WO2018129714A1 - 抗pd-1的单克隆抗体及其应用 - Google Patents

抗pd-1的单克隆抗体及其应用 Download PDF

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WO2018129714A1
WO2018129714A1 PCT/CN2017/071125 CN2017071125W WO2018129714A1 WO 2018129714 A1 WO2018129714 A1 WO 2018129714A1 CN 2017071125 W CN2017071125 W CN 2017071125W WO 2018129714 A1 WO2018129714 A1 WO 2018129714A1
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antibody
antigen
binding
polynucleotide
binding fragment
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PCT/CN2017/071125
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French (fr)
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张发明
席甘
黄莺
夏瑜
李百勇
***
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杭州翰思生物医药有限公司
中山康方生物医药有限公司
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Priority to ES17891529T priority Critical patent/ES2902670T3/es
Priority to JP2019538373A priority patent/JP7181878B2/ja
Priority to PCT/CN2017/071125 priority patent/WO2018129714A1/zh
Priority to EP17891529.4A priority patent/EP3569616B1/en
Publication of WO2018129714A1 publication Critical patent/WO2018129714A1/zh
Priority to US16/510,449 priority patent/US10858433B2/en
Priority to JP2021139320A priority patent/JP2022003030A/ja

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Definitions

  • the present invention relates to the field of immunology and antibody engineering, and in particular to monoclonal antibodies against PD-1 and uses thereof.
  • PD-1 Programmed death factor 1
  • CD279 also known as CD279; gene name PDCD1; accession number NP_005009
  • PD-1 is essential for regulating the balance between stimulatory and inhibitory signals of the immune system and maintaining peripheral tolerance The role of cell surface receptors. It is an inhibitory member of the immunoglobulin superfamily that shares homology with CD28.
  • the structure of PD-1 is a monomeric type I transmembrane protein that is transformed by an immunoglobulin variable region-like extracellular domain and an immunoreceptor tyrosine inhibition motif (ITIM) and an immunoreceptor tyrosine.
  • ITIM immunoreceptor tyrosine inhibition motif
  • ITMS immunoreceptor tyrosine
  • PD-1 expression is inducible on T cells, B cells, natural killer (NK) cells, and monocytes, for example, by T cell receptor (TCR) or B cell receptor (BCR) signaling. After lymphocytes.
  • TCR T cell receptor
  • BCR B cell receptor
  • PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), which are members of the cell surface expression of the B7 family.
  • phosphatases such as SHP-1 and SHP-2 to its intracellular tyrosine motif, which then detaches the effector molecule activated by TCR or BCR signaling. Phosphoric acid.
  • PD-1 can transduce inhibitory signals into T cells and B cells only when they are simultaneously engaged with TCR or BCR.
  • antibodies that specifically recognize PD-1 are still to be improved.
  • antibodies that specifically recognize PD-1 ie, monoclonal antibodies against PD-1) such as IgG antibodies are commonly used in therapy, and one of the key problems in the therapeutic use of natural IgG antibodies is that they are in the blood circulation.
  • the low persistence, short serum half-life, and the rate of antibody clearance directly affect the efficacy of the treatment, and thus the frequency and amount of drug administration that causes side effects in the patient and also increases the cost of treatment. Therefore, prolonging the serum half-life of existing IgG-like antibodies (especially natural IgG-like antibodies) and increasing their binding affinity to FcRn, a novel IgG-like antibody with enhanced binding affinity to FcRn and prolonged serum half-life is obtained. major.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a novel monoclonal antibody against PD-1 having enhanced binding affinity to FcRn and prolonged serum half-life. It should be noted that the present invention has been completed based on the following findings and work of the inventors:
  • the neonatal Fc receptor is a receptor responsible for the active transport of immunoglobulin IgG by epithelial cells, and is a heterodimer composed of two subunits of the alpha chain and the beta chain in a non-covalent bond.
  • the Fc portion of an IgG molecule comprises two identical polypeptide chains, wherein each polypeptide chain binds to a single FcRn molecule through its FcRn binding site. In adult mammals, IgG binds to FcRn via the Fc portion, thereby protecting IgG antibodies from degradation and plays a key role in maintaining serum antibody levels.
  • IgG molecules After IgG molecules are endocytosed by endothelial cells, if they bind to FcRn, they are recycled into the circulation. In contrast, IgG molecules that do not bind to FcRn enter the cell and are degraded by lysosomes. Thus, the FC region is also critical for the binding affinity of IgG antibodies to FcRn.
  • the inventors attempted to extend the serum half-life and increase the binding affinity to FcRn by changing the sequence of the IgG antibody heavy chain constant region (ie, the FC region) by targeting the IgG class antibody H2L2 which specifically recognizes PD-1. . That is, the inventors aimed to achieve the purpose of changing the serum half-life thereof and the binding affinity to FcRn by mutating the Fc end of the IgG antibody H2L2. After a series of experimental designs and explorations, the inventors were consciously surprised to find that these mutations alter the affinity of the IgG molecule for FcRn, thereby altering the serum half-life of the antibody, based on several mutations introduced in the constant region of the human IgG molecule.
  • the sequence of the IgG antibody heavy chain constant region ie, the FC region
  • amino acid residues other than unmodified antibodies are introduced in amino acid residues 254, 308, 434 of the heavy chain constant region of an IgG class antibody, such mutations such that the optimized antibody has a longer serum than the wild type antibody Half-life, and its binding affinity and recognition specificity for antigen PD-1 is not reduced.
  • the present invention provides a monoclonal antibody against PD-1 or an antigen-binding fragment thereof.
  • the antibody comprises an FcRn binding site region having the amino acid sequence set forth in SEQ ID NO:5.
  • the box-labeled amino acid is amino acids 254, 308, and 434 of the FcRn binding site region of the heavy chain constant region of the anti-PD-1 monoclonal antibody.
  • the anti-PD-1 monoclonal antibody (IgG-like antibody) of the present invention has a threonine at positions 254, 308, and 434 of the FcRn-binding site region of the heavy chain constant region, respectively. , proline and alanine.
  • the inventors have surprisingly found that the antibody has strong binding affinity for FcRn, long serum half-life, and good binding affinity and recognition specificity for antigen PD-1.
  • the antibody comprises: a heavy chain having the amino acid sequence shown in SEQ ID NO: 1 and a light chain having the amino acid sequence shown in SEQ ID NO: 3.
  • the monoclonal antibody against PD-1 is named H8L2.
  • the amino acids 254, 308, and 434 of the FcRn binding site region of the heavy chain constant region were mutated to threonine, proline, and alanine, respectively, relative to the wild-type H2L2 antibody.
  • the anti-PD-1 monoclonal antibody (H8L2) of the present invention has enhanced binding affinity to FcRn, prolonged serum half-life, and its binding affinity to antigen PD-1 and recognition specificity relative to the wild-type H2L2 antibody. No reduction.
  • the invention provides an isolated polynucleotide.
  • the polynucleotide encodes an antibody or antigen-binding fragment thereof as described above.
  • the antibody encoded by the isolated polynucleotide has strong binding affinity to FcRn, long serum half-life, and good binding affinity and recognition specificity to antigen PD-1.
  • the polynucleotide comprises the following nucleotide sequence encoding an amino acid sequence such as the FcRn binding site region of the antibody set forth in SEQ ID NO: 5: the nucleotide sequence set forth in SEQ ID NO: Or its complementary sequence.
  • the antibody encoded by the isolated polynucleotide has the amino acids 254, 308, and 434 of the FcRn binding site region of the heavy chain constant region as threonine, proline, and alanine, respectively, and the antibody It has strong binding affinity to FcRn, long serum half-life, and good binding affinity and recognition specificity for antigen PD-1.
  • the sequence of the polynucleotide is set forth in SEQ ID NO: 2.
  • the antibody encoded by the isolated polynucleotide is mutated to threonine, proline and c, respectively, in the FcRn binding site region of the heavy chain constant region relative to the wild-type H2L2 antibody.
  • the monoclonal antibody against PD-1 of the present invention has enhanced binding affinity to FcRn, prolonged serum half-life, and is resistant to the wild-type H2L2 antibody. The binding affinity and recognition specificity of the original PD-1 were not reduced.
  • the invention provides an expression vector.
  • the expression vector comprises a polynucleotide as described above.
  • the invention provides a recombinant cell.
  • the recombinant cell comprises an expression vector as described above.
  • the invention provides a method of making an antibody or antigen-binding fragment thereof as described above.
  • the method comprises culturing the recombinant cells described above.
  • the invention also provides the use of a polynucleotide, expression vector, or recombinant cell as described above for the preparation of an antibody or antigen-binding fragment thereof, which antibody specifically binds to PD-1.
  • a polynucleotide, expression vector, or recombinant cell as described above for the preparation of an antibody or antigen-binding fragment thereof, which antibody specifically binds to PD-1.
  • the inventors have found that an antibody or an antigen-binding fragment thereof capable of specifically binding to PD-1 can be efficiently produced by using the above polynucleotide, expression vector, or recombinant cell, and the antibody has a long serum half-life, FcRn has strong binding affinity and good binding affinity and recognition specificity for antigen PD-1.
