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WO2021209049A1 - 一种pd-1突变体多肽及其制备和用途 - Google Patents

一种pd-1突变体多肽及其制备和用途 Download PDF

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WO2021209049A1
WO2021209049A1 PCT/CN2021/087864 CN2021087864W WO2021209049A1 WO 2021209049 A1 WO2021209049 A1 WO 2021209049A1 CN 2021087864 W CN2021087864 W CN 2021087864W WO 2021209049 A1 WO2021209049 A1 WO 2021209049A1
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variable region
chain variable
amino acid
acid sequence
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王峰
郑花鸯
张雨菡
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南通壹宸生物医药科技有限公司
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Priority to CN202180028808.9A priority Critical patent/CN115708411A/zh
Priority to US17/918,919 priority patent/US20230287081A1/en
Priority to EP21789230.6A priority patent/EP4137510A4/en
Publication of WO2021209049A1 publication Critical patent/WO2021209049A1/zh

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Definitions

  • the term of the present invention refers to the field of biopharmaceuticals, and specifically relates to a PD-1 mutant polypeptide, an antibody fusion protein containing the PD-1 mutant polypeptide, and preparation and application thereof.
  • PD-1 is an immunosuppressive molecule, which belongs to the CD28 family. Its structure includes: extracellular immunoglobulin variable region (IgV)-like domain, hydrophobic transmembrane region, and intracellular. PD-1 is expressed on CD4-CD8-thymocytes, and inducibly expressed on activated T cells, B cells, bone marrow cells, dendritic cells, natural killer cells, monocytes, etc. The continuous expression of PD-1 on T cells induces T cell exhaustion. The expression of PD-1 by tumor infiltrating lymphocytes can affect T cell function, weaken cytokine secretion, and weaken the tumor-killing effect of T cells. It is closely related to the poor prognosis and high tumor recurrence rate of patients with renal cell carcinoma and non-small cell lung cancer (Pan Jiajia, etc.; Journal of China Pharmaceutical University 2016,47(1):9-18).
  • PD-1 has two ligands, PD-L1 and PD-L2 ligands, both of which belong to the B7 family.
  • PD-L1 is widely expressed in activated B cells, T cells, macrophages, DCs, and NK cells.
  • PD-L1 is also expressed on the surface of many tumor cells, such as lung cancer, breast cancer, malignant melanoma, esophageal cancer, gastric cancer, pancreatic cancer and other tumor cells.
  • the surface of tumor cells expresses PD-L1 or PD-L2 molecules highly, and binds to the receptor PD-1 on T cells to transmit negative regulatory signals, leading to immune apoptosis and immune incompetence of tumor antigen-specific T cells, and making tumor cells Escape the body's immune surveillance and killing (Pan Jiajia et al.; Journal of China Pharmaceutical University 2016,47(1):9-18). Therefore, with the PD-1/PD-Ls signaling pathway as the target, the development of blockers against PD-1 or PD-Ls can enhance the killing of tumor cells by T cells.
  • the PD-1 mutant polypeptide of the present invention Compared with the affinity (nM order of magnitude) of wild-type PD-1 and PD-L1, the PD-1 mutant polypeptide of the present invention has only 2 amino acid mutations, and not only retains the similarity to wild-type PD-1 Physical and chemical properties, the epitope that binds to PD-L1 is the same as that of wild-type PD-1, and the affinity to PD-L1 is greatly improved (pM order of magnitude), and the possibility of off-target is low; in addition, the PD-1 mutant polypeptide has a potential for human The cross-species activity of monkeys, mice, and monkeys provides great convenience for subsequent studies on pharmacodynamics and toxicological doses in animal models.
  • the PD-1 mutant polypeptide Compared with the antibody targeting PD-L1, the PD-1 mutant polypeptide has a single domain and is easy to construct double antibodies.
  • the antibody fusion protein formed by fusing the PD-1 mutant polypeptide with the antibody targeting the tumor antigen on the one hand, can be specific for tumor antigen-specific antibodies with high affinity (higher than the affinity of the PD-1 mutant polypeptide and PD-L1) Aggregate PD-1 mutant polypeptides on tumor cells, or its vicinity, and in the tumor microenvironment, greatly reducing the extensive immune activation caused by the use of conventional immune checkpoint inhibitors in the body; on the other hand, PD-1 mutants
  • the fast binding and fast dissociation characteristics of peptides enable it to block the PD-1/PD-L1 signaling pathway while avoiding the possibility of continuous immune cell activation due to the fast binding and slow dissociation properties of conventional immune checkpoint inhibitors.
  • the in vivo metabolism of PD-1 mutants is faster than conventional antibody
  • a PD-1 mutant polypeptide in one aspect of the present invention, has a nucleic acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence as set forth in SEQ ID NO: 2.
  • the antibody fusion protein containing a PD-1 mutant polypeptide.
  • the antibody fusion protein comprises the PD-1 mutant polypeptide and an antibody targeting tumor cell surface antigen.
  • the tumor cell surface antigen is selected from EGFR, HER2, HER3, GPC3, PSMA, CD27, CD33, CD37, CD38, CD51, CD56, mesothelin, HGFR, Ne ctin-4, Mucin 5AC, folate receptor ⁇ , Carbonic anhydrase IX (CAIX), DLL1, Notch2, Notch3, endoglin, etc.
  • the tumor antigen is selected from EGFR.
  • the antibody that targets the tumor cell surface antigen EGFR has the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and the sequence shown in SEQ ID The LCDR1, LCDR2, and LCDR3 sequences shown in NO: 16, SEQ ID NO: 17, SEQ ID NO: 18.
  • the antibody targeting the tumor antigen EGFR has a heavy chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 34, and a light chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 36. Chain variable region (VL).
  • the antibody targeting the tumor cell surface antigen EGFR has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 65, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 66 Light chain variable region (VL).
  • VH heavy chain variable region
  • VL Light chain variable region
  • the antibody targeting the tumor antigen EGFR has a heavy chain (H C) with the amino acid sequence shown in SEQ ID NO: 4, and a light chain (LC) with the amino acid sequence shown in SEQ ID NO: 6 ).
  • the antibody fusion protein containing the PD-1 mutant polypeptide and the EGFR targeting tumor cell surface antigen has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4 and a heavy chain as shown in SEQ ID NO: 28. Shows the amino acid sequence of the light chain.
  • the tumor antigen is selected from HER2.
  • the antibody targeting the tumor cell surface antigen HER2 has the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and the sequence shown in SEQ ID The LCDR1, LCDR2, and LCDR3 sequences shown in NO: 22, SEQ ID NO: 23, SEQ ID NO: 24.
  • the antibody targeting the tumor cell surface antigen HER2 has a VH with an amino acid sequence as shown in SEQ ID NO: 38 and a VL with an amino acid sequence as shown in SEQ ID NO: 40.
  • the antibody targeting the tumor cell surface antigen HER2 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 67, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 68 Light chain variable region (VL).
  • VH heavy chain variable region
  • VL Light chain variable region
  • the antibody targeting the tumor antigen HER2 has an HC with the amino acid sequence shown in SEQ ID NO: 8 and an L C with the amino acid sequence shown in SEQ ID NO: 8.
  • the fusion protein containing the PD-1 mutant polypeptide and the antibody fusion protein targeting tumor antigen HER2 has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 10 and an amino acid sequence as shown in SEQ ID NO: 32 Light chain.
  • the tumor cell surface antigen is selected from GPC-3.
  • the antibody targeting the tumor cell surface antigen GPC3 has the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID N O: 47, and sequences such as SEQ ID The LCDR1, LCDR2, and LCDR3 sequences contained in the light chain amino acid sequence shown in NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.
  • the antibody targeting the tumor cell surface antigen GPC-3 has a VH with an amino acid sequence as shown in SEQ ID NO: 42 and a VL with an amino acid sequence as shown in SEQ ID NO: 44.
  • the antibody targeting the tumor antigen GPC-3 has an HC with the amino acid sequence shown in SEQ ID NO: 58 and an LC with the amino acid sequence shown in SEQ ID NO: 60.
  • the antibody fusion protein containing the PD-1 mutant polypeptide and the tumor antigen GPC-3 targeting tumor antigen has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 52 and an amino acid as shown in SEQ ID NO: 60 Sequence of the light chain.
  • the fusion protein containing the PD-1 mutant polypeptide and the antibody fusion protein targeting tumor antigen GPC-3 has a heavy chain with the amino acid sequence shown in SEQ ID NO: 58 and the amino acid shown in SEQ ID NO: 54 Sequence of the light chain.
  • the antibody fusion protein containing the PD-1 mutant polypeptide and the tumor antigen GPC-3 targeting tumor antigen has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 58 and an amino acid as shown in SEQ ID NO: 56 Sequence of the light chain.
