WO2022258011A1 - 抗pd-1人源化抗体或其抗原结合片段及其应用 - Google Patents

抗pd-1人源化抗体或其抗原结合片段及其应用 Download PDF

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WO2022258011A1
WO2022258011A1 PCT/CN2022/097854 CN2022097854W WO2022258011A1 WO 2022258011 A1 WO2022258011 A1 WO 2022258011A1 CN 2022097854 W CN2022097854 W CN 2022097854W WO 2022258011 A1 WO2022258011 A1 WO 2022258011A1
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antibody
antigen
binding fragment
seq
nucleic acid
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French (fr)
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霍永庭
符俊
芦迪
张喆
路力生
李凡
龚春喜
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广东菲鹏制药股份有限公司
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Priority to EP22819608.5A priority patent/EP4353746A1/en
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the invention belongs to the technical field of biomedicine. More specifically, it relates to an anti-PD-1 humanized antibody or an antigen-binding fragment thereof and applications thereof.
  • PD1 Programmed death factor-1
  • Pdcd1 located at 2q37.3, with a total length of 9.6kb, consisting of 5 exons and 4 introns, and its upstream contains a 663bp promoter.
  • PD1 is a 55KDa type I transmembrane protein. Its molecular structure consists of an extracellular region, a transmembrane region and an intracellular region.
  • the extracellular region contains an immunoglobulin variable region IgV domain, and the intracellular region contains the immunoreceptor tyrosine Acid Inhibition Motif (ITIM) and Immunoreceptor Tyrosine Switching Module (ITSM).
  • ITIM immunoreceptor tyrosine Acid Inhibition Motif
  • ITMS Immunoreceptor Tyrosine Switching Module
  • PD-1 has two ligands: PD-L1 and PD-L2. Both PD-L1 and PD-L2 are B7 homologues.
  • the PDL gene is located at the 9P24.2 site of human chromosome, with a size of 42kb. Its molecular structure contains an immunoglobulin-like variable region domain and a constant region-like structure. domain, a transmembrane region and a short cytoplasmic tail.
  • the combination of PD-1 and PD-L1 and PD-L2 can down-regulate the activation of T cells.
  • PD-L1 is expressed on the surface of a variety of tumor cells, including: lung cancer, gastric cancer, liver cancer, esophagus cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, skin cancer, colon cancer, bladder cancer, glial cancer , head and neck cancer, oral squamous cell carcinoma.
  • a large number of CD8+ T cells expressing PD-L1 were found around these cancers. The statistics of clinical results show that the high expression level of PD-L1 on tumor cells is related to the poor prognosis of cancer patients.
  • the technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing antibodies with low binding affinity and specificity to PD-1, and provide anti-PD-1 humanized antibodies or antigen-binding fragments thereof and applications thereof.
  • the purpose of the present invention is to provide an anti-PD-1 humanized antibody or an antigen-binding fragment thereof.
  • the antibody comprises a light chain CDR region and a heavy chain CDR region.
  • the heavy chain CDR region is composed of HCDR1, HCDR2, and HCDR3.
  • the light chain CDR region Composed of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 8-10, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 11-13;
  • the amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NO: 3-5.
  • amino acid sequence of the light chain variable region of the antibody is shown in any one of SEQ ID NO: 6-7.
  • the present invention also relates to nucleic acids, vectors, cells or pharmaceutical compositions related to the antibodies or antigen-binding fragments thereof.
  • the present invention also relates to the use of the antibody or its antigen-binding fragment, and its related nucleic acid, carrier, cell or pharmaceutical composition in the preparation of medicines for treating PD-1-mediated diseases or disorders.
  • the present invention also relates to a method for treating a disease or disorder mediated by PD-1, the method comprising: ingesting an effective amount of the above-mentioned antibody or its antigen-binding fragment, nucleic acid, carrier, cell or pharmaceutical composition to a subject.
  • the present invention also relates to the above-mentioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells or pharmaceutical compositions for use in therapy.
  • the present invention also relates to the above-mentioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells or pharmaceutical compositions for treating PD-1-mediated diseases or disorders.
  • Figure 1 is a graph showing the effects of different concentrations of PD-1-112-C2 on the secretion of IL-2/IFN- ⁇ .
  • Figure 2 is a graph showing the effects of different concentrations of PD-1-112-C2 on the proliferation of T cells and the secretion of cytokine IL-2 by T cells.
  • Figure 3 is a graph showing the effects of different concentrations of PD-1-112-C2 on the proliferation of T cells and the secretion of cytokine IFN- ⁇ by T cells.
  • Figure 4 is a graph showing the effect of anti-PD-1 humanized antibody c53 on tumor volume.
  • Figure 5 is a graph showing the effect of anti-PD-1 humanized antibody c53 on the survival of mice.