  • the prepared antibody or antigen-binding fragment thereof is used to effectively block the binding of PD-1 to its receptor, thereby blocking the PD-1 receptor, such as the SHP1/2-related signaling pathway, thereby effectively inhibiting tumor growth.
  • the invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, polynucleotide, expression vector, or recombinant cell for the preparation of a medicament for promoting T cell activation And proliferate, and regulate the expression and secretion of cytokines, used to stimulate anti-tumor cells to produce a stronger immune response.
  • the invention provides a pharmaceutical composition.
  • the antibody or antigen-binding fragment thereof, polynucleotide, expression vector, or recombinant cell thereof is described.
  • the pharmaceutical composition can be effectively used to promote activation and proliferation of T cells, and regulate the expression and secretion of cytokines, which are used to stimulate anti-tumor cells to produce a stronger immune response.
  • the invention provides a method for identifying a drug capable of binding to PD-1.
  • the method comprises: contacting an antibody or antigen-binding fragment thereof as described above with an antigen in the presence of a drug candidate, and determining a first binding of the antibody or antigen-binding fragment thereof to the antigen And wherein the antigen is PD-1 or a fragment thereof; and in the absence of the candidate drug, contacting the antibody or antigen-binding fragment thereof as described above with an antigen, and determining the antibody or antigen-binding fragment thereof A second amount of binding to the antigen, wherein the antigen is PD-1 or a fragment thereof, wherein the second amount is greater than the first amount is an indication that the candidate drug is capable of binding to PD-1.
  • a candidate drug that binds to PD-1 can be screened.
  • the combined blockade of PD-1 and CTLA-4 will be used in combination with standard tumor therapy.
  • the combined blockade of PD-1 and CTLA-4 will effectively bind to the tissue of chemotherapy.
  • Tests have shown that by combining anti-PD-1 antibodies with anti-CTLA-4 antibodies, the same effect can be achieved by reducing the dose of chemotherapeutic drugs.
  • An example of published literature Anti-PD-1 antibodies and anti-CTLA-4 antibodies were used in combination with decarbazine (docetaxel, an anticancer drug) to document melanoma.
  • Another example uses anti-PD-1 antibody and anti-CTLA-4 antibody and IL-2 (interleukin-2) to treat black Prime tumor.
  • the principle of the above combination is that cell death is the result of cytotoxic effects in many chemotherapeutic drugs, and the level of pathways in which tumor cells express antigens is increased.
  • Another combination therapy is a combination of anti-PD-1 antibody and anti-CTLA-4 antibody to increase the synergistic effect of radiation therapy, surgery, hormone therapy, and the like. Each method increases the source of the antigen in the body.
  • Angiogenesis inhibitors can also be used in combination with anti-PD-1 antibodies and anti-CTLA-4 antibody blockers to inhibit vascular proliferation and thereby inhibit tumor cell growth. This may also be by increasing the expression of tumor cell antigens in the body.
  • the invention provides a pharmaceutical combination.
  • the pharmaceutical combination comprises:
  • the immunopotentiating drug different from (1) comprises at least one selected from the group consisting of an anti-CTLA-4 antibody, an anti-CD40 antibody, Budesonide, a salicylate, optionally the water.
  • Salicylates include at least one of sulfasalazine, olsalazine, balsalazide, and mesalamine.
  • amino acid as used herein means one of 20 natural amino acids or any non-natural analog which may be present at a specific, defined position. Natural amino acids can be abbreviated with a three-letter code or with a one-letter code:
  • n-th amino acid refers to a position in the sequence of a protein.
  • the position can be numbered according to the EU index in Kabat.
  • FIG. 1 is a graph showing the results of binding ELISA of H8L2 and H2L2 to PD1 according to an embodiment of the present invention
  • FIG. 2 is a graph showing the results of competition Elisa for inhibition of Pd1 and PdL1 by H8L2 and H2L2, in accordance with an embodiment of the present invention
  • 3 is a graph showing the results of competition Elisa for inhibition of Pd1 and PdL2 by H8L2 and H2L2, in accordance with an embodiment of the present invention
  • FIG. 4 is a graph showing detection results of dynamic characteristic parameters of H8L2 and H2L2 according to an embodiment of the present invention
  • Figure 5 is a graph showing that antibodies H8L2 and H2L2 stimulate IL-secreting IL-2 levels by blocking PD-1 protein function activation, in accordance with an embodiment of the present invention
  • Figure 6 is a graph showing the levels of IFNgamma secreted by T cells stimulated by antibodies H8L2 and H2L2 by blocking PD-1 protein function activation, in accordance with an embodiment of the present invention
  • FIG. 7 is a blood concentration time curve of H8L2 and H2L2 measured by ELISA in a cynomolgus monkey serum concentration study according to an embodiment of the present invention
  • Figure 8 is a graph showing blood plasma concentration of 1 mg/kg of variant H8L2 in a pharmacokinetic study of cynomolgus monkeys according to an embodiment of the present invention
  • Figure 9 is a graph showing blood drug concentration of 3 mg/kg of variant H8L2 in a pharmacokinetic study of cynomolgus monkeys according to an embodiment of the present invention.
  • Figure 10 is a graph showing blood drug concentration of 10 mg/kg of variant H8L2 in a pharmacokinetic study of cynomolgus monkeys according to an embodiment of the present invention.
  • Figure 11 is a graph showing blood drug concentration of 10 mg/kg of wild-type H2L2 in a cynomolgus monkey pharmacokinetic study according to an embodiment of the present invention
  • Figure 12 is an average effective half-life of variant H8L2 and wild-type H2L2 in a cynomolgus monkey pharmacokinetic study, in accordance with an embodiment of the present invention
  • Figure 13 is a result of SDS-PAGE identification of H8L2 according to an embodiment of the present invention.
  • Figure 14 is a SEC-HPLC identification result of H8L2 according to an embodiment of the present invention.
  • Figure 15 is a graph showing the results of detecting the binding activity of H8L2 to PD-1 by the FACS method according to an embodiment of the present invention.
  • Figure 16 is a graph showing the results of FACS method for detecting the activity of H8L2 blocking the binding of PDL-1 to PD-1, according to an embodiment of the present invention
  • Figure 17 is a graph showing the results of detection of affinity constants of H8L2 and Fc ⁇ RIIIa in the ADCC and CDC effect studies, in accordance with an embodiment of the present invention.
  • Figure 18 is a graph showing the results of detection of affinity constants of H8L2 and C1q in the ADCC and CDC effect studies, in accordance with an embodiment of the present invention.
  • H2L2 anti-PD-1 IgG antibody
  • amino acids 254, 308, and 434 of the FcRn binding site region of the antibody heavy chain constant region were mutated to threonine and valine, respectively.
  • alanine named H8L2 (anti-PD-1 IgG class antibody variant).
  • the target antibody H8L2 mutant which is mutated to threonine, proline and alanine, respectively, with respect to the humanized antibody H2L2, the FcRn binding site region of the antibody heavy chain constant region, 254, 308, and 434 amino acids, respectively. Acid, the sequence of other regions is unchanged.
  • the nucleic acid sequence encoding the humanized antibody H8L2 is fully gene-generated and constructed into an expression vector.
  • the expression vector DNA was extracted and transfected into mammalian cell 293 cells. After transfection, the antibody is expressed in mammalian cells and secreted outside the cell. Then, the expressed antibody is purified by an antibody A affinity chromatography column to obtain a humanized antibody H8L2 protein. It was identified by SDS-PAGE and SEC-HPLC standard analytical techniques for subsequent pharmacodynamic studies.
  • the humanized antibody H2L2 and H8L2 antibodies differ only in amino acid positions 254, 308, and 434 of the FcRn binding site region of the heavy chain constant region, and thus only the H8L2 sequence of the H8L2 mutant is provided below for reference.
  • the underlined portion is the antibody variable region, and the box is labeled as the mutation site of H8L2 relative to the antibody H2L2, that is, the amino acids 254, 308, and 434 of the FcRn binding site region of the heavy chain constant region.
  • the 254th amino acid of the FcRn binding site region of the heavy chain constant region of the H8L2 mutant is mutated from serine to threonine, and the amino acid at position 308 is mutated from proline to pro-ammonium.
  • the 254th amino acid is mutated from asparagine to alanine.
  • the portion underlined is the antibody variable region.
  • the portion underlined is the antibody variable region.
  • the portion underlined is the antibody variable region.
  • H8L2 and H2L2 to calculate PD-1 EC 50 values were 0.04nM and 0.05nM.
  • the mutation on the FcRN binding region has no effect on the affinity of the antibody for PD-1.
  • Ligand Add PDL1-mIgG2aFc solution 0.6 ⁇ g/ml, 50 ⁇ l/well, and incubate at 37 ° C for 1 hour;
  • Ligand Add PDL2-his tag solution 1.0 ⁇ g/ml, 50 ⁇ l/well, and incubate at 37 ° C for 1 hour;
  • ELISA was used to determine whether H8L2 binds to other non-human-derived antigens: whether monkey PD1 binds to indirectly investigate the cross-reactivity of H8L2 with non-human antigens.
  • the 450 nm reading in the above table shows that the binding EC50 of H8L2 to monkey PD1-hFc is 0.219 nM.
  • the ELISA method was used to determine whether H8L2 binds to other non-human-derived antigens: whether rat PD1 binds to indirectly investigate the cross-reactivity of H8L2 with non-human antigen.
