WO2022068919A1 - Peptide binding to pd-1 antibody and application thereof - Google Patents

Peptide binding to pd-1 antibody and application thereof Download PDF

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WO2022068919A1
WO2022068919A1 PCT/CN2021/122062 CN2021122062W WO2022068919A1 WO 2022068919 A1 WO2022068919 A1 WO 2022068919A1 CN 2021122062 W CN2021122062 W CN 2021122062W WO 2022068919 A1 WO2022068919 A1 WO 2022068919A1
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seq
amino acids
protein
fab
positions
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夏瑜
王忠民
张鹏
李百勇
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正大天晴康方(上海)生物医药科技有限公司
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    • 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
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention belongs to the field of immunology. Specifically, it relates to peptides that bind to PD-1 antibodies and uses thereof.
  • Cell surface glycans play important roles in cancer, such as cell signaling and communication, tumor cell dissociation and invasion, cell-matrix interaction, tumor angiogenesis, immune regulation and metastasis formation, and immune surveillance (Varki A.( 2017). Glycobiology, 27, 3-49.). Glycosylation helps tumor cells escape immune surveillance (Okada M. et al. (2017). Cell Rep., 20, 1017-1028.) (Liu CY. et al. (2011). Proc. Natl. Acad. Sci. USA, 108, 11332-11337.) (Potapenko O.I. et al. (2010). Mol. Oncol., 4, 98-118.).
  • the core part of the glycan is composed of two N-acetylglucosamine (GlcNac, NAG) and one fucose (Fucose, FUC). Fucosylation is associated with cancer (Pinho, 2015), and depleted T cells in tumors tend to be highly core-fucosylated (Okada M. et al. (2017). Cell Rep., 20, 1017-1028 ). In some cancers, such as lung and breast cancer, overexpression of core fucosylation (FUT8) has been observed (Liu CY. et al. (2011). Proc. Natl. Acad. Sci. USA, 108, 11332- 11337) (Potapenko O.I. et al.
  • glycosyl groups on the surface of PD-1 may interact with anti-PD-1 antibodies, enhancing the binding activity of the two, which in turn leads to better clinical outcomes (Fessas H. et al. (2017). Semin. Oncol., 44, 136-140).
  • the present invention has been completed.
  • the present invention relates to the following aspects:
  • a peptide preferably binding an anti-PD-1 antibody or an antigen-binding fragment thereof, said peptide comprising building block 1 and building block 2, wherein building block 1 comprises a PD-1 protein fragment selected from the group consisting of: PD
  • building block 1 comprises a PD-1 protein fragment selected from the group consisting of: PD
  • Structural unit 2 comprises a PD-1 protein fragment selected from the group consisting of amino acids at positions 127-133 of PD-1 protein (as shown in SEQ ID NO: 18), amino acids at positions 127-132 (show in SEQ ID NO: 19), amino acids 128-133 (shown in SEQ ID NO: 20), and amino acids 128-132 (shown in SEQ ID NO: 21).
  • the peptide of claim 1, comprising structural unit 1 and structural unit 2, wherein structural unit 1 comprises amino acids at positions 29-85 of PD-1 protein (as shown in SEQ ID NO: 11) or Amino acids at positions 58-85 (as shown in SEQ ID NO: 15), and structural unit 2 comprises amino acids at positions 128-132 of PD-1 protein (as shown in SEQ ID NO: 21) or 130-132.
  • Amino acids (as shown in SEQ ID NO: 3) preferably structural unit 1 comprises amino acids at positions 58 to 85 of PD-1 protein (as shown in SEQ ID NO: 15), and structural unit 2 comprises 130 of PD-1 protein - amino acid at position 132 (as shown in SEQ ID NO: 3).
  • structural unit 1 comprises the sequence shown in any one of SEQ ID NO: 2 or SEQ ID NO: 10-17
  • structural unit 2 comprises SEQ ID NO: 3 or The sequence shown in any one of SEQ ID NOs: 18-21, preferably structural unit 1 comprises the sequence shown in SEQ ID NO: 2, and structural unit 2 comprises the sequence shown in SEQ ID NO: 3.
  • the antigen-binding fragment of the anti-PD-1 antibody is a Fab fragment, Fab', (Fab') 2 , Fab'-SH, Fab/c, Fv, Single chain antibodies (eg, scFv).
  • glycosylated side chain comprises mannose, N-acetylglucosamine; fucose and ⁇ -D-mannose.
  • D29, T59, E61, S62, K78, E84, D85, L128, P130, A132 or their combination of the PD-1 protein shown in SEQ ID NO: 1 is used for screening for antibodies or antigen-binding fragments thereof that bind to PD-1 use in.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 -917; definition by Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited by any particular method of producing an antibody.
  • it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies are typically obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (eg, see U.S. Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody)
  • the antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • antigen-binding fragments include, but are not limited to: Fab fragments, Fab', (Fab') 2 , Fab/c, Fv, Fab'-SH, single chain antibodies (eg, scFv).
  • Fab fragment consists of a light chain and the variable regions of CH1 and a heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • Fab'fragment contains part of one light chain and one heavy chain (which contains the VH and CH1 domains and also the region between the CH1 and CH2 domains) part) so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab') 2 molecule.
  • F(ab') 2 fragment contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that the Interchain disulfide bonds are formed between the chains.
  • the F(ab') 2 fragment thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • Fv region includes variable regions from heavy and light chains, but lacks constant regions.
  • Fab'-SH is the designation herein for Fab' wherein one or more cysteine residues of the constant domains carry free thiol groups.
  • Figure 1 Overall structural model diagram of PD-1 protein complex with 14C12H1L1-Fab.
  • FAB-HC 14C12H1L1-Fab heavy chain portion;
  • FAB-LC 14C12H1L1-Fab light chain portion.
  • Figure 2 Schematic diagram of the interaction between PD-1 protein and 14C12H1L1-Fab heavy chain amino acids.
  • FAB-HC 14C12H1L1-Fab heavy chain portion.
  • Figure 3 Schematic diagram of the interaction between PD-1 protein and 14C12H1L1-Fab light chain amino acids.
  • FAB-LC 14C12H1L1-Fab light chain portion.
  • Figure 4 Schematic diagram of the interaction between the glycosylated side chain of PD-1 protein Asn58 and the heavy chain amino acids of 14C12H1L1-Fab.
  • MAN mannose, mannose
  • NAG N Acetyl Glucosamine, acetylglucosamine
  • FUC Fucose, fucose
  • BMA ⁇ -D-mannose, ⁇ -D-mannose.
  • FAB-HC 14C12H1L1-Fab heavy chain portion
  • FAB-LC 14C12H1L1-Fab light chain portion.
  • Figure 5 Binding surface of PD-1 to 14C12H1L1-Fab, Pembrolizumab or nivolumab.
  • the residues in contact with 14C12H1L1-Fab are E61, S62 amino acid residues
  • the residues in contact with nivolumab are V64, N66, Y68, Q75, T76, D77, K78, P83 amino acid residues
  • the overlapping residue bound by 14C12H1L1-Fab and nivolumab is the amino acid residue at position E84.
  • the residues in contact with pembrolizumab are L128, A129 amino acid residues, and the overlapping residues bound by 14C12H1L1-FAB and pembrolizumab are T59, P130, A131, K132 amino acid residues.
  • the amino acid residue at position D85 was not shown due to the occlusion of the steric structure.
  • Figure 6 The binding activity of anti-PD-1 antibody to human PD-1 protein mutant hPD1(D29A)-mFc and hPD1(E61A)-mFc was detected by combined ELISA method.
  • Figure 7 The binding activity of anti-PD-1 antibody to human PD-1 protein mutant hPD1(K78A)-mFc and hPD1(E84A)-mFc was detected by combined ELISA method.
  • Figure 8 Binding ELISA method to detect the binding activity of anti-PD-1 antibody to human PD-1 protein mutant hPD1(L128A)-mFc.
  • Figure 9 Detection results of kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein hPD1-mFc.
  • Fig. 10 The detection results of kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(D29A)-mFc.
  • Figure 11 The results of the kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(E61A)-mFc.
  • Figure 12 The results of the kinetic characteristic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(K78A)-mFc.
  • Figure 13 The results of the kinetic parameter detection of anti-PD-1 antibody binding to human PD-1 protein mutant hPD1(E84A)-mFc.