  • the present invention will be further described below in conjunction with specific examples, but the examples do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the present invention relates to an anti-PD-1 humanized antibody or an antigen-binding fragment thereof, the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2, and HCDR3, and the light chain CDR region consists of LCDR1, LCDR2 and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 8-10 in turn, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 11-13 in turn, and the weight of the antibody
  • the amino acid sequence of the chain variable region is shown in any one of SEQ ID NO: 3-5; the amino acid sequence of the light chain variable region of the antibody is shown in any one of SEQ ID NO: 6-7.
  • the present invention uses the Kabat numbering system to mark the CDR region, but the CDR region marked by other methods also belongs to the protection scope of the present invention.
  • the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 3, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6, or the heavy chain of the antibody can be The amino acid sequence of the variable region is shown in SEQ ID NO: 4, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 5, the light chain variable region The amino acid sequence of the chain variable region is shown in SEQ ID NO: 7.
  • the antibody contains a heavy chain constant region and a light chain constant region, and the heavy chain constant region is any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM ;
  • the light chain constant region is a ⁇ chain or a ⁇ chain.
  • the species source of the heavy chain constant region and the light chain constant region is selected from human, mouse or monkey.
  • the antibody is a chimeric antibody or a multispecific antibody (eg, a bispecific antibody).
  • multispecific antibody is an antigen binding protein or antibody that targets more than one antigen or epitope.
  • bispecific antibody is a multispecific antigen binding protein or multispecific antibody, and can be produced by various methods including, but not limited to fusion of hybridomas or linking of Fab' fragments . See, eg, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.
  • the two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes, present on the same or different protein targets.
  • the term "specific binding” or similar expressions thereof refers to the binding of an antibody or antigen-binding fragment thereof to a predetermined epitope on an antigen.
  • the antibody or antigen-binding fragment thereof binds with an affinity ( KD ) of about less than 10" 6M , eg, about less than 10" 7M , 10" 8M , 10" 9M or 10" 10M or less.
  • KD refers to the ratio of the off-rate to the on-rate (koff/kon), which quantity can be measured by methods familiar to those skilled in the art.
  • the antigen-binding fragment is any one or more of F(ab') 2 , Fab, scFv, Fv and single domain antibody.
  • the term "F(ab') 2" contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that an interchain chain is formed between the two heavy chains. disulfide bond.
  • the F(ab') 2 fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • Fab consists of a light chain and CH1 and the variable region of a heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • scFv is an Fv molecule in which the variable regions of the heavy and light chains are linked by a flexible linker to form a single polypeptide chain (which forms the antigen-binding region) (see, e.g., Bird et al., Science. 242 : 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA. 90: 5879-5883 (1988)).
  • Fv comprises variable regions from heavy and light chains, but lacks constant regions.
  • single domain antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other as artificially engineered single chain antibody (scFv) , and even gather into blocks. More importantly, the VHH structure cloned and expressed separately has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind the target antigen.
  • VHH heavy chain variable region
  • scFv artificially engineered single chain antibody
  • the present invention also relates to a nucleic acid encoding the anti-PD-1 humanized antibody or an antigen-binding fragment thereof.
  • the nucleic acid comprises: a first nucleic acid encoding a heavy chain variable region of the antibody or an antigen-binding fragment thereof, and/or, encoding a light chain variable region of the antibody or an antigen-binding fragment thereof of the second nucleic acid.
  • the nucleic acid is usually RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA is preferably used when it is ligated into a vector.
  • the nucleic acid since antibodies are membrane proteins, the nucleic acid usually carries a signal peptide sequence.
  • the invention also relates to a vector carrying said nucleic acid.
  • the term "vector" is a nucleic acid delivery tool into which a polynucleotide can be inserted.
  • the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • herpesviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • the invention also relates to a cell carrying said nucleic acid, containing said vector or capable of expressing said antibody or antigen-binding fragment thereof.
  • the present invention also relates to a pharmaceutical composition containing the antibody or antigen-binding fragment thereof, the nucleic acid, the vector or the cell.
  • the term "pharmaceutical composition” is in a form that allows the biological activity of the active ingredients to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered.
  • the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients.
  • the term "pharmaceutically acceptable carrier” may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible, used for Extend the shelf life or potency of antibodies.
  • the use of the antibody or its antigen-binding fragment, the nucleic acid, the carrier, the cell or the pharmaceutical composition in the preparation of a medicament for treating a PD-1-mediated disease or disorder should also be within the protection scope of the present invention.
  • the pharmaceutical composition or medicament is in a form suitable for injection.
