ID NO：34、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3，且在FR区包含2G氨基酸回复突变。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体包含选自以下的重链可变区和轻链可变区：
重链可变区包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，且在FR区包含1K和/或94S氨基酸回复突变，和
轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3，且在FR区包含选自42G、44V和71Y中的一个或更多个氨基酸回复突变；上述氨基酸回复突变的位置符合Kabat编号规则。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体包含选自以下的重链可变区和轻链可变区：
重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，且在FR区包含选自27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变，和
轻链可变区包含序列分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3，且在FR区包含2G氨基酸回复突变。
在一些实施方案中，前述融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段的重链可变区和轻链可变区选自如下(g)至(k)中的任一项：
g)重链可变区序列如SEQ ID NO：4所示或与其具有至少90％序列同一性和轻链可变区序列如SEQ ID NO：5所示或与其具有至少90％序列同一性；
h)重链可变区序列如SEQ ID NO：6所示或与其具有至少90％序列同一性和轻链可变区序列如SEQ ID NO：7所示或与其具有至少90％的序列同一性；
j)重链可变区序列如SEQ ID NO：20、23、24或25所示或分别与SEQ ID NO：20、23、24或25具有至少90％序列同一性，和轻链可变区序列如SEQ ID NO：21、22、38、39、40、41、42、43、44、45、46、47、48或49所示或分别与SEQ ID NO：21、22、38、39、40、41、42、43、44、45、46、47、48或49具有至少90％序列同一性；和
k)重链可变区序列如SEQ ID NO：26、30或31所示或分别与SEQ ID NO：26、30或31具有至少90％序列同一性，和轻链可变区序列如SEQ ID NO：27、28或29所示或分别与SEQ ID NO：27、28或29具有至少90％序列同一性；
优选地，所述的抗PD-1抗体或其抗原结合片段包含：
重链可变区序列如SEQ ID NO：20所示和轻链可变区序列如SEQ ID NO：40所示；或
重链可变区序列如SEQ ID NO：30所示和轻链可变区序列如SEQ ID NO：28所示；或
重链可变区序列如SEQ ID NO：31所示和轻链可变区序列如SEQ ID NO：28所示。
上述具有至少90％包含具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％和99％的序列一性，序列同一性可以通过常规的序列比对软件获得。
在一些实施方式中，前述的融合蛋白，其中所述的抗PD-1抗体包含SEQ ID NO：20所示的重链可变区或其变体，所述的变体为在SEQ ID NO：20中具有选自如G27Y、M48I、V67T、I69L、L82F和A93T中一个或更多个回复突变；和所述的抗PD-1抗体包含如SEQ ID NO：21所示的轻链可变区或其变体，其中所述的变体为在SEQ ID NO：21中具有I2G回复突变。
在一些实施方式中，前述的融合蛋白，其中所述的抗PD-1抗体包含SEQ ID NO：26所示的重链可变区或其变体，所述的变体为在SEQ ID NO：26中具有R94S和/或E1K回复突变；和所述的抗PD-1抗体包含如SEQ ID NO：27所示的轻链可变区或其变体，其中所述的变体为在SEQ ID NO：27中具有选自K42G、P44V和F71Y中一个或更多个回复突变。
在一些实施方式中，前述的融合蛋白，其中所述的抗PD-1抗体包含SEQ ID NO：20所示的重链可变区或其变体，所述的变体为在SEQ ID NO：20中具有选自如G27Y、M48I、V67T、I69L、L82F和A93T中一个或更多个回复突变；和所述的抗PD-1抗体包含如SEQ ID NO：40所示的轻链可变区或其变体，其中所述的变体为在SEQ ID NO：40中具有I2G回复突变。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含如SEQ ID NO：20所示的重链可变区和如SEQ ID NO：40所示的轻链可变区。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合 片段包含如SEQ ID NO：30所示的重链可变区和如SEQ ID NO：28所示的轻链可变区。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含如SEQ ID NO：31所示的重链可变区和如SEQ ID NO：28所示的轻链可变区。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段进一步包含抗体重链恒定区和轻链恒定区；所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；优选地，所述抗PD-1抗体或其抗原结合片段包含序列如SEQ ID NO：50或51所示的重链恒定区和/或序列如SEQ ID NO：52所示的轻链恒定区。
在一些实施方案中，前述的融合蛋白，其中所述抗PD-1抗体包含选自以下(l)-(n)中任一项的重链和轻链：
(l)重链序列如SEQ ID NO：53或54所示或与SEQ ID NO：53或54具有至少85％的序列同一性；和轻链序列如SEQ ID NO：55所示或与SEQ ID NO：55具有至少85％的序列同一性；
(m)重链序列如SEQ ID NO：56或57所示或与SEQ ID NO：56或57具有至少85％的序列同一性；和轻链序列如SEQ ID NO：58所示或与SEQ ID NO：58具有至少85％的序列同一性；和
(n)重链序列如SEQ ID NO：59或60所示或与SEQ ID NO：59或60具有至少85％的序列同一性；和轻链序列如SEQ ID NO：58所示或与SEQ ID NO：58具有至少85％的序列同一性；
上述具有至少85％包含具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％和99％的序列一性，序列同一性可以通过常规的序列比对软件获得。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体包含：SEQ ID NO：53的重链和SEQ ID NO：55的轻链。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体包含：SEQ ID NO：56的重链和SEQ ID NO：58的轻链。
在一些实施方案中，前述的融合蛋白，其中所述的抗PD-1抗体包含：SEQ ID NO：59的重链和SEQ ID NO：58的轻链。
在一些实施方案中，前述的融合蛋白，其中所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
在一些实施方案中，前述的融合蛋白，其中所述的TGF-βRII胞外区可通过接头融合至所述的抗PD-1抗体或其抗原结合片段的重链的羧基端(C-端)、重链的氨基端(N-端)、轻链的C-端或轻链的N端，优选融合至重链的C-端。
在一些实施方案中，前述的融合蛋白，其中所述的接头可为本领域内的常用接头，包括但不限于(ASTKGP)n、(ASTKGPSVFPLAP)n、(TVAAP)n、(TVAAPSVFIFPP)n或(G 4S)nG，其中n为1、2、3、4、5或6，优选为4或5。
在一些实施方案中，前述的融合蛋白包含：
-第一链，其包含融合至抗PD-1抗体重链的TGF-βRII胞外区；和
-第二链，其包含抗PD-1抗体轻链，
其中：
o)所述的第一链的序列如SEQ ID NO：69或71所示或与SEQ ID NO：69或71所示的氨基酸序列具有至少85％的同一性，和第二链序列如SEQ ID NO：55所示或与SEQ ID NO：55所示氨基酸序列具有至少85％的同一性；或
p)所述的第一链的序列如SEQ ID NO：70、72、73或74所示或与SEQ ID NO：70、72、73或74所示的氨基酸序列具有至少85％的同一性，和第二链序列如SEQ ID NO：58所示或与SEQ ID NO：58所示氨基酸序列具有至少85％的同一性；
上述具有至少85％包含具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％和99％的序列一性，序列同一性可以通过常规的序列比对软件获得。
在一些实施方案中，前述的融合蛋白包含两条序列相同的第一链和第二链，其中：
-第一链，其包含融合至抗PD-1抗体重链的TGF-βRII胞外区；和
-第二链，其包含抗PD-1抗体轻链，
其中：
o)所述的第一链的序列如SEQ ID NO：69或71所示，和第二链序列如SEQ ID NO：55所示；或
p)所述的第一链的序列如SEQ ID NO：70、72、73或74所示，和第二链序列如SEQ ID NO：58所示。
在一些实施方式中，前述的融合蛋白包含两条序列相同的第一链和第二链，其中：
所述的第一链的序列如SEQ ID NO：71所示，和第二链的序列如SEQ ID NO：55所示；或
所述的第一链的序列如SEQ ID NO：72所示，和第二链的序列如SEQ ID NO：58所示。
在一些实施方案中，前述的融合蛋白，其与人PD-1(或其表位)的亲和力的KD值小于1×10 -8M，小于5×10 -9M，小于3×10 -9M或小于1×10 -9M；其中所述的KD值可通过Biacore检测获得。
在一些实施方案中，前述的融合蛋白，其与人TGF-β1的亲和力的KD值小于 1×10 -11M，小于5×10 -11M，小于7×10 -12M或小于5×10 -12M；其中所述的KD值可通过Biacore检测获得。
在一些实施方案中，前述的融合蛋白结合细胞表面表达的PD-1；在一些实施方案中，其结合EC50小于10nM，小于8nM，小于5nM或小于2nM，其中结合EC50值可通过测试例8的方法检测获得。
在一些实施方案中，前述的融合蛋白可以阻断PD-1与其配体(如PD-L1)的结合；在一些实施方案中，其阻断IC50值小于15nM，小于13nM，小于10nM或小于5nM；其中阻断IC50值可通过测试例9的方法检测获得。
在一些实施方案中，前述的融合蛋白可以抑制TGF-β1诱导Smad3活化；在一些实施方案中，其抑制IC50小于5nM，小于3nM，小于1nM，小于0.5nM或小于0.2nM；其中阻断IC50值可通过测试例10的方法检测获得。
在一些实施方案中，前述的融合蛋白可以刺激T淋巴细胞分泌细胞因子，特别是IFN-γ的分泌；在一些实施方案中，刺激IFN-γ的分泌增加至少5倍，至少8倍，至少10倍，至少11倍或至少12倍。
在一些实施方案中，前述的融合蛋白可以阻断PD-1与其配体的结合；在一些实施方案中，前述的融合蛋白阻断PD-1与PD-L1结合的IC50值小于5nM，小于2.5nM或小于2nM、或小于1.7nM、或小于1.4nM，其中所述的IC50值可通过测试例12的方案检测获得。
在一些实施方案中，前述的融合蛋白可以阻断PD-1与其配体的结合；在一些实施方案中，前述的融合蛋白阻断PD-1与PD-L2结合的IC50值小于10nM，小于8nM或小于5nM、或小于4.0nM、或小于3.5nM，其中所述的IC50值可通过测试例12的方案检测获得。