  • Fig. 14 The detection results of kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(L128A)-mFc.
  • hPD1-mFc produced by Kangfang Bio, batch number: 20181025 (Genbank ID of hPD-1: NP_005009, Genbank ID of mFc: P01863).
  • hPD1(D29A)-mFc produced by Kangfang Biotechnology, batch number 20191113.
  • hPD1(E61A)-mFc produced by Kangfang Biotechnology, batch number 20191113.
  • hPD1(K78A)-mFc produced by Kangfang Biotechnology, batch number 20191113.
  • hPD1(E84A)-mFc produced by Kangfang Biotechnology, batch number 20191113.
  • hPD1(L128A)-mFc produced by Kangfang Biotechnology, batch number 20191113.
  • the sequence of human PD-1 was searched through the NCBI protein database (PD-1 Genbank ID: NP_005009), and the amino acid sequence of the extracellular region of human PD-1 (amino acids 1-170) was combined with his purification tag sequence (SEQ ID NO: 6 ) for fusion design, and the fusion protein was abbreviated as "PD1-his” (SEQ ID NO: 7), also expressed as "hPD1-his”.
  • the cDNA sequence encoding the fusion protein was derived from the amino acid codon optimization and gene synthesis commissioned by Nanjing GenScript Biotechnology. Referring to the standard techniques introduced in "Molecular Cloning Experiment Guide (Second Edition)", PCR, enzyme digestion, gel recovery, Standard molecular cloning techniques such as ligation transformation, colony PCR or enzyme digestion identification are used to subclon the target gene into a mammalian cell expression vector, and the target gene of the recombinant expression vector is further sequenced and analyzed. After the sequencing was verified to be correct, the endotoxin-free expression plasmid was prepared in large quantities and the plasmid was transiently transfected into HEK293 cells for protein expression. After 7 days of culture, the cell culture medium was collected and affinity purified using MabSelect SuRe column (GE Healthcare).
  • the inventors based on the reported PD-1-nivolumab Fab (Tan SH, et al. (2017). Nat Commun., 8, 14369) and PD-1-Pembrolizumab Fab (Horita S et al. (2016). Sci Rep. ., 6, 35297) complex structure, using the method of X-ray crystal diffraction, to The Fab portion of antibody 14C12H1L1 (14C12H1L1-Fab) interacted with the PD-1 antigen at the resolution of 14C12H1L1 and compared the differences among the three.
  • amino acid sequence of the heavy chain portion of the antibody 14C12H1L1-Fab is as follows, the underlined portion is CH1, the bold bold is the CDR region, and the underlined bold italic is his-tag:
  • amino acid sequence of the light chain in the antibody 14C12H1L1-Fab is as follows, the underlined part is CL, and the bolded bold is the CDR region:
  • 14C12H1L1-Fab binds to PD-1 in a manner distinct from pembrolizumab and nivolumab. Although all three block the binding between PD-1 and PD-L1, according to literature reports, pembrolizumab and nivolumab bind to PD-1 in a glycosylation-independent manner (Tan SH, et al. (2017). ).Nat Commun., 8, 14369.) and PD-1-Pembrolizumab Fab (Horita S et al. (2016). Sci Rep., 6, 35297), 14C12H1L1-Fab showed on the BC loop of PD-1 Numerous interactions with sugar side chains linked at the N58 position.
  • PD1 protein such as PD-1-His
  • 14C12H1L1-Fab-his was mixed with 14C12H1L1-Fab-his at a molar ratio of 1:2, and incubated on ice for 2 hours.
  • the mixture was then purified by molecular sieves (Superdex 200 10/300 column, GE Healthcare) to obtain a complex of PD-1 protein and 14C12H1L1-Fab-his using buffer conditions of 20 mM HEPEs, pH 7.5, 100 mM NaCl, 5 mM DTT.
  • the complex peak was collected and concentrated by centrifugation (Millipore, MWCO 10 kDa), and the final complex concentration was about 10 mg/ml.
  • the concentrated complex was used for the primary screening of crystals and kept in a crystal room at 20°. Two weeks later, the crystal plate was observed under a microscope, and the crystallographic conditions were selected to repeat and optimize the crystallographic conditions.
  • the crystals were grown in PEG II suit F2 (0.1M MES, pH 6.5, 20% PEG4000, 0.6M sodium chloride) with 30% EG antifreeze. SDS-PAGE showed that the crystal contents were PD1-His+14C12H1L1-Fab-His complex. After acquiring available crystals, the resolution data was collected by using the Shanghai Synchrotron Radiation Light Source for diffraction to diffraction pattern.
  • the data analysis process is as follows: DIALS is used to index and integrate the data. Aimless was used to analyze and merge the processed data, and 5% of the data were randomly selected for Rfree estimation.
  • the Molrep molecular replacement procedure was used to find the phase solution in two steps. After sequence alignment, the sequence homology between 14C12H1L1-Fab and 6foe in the PDB database is as high as 85%, so use 6foe to find the solution of 14C12H1L1-Fab-his, then fix the position of 14C12H1L1-Fab-his, and use PD-1 monomer The structure of (PDB: 3rre) to find the solution of PD-1.
  • the analytical result is that an asymmetric unit contains a PD1-his monomer and a 14C12H1L1-Fab-his fragment.
  • the model is then revised in the reciprocal space using REFMAC5.
  • the protein model was corrected in real space using COOT.
  • the model is in good agreement with the electron density map, the crystallographic R-factor and Rfree are 0.21 and 0.27, respectively (Fig. 5), and the stereochemical parameters of the structural model are within a reasonable range.
  • 5 amino acid site mutations of human PD-1 protein were selected as 29 aspartic acid, 61 glutamic acid, 78 lysine, 84 glutamic acid and 128 leucine. is alanine [Lee, J.Y., et al., Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy.
  • Dilute PD-1 antigen to 1 ⁇ g/mL with coating buffer add 50 ⁇ L per well to the ELISA plate, and incubate at 4°C overnight. After washing the plate with PBST, 300 ⁇ L of 1% BSA (PBS) was added to each well, and the plate was blocked at 37° C. for 2 h.
  • PBS 1% BSA
  • the anti-PD-1 antibody 14C12H1L1(hG1TM) was diluted with PBST to 0.3333 ⁇ g/mL as the starting concentration, and diluted 1:3 down to 0.1111 ⁇ g/mL, 0.0370 ⁇ g/mL on the ELISA plate , 0.0123 ⁇ g/mL, 0.0041 ⁇ g/mL, 0.0014 ⁇ g/mL, 0.0005 ⁇ g/mL, a total of 7 gradient concentrations, a blank control was set up, and 2 duplicate wells were made, each well 100 ⁇ L, mixed and incubated at 37°C 30min.
  • the detection results are shown in Table 1 and Figures 6-8.
  • the five human PD-1 protein mutants D29A, E61A, K78A, E84A, L128A bind to the anti-PD-1 antibody with EC50 of 0.021nM, 2.47 ⁇ 10 12 nM, 0.023nM, 0.022nM, 4.207nM.
  • the binding ability of E61A and L128A mutants to anti-PD-1 antibody was significantly reduced.
  • the anti-PD-1 antibody was diluted to 5 ⁇ g/mL with PBS (containing 0.02% Tween-20, 0.1% BSA, pH 7.4) and then immobilized on the surface of AHC sensor (Fortebio) for 120 s, and the sensor was equilibrated in the buffer
  • the anti-PD-1 antibody immobilized on the sensor binds to each PD1-mFc mutant at a concentration of 1.24-100nM (three-fold dilution) for 120s, and the protein dissociates in the buffer for 300s.
  • the detection temperature was 37° C.
  • the detection frequency was 0.3 Hz
  • the vibration rate of the sample plate was 1000 rpm. Data were analyzed with a 1:1 model fit to yield affinity constants.
  • the binding results of anti-PD-1 antibody and each PD-1 mutant are shown in Figure 9-14 and Table 2.
  • Five human PD-1 protein mutants D29A, E61A, K78A, E84A, L128A bind to anti-PD-1 antibody
  • the affinity constants were 4.25E-10M, 3.30E-08M, 1.31E-10M, 6.65E-10M, 7.68E-09M, respectively. Consistent with the binding ELISA results, the binding ability of the two mutants E61A and L128A to anti-PD-1 antibodies was significantly reduced.