  • the pharmaceutical composition or medicament is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
  • the anti-PD-1 humanized antibody or antigen-binding fragment thereof provided by the present invention can bind to CHO-hPD1 cells, CHO-cyno cells and activated PBMCs with high affinity, and its affinity is significantly higher than that of the positive control, and can bind efficiently and specifically PD-1, effectively blocks the binding of ligand PD-L1/PD-L2 to CHO-hPD1, in MLR, can block the binding of PD-1 and ligand, inhibit PD-1 signaling pathway, thereby promoting T cell proliferation and secrete IL-2, IFN- ⁇ cytokines; therefore, the antibody or its antigen-binding fragment, and its related nucleic acid, vector, cell or pharmaceutical composition are prepared for the treatment of PD-1-mediated diseases or disorders
  • the application prospect in the medicine is extensive.
  • mice 125ug of hPD-1 recombinant protein with a concentration of 1.23mg/ml was mixed with the same amount of immune adjuvant Freund's adjuvant (Sigma-Aldrich F5881) as an antigen, and five 6-week-old female BAL b/C mice were subcutaneously immunized. The amount of immunized antigen per mouse was 25ug. After the primary immunization, a booster immunization of the same dose was carried out once a week. After a total of 5 immunizations, the immune response was monitored by collecting mouse tail blood. Mice with sufficient anti-hPD-1 immunoglobulin titers were used for fusions by FACS screening (described below). Three days after the intraperitoneal boost with antigen, mice were sacrificed and spleens were removed for cell fusion.
  • immune adjuvant Freund's adjuvant Sigma-Aldrich F5881
  • mice that produce anti-hPD-1 antibodies were tested by FACS. Serum dilutions from mice immunized with hPD-1 recombinant protein were incubated with hPD1-transfected CHO cells at 4°C for 30 minutes, washed 3 times with PBS, and then 0.4ug/ml PE goat anti-mouse IgG (Biolegend 405307 ) and incubated at 4°C for 30 minutes.
  • Splenocytes from immunized BAL b/c mice were fused with mouse myeloma cells, and then the resulting hybridomas were screened for antigen-specific antibodies.
  • Single-cell suspensions of splenocytes from immunized mice were incubated with one-fifth the number of non-immunoglobulin-secreting mouse myeloma cells (SP2/0, ATCC CRL1581) using PEG 1500 (Roche 10783641001). fusion.
  • the fused cells were plated in a 96-well cell culture plate at about 1 ⁇ 10 5 cells/well, and placed in an incubator (Panasonic MCO-18AIC), and the culture conditions were 37° C., 5% CO 2 .
  • HAT selective medium containing 1X penicillin and streptomycin double antibody (Gibco 15140122), 1X HAT (Sigma CRLP-7185) and 20% fetal calf serum in 1640 medium for about one week (Royacel RY-F11-01).
  • HT medium 1640 medium containing 1X penicillin-streptomycin double antibody (gibco 15140122), 1X HT (gibco 11067030) and 20% fetal bovine serum (Royacel RY-F11-01)
  • the culture medium was cultured, and then the cell culture supernatant of the fusion plate was detected by FACS, and hybridomas secreting antibodies that could bind to hPD-1 protein were screened out.
  • Hybridomas secreting hPD-1 protein antibodies were re-plated and screened again.
  • the screened hybridomas that are positive for binding hPD-1 protein antibodies they are subcloned at least twice by the limited release method. Stable subclones were then cultured in vitro and produced small amounts of antibodies for further analysis.
  • the hybridoma clone PD1-112-C2 was selected for further analysis.
  • a CHO (Chinese hamster ovary cell) cell line (CHO-hPD1) expressing recombinant human PD-1 on the cell surface and a CHO cell line (CHO-cynoPD1) expressing monkey PD1 (Uniprot: BOLAJ2) were prepared using recombinant technology , a CHO cell line (CHO-mousePD1) expressing mouse PD1 (Uniprot: Q02242), the cell line will be used to measure the binding of anti-PD-1 murine mAb PD-1-112-C2 by flow cytometry (FCM) characterization.
  • FCM flow cytometry
  • CHO-hPD1 In order to evaluate the binding of anti-PD-1 mouse monoclonal antibody to CHO-hPD1, add 2 ⁇ 10 5 CHO-hPD1 cells and anti-PD-1 cells diluted in a concentration gradient (initial concentration is 10 ⁇ g/ml, three-fold dilution) in a 96-well plate.
  • PD-1 mouse monoclonal antibody incubate at 4°C for 30 minutes, wash cells once with buffer (PBS containing 3% BSA), add PE-labeled anti-mouse IgG (Fc) Ab (Biolegend) fluorescent secondary antibody, incubate at 4°C for 30 minutes After the cells were washed once with the buffer solution and resuspended in PBS, the cell suspension was subjected to flow cytometry analysis by CytoFlex (Beckman flow cytometer), and the amount of antibody bound to the cells was measured according to the mean fluorescence intensity (MFI) of the staining; The same method was used to evaluate the binding of this anti-PD-1 murine monoclonal antibody to CHO-cyno cells, CHO-mousePD1 (sometimes abbreviated as "CHO-mPD1" in the present invention) cells.