  • Rat microplates of 1 ⁇ g/ml of rat PD1 were coated with 50 ⁇ l per well and incubated overnight at 4 °C. After washing the plate once, each well was blocked with 300 ⁇ l of 1% BSA solution and incubated at 37 ° C for 2 hours. After washing the plate three times, the antibody was diluted to 7 nM as the starting concentration, and a 1:3 gradient dilution was performed on the plate, and a blank control was additionally prepared, and two duplicate wells were made, each volume was 100 ⁇ l, and incubated at 37 ° C for 30 minutes.
  • H8L2 obtained in Example 1
  • H2L2 The kinetic parameters of H8L2 (obtained in Example 1) and H2L2 were determined and compared using a Fortebio molecular interaction instrument, as follows:
  • the biotin-labeled antigen PD-1 was immobilized on the surface of the SA sensor, and after equilibration in PBST, it was bound to the antibody H8L2, and H8L2 was diluted three times with PBST at concentrations of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0 nM. Dissociated in PBST.
  • the detection method of H2L2 is the same as that of H8L2.
  • the results of H8L2 and H2L2 kinetic parameters are shown in Figure 4. As can be seen from Figure 4, the mutation on FcRN has no effect on the kinetic parameters of the antibody.
  • the binding and competitive activity of H8L2 (obtained in the preparation of Example 1) was measured by the FACS method, as follows:
  • Each group of cells was taken 100 ⁇ l of the cell suspension into a 1.5 ml EP tube, i.e., 10 5 cells per group.
  • 18A10H8L2 PDL1 blockade of the PD1 binding EC 50 is 19.65nM.
  • the mixed lymphocyte reaction (MLR) assay was used to detect and compare the secretion of IL-2 and IFNgamma by H8L2 (obtained in Example 1) and H2L2 to stimulate T lymphocytes, as follows:
  • MLR experiments were performed by mixing human-derived T cells (TC) and dendritic cells (DC), and using DC cell antibody-presenting ability to stimulate T cells to secrete IL-2 and IFNgamma.
  • TC human-derived T cells
  • DC dendritic cells
  • the monocytes in the blood were induced to differentiate into dendritic cells with the cytokines GM-CSF and IL-4, and then stimulated with TNFa to immature DC cells.
  • the mature DC was mixed with the allogeneic TC cells for 5 days, the secretion levels of IL-2 and IFNgamma in the cell supernatant were examined.
  • TC and DC were mixed in a 96-well plate, TC 1 ⁇ 10 5 and DC 1 ⁇ 10 4 were added per well, and 8 gradients of antibody concentration from 10 ⁇ M to 0.09765625 nM were set.
  • IL-2 detection reagent was used. The box quantitatively detects the supernatant IL-2 content. A total of 5 gradients of antibody concentration from 300 nM to 0.1 nM were set. After 5 days of the mixed reaction, the IFNgamma content was quantitatively detected using an IFNgamm detection kit.
  • the levels of IL-2 secretion secreted by T cells by antibodies H8L2 and H2L2 are shown in Figure 5. As can be seen from Figure 5, the antibodies H8L2 and H2L2 can effectively stimulate the secretion of IL-2 by T cells, and the mutation of the FcRN site has no effect on the ability of the antibody to stimulate T cells to secrete IL-2.
  • IFNgamma secreted by T cells stimulated by antibodies H8L2 and H2L2 are shown in Figure 6.
  • the antibodies H8L2 and H2L2 can effectively stimulate the secretion of IFNgamma by T cells, and the mutation of the FcRN site has no effect on the ability of the antibody to stimulate T cells to secrete IFNgamma.
  • "IgG" in Fig. 6 is an isotype antibody control.
  • H8L2 (1 mg/kg) group H8L2 (1 mg/kg) dose group, with 2 rats in each group.
  • H8L2 (1 mg/kg) group
  • H2L2 (1 mg/kg) dose group
  • Each intravenous administration Whole blood was collected from each dose group before administration, 5 minutes after administration, and 5, 24, 72, 168, and 240 hours, and serum was separated by centrifugation.
  • concentrations of H8L2 and H2L2 in the serum of cynomolgus monkeys were determined by ELISA (see table below and Figure 7).
  • cynomolgus monkeys were randomly divided into 4 groups according to their body weight: wild type H2L2 group (10 mg/kg) and variant (H8L2) low, medium and high (1, 3, 10 mg/kg) dose groups. 6 males and half females. Each intravenous administration. Whole blood was collected from each dose group before administration, 5, 30 minutes and 1, 2, 4, 8, 24, 48, 144, and 216 hours after administration, and the serum was separated by centrifugation. The concentration of wild type and variant in cynomolgus monkey serum was determined by ELISA, and the relevant pharmacokinetic parameters were calculated using Phoenix WinNonlin (Pharsight) 6.4.
  • the serum drug concentration level of all individuals before a single administration of cynomolgus monkeys was below the lower limit of quantitation.
  • the serum drug concentration level in the cynomolgus monkey increased with the dose of the drug; the average effective half-life of the low, medium and high (1, 3, 10 mg/kg) dose groups was 215.52 ( See Figures 8), 288.78 (see Figure 9), and 268.92h (see Figure 10).
  • the wild type (10 m g/kg) has an average effective half-life of 224 h (see Figure 11). At the same dose (10 mg/kg), the variant had a longer mean effective half-life than the wild type (see Figure 12).
  • a human tumor transplantation model was established using NSG mice to study the anti-tumor effect of H8L2 (obtained in Example 1) in the subcutaneous transplantation of MiXeno model of HCC827 human non-small cell lung cancer, as follows:
  • NSG mice with NOD, Prkdc scid, IL2rg null deletion / variability is the highest degree of immunity defects, the most suitable tool for human cell transplantation in mice, human cells and tissues of almost no rejection. Therefore, the inventors selected a graft-versus-host response (GVHD) model that was designed to transfer human peripheral blood mononuclear cells (PBMC) to NSG mice, and thereby measured the in vivo pharmacodynamics of H8L2.
  • PBMC peripheral blood mononuclear cells
  • the drug was administered by tail vein injection, and administered on the 6th, 9th, 13th, 16th, 19th and 22nd day after inoculation of the tumor cells, respectively, for a total of six doses (see Table 1). Efficacy was evaluated based on the relative rate of tumor inhibition (TGI RTV), safety evaluation performed in accordance with changes in animal body weight and death.
  • TGI RTV relative rate of tumor inhibition
  • the administration volume is 10 ul / g; n: the number of animals; Day 0 is the day of tumor cell inoculation; i.v.: tail vein administration.
  • the same type of antibody (Human lgG4) was inoculated with PBMC, and the test drug H8L2 (10 mg/kg) showed significant tumor growth inhibition on the 9th and 13th day after inoculation of tumor cells.
  • Opdivo did not show significant tumor growth inhibition.
  • Table 2 The results showed that H8L2 significantly inhibited tumor growth in the HCC827 human non-small cell lung cancer Mixeno tumor model, and the drug efficacy was better than the positive control Opdivo.
  • H8L2 (10 mg/kg, 5 mg/kg) and Opdivo (5 mg/kg) treatment groups did not develop drug-related toxicity (such as severe weight loss or death) within 16 days of the start of dosing (ie, 22 days after tumor inoculation). , indicating that the treatment is well tolerated.
  • Anti-PD-1 monoclonal antibody injection significantly inhibited tumor growth in HCC827 human non-small cell lung cancer Mixeno tumor model at 10 mg/kg, 5 mg/kg dose, 10 mg/kg anti-PD-1 monoclonal antibody The effect of injection inhibiting tumor growth was the most obvious, and the efficacy was better than that of the positive control Opdivo of 5 mg/kg. Tumor-bearing mice were well tolerated against PD-1 monoclonal antibody injection at the test dose.
  • MC38 cells are murine colon cancer cells derived from C57BL/6 mice.
  • PD-1 HuGEMM mice are mouse models of genetically engineered mouse parts of the mouse PD-1 protein that interact with PD-L1 protein molecules in C57BL/6 mice.
  • MC38 tumor cells (1 ⁇ 10 6 /po) were inoculated subcutaneously in the right side of the test mice.
  • the mice were randomly divided into 4 experimental groups according to the tumor volume, 8 in each group. 4 per cage.
  • the four experimental components were the test drugs H8L2 5 mg/kg and 10 mg/kg, the control Keytruda 10 mg/kg group and the homologous antibody Human lgG4 5 mg/kg control group.
  • the drug was administered by tail vein injection for a total of six doses (see Table 3).
  • Dosage dose (mg/kg) Route of administration Dosing schedule 1 8 Isotype control 10 I.p. BIW ⁇ 3 2 8 Keytruda 10 I.p. BIW ⁇ 3 3 8 H8L2 5 I.p. BIW ⁇ 3 4 8 H8L2 10 I.p. BIW ⁇ 3
  • the average tumor volume of the first group of mice reached 1933.67 mm 3 ; at this time point, Keytruda treated group 2 (high dose group) and H8L2 treated group 3 (low dose group), The TGI (%) of Group 4 (high dose group) was 85%, 93%, and 90%, respectively (see Table 4).