Abstract

Provided is a peptide binding to an anti-PD-1 antibody or an antigen binding fragment thereof, comprising structural units 1 and 2, wherein the structural unit 1 comprises amino acid at positions 58-85 of a PD-1 protein, and the structural unit 2 comprises amino acid at positions 130-132 of the PD-1 protein.

Description

结合PD-1抗体的肽及其应用Peptides that bind to PD-1 antibodies and their applications 技术领域technical field
本发明属于免疫学领域。具体涉及结合PD-1抗体的肽及其应用。The present invention belongs to the field of immunology. Specifically, it relates to peptides that bind to PD-1 antibodies and uses thereof.
背景技术Background technique
细胞表面聚糖在癌症中起着重要作用，例如细胞信号和通讯，肿瘤细胞的解离和侵袭，细胞与基质的相互作用，肿瘤血管生成，免疫调节和转移形成以及免疫监测(Varki A.(2017).Glycobiology，27，3-49.)。糖基化有助于肿瘤细胞逃脱免疫监视(Okada M.et al.(2017).Cell Rep.，20，1017-1028.)(Liu CY.et al.(2011).Proc.Natl.Acad.Sci.USA，108，11332-11337.)(Potapenko O.I.et al.(2010).Mol.Oncol.，4，98-118.)。Cell surface glycans play important roles in cancer, such as cell signaling and communication, tumor cell dissociation and invasion, cell-matrix interaction, tumor angiogenesis, immune regulation and metastasis formation, and immune surveillance (Varki A.( 2017). Glycobiology, 27, 3-49.). Glycosylation helps tumor cells escape immune surveillance (Okada M. et al. (2017). Cell Rep., 20, 1017-1028.) (Liu CY. et al. (2011). Proc. Natl. Acad. Sci. USA, 108, 11332-11337.) (Potapenko O.I. et al. (2010). Mol. Oncol., 4, 98-118.).
PD-1的细胞外免疫球蛋白可变(IgV)域中有四个报道的糖基化位点，分别是N49，N58，N74和N116(Chen D.et al.(2019).iScience，14，113-124.)(Na Z.et al.(2017).Cell Res.，27，147-150.)(Okada M.et al.(2017).Cell Rep.，20，1017-1028)。据报道，在哺乳动物(Tan SH.et al.(2017).Nat Commun.，8，14369)细胞的PD-1的N58氨基酸残基位于PD-1的BC环上，其糖基化程度很高，多数情况下其核心部分的聚糖是由两个N-乙酰氨基葡萄糖(GlcNac，NAG)和一个岩藻糖(Fucose，FUC)组成。岩藻糖糖基化与癌症有关(Pinho，2015)，肿瘤中耗竭T细胞往往为高度核心岩藻糖基化的(Okada M.et al.(2017).Cell Rep.，20，1017-1028)。在一些癌症，例如肺癌和乳腺癌中，观察到核心岩藻糖基化(FUT8)的过表达(Liu CY.et al.(2011).Proc.Natl.Acad.Sci.USA，108，11332-11337)(Potapenko O.I.et al.(2010).Mol.Oncol.，4，98-118)。核心岩藻糖基化的丧失可降低细胞表面PD-1的表达，进而增强T细胞活化(Okada M.et al.(2017).Cell Rep.，20，1017-1028)。There are four reported glycosylation sites in the extracellular immunoglobulin variable (IgV) domain of PD-1, which are N49, N58, N74 and N116 (Chen D. et al. (2019). iScience, 14 , 113-124.) (Na Z. et al. (2017). Cell Res., 27, 147-150.) (Okada M. et al. (2017). Cell Rep., 20, 1017-1028). It has been reported that the N58 amino acid residue of PD-1 in mammalian (Tan SH. et al. (2017). Nat Commun., 8, 14369) cells is located on the BC loop of PD-1, and its glycosylation degree is very high. In most cases, the core part of the glycan is composed of two N-acetylglucosamine (GlcNac, NAG) and one fucose (Fucose, FUC). Fucosylation is associated with cancer (Pinho, 2015), and depleted T cells in tumors tend to be highly core-fucosylated (Okada M. et al. (2017). Cell Rep., 20, 1017-1028 ). In some cancers, such as lung and breast cancer, overexpression of core fucosylation (FUT8) has been observed (Liu CY. et al. (2011). Proc. Natl. Acad. Sci. USA, 108, 11332- 11337) (Potapenko O.I. et al. (2010). Mol. Oncol., 4, 98-118). Loss of core fucosylation reduces cell surface PD-1 expression, which in turn enhances T cell activation (Okada M. et al. (2017). Cell Rep., 20, 1017-1028).
PD-1表面的糖基可能会与抗PD-1抗体相互作用，增强的两者的结合活性，进而表现为更好的临床结果(Fessas H.et al.(2017).Semin.Oncol.，44，136-140)。The glycosyl groups on the surface of PD-1 may interact with anti-PD-1 antibodies, enhancing the binding activity of the two, which in turn leads to better clinical outcomes (Fessas H. et al. (2017). Semin. Oncol., 44, 136-140).
发明内容SUMMARY OF THE INVENTION
本发明人发现，PD-1蛋白(PD-1Genbank ID：NP_005009，SEQ ID NO：1)的T59、E61、S62、E84、D85位氨基酸与抗PD-1抗体例如14C12H1L1-Fab重链的Y32、D33、S52、G54、Y57、Y100位氨基酸，PD-1蛋白的P130、K131、A132位氨基酸与抗PD-1抗体例如14C12H1L1-Fab轻链的F325、E324、D323位氨基酸发生相互作用。由此完成了本发明。The inventors found that the amino acids at positions T59, E61, S62, E84, and D85 of PD-1 protein (PD-1 Genbank ID: NP_005009, SEQ ID NO: 1) were associated with anti-PD-1 antibodies such as Y32, Amino acids at positions D33, S52, G54, Y57, and Y100, and amino acids at positions P130, K131, and A132 of PD-1 protein interact with amino acids at positions F325, E324, and D323 of anti-PD-1 antibodies such as 14C12H1L1-Fab light chain. Thus, the present invention has been completed.
具体地，本发明涉及以下几个方面：Specifically, the present invention relates to the following aspects:
1.肽，优选结合抗PD-1抗体或其抗原结合片段，所述肽包含结构单元1和结构单元2，其中结构单元1包含选自以下各项组成的组的PD-1蛋白片段：PD-1蛋白的28位-86位的氨基酸(如SEQ ID NO：10所示)，28位-85位的氨基酸(如SEQ ID NO：11所示)，29位-86位的氨基酸(如SEQ ID NO：12所示)，29位-85位的氨基酸(如SEQ ID NO：13所示)，58位-86位的氨基酸(如SEQ ID NO：14所示)、58位-85位的氨基酸(如SEQ ID NO：15所示)，59位-86位的氨基酸(如SEQ ID NO：16所示)和59位-85位的氨基酸(如SEQ ID NO：17所示)，1. A peptide, preferably binding an anti-PD-1 antibody or an antigen-binding fragment thereof, said peptide comprising building block 1 and building block 2, wherein building block 1 comprises a PD-1 protein fragment selected from the group consisting of: PD The amino acids from positions 28 to 86 of the -1 protein (as shown in SEQ ID NO: 10), the amino acids from positions 28 to 85 (as shown in SEQ ID NO: 11), the amino acids from positions 29 to 86 (as shown in SEQ ID NO: 11) ID NO: 12), amino acids from positions 29 to 85 (as shown in SEQ ID NO: 13), amino acids from positions 58 to 86 (as shown in SEQ ID NO: 14), amino acids from positions 58 to 85 amino acids (as shown in SEQ ID NO: 15), amino acids at positions 59-86 (as shown in SEQ ID NO: 16) and amino acids at positions 59-85 (as shown in SEQ ID NO: 17),
结构单元2包含选自以下各项组成的组的PD-1蛋白片段：PD-1蛋白的127位-133位的氨基酸(如SEQ ID NO：18所示)，127位-132位的氨基酸(如SEQ ID NO：19所示)，128位-133位的氨基酸(如SEQ ID NO：20所示)，和128位-132位的氨基酸(如SEQ ID NO：21所示)。 Structural unit 2 comprises a PD-1 protein fragment selected from the group consisting of amino acids at positions 127-133 of PD-1 protein (as shown in SEQ ID NO: 18), amino acids at positions 127-132 ( shown in SEQ ID NO: 19), amino acids 128-133 (shown in SEQ ID NO: 20), and amino acids 128-132 (shown in SEQ ID NO: 21).