在一些实施方案中，前述的融合蛋白可以抑小鼠移植瘤的生长。在一些实施方案中，给药量为1mpk时，所述融合蛋白对MC38的移植瘤的抑瘤率大于50％，大于70％，大于80％，或大于85％。
在一些具体的实施方案中，前述的融合蛋白是单体；例如当靶向部分呈现为单链结构时，TGF-βRII胞外区通过接头融合至单链抗体的羧基端或氨基端。
在另一些具体的实施方案中，前述的融合蛋白是二聚体，例如同二聚体或异二聚体；优选同二聚体。
本披露还提供一种药物组合物，其含有治疗有效量的前述的融合蛋白，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
在一些实施方案中，所述治疗有效量为单位剂量的组合物中含有0.1mg-3000mg的如前所述的融合蛋白。
在一些实施方案中，本披露还提供一种核酸分子，其编码前述任一项所述的融合蛋白。
在一些实施方案中，本披露还提供一种宿主细胞，其包含前述的核酸分子； 所述宿主细胞选自细菌、酵母菌和哺乳动物细胞；优选哺乳动物细胞。更优选地，所述哺乳动物细胞为非人细胞。
在一些实施方案中，本披露还提供一种治疗或预防肿瘤的方法，所述方法包括给予所需患者治疗有效量或预防有效量的前述的融合蛋白，或前述的药物组合物，或前述的核酸分子；优选地，其中所述的肿瘤为PD-1相关的癌症。
在一些实施方案中，本披露还提供一种前述的融合蛋白，或前述的药物组合物，或前述的核酸分子在制备用于治疗或预防肿瘤的药物中的用途，优选地，其中所述的肿瘤为PD-1相关的癌症。
在一些实施方案中，本披露还提供一种前述的融合蛋白，或前述的药物组合物，或前述的核酸分子用作药物，优选用作治疗肿瘤的药物，更优选用作治疗PD-1相关的癌症的药物。
在一些实施方案中，前述的肿瘤选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；优选地，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；所述肺癌选自：非小细胞肺癌和小细胞肺癌；所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；最优选的，所述疾病选自：黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌、食管癌、肝癌和结肠癌。
附图说明
图1：TGF-β受体融合蛋白结构示意图；
图2：抗PD-1抗体阻断PD-1与其配体的结合测试结果；
图3：抗PD-1抗体对PBMC细胞分泌IFNγ的影响；
图4：抗PD-1抗体对小鼠结肠癌MC38移植瘤的疗效；
图5：抗PD-1抗体对转人PD-1基因小鼠结肠癌MC38移植瘤体积的影响；
图6：融合蛋白与稳定转染PD-1的CHO-S细胞的亲和力检测结果；
图7：融合蛋白的体内药效试验。
具体实施方式
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本领域的一般技术人员通常理解的含义。
术语“程序性死亡1”、“细胞程序性死亡1”、“蛋白PD-1”、“PD-1”、“PDCD1”和“hPD-1”可互换使用，且包括人PD-1的变体、同种型、物种同源物、以及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以GenBank登录号U64863找到。
术语“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种为PD-L2)，它在与PD-1结合时下调T细胞活化和细胞因子分泌。如本文中使用的术语“PD-L1”包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型、和种间同源物，以及与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列可以用GenBank登录号Q9NZQ7查到。
术语“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的一般术语。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：IL-2、IFN-γ、IL-6、TNFα、IL-17和IL-5。
本披露所述的“免疫调节分子”可用于削弱癌细胞的免疫耐受性。本披露采用TGF-βRII胞外结构域(也称TGF-βRII胞外区)，作为融合蛋白中免疫调节分子部分。本披露中所述的TGF-βRII胞外结构域包含全长形式和N端的截短形式。在一些实施方案中，本披露采用TGF-βRII胞外结构域的N端的截短形式作为融合蛋白中免疫调节分子部分。
“TGF-β受体II(TGF-βRII)”是指可结合配体(包括但不限于TGF-β1、TGF-β2和TGF-β3)细胞表面受体。TGF-βRII/TGF-β复合物招募TGF-βRI以形成信号转导复合物(Won等，Cancer Res.1999；59：1273-7)。
全长TGF-βRII的胞外结构域是TGF-βRII细胞外自N端开始的一段长136个氨基酸残基的肽段。其他长度约为136个氨基酸，并且来源于人的具有TGF-βRII的胞外区功能，能够与TGF-β1、TGF-β2和TGF-β3相结合的变体同样属于本披露的TGF-βRII的胞外结构域的范围。本披露所述的“TGF-βRII胞外结构域N端截短形式”或“TGF-βRII胞外区的N端截短形式”为从TGF-βRII胞外结构域的N端开始截短，即从N端开始的连续的氨基酸缺失后获得的TGF-βRII胞外结构域，优选从TGF-βRII胞外结构域的N端开始连续的26个以下的氨基酸缺失，优选14-26个氨基酸的缺失，更优选N端14-21个氨基酸的缺失，最优选N端19或21个连续氨基酸缺失。
本披露中的“TGF-βRII胞外结构域”与“TGF-βRII胞外区”可相互替换。
本披露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。
本披露所述的“抗体”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(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(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。
本披露的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人抗体，优选人源化抗体。
术语“鼠源抗体”在本披露中为根据本领域知识和技能制备的针对人PD-1(或其表位)的单克隆抗体。制备时用PD-1抗原(或其表位)注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本披露一个优选的实施方案中，所述的鼠源抗PD-1抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种抗体的恒定区融合而成的抗体，可以减轻异源抗体诱发的免疫应答反应。例如，建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本披露一个优选的实施方案中，所述的抗PD-1嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的抗PD-1嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人物种的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的 公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。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。本披露的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变、L234A和/或L235A突变、S228P突变、和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座的元素元件引入到源于胚胎干细胞系的生物体的细胞株中，这些细胞系中的内源性重链和轻链基因座被靶向破坏。转基因生物可以合成对人抗原特异的人抗体，并且该生物可以用于产生人抗体-分泌杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或更多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指重链包含Fc区的抗体。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(或其表位)的能力的一个或更多个片段。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区的二硫桥连接的两个Fab片段的二价片段，(iii)Fd片段，通常由VH和CH1结构域组成；(iv)Fv片段，通常由抗体的单臂VH和VL结构域组成；(v)单结构域或dAb片段(Ward等人，(1989)Nature341：544-546)，其由VH结构域组成；(vi)单链Fv(scFv)。虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使 得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如Bird等人(1988)Science 242：423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85：5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
本披露的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)等。
Fab是通过用酶处理IgG抗体分子所获得的具有约50，000分子量的片段，并具有抗原结合活性，其中重链N端侧的约一半和整个L链通过二硫键结合在一起。
本披露的Fab可以通过用酶处理本披露单克隆抗体来生产。此外，可以通过将编码所述Fab的DNA插入到原核生物表达载体或真核生物表达载体中，并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab')2是通过用酶消化IgG铰链区中二硫键的下游部分而获得的片段，并具有抗原结合活性，并包含在铰链位置相连的两个Fab区。
本披露的F(ab')2可以通过用酶处理本披露的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接Fab'来生产所述F(ab')2。
Fab'是通过切割上述F(ab')2中铰链区的二硫键而获得的片段，并具有抗原结合活性。本披露的Fab'可以通过用还原剂处理本披露的F(ab')2来生产。
此外，可以通过将编码Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中，并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成(例如Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90：6444-6448)。可用于本披露的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immuno l.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。
本披露的scFv可以通过以下步骤来生产，例如：获得本披露的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个 抗原可以是相同或不同的。