  • the tumor cell surface antigen is selected from PSMA.
  • the antibody targeting the tumor cell surface antigen PSMA has a heavy chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 64.
  • VH heavy chain variable region
  • VL light chain variable region
  • the tumor cell surface antigen is selected from CD27.
  • the antibody targeting the tumor cell surface antigen CD27 has a heavy chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 69, and a light chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 70.
  • VH heavy chain variable region
  • VL light chain variable region
  • the tumor cell surface antigen is selected from CD33.
  • the antibody targeting the tumor cell surface antigen CD33 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 71, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 72 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from CD37.
  • the antibody targeting the tumor cell surface antigen CD37 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 73, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 74 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from CD38.
  • the antibody that targets the tumor cell surface antigen CD38 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 75, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 76 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from CD51.
  • the antibody targeting the tumor cell surface antigen CD51 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 77, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 78 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from CD56.
  • the antibody targeting the tumor cell surface antigen CD56 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 79, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 80 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from mesothelin.
  • the antibody targeting the tumor cell surface antigen mesothelin has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 81, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 82 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from HGFR.
  • the antibody targeting the tumor cell surface antigen HGFR has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 83, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 84 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from Nectin-4.
  • the antibody targeting tumor cell surface antigen Nectin-4 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 85, and an amino acid as shown in SEQ ID NO: 86 Sequence of the light chain variable region (VL).
  • the tumor cell surface antigen is selected from Mucin5AC.
  • the antibody targeting the tumor cell surface antigen Mucin5AC has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 87, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 88 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from folate receptor alpha.
  • the antibody targeting the tumor cell surface antigen folate receptor ⁇ has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 89, and an amino acid sequence as shown in SEQ ID NO: 90 The light chain variable region (VL).
  • the tumor cell surface antigen is carbonic anhydrase IX (CAIX).
  • the antibody targeting the tumor cell surface antigen carbonic anhydrase IX (CAIX) has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 91, and a heavy chain variable region (VH) as shown in SEQ ID NO: 92 The light chain variable region (VL) of the amino acid sequence shown.
  • the tumor cell surface antigen is selected from DLL1.
  • the antibody targeting the tumor cell surface antigen DLL1 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 93, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 94 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from Notch 2/3.
  • the antibody targeting the tumor cell surface antigen Notch 2/3 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 95, and a heavy chain variable region (VH) as shown in SEQ ID NO: 96 The amino acid sequence of the light chain variable region (VL).
  • the tumor cell surface antigen is selected from endoglin.
  • the antibody that targets the tumor cell surface antigen endoglin has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 97, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 98 Light chain variable region (VL).
  • the tumor cell surface antigen is selected from HER3.
  • the antibody targeting the tumor cell surface antigen HER3 has a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 99, and a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO: 100 Light chain variable region (VL).
  • a polynucleotide which encodes the aforementioned PD-1 mutant polypeptide or an antibody fusion protein containing the PD-1 mutant polypeptide.
  • the polynucleotide is selected from the nucleotide sequence of the following group: SEQ ID NO: 1, 3, 5, 7, 9, 11, 27, 31, 51, 53, 55, 57, 59 .
  • One aspect of the present invention provides a vector containing the PD-1 mutant polypeptide or the antibody fusion protein sequence containing the PD-1 mutant polypeptide as described above.
  • a host cell which contains the vector as described above.
  • a pharmaceutical composition which comprises a pharmaceutically acceptable carrier or preparation and the above-mentioned PD-1 mutant polypeptide or an antibody fusion protein containing the PD-1 mutant polypeptide.
  • One aspect of the present invention provides the use of a PD-1 mutant polypeptide or an antibody fusion protein containing a PD-1 mutant polypeptide as a PD-1-PD-L1 interaction inhibitor.
  • One aspect of the present invention provides the use of a PD-1 mutant polypeptide or an antibody fusion protein containing the PD-1 mutant polypeptide as an immunomodulator.
  • the use of the treatment of tumor or cancer includes: B-cell lymphoma, breast cancer, ovarian cancer, NSCLC, low-grade glioma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), Multiple myeloma, metastatic colorectal cancer, Merkel cell tumor, NSCLC, squamous NSCLC, pancreatic cancer, neuromesothelioma, breast cancer, HER2-positive metastatic breast cancer, metastatic gastric cancer, liver cancer, urinary tract Skin cell carcinoma, bladder cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, multiple myeloma, soft tissue sarcoma, primary peritoneal cancer, etc.
  • the affinity of the high-affinity PD-1 mutant polypeptide of the present invention with PD-L1 is greatly improved.
  • the affinity of the antibody fusion protein containing the PD-1 mutant polypeptide to recombinant PD-L1 is 39-109 times higher than the affinity of the antibody fusion protein containing wild-type PD-1 to recombinant PD-L1.
  • the affinity of the antibody fusion protein containing the PD-1 mutant polypeptide to cell surface PD-L1 is higher than the affinity of the antibody fusion protein containing wild-type PD-1 to cell surface PD-L1 (almost no binding ) Greatly enhanced. Therefore, the PD-1 mutant of the present invention can be used as a competitive inhibitor of PD-1-PD-L1 interaction.
  • the PD-1 mutant of the present invention can also be used as an immunomodulator for, for example, the immunotherapy of tumors.
  • Figure 1 is the SDS-PAGE electrophoresis diagram of the antibody fusion protein, where M represents protein marker; "-" represents loading without DTT; “+” represents loading after adding DTT, lane 1 in Figure 1 is PD-1-aEGFRL , Lane 2 is PD-1m-aEGFRL, Lane 3 is aEGFR, Lane 4 is aPDL1, Lane 5 is aHER2, Lane 6 is PD-1-aHER2L, Lane 7 is PD-1m-aHER2L, Lane 8 is aGPC-3, Lane 9 is PD-1-aGPC-3L, lane 10 is PD-1m-aGPC-3L, and lane 11 is aEGFR.
  • Figure 2 is a SEC detection of protein monomer peaks of the fusion protein of PD-1 mutant polypeptide and aEGFR and aHER2.
  • Figure 3 shows the binding between the PD-1 mutant polypeptide and aEGFR antibody fusion protein, as well as the scaffold antibody and antigen hEGFR in an ELISA experiment.
  • Figure 4 shows the binding of the PD-1 mutant polypeptide and aHER2 antibody fusion protein and the scaffold antibody to the antigen hHER2 in an ELISA experiment.
  • Figure 5 shows the binding of the PD-1 mutant polypeptide and aGPC3 antibody fusion protein and the scaffold antibody to the antigen hGPC3 in an ELISA experiment.
  • Figure 6 shows the affinity detection of PD-1 mutant polypeptide and antibody fusion protein and control antibody and soluble hPD-L1 protein in an ELISA experiment.
  • Figure 7 shows the affinity detection of PD-1 mutant polypeptide and antibody fusion protein and control antibody and soluble mPD-L1 protein in an ELISA experiment.
  • FIG 8 is a PD-1 mutant polypeptide aEGFR antibody fusion binding of proteins associated with cell line, wherein 8A is an antibody fusion protein HepG2 (hEGFR + hPDL1 -) cell binding, 8B fusion protein and CHO-hPDL1 is an antibody ( hEGFR - hPDL1 + ) cells, 8C is the binding of antibody fusion protein to MC38-hEGFR (hEGFR + mPDL1 + ) cells, and 8D is the binding of antibody fusion protein to A431 (hEGFR + hPDL1 + ) cells.
  • 8A is an antibody fusion protein HepG2 (hEGFR + hPDL1 -) cell binding
  • 8B fusion protein and CHO-hPDL1 is an antibody ( hEGFR - hPDL1 + ) cells
  • 8C is the binding of antibody fusion protein to MC38-hEGFR (hEGFR + mPDL1 + ) cells
  • 8D
  • FIG 9 is a PD-1 mutant polypeptide aGPC3 antibody fusion binding of proteins related to cell lines in which 9A is the HepG2 (hGPC3 + hPDL1 -) cell binding, 9B with the A431 (hGPC3 - hPDL1 +) binding cells , 9C is the combination with Hepa1-6 (hGPC3 - mPDL1 + ) cells.
  • Figure 10 shows that the soluble hEGFR protein competitively inhibits the binding of the PD-1 mutant polypeptide to aEGFR antibody fusion protein and the scaffold antibody to hEGFR on the cell surface.
  • 9A is the PD-1 mutant polypeptide and aEGFR antibody fusion protein and the scaffold antibody and MC38 -hEGFR (hEGFR + mPDL1 + ) cell binding
  • 9B is the binding of PD-1 mutant polypeptide and aEGFR antibody fusion protein and skeleton antibody to MC38-hEGFR (hEGFR + mPDL1 + ) cell in the presence of EGFR.