2.权利要求1所述的肽，所述肽包含结构单元1和结构单元2，其中结构单元1包含PD-1蛋白的29位-85位的氨基酸(如SEQ ID NO：11所示)或58位-85位的氨基酸(如SEQ ID NO：15所示)，结构单元2包含PD-1蛋白的128位-132位的氨基酸(如SEQ ID NO：21所示)或130-132位的氨基酸(如SEQ ID NO：3所示)，优选结构单元1包含PD-1蛋白的58位-85位的氨基酸(如SEQ ID NO：15所示)，结构单元2包含PD-1蛋白的130-132位的氨基酸(如SEQ ID NO：3所示)。2. The peptide of claim 1, comprising structural unit 1 and structural unit 2, wherein structural unit 1 comprises amino acids at positions 29-85 of PD-1 protein (as shown in SEQ ID NO: 11) or Amino acids at positions 58-85 (as shown in SEQ ID NO: 15), and structural unit 2 comprises amino acids at positions 128-132 of PD-1 protein (as shown in SEQ ID NO: 21) or 130-132. Amino acids (as shown in SEQ ID NO: 3), preferably structural unit 1 comprises amino acids at positions 58 to 85 of PD-1 protein (as shown in SEQ ID NO: 15), and structural unit 2 comprises 130 of PD-1 protein - amino acid at position 132 (as shown in SEQ ID NO: 3).
3.项目1或2所述的肽，其中PD-1蛋白的氨基酸序列如SEQ ID NO：1所示。3. The peptide of item 1 or 2, wherein the amino acid sequence of the PD-1 protein is shown in SEQ ID NO: 1.
4.项目1-3任一项所述的肽，其中结构单元1包含SEQ ID NO：2 或SEQ ID NO：10-17任一项所示的序列，结构单元2包含SEQ ID NO：3或SEQ ID NO：18-21任一项所示的序列，优选结构单元1包含SEQ ID NO：2所示的序列，结构单元2包含SEQ ID NO：3所示的序列。4. The peptide of any one of items 1-3, wherein structural unit 1 comprises the sequence shown in any one of SEQ ID NO: 2 or SEQ ID NO: 10-17, and structural unit 2 comprises SEQ ID NO: 3 or The sequence shown in any one of SEQ ID NOs: 18-21, preferably structural unit 1 comprises the sequence shown in SEQ ID NO: 2, and structural unit 2 comprises the sequence shown in SEQ ID NO: 3.
5.项目1-4任一项所述的肽，其中所述抗PD-1抗体的抗原结合片段为Fab片段、Fab’、(Fab’) 2、Fab’-SH、Fab/c、Fv、单链抗体(例如，scFv)。 5. The peptide of any one of items 1-4, wherein the antigen-binding fragment of the anti-PD-1 antibody is a Fab fragment, Fab', (Fab') 2 , Fab'-SH, Fab/c, Fv, Single chain antibodies (eg, scFv).
6.项目5所述的肽，其中所述Fab片段的重链氨基酸序列如SEQ ID NO：4所示，轻链氨基酸序列如SEQ ID NO：5所示。6. The peptide of item 5, wherein the heavy chain amino acid sequence of the Fab fragment is shown in SEQ ID NO:4, and the light chain amino acid sequence is shown in SEQ ID NO:5.
7.项目1-6任一项所述的肽，其中PD-1蛋白的T59、E61、S62、E84、D85氨基酸与抗PD-1抗体(例如14C12H1L1-Fab)重链的Y32、D33、S52、G54、Y57、Y100位氨基酸结合，PD-1蛋白的P130、K131、A132氨基酸与抗PD-1抗体轻链的F325、E324、D323位氨基酸结合，PD-1蛋白的N58糖基化侧链与抗PD-1抗体(例如14C12H1L1-Fab)重链氨基酸(例如S31、G53、G54和R56)结合。7. The peptide of any one of items 1-6, wherein amino acids T59, E61, S62, E84, D85 of PD-1 protein are associated with Y32, D33, S52 of the heavy chain of an anti-PD-1 antibody (eg, 14C12H1L1-Fab) , G54, Y57, Y100 amino acids are combined, P130, K131, A132 amino acids of PD-1 protein are combined with F325, E324, D323 amino acids of anti-PD-1 antibody light chain, N58 glycosylated side chain of PD-1 protein Binds to heavy chain amino acids (eg S31, G53, G54 and R56) of anti-PD-1 antibodies (eg 14C12H1L1-Fab).
8.项目7所述的肽，其中所述糖基化侧链包含甘露糖，N-乙酰氨基葡萄糖；岩藻糖和β-D-甘露糖。8. The peptide of item 7, wherein the glycosylated side chain comprises mannose, N-acetylglucosamine; fucose and β-D-mannose.
9.项目1-8任一项所述的肽在筛选结合PD-1的抗体或其抗原结合片段中的用途。9. Use of the peptide of any one of items 1 to 8 for screening an antibody or antigen-binding fragment thereof that binds to PD-1.
10.SEQ ID NO：1所示的PD-1蛋白的D29，T59，E61，S62，K78，E84，D85，L128，P130，A132或其组合在筛选结合PD-1的抗体或其抗原结合片段中的用途。10. D29, T59, E61, S62, K78, E84, D85, L128, P130, A132 or their combination of the PD-1 protein shown in SEQ ID NO: 1 is used for screening for antibodies or antigen-binding fragments thereof that bind to PD-1 use in.
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构 域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987 and 1991))，或Chothia & Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。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。As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain . Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair, respectively, form the antibody binding site. The assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 -917; definition by Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies are typically obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (eg, see U.S. Patent 4,816,567).
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature，321：522525(1986)；Reichmann et al.，Nature，332：323 329(1988)；Presta，Curr.Op. Struct.Biol.，2：593 596(1992)；和Clark，Immunol.Today 21：397 402(2000)。As used herein, the term "humanized antibody" refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody) The antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, eg, Jones et al., Nature, 321:522525 (1986); Reichmann et al., Nature, 332:323329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).
在本发明中，抗原结合片段包括，但不限于：Fab片段、Fab’、(Fab’) 2、Fab/c、Fv、Fab’-SH、单链抗体(例如，scFv)。 In the present invention, antigen-binding fragments include, but are not limited to: Fab fragments, Fab', (Fab') 2 , Fab/c, Fv, Fab'-SH, single chain antibodies (eg, scFv).
如本发明中所使用的，术语“Fab片段”由一条轻链和C H1以及一条重链的可变区组成。Fab分子的重链不能与另一条重链分子形成二硫键。 As used in the present invention, the term "Fab fragment" consists of a light chain and the variable regions of CH1 and a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
如本发明中所使用的，术语“Fab’片段”含有一条轻链和一条重链的部分(其含有V H结构域和C H1结构域以及还有C H1与C H2结构域之间的区域的部分)，以便可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’) 2分子。 As used in the present invention, the term "Fab'fragment" contains part of one light chain and one heavy chain (which contains the VH and CH1 domains and also the region between the CH1 and CH2 domains) part) so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab') 2 molecule.
如本发明中所使用的，术语“F(ab’) 2片段”含有两条轻链和两条含有C H1与C H2结构域之间的恒定区的部分的重链，以便在两条重链之间形成链间二硫键。F(ab’) 2片段从而由通过两条重链之间的二硫键保持在一起的两个Fab’片段组成。 As used in the present invention, the term "F(ab') 2 fragment" contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that the Interchain disulfide bonds are formed between the chains. The F(ab') 2 fragment thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
如本发明中所使用的，术语“Fv区”包含来自重链和轻链的可变区，但缺乏恒定区。As used in the present invention, the term "Fv region" includes variable regions from heavy and light chains, but lacks constant regions.
如本发明中所使用的，术语“Fab’-SH”是本文对Fab’的命名，其中恒定结构域的一个或多个半胱氨酸残基携带游离硫醇基团。As used in the present invention, the term "Fab'-SH" is the designation herein for Fab' wherein one or more cysteine residues of the constant domains carry free thiol groups.