  • MFI mean fluorescence intensity
  • the results are shown in Table 1.
  • the data show that the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 can bind to CHO-hPD1 cells and CHO-cyno cells with high affinity; at the same time, the mouse monoclonal antibody does not bind to CHO- mousePD1 cells.
  • PBMC peripheral blood mononuclear cells
  • PBMCs were obtained from fresh human peripheral blood by gradient centrifugation of lymph separation fluid, and then adjusted to a density of 1 ⁇ 10 6 cells/ml and inoculated into T75, while adding PHA-L (Sigma) at a final concentration of 1 ⁇ g/ml to stimulate lymphocyte proliferation.
  • the anti-PD1 antibody can bind to activated lymphocytes with high affinity.
  • the anti-PD-1 murine monoclonal antibody was combined with four different CD28 family member proteins to verify the specificity of the antibody binding to PD-1.
  • immobilize PD-1, CD28, CTLA-4, ICOS (ACRO) at a concentration of 1 ⁇ g/ml on the ELISA plate immobilize PD-1, CD28, CTLA-4, ICOS (ACRO) at a concentration of 1 ⁇ g/ml on the ELISA plate, add anti-human PD-1 mouse monoclonal antibody at a concentration of 10 ⁇ g/ml, and couple the Anti-mouse IgG with peroxidase (HRP) was used as the secondary antibody (Sigma).
  • TMB color development after termination, read the OD450 value with a microplate reader.
  • the results are shown in Table 2.
  • the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 can specifically bind to PD-1, but not to other CD28 family members.
  • Anti-PD-1 mouse monoclonal antibody blocks the binding of ligand PD-L1/PD-L2 to CHO-hPD1
  • the ability of anti-PD-1 murine mAb to block ligand binding to PD-1 stably expressed on the surface of transfected CHO cells was analyzed by flow cytometry.
  • the ligand protein used in the experiment is the fusion protein of recombinant PD-L1/PD-L2 extracellular segment connected with human IgG1 Fc segment: PD-L1-hFc (ACRO), PD-L2-hFc (ACRO).
  • CHO-PD1 cells were resuspended in buffer (PBS containing 3% BSA), adjusted to a density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well of the cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, and removed supernatant.
  • buffer PBS containing 3% BSA
  • the follow-up process can be divided into two blocking modes: Mode 1, add PD-L1-hFc/PD-L2-hFc at a concentration of 3 ⁇ g/ml to the cell well, incubate at 4°C for 30 minutes, then add 30 ⁇ g/ml Start with ml, 3-fold serial dilution, a total of 10 concentrations of anti-PD-1 mouse monoclonal antibody, incubate at 4°C for 30 minutes; mode 2, add 3-fold serial dilution starting from 30 ⁇ g/ml to the cell well, A total of 10 concentration gradients of anti-PD-1 mouse monoclonal antibody were incubated at 4°C for 30 minutes, then PD-L1-hFc/PD-L2-hFc protein at a concentration of 3 ⁇ g/ml was added, and incubated at 4°C for 30 minutes.
  • Anti-PD-1 murine monoclonal antibody PD-1-112-C2 can effectively block the binding of PD-L1/PD-L2 to CHO-PD1 cells in both modes.
  • PBMCs peripheral blood mononuclear cells
  • SEB superantigen Staphylococcus aureus enterotoxin B
  • PBMCs peripheral mononuclear cells
  • LONZA X-VIVO 15 medium
  • FBS X-VIVO 15 medium
  • SEB superantigen SEB superantigen
  • the presence or absence of anti-PD-1 antibodies can demonstrate T cell proliferation and T cell secreted cytokine levels in the presence of PD1 signaling blockade.
  • CD14 MicroBeads human (Miltenyi) to separate CD14 + monocytes from fresh PBMCs, in the presence of GM-CSF/IL-4, after 6 days of induction, add TNF- ⁇ , and induce DC maturation after 3 days;
  • T cells in PBMC were purified with EasySep TM Human T Cell Enrichment Kit (StemCell), 1 ⁇ 10 4 DC cells were mixed with 1 ⁇ 10 5 T cells, and different concentration gradients of anti-T cells were added to the mixed cells.
  • PD-1 antibody additionally set isotype control antibody (mIgG1 isotype control antibody and hIgG4 isotype control antibody (Biolegend)), no antibody control well. After 3 days of mixed culture, the supernatant was taken for the detection of IL-2, and after another 2 days of culture, the supernatant was taken for the detection of IFN- ⁇ .