  • the percentage change in body weight of tumor-bearing mice was 8.72%, 0.94%, -2.07%, and 1.68%, respectively. There was no significant weight loss or death in the tumor-bearing mice of each treatment group. There was a statistically significant difference between the 2-4 groups and the first group (P ⁇ 0.05).
  • the TC of groups 2-4 (when the tumor volume reached 1000 mm 3 ) were 10 days, 14 days, and > 14 days, respectively.
  • mice in groups 2-4 had complete tumor regression and continued for more than one month (see Table 5).
  • H8L2 (5 mg/kg, 10 mg/kg) showed statistically significant anti-tumor effects in the PD-1 HuGEMM mouse MC38 tumor model; compared with Keytruda, H8L2 was more effective in completely resolving tumors in mice.
  • the Fc receptor Fc ⁇ RIIIa (aka CD16a) binds to the Fc end of an IgG antibody and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the ability of a therapeutic monoclonal antibody to bind to an Fc receptor affects the safety and efficacy of the antibody.
  • the affinity constant of H8L2 and Fc ⁇ RIIIa was detected using the Fortebio molecular interaction system to evaluate the binding ability of H8L2 to Fc receptor.
  • the affinity constant of the antibody to Fc ⁇ RIIIa was detected using a Fortebio Octet molecular interaction instrument. 1 ⁇ g/ml Fc ⁇ RIIIa-Biotin was immobilized on the surface of the SA sensor with PBST as a buffer for 300 s.
  • the antibody concentration in the H8L2 experiment was 4000 nM
  • the binding time was 120 s
  • the dissociation time was 180 s.
  • Data acquisition was performed using Fortebio Data Acquisition 7.0 software, and data analysis was performed by Fortebio Data Analysis 7.0 software.
  • H8L2 did not bind to Fc ⁇ RIIIa.
  • Serum complement C1q binds to the Fc end of an IgG antibody and is involved in complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the ability of a therapeutic monoclonal antibody to bind to C1q affects the safety and efficacy of the antibody.
  • the affinity constant of H8L2 and C1q was detected using the Fortebio molecular interaction instrument system to evaluate the binding ability of H8L2 to C1q.
  • the affinity constant of the antibody to C1q was determined using a Fortebio Octet molecular interaction instrument. Using PBST as a buffer, 100 ⁇ g/ml of antibody was immobilized on the surface of the FAB2G sensor for 300 s. In the step of binding the antibody to the C1q, the concentration of C1q is 3.13 to 200 nM, the binding time is 120 s, and the dissociation time is 180 s. Data acquisition was performed using Fortebio Data Acquisition 7.0 software and data analysis was performed by Fortebio Data Analysis 7.0 software.
  • the anti-PD-1 monoclonal antibody of the invention has strong binding affinity to FcRn, long serum half-life, and good binding affinity and recognition specificity to antigen PD-1, and further, the antibody or antigen-binding fragment thereof can It effectively blocks the binding of PD-1 to its receptor, thereby blocking the PD-1 receptor, such as the SHP1/2-related signaling pathway, thereby effectively inhibiting tumor growth.

Abstract

提供了抗PD-1的单克隆抗体及其应用。其中，该抗PD-1的单克隆抗体包括具有SEQ ID NO：5所示氨基酸序列的FcRn结合位点区域。

Description

抗PD-1的单克隆抗体及其应用
优先权信息
无
技术领域
本发明涉及免疫学和抗体工程技术领域，具体涉及抗PD-1的单克隆抗体及其应用。
背景技术
然而，目前延长IgG类抗体(尤其是天然IgG类抗体)的血清半衰期并提高其对FcRn的结合亲和力的方法仍有待进一步研究。
程序化死亡因子1(PD-1)，也称为CD279；基因名称PDCD1；登录号NP_005009为在调节免疫***的刺激性与抑制性信号之间的平衡和维持外周耐受性中具有至关重要的作用的细胞表面受体。其为与CD28具有同源性的免疫球蛋白超家族的抑制性成员。PD-1的结构为单体I型跨膜蛋白，其由一个免疫球蛋白可变区样细胞外结构域以及含有免疫受体酪氨酸抑制基序(ITIM)和免疫受体酪氨酸转换基序(ITSM)的细胞质结构域组成。PD-1的表达在T细胞、B细胞、天然杀伤(NK)细胞和单核细胞上是可诱导的，例如在通过T细胞受体(TCR)或B细胞受体(BCR)信号转导活化淋巴细胞后。PD-1具有两个已知的配体，PD-L1(B7-H1,CD274)和PD-L2(B7-DC,CD273)，它们为B7家族的细胞表面表达的成员。当衔接配体时，PD-1将磷酸酶例如SHP-1和SHP-2招募至其细胞内酪氨酸基序，所述基序随后使被TCR或BCR信号转导激活的效应分子脱去磷酸。由此，仅当其同时与TCR或BCR衔接时，PD-1才可将抑制性信号转导入T细胞和B细胞。
然而，目前特异性识别PD-1的抗体仍有待改进。并且，特异性识别PD-1的抗体(即抗PD-1的单克隆抗体)如IgG类抗体常用于治疗中，而天然IgG类抗体在治疗用途中的关键问题之一是它们在血液循环中的持久性低、血清半衰期短，以至抗体清除的速度直接影响治疗的功效，并因此影响在患者中引起副作用且还增加治疗费用的药物施用的频率和量。因此，延长现有IgG类抗体(尤其是天然IgG类抗体)的血清半衰期并提高其对FcRn的结合亲和力，获得一种新的、对FcRn的结合亲和力增强且血清半衰期延长的IgG类抗体，意义重大。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提供一种新的、对FcRn的结合亲和力增强且血清半衰期延长的抗PD-1的单克隆抗体。需要说明的是，本发明是基于发明人的下列发现和工作而完成的：
用木瓜蛋白酶水解IgG抗体，可将其从铰链区二硫键的N端部位切断，得到三个片段： 二个相同的可与抗原结合的片段称为Fab片段；一个可结晶的片段称为Fc片段。抗体的Fc区与大量Fc受体和配体相互作用，赋予抗体一些重要的效应器功能的性能。效应器功能包括启动补体依赖的细胞毒性(CDC)、启动吞噬作用和抗体依赖的细胞介导的细胞毒性(ADCC)并通过胞转作用转运抗体通过细胞屏障。此外，FC区域对维持IgG类抗体血清的半衰期非常重要。