附图说明：Description of drawings:
图1：PD-1蛋白与14C12H1L1-Fab复合物的整体结构模型图。FAB-HC：14C12H1L1-Fab重链部分；FAB-LC：14C12H1L1-Fab轻链部分。Figure 1: Overall structural model diagram of PD-1 protein complex with 14C12H1L1-Fab. FAB-HC: 14C12H1L1-Fab heavy chain portion; FAB-LC: 14C12H1L1-Fab light chain portion.
图2：PD-1蛋白与14C12H1L1-Fab重链氨基酸相互作用示意图。FAB-HC：14C12H1L1-Fab重链部分。Figure 2: Schematic diagram of the interaction between PD-1 protein and 14C12H1L1-Fab heavy chain amino acids. FAB-HC: 14C12H1L1-Fab heavy chain portion.
图3：PD-1蛋白与14C12H1L1-Fab轻链氨基酸相互作用示意图。FAB-LC：14C12H1L1-Fab轻链部分。Figure 3: Schematic diagram of the interaction between PD-1 protein and 14C12H1L1-Fab light chain amino acids. FAB-LC: 14C12H1L1-Fab light chain portion.
图4：PD-1蛋白Asn58糖基化侧链与14C12H1L1-Fab重链氨基酸相互作用示意图。MAN：mannose，甘露糖；NAG：N Acetyl Glucosamine，乙酰氨基葡萄糖；FUC：Fucose，岩藻糖；BMA：β-D-mannose，β-D-甘露糖。FAB-HC：14C12H1L1-Fab重链部分；FAB-LC：14C12H1L1-Fab轻链部分。Figure 4: Schematic diagram of the interaction between the glycosylated side chain of PD-1 protein Asn58 and the heavy chain amino acids of 14C12H1L1-Fab. MAN: mannose, mannose; NAG: N Acetyl Glucosamine, acetylglucosamine; FUC: Fucose, fucose; BMA: β-D-mannose, β-D-mannose. FAB-HC: 14C12H1L1-Fab heavy chain portion; FAB-LC: 14C12H1L1-Fab light chain portion.
图5：PD-1与14C12H1L1-Fab，Pembrolizumab或nivolumab的结合表面。PD-1蛋白中，与14C12H1L1-Fab接触的残基是E61、S62位氨基酸残基，而与nivolumab接触的残基是V64、N66、Y68、Q75、T76、D77、K78、P83位氨基酸残基，由14C12H1L1-Fab和nivolumab结合的重叠残基是E84位氨基酸残基。与pembrolizumab接触的残基是L128、A129位氨基酸残基，由14C12H1L1-FAB和pembrolizumab结合的重叠残基是T59、P130、A131、K132位氨基酸残基。D85位氨基酸残基由于立体结构遮挡未能显示。Figure 5: Binding surface of PD-1 to 14C12H1L1-Fab, Pembrolizumab or nivolumab. In PD-1 protein, the residues in contact with 14C12H1L1-Fab are E61, S62 amino acid residues, while the residues in contact with nivolumab are V64, N66, Y68, Q75, T76, D77, K78, P83 amino acid residues , the overlapping residue bound by 14C12H1L1-Fab and nivolumab is the amino acid residue at position E84. The residues in contact with pembrolizumab are L128, A129 amino acid residues, and the overlapping residues bound by 14C12H1L1-FAB and pembrolizumab are T59, P130, A131, K132 amino acid residues. The amino acid residue at position D85 was not shown due to the occlusion of the steric structure.
图6：结合ELISA方法检测抗PD-1抗体与人PD-1蛋白突变体hPD1(D29A)-mFc、hPD1(E61A)-mFc的结合活性。Figure 6: The binding activity of anti-PD-1 antibody to human PD-1 protein mutant hPD1(D29A)-mFc and hPD1(E61A)-mFc was detected by combined ELISA method.
图7：结合ELISA方法检测抗PD-1抗体与人PD-1蛋白突变体hPD1(K78A)-mFc、hPD1(E84A)-mFc的结合活性。Figure 7: The binding activity of anti-PD-1 antibody to human PD-1 protein mutant hPD1(K78A)-mFc and hPD1(E84A)-mFc was detected by combined ELISA method.
图8：结合ELISA方法检测抗PD-1抗体与人PD-1蛋白突变体hPD1(L128A)-mFc的结合活性。Figure 8: Binding ELISA method to detect the binding activity of anti-PD-1 antibody to human PD-1 protein mutant hPD1(L128A)-mFc.
图9：抗PD-1抗体与人PD-1蛋白hPD1-mFc结合的动力学特征参数检测结果。Figure 9: Detection results of kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein hPD1-mFc.
图10：抗PD-1抗体与人PD-1蛋白突变体hPD1(D29A)-mFc结合的动力学特征参数检测结果。Fig. 10: The detection results of kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(D29A)-mFc.
图11：抗PD-1抗体与人PD-1蛋白突变体hPD1(E61A)-mFc结合的动力学特征参数检测结果。Figure 11 : The results of the kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(E61A)-mFc.
图12：抗PD-1抗体与人PD-1蛋白突变体hPD1(K78A)-mFc结合的动力学特征参数检测结果。Figure 12 : The results of the kinetic characteristic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(K78A)-mFc.
图13：抗PD-1抗体与人PD-1蛋白突变体hPD1(E84A)-mFc结合的动力学特征参数检测结果。Figure 13 : The results of the kinetic parameter detection of anti-PD-1 antibody binding to human PD-1 protein mutant hPD1(E84A)-mFc.
图14：抗PD-1抗体与人PD-1蛋白突变体hPD1(L128A)-mFc结合的动力学特征参数检测结果。Fig. 14: The detection results of kinetic parameters of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(L128A)-mFc.
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的 范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. If the reagents or instruments used do not indicate the manufacturer, they are conventional products that can be purchased in the market.
主要仪器与试剂Main instruments and reagents
PD-1-his(康方生物生产)PD-1-his (produced by Kangfang Bio)
14C12H1L1-Fab-his(康方生物生产)14C12H1L1-Fab-his (produced by Kangfang Bio)
14C12H1L1(hG1TM)(康方生物生产)14C12H1L1(hG1TM) (manufactured by Kangfang Bio)
hPD1-mFc：康方生物生产，批号：20181025(hPD-1的Genbank ID：NP_005009，mFc的Genbank ID：P01863)。hPD1-mFc: produced by Kangfang Bio, batch number: 20181025 (Genbank ID of hPD-1: NP_005009, Genbank ID of mFc: P01863).
hPD1(D29A)-mFc：康方生物生产，批号20191113。hPD1(D29A)-mFc: produced by Kangfang Biotechnology, batch number 20191113.
hPD1(E61A)-mFc：康方生物生产，批号20191113。hPD1(E61A)-mFc: produced by Kangfang Biotechnology, batch number 20191113.
hPD1(K78A)-mFc：康方生物生产，批号20191113。hPD1(K78A)-mFc: produced by Kangfang Biotechnology, batch number 20191113.
hPD1(E84A)-mFc：康方生物生产，批号20191113。hPD1(E84A)-mFc: produced by Kangfang Biotechnology, batch number 20191113.
hPD1(L128A)-mFc：康方生物生产，批号20191113。hPD1(L128A)-mFc: produced by Kangfang Biotechnology, batch number 20191113.
制备例1.人PD1-his融合蛋白的制备Preparation Example 1. Preparation of human PD1-his fusion protein
通过NCBI蛋白质数据库查找人PD-1的序列(PD-1Genbank ID：NP_005009)，将人PD-1胞外区氨基酸序列(1位氨基酸-170位氨基酸)与his纯化标签序列(SEQ ID NO：6)进行融合设计，融合蛋白简写命名为“PD1-his”(SEQ ID NO：7)，也表示为“hPD1-his”。The sequence of human PD-1 was searched through the NCBI protein database (PD-1 Genbank ID: NP_005009), and the amino acid sequence of the extracellular region of human PD-1 (amino acids 1-170) was combined with his purification tag sequence (SEQ ID NO: 6 ) for fusion design, and the fusion protein was abbreviated as "PD1-his" (SEQ ID NO: 7), also expressed as "hPD1-his".