  • the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 8-10, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in sequence:
  • the sequence of the heavy chain variable region shown in SEQ ID NO: 11-13, the sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the sequence of the light chain variable region is shown in SEQ ID NO: 15) for humanization the specific method is:
  • the human PD-1 sequence (NCBI NP 005009) was artificially synthesized, cloned into the PCDNA3.4A eukaryotic expression system, and 293 cells were transfected with this plasmid, and the human PD-1 recombinant protein was purified after harvesting the supernatant.
  • the screened hybridomas positive for binding to hPD-1 protein antibody were subcloned at least twice by limiting dilution method, and the stable subclones were cultured in vitro to generate a small amount of antibodies, and PD-1-112-C2 clones were obtained after further screening.
  • SEQ ID NO: 8 TYYMY
  • SEQ ID NO: 10 RDSNYDGGFDY
  • the template of the heavy chain of the antibody is IGHV1
  • the template of the light chain of the antibody is IGKV1, so as not to affect the structural stability of the antibody, the binding of the antibody to the antigen, the introduction of glycosylation, phosphorylation and other protein modification sites, and the introduction of existing Under the principle of oxidative amination of all sites and enhancing structural stability, the humanized sequence of the heavy chain was designed as VH1-5, and the humanized sequence of the light chain was designed as VL1-3, and the light chain was IGKV7 with the highest homology -3*01 is another group of humanized design templates, the humanized sequence VL-4 is designed.
  • the pairing form of light and heavy chains is designed to be IGHV1/IGKV2 as a common pairing form to obtain humanized antibodies.
  • the antibody was purified according to conventional methods to obtain the anti-PD-1 humanized antibody (c11, c21, c31, c41, c51, c12, c22, c32, c42, c52, c43, c53 , c44, c54); wherein, the amino acid sequences of the three anti-PD-1 humanized antibodies (c22, c43, c53) are shown in Table 5.
  • the affinity characterization of the anti-PD-1 humanized antibody is shown in Table 6. The results show that the anti-PD-1 humanized antibody of the present invention can bind to CHO-hPD1 cells and CHO-cynoPD1 cells with high affinity.
  • PBMC peripheral blood mononuclear cells
  • PBMCs were obtained from fresh human peripheral blood by gradient centrifugation of lymph separation fluid, and then inoculated into T75 at a density of 1 ⁇ 10 6 cells/ml, and PHA-L (Sigma) was added at a final concentration of 1 ⁇ g/ml to stimulate lymphocyte proliferation. After standing in a 5% CO2 incubator at 37°C for 3 days, the cell suspension was taken out, centrifuged to remove the supernatant, resuspended in buffer (PBS containing 3% BSA), and added to a 96-well U-shaped plate according to 2E5/well.
  • PBS buffer containing 3% BSA
  • the anti-PD-1 humanized antibodies (c22, c43, c53) of the present invention and CHO-hPD1 Cells, CHO-cyno cells and activated PBMCs had comparable binding abilities.
  • the anti-PD-1 humanized antibody (c22, c43, c53) of the present invention was combined with four different CD28 family member proteins to verify the specificity of the anti-PD-1 humanized antibody binding to PD-1.
  • fix PD-1(Acro), CD28(Acro), CTLA-4(Acro) and ICOS at a concentration of 1 ⁇ g/ml on the ELISA plate add humanized antibodies at a concentration of 10 ⁇ g/ml, and Anti-human IgG (Fab) conjugated to peroxidase (HRP) was used as the secondary antibody.
  • TMB color development after termination, read the OD450 value with a microplate reader.
  • the binding specificity results of the anti-PD-1 humanized antibodies are shown in Table 7.
  • the results show that the anti-PD-1 humanized antibodies of the present invention can specifically bind to PD-1, but not to other CD28 family members.
  • the affinity determination results of the anti-PD-1 humanized antibodies are shown in Table 8, and the results show that all the anti-PD-1 humanized antibodies of the present invention can bind to PD-1 with high affinity.
  • the affinity of the anti-PD-1 humanized antibody of the present invention is comparable to it; compared with the positive control Opdivo (Table 3) 3), the affinity of the anti-PD-1 humanized antibody of the present invention is significantly improved.
  • Antibody to be tested kon(1/Ms) kdis(1/s) KD(M) c22 9.85E+05 ⁇ 1.0E-07 ⁇ 1.0E-12 c43 8.84E+05 ⁇ 1.0E-07 ⁇ 1.0E-12 c53 9.79E+05 ⁇ 1.0E-07 ⁇ 1.0E-12
  • Anti-PD-1 humanized antibody blocks the binding of ligand PD-L1/PD-L2 to CHO-hPD1
  • the ability of anti-PD-1 humanized antibodies to block ligand binding to PD-1 stably expressed on the surface of transfected CHO cells was analyzed by flow cytometry.