新生儿Fc受体(FcRn)是负责上皮细胞主动转运免疫球蛋白IgG的受体，是由α链和β链两个亚基以非共价键的形式组成的异源二聚体。IgG分子的Fc部分包括2条相同的多肽链，其中每条多肽链通过其FcRn结合位点结合单个FcRn分子。在成年哺乳动物中，IgG通过Fc部分与FcRn结合，从而保护IgG抗体免受降解，在维持血清抗体水平中起关键作用。IgG分子被内皮细胞内吞后，如果它们与FcRn结合，那么再循环进入循环内。相比之下，不与FcRn结合的IgG分子进入细胞，被溶酶体降解。因而，FC区域对于IgG抗体对FcRn的结合亲和力的强弱也至关重要。
因而，发明人以已知特异性识别PD-1的IgG类抗体H2L2为对象，尝试通过改变IgG抗体重链恒定区(即FC区)的序列，延长其血清半衰期并提高其对FcRn的结合亲和力。也即，发明人旨在通过突变IgG抗体H2L2的Fc端，达到改变其血清半衰期，以及对FcRn的结合亲和力的目的。经过一系列的实验设计和探索，发明人惊喜地发现，以在人IgG分子恒定区中引入的几个突变作为基础，这些突变改变了IgG分子对FcRn的亲和力，从而改变了抗体的血清半衰期。具体地，在IgG类抗体的重链恒定区的氨基酸残基254、308、434中引入不同于未修饰抗体中的氨基酸残基，这种突变使得优化的抗体具有比野生型抗体更长的血清半衰期，并且其对抗原PD-1的结合亲和力和识别特异性没有降低。
进而，在本发明的第一方面，本发明提供了一种抗PD-1的单克隆抗体或其抗原结合片段。根据本发明的实施例，该抗体包括具有SEQ ID NO：5所示氨基酸序列的FcRn结合位点区域。
Figure PCTCN2017071125-appb-000001
Figure PCTCN2017071125-appb-000002
其中，方框标记的氨基酸为该抗PD-1的单克隆抗体的重链恒定区的FcRn结合位点区域的第254、308、434位氨基酸。换言之，根据本发明的实施例，本发明的抗PD-1的单克隆抗体(IgG类抗体)，其重链恒定区的FcRn结合位点区域第254、308、434位氨基酸分别为苏氨酸、脯氨酸和丙氨酸。发明人惊奇地发现，该抗体对FcRn的结合亲和力强，血清半衰期长，并且，其对抗原PD-1的结合亲和力和识别特异性好。
根据本发明的实施例，该抗体包括：具有SEQ ID NO：1所示氨基酸序列的重链和具有SEQ ID NO：3所示氨基酸序列的轻链。在本文中，将此抗PD-1的单克隆抗体命名为H8L2， 其相对于野生型H2L2抗体，重链恒定区的FcRn结合位点区域第254、308、434位氨基酸分别突变为苏氨酸、脯氨酸和丙氨酸。由此，相对于野生型H2L2抗体，本发明的抗PD-1的单克隆抗体(H8L2)对FcRn的结合亲和力增强、血清半衰期延长，并且，其对抗原PD-1的结合亲和力和识别特异性没有降低。
在本发明的第二方面，本发明提供了一种分离的多核苷酸。根据本发明的实施例，该多核苷酸编码前面所述的抗体或其抗原结合片段。根据本发明的实施例，该分离的多核苷酸编码的抗体，对FcRn的结合亲和力强、血清半衰期长，并且，其对抗原PD-1的结合亲和力和识别特异性好。
根据本发明的实施例，所述多核苷酸包含下列编码氨基酸序列如SEQ ID NO：5所示的抗体FcRn结合位点区域的核苷酸序列：SEQ ID NO：6所示的核苷酸序列或其互补序列。
Figure PCTCN2017071125-appb-000003
由此，该分离的多核苷酸编码的抗体，其重链恒定区的FcRn结合位点区域第254、308、434位氨基酸分别为苏氨酸、脯氨酸和丙氨酸，并且，该抗体对FcRn的结合亲和力强，血清半衰期长，对抗原PD-1的结合亲和力和识别特异性好。
根据本发明的实施例，所述多核苷酸的序列如SEQ ID NO：2所示。由此，该分离的多核苷酸编码的抗体，相对于野生型H2L2抗体，重链恒定区的FcRn结合位点区域第254、308、434位氨基酸分别突变为苏氨酸、脯氨酸和丙氨酸；且相对于野生型H2L2抗体，本发明的抗PD-1的单克隆抗体对FcRn的结合亲和力增强，血清半衰期延长，并且，其对抗 原PD-1的结合亲和力和识别特异性没有降低。
在本发明的第三方面，本发明提供了一种表达载体。根据本发明的实施例，该表达载体包含前面所述的多核苷酸。
在本发明的第四方面，本发明提供了一种重组细胞。根据本发明的实施例，该重组细胞包含前面所述的表达载体。
发明人发现，通过培养上述重组细胞能够有效地合成根据本发明实施例的特异性识别PD-1的抗体或其抗原结合片段。因而，在本发明的第五方面，本发明提供了一种制备前面所述的抗体或其抗原结合片段的方法。根据本发明的实施例，该方法包括培养前面所述的重组细胞。关于特异性识别PD-1的抗体或其抗原结合片段，前面所描述的特征和优点同样适用该方法，在此不再赘述。
在本发明的第六方面，本发明还提供了前面所述的多核苷酸、表达载体、或重组细胞在制备抗体或其抗原结合片段的用途，所述抗体与PD-1特异性结合。由此，发明人发现，利用上述的多核苷酸、表达载体、或重组细胞能够有效地制备得到能够与PD-1特异性结合的抗体或其抗原结合片段，并且该抗体的血清半衰期长、对FcRn的结合亲和力强，对抗原PD-1的结合亲和力和识别特异性好。进而利用制备得到的抗体或其抗原结合片段有效地阻断PD-1与其受体的结合，进而阻断PD-1受体例如SHP1/2相关信号通路，从而可以有效地抑制肿瘤的增长。
在本发明的第七方面，本发明提供了前面所述的抗体或其抗原结合片段、多核苷酸、表达载体、或重组细胞在制备药物中的用途，所述药物用于促进T细胞的活化和增殖，并调节细胞因子的表达和分泌，使用来刺激抗肿瘤细胞产生更强的免疫应答。
在本发明的第八方面，本发明提供了一种药物组合物。根据本发明的实施例，该包含掐面所述的抗体或其抗原结合片段、多核苷酸、表达载体、或重组细胞。由此，该药物组合物能够有效用于促进T细胞的活化和增殖，并调节细胞因子的表达和分泌，使用来刺激抗肿瘤细胞产生更强的免疫应答。
在本发明的第九方面，本发明提供了一种用于鉴定能够与PD-1结合的药物的方法。根据本发明的实施例，该方法包括：在候选药物存在时，使前面所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第一结合量，其中，所述抗原为PD-1或其片段；以及在不存在所述候选药物时，使前面所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第二结合量，其中，所述抗原为PD-1或其片段，其中，所述第二量大于所述第一量是所述候选药物能够与PD-1结合的指示。由此，通过采用该方法，能够筛选与PD-1结合的候选药物。
需要说明的是，PD-1和CTLA-4联合阻断将会与标准肿瘤治疗联合使用。例如PD-1和CTLA-4联合阻断会与化疗的组织有效的结合。试验表明，通过与抗PD-1抗体和抗CTLA-4抗体联合使用，降低化疗药物的剂量即可达到相同的疗效。文献发布的实例：用抗PD-1抗体和抗CTLA-4抗体与decarbazine(多西紫杉醇，一种抗癌药)联合使用来资料黑色素瘤。另一个实例用抗PD-1抗体和抗CTLA-4抗体与IL-2(白细胞介素-2)来治疗黑色 素瘤。以上联合使用的原理为：细胞死亡是很多化疗药物中细胞毒素作用的结果，会使肿瘤细胞表达抗原的途径的水平增加。另外一个联合治疗是用抗PD-1抗体和抗CTLA-4抗体联合阻断来增加放射治疗、手术治疗、激素治疗等的协同作用。每一种方法都是增加了抗原在机体中的来源。血管增生抑制剂也可和抗PD-1抗体和抗CTLA-4抗体阻断联合使用，抑制血管增生进而抑制肿瘤细胞的增长。这可能也是通过增加机体内肿瘤细胞抗原的表达。
因而，在本发明的第十方面，本发明提供了一种药物联合。根据本发明的实施例，该药物联合包括：
(1)前面所述的抗体或其抗原结合片段、多核苷酸、表达载体、或重组细胞；以及
(2)与(1)不同的免疫增强药物。
由此，对肿瘤治疗的效果更好。
根据本发明的实施例，所述与(1)不同的免疫增强药物包括选自下列的至少之一：抗CTLA-4抗体，抗CD40抗体，Budesonide，水杨酸盐，任选地所述水杨酸盐类包括sulfasalazine、olsalazine、balsalazide以及mesalamine的至少之一。
其中，需要说明的是，本文中使用的术语“氨基酸”意指可以存在于具体的、确定的位置上的20种天然氨基酸或任意非天然类似物之一。可以用三字母密码或用单字母密码缩写天然氨基酸：
丙氨酸 ala A
精氨酸 arg R
天冬酰胺 asn N
天冬氨酸 asp D
天冬酰胺或天冬氨酸 asx B
半胱氨酸 cys C
谷氨酸 glu E
谷氨酰胺 gln Q
谷氨酰胺或谷氨酸 glx Z
甘氨酸 gly G
组氨酸 his H
异亮氨酸 ile I
亮氨酸 leu L
赖氨酸 lys K
甲硫氨酸 met M
苯丙氨酸 phe F
脯氨酸 pro P
丝氨酸 ser S
苏氨酸 thr T
色氨酸 try W
酪氨酸 tyr Y
缬氨酸 val V
本文中使用的表达方式“第n位氨基酸”(例如第254、308、434位氨基酸)是指蛋白质的序列中的位置。对于本发明中针对的Fc区，可以根据Kabat中的EU索引编号位置。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明的实施例，H8L2与H2L2对PD1的结合ELISA的结果图；
图2是根据本发明的实施例，H8L2与H2L2对Pd1和PdL1抑制的竞争Elisa的结果图；
图3是根据本发明的实施例，H8L2与H2L2对Pd1和PdL2抑制的竞争Elisa的结果图；
图4是根据本发明的实施例，H8L2和H2L2动力学特征参数检测结果图；
图5是根据本发明的实施例，抗体H8L2和H2L2通过阻断PD-1蛋白功能活化刺激T细胞分泌IL-2水平图；