  • Figure 11 is the metabolic curve of the PD-1 mutant polypeptide and the antibody fusion protein in rats, where 11A is the metabolic curve obtained by detecting the intact antibody fusion protein, and 11B is the metabolic curve obtained by detecting the antibody backbone in the antibody fusion protein.
  • Figure 12 is the evaluation of the effectiveness of the PD-1 mutant polypeptide and antibody fusion protein in mouse tumor models, where 12A is the change in mouse tumor growth after the administration of different protein samples, and Figure 12B is after the administration of different protein samples Changes in mouse body weight.
  • CDR refers to the complementarity determining region within the immunoglobulin variable region sequence.
  • CDR1, CDR2, and CDR3 CDR1, CDR2, and CDR3.
  • CDR set refers to a set of three CDRs that appear in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody or binding protein, but also provides a definition of each heavy chain or light chain The precise residue boundaries of the three CDRs in the sequence.
  • CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883) found that certain sub-parts of Kabat CDR adopt almost the same peptide bone structure. Although there is great diversity at the amino acid sequence level. These sub-portions are named L1, L2, and L3 or H1, H2, and H3, where "L” and “H” refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have borders that overlap with Kabat CDRs.
  • tumor cell surface antigen refers to an antigen that is expressed on the surface of tumor cells and can be targeted for disease treatment.
  • the target molecule is an antigen that is preferentially expressed on the surface of selected tumor cells (such as solid tumors or hematological tumor cells): non-limiting examples of specific tumor-associated antigens include, for example, EGFR, HER2, HER3, HER4, GPC3, PSMA, CD27, CD33, CD37, CD38, CD51, CD56, mesothelin, HGFR, Nectin-4, Mucin 5AC, folate receptor ⁇ , carbonic anhydrase IX (CAIX), DLL1, Notch2, Notch3, endoglin, MUC1, MUC2, MUC3A , MUC3B, MUC4, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, VEGFR-1
  • SSX-4 Survivin, SYT-SSX1 or -SSX2, TAG-1, TAG-2, Telomerase, TGF- ⁇ , TGF-beta RII, TRAG-3, TRP-2, TRP2-INT2, VEGF , WT1, TRPM4, CRIPTO, glycoprotein IIb/IIIa receptor, glycolipid GD2, GD3, folate receptor 1 (FOLR1), IFN ⁇ , IFN ⁇ , ⁇ , ⁇ receptor 1, TROP-2, Glyco-protein NMB, MMP9, GM3, mesothelin, fibronectin extra-domain B, endoglin, Rhesus D, plasma kallikrein, CS, thymic stromal lymphopoietin, mucosal addressin cell adhesion molecule, nectin 4, N GcGM3, DLL3, DLL4, CLEC12A, KLB, FGFR1C, CEA, BCMA, p-cadherin, FAP, DR1, DR
  • aEGFR VH anti-human EGFR antibody heavy chain variable region
  • aEGFR VL EGFR antibody light chain variable region
  • aHER2 VH Anti-human HER2 antibody heavy chain variable region
  • aHER2VL Anti-human HER2 antibody light chain variable region
  • the amplified aEGFR VH and aHER2 VH genes were cloned into the pFuse-hIgG1-Fc2 vector (InvivoGen) by enzyme digestion and ligation (the hIgG1-Fc on the vector contains 9 mutations: E233P, L234V, L235A, ⁇ G236, A327G, A330S, P331S, E356D, and M358L were all completed by our laboratory), aEGFR VL and aHER2 VL were cloned into pFuse2-CLIg-Hk vector (InvivoGen).
  • the amplified PD-1m gene was cloned into the N-terminus of the aEGFR antibody light chain or the N-terminus of the aHER2 antibody light chain constructed as described above by the method of restriction enzyme digestion and ligation. All constructed vectors were verified by sequencing.
  • PD-1 human PD-1
  • PD-1m Human PD-1 mutant
  • the heavy chain and light chain of the fusion protein expression vector constructed in Example 1 were transiently transfected into FreeStyle HEK293 cells (ThermoFisher), and the amount of the plasmid of the heavy chain and the plasmid of the light chain during transfection was a molar ratio of 1:1.
  • the plasmid-293Fectin mixture was added to the cell culture medium at 125 rpm, 37°C, and 5% CO2.
  • the cell culture supernatant was collected 96h after transfection, purified by Protein A Resin (Genscript), and detected by SDS-PAGE.
  • the SDS-PAGE chart is shown in Figure 1, indicating that the antibody fusion protein was successfully expressed and the molecular weight was in line with expectations.
  • the obtained protein A resin purified antibody fusion protein was analyzed by GE's AKTA chromatography.
  • the column used is: Superdex 200 Increase 10/300GL gel exclusion chromatography column, which is used for gel exclusion chromatography
  • the solution is PBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH 7.4). From the chromatogram in Figure 2, the expression of the antibody fusion protein of PD-1 and aEGFR or aHER2 has considerable purity.
  • hEGFR-hIGg1Fc (SinoBiological) (100ng/well) in a 96-well plate, incubate overnight at 4°C; PBST (0.5% Tween-20 in PBS) containing 2% skimmed milk powder was blocked at room temperature for 1 hour, and then added to the gradient dilution (10pM) -1.2nM) antibody (aEGFR) fusion protein incubate at room temperature for 2h, wash 4-5 times with PBST containing 2% skimmed milk powder, add anti-human kappa light chain-HRP (Sigma A7146, 1:3000) secondary antibody and incubate at room temperature After washing 4-5 times in PBST containing 2% skimmed milk powder for 1 hour, QuantaBlu fluorescent peroxidase substrate (Life technologies, Cat.15169) develops color and reads at 325nm and 420nm, or use TMB color reagent (BioLegend ,Cat.421101), read at 650n
  • the results of the combination with human PD-L1 are shown in Figure 6.
  • the affinity of PD-1m antibody fusion protein (PD-1m-aEGFRL, PD-1m-aHER2L or PD-1m-aGPC-3L) to human PD-L1 is similar to that of the positive control aPD-L to human PD-L1, and It is much higher than the affinity of PD-1 antibody fusion protein to human PD-L1 (25-35 times).
  • the results of binding to mouse PD-L1 are shown in Figure 7.
  • the affinity of PD-1m antibody fusion protein (PD-1m-aEGFRL, PD-1m-aHER2L or PD-1m-aGPC-3L) to mouse PD-L1 is similar to that of the positive control aPD-L to mouse PD-L1, and It is much higher than the affinity of PD-1 antibody fusion protein to mouse PD-L1.
  • Cultivate A431, MC38-hEGFR, CHO-PD-L1, HepG2, Hepa1-6 cells (DMEM medium containing 10% FBS, 1% double antibody), after trypsinization, take 2*10 5 cells with pre-cooled PBS After washing 3 times, after blocking in PBS containing 2% FBS at 4°C, centrifuge to discard the supernatant, add 100ul of antibody fusion proteins of different concentrations diluted in 2% FBS in PBS solution and incubate at 4°C for 1 hour, and use PBS containing 2% FBS The unbound antibody fusion protein was washed away, and the secondary antibody Mouse Anti-Human IgG Fc-APC (Southern biotech) was added and incubated at 4°C for 1 hour. After washing three times with 2% FBS in PBS, the fluorescence intensity was measured by flow cytometry.
  • PD-1m-aGPC-3L The results of PD-1m-aGPC-3L binding to cells are shown in Figure 9.
  • PD-1m-a GPC-3L and PD-1-aGPC-3L have reduced binding to GPC-3 on the surface of HepG2 cells, but still have good binding activity (Figure 9A).
  • PD-1m-aGPC-3L can bind hPD-L1 (9B) on the surface of A431 cells and mPDL1 (9C) on the surface of Hepa1-6 cells.
  • MC38-hEGFR Cultivate human EGFR MC38 cells (MC38-hEGFR, constructed by our laboratory) (DMEM medium containing 10% FBS, 1% double antibody), after trypsinization, take 2*10 5 cells and wash 3 times with pre-cooled PBS Then, after blocking in PBS containing 2% FBS at 4°C, centrifuge to discard the supernatant, add 100ul of antibody fusion proteins of different concentrations diluted in 2% FBS in PBS solution, and incubate at 4°C for 1 hour, and wash off with PBS containing 2% FBS.
  • the bound antibody fusion protein was added with the secondary antibody Mouse Anti-Human IgG Fc-APC (Southern biotech), incubated at 4°C for 1 hour, washed three times with 2% FBS in PBS, and then the fluorescence intensity was measured by flow cytometry.