编码融合蛋白的cDNA序列来源于委托南京金斯瑞生物进行的氨基酸密码子优化和基因合成，参照《分子克隆实验指南(第二版)》介绍的标准技术，采用PCR、酶切、胶回收、连接转化、菌落PCR或酶切鉴定等标准的分子克隆技术将目的基因亚克隆到哺乳动物细胞表达载体，并进一步对重组表达载体的目的基因进行测序分析。测序验证正确后，中大量制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行蛋白表达。培养7天后收集细胞培养液，采用MabSelect SuRe柱料(GE Healthcare)进行亲和纯化。The cDNA sequence encoding the fusion protein was derived from the amino acid codon optimization and gene synthesis commissioned by Nanjing GenScript Biotechnology. Referring to the standard techniques introduced in "Molecular Cloning Experiment Guide (Second Edition)", PCR, enzyme digestion, gel recovery, Standard molecular cloning techniques such as ligation transformation, colony PCR or enzyme digestion identification are used to subclon the target gene into a mammalian cell expression vector, and the target gene of the recombinant expression vector is further sequenced and analyzed. After the sequencing was verified to be correct, the endotoxin-free expression plasmid was prepared in large quantities and the plasmid was transiently transfected into HEK293 cells for protein expression. After 7 days of culture, the cell culture medium was collected and affinity purified using MabSelect SuRe column (GE Healthcare).
实施例1.人PD-1抗原与抗PD-1抗体结合表位分析Example 1. Analysis of human PD-1 antigen and anti-PD-1 antibody binding epitope
发明人基于已报道的PD-1-nivolumab Fab(Tan SH，et al.(2017).Nat Commun.，8，14369)和PD-1-Pembrolizumab Fab(Horita S et al.(2016).Sci Rep.，6，35297)的复杂结构，采用X线晶体衍射的方法，以
Figure PCTCN2021122062-appb-000001
的分辨率研究了抗体14C12H1L1的Fab部分(14C12H1L1-Fab)和PD-1抗原相互作用，并比较了三者的差异。 The inventors based on the reported PD-1-nivolumab Fab (Tan SH, et al. (2017). Nat Commun., 8, 14369) and PD-1-Pembrolizumab Fab (Horita S et al. (2016). Sci Rep. ., 6, 35297) complex structure, using the method of X-ray crystal diffraction, to
Figure PCTCN2021122062-appb-000001
The Fab portion of antibody 14C12H1L1 (14C12H1L1-Fab) interacted with the PD-1 antigen at the resolution of 14C12H1L1 and compared the differences among the three.
抗体14C12H1L1-Fab中的重链部分的氨基酸序列如下，下划线部分为CH1，加粗黑体的为CDR区域，下划线粗斜体的为组氨酸标签(his-tag)：The amino acid sequence of the heavy chain portion of the antibody 14C12H1L1-Fab is as follows, the underlined portion is CH1, the bold bold is the CDR region, and the underlined bold italic is his-tag:
Figure PCTCN2021122062-appb-000002
Figure PCTCN2021122062-appb-000002
抗体14C12H1L1-Fab中的轻链的氨基酸序列如下，下划线部分为CL，加粗黑体的为CDR区域：The amino acid sequence of the light chain in the antibody 14C12H1L1-Fab is as follows, the underlined part is CL, and the bolded bold is the CDR region:
Figure PCTCN2021122062-appb-000003
Figure PCTCN2021122062-appb-000003
发明人出乎意料地发现，14C12H1L1-Fab通过区别于pembrolizumab和nivolumab的方式与PD-1结合。尽管三者都阻断了PD-1和PD-L1之间的结合，但是，根据文献报道，pembrolizumab和nivolumab以不依赖糖基化的方式结合至PD-1(Tan SH，et al.(2017).Nat Commun.，8，14369.)和PD-1-Pembrolizumab Fab(Horita S et al.(2016).Sci Rep.，6，35297)，14C12H1L1-Fab在PD-1的BC环上显示出与N58位链接的糖侧链的大量相互作用。The inventors unexpectedly discovered that 14C12H1L1-Fab binds to PD-1 in a manner distinct from pembrolizumab and nivolumab. Although all three block the binding between PD-1 and PD-L1, according to literature reports, pembrolizumab and nivolumab bind to PD-1 in a glycosylation-independent manner (Tan SH, et al. (2017). ).Nat Commun., 8, 14369.) and PD-1-Pembrolizumab Fab (Horita S et al. (2016). Sci Rep., 6, 35297), 14C12H1L1-Fab showed on the BC loop of PD-1 Numerous interactions with sugar side chains linked at the N58 position.
实验方法：将PD1蛋白，如PD-1-His，与14C12H1L1-Fab-his按照1∶2摩尔比混合，冰上孵育2小时。混合物随后进行分子筛(Superdex 200  10/300 column，GE Healthcare)纯化得到PD-1蛋白与14C12H1L1-Fab-his的复合物，所使用的缓冲液条件为20mM HEPEs，pH 7.5，100mM NaCl，5mM DTT。收取复合物峰进行离心浓缩(Millipore，MWCO 10kDa)，最终复合物浓度约为10mg/ml。将浓缩后的复合物用于晶体初筛，于20°晶体房静置。两周后，在显微镜下观察晶体板，选取晶体外形较好的条件进行结晶条件的重复及优化。晶体的生长条件为PEG II suit F2(0.1M MES，pH 6.5，20％PEG4000，0.6M sodium chloride)，防冻剂为30％EG。SDS-PAGE显示晶体内容物为PD1-His+14C12H1L1-Fab-His复合物。在获取可用晶体后，通过使用上海同步辐射光源收集分辨率数据为衍射到
Figure PCTCN2021122062-appb-000004
的衍射图形。 Experimental method: PD1 protein, such as PD-1-His, was mixed with 14C12H1L1-Fab-his at a molar ratio of 1:2, and incubated on ice for 2 hours. The mixture was then purified by molecular sieves (Superdex 200 10/300 column, GE Healthcare) to obtain a complex of PD-1 protein and 14C12H1L1-Fab-his using buffer conditions of 20 mM HEPEs, pH 7.5, 100 mM NaCl, 5 mM DTT. The complex peak was collected and concentrated by centrifugation (Millipore, MWCO 10 kDa), and the final complex concentration was about 10 mg/ml. The concentrated complex was used for the primary screening of crystals and kept in a crystal room at 20°. Two weeks later, the crystal plate was observed under a microscope, and the crystallographic conditions were selected to repeat and optimize the crystallographic conditions. The crystals were grown in PEG II suit F2 (0.1M MES, pH 6.5, 20% PEG4000, 0.6M sodium chloride) with 30% EG antifreeze. SDS-PAGE showed that the crystal contents were PD1-His+14C12H1L1-Fab-His complex. After acquiring available crystals, the resolution data was collected by using the Shanghai Synchrotron Radiation Light Source for diffraction to
Figure PCTCN2021122062-appb-000004
diffraction pattern.
数据解析流程如下：采用DIALS对数据进行指标化、积分处理。用Aimless分析、合并处理数据，随机选取5％数据进行Rfree估测。采用Molrep分子置换程序分两步来寻找相位解。经过序列比对，14C12H1L1-Fab与PDB数据库中6foe的序列同源性高达85％，所以用6foe寻找14C12H1L1-Fab-his的解，然后固定14C12H1L1-Fab-his的位置，用PD-1单体的结构(PDB：3rre)来寻找PD-1的解。解析结果为一个不对称单位里包含一个PD1-his单体和一个14C12H1L1-Fab-his片段。随后采用REFMAC5在倒易空间中进行模型修正。采用COOT对蛋白模型在实空间进行修正。模型与电子密度图吻合良好，晶体学R因子和Rfree分别为0.21和0.27(图5)，结构模型的立体化学参数处在合理范围内。The data analysis process is as follows: DIALS is used to index and integrate the data. Aimless was used to analyze and merge the processed data, and 5% of the data were randomly selected for Rfree estimation. The Molrep molecular replacement procedure was used to find the phase solution in two steps. After sequence alignment, the sequence homology between 14C12H1L1-Fab and 6foe in the PDB database is as high as 85%, so use 6foe to find the solution of 14C12H1L1-Fab-his, then fix the position of 14C12H1L1-Fab-his, and use PD-1 monomer The structure of (PDB: 3rre) to find the solution of PD-1. The analytical result is that an asymmetric unit contains a PD1-his monomer and a 14C12H1L1-Fab-his fragment. The model is then revised in the reciprocal space using REFMAC5. The protein model was corrected in real space using COOT. The model is in good agreement with the electron density map, the crystallographic R-factor and Rfree are 0.21 and 0.27, respectively (Fig. 5), and the stereochemical parameters of the structural model are within a reasonable range.