图6是根据本发明的实施例，抗体H8L2和H2L2通过阻断PD-1蛋白功能活化刺激T细胞分泌IFNgamma水平图；
图7是根据本发明的实施例，食蟹猴血清浓度研究中，ELISA测定的H8L2与H2L2的血药浓度时间曲线；
图8是根据本发明的实施例，食蟹猴药代动力学研究中，变体H8L2的1mg/kg个体血药浓度数据；
图9是根据本发明的实施例，食蟹猴药代动力学研究中，变体H8L2的3mg/kg个体血药浓度数据；
图10是根据本发明的实施例，食蟹猴药代动力学研究中，变体H8L2的10mg/kg个体血药浓度数据
图11是根据本发明的实施例，食蟹猴药代动力学研究中，野生型H2L2的10mg/kg个体血药浓度数据；
图12是根据本发明的实施例，食蟹猴药代动力学研究中，变体H8L2和野生型H2L2平均有效半衰期；
图13是根据本发明的实施例，H8L2的SDS-PAGE鉴定结果；
图14是根据本发明的实施例，H8L2的SEC-HPLC鉴定结果；
图15是根据本发明的实施例，FACS方法检测H8L2与PD-1的结合活性的结果图；
图16是根据本发明的实施例，FACS方法检测H8L2阻断PDL-1与PD-1结合的活性的结果图；
图17是根据本发明的实施例，ADCC和CDC效应研究中，H8L2与FcγRIIIa亲和力常数检测结果图；
图18是根据本发明的实施例，ADCC和CDC效应研究中，H8L2与C1q亲和力常数检测结果图。
发明详细描述
下面将结合实施例对本发明的方案进行解释。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的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1 H8L2突变体的蛋白表达
在人源化抗体H2L2(抗PD-1的IgG类抗体)基础上，使其抗体重链恒定区的FcRn结合位点区域第254、308、434位氨基酸分别突变为苏氨酸、脯氨酸和丙氨酸，命名为H8L2(抗PD-1的IgG类抗体变体)。
也即，目的抗体H8L2突变体，其相对于人源化抗体H2L2，抗体重链恒定区的FcRn结合位点区域第254、308、434位氨基酸分别突变为苏氨酸、脯氨酸和丙氨酸，其他区域序列不变。
实际操作中，全基因合成编码人源化抗体H8L2的核酸序列，并构建到表达载体。提取表达载体DNA，转染哺乳动物细胞293细胞。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过抗体A亲和层析柱，纯化表达的抗体，即获得人源化抗体H8L2蛋白。用SDS-PAGE和SEC-HPLC标准分析技术对其进行质量鉴定后用于后续的药效学研究。
其中，H8L2的SDS-PAGE和SEC-HPLC鉴定结果分别见图13和图14。
H8L2的SDS-PAGE鉴定结果见图13。如图13所示，泳道1：H8L2非还原；泳道2：H8L2还原；泳道M：DNA标准品(14.4KDa 18.4KDa 25KDa 35KDa 45KDa 66.2KDa 116KDa)；泳道3：BSA。由图13可知，候选抗体18A10H8L2样品总体纯度较高。
H8L2的SEC-HPLC鉴定结果见图14。如图14所示，积分定量确认该抗体总纯度为98.19％。
如前所述，人源化抗体H2L2与H8L2抗体，仅重链恒定区的FcRn结合位点区域第254、308、434位氨基酸不同，因而下面仅提供H8L2突变体的H8L2序列以供参考。
H8L2重链氨基酸序列：
Figure PCTCN2017071125-appb-000004
Figure PCTCN2017071125-appb-000005
Figure PCTCN2017071125-appb-000006
其中，下划线标注的部分为抗体可变区，方框标记的为H8L2相对于抗体H2L2的突变位点，即重链恒定区的FcRn结合位点区域的第254、308、434位氨基酸。
其中，相对于人源化抗体H2L2，H8L2突变体的抗体重链恒定区的FcRn结合位点区域的第254位氨基酸由丝氨酸突变为苏氨酸，第308位氨基酸由缬氨酸突变为脯氨酸，第254位氨基酸由天冬酰胺突变为丙氨酸。
编码H8L2重链的核酸序列：
Figure PCTCN2017071125-appb-000007
Figure PCTCN2017071125-appb-000008
其中，下划线标注的部分为抗体可变区。
H8L2轻链氨基酸序列：
Figure PCTCN2017071125-appb-000009
Figure PCTCN2017071125-appb-000010
其中，下划线标注的部分为抗体可变区。
编码H8L2轻链的核酸序列：
Figure PCTCN2017071125-appb-000011
Figure PCTCN2017071125-appb-000012
其中，下划线标注的部分为抗体可变区。
实施例2 H8L2突变体重组人源化抗体ELISA实验
针对H2L2抗体和实施例1制备获得的H8L2抗体，进行ELISA结合实验和竞争ELISA实验比较研究，具体如下：
1、18A10 H8L2、18A10 H2L2 ELISA结合实验
具体步骤如下：
1)包被抗原：PD-1-his抗原0.25μg/ml，100μl/孔，4℃包被过夜；
2)1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤3次，轻轻拍干；
3)一抗：2μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，37℃孵育1小时；
4)二抗：PBST洗涤3次，轻轻拍干，每孔100μl加入1:10000稀释的HRP酶标羊抗人IgG(H+L)二抗，37℃孵育1小时；
5)显色：PBST洗涤3次，轻轻拍干；每孔100μl加入TMB显色剂，室温反应5～10min；
6)显色终止：50μl/孔加入2M H2SO4溶液终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
结果见图1，可计算出H8L2和H2L2对PD-1的EC50值分别为0.04nM和0.05nM。由图1可知，FcRN结合区域上的突变对抗体与PD-1亲和力没有影响。
Figure PCTCN2017071125-appb-000013
2、18A10 H8L2、18A10 H2L2与PDL1竞争ELISA实验
具体步骤如下：
1)包被抗原：在96孔酶标板上包被PD-1-hIgGFc抗原0.5μg/ml，50μl/孔，4℃包被过夜；
2)PBST洗板3次，轻轻拍干，加入1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤3次；
3)一抗：6μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，50μl/孔加入到包被好的酶标板上，室温孵育10min；
4)配体：加入PDL1-mIgG2aFc溶液0.6μg/ml，50μl/孔，37℃孵育1小时；
5)二抗：PBST洗涤3次，轻轻拍干；每孔50μl加入1:5000稀释的HRP酶标羊抗鼠IgG(H+L)二抗，37℃孵育1小时；
5)显色：PBST洗涤3次，轻轻拍干；每孔50μl加入TMB显色剂，室温反应5-10min；
6)终止：50μl/孔加入2M H2SO4终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
结果见图2，并且H8L2和H2L2抑制Pd-1与PdL1的EC50值分别为0.474nM和0.783nM。由此可知，FcRN上的突变对抗体抑制Pd-1与PdL1的结合没有影响。
Figure PCTCN2017071125-appb-000014
3、18A10 H2L2与PDL2竞争ELISA实验
具体步骤如下：
1)包被抗原：在96孔酶标板上包被PD-1-hIgGFc抗原1.0μg/ml，100μl/孔，4℃包被过夜；
2)PBST洗板3次，轻轻拍干，加入1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤4次；
3)一抗：20μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，50μl/孔加入到包被好的酶标板上，室温孵育10min；
4)配体：加入PDL2-his tag溶液1.0μg/ml，50μl/孔，37℃孵育1小时；
5)二抗：PBST洗涤5次，轻轻拍干；每孔50μl加入1:750稀释的HRP酶标抗his tag小鼠单克隆抗体二抗，37℃孵育1小时；
5)显色：PBST洗涤6次，轻轻拍干；每孔100μl加入TMB显色剂，室温反应30min；
6)终止：50μl/孔加入2M H2SO4终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
结果见图3，并且H8L2和H2L2抑制Pd-1与PdL2的EC50值分别为1.83nM和1.58nM.由此可知，FcRN上的突变对抗体抑制Pd-1与PdL2的结合没有影响。
Figure PCTCN2017071125-appb-000015
4、H8L2与猴子PD1-hFc结合的活性研究
本实验应用ELISA的方法测定H8L2与其他非人源来源的抗原：猴子PD1是否有结合，以间接考察H8L2与非人源抗原的交叉反应。
以0.125μg/ml的猴子PD1-hFc，每孔50μl包被酶标板，4℃孵育过夜。洗板一次拍干后，每孔用300μl 1％BSA溶液封闭，37℃孵育2小时。洗板一次拍干后，抗体稀释至7nM作为起始浓度，在板上进行1:3的梯度稀释，另设空白对照，均做2个复孔，每孔体积100μl，37℃孵育30分钟。洗板三次后，每孔加入50μl辣根过氧化物酶标记的羊抗人IgG F(ab')2二抗工作液，37℃孵育30分钟。洗板四次后，每孔加入50μl TMB显色液，室温避光显色5分钟后，每孔加入50μl终止液终止显色反应。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 Pro 6.2.1软件对数据进行分析处理。