本披露的双抗体可以通过以下步骤来生产，例如：获得本披露的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv例如可以通过以下方式获得，例如：将VH和VL中的一个氨基酸残基取代为半胱氨酸残基，通过半胱氨酸残基之间形成二硫键而获得的片段。可以按照已知方法(Protein Engineering，7，697(1994))来选择被半胱氨酸残基取代的氨基酸残基。
本披露的dsFv可以通过以下步骤来生产，例如：获得本披露的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变、突变、或修饰，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失、替换、或修饰。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.，Immunologist，7，132-136(1999))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为27-38(CDR1)、56-65(CDR2)和105-117(CDR3)，VL中的CDR氨基酸残基编号大致为27-38(CDR1)、56-65(CDR2)和105-117(CDR3)。
术语“表位”或“抗原决定簇”是指抗原上被免疫球蛋白(或抗体)所特异性结合的部位(例如，PD-1分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合PD-1，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
“Linker”或“接头”或“连接子”或“连接片段”指用于连接蛋白质结构域的连接性多肽序列，通常具有一定的柔性，接头的使用不会使蛋白质结构域原有的功能丧失。
本文中使用的术语“核酸分子”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或单链mRNA或修饰的mRNA。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“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人PD-1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http：//imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢 杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本披露工程化的融合蛋白或抗体及其抗原结合片段可用常规方法制备和纯化。比如，编码第一链和第二链的cDNA序列，可以克隆并重组至表达载体。重组的表达载体可以稳定地转染细胞。作为一种更推荐的现有技术，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端位点。通过筛选获得阳性克隆，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。分泌了融合蛋白的培养液可以用常规技术纯化。比如，用A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，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。
“施用”、“给予”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。“施用”、“给予”和“处理”可以指例如治疗、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。“施用”、“给予”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。“处理”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。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本披露的任一种融合蛋白的组合物，所述受试者具有一种或多种疾病症状，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，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。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。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个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。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频 繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
表1.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指获得任一种或多种有益的或所需的预防/治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本披露靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本披露靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或体外产生的物质。
“内源性”指根据情况在生物、细胞或体内产生的物质。
“同源性”或“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碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的(或同一的)。两个序列之间的同源性(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％同源或同一；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源或同一。通常，当比对两个序列时进行比较以给出最大百分比同源性或同一性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS)：Altschul，S.F.等人，(1990)J.Mol.Biol.215：403-410；Gish，W.等人，(1993)Nature Genet.3：266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266：131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25：3389-3402；Zhang，J.等人，(1997)Genome Res.7：649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。本披露中的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。
本文使用的表述“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后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑传代数目。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。在意指不同名称的情况下，其由上下文清楚可见。
“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。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示一种混合物，其含有一种或多种本文所述的融合蛋白或其生理学上/可药用的盐或前体药物、与其他化学组分；所述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“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本披露包括用于治疗与目标抗原(例如PD-1)阳性细胞相关的疾病的药剂，所述药剂包含本披露的融合蛋白作为活性成分。
本披露中与PD-1相关的癌症没有限制，只要它是与PD-1相关的疾病即可， 例如利用本披露的融合蛋白诱导的治疗反应可通过结合PD-1，然后阻遏PD-1与其配体的结合，或杀伤肿瘤细胞。因此，当处于适于治疗应用的制备物和制剂中时，本披露的融合蛋白对这样一些受试者是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌、肝癌、食管癌等。
上述PD-1相关的癌症可以通过用的抗PD-1抗体或其抗原结合片段检测或测定表达PD-1的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本披露中，对用于检测或测定目标抗原(例如PD-1)的活体样品没有特别限制，只要它具有包含表达目标抗原(例如PD-1)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
实施例
以下结合实施例进一步描述本披露，但这些实施例并非限制着本披露的范围。本披露实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗人PD-1鼠源抗体获得
抗原的制备
抗原构建和筛选：
设计并合成人PD-1-IgG1Fc融合蛋白，N端为人PD-1胞外区150个氨基酸，C端为人IgG1的Fc段(hIgG1Fc)。经Protein A的亲和柱纯化，可获得高纯度的重组PD-1-Fc蛋白，用于检测抗PD-1抗体与抗原的结合。
人PD-1-IgG1Fc(SEQ ID NO：1；信号肽+胞外区+hIgG1Fc)：
Figure PCTCN2020128045-appb-000001
注释：下划线部分为信号肽，正体部分为人PD-1胞外区，斜体部分为hIgG1Fc。
人PD-1-his(SEQ ID NO：2)：
Figure PCTCN2020128045-appb-000002
转染细胞核酸编码的PD-1抗原(SEQ ID NO：3)：
Figure PCTCN2020128045-appb-000003
抗体的制备
抗人PD-1抗体可通过免疫小鼠产生，也可通过抗人PD-1噬菌体小鼠免疫文库获得。
通过免疫小鼠制备抗人PD-1抗体的方法如下：
1.免疫：实验用SJL白小鼠，雌性，6-8周龄和Balb/c白小鼠，雌性，6-8周龄。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按不同方案免疫，每组6-10只。
免疫抗原可以是纯化的重组蛋白PD-1-IgG1Fc(见SEQ ID NO：1)、PD-1-his(见SEQ ID NO：2)、或PD-1作为抗原(见SEQ ID NO：3)转染的Jurkat/CHO-PD-1细胞，可以单独一种试剂配合不同的免疫佐剂或者不同类型免疫原交叉免疫。免疫部位可以是腹腔或者背部皮下，或者两种位置交替免疫。
免疫佐剂
Figure PCTCN2020128045-appb-000004
Gold Adjuvant(以下简称Titermax，购自Sigma货号T2684)与Imject Alum Adjuvant(以下简称Alum，购自Pierce货号77161)交叉免疫。抗原与佐剂(Titermax)比例为1：1，抗原与佐剂(Alum)比例为3：1，25-50μg/只(首次免疫)，50μg/只(加强免疫)，或是1×10 7个Jurkat/CHO-PD-1细胞/只。第0天腹膜内注射25-50μg/只的乳化后抗原，首次免疫后每周一次或是每两周一次，Titermax和Alum交替使用，共5-8次。