结构分析结果如图1-5所示。The structural analysis results are shown in Figure 1-5.
实施例2.抗PD-1抗体14C12H1L1(hG1TM)抗原结合表位的氨基酸定点突变研究Example 2. Amino acid site-directed mutagenesis of the antigen-binding epitope of anti-PD-1 antibody 14C12H1L1 (hG1TM)
根据Nivolumab抗体结合位点，选择了人PD-1蛋白29位天冬氨酸、61位谷氨酸、78位赖氨酸、84位谷氨酸、128位亮氨酸5个氨基酸位点突变为丙氨酸[Lee，J.Y.，et al.，Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy.Nature Communications，2016.7(1)：p.13354.]，这些突变体分别记为hPD1(D29A)-mFc、hPD1(E61A)-mFc、hPD1(K78A)-mFc、hPD1(E84A)-mFc、hPD1(L128A)-mFc采用结合ELISA以及Fortebio Kinetics方法检测抗PD-1抗体与这些突变 体的结合活性，实验用未突变的PD1-mFc蛋白作为对照对比。According to the binding site of Nivolumab antibody, 5 amino acid site mutations of human PD-1 protein were selected as 29 aspartic acid, 61 glutamic acid, 78 lysine, 84 glutamic acid and 128 leucine. is alanine [Lee, J.Y., et al., Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy. Nature Communications, 2016.7(1): p.13354.], and these mutants were denoted as hPD1(D29A)- mFc, hPD1(E61A)-mFc, hPD1(K78A)-mFc, hPD1(E84A)-mFc, hPD1(L128A)-mFc were used to detect the binding activity of anti-PD-1 antibodies to these mutants by binding ELISA and Fortebio Kinetics methods. The experiment used unmutated PD1-mFc protein as a control.
1、结合ELISA方法检测抗PD-1抗体14C12H1L1(hG1TM)与人PD-1蛋白突变体的结合活性1. The binding activity of anti-PD-1 antibody 14C12H1L1 (hG1TM) to human PD-1 protein mutant was detected by combined ELISA method
用包被缓冲液将PD-1抗原稀释成1μg/mL，每孔50μL加入酶标板中，置于4℃孵育过夜。用PBST洗板后每孔加入300μL 1％BSA(PBS)，37℃封闭2h。洗板后用PBST将抗PD-1抗体(14C12H1L1(hG1TM))稀释成0.3333μg/mL作为起始浓度，在酶标板上进行1∶3往下稀释为0.1111μg/mL，0.0370μg/mL，0.0123μg/mL，0.0041μg/mL，0.0014μg/mL，0.0005μg/mL共7个梯度浓度，另设空白对照，均做2个复孔，每孔100μL，混匀后置于37℃孵育30min。洗板后每孔加入50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗(Jackson)工作液，置于37℃孵育30min，用PBST洗板后加入每孔50μL TMB，置于室温避光显色5min后，每孔加入50μL终止液(2M H 2SO 4溶液)终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro6.2.1软件对数据进行分析处理。以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，得到抗体的结合EC50值。 Dilute PD-1 antigen to 1 μg/mL with coating buffer, add 50 μL per well to the ELISA plate, and incubate at 4°C overnight. After washing the plate with PBST, 300 μL of 1% BSA (PBS) was added to each well, and the plate was blocked at 37° C. for 2 h. After washing the plate, the anti-PD-1 antibody (14C12H1L1(hG1TM)) was diluted with PBST to 0.3333μg/mL as the starting concentration, and diluted 1:3 down to 0.1111μg/mL, 0.0370μg/mL on the ELISA plate , 0.0123μg/mL, 0.0041μg/mL, 0.0014μg/mL, 0.0005μg/mL, a total of 7 gradient concentrations, a blank control was set up, and 2 duplicate wells were made, each well 100μL, mixed and incubated at 37°C 30min. After washing the plate, add 50 μL of horseradish peroxidase-labeled goat anti-human IgG (H+L) secondary antibody (Jackson) working solution to each well, incubate at 37°C for 30 min, wash the plate with PBST and add 50 μL TMB to each well, After 5 minutes of color development at room temperature in the dark, 50 μL of stop solution (2M H 2 SO 4 solution) was added to each well to stop the color development reaction. Immediately put the ELISA plate into the microplate reader, and select the 450nm light wavelength to read the OD value of each well of the ELISA plate. The data were analyzed and processed with SoftMax Pro6.2.1 software. Taking the antibody concentration as the abscissa and the absorbance value as the ordinate, a 4-parameter fitting curve was drawn to obtain the binding EC50 value of the antibody.
检测结果见表1和图6-8所示，人PD-1蛋白五个突变体D29A、E61A、K78A、E84A、L128A与抗PD-1抗体结合EC50分别为0.021nM、2.47×10 12nM、0.023nM、0.022nM、4.207nM。E61A和L128A两个突变体与抗PD-1抗体的结合能力明显降低。 The detection results are shown in Table 1 and Figures 6-8. The five human PD-1 protein mutants D29A, E61A, K78A, E84A, L128A bind to the anti-PD-1 antibody with EC50 of 0.021nM, 2.47×10 12 nM, 0.023nM, 0.022nM, 4.207nM. The binding ability of E61A and L128A mutants to anti-PD-1 antibody was significantly reduced.
表1 抗PD-1抗体与人PD-1突变体的结合ELISAEC50结果Table 1 ELISA EC50 results of binding of anti-PD-1 antibodies to human PD-1 mutants
Figure PCTCN2021122062-appb-000005
Figure PCTCN2021122062-appb-000005
2.Fortebio Kinetics方法检测抗PD-1抗体14C12H1L1(hG1TM)与人PD-1蛋白突变体的结合活性2. Fortebio Kinetics method to detect the binding activity of anti-PD-1 antibody 14C12H1L1 (hG1TM) to human PD-1 protein mutants
首先将抗PD-1抗体用PBS(含0.02％Tween-20，0.1％BSA，pH7.4)稀释至5μg/mL后固定于AHC传感器(Fortebio)表面，时间为120s，传感器在缓冲液中平衡60s，固定在传感器上的抗PD-1抗体与各PD1-mFc突变体结合，浓度为1.24-100nM(三倍稀释)，时间120s，蛋白在缓冲液中解离，时间300s。检测温度为37℃，检测频率为0.3Hz，样品板震动速率为1000rpm。数据以1∶1模型拟合分析，得到亲和力常数。First, the anti-PD-1 antibody was diluted to 5 μg/mL with PBS (containing 0.02% Tween-20, 0.1% BSA, pH 7.4) and then immobilized on the surface of AHC sensor (Fortebio) for 120 s, and the sensor was equilibrated in the buffer For 60s, the anti-PD-1 antibody immobilized on the sensor binds to each PD1-mFc mutant at a concentration of 1.24-100nM (three-fold dilution) for 120s, and the protein dissociates in the buffer for 300s. The detection temperature was 37° C., the detection frequency was 0.3 Hz, and the vibration rate of the sample plate was 1000 rpm. Data were analyzed with a 1:1 model fit to yield affinity constants.
抗PD-1抗体与各PD-1突变体结合结果见图9-14和表2所示，人PD-1蛋白五个突变体D29A、E61A、K78A、E84A、L128A与抗PD-1抗体结合亲和力常数分别为4.25E-10M、3.30E-08M、1.31E-10M、6.65E-10M、7.68E-09M。与结合ELISA结果一致，E61A和L128A两个突变体与抗PD-1抗体的结合能力明显降低。The binding results of anti-PD-1 antibody and each PD-1 mutant are shown in Figure 9-14 and Table 2. Five human PD-1 protein mutants D29A, E61A, K78A, E84A, L128A bind to anti-PD-1 antibody The affinity constants were 4.25E-10M, 3.30E-08M, 1.31E-10M, 6.65E-10M, 7.68E-09M, respectively. Consistent with the binding ELISA results, the binding ability of the two mutants E61A and L128A to anti-PD-1 antibodies was significantly reduced.