  • the ligand protein used in the experiment is the fusion protein of recombinant PD-L1/PD-L2 extracellular segment connected with mouse IgG1 Fc segment: PD-L1-mFc, PD-L2-mFc.
  • CHO-PD1 cells were resuspended in buffer (PBS containing 3% BSA), adjusted to a density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well of the cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, and removed supernatant.
  • the affinity determination results of the anti-PD-1 humanized antibody are shown in Table 9, and the results show that the anti-PD-1 humanized antibody of the present invention can effectively block the binding of PD-L1/PD-L2 to the cell CHO-PD1 .
  • MLR mixed lymphocyte reactions
  • human Miltenyi was used to isolate CD14 + monocytes from fresh PBMCs.
  • TNF- ⁇ was added to induce DC maturation after 3 days; experiment On the same day, use EasySep TM Human T Cell Enrichment Kit (StemCell) to purify T cells in PBMC, mix 1 ⁇ 10 4 cells/well DC cells with 1 ⁇ 10 5 cells/well T cells, and add Anti-PD-1 humanized antibodies (c22, c43, c53) with different concentration gradients, additional isotype control antibodies, and no antibody control wells.
  • the supernatant was taken for the detection of IL-2, and after another 2 days of culture, the supernatant was taken for the detection of IFN- ⁇ .
  • the effect of the anti-PD-1 humanized antibody in the mixed lymphocyte reaction is shown in Table 10.
  • the results show that the anti-PD-1 humanized antibody of the present invention can block the binding of PD1 to the ligand in the MLR experiment. Inhibit the PD1 signaling pathway, thereby promoting the proliferation of T cells, and promoting the secretion of IL-2 and IFN- ⁇ cytokines by T cells.
  • Example 16 In vivo anti-tumor efficacy evaluation of anti-PD-1 humanized antibody on mouse colon cancer cells
  • mice hPD1 Knock in mice, female, 6-8 weeks old (C57BL/6 background, source: Beijing Weitongda Biotechnology Co., Ltd.); MC38 cells (National Experimental Cell Sharing Resource Platform); FBS (Gibco, 10091-148 ), 0.25% trypsin-EDTA (Gibco, 25200056), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02), penicillin-streptomycin (Gibco, 15140122), DMEM high glucose medium (Gibco, 11965084 ).
  • Fetal Bovine Serum Gibco
  • Glutamine Gibco
  • MC38 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1% glutamine and 1% penicillin-streptomycin (1 : 1).
  • Inoculation collect MC38 cells in the logarithmic growth phase, and adjust the cell concentration to 3 ⁇ 10 6 /mL.
  • 40 female hPD1 mice were taken and subcutaneously inoculated with MC38 cells at a volume of 0.1 mL/mouse, ie 3 ⁇ 10 5 /mouse.
  • the tumor volume was measured and recorded, and then the long diameter and short diameter of the tumor were measured with a vernier caliper twice a week.
  • the tumor volume was calculated by the formula: (1/2) ⁇ long diameter ⁇ (short diameter) 2 .
  • the mice were sacrificed by cervical dislocation, and the survival curve was recorded.
  • anti-PD-1 humanized antibody The effect of anti-PD-1 humanized antibody on the survival period of mice is shown in Figure 5. It can be seen that compared with the Isotype group, anti-PD-1 humanized antibody (c53) can significantly prolong the survival period of mice .
  • the anti-PD-1 humanized antibody (c53) provided by the present invention can significantly inhibit the growth of MC38 cells, effectively prolong the survival period of mice, and has a significant effect on the treatment of colon cancer in mice.