  • the antibody fusion protein at a dose of 10 mg/kg was injected intraperitoneally (I.P.) and tail vein injection (I.V.) into SD male rats (3 rats/group). Heparin anticoagulant blood was collected from the tail vein, and the blood collection time was as follows: 15min, 30min, 1h, 4h, 8h, 1h, 2d, 3d, 5d, 7d and 14d. After centrifugation, the plasma was collected and stored at -80°C for later use. The content detection of antibody fusion protein and framework antibody in plasma was performed with reference to Example 4.1.
  • the results are shown in Figure 12.
  • the antibody fusion protein can effectively inhibit tumor growth without significant side effects such as weight loss.
  • the control double antibody without EGFR targeting (PD1m-aGPC3) cannot inhibit tumor growth at all, proving tumor targeting.
  • the BIC-02 has good selectivity and safety potential.

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Abstract

本发明提供了高亲和力PD-1突变体多肽及PD-1突变体多肽与抗体(特异结合肿瘤抗原)的融合蛋白及其制备与应用。

Description

一种PD-1突变体多肽及其制备和用途 技术领域
本发明术语生物制药领域，具体涉及一种PD-1突变体多肽、含有PD-1突变体多肽的抗体融合蛋白及其制备和应用。
背景技术
PD-1是一种免疫共抑制分子，属于CD28家族成员。其结构包括：胞外免疫球蛋白可变区(IgV)样结构域、疏水的跨膜区、以及胞内。PD-1表达在CD4-CD8-胸腺细胞上，诱导性表达于活化的T细胞、B细胞、骨髓细胞、树突状细胞、自然杀伤细胞、单核细胞等。PD-1持续表达于T细胞上会诱导T细胞的衰竭。肿瘤浸润淋巴细胞表达PD-1会影响T细胞功能，减弱细胞因子分泌，减弱T细胞的杀瘤效应，与肾细胞癌、非小细胞肺癌患者预后差、肿瘤复发率高密切相关(潘佳佳等；中国药科大学学报2016,47(1):9-18)。
PD-1有两个配体，即PD-L1和PD-L2配体，均属于B7家族成员。PD-L1在活化的B细胞、T细胞、巨噬细胞、DCs、NK细胞等广泛表达。在许多肿瘤细胞表面，如肺癌、乳腺癌、恶性黑色素瘤、食管癌、胃癌、胰腺癌等肿瘤细胞中也有PD-L1的表达。肿瘤细胞表面通过高表达PD-L1或PD-L2分子，与T细胞上的受体PD-1结合，传递负调控信号，导致肿瘤抗原特异性T细胞的免疫凋亡和免疫无能，使肿瘤细胞逃避机体的免疫监控和杀伤(潘佳佳等；中国药科大学学报2016,47(1):9-18)。因此，以PD-1/PD-Ls信号通路为靶标，研发针对PD-1或PD-Ls的阻断剂，能够增强T细胞对肿瘤细胞的杀伤。
目前已经有三个针对PD-L1的免疫检查点抑制剂上市，但这些已知PD-L1抗体的抗原表位和PD-L1与PD-1结合的关键结构域并不完全一致，虽然可以阻断PD-1和PD-L1相互作用，但是对肿瘤细胞产生的影响和特异性结合PD-L1的PD-1及其衍生物不相同。此外PD-1胞外区结构域的蛋白折叠、结构、翻译后修饰和由此产生蛋白质理化性质以及体内代谢过程和抗体蛋白结构域不同。这些差异会使得包含PD-1胞外结构域的药物 具有抗体无法替代的功能特点。因此获得可以和PD-L1产生特异性相互作用的高亲和力PD-1类似物对于抗肿瘤免疫具有重大的临床应用前景。
本发明简述
与野生型PD-1和PD-L1的亲和力(nM数量级)相比，本发明的PD-1突变体多肽在仅有2个氨基酸突变的基础上，不仅保留了与野生型PD-1类似的理化性质，结合PD-L1的表位与野生型PD-1一致，而且极大提高了与PD-L1的亲和力(pM数量级)，脱靶可能性低；另外，PD-1突变体多肽具有对人、猴、鼠的物种交叉活性，为后续动物模型上的药效和毒理剂量研究提供了极大的便利。
与靶向PD-L1的抗体相比，PD-1突变体多肽为单一结构域、易于构建双抗。将PD-1突变体多肽与靶向肿瘤抗原的抗体融合形成的抗体融合蛋白，一方面可以通过选择高亲和力(高于PD-1突变体多肽与PD-L1亲和力)的肿瘤抗原特异性抗体特异性将PD-1突变体多肽聚集到肿瘤细胞上、或其附近以及肿瘤微环境内，大大减少常规免疫检查点抑制剂在体内使用带来的广泛免疫激活；另一方面，PD-1突变体多肽的快结合、快解离特性使其在发挥阻断PD-1/PD-L1信号通路的同时，避免了常规免疫检查点抑制剂由于快结合、慢解离性质导致的免疫细胞持续激活可能产生的不良反应。此外PD-1突变体的体内代谢速度比常规抗体片段快，有助于强化肿瘤部位的活性的同时减少在正常组织和血浆中的存留时间，有助于提高治疗指数(TI，therapeutic Index)。
本发明的一个方面，提供了一种PD-1突变体多肽。在一些实施方案中，所述PD-1突变体多肽具有如SEQ ID NO:1所述的核酸序列，或者具有如SEQ ID NO:2所示的氨基酸序列。
本发明的一个方面，提供了一种含有PD-1突变体多肽的抗体融合蛋白。在一些实施方案中，所述抗体融合蛋白包含所述PD-1突变体多肽以及靶向肿瘤细胞表面抗原的抗体。在一些实施方案中，所述肿瘤细胞表面抗原选自EGFR、HER2、HER3、GPC3、PSMA、CD27、CD33、CD37、CD38、CD51、CD56、mesothelin、HGFR、Ne ctin-4、Mucin 5AC、folate receptorα、carbonic anhydrase IX(CAIX)、DLL1、Notch2、Notch3、endoglin等。
在一些实施方案中，所述肿瘤抗原选自EGFR。在一些实施方案中，所述靶向肿瘤细胞表面抗原EGFR的抗体具有如SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3序列。在一些实施方案中，所述靶向肿瘤抗 原EGFR的抗体具有如SEQ ID NO:34所示氨基酸序列的重链可变区(VH)，以及如S EQ ID NO:36所示氨基酸序列的轻链可变区(VL)。在一些实施方案中，所述靶向肿瘤细胞表面抗原EGFR的抗体具有如SEQ ID NO:65所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:66所示氨基酸序列的轻链可变区(VL)。在一些实施方案中，所述靶向肿瘤抗原EGFR的抗体具有如SEQ ID NO:4所示氨基酸序列的重链(H C)，以及如SEQ ID NO:6所示氨基酸序列的轻链(LC)。在一些实施方案中，所述含有PD-1突变体多肽和靶向肿瘤细胞表面抗原EGFR的抗体融合蛋白具有如SEQ ID N O:4所示氨基酸序列的重链和如SEQ ID NO:28所示氨基酸序列的轻链。
在一些实施方案中，所述肿瘤抗原选自HER2。在一些实施方案中，所述靶向肿瘤细胞表面抗原HER2的抗体具有如SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24所示的LCDR1、LCDR2和LCDR3序列。在一些实施方案中，所述靶向肿瘤细胞表面抗原HER2的抗体具有如SEQ ID NO:38所示氨基酸序列的VH，以及如SEQ ID NO:40所示氨基酸的VL。在一些实施方案中，所述靶向肿瘤细胞表面抗原HER2的抗体具有如SEQ ID NO:67所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:68所示氨基酸序列的轻链可变区(VL)。在一些实施方案中，所述靶向肿瘤抗原HER2的抗体具有如SEQ ID NO:8所示氨基酸序列的HC和如SEQ ID NO:8所示氨基酸序列的L C。在一些实施方案中，所述含有PD-1突变体多肽和靶向肿瘤抗原HER2的抗体融合蛋白具有如SEQ ID NO:10所示氨基酸序列的重链和如SEQ ID NO:32所示氨基酸序列的轻链。
在一些实施方案中，所述肿瘤细胞表面抗原选自GPC-3。在一些实施方案中，所述靶向肿瘤细胞表面抗原GPC3的抗体具有SEQ ID NO:45、SEQ ID NO:46、SEQ ID N O:47所示的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。在一些实施方案中，所述靶向肿瘤细胞表面抗原GPC-3的抗体具有如SEQ ID NO:42所示氨基酸序列的VH，以及如SEQ ID NO:44所示氨基酸序列的VL。在一些实施方案中，所述靶向肿瘤抗原GPC-3的抗体具有如SEQ ID NO:58所示氨基酸序列的HC和如SEQ ID NO:60所示氨基酸序列的LC。在一些实施方案中，所述含有PD-1突变体多肽和靶向肿瘤抗原GPC-3的抗体融合蛋白具有如SEQ ID NO:52所示氨基酸序列的重链和SEQ ID NO:60所示氨基酸序列的轻链。在一些实施方案中，所述含有PD-1突变体多 肽和靶向肿瘤抗原GPC-3的抗体融合蛋白具有如SEQ ID NO:58所示氨基酸序列的重链和SEQ ID NO:54所示氨基酸序列的轻链。在一些实施方案中，所述含有PD-1突变体多肽和靶向肿瘤抗原GPC-3的抗体融合蛋白具有如SEQ ID NO:58所示氨基酸序列的重链和SEQ ID NO:56所示氨基酸序列的轻链。