2.细胞融合：选择血清中抗体滴度高的小鼠进行脾细胞融合，将冲刺免疫72小时后的小鼠眼球放血，拉颈处死，放入75％乙醇中消毒。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(中国科学院)进行融合得到杂交瘤细胞。
融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×10 5/150μl/孔)，37℃，5％CO 2孵育，种板10-30块左右。融合后的第5天加入HAT完全培养基，50μl/孔，37℃，5％CO 2孵育。融合后第7天至8天，根据细胞生长密度，换液，200μl/ 孔，37℃，5％CO 2孵育。
3.杂交瘤细胞筛选：融合后第7-9天，根据细胞生长密度，进行抗体与PD-1结合的ELISA方法检测，并将检测的阳性孔细胞进行PD-1/PDL1结合的阻断ELISA检测，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选为阳性的进行保种，并进行第二次或第三次亚克隆，直至获得单细胞克隆。多次融合获得有阻断PD-1与PDL1结合效果的杂交瘤细胞。
4.通过抗人PD-1噬菌体小鼠免疫文库获得抗人PD-1抗体的方法如下：
构建抗人PD-1噬菌体小鼠免疫文库：选择血清中抗体滴度高的小鼠的脾脏，用Trizol(Invitrogen Cat No.15596-018)提取组织总RNA。使用PrimeScript TMII 1st Strand cDNA Synthesis Kit试剂盒(Takara Cat No.6210A)进行反转录获得cDNA。根据IMGT数据库设计并合成构建文库的引物。通过三轮PCR反应，获得单链抗体片段。将单链抗体片段和经过改造的建库载体pCantab5E(Amersham Biosciences/GE Cat No.27-9400-01)用Sfi1(NEB Cat No.#R0123L)进行酶切，电泳后用
Figure PCTCN2020128045-appb-000005
Gel Extraction Kit(Omega Cat No.D2500-02)进行纯化回收。然后用T4DNA连接酶(NEB Cat No.#M0202L)16℃连接16-18小时，再用上述试剂盒进行纯化回收，最后用去离子水洗脱。取1μg连接产物与1支电转化感受态TG1(Lucigen Cat No.60502-2)混合，电转化仪(Bio Rad Micropulser)参数设至2.5kV，200Ω，25uF，进行电转化。重复转化10次，涂平板，37℃倒置培养16-18小时。将所有菌落刮洗下来混和在一起，加入终浓度为15％的甘油，-80℃保存备用。
5.抗人PD-1噬菌体小鼠免疫文库的筛选：将包装好的抗人PD-1噬菌体免疫文库(1×10 12-1×10 13)与100μl链菌素微珠(Mi1envi Biotec，Auburn，CA)加入1m1含2％脱脂牛奶-磷酸盐缓冲液(缩写MPBS)中于室温下孵育1小时，放置在磁力架上，取上清。上清加入10μg/ml生物素化的人PD-1-ECD-his蛋白(购自Sino Biological)中于室温下孵育1小时，再加入100μl链霉亲和素包被的磁珠(1ml MPBS预孵育)于室温下孵育1小时。并使其负载于磁力架系统上用于分选，吸去上清。加入1ml PBST(含0.1％Tween-20的磷酸盐缓冲液)，翻转多次，吸尽后再加入新鲜洗液，重复11次，以去除未结合的抗体片段，加入0.5ml洗脱液(50μl10mg/ml胰蛋白酶存储液加入450μl PBS中)。室温下摇晃15min。放置在磁力架上，吸出上清至一新EP管中。TG1接入2YT培养基中扩增至培养细菌密度OD600＝0.4时。每管加入1.75ml TG1(OD600＝0.4)，并加入250μl洗脱后phage(噬菌体)，37℃水浴中静置孵育30min，梯度稀释涂板，用于测试滴度。其余TG1溶液离心，涂板，37℃过夜孵育。
噬菌体小鼠免疫文库利用生物素化的人PD-1-ECD-his抗原，经过2-3轮MACS筛选(链霉素磁珠，Invitrogen)，最终获得具有结合PD-1和阻断PD-1与PD-L1 结合的单克隆，测序验证，得到抗体的可变区序列。
重组抗原蛋白/抗体的纯化
1.杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清纯化首选ProteinG进行亲和层析，将培养所得杂交瘤离心取上清，根据上清体积加入10-15％体积的1M Tris-HCl(pH8.0-8.5)调节上清pH。ProteinG柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M醋酸/醋酸钠(pH3.0)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换(如，利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻替换成所需的缓冲体系，或者利用分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度)。
2.Protein A亲和层析纯化蛋白或抗体：
首先将表达抗原蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换(同上)。
将经前述方法获得的抗人PD-1鼠源抗体进行抗原结合实验，筛选得到2株活性良好的克隆：M23和M33，将单细胞克隆扩培养，提取RNA，利用小鼠-Ig的简并引物进行反转录扩增(RT-PCR)，得到抗体的可变区序列。将该鼠抗体可变区序列与人抗体恒定区序列连接，克隆并重组表达出该鼠单克隆抗体的嵌合抗体，进行体外活性实验，确认所得到的单克隆抗体可变区序列正确。
测得鼠源抗体M23和M33的可变区序列如下：
鼠源抗体M23的重链可变区(SEQ ID NO：4)：
Figure PCTCN2020128045-appb-000006
鼠源抗体M23的轻链可变区(SEQ ID NO：5)：
Figure PCTCN2020128045-appb-000007
鼠源抗体M33的重链可变区：(SEQ ID NO：6)
Figure PCTCN2020128045-appb-000008
鼠源抗体M33的轻链可变区：(SEQ ID NO：7)
Figure PCTCN2020128045-appb-000009
备注：上述抗体的重链可变区和轻链可变区序列中，下划线为Kabat编号系统确定的CDR序列，依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
表2.鼠源抗体M23和M33重链及轻链CDR区序列
Figure PCTCN2020128045-appb-000010
备注：表中的抗体CDR序列是根据Kabat编号系统确定。
实施例2.抗人PD-1单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与M23，M33的轻重链序列同一性高的重轻链可变区种系基因作为模板，将这2个鼠源抗体的CDR分别移植到相应的人源抗体模板中，分别构建其对应的人源化抗体。
1.鼠源抗体M23人源化
1.1鼠源抗体M23人源化构架选择
鼠源抗体M23的人源化轻链模板为IGKV2-40*01和IGKJ4*01，人源化重链模板为IGHV1-69*02和IGHJ6*01，人源化改造后可变区序列如下(下划线为CDR序列)：
Hu23VH-CDR移植：(SEQ ID NO：20)
Figure PCTCN2020128045-appb-000011
Figure PCTCN2020128045-appb-000012
Hu23VL-CDR移植：(SEQ ID NO：21)
Figure PCTCN2020128045-appb-000013
1.2人源化抗体的回复突变设计
表3.人源化抗体回复突变设计
Figure PCTCN2020128045-appb-000014
注：移植(Grafted)代表鼠抗体CDR植入人种系FR区序列；氨基酸残基由Kabat编号系统确定并注释，如I2G表示依照Kabat编号系统，将Kabat编号的第2位I突变回G。
M23的人源化抗体轻/重链可变区序列如下：
>Hu23VL1(同Hu23VL-CDR移植)：(SEQ ID NO：21)
Figure PCTCN2020128045-appb-000015
>Hu23VL2(SEQ ID NO：22)
Figure PCTCN2020128045-appb-000016
>Hu23VH1(同Hu23VH-CDR移植)：(SEQ ID NO：20)
Figure PCTCN2020128045-appb-000017
>Hu23VH2(SEQ ID NO：23)
Figure PCTCN2020128045-appb-000018
>Hu23VH3(SEQ ID NO：24)
Figure PCTCN2020128045-appb-000019
>Hu23VH4(SEQ ID NO：25)
Figure PCTCN2020128045-appb-000020
1.3鼠源抗体M23的人源化序列组合
鼠源抗体M23的人源化模板进行回复突变设计后，不同的可变区回复突变组合成不同的人源化抗体可变区，具体见下表。
表4.Hu23人源化抗体可变区组合
Figure PCTCN2020128045-appb-000021
注：表中例如“Hu23-1”指代抗体轻链可变区为Hu23VL1，且重链可变区为Hu23VH1，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23-1)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：52所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：50或51所示的IgG4-AA重链恒定区连接形成抗体重链。
2.鼠源抗体M33人源化
2.1鼠源抗体M33人源化构架选择
鼠源抗体M33的人源化轻链模板为IGKV1-39*01和IGKJ4*01，人源化重链模板为IGHV3-7和IGHJ6*01，人源化可变区序列如下：
Hu33VH-CDR移植(SEQ ID NO：26)：
Figure PCTCN2020128045-appb-000022
Hu33VL-CDR移植(SEQ ID NO：27)：
Figure PCTCN2020128045-appb-000023
2.2人源化抗体的回复突变设计
表5.鼠源抗体M33人源化抗体轻/重链可变区序列
Figure PCTCN2020128045-appb-000024
注：移植(Grafted)代表鼠抗体CDR植入人种系FR区序列。氨基酸残基由 Kabat编号系统确定并注释，如F71Y表示依照Kabat编号系统，将Kabat编号的第71位F突变回Y。
鼠源抗体M33的人源化抗体轻链可变区和重链可变区序列如下：
>Hu33VL1(同Hu33VL-CDR移植)：(SEQ ID NO：27)
Figure PCTCN2020128045-appb-000025
>Hu33VL2(SEQ ID NO：28)
Figure PCTCN2020128045-appb-000026
>Hu33VL3(SEQ ID NO：29)
Figure PCTCN2020128045-appb-000027
>Hu33VH1(同Hu33VH-CDR移植)：(SEQ ID NO：26)
Figure PCTCN2020128045-appb-000028
>Hu33VH2(SEQ ID NO：30)
Figure PCTCN2020128045-appb-000029
>Hu33VH3(SEQ ID NO：31)
Figure PCTCN2020128045-appb-000030
3.鼠源抗体M33的人源化序列组合
表6.人源化抗体轻重链可变区组合
Figure PCTCN2020128045-appb-000031