450nm波长下测定的H8L2与猴子PD1-hFc的结合活性检测结果见下表：
Figure PCTCN2017071125-appb-000016
上表中的450nm读数结果显示H8L2与猴子PD1-hFc的结合EC50为0.219nM。
5、H8L2与大鼠PD1结合的活性研究(ELISA法)
本实验应用ELISA的方法测定H8L2与其他非人源来源的抗原：大鼠PD1是否有结合，以间接考察H8L2与非人源抗原的交叉反应。
以1μg/ml的大鼠PD1，每孔50μl包被酶标板，4℃孵育过夜。洗板一次拍干后，每孔用300μl 1％BSA溶液封闭，37℃孵育2小时。洗板三次拍干后，抗体稀释至7nM作为起始浓度，在板上进行1:3的梯度稀释，另设空白对照，均做2个复孔，每孔体积100μl，37℃孵育30分钟。洗板三次后，每孔加入50μl辣根过氧化物酶标记的羊抗人IgG二抗工作液，37℃孵育30分钟。洗板四次后，每孔加入50μl TMB显色液，室温避光显色5分钟后，每孔加入50μl终止液终止显色反应。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 Pro 6.2.1软件对数据进行分析处理。
450nm波长下测定的H8L2与大鼠PD1的结合活性检测结果见下表：
Figure PCTCN2017071125-appb-000017
上表中的450nm读数结果显示H8L2与大鼠PD1无结合活性。
实施例3 使用Fortebio分子相互作用仪测定H8L2和H2L2动力学参数
使用Fortebio分子相互作用仪测定并比较H8L2(实施例1制备获得)和H2L2的动力学参数，具体如下：
生物素标记的抗原PD-1固定于SA传感器表面，在PBST中平衡后，与抗体H8L2结合，H8L2用PBST三倍稀释，浓度为200、66.67、22.22、7.41、2.47、0.82、0.27、0nM，于PBST中解离。H2L2的检测方法与H8L2相同。H8L2、H2L2动力学参数结果图见图4。由图4可见，FcRN上的突变对抗体的动力学参数没有影响。
实施例4 使用FACS方法检测H8L2的结合及竞争活性
使用FACS方法检测H8L2(实施例1制备获得)的结合及竞争活性，具体如下：
1、FACS方法检测H8L2与PD1的结合活性
本试验采用293T-PD1稳定转染细胞株细胞作为实验细胞，采用FACS方法检测H8L2与细胞膜表面PD1蛋白的结合情况。具体方法如下：
(1)将表达PD-1的293T细胞消化下来，计数，将其最终浓度配成106细胞/ml。
(2)每组取100μl的细胞混悬液到1.5ml的EP管中，即每组105个细胞。
(3)加入配好的各浓度的抗体，冰上孵育1小时。
(4)1小时后，离心，用PBS洗涤一次。
(5)每组加入配好的FITC Goat Anti-human IgG二抗，冰上避光孵育1小时。
(6)1h后，低温高速离心(4000r/min，5min)，用PBS洗涤一次，最终用200μl的PBS混悬，细胞上机检测。
通过FACS方法检测H8L2与细胞膜表面PD1的结合活性情况，结果如图15和下表所示。
Figure PCTCN2017071125-appb-000018
由图15和上表的统计结果可知，H8L2与PD-1的结合EC50为3.40nM。
2、FACS方法检测H8L2阻断PDL1与PD1结合的活性
本试验采用293T-PD1稳定转染细胞株细胞作为实验细胞，PDL1蛋白为竞争蛋白，采用FACS方法检测PDL1与细胞膜表面PD1蛋白的结合情况，以间接反应H8L2阻断PDL1与PD1结合的情况。具体方法如下：
(1)将表达PD1的293T细胞消化下来，计数，将其最终浓度配成106细胞/ml。
(2)每组细胞取100μl细胞混悬液到1.5ml的EP管中，即每组105个细胞。
(3)加入配好的各浓度的抗体，冰上孵育0.5小时。
(4)在所有组中加入配体PDL1-mFc使其最终浓度为20nM。
(5)加入配体0.5小时后，离心，用PBS洗涤一次。
(6)每组加入配好的FITC Goat Anti-mouse IgG/IgM二抗，冰上避光孵育1小时。
(7)1小时后，低温高速离心(4000r/min，5min)，用PBS洗涤一次，最终用200μl的PBS混悬，细胞上机检测。
通过FACS方法检测PDL1与细胞膜表面PD1的结合活性情况，结果如图16和下表所示。
Figure PCTCN2017071125-appb-000019
由图16和上表的统计结果可知，18A10H8L2阻断PDL1与PD1结合的EC50是19.65nM。
实施例5 混合淋巴反应检测PD1抗体生物学活性
利用混合淋巴细胞反应(MLR)实验检测并比较H8L2(实施例1制备获得)和H2L2刺激T淋巴细胞分泌IL-2和IFNgamma分泌能力，具体如下：
MLR实验采用不同人来源的T细胞(TC)和树突状细胞(DC)进行混合，利用DC细胞抗体提呈能力刺激T细胞分泌IL-2和IFNgamma。首先用细胞因子GM-CSF和IL-4诱导血液中单核细胞分化成树突状细胞，然后用TNFa刺激是未成熟的DC细胞成熟。成熟后的DC与同种异源的TC细胞进行混合5天后，检测细胞上清中的IL-2和IFNgamma的分泌水平。在96孔板中混合TC和DC，按每孔加入TC 1×105和DC 1×104，设置抗体浓度从10μM到0.09765625nM共8个梯度，混合反应5天后，用IL-2检测试剂盒定量检测上清IL-2含量。设置抗体浓度300nM到0.1nM共5个梯度。混合反应5天后，用IFNgamm检测试剂盒定量检测IFNgamma含量。
抗体H8L2和H2L2刺激T细胞分泌IL-2分泌水平如图5所示。由图5可见，抗体H8L2和H2L2能有效地刺激T细胞分泌IL-2，FcRN位点的突变对抗体刺激T细胞分泌IL-2的能力没有影响。
抗体H8L2和H2L2刺激T细胞分泌IFNgamma分泌水平如图6所示。由图6可见，抗体H8L2和H2L2能有效地刺激T细胞分泌IFNgamma，FcRN位点的突变对抗体刺激T细胞分泌IFNgamma的能力没有影响。其中，图6中的“IgG”为同型抗体对照。
实施例6 食蟹猴血清浓度研究
对H8L2(实施例1制备获得)和H2L2进行食蟹猴血清浓度比较研究，具体如下：
4只食蟹猴根据体重随机分为2组，分别为H8L2(1mg/kg)组和H2L2(1mg/kg)剂量组，每组2只。每次静脉注射给药。各剂量组分别于给药前，给药后5分钟及5、24、72、168、240小时采集全血，离心分离血清。采用ELISA法测定食蟹猴血清中H8L2和H2L2的浓度(见下表和图7)。
Figure PCTCN2017071125-appb-000020
Figure PCTCN2017071125-appb-000021
实施例7 食蟹猴药代动力学研究
对H8L2(实施例1制备获得)和H2L2进行食蟹猴药代动力学比较研究，具体如下：
24只食蟹猴根据体重随机分为4组，分别为野生型H2L2组(10mg/kg)，以及变体(H8L2)低、中、高(1、3、10mg/kg)剂量组，每组6只，雌雄各半。每次静脉注射给药。各剂量组分别于给药前，给药后5、30分钟及1、2、4、8、24、48、144、216小时采集全血，离心分离血清。采用ELISA法测定食蟹猴血清中野生型和变体的浓度，并采用PhoenixWinNonlin(Pharsight)6.4计算相关的药代动力学参数。
食蟹猴单次给药前所有个体的血清药物浓度水平均低于定量下限。变体组三个剂量组，食蟹猴体内血清药物浓度水平随给药剂量增加而增加；变体低、中、高(1、3、10mg/kg)剂量组的平均有效半衰期分别为215.72(见图8)、288.78(见图9)、268.92h(见图10)，野生型(10m g/kg)的平均有效半衰期为224h(见图11)。在同等剂量(10mg/kg)下，变体比野生型具有更长的平均有效半衰期(见图12)。
实施例8 H8L2在HCC827人非小细胞肺癌皮下移植MiXeno模型中的抗肿瘤作用
利用NSG小鼠建立人肿瘤移植模型，研究H8L2(实施例1制备获得)在HCC827人非小细胞肺癌皮下移植MiXeno模型中的抗肿瘤作用，具体如下：
NSG小鼠具有NOD，Prkdcscid，IL2rgnull缺失/变异特征，是目前免疫缺陷程度最高、最适合人源细胞移植的工具小鼠，对人源细胞和组织几乎没有排斥反应。因此，发明人选择过继转输人外周血单核细胞(PBMC)至NSG小鼠所构建的移植物抗宿主反应(GVHD)模型，并由此来衡量H8L2的体内药效学。发明人运用NSG小鼠建立人肿瘤移植模型(Mixeno模型)，研究H8L2在HCC827人非小细胞肺癌皮下移植MiXeno模型中的抗肿瘤作用。
第0天(Day 0)在右侧背部皮下接种5×106HCC827细胞于40只NCG小鼠(32只小鼠加上8只富余量)中，接种肿瘤细胞后6天(Day 6)当平均瘤体积达到66mm3，选择肿瘤体积较好的32只小鼠均匀分成4组，每组8只小鼠。自尾静脉移植PBMC于32只NCG小鼠(第1-4组小鼠)中，细胞重悬在PBS中(0.1ml接种体积)。试验分为测试药H8L2 5mg/kg和10mg/kg、阳性对照Opdivo 5mg/kg组及同型抗体Human IgG4 5mg/kg对照组。尾静脉注射给药，分别于接种肿瘤细胞后第6，9，13，16，19，22天给药，共给药六次(见表1)。根据相对肿瘤抑制率(TGIRTV)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
表1 测试药H8L2在HCC827人非小细胞肺癌Mixeno肿瘤模型中的抗肿瘤作用实验设计
Figure PCTCN2017071125-appb-000022
注：给药体积为10ul/g；n：动物只数；Day 0为肿瘤细胞接种当天；i.v.：尾静脉给药。