在一些实施方案中，所述肿瘤细胞表面抗原选自PSMA。在一些实施方案中，所述靶向肿瘤细胞表面抗原PSMA的抗体具有SEQ ID NO:62所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:64所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自CD27。在一些实施方案中，所述靶向肿瘤细胞表面抗原CD27的抗体具有SEQ ID NO:69所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:70所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自CD33。在一些实施方案中，所述靶向肿瘤细胞表面抗原CD33的抗体具有如SEQ ID NO:71所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:72所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自CD37。在一些实施方案中，所述靶向肿瘤细胞表面抗原CD37的抗体具有如SEQ ID NO:73所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:74所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自CD38。在一些实施方案中，所述靶向肿瘤细胞表面抗原CD38的抗体具有如SEQ ID NO:75所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:76所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自CD51。在一些实施方案中，所述靶向肿瘤细胞表面抗原CD51的抗体具有如SEQ ID NO:77所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:78所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自CD56。在一些实施方案中，所述靶向肿瘤细胞表面抗原CD56的抗体具有如SEQ ID NO:79所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:80所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自mesothelin。在一些实施方案中，所述靶向肿瘤细胞表面抗原mesothelin的抗体具有如SEQ ID NO:81所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:82所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自HGFR。在一些实施方案中，所述靶向肿瘤细胞表面抗原HGFR的抗体具有如SEQ ID NO:83所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:84所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自Nectin-4。在一些实施方案中，所述靶向肿瘤细胞表面抗原Nectin-4的抗体具有如SEQ ID NO:85所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:86所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自Mucin5AC。在一些实施方案中，所述靶向肿瘤细胞表面抗原Mucin5AC的抗体具有如SEQ ID NO:87所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:88所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自folate receptorα。在一些实施方案中，所述靶向肿瘤细胞表面抗原folate receptorα的抗体具有如SEQ ID NO:89所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:90所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选carbonic anhydrase IX(CAIX)。在一些实施方案中，所述靶向肿瘤细胞表面抗原carbonic anhydrase IX(CAIX)的抗体具有如SEQ ID NO:91所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:92所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自DLL1。在一些实施方案中，所述靶向肿瘤细胞表面抗原DLL1的抗体具有如SEQ ID NO:93所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:94所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自Notch 2/3。在一些实施方案中，所述靶向肿瘤细胞表面抗原Notch 2/3的抗体具有如SEQ ID NO:95所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:96所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自endoglin。在一些实施方案中，所述靶向肿瘤细胞表面抗原endoglin的抗体具有如SEQ ID NO:97所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:98所示氨基酸序列的轻链可变区(VL)。
在一些实施方案中，所述肿瘤细胞表面抗原选自HER3。在一些实施方案中，所述靶向肿瘤细胞表面抗原HER3的抗体具有如SEQ ID NO:99所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:100所示氨基酸序列的轻链可变区(VL)。
本发明的一个方面，提供了一种多核苷酸，其编码如上所述的PD-1突变体多肽或含有PD-1突变体多肽的抗体融合蛋白。在一些实施方案中，所述多核苷酸选自下组的核苷酸序列：SEQ ID NO:1、3、5、7、9、11、27、31、51、53、55、57、59。本发明的一个方面，提供了一种含有如上所述PD-1突变体多肽或含有PD-1突变体多肽的抗体融合蛋白序列的载体。
本发明的一个方面，提供了一种宿主细胞，其包含如上所述的载体。
本发明的一个方面，提供了一种药物组合物，其包含药学上可接受的载体或制剂以及如上所述的PD-1突变体多肽或含有PD-1突变体多肽的抗体融合蛋白。
本发明的一个发面，提供了PD-1突变体多肽或含有PD-1突变体多肽的抗体融合蛋白作为PD-1-PD-L1相互作用抑制剂的用途。
本发明的一个方面，提供了PD-1突变体多肽或含有PD-1突变体多肽的抗体融合蛋白作为免疫调节剂的用途。
本发明的另一个方面，提供了PD-1突变体多肽或含有PD-1突变体多肽的抗体融合蛋白用于治疗肿瘤或癌症的用途。在一些实施方案中，所述治疗肿瘤或癌症的用途包括：B细胞淋巴瘤、乳腺癌、卵巢癌、NSCLC、低度恶性胶质瘤、霍奇金淋巴瘤、慢性淋巴细胞白血病(CLL)、多发性骨髓瘤、转移性结直肠癌、默克尔细胞瘤、NSCLC、鳞状NSCLC、胰腺癌、神经性间皮瘤、乳腺癌、HER2阳性转移性乳腺癌、转移性胃癌、肝癌、尿路上皮细胞癌、膀胱癌、胰腺癌、卵巢癌、肾细胞癌、多发性骨髓瘤、软组织肉瘤、原发性腹膜癌等。
和野生型PD-1与PD-L1的亲和力(nM数量级)相比，本发明的高亲和力PD-1突变体多肽与PD-L1的亲和力(pM数量级)大大提高。在一些实施方案中，所述含有PD-1突变体多肽的抗体融合蛋白与重组PD-L1的亲和力比含有野生型PD-1的抗体融合蛋白与重组PD-L1的亲和力提高39-109倍。在一些实施方案中，所述含有PD-1突变体多肽的抗体融合蛋白与细胞表面PD-L1的亲和力比含有野生型PD-1的抗体融合蛋白与细胞表面PD-L1的亲和力(几乎无结合)大大增强。因此，本发明的PD-1突变体可以用作PD-1—PD-L1相互作用的竞争性抑制剂。本发明的PD-1突变体亦可用作免疫调节剂，用于例如肿瘤的免疫治疗。
附图说明
构成本申请的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定，在附图中：
图1为抗体融合蛋白的SDS-PAGE电泳图，其中M表示蛋白marker；“-”表示不加DTT上样；“+”表示加DTT后上样，图1的泳道1为PD-1-aEGFRL、泳道2为PD-1m-aEGFRL、泳道3为aEGFR、泳道4为aPDL1、泳道5为aHER2、泳道6为PD-1-aHER2L、泳道7为PD-1m-aHER2L、泳道8为aGPC-3、泳道9为PD-1-aGPC-3L、泳道10为PD-1m-aGPC-3L、泳道11为aEGFR。
图2为PD-1突变体多肽与aEGFR、aHER2的融合蛋白SEC检测的蛋白单体峰图。
图3为PD-1突变体多肽与aEGFR抗体融合蛋白以及骨架抗体与抗原hEGFR在ELISA实验中的结合。
图4为PD-1突变体多肽与aHER2抗体融合蛋白以及骨架抗体与抗原hHER2在ELISA实验中的结合。
图5为PD-1突变体多肽与aGPC3抗体融合蛋白以及骨架抗体与抗原hGPC3在ELISA实验中的结合。
图6为PD-1突变体多肽与抗体融合蛋白以及对照抗体与可溶性的hPD-L1蛋白在ELISA实验中的亲和力检测。
图7为PD-1突变体多肽与抗体融合蛋白以及对照抗体与可溶性的mPD-L1蛋白在ELISA实验中的亲和力检测。
图8为PD-1突变体多肽与aEGFR抗体融合蛋白与相关细胞系的结合情况，其中8A为抗体融合蛋白与HepG2(hEGFR +hPDL1 -)细胞的结合，8B为抗体融合蛋白与CHO-hPDL1(hEGFR -hPDL1 +)细胞的结合，8C为抗体融合蛋白与MC38-hEGFR(hEGFR +mPDL1 +)细胞的结合，8D为抗体融合蛋白与A431(hEGFR +hPDL1 +)细胞的结合。
图9为PD-1突变体多肽与aGPC3抗体融合蛋白与相关细胞系的结合情况，其中9A为与HepG2(hGPC3 +hPDL1 -)细胞的结合，9B为与A431(hGPC3 -hPDL1 +)细胞的结合，9C为与Hepa1-6(hGPC3 -mPDL1 +)细胞的结合。
图10为可溶性的hEGFR蛋白竞争性抑制PD-1突变体多肽与aEGFR抗体融合蛋白以及骨架抗体与细胞表面hEGFR的结合，其中9A为PD-1突变体多肽与aEGFR抗体融合蛋白以及骨架抗体与MC38-hEGFR(hEGFR +mPDL1 +)细胞的结合，9B为在EGFR存在的情况下，PD-1突变体多肽与aEGFR抗体融合蛋白以及骨架抗体与MC38-hEGFR(hEGFR +mPDL1 +)细胞的结合。