注：表中例如“Hu33-6”指代抗体轻链可变区为Hu33VL2且重链可变区为Hu33VH3，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu33-6)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻 链可变区与SEQ ID NO：52所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：50或51所示的IgG4-AA重链恒定区连接形成抗体重链。
4.人源化抗体的突变体
Hu23人源化抗体的突变抗体
通过计算机模拟，对Hu23人源化抗体的轻链LCDR1(SEQ ID NO：11)的特定位点氨基酸进行定点突变，具体突变见表7：
表7.Hu23轻链LCDR1的突变序列：
Figure PCTCN2020128045-appb-000032
注：Hu23LCDR1(N28Q)表示对Hu23人源化抗体轻链可变区Hu23VL1或Hu23VL2的第28位N突变为Q后可变区的LCDR1突变序列，Hu23LCDR1(G29A)表示对Hu23人源化抗体轻链可变区Hu23VL1或Hu23VL2的第29位G突变为A后可变区的LCDR1突变序列(氨基酸位置和CDR通过Kabat编号系统确定)，其他类推。
LCDR1突变后的Hu23人源化抗体轻链可变区序列如下：
>Hu23VL1(N28Q)序列为：
Figure PCTCN2020128045-appb-000033
>Hu23VL1(N28L)序列为：
Figure PCTCN2020128045-appb-000034
>Hu23VL1(N28T)序列为：
Figure PCTCN2020128045-appb-000035
>Hu23VL1(N28D)序列为：
Figure PCTCN2020128045-appb-000036
Figure PCTCN2020128045-appb-000037
>Hu23VL1(G29A)序列为：
Figure PCTCN2020128045-appb-000038
>Hu23VL1(G29V)序列为：
Figure PCTCN2020128045-appb-000039
>Hu23VL2(N28Q)序列为：
Figure PCTCN2020128045-appb-000040
>Hu23VL2(N28L)序列为：
Figure PCTCN2020128045-appb-000041
>Hu23VL2(N28T)序列为：
Figure PCTCN2020128045-appb-000042
>Hu23VL2(N28D)序列为：
Figure PCTCN2020128045-appb-000043
>Hu23VL2(G29A)序列为：
Figure PCTCN2020128045-appb-000044
>Hu23VL2(G29V)序列为：
Figure PCTCN2020128045-appb-000045
表8.Hu23人源化抗体轻/重链可变区组合
Figure PCTCN2020128045-appb-000046
Figure PCTCN2020128045-appb-000047
备注：表中例如“Hu23-11”指代抗体轻链可变区为Hu23VL1(N28T)且重链可变区为Hu23VH1，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23-11)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：52所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：50或51所示的IgG4-AA重链恒定区连接形成抗体重链。
对突变后得到的抗体进行亲和力检测，实验结果显示，Hu23LCDR1(N28Q)、Hu23LCDR1(N28L)、Hu23LCDR1(N28T)、Hu23LCDR1(N28D)、Hu23LCDR1(G29A)、Hu23LCDR1(G29V)位点突变后的人源化抗体均保持与PD-1的结合能力(表9)。
5.人源化抗体的筛选
通过Biacore进行不同人源化抗体的亲和力检测(方法参见测试例3)，结果见表9，结果显示不同的人源化抗体保持了对于PD-1的结合能力，部分人源化抗体的亲和力甚至和其鼠源抗体基本接近。
表9.Hu23人源化抗体与人PD-1的亲和力
Figure PCTCN2020128045-appb-000048
Figure PCTCN2020128045-appb-000049
实施例3.构建和表达PD-1人源化抗体
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-Fc-pHr/VK-CL-pHr。IgG4-AA代表F234A(对应于序列SEQ ID NO：50的第114位)、L235A(对应于序列SEQ ID NO：50第115位)突变和S228P(对应于序列SEQ ID NO：50的第108位)，IgG4-AA可以通过IgG4抗体形式简单点突变获得。此外，可选的，将IgG4恒定区的最后一位氨基酸K突变为A，该突变并不会影响抗体的活性。示例性的轻链恒定区为Kappa链的恒定区。
IgG4-AA重链恒定区变体序列如下(SEQ ID NO：50)：
Figure PCTCN2020128045-appb-000050
IgG4-AA重链恒定区序列如下(SEQ ID NO：51)：
Figure PCTCN2020128045-appb-000051
Kappa链恒定区序列如下(SEQ ID NO：52)：
Figure PCTCN2020128045-appb-000052
构建的IgG4AA形式全长抗体序列示例性列举如下：
Hu23-11(A)抗体重链(SEQ ID NO：53)：
Figure PCTCN2020128045-appb-000053
Hu23-11抗体重链(SEQ ID NO：54)：
Figure PCTCN2020128045-appb-000054
Hu23-11(A)/Hu23-11抗体轻链(SEQ ID NO：55)：
Figure PCTCN2020128045-appb-000055
Hu33-5(A)抗体重链(SEQ ID NO：56)：
Figure PCTCN2020128045-appb-000056
Hu33-5抗体重链(SEQ ID NO：57)：
Figure PCTCN2020128045-appb-000057
Figure PCTCN2020128045-appb-000058
Hu33-5(A)/Hu33-5抗体轻链(SEQ ID NO：58)：
Figure PCTCN2020128045-appb-000059
Hu33-6(A)抗体重链(SEQ ID NO：59)：
Figure PCTCN2020128045-appb-000060
Hu33-6抗体重链(SEQ ID NO：60)：
Figure PCTCN2020128045-appb-000061
Hu33-6(A)/Hu33-6抗体轻链(SEQ ID NO：58)：
Figure PCTCN2020128045-appb-000062
实施例4.融合蛋白PD-1/TGF-β trap克隆和表达
采用TGF-βRII胞外结构域(也称TGF-βRII胞外区)作为融合蛋白中免疫调节分子部分，将PD-1抗体作为融合蛋白的靶向部分，形成PD-1抗体/TGF-βRII 胞外区的融合蛋白(PD-1/TGF-β trap)。研究发现，包含TGF-βRII胞外结构域截短形式的融合蛋白的结构和功能较为稳定，尤其是在TGF-βRII胞外结构域的N端作19个氨基酸的截短后，其融合蛋白具有更高的表达量和稳定的结构。本披露中的TGF-βRII胞外结构域及其截短形式的非限制性实施例序列如下：
TGF-βRII胞外结构域全长序列：ECD(1-136)
Figure PCTCN2020128045-appb-000063
TGF-βRII胞外结构域序列在N端有19个氨基酸的截短：ECD(20-136)
Figure PCTCN2020128045-appb-000064
TGF-βRII胞外结构域序列在N端有21个氨基酸的截短：ECD(22-136)
Figure PCTCN2020128045-appb-000065
TGF-βRII胞外结构域序列在N端有14个氨基酸的截短：ECD(15-136)
Figure PCTCN2020128045-appb-000066
利用同源重组技术将本披露抗PD-1抗体的重链C末端氨基酸通过常规的接头连接不同长度TGF-βRII胞外区，与轻链一起，通过293表达系统进行常规表达，得到PD-1/TGF-β trap融合蛋白，常见的接头包括但不限于：
(ASTKGP)n          SEQ ID NO：65；
(ASTKGPSVFPLAP)n   SEQ ID NO：66；
(TVAAP)n           SEQ ID NO：67；
(TVAAPSVFIFPP)n    SEQ ID NO：68；或
(G 4S)nG；
其中n为1-6的整数，优选为4或5，非限制性的实施例如表10所示，本披露的融合蛋白结构如图1所示。
表10：PD-1/TGF-β trap融合蛋白
融合蛋白例 序列描述 N端连续氨基酸缺失数
融合蛋白1 Hu23-11(A)-(G 4S) 4G-ECD(1-136) 未缺失
融合蛋白2 Hu33-5(A)-(G 4S) 4G-ECD(1-136) 未缺失
融合蛋白3 Hu23-11(A)-(G 4S) 4G-ECD(20-136) 19
融合蛋白4 Hu33-5(A)-(G 4S) 4G-ECD(20-136) 19
融合蛋白5 Hu33-6(A)-(G 4S) 4G-ECD(1-136) 未缺失
融合蛋白6 Hu33-6(A)-(G 4S) 4G-ECD(20-136) 19
融合蛋白7 Hu23-11(A)-(G 4S) 4G-ECD(15-136) 14
融合蛋白8 Hu23-11(A)-(G 4S) 4G-ECD(22-136) 21
融合蛋白9 Hu33-5(A)-(G 4S) 4G-ECD(15-136) 14
融合蛋白10 Hu33-5(A)-(G 4S) 4G-ECD(22-136) 21
融合蛋白11 Hu33-6(A)-(G 4S) 4G-ECD(15-136) 14
融合蛋白12 Hu33-6(A)-(G 4S) 4G-ECD(22-136) 21
注：Hu23-11(A)、Hu33-5(A)和Hu33-6(A)为本披露所述抗PD-1抗体，序列描述中ECD(n-136)为TGF-βRII胞外区的全长或截短形式，n为TGF-βRII胞外区截短后的氨基酸起始位数。
本披露中的非限制性的融合蛋白的序列如下所示：
融合蛋白1的第一链(即抗Hu23-11(A)抗体重链-(G 4S) 4G-ECD(1-136))：
Figure PCTCN2020128045-appb-000067
融合蛋白1的第二链(同Hu23-11抗体轻链序列)：
Figure PCTCN2020128045-appb-000068
融合蛋白2的第一链(即抗Hu33-5(A)抗体重链-(G 4S) 4G-ECD(1-136))：
Figure PCTCN2020128045-appb-000069
Figure PCTCN2020128045-appb-000070
融合蛋白2的第二链(同Hu33-5抗体轻链序列)：
Figure PCTCN2020128045-appb-000071
融合蛋白3的第一链(即抗Hu23-11(A)抗体重链-(G 4S) 4G-ECD(20-136))：
Figure PCTCN2020128045-appb-000072
融合蛋白3的第二链(同Hu23-11抗体轻链序列)：
Figure PCTCN2020128045-appb-000073
融合蛋白4的第一链(即抗Hu33-5(A)抗体重链-(G 4S) 4G-ECD(20-136))：
Figure PCTCN2020128045-appb-000074