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Abstract

属于生物医药技术领域，具体而言，提供抗PD-1人源化抗体或其抗原结合片段及其应用。该抗体能高效、特异性结合PD-1，并能有效阻断PD-1与PD-L1、PD-L2结合；因此，该抗体或其抗原结合片段、及其相关的核酸、载体、细胞或药物组合物能够制备用于治疗PD-1介导的疾病或病症的药物。

Description

抗PD-1人源化抗体或其抗原结合片段及其应用 技术领域
本发明属于生物医药技术领域。更具体地，涉及抗PD-1人源化抗体或其抗原结合片段及其应用。
背景技术
程序死亡因子-1(PD1)是CD28家族成员，在活化的B细胞，T细胞及髓样细胞上表达。人PD1由基因Pdcd1编码，位于2q37.3，全长9.6kb，由5个外显子和4个内含子组成，其上游包含663bp的启动子。PD1为55KDa的I型跨膜蛋白，分子结构由胞外区，跨膜区及胞内区组成，胞外区含有一个免疫球蛋白可变区IgV结构域，胞内区含有免疫受体酪氨酸抑制基序(ITIM)和免疫受体酪氨酸转换作用模块(ITSM)。PD-1胞外区氨基酸序列和CTLA-4有24％的同源性，与CD28有28％的同源性。在T细胞被激活后，PD-1主要通过ITIM将酪氨酸磷脂酶SHP2集合，导致下游效应分子去磷酸化。
PD-1有两个配体：PD-L1和PD-L2。PD-L1和PD-L2都是B7同源物，PDL基因位于人染色体9P24.2位点，大小为42kb，其分子结构均包含一个免疫球蛋白样可变区结构域，一个恒定区样结构域，一个跨膜区和一个短的胞质尾巴。
PD-1与PD-L1和PD-L2结合后可下调T细胞的活化。PD-L1表达于多种肿瘤细胞表面，这些肿瘤细胞包括：肺癌，胃癌，肝癌，食道癌，肾癌，卵巢癌，宫颈癌，乳腺癌，皮肤癌，结肠癌，膀胱癌，神经胶质癌，头颈癌，口腔鳞状细胞癌。而且这些癌症周边发现大量表达PD-L1的CD8+T细胞，临床结果统计显示，PD-L1在肿瘤细胞上的高表达水平与癌症患者不良预后有关。
发明内容
本发明要解决的技术问题是克服现有抗体结合PD-1的亲和力和特异性不高的缺陷和不足，提供抗PD-1人源化抗体或其抗原结合片段及其应用。
本发明的目的是提供抗PD-1人源化抗体或其抗原结合片段，所述抗体包含轻链CDR区和重链CDR区，重链CDR区由HCDR1、HCDR2、HCDR3组成，轻链CDR区由LCDR1、LCDR2、LCDR3组成，HCDR1、HCDR2、HCDR3的氨基酸序列依次如SEQ ID NO：8～10所示，LCDR1、LCDR2、LCDR3的氨基酸 序列依次如SEQ ID NO：11～13所示；所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3～5任一项所示。
所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：6～7任一项所示。
本发明还涉及所述抗体或其抗原结合片段相关的核酸、载体、细胞或药物组合物。
本发明还涉及所述抗体或其抗原结合片段、及其相关的核酸、载体、细胞或药物组合物在制备用于治疗PD-1介导的疾病或病症的药物中的用途。
本发明还涉及治疗PD-1介导的疾病或病症的方法，该方法包括：给受试者摄入有效量的上述抗体或其抗原结合片段、核酸、载体、细胞或药物组合物。
本发明还涉及用于治疗的上述抗体或其抗原结合片段、核酸、载体、细胞或药物组合物。
本发明还涉及用于治疗PD-1介导的疾病或病症的上述抗体或其抗原结合片段、核酸、载体、细胞或药物组合物。
附图说明
图1是不同浓度的PD-1-112-C2对IL-2/IFN-γ分泌的影响结果图。
图2是不同浓度的PD-1-112-C2对T细胞增殖和T细胞分泌细胞因子IL-2的影响结果图。
图3是不同浓度的PD-1-112-C2对T细胞增殖和T细胞分泌细胞因子IFN-γ的影响结果图。
图4是抗PD-1人源化抗体c53对肿瘤体积的影响结果图。
图5是抗PD-1人源化抗体c53对小鼠生存期的影响结果图。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本发明涉及抗PD-1人源化抗体或其抗原结合片段，所述抗体包含轻链CDR区和重链CDR区，重链CDR区由HCDR1、HCDR2、HCDR3组成，轻链CDR区由LCDR1、LCDR2、LCDR3组成，HCDR1、HCDR2、HCDR3的氨基酸序列 依次如SEQ ID NO：8～10所示，LCDR1、LCDR2、LCDR3的氨基酸序列依次如SEQ ID NO：11～13所示，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3～5任一项所示；所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：6～7任一项所示。
本发明采用Kabat编号系统标示CDR区，但其他方法标示的CDR区也属于本发明的保护范围。
在一些实施方式中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3、轻链可变区的氨基酸序列如SEQ ID NO：6所示，或所述抗体的重链可变区的氨基酸序列如SEQ ID NO：4、轻链可变区的氨基酸序列如SEQ ID NO：7所示，或所述抗体的重链可变区的氨基酸序列如SEQ ID NO：5、轻链可变区的氨基酸序列如SEQ ID NO：7所示。
在一些实施方式中，所述抗体含有重链恒定区和轻链恒定区，所述重链恒定区为IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种；所述轻链恒定区为κ链或λ链。
在一些实施方式中，所述重链恒定区和轻链恒定区的种属来源选自人、鼠或猴。
在一些实施方式中，所述抗体为嵌合抗体或多特异性抗体(例如双特异性抗体)。