图11为PD-1突变体多肽与抗体融合蛋白在大鼠中的代谢曲线，其中11A为检测完整抗体融合蛋白得到的代谢曲线，11B为检测抗体融合蛋白中的抗体骨架得到的代谢曲线。
图12为PD-1突变体多肽与抗体融合蛋白在小鼠肿瘤模型上的有效性评价，其中12A为不同蛋白样品给药后小鼠肿瘤生长的变化情况，图12B为不同蛋白样品给药后小鼠体重的变化情况。
本发明详述
本发明在此通过对使用下述定义和实施例的引用进行详细描述。所有在本文中提及的专利和公开文献的内容，包括在这些专利和公开中披露的所有序列，明确地通过提述并入本文。
术语“CDR”是指在免疫球蛋白可变区序列内的互补决定区。对于各重链和轻可变区，在重链和轻链的各可变区中存在三个CDR，其被命名为CDR1、CDR2和CDR3。术语“CDR组”是指在能够结合抗原的单一可变区中出现的三个CDR的组。这些CDR的确切边界已根据不同系统不同地定义。由Kabat(Kabat等(1987)和(1991))描述的系统，不仅提供了可适用于抗体或结合蛋白的任何可变区的明确残基编号系统，而且还提供了定义各重链或轻链序列中的三个CDR的精确残基边界。这些CDR可以被称为Kabat CDR。Chothia和同事(Chothia和Lesk(1987)J.Mol.Biol.196:901-917；Chothia等(1989)Nature342:877-883)发现Kabat CDR内的某些亚部分采取几乎相同的肽骨架构象，尽管在氨基酸序列水平上具有大的多样性。这些亚部分被命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为Chothia CDR，所述Chothia CDR具有与Kabat CDR重叠的边界。定义与Kabat CDR重叠的CDR的其它边界已由Padlan(1995)FASEB J.9:133-139和MacCallum(1996)J.Mol.Biol.262(5):732-45)描述。还有其它CDR边界定义可以不严格遵循本文系统之一，但仍将与Kabat CDR重叠，尽管鉴于特定残基或残基组或甚至整个CDR不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。本文采用了Kabat定义CDR的方法(详见 http://www.bioinf.org.uk/abs/)。
本发明的“肿瘤细胞表面抗原”指表达在肿瘤细胞表面的、可以被靶向以进行疾病治疗的抗原。在某些实施方案中，靶分子是在选自肿瘤细胞(如实体瘤或血液肿瘤细胞)表面上优先表达的抗原：特定肿瘤相关抗原的非限制性示例包括例如EGFR、HER2、HER3、HER4、GPC3、PSMA、CD27、CD33、CD37、CD38、CD51、CD56、mesothelin、HGFR、Nectin-4、Mucin 5AC、folate receptorα、carbonic anhydrase  IX(CAIX)、DLL1、Notch2、Notch3、endoglin、MUC1、MUC2,MUC3A,MUC3B、MUC4、MUC5B、MUC6、MUC7、MUC8、MUC12、MUC13、MUC15、MUC16、MUC17、MUC19、MUC20、VEGFR-1(FLT1)、VEGFR-2(KDR/FIK-1)、VEGFR-3、PDGF-RA、PDGF-RB、IGF-1R、IGF2B3、K-RAS、N-RAS、BAFF-R、EpCAM、SAGE、XAGE-1b、BAGE、MAGE蛋白(如MAGE-1、MAGE-2、MAGE-3、MAGE-4、MAGE-6、MAGE-9、MAGE-10、MAGE-12)、GAGE-1、GAGE-2、GAGE-8、GAGE-3、GAGE-4、GAGE-5、GAGE-6、GAGE-7、XAGE-1b/GAGED2a、RAGE-1、RBAF600、CD-11α、CD16A、CD21、CD22、dipeptidyl-peptidase 4(CD26)、CD30、CD32B、CD45、CD52、CD70、CD80、CD60、CD62、CD72、CD79a、CD79B、CD123、Ly6D、Ly6E、Ly6K、gp100/Pmel17、EDAR、GFRA1(GDNF-Ra1)、MRP4、RET、STEAP1、STEAP2、TENB2、E16(LAT1、SLC7A5)、SLC35D3、MPF、SCL34A2、Sema 5b、PSCAhIg、ETBR、MSG783、FcRH1、FcRH2、NCA、MDP、IL20Ra、EphA2、EphA3、EphB2R、ASLG659、GEDA、CXCR5、P2X5、LY64、IRTA2、TMEF1、TMEM46、TMEM118、LGR5、GPR19、GPR172A、CLL1、RNF43、KISS1R、ASPHD1、CXORF61、HAVCR1、表皮调节素、双调蛋白、亲脂素、AIM-2、ALDH1A1、a-辅肌动蛋白-4、ARTC1、BING-4、CALCA、CASP-5、CASP-8、cdc27、CDK4、CDKN2A、CLPP、COA-1、CPSF、Cw6、RANKL、DEK-CAN、DKK1、、EFTUD2、延伸因子2、ENAH(hMena)、ETV6-AML1、EZH2、FLT3-ITD、FN1、G250、MN、CAIX、GnTVf、GPNMB、HERV-K-MEL、hsp70-2、IDO1、、IL13Ra2、KIF20A、KK-LC-1、KM-HN-1、LAGE-1、Lengsin、M-CSF、MART-1、Melan-A/MART-1、MART2、MCSP、mdm-2、ME-1、Meloe、MMP-2、MMP-7、粘蛋白、MUM-1、MUM-2、MUM-3、肌球蛋白I类、NA88-A、PAP、neo-PAP、NFYC、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、NY-ESO-1/LAGE-2、RAB38/NY-MEL-1、OA1、OGT、OS-9、p53、PAX3、PAX5、PBF、PML-RARa、PRAME、PRDX5、PSMA(FOLH1)、PTPRK、RGS5、Rho、RhoC、RNF43、RU2AS、分离蛋白1、SIRT2、SNRPD1、SOX10、Sp17、SSX-2、SSX-4、生存素、SYT-SSX1或-SSX2、TAG-1、TAG-2、端粒酶、TGF-β、TGF-beta RII、TRAG-3、TRP-2、TRP2-INT2、VEGF、WT1、TRPM4、CRIPTO、glycoprotein IIb/IIIa receptor、glycolipid GD2、GD3、folate receptor 1(FOLR1)、IFNγ、IFNα,β,ωreceptor 1、TROP-2、Glyco-protein NMB、MMP9、GM3、mesothelin、fibronectin extra-domain B、endoglin、Rhesus D、plasma kallikrein、CS、thymic stromal lymphopoietin、mucosal addressin cell adhesion  molecule、nectin 4、NGcGM3、DLL3、DLL4、CLEC12A、KLB、FGFR1C、CEA、BCMA、p-cadherin、FAP、DR1、DR5、DR13、PLK、B7-H3、c-Met、gpA33、gp100/Pmel17、gp100、TRP-1/gp75、BCR-ABL、AFP、ALK、β-链蛋白、BRCA1、BORIS、CA9、胱天蛋白酶-8、CDK4、CTLA4、细胞周期蛋白-B1、细胞周期蛋白D1、细胞周期蛋白-A1、CYP1B1、Fra-1、GloboH、磷脂酰肌醇蛋白聚糖-3、GM3、HLA/B-RAF、hTERT、LMP2、间皮素、ML-IAP、、NA17、OX40、p15、PPLR、PCTA-1、PLAC1、PRLR、PRAME、SART-1、SART-3、TAG-72、TMPRSS2、Tn、酪氨酸酶和尿空斑蛋白-3。CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CCL27、CCL28、CX3CR1、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、雄激素受体(AR)、骨化三醇受体(CR)、雌激素受体(ER)、促肾上腺素释放激素受体(CRHR)、胰高血糖素受体(GCGR)、促性腺激素受体(FSHR、LHR)或黑皮质素1受体(MC1R,MSHR)
实施例
需要说明的是，在不冲突的情况下，本申请中的实施例仅为举例说明，不旨在对本发明造成任何方式上的限制。
实施例
实施例1抗体融合蛋白真核表达载体的构建
PCR扩增抗人EGFR抗体重链可变区(aEGFR VH)(SEQ ID No.3的1-357bp)、EGFR抗体轻链可变区(aEGFR VL)(SEQ ID NO.5的1-321bp)、抗人HER2抗体重链可变区(aHER2 VH)(SEQ ID No.9的1-360bp)、抗人HER2抗体轻链可变区(aHER2VL)(SEQ ID NO.7的1-321bp)、抗人GPC-3抗体重链可变区(aGPC-3VH)(SEQ ID NO.41)、抗人GPC-3抗体轻链可变区(aGPC-3VL)(SEQ ID NO.43)、人PD-1突变体多肽PD-1m(SEQ ID No.1)基因。通过酶切连接的方法将扩增的aEGFR VH和aHER2 VH基因分别克隆进pFuse-hIgG1-Fc2载体(InvivoGen)(其中载体上的hIgG1-Fc含有的9个突变：E233P,L234V,L235A,ΔG236,A327G,A330S,P331S,E356D,M358L均由本实验室完成)，将aEGFR VL和aHER2 VL分别克隆进pFuse2-CLIg-Hk载体(InvivoGen)。通过酶切连接的方法将扩增的PD-1m基因通过连接肽克隆进上述构建好的aEGFR抗体轻链的N端或aHER2抗体轻链的N端。所有构建的载体均测序验证。
表1序列表
Figure PCTCN2021087864-appb-000001
Figure PCTCN2021087864-appb-000002
Figure PCTCN2021087864-appb-000003
注：PD-1：人PD-1
PD-1m：人PD-1突变型
实施例2抗体融合蛋白的表达、纯化和SEC检测