Figure PCTCN2020128045-appb-000075
融合蛋白4的第二链(同Hu33-5抗体轻链序列)：
Figure PCTCN2020128045-appb-000076
融合蛋白5的第一链(即抗Hu33-6(A)抗体重链-(G 4S) 4G-ECD(1-136))：
Figure PCTCN2020128045-appb-000077
融合蛋白5的第二链(同Hu33-6抗体轻链序列)：
Figure PCTCN2020128045-appb-000078
融合蛋白6的第一链(即抗Hu33-6(A)抗体重链-(G 4S) 4G-ECD(20-136))：
Figure PCTCN2020128045-appb-000079
Figure PCTCN2020128045-appb-000080
融合蛋白6的第二链(同Hu33-6抗体轻链序列)：
Figure PCTCN2020128045-appb-000081
备注：上述融合蛋白的第一链序列中，划线部分为PD-1抗体重链，斜体为接头，点划线部分为TGF-βRII胞外区。
编码抗PD-1抗体的核苷酸序列、编码TGF-βRII胞外区的核苷酸序列、接头蛋白片段((G 4S) 4G)n的核苷酸序列通过所属领域常规技术手段获得。利用同源重组技术将抗PD-1抗体的重链的C末端核苷酸通过接头蛋白连接不同长度TGF-βRII胞外区的N末端核苷酸，克隆到Phr-BsmbI载体上。重组的PD-1/TGF-β trap在293细胞等其他工程化的细胞中进行表达，通过实施例5进行纯化。纯化的蛋白可用于下述各实施例实验中。
实施例5：PD-1/TGF-β trap融合蛋白纯化
细胞培养液高速离心后收集上清，利用亲和层析进行第一步纯化。层析介质为与Fc相互作用的Protein A或者衍生填料，如GE的MabSelect SuRe。平衡缓冲液为1×PBS(137mmol/L NaCl，2.7mmol/L KCl，10mmol/L Na 2HPO 4，2mmol/L KH 2PO 4，pH7.4)，平衡5倍柱体积后，将细胞上清上样结合，流速控制为样品在柱上保留时间≧1min。上样结束后，用1×PBS(pH7.4)冲洗柱子，直至A280紫外吸收降至基线。然后用0.1M乙酸(pH3.5)的洗脱缓冲液冲洗层析柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样品用1M Tris(pH8.0)中和。
将上述中和后的洗脱样品超滤浓缩后进行体积排阻层析，缓冲液为1×PBS，层析柱为XK26/60 Superdex200GE)，流速控制在4mL/min，上样体积小于5mL，根据A280紫外吸收合并目的蛋白峰。收集的蛋白经SEC-HPLC鉴定纯度大于95％，经LC-MS鉴定为正确后分装备用，得到PD-1/TGF-β trap融合蛋白。
测试例
测试例1.抗PD-1抗体在体外阻断PD-1对其配体的结合的ELISA实验
肿瘤细胞表面的PD-L1通过和T细胞表面的PD-1的结合，从而对T细胞的增殖起到抑制的效果。抗PD-1的抗体能通过和PD-1结合，阻断PD-L1/PD-1的信号通路，进而刺激T细胞的增殖。阻断PD-1/PD-L1的结合实验用于检测抗PD-1抗体对于信号通路的阻断活性。
本实验中，将胞外区与Fc融合的PD-1蛋白(PD-1-Fc，序列见SEQ ID NO：1)包被96孔板后，分别加入待测的抗PD-1抗体(包括抗体：Hu23-11、Hu33-6，阳性对照抗体：H005-1(参见WO2015085847中H005-1抗体)，进行孵育反应；稍后再加入生物素标记的PD-L1/PD-L2，孵育反应。洗板后，检测生物素标记的PD-L1/PD-L2结合量，计算得到抗PD-1抗体对配体PD-L1/PD-L2结合阻断的IC 50值。
用pH 9.6CB缓冲液(1.59g Na 2CO 3和2.93g NaHCO 3溶于1L蒸馏水)将PD-1-Fc稀释至1μg/mL，以100μL/孔的体积加于96孔板中，于4℃放置16h-20h。将96孔板中PBS缓冲液吸掉，用PBST(pH7.4 PBS含0.05％tween20)缓冲液洗板1次后，加入120μL/孔PBST/1％脱脂乳，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板1次后，加入90μl用样品稀释液(pH7.4 PBS含5％BSA，0.05％Tween20)稀释至合适浓度的待测抗PD-1抗体，置4℃预孵育1h。以10μL/孔的体积加入10×浓度的生物素标记PD-L1/PD-L2(北京义翘神州生物技术有限公司)(10μg/ml)，在振荡器上振荡、混匀后，置37℃孵育1h。移去反应体系，用PBST洗板6次后，加入100μL/孔用PBST缓冲液1：400稀释的链霉亲和素-过氧化物酶聚合物，室温振荡孵育50分钟。用PBST洗板6次后，加入100μLl/孔TMB，于室温孵育5-10min。加入100μL/孔1M H 2SO 4终止反应。用酶标仪在450nm处读取吸收值，计算抗PD-1抗体阻断配体PD-L1/PD-L2的结合的IC 50值。数据详见下表。
表11.抗PD-1抗体与PD-1结合及对PD-L1/PD-L2结合的阻断
Figure PCTCN2020128045-appb-000082
本披露示例性抗PD-1抗体Hu23-11和Hu33-6都能够有效阻断PD-1与PD-L1/PD-L2的结合，其阻断活性与阳性对照抗体H005-1相似。
测试例2.示例性抗体和配体的阻断试验
研究抗体对PD-1与PD-L1结合的阻断作用。实验过程简单描述如下：消化 CHOK1/PD-L1细胞(Promega)，按照100μL/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养24小时。使用PBS稀释对照品和样品至所需浓度。计数Jurkat/PD-1细胞(稳定转染有PD-1的Jurkat细胞)，按一定比例种CHOK1/PD-L1细胞的细胞培养板中(90μL/孔)同时加入10μL/孔加入稀释后的抗体(抗体：Hu23-11和Hu33-6，阳性对照抗体：H005-1)，阴性对照IgG4蛋白，抗体梯度稀释浓度为0.3mg/mL、3mg/mL、30mg/mL，置于37℃，5％CO 2培养箱培养5小时。取出细胞培养板，置于室温放置5分钟，然后每孔加入50μl Bio-Glo TMReagent，室温孵育5分钟，读板。实验结果见附图2。
结果表明，本披露中示例性的抗PD-1抗体Hu23-11和Hu33-6能够有效阻断PD-1与PD-L1的结合，其阻断活性与阳性对照抗体H005-1相似。
测试例3.示例性抗体的BIAcore抗体亲和力实验
用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获IgG，人PD-1抗原(Cat.#10377H08H，Sino Biological)、Cyno PD-1抗原(购自Sino Biological)流过芯片表面，Biacore T200仪器实时检测PD-1抗体和抗原PD-1反应信号获得结合和解离曲线。在每个实验循环解离完成后，用10mM甘氨酸-HCl pH1.5的缓冲液将生物传感芯片洗净再生。实验缓冲体系为1×HBS-EP缓冲溶液(Cat#BR-1001-88，GE)。实验结束后用GE Biacore T200 Evaluation 3.0版软件以(1：1)Langmuir模型拟合数据，得出亲和力数值，结果见表12。
表12.抗PD-1抗体与人PD-1和食蟹猴PD-1的亲和力
Figure PCTCN2020128045-appb-000083
结果显示，本披露示例性的抗PD-1抗体Hu23-11和Hu33-6A均能够与人PD-1和猴PD-1结合。
测试例4.在PBMC-T淋巴细胞激活实验中，抗体对细胞分泌IFNγ的作用
为了研究抗PD-1抗体对人原代T淋巴细胞功能的影响，收集和纯化人外周血单核细胞(PBMC)，采用结核菌素(TB)体外刺激5天后，检测细胞因子IFNγ分泌水平。实验过程简单描述如下：
新鲜血液利用Ficoll-Hypaque(17-5442-02，GE)，密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI 1640(SH30809.01，GE)培养基中培养，该培养基中添加10％(v/v)FBS(10099-141，Gibco)，37℃，5％CO 2条件下培养。
新鲜分离纯化的PBMC以RPMI 1640培养基调整密度为2×10 6个/mL，20mL 细胞悬液中加入40μL结核菌素(97-8800，Synbiotics)，37℃，5％CO 2培养箱培养5天。第5天，收集上述培养的细胞离心，重悬至新鲜的RPMI 1640培养基中，调整密度为1.1×10 6个/mL，接种至96孔细胞培养板，每孔90μL。同时加入梯度稀释的抗体样品(包括本披露的抗体：Hu23-11和Hu33-6，阳性对照抗体H005-1，和阴性对照IgG4蛋白，抗体梯度稀释浓度为0.3mg/mL、3mg/mL、30mg/mL)，用PBS(B320，上海源培生物科技股份有限公司)稀释，每孔10μL。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)收集细胞培养上清，采用ELISA的方法(人IFN-γ检测试剂盒(EHC102g.96，欣博盛)，检测IFN-γ的水平。具体操作参考试剂说明书。
试验结果见图3，结果表明本披露的抗PD-1抗体Hu23-11和Hu33-6均能有效激活IFN-γ的分泌，其激活IFN-γ分泌的能力与阳性H005-1对照相似。
测试例5.抗PD-1抗体在转基因PD-1小鼠结肠癌模型MC38中的作用
将MC38细胞5×10 5细胞/小鼠/100μL接种于90只hPD-1TG小鼠(百奥赛图)右肋部皮下，10天后去除肿瘤体积过大过小的动物，按平均肿瘤体积约120mm 3将小鼠随机分为：空白对照(PBS)、阳性对照H005-1 3mpk、Hu23-11 1mpk、Hu23-11 3mpk、Hu33-6 3mpk共5组，每组8只。Day0(第0天)起每周三次腹腔注射各组抗体，第一周给药结束后发现肿瘤被明显抑制，第二、三周调整给药频率为每周一次，共给药5次。每周2次监测肿瘤体积、动物重量并记录数据。当肿瘤体积超过2000mm 3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率(T/C％)＝(T-T 0)/(C-C 0)×100％
抑瘤率(TGI％)＝1-T/C％
其中，T、T 0分别表示抗体给药组试验结束和试验开始时的肿瘤体积，C、C 0分别表示空白对照组试验结束和试验开始时的肿瘤体积。
试验结果见表13和附图4，试验结果表明，与空白对照相比，本披露的抗体均能显著抑制小鼠结肠癌MC38移植瘤的生长。当给药频率为一周给药3次，在第七天检测时，结果显示本披露的抗体的抑瘤率均明显优于阳性对照抗体H005-1；其后给药频率降为一周一次，给药2次后(第21天)，本披露的抗体间药效逐渐拉开差距，且表现出剂量依赖性。而且荷瘤小鼠对抗PD-1抗体均能很好耐受，在整个给药过程中体重平稳上升，无明显药物致体重减轻等症状发生。
表13.抗PD-1抗体对小鼠结肠癌MC38的抑瘤率
Figure PCTCN2020128045-appb-000084
测试例6.抗PD-1抗体在转基因PD-1小鼠结肠癌模型MC38中的作用
转基因PD-1小鼠来源于购买的转基因PD-1小鼠(ISIS INNOVATION LIMITED，University Offices，Wellington Square，Oxford OX1 2JD，England)在Cephrim Biosciences，Inc.培育的第五代小鼠。将MC38细胞以5×10 5个/100μl/只接种到hPD-1转基因小鼠(雌雄各半)右肋后部皮下，待小鼠平均肿瘤体积达到80-100mm 3之间时，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为5组(每组8只)：阴性对照hIgG 30mpk、H005-1 10mpk、H005-1 30mpk、Hu33-6 10mpk、Hu33-6 30mpk。分组给药日期设定为D0(第0天)。分组后腹腔给予各药物，给药周期22天，每两天给药一次，共11次。每周测2次瘤体积，称体重，记录数据。各组动物体重、肿瘤体积均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 5和Excel软件作图，使用学生T检验统计分析。