表2 抗PD-1抗体与人PD-1突变体的Fortebio Kinetics结果Table 2 Fortebio Kinetics results of anti-PD-1 antibody and human PD-1 mutant
Figure PCTCN2021122062-appb-000006
Figure PCTCN2021122062-appb-000006
Figure PCTCN2021122062-appb-000007
Figure PCTCN2021122062-appb-000007
Figure PCTCN2021122062-appb-000008
Figure PCTCN2021122062-appb-000008
Figure PCTCN2021122062-appb-000009
Figure PCTCN2021122062-appb-000009
Figure PCTCN2021122062-appb-000010
Figure PCTCN2021122062-appb-000010

Claims (10)

  1. 肽，优选结合抗PD-1抗体或其抗原结合片段，所述肽包含结构单元1和结构单元2，其中结构单元1包含选自以下各项组成的组的PD-1蛋白片段：PD-1蛋白的28位-86位的氨基酸(如SEQ ID NO：10所示)，28位-85位的氨基酸(如SEQ ID NO：11所示)，29位-86位的氨基酸(如SEQ ID NO：12所示)，29位-85位的氨基酸(如SEQ ID NO：13所示)，58位-86位的氨基酸(如SEQ ID NO：14所示)、58位-85位的氨基酸(如SEQ ID NO：15所示)，59位-86位的氨基酸(如SEQI ID NO：16所示)和59位-85位的氨基酸(如SEQ ID NO：17所示)，A peptide, preferably binding an anti-PD-1 antibody or antigen-binding fragment thereof, said peptide comprising building block 1 and building block 2, wherein building block 1 comprises a PD-1 protein fragment selected from the group consisting of: PD-1 The amino acids at positions 28-86 of the protein (as shown in SEQ ID NO: 10), the amino acids at positions 28-85 (as shown in SEQ ID NO: 11), the amino acids at positions 29-86 (as shown in SEQ ID NO: 11) : shown in SEQ ID NO: 12), amino acids at positions 29-85 (shown in SEQ ID NO: 13), amino acids at positions 58-86 (shown in SEQ ID NO: 14), amino acids at positions 58-85 ( as shown in SEQ ID NO: 15), amino acids from positions 59 to 86 (as shown in SEQ ID NO: 16) and amino acids from positions 59 to 85 (as shown in SEQ ID NO: 17),
    结构单元2包含选自以下各项组成的组的PD-1蛋白片段：PD-1蛋白的127位-133位的氨基酸(如SEQ ID NO：18所示)，127位-132位的氨基酸(如SEQ ID NO：19所示)，128位-133位的氨基酸(如SEQ ID NO：20所示)，和128位-132位的氨基酸(如SEQ ID NO：21所示)。Structural unit 2 comprises a PD-1 protein fragment selected from the group consisting of amino acids at positions 127-133 of PD-1 protein (as shown in SEQ ID NO: 18), amino acids at positions 127-132 ( shown in SEQ ID NO: 19), amino acids 128-133 (shown in SEQ ID NO: 20), and amino acids 128-132 (shown in SEQ ID NO: 21).
  2. 权利要求1所述的肽，所述肽包含结构单元1和结构单元2，其中结构单元1包含PD-1蛋白的29位-85位的氨基酸(如SEQ ID NO：11所示)或58位-85位的氨基酸(如SEQ ID NO：15所示)，结构单元2包含PD-1蛋白的128位-132位的氨基酸(如SEQ ID NO：21所示)或130-132位的氨基酸(如SEQ ID NO：3所示)，优选结构单元1包含PD-1蛋白的58位-85位的氨基酸(如SEQ ID NO：15所示)，结构单元2包含PD-1蛋白的130-132位的氨基酸(如SEQ ID NO：3所示)。The peptide of claim 1, which comprises structural unit 1 and structural unit 2, wherein structural unit 1 comprises amino acids at positions 29-85 of PD-1 protein (as shown in SEQ ID NO: 11) or at position 58 - amino acid at position 85 (as shown in SEQ ID NO: 15), and structural unit 2 comprises amino acids at positions 128 to 132 of PD-1 protein (as shown in SEQ ID NO: 21) or amino acids at positions 130-132 ( As shown in SEQ ID NO: 3), preferably structural unit 1 comprises amino acids from positions 58 to 85 of PD-1 protein (as shown in SEQ ID NO: 15), and structural unit 2 comprises 130-132 of PD-1 protein amino acid at position (as shown in SEQ ID NO: 3).
  3. 权利要求1或2所述的肽，其中PD-1蛋白的氨基酸序列如SEQ ID NO：1所示。The peptide of claim 1 or 2, wherein the amino acid sequence of PD-1 protein is shown in SEQ ID NO: 1.
  4. 权利要求1-3任一项所述的肽，其中结构单元1包含SEQ ID NO：2或SEQ ID NO：10-17任一项所示的序列，结构单元2包含SEQ ID NO：3或SEQ ID NO：18-21任一项所示的序列，优选结构单元1包含SEQ ID NO：2所示的序列，结构单元2包含SEQ ID NO：3所示的序列。The peptide of any one of claims 1-3, wherein structural unit 1 comprises the sequence shown in any one of SEQ ID NO: 2 or SEQ ID NO: 10-17, and structural unit 2 comprises SEQ ID NO: 3 or SEQ ID NO: 3 The sequence shown in any one of ID NO: 18-21, preferably the structural unit 1 comprises the sequence shown in SEQ ID NO: 2, and the structural unit 2 comprises the sequence shown in SEQ ID NO: 3.
  5. 权利要求1-4任一项所述的肽，其中所述抗PD-1抗体的抗原结合片段为Fab片段、Fab’、(Fab’) 2、Fab’-SH、Fab/c、Fv、单链抗体(例如，scFv)。 The peptide of any one of claims 1-4, wherein the antigen-binding fragment of the anti-PD-1 antibody is a Fab fragment, Fab', (Fab') 2 , Fab'-SH, Fab/c, Fv, mono Chain antibodies (eg, scFv).
  6. 权利要求5所述的肽，其中所述Fab片段的重链氨基酸序列如SEQ ID NO：4所示，轻链氨基酸序列如SEQ ID NO：5所示。The peptide of claim 5, wherein the heavy chain amino acid sequence of the Fab fragment is shown in SEQ ID NO: 4, and the light chain amino acid sequence is shown in SEQ ID NO: 5.
  7. 权利要求1-6任一项所述的肽，其中PD-1蛋白的T59、E61、S62、E84、D85氨基酸与抗PD-1抗体(例如14C12H1L1-Fab)重链的Y32、D33、S52、G54、Y57、Y100位氨基酸结合，PD-1蛋白的P130、K131、A132氨基酸与抗PD-1抗体轻链的F325、E324、D323位氨基酸结合，PD-1蛋白的N58糖基化侧链与抗PD-1抗体(例如14C12H1L1-Fab)重链氨基酸(例如S31、G53、G54和R56)结合。The peptide of any one of claims 1-6, wherein T59, E61, S62, E84, D85 amino acids of PD-1 protein are associated with Y32, D33, S52, G54, Y57, Y100 amino acids are combined, P130, K131, A132 amino acids of PD-1 protein are combined with F325, E324, D323 amino acids of anti-PD-1 antibody light chain, N58 glycosylated side chain of PD-1 protein is combined with Anti-PD-1 antibodies (eg 14C12H1L1-Fab) bind heavy chain amino acids (eg S31, G53, G54 and R56).
  8. 权利要求7所述的肽，其中所述糖基化侧链包含甘露糖，N-乙酰氨基葡萄糖；岩藻糖和β-D-甘露糖。7. The peptide of claim 7, wherein the glycosylated side chain comprises mannose, N-acetylglucosamine; fucose and beta-D-mannose.
  9. 权利要求1-8任一项所述的肽在筛选结合PD-1的抗体或其抗原结合片段中的用途。Use of the peptide of any one of claims 1-8 in screening for an antibody or antigen-binding fragment thereof that binds to PD-1.
  10. SEQ ID NO：1所示的PD-1蛋白的D29，T59，E61，S62，K78，E84，D85，L128，P130，A132或其组合在筛选结合PD-1的抗体或其抗原结合片段中的用途。D29, T59, E61, S62, K78, E84, D85, L128, P130, A132 of the PD-1 protein shown in SEQ ID NO: 1 or their combination in screening for antibodies that bind to PD-1 or their antigen-binding fragments use.
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