将实施例1构建好的融合蛋白表达载体重链和轻链瞬时转染FreeStyle HEK293细胞(ThermoFisher)，转染时重链的质粒和轻链的质粒用量为摩尔比1:1。将28ml FreeStyle HEK 293(3×10 7细胞/ml)接种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl 293Fectin(Invitrogen)的Opti-MEM中，室温静置30min，将质粒-293Fectin mixture加至细胞培养液中125rpm，37℃，5％CO2培养。于 转染后96h收集细胞培养上清，Protein A Resin(Genscript)纯化后，SDS-PAGE检测。SDS-PAGE图如图1所示，说明成功表达出了抗体融合蛋白，且分子量大小符合预期。
将获得的Protein A resin纯化后的抗体融合蛋白用GE的AKTA chromatography过柱分析，所用的层析柱为：Superdex 200 Increase 10/300GL凝胶排阻层析柱，凝胶排阻层析所用的溶液为PBS缓冲液(0.010M phosphate buffer,0.0027M KCl,0.14M NaCl,pH7.4)。从图2的色谱图看，PD-1与aEGFR或aHER2的抗体融合蛋白的表达具有相当的纯度。
实施例4抗体融合蛋白体外活性检测
4.1结合人EGFR ELISA检测
包被hEGFR-hIGg1Fc(SinoBiological)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20 in PBS)室温封闭1小时，分别加入梯度稀释(10pM-1.2nM)的抗体(aEGFR)融合蛋白室温孵育2h，含2％脱脂奶粉的PBST洗4-5次后，加入anti-human kappa light chain-HRP(Sigma A7146,1:3000)二抗室温孵育1h，含2％脱脂奶粉的PBST洗4-5次后，QuantaBlu荧光过氧化物酶底物(Life technologies,Cat.15169)显色后于325nm和420nm处读数，或者采用TMB显色试剂(BioLegend,Cat.421101)显色后于650nm(未终止)或450nm(终止)处读数。Prizm Graphpad软件用specific binding model对数据进行非线性回归。
结果见图3。抗体融合蛋白PD-1m-aEGFR和PD-1-aEGFR对EGFR的亲和力类似。
4.2结合人HER2ELISA检测
包被hHER2-His(Acro)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，分别加入梯度稀释(10pM-1.2nM)的抗体(aHER2)融合蛋白室温孵育2h，含2％脱脂奶粉的PBST洗4-5次后，加入anti-human kappa light chain-HRP(Sigma A7146,1:3000)二抗室温孵育1h，含2％脱脂奶粉的PBST洗4-5次后，QuantaBlu荧光过氧化物酶底物(Life technologies,Cat.15169)显色后于325nm和420nm处读数，或者采用TMB显色试剂(BioLegend,Cat.421101)显色后于650nm(未终止)或450nm(终止)处读数。Prizm Graphpad软件用specific binding model对数据进行非线性回归。
结果如图4所示，PD-1-aHER2和PD-1m-aHER2对HER2的亲和力类似，与阳性对照aHER2相比，PD-1或PD-1m的融合对骨架抗体与HER2的结合影响很小。
4.3结合人GPC-3ELISA检测
分别包被hGPC-3(BioLegend)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1h，加入梯度稀释(25pM-3nM)的抗体(aGPC-3)融合蛋白室温孵育2h，含2％脱脂奶粉的PBST洗4-5次后，加入anti-human kappa light chain-HRP(Sigma A7146,1:3000)二抗室温孵育1h，含2％脱脂奶粉的PBST洗4-5次后，QuantaBlu fluoregenic peroxidase substrate(Life technologies,Cat.15169)显色后于325nm和420nm处读数，或者采用TMB显色试剂(BioLegend,Cat.421101)显色后于650nm(未终止)或450nm(终止)处读数。PrizmGraphpad软件用specific binding model对数据进行非线性回归。
结果见图5所示，PD-1-aGPC-3和PD-1m-aGPC-3对GPC3的亲和力与骨架抗体aGPC-3的亲和力类似，提示PD-1或PD-1m的融合对骨架抗体与GPC-3的结合影响很小。
4.4结合人PD-L1或鼠PD-L1ELISA检测
分别包被hPD-L1-hIGg1Fc(SinoBiological)、鼠PD-L1-Fc(SinoBiological)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1h，加入梯度稀释(25pM-3nM)的抗体融合蛋白室温孵育2h，含2％脱脂奶粉的PBST洗4-5次后，加入anti-human kappa light chain-HRP(Sigma A7146,1:3000)二抗室温孵育1h，含2％脱脂奶粉的PBST洗4-5次后，QuantaBlu fluoregenic peroxidase substrate(Life technologies,Cat.15169)显色后于325nm和420nm处读数，或者采用TMB显色试剂(BioLegend,Cat.421101)显色后于650nm(未终止)或450nm(终止)处读数。PrizmGraphpad软件用specific binding model对数据进行非线性回归。
与人PD-L1的结合结果见图6。PD-1m抗体融合蛋白(PD-1m-aEGFRL、PD-1m-aHER2L或PD-1m-aGPC-3L)对人PD-L1的亲和力与阳性对照aPD-L对人PD-L1的亲和力类似，且远远高于PD-1抗体融合蛋白与人PD-L1的亲和力(25-35倍)。
与鼠PD-L1的结合结果见图7。PD-1m抗体融合蛋白(PD-1m-aEGFRL、PD-1m-aHER2L或PD-1m-aGPC-3L)对鼠PD-L1的亲和力与阳性对照aPD-L对鼠PD-L1的亲和力类似，且远远高于PD-1抗体融合蛋白与鼠PD-L1的亲和力。
4.5抗体融合蛋白与细胞的结合
培养A431、MC38-hEGFR、CHO-PD-L1、HepG2、Hepa1-6细胞(DMEM培养基含10％FBS，1％双抗)，胰酶消化后，取2*10 5细胞用预冷的PBS洗3次后，含2％FBS的PBS 4℃封闭后，离心弃上清，加入100ul 2％FBS的PBS溶液稀释的不同浓度的抗体融合蛋白于4℃孵育1h，用含2％FBS的PBS洗去未结合的抗体融合蛋白，加入二抗Mouse  Anti-Human IgG Fc-APC(Southern biotech)在4℃孵育1h，2％FBS的PBS洗三次后，流式细胞仪检测荧光强度。
PD-1m-aEGFRL与细胞结合的结果如图8。与aEGFR相比，PD-1m-aEGFRL和PD-1-aEGFRL与HepG2细胞表面EGFR结合降低，但依然结合活性很好(图8A)，与ELISA一致。相比PD-1-aEGFRL，PD-1m-aEGFRL可以结合CHO-hPD-L1细胞表面的hPD-L1，结合活性与aPD-L1相近(图8B)，与ELISA一致。与aEGFR相比，PD-1m-aEGFRL或PD-1-aEGFRL对MC38-hEGFR拥有略好的细胞结合活性(图8C)，即协同效应。
PD-1m-aGPC-3L与细胞结合的结果如图9。与aGPC-3相比，PD-1m-a GPC-3L和PD-1-aGPC-3L与HepG2细胞表面GPC-3结合降低，但依然结合活性很好(图9A)。相比PD-1-aGPC-3L，PD-1m-aGPC-3L可以结合A431细胞表面的hPD-L1(9B)和Hepa1-6细胞表面的mPDL1(9C)。
4.6竞争抑制试验
培养人EGFR的MC38细胞(MC38-hEGFR，本实验室构建)(DMEM培养基含10％FBS，1％双抗)，胰酶消化后，取2*10 5细胞用预冷的PBS洗3次后，含2％FBS的PBS 4℃封闭后，离心弃上清，加入100ul 2％FBS的PBS溶液稀释的不同浓度的抗体融合蛋白于4℃孵育1h，用含2％FBS的PBS洗去未结合的抗体融合蛋白，加入二抗Mouse Anti-Human IgG Fc-APC(Southern biotech)在4℃孵育1h，2％FBS的PBS洗三次后，流式细胞仪检测荧光强度。
PBS洗3次后，离心弃上清后加入2％FBS的PBS溶液4℃孵育1h。孵育结束后，离心弃上清，加入100ul 2％FBS的PBS溶液稀释的不同浓度的抗体融合蛋白进行细胞结合，或者将不同浓度的抗体融合蛋白与500nM的EGFR-His和细胞孵育进行竞争结合。用含2％FBS的PBS洗去未结合的抗体融合蛋白，加入二抗Mouse Anti-Human IgG Fc-APC(southern biotech)在4℃孵育1h，2％FBS的PBS洗三次后，流式细胞仪检测荧光强度。
结果如图10所示。PD-1m-aEGFRL与MC38-hEGFR稳转株细胞表面hEGFR的结合可被溶液中游离的hEGFR-his减弱，但是在有PD-1m与细胞表面mPD-L1结合发生的情况下，其减弱程度小于对照aEGFR或PD-1-aEGFR与MC38-hEGFR稳转株细胞的结合。
4.7大鼠PK实验
将10mg/kg剂量的抗体融合蛋白通过腹腔注射(I.P.)和尾静脉注射(I.V.)给SD雄性大鼠(3只/组)。尾静脉采集肝素抗凝血，采血时间如下：15min、30min、1h、4h、 8h、1h、2d、3d、5d、7d和14d。离心后取血浆，-80℃保存备用。血浆中抗体融合蛋白及骨架抗体的含量检测参照实施例4.1进行。
结果如图11所示，抗体融合蛋白的骨架蛋白部分拥有更长的半衰期，相比之下，抗体融合蛋白的全蛋白部分拥有较短的半衰期。
4.8小鼠体内活性研究
PD1m-aEGFR抑制荷瘤小鼠肿瘤生长能力的检测在C57BL/6小鼠上进行。将5x10 6/100ul MC38-hEGFR细胞重悬于200ul无血清培养基后皮下注射小鼠(Day0)，肿瘤大小50-200mm3时候开始进行治疗给药或对照给药，并记录肿瘤和体重测量数据。在12mg/kg的给药剂量下，给药频率如图12中箭头所示，用卡钳测量实验小鼠肿瘤块大小。按下式计算肿瘤体积：肿瘤体积＝宽度*宽度*长度/2。
结果如图12所示，抗体融合蛋白可以有效抑制肿瘤生长，且无明显的体重减轻等副作用，没有EGFR靶向性的对照双抗(PD1m-aGPC3)完全无法抑制肿瘤生长，证明肿瘤靶向性的BIC-02具有良好的选择性和安全性潜力。