肿瘤体积(TV)＝0.5236×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T 0)/(C-C 0)×100％
抑瘤率％TGI＝1-T/C％
其中，T、T 0分别表示抗体给药组试验结束和试验开始时的肿瘤体积，C、C 0分别表示空白对照组试验结束和试验开始时的肿瘤体积。
试验结果见表14和附图5所示，试验结果表明，与对照组相比，本披露的抗体能显著抑制小鼠结肠癌MC38移植瘤的生长，其中抑瘤率最高的是Hu33-6 30mpk组，第20天测量时抑瘤率为80.4％。在低剂量组(10mpk)，Hu33-6 10mpk的药效好于阳性对照H005-1-10mpk。
表14.抗PD-1抗体对小鼠结肠癌MC38肿瘤体积影响
Figure PCTCN2020128045-appb-000085
备注：表中各组肿瘤平均体积的单位为：mm 3；
“/”表示：对照组23天后小鼠因肿瘤过大死亡。
测试例7：Biacore检测PD-1/TGF-β trap融合蛋白的体外结合和动力学实验
通过Biacore T200(GE)测定待测分子与人TGF-β1或人PD-1蛋白的亲和力，实验过程描述如下：
用Protein A生物传感芯片亲和捕获PD-1/TGF-β trap，然后于芯片表面流经高浓度的抗原1(人PD1(Sino Biological，Cat.#10377-H08H)或人TGF-β1(Acro，Cat.1G1-H4212#))120s，将抗体上针对抗原1的位点饱和，再上样抗原2(人 TGF-β1或者人PD1)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸(pH 1.5，GE)将生物传感芯片洗净再生。实验中用到的缓冲溶液为HBS-EP缓冲液(GE)。实验得到的数据用BIAevaluation 4.1版软件(GE)以(1：1)Langmuir模型进行拟合，得出如表15所示的亲和力数值。
表15：融合蛋白与TGF-β1或人PD-1的体外亲和力
Figure PCTCN2020128045-appb-000086
*对应序号的融合蛋白见表10。
结果表明，本披露的融合蛋白1，2，3和4均对人TGF-β1以及人PD-1具极高的亲和力。
测试例8：PD-1/TGF-β trap的体外细胞结合实验
待检测融合蛋白或抗体：
(1)融合蛋白3或融合蛋白4。
(2)阴性对照C25：HIV抗体(参见专利US6114143制备)
C25抗体轻链的氨基酸序列：
Figure PCTCN2020128045-appb-000087
C25抗体重链的氨基酸序列
Figure PCTCN2020128045-appb-000088
Figure PCTCN2020128045-appb-000089
实验过程：
将稳定转染有PD-1的CHO-S细胞以300g的转速离心5分钟，丢掉上清，沉淀用预冷封闭缓冲液1％BSA(Beyotime，ST023)重悬，密度为1.0×10 6细胞/毫升。于4℃孵育30分钟后，重悬以每孔100μL加入到96孔板。96孔板在300g的转速下离心5分钟后，弃上清。向每个孔加入100μL一抗(融合蛋白或阴性对照C25)，将细胞重悬，室温孵育30分钟。离心弃上清，加入50μL 1：200稀释的PE标记二抗(Life Technologies，H10104)。将细胞重悬，室温避光孵育30分钟。用流式缓冲液1％BSA(Beyotime，ST023)洗两次细胞，并用1％的多聚甲醛重悬细胞进行固定，进行流式检测，结果见表16和图6。
表16：融合蛋白与稳定转染有PD-1的CHO-S细胞的体外亲和力
抗体 EC50(nM)
C25 /
融合蛋白3 7.4
融合蛋白4 1.36
结果显示，融合蛋白3，融合蛋白4能够剂量依赖地结合稳定转染有PD-1的CHO-S细胞(图6)。
测试例9：体外检测PD-1/PD-L1通路阻断实验
1、测试目的：
为了研究PD-1/TGF-β trap对PD-1/PD-L1信号通路的阻断作用，采用来自Promega公司构建的分别带有人源PD-1和PD-L1受体分子的细胞，进行基于细胞水平上的抗体阻断实验。
2、测试样品：
①抗PD-1抗体：Hu23-11，Hu33-5；
②融合蛋白3，融合蛋白4；
③阴性对照C25；
3、测试过程
取CHO/PD-L1细胞(CS187108，Promega)，消化并用F-12 Nutrient Mixture(Gibco，22400-089)完全培养基重悬细胞，根据细胞计数结果使用完全培养基调整细胞密度至4×10 5/mL，将细胞悬液转移至加样槽中，使用多道移液器以100μL/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养20至24h；第二天制备Jurkat/PD-1(CS187102，Promega)细胞悬液，根据细胞计数结果使用分析培养基重悬细胞，并调整细胞密度至1.25×10 6/mL；将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出，使用多道移液器每孔取出95μL培养液，按照410μL/孔加入梯度稀释的融合蛋白，以及PD-1抗体，然后将Jurkat/PD-1细胞悬液转移至加样 槽中，以490μL/孔加入到细胞培养板中，置于37℃，5％CO 2培养箱培养5至6h。在蛋白孵育期间，将Bio-Glo TM试剂(Promega，G7940)取出使其温度恢复至室温。取出细胞培养板，置于室温放置5至10min，然后每孔加入450μL Bio-Glo TM试剂，置于安全柜中孵育5至10min，使用多功能酶标仪读取化学发光信号值。
4、结果
如表17所示，本披露的融合蛋白3，融合蛋白4均能够有效地阻断表达有PD-1分子的Jurkat细胞同CHO/PD-L1细胞结合。
表17：融合蛋白对PD-1/PD-L1通路的阻断能力
抗体 IC50(nM) 最大阻断倍数
C25 / /
融合蛋白3 12.32 3.56
Hu23-11 6.00 3.6
融合蛋白4 3.03 3.63
Hu33-5 1.82 3.63
注：最大阻断倍数为较空白荧光增强的倍数。
测试例10：SMAD3报告基因抑制实验
1、测试目的
该实验通过HepG2细胞表达带荧光素酶报告基因的Smad3结合原件(SBE)来研究PD-1/TGF-β trap对TGF-β1诱导Smad3活化的抑制作用，根据IC50大小评价PD-1/TGF-β trap的体外活性。
2、测试样品：融合蛋白3、融合蛋白4、阴性对照(C25)。
3、测试过程
HepG2细胞(ATCC，HB-8065 TM)使用含有10％FBS的MEM完全培养基(GE，SH30243.01)培养，每3天传代一次。实验第一天以每孔25,000个细胞的密度接种于96孔板(Corning，3903)，在37℃，5％CO 2条件下培养24小时。第二天，弃去细胞培养板中的培养基，每孔转染100ng 3TP-Lux质粒(普如汀生物技术(北京)有限公司，货号11767)。细胞在37℃，5％CO 2条件下继续培养24小时。加入待测样品前6小时，弃去96孔板中完全培养基，每孔加入80μL不完全培养基(MEM+0.5％FBS)。6小时后再加入10μL使用不完全培养基配制的人TGF-β1(R&D，240-B-010)溶液，终浓度为2ng/mL和10μL待测样品，待测样品的终浓度为500、50、5、0.5、0.05、0.005、0.0005、0.00005、0.000005和0nM，以人TGF-β1溶剂为对照，细胞在37℃，5％CO 2条件下继续培养18h。然后每孔加入100μL配制好的萤光素酶底物ONE-GloTM Luciferase Assay system(promega，E6110)，室温避光放置10分钟，然后使用Victor3多功能酶标仪(Perkin Elmer)读取发光信号值。待测样品的IC50值使用数据处理软件Graphpad Prism5.0计算得到，实验结果表18，将不加TGF-β1的孔作为抑制100％，计算获得Emax值。
结果显示融合蛋白3，融合蛋白4可明显抑制TGFβ诱导的pSMAD3报告物活性。
表18：融合蛋白对SMAD报告基因通路的抑制能力
抗体 IC50(nM) Emax(％)
C25 / 21.2
融合蛋白3 0.130 88.5
融合蛋白4 0.124 88.6
测试例11：体外检测结核杆菌素(TB)刺激PBMC释放IFNγ实验
1、测试目的
为了研究PD-1/TGF-β trap对T淋巴细胞的激活作用，收集和纯化人外周血单核细胞(PBMC)，采用结核杆菌素(TB)体外刺激5天，检测IFNγ细胞因子的分泌水平。
2、测试样品：①C25；②PD-1抗体；③融合蛋白3；④融合蛋白4。
3、测试过程
新鲜分离纯化的PBMC，15mL约3×10 7个，加入20μL结核菌素，37℃，5％CO 2培养箱培养5天。第6天，收集上述培养的细胞离心，用PBS洗一次，重悬至新鲜的培养基中，调整密度为1×10 6个每毫升，接种至96孔细胞培养板，每孔90μL。将不同浓度的抗体分别加入上述96孔细胞培养板的对应孔中，每孔10μL，对照组和空白组分别加入10μL PBS。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)每孔取上清，10倍稀释后，采用ELISA的方法(人IFN-γ检测试剂盒，欣博盛，EHC102g.96)检测IFN-γ的水平。具体操作参考试剂说明书。结果如表19所示。
表19.PD-1/TGF-β trap对T淋巴细胞分泌IFN-γ的影响
Figure PCTCN2020128045-appb-000090
4、结果
如表19所示，融合蛋白3，融合蛋白4能够剂量依赖地增强激活的T淋巴细胞分泌细胞因子IFN-γ，并且具有比抗PD-1抗体更强的激活作用。
测试例12：PD-1/PD-L1，PD-1/PD-L2阻断实验
将PD-1-IgG1Fc用PBS稀释至0.5μg/mL，以100μL/孔加于96孔板中，于4℃孵育过夜。将96孔板中PBS弃去，用PBST(pH7.4 PBS含0.05％tween20)洗板 3次后，加入100μL/孔PBS/3％BSA，室温孵育1h进行封闭。弃去封闭液，用PBST洗板3次后，加入100μL用PBS/3％BSA稀释至合适浓度的融合蛋白或对照，4℃预孵育1h。以100μL/孔加入1μg/mL生物素标记PD-L1或PD-L2(北京义翘神州生物技术有限公司)，37℃孵育1h。用PBST洗板6次后，以100μL/孔加入用PBS/3％BSA 1：3000稀释的链霉亲和素-过氧化物酶聚合物(Jackson，016-030-084)，室温孵育1h。用PBST洗板6次后，加入100μL/孔TMB显色。加入100μL/孔1M H 2SO 4终止反应。用酶标仪在450nm处读取吸收值。结果见下表。
表20：PD-L1/PD-L2阻断实验结果
样品 PD-L1的阻断IC50(nM) PD-L2的阻断IC50(nM)
Hu23-11 2.85 3.88
Hu33-5 1.94 3.06
融合蛋白3 1.61 4.94
融合蛋白4 1.38 3.41
测试例13：融合蛋白体内药效试验
人PD1转基因C57BL/6J小鼠，购自百奥赛图有限责任公司。于实验室环境适应性饲养7天，给予12/12小时光/暗周期调节，温度23±1℃，湿度40至50％，动物均给予标准灭菌鼠饲料，自由进食饮水。
人PD1转基因C57BL/6J在右肋部皮下接种MC38细胞(1×10 5个/只，购自南京银河)，8天后分4组，10只/组，每组平均肿瘤体积为59.08mm 3。