相对对照组接种了PBMC的同型抗体(Human lgG4)，测试药H8L2(10mg/kg)在接种肿瘤细胞后第9、13天都表现显著的肿瘤生长抑制作用，相对肿瘤抑制率TGIRTV(％)分别为30％(p＝0.007)，30％(p＝0.039))；H8L2(5mg/kg)在接种肿瘤细胞后第9、13天都表现显著的肿瘤生长抑制作用，相对肿瘤抑制率TGIRTV(％)分别为18％(p＝0.049)，25％(p＝0.041)。对应的，Opdivo没有表现出显著的肿瘤生长抑制作用，在接种肿瘤细胞后第9、13天相对肿瘤抑制率TGIRTV(％)分别为17％(p＝0.084)，23％(p＝0.073)(表2)。试验结果证明H8L2对HCC827人源非小细胞肺癌Mixeno肿瘤模型有显著抑制肿瘤生长的作用，药效相对阳性对照品Opdivo更好。H8L2(10mg/kg，5mg/kg)和Opdivo(5mg/kg)各治疗组在给药开始16天内(即肿瘤接种22天内)均未发生药物治疗相关的毒性反应(如严重体重下降或死亡)，说明治疗耐受良好。
表2.在HCC827人非小细胞肺癌Mixeno肿瘤模型中的药效分析
Figure PCTCN2017071125-appb-000023
注：P value1是与同型对照组(G1 Human IgG4)比较
抗PD-1单克隆抗体注射液在10mg/kg，5mg/kg剂量下对HCC827人源非小细胞肺癌Mixeno肿瘤模型均有显著抑制肿瘤生长的作用，其中10mg/kg抗PD-1单克隆抗体注射液抑制肿瘤生长效果最明显，药效相对5mg/kg的阳性对照品Opdivo更好。荷瘤小鼠对抗PD-1单克隆抗体注射液在测试剂量下耐受良好。
实施例9 H8L2在MC38小鼠结直肠癌HuGEMM模型中的抗肿瘤作用
临床前验证在PD-1 HuGEMM小鼠中使用H8L2(实施例1制备获得)治疗小鼠MC38肿瘤模型的药效，具体如下：
MC38细胞为源自C57BL/6小鼠的鼠源结肠癌细胞。PD-1 HuGEMM小鼠是经过基因工程技术，将C57BL/6小鼠体内与PD-L1蛋白分子相互作用的鼠源PD-1蛋白分子部分替换为人源蛋白的小鼠模型。
在受试小鼠右侧皮下接种MC38肿瘤细胞(1×106/只)，当平均肿瘤体积达到约134mm3时，根据肿瘤体积将小鼠随机分入4个实验组，每组8只，每笼4只。4个实验组分为测试药H8L2 5mg/kg和10mg/kg、对照Keytruda 10mg/kg组及同型抗体Human lgG4 5mg/kg对照组。尾静脉注射给药，共给药六次(见表3)。
表3.药效学实验设计
组号 动物数 治疗 给药剂量(mg/kg) 给药途径 给药安排
1 8 Isotype control 10 i.p. BIW×3
2 8 Keytruda 10 i.p. BIW×3
3 8 H8L2 5 i.p. BIW×3
4 8 H8L2 10 i.p. BIW×3
在分组后第13天，第1组小鼠的平均肿瘤体积达到1933.67mm3；在该时间点，Keytruda治疗的第2组(高剂量组)以及H8L2治疗的第3组(低剂量组)、第4组(高剂量组)的TGI(％)分别为85％、93％、90％(见表4)。荷瘤鼠体重改变百分比分别为8.72％,0.94％,-2.07％,1.68％。各治疗组荷瘤鼠无明显体重降低或死亡，第2-4组与第1组相比均有统计学显著差异(P<0.05)。
表4.H8L2在PD-1HuGEMM MC38荷瘤鼠中的抑瘤效果
Figure PCTCN2017071125-appb-000024
注释：a.数据以“平均值±标准误差”表示；
b.利用单因素方差分析(one-way ANOVA)方法进行各治疗组肿瘤体积间的显著性差异分析；第2-4组与第1组相比在统计学上均有显著性差异(P<0.05)。
第2-4组的T-C(当肿瘤体积达到1000mm3时)分别为10天、14天、>14天。当本实 验在第55天结束时，第2-4组分别有3、5、5只小鼠肿瘤完全消退且持续一个月以上(见表5)。
表5.MC38肿瘤体积的原始数据
Figure PCTCN2017071125-appb-000025
H8L2(5mg/kg、10mg/kg)在PD-1HuGEMM小鼠MC38肿瘤模型中显示有统计学显著的抗肿瘤作用；与Keytruda相比，H8L2能更有效的使小鼠体内肿瘤完全消退。
实施例10 H8L2的ADCC和CDC效应研究
对H8L2(实施例1制备获得)进行ADCC和CDC效应研究，具体如下：
1、H8L2与FcγRIIIa的亲和力常数测定
Fc受体FcγRIIIa(又名CD16a)可与IgG抗体的Fc端结合，参与抗体依赖细胞介导的细胞毒作用(ADCC)。治疗性单克隆抗体与Fc受体结合的能力影响到该抗体的安全性和有效性。本实验使用Fortebio分子相互作用仪***检测H8L2与FcγRIIIa的亲和力常数，以评价H8L2与Fc受体的结合能力。
采用Fortebio Octet分子相互作用仪检测抗体与FcγRIIIa的亲和力常数。以PBST作为缓冲液，1μg/ml FcγRIIIa-Biotin固定于SA传感器表面，时间300s。抗体与FcγRIIIa结合步骤中，H8L2实验中抗体浓度为4000nM，结合时间为120s，解离时间为180s。使用FortebioData Acquisition 7.0软件进行数据采集，Fortebio Data Analysis 7.0软件进行数据分析。
结果如图17所示，H8L2与FcγRIIIa没有结合。
2、AK103与C1q的亲和力常数测定
血清补体C1q可与IgG抗体的Fc端结合，参与补体依赖的细胞毒性作用(CDC)。治疗性单克隆抗体与C1q结合的能力影响到该抗体的安全性和有效性。本实验使用Fortebio分子相互作用仪***检测了H8L2与C1q的亲和力常数，以评价H8L2与C1q的结合能力。
采用Fortebio Octet分子相互作用仪检测抗体与C1q的亲和力常数。以PBST作为缓冲液，100μg/ml的抗体固定于FAB2G传感器表面，时间300s。抗体与C1q结合步骤中，C1q浓度为3.13-200nM，结合时间为120s，解离时间180s。使用Fortebio Data Acquisition 7.0软件进行数据采集，Fortebio Data Analysis 7.0软件进行数据分析。
结果如图18所示，H8L2与C1q没有结合。
工业实用性
本发明的抗PD-1的单克隆抗体，对FcRn的结合亲和力强，血清半衰期长，并且，其对抗原PD-1的结合亲和力和识别特异性好，进而，该抗体或其抗原结合片段能够有效地阻断PD-1与其受体的结合，进而阻断PD-1受体例如SHP1/2相关信号通路，从而可以有效地抑制肿瘤的增长。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (14)

  1. 一种抗PD-1的单克隆抗体或其抗原结合片段，其特征在于，包括具有SEQ ID NO：5所示氨基酸序列的FcRn结合位点区域。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，包括：
    具有SEQ ID NO：1所示氨基酸序列的重链和具有SEQ ID NO：3所示氨基酸序列的轻链。
  3. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1或2所述的抗体或其抗原结合片段。
  4. 根据权利要求3所述的多核苷酸，其特征在于，所述多核苷酸包含下列编码氨基酸序列如SEQ ID NO：5所示的抗体FcRn结合位点区域的核苷酸序列：
    SEQ ID NO：6所示的核苷酸序列或其互补序列。
  5. 根据权利要求3所述的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO：2所示。
  6. 一种表达载体，其特征在于，包含权利要求3-5任一项所述的多核苷酸。
  7. 一种重组细胞，其特征在于，包含权利要求6所述的表达载体。
  8. 一种制备权利要求1或2所述的抗体或其抗原结合片段的方法，其特征在于，包括培养权利要求7所述的重组细胞。
  9. 权利要求3-5任一项所述的多核苷酸、权利要求6所述的表达载体、或权利要求7所述的重组细胞在制备抗体或其抗原结合片段的用途，所述抗体与PD-1特异性结合。
  10. 权利要求1或2所述的抗体或其抗原结合片段、权利要求3-5任一项所述的多核苷酸、权利要求6所述的表达载体、或权利要求7所述的重组细胞在制备药物中的用途，所述药物用于促进T细胞的活化和增殖，并调节细胞因子的表达和分泌，使用来刺激抗肿瘤细胞产生更强的免疫应答。
  11. 一种药物组合物，其特征在于，包含权利要求1或2所述的抗体或其抗原结合片段、权利要求3-5任一项所述的多核苷酸、权利要求6所述的表达载体、或权利要求7所述的重组细胞。
  12. 一种用于鉴定能够与PD-1结合的药物的方法，其特征在于，包括：
    在候选药物存在时，使权利要求1或2所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第一结合量，其中，所述抗原为PD-1或其片段；以及
    在不存在所述候选药物时，使权利要求1或2所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第二结合量，其中，所述抗原为PD-1或其片段，
    其中，所述第二量大于所述第一量是所述候选药物能够与PD-1结合的指示。
  13. 一种药物联合，其特征在于，包括：
    (1)权利要求1或2所述的抗体或其抗原结合片段、权利要求3-5任一项所述的多核苷酸、权利要求6所述的表达载体、或权利要求7所述的重组细胞；以及
    (2)与(1)不同的免疫增强药物。
  14. 根据权利要求13所述的药物联合，其特征在于，所述与(1)不同的免疫增强药物包括选自下列的至少之一：
    抗CTLA-4抗体，抗CD40抗体，Budesonide，水杨酸盐，
    任选地，所述水杨酸盐包括sulfasalazine、olsalazine、balsalazide以及mesalamine的至少之一。
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