Claims (32)

  1. 一种PD-1突变体多肽，其中所述突变体多肽具有如SEQ ID NO:2所示的氨基酸序列。
  2. 包含权利要求1所述PD-1突变体多肽的抗体融合蛋白。
  3. 权利要求2的抗体融合蛋白，其中所述融合蛋白包含：
    a)PD-1突变体多肽；
    b)靶向肿瘤细胞表面抗原的抗体，其中所述肿瘤细胞表面抗原选自EGFR、HER2、HER3、GPC3、PSMA、CD27、CD33、CD37、CD38、CD51、CD56、mesothelin、HGFR、Nectin-4、Mucin 5AC、folate receptorα、carbonic anhydrase IX(CAIX)、DLL1、Notch2、Notch3、endoglin。
  4. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原EGFR的抗体具有：如SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3序列；或具有如SEQ ID NO:34所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:36所示氨基酸序列的轻链可变区(VL)；
  5. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原EGFR的抗体具有如SEQ ID NO:65所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:66所示氨基酸序列的轻链可变区(VL)。
  6. 权利要求3的抗体融合蛋白，其中所述靶向细胞表面肿瘤抗原HER2的抗体具有如SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24所示的LCDR1、LCDR2和LCDR3序列；或具有如SEQ ID NO:38所示氨基酸序列的VH，以及如SEQ ID NO:40所示氨基酸序列的VL。
  7. 权利要求3的抗体融合蛋白，其中所述靶向细胞表面肿瘤抗原HER2的抗体具有如SEQ ID NO:67所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:68所示氨基酸序列的轻链可变区(VL)。
  8. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原GPC3的抗体具有SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47所示的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的LCDR1、LCDR2和LCDR 3序列；或具有如SEQ ID NO:42所示氨基酸序列的VH，以及如SEQ ID NO:44所示氨基酸序列的VL。
  9. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原PSMA的抗体具有如SEQ ID NO:62所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:64所示氨基酸序列的轻链可变区(VL)。
  10. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原CD27的抗体具有如SEQ ID NO:69所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:70所示氨基酸序列的轻链可变区(VL)。
  11. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原CD33的抗体具有如SEQ ID NO:71所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:72所示氨基酸序列的轻链可变区(VL)。
  12. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原CD37的抗体具有如SEQ ID NO:73所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:74所示氨基酸序列的轻链可变区(VL)。
  13. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原CD38的抗体具有如SEQ ID NO:75所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:76所示氨基酸序列的轻链可变区(VL)。
  14. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原CD51的抗体具有如SEQ ID NO:77所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:78所示氨基酸序列的轻链可变区(VL)。
  15. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原CD56的抗体具有如SEQ ID NO:79所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:80所示氨基酸序列的轻链可变区(VL)。
  16. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原mesothelin的抗体具有如SEQ ID NO:81所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:82所示氨基酸序列的轻链可变区(VL)。
  17. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原HGFR的抗体具有如SEQ ID NO:83所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:84所示氨基酸序列的轻链可变区(VL)。
  18. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原Nectin-4的抗体具有如SEQ ID NO:85所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:86所示氨基酸序列的轻链可变区(VL)。
  19. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原Mucin5AC的抗体具有如SEQ ID NO:87所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:88所示氨基酸序列的轻链可变区(VL)。
  20. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原folate receptorα的抗体具有如SEQ ID NO:89所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:90所示氨基酸序列的轻链可变区(VL)。
  21. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原carbonic anhydrase IX(CAIX)的抗体具有如SEQ ID NO:91所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:92所示氨基酸序列的轻链可变区(VL)。
  22. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原DLL1的抗体具有如SEQ ID NO:93所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:94所示氨基酸序列的轻链可变区(VL)。
  23. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原Notch 2/3的抗体具有如SEQ ID NO:95所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:96所示氨基酸序列的轻链可变区(VL)。
  24. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原endoglin的抗体具有如SEQ ID NO:97所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:98所示氨基酸序列的轻链可变区(VL)。
  25. 权利要求3的抗体融合蛋白，其中所述靶向肿瘤细胞表面抗原endoglin的抗体具有如SEQ ID NO:99所示氨基酸序列的重链可变区(VH)，以及如SEQ ID NO:100所示氨基酸序列的轻链可变区(VL)。
  26. 权利要求2-25任一项的抗体融合蛋白，其中所述融合蛋白包含具有如下氨基酸序列的重链和轻链：SEQ ID NO:4所示的重链和SEQ ID NO:28所示的轻链；SEQ ID NO:10所示的重链和SEQ ID NO:32所示的轻链；SEQ ID NO:52所示的重链和SEQ ID NO:60所示的轻链；SEQ ID NO:58所示的重链和SEQ ID NO:54所示的轻链；SEQ ID NO:58所示的重链和SEQ ID NO:56所示的轻链。
  27. 一种多核苷酸，其编码权利要求1所述的PD-1突变体多肽或权利要求2-26中任一项所述的抗体融合蛋白，优选地，所述多核苷酸包含选自下组的核苷酸序列：SEQ ID NO:1、3、5、7、9、11、27、31、51、53、55、57、59。
  28. 一种载体，其包含如权利要求27所述的多核苷酸。
  29. 一种宿主细胞，其包含如权利要求28所述的载体。
  30. 一种药物组合物，其包含药学上可接受的载体或制剂和如权利要求1-26任一项所述的PD-1突变体多肽或抗体融合蛋白。
  31. 权利要求1-26中任一项所述的PD-1突变体多肽或抗体融合蛋白作为PD-1-PD-L1相互作用抑制剂的用途。
  32. 权利要求1-26中任一项所述的PD-1突变体多肽或抗体融合蛋白作为免疫调节剂的用途。
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