腹腔注射等摩尔量的C25(2.48mpk)，Hu23-11(0.83mpk)和融合蛋白3(3mpk和1mpk)，共给药4次，于肿瘤接种后7天，9天，11天和14天给药。每周测量2次瘤体积和体重，记录数据。使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对肿瘤增殖率T/C(％)＝(T-T 0)/(C-C 0)×100％
抑瘤率TGI(％)＝1-T/C(％)。
其中T、C为实验结束时治疗组和对照组的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
本次实验腹腔注射给药四次，观察至第26天，采用第19天数据进行统计，结果见下表和图7。
表21：给药抗体对荷瘤裸鼠MC38-H11移植瘤的疗效
样品 给药量(mpk) 抑瘤率(％)
C25 2.48 /
Hu23-11 0.83 68.25
融合蛋白3 3 89.52
融合蛋白3 1 86.78
结果显示，抗PD-1抗体Hu23-11和融合蛋白3均能有效抑制MC38移植瘤的增长，其中等摩尔给药时，Hu23-11(0.83mpk)的抑瘤率为68.25％，融合蛋白3(1mpk)的抑瘤率达到86.78％；增大给药剂量后，融合蛋白3(3mpk)的抑瘤率高达89.52％。给药过程中各组动物包括对照组体重均无明显下降，说明受试抗体和融合蛋白无明显毒副作用。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本披露的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地并入。

Claims (22)

  1. 一种融合蛋白，其包含靶向部分和TGF-β受体部分，其中，所述的TGF-β受体部分为TGF-βRII胞外区，所述的靶向部分为抗PD-1抗体或其抗原结合片段。
  2. 根据权利要求1所述的融合蛋白，其中所述的TGF-βRII胞外区为TGF-βRII胞外区的N端截短形式。
  3. 根据权利要求2所述的融合蛋白，其中所述的TGF-βRII胞外区的N端截短形式为在TGF-βRII胞外区的N端缺失1至26个连续的氨基酸，优选N端缺失14至26个氨基酸，更优选缺失14至21个氨基酸，最优选缺失14、19或21个氨基酸。
  4. 根据权利要求1至3中任一项所述融合蛋白，其中所述的TGF-βRII胞外区的序列如SEQ ID NO：61所示。
  5. 根据权利要求2或3所述的融合蛋白，其中所述的TGF-βRII胞外区的N端截短形式的序列如SEQ ID NO：62、63或64所示。
  6. 根据权利要求1至5中任一项所述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中
    a)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和
    所述轻链可变区包含序列如SEQ ID NO：11、32、33、34、35、36或37所示的LCDR1，和序列分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3；或
    b)所述重链可变区包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和
    所述轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3；
    优选地，其中所述的抗PD-1抗体或其抗原结合片段包含：
    重链可变区，其包含分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：34、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3。
  7. 根据权利要求1至5中任一项所述的融合蛋白，其中所述的抗PD-1抗体或 其抗原结合片段包含选自以下的重链可变区和轻链可变区：
    c)所述重链可变区包含与如SEQ ID NO：4所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：5所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3；或
    d)所述重链可变区包含与如SEQ ID NO：6所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：7所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3。
  8. 根据权利要求1至7中任一项中所述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或其抗原结合片段。
  9. 根据权利要求8所述的融合蛋白，其中所述抗PD-1抗体为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多6个氨基酸的回复突变；
    优选地，所述的回复突变选自：
    e)轻链框架区中的2G氨基酸回复突变，和/或
    重链框架区中的氨基酸回复突变，其选自：27Y、48I、67T、69L、82F和93T中的一个或更多个；或
    f)轻链框架区中的氨基酸回复突变，选自：42G、44V和71Y中的一个或更多个，和/或
    重链框架区中的氨基酸回复突变，其选自：1K和/或94S。
  10. 根据权利要求8所述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段包含选自如下g)至k)中任一项所示的重链可变区和轻链可变区：
    g)重链可变区序列如SEQ ID NO：4所示或与SEQ ID NO：4具有至少90％序列同一性；和轻链可变区序列如SEQ ID NO：5所示或与SEQ ID NO：5具有至少90％序列同一性；
    h)重链可变区序列如SEQ ID NO：6所示或与SEQ ID NO：6具有至少90％序列同一性；和轻链可变区序列如SEQ ID NO：7所示或与SEQ ID NO：7具有至少90％的序列同一性；
    j)重链可变区序列如SEQ ID NO：20、23、24或25所示或分别与SEQ ID NO：20、23、24或25具有至少90％序列同一性，和轻链可变区序列如SEQ ID NO：21、22、38、39、40、41、42、43、44、45、46、47、48或49所示或分别与SEQ ID NO：21、22、38、39、40、41、42、43、44、45、46、47、48或49具有至少90％序列同一性；和
    k)重链可变区序列如SEQ ID NO：26、30或31所示或分别与SEQ ID NO： 26、30或31具有至少90％序列同一性，和轻链可变区序列如SEQ ID NO：27、28或29所示或分别与SEQ ID NO：27、28或29具有至少90％序列同一性；
    优选地，所述的抗PD-1抗体或其抗原结合片段包含：
    重链可变区序列如SEQ ID NO：20所示和轻链可变区序列如SEQ ID NO：40所示；或
    重链可变区序列如SEQ ID NO：30所示和轻链可变区序列如SEQ ID NO：28所示；或
    重链可变区序列如SEQ ID NO：31所示和轻链可变区序列如SEQ ID NO：28所示。
  11. 根据权利要求1至10中任一项所述的融合蛋白，其中所述的抗PD-1抗体或其抗原结合片段进一步包含抗体重链恒定区和轻链恒定区；
    所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，
    所述轻链恒定区选自人抗体κ或λ链恒定区及其常规变体；
    优选地，所述抗PD-1抗体或其抗原结合片段包含序列如SEQ ID NO：50或51所示的重链恒定区和/或序列如SEQ ID NO：52所示的轻链恒定区。
  12. 根据权利要求1至11中任一项所述的融合蛋白，其中所述抗PD-1抗体包含选自以下l)-n)中任一项所示的重链和轻链：
    l)重链序列如SEQ ID NO：53或54所示或与SEQ ID NO：53或54具有至少85％的序列同一性；和轻链序列如SEQ ID NO：55所示或与SEQ ID NO：55具有至少85％的序列同一性；
    m)重链序列如SEQ ID NO：56或57所示或与SEQ ID NO：56或57具有至少85％的序列同一性；和轻链序列如SEQ ID NO：58所示或与SEQ ID NO：58具有至少85％的序列同一性；和
    n)重链序列如SEQ ID NO：59或60所示或与SEQ ID NO：59或60具有至少85％的序列同一性；和轻链序列如SEQ ID NO：58所示或与SEQ ID NO：58具有至少85％的序列同一性。
  13. 根据权利要求1至11中任一项所述的所述的融合蛋白，其中所述抗原结合片段选自Fab、Fab'、F(ab') 2、scFv、双抗体和dsFv。
  14. 根据权利要求1至13中任一项所述的融合蛋白，其中所述的TGF-βRII胞外区通过接头融合至所述的抗PD-1抗体或其抗原结合片段的重链的羧基端、重链的氨基端、轻链的羧基端或轻链的氨基端；优选融合至重链的羧基端。
  15. 根据权利要求14所述的融合蛋白，其中所述的接头为(ASTKGP)n、 (ASTKGPSVFPLAP)n、(TVAAP)n、(TVAAPSVFIFPP)n或(G 4S) nG，其中n为1-6的整数，优选为4或5。
  16. 根据权利要求1至15中任一项所述的融合蛋白，其包含：
    -第一链，其包含与抗PD-1抗体的重链融合的TGF-βRII胞外区、和
    -第二链，其包含抗PD-1抗体的轻链；
    其中：
    o)所述的第一链的序列如SEQ ID NO：69或71所示或与SEQ ID NO：69或71所示的氨基酸序列具有至少85％的同一性，和第二链的序列如SEQ ID NO：55所示或与SEQ ID NO：55所示氨基酸序列具有至少85％的同一性；或
    p)所述的第一链的序列如SEQ ID NO：70、72、73或74所示或与SEQ ID NO：70、72、73或74所示的氨基酸序列具有至少85％的同一性，和第二链的序列如SEQ ID NO：58所示或与SEQ ID NO：58所示氨基酸序列具有至少85％的同一性。
  17. 一种药物组合物，其含有：
    -预防有效量或治疗有效量的权利要求1至16中任一项所述的融合蛋白，以及
    -一种或多种药学上可接受的载体、稀释剂或赋形剂。
  18. 一种核酸分子，其编码权利要求1至16中任一项所述的融合蛋白。
  19. 一种表达载体，其含有权利要求18所述的核酸分子。
  20. 一种宿主细胞，其含有权利要求19所述的表达载体，所述宿主细胞选自细菌、酵母菌和哺乳动物细胞；优选哺乳动物细胞。
  21. 一种治疗或预防肿瘤的方法，所述方法包括：
    向所需患者施用预防有效量或治疗有效量的权利要求1至16中任一项所述的融合蛋白，或权利要求17所述的药物组合物，或权利要求18所述的核酸分子；优选地，所述的肿瘤为PD-1相关的癌症。
  22. 根据权利要求21所述的方法，其中所述的肿瘤选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、 骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；
    优选地，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；
    所述肺癌选自：非小细胞肺癌和小细胞肺癌；
    所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；
    最优选的，所述肿瘤选自：黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌、食管癌、肝癌和结肠癌。
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