在本发明中，术语“多特异性抗体”是靶向不止一种抗原或表位的抗原结合蛋白或抗体。
在本发明中，术语“双特异性抗体”是一种多特异性抗原结合蛋白或多特异性抗体，并且可通过多种方法产生，包括，但不限于杂交瘤的融合或Fab’片段的连接。参见，例如，Songsivilai和Lachmann，1990，Clin.Exp.Immunol.79：315-321；Kostelny等人，1992，J.Immunol.148：1547-1553。双特异性抗原结合蛋白或抗体的两个结合位点将结合两个不同的表位，所述表位存在于相同或不同的蛋白质靶标上。
在本发明中，术语“特异性结合”或其类似表述是指抗体或其抗原结合片段对预先确定的抗原上的表位的结合。通常，抗体或其抗原结合片段以大约小于10 -6M，例如大约小于10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合。KD是指解离速率与结合速率的比率(koff/kon)，该量可通过本领域技术人员熟悉 的方法测量。
在一些实施方式中，所述抗原结合片段为F(ab’) 2、Fab、scFv、Fv及单域抗体中的任一种或几种。
在本发明中，术语“F(ab’) 2”含有两条轻链和两条含有CH1与CH2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。
在本发明中，术语“Fab”由一条轻链和CH1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。
在本发明中，术语“scFv”是其中重链与轻链可变区通过柔性接头连接来形成单条多肽链(其形成抗原结合区)的Fv分子(参见，例如，Bird等人，Science.242：423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA.90：5879-5883(1988))。
在本发明中，术语“Fv”包含来自重链和轻链的可变区，但缺乏恒定区。
在本发明中，术语“单域抗体”只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，但却不像人工改造的单链抗体(scFv)那样容易相互沾粘，甚至聚集成块。更重要的是单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。
本发明还涉及一种核酸，编码所述抗PD-1人源化抗体或其抗原结合片段。
在优选实施方式中，所述核酸包括：编码所述抗体或其抗原结合片段的重链可变区的第一核酸，和/或，编码所述抗体或其抗原结合片段的轻链可变区的第二核酸。
在本发明中，核酸通常是RNA或DNA，核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时优选采用DNA。此外，鉴于抗体为膜蛋白，所以核酸通常带有信号肽序列。
本发明还涉及一种载体，所述载体携带所述核酸。
在本发明中，术语“载体(vector)”是可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿 主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。
本发明还涉及一种细胞，所述细胞携带所述核酸、含有所述载体或能够表达所述抗体或其抗原结合片段。
本发明还涉及一种药物组合物，所述组合物含有所述抗体或其抗原结合片段、所述核酸、所述载体或所述细胞。
在本发明中，术语“药物组合物”是以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。
在一些实施方式中，所述药物组合物还包括药学上可接受的载体和/或赋形剂。
在本发明中，术语“药学可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等，用来延长抗体的保存限期或效力。
另外，所述抗体或其抗原结合片段、所述核酸、所述载体、所述细胞或所述药物组合物在制备用于治疗PD-1介导的疾病或病症的药物中的用途，也应在本发明的保护范围之内。
在一些实施方式中，所述药物组合物或药物为适于注射的形式。
在优选实施方式中，所述药物组合物或药物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
本发明具有以下有益效果：
本发明提供的抗PD-1人源化抗体或其抗原结合片段能高亲和力结合CHO-hPD1细胞、CHO-cyno细胞和活化的PBMC，其亲和力相比阳性对照显著提高，能高效、特异性结合PD-1，有效阻断配体PD-L1/PD-L2与CHO-hPD1结合，在MLR中，能够阻断PD-1与配体的结合，抑制PD-1信号通路，从而促进T细胞增殖和分泌IL-2、IFN-γ细胞因子；因此，该抗体或其抗原结合片段、及 其相关的核酸、载体、细胞或药物组合物在制备用于治疗PD-1介导的疾病或病症的药物中的应用前景广泛。
实施例1抗PD-1抗体的制备
1.免疫原
人工合成人PD-1序列(NCBI NP 005009)，上游引物：5’-CCGCAAGCTTGCCGCCACCATG-3’(SEQ ID NO：1)、下游引物：5’-CCGGAATTCTCATTAATGGTGATGGTGATGATGCTGGAACTGGCCGGCAGGTC-3’(SEQ ID NO：2)，PCR扩增胞外端，经Hind III和EcoRI双酶切后克隆到pCDNA3.4A真核表达系统，以此质粒转染293细胞，收获上清后纯化获得人PD-1重组蛋白(hPD-1)。
2.免疫动物
将125ug浓度为1.23mg/ml的hPD-1重组蛋白作为抗原与等量免疫佐剂弗氏佐剂(Sigma-Aldrich F5881)混合，取5只6周雌性BAL b/C小鼠进行皮下免疫，每只小鼠免疫抗原量为25ug。在初次免疫以后，每周进行一次相同剂量的加强免疫。总共5次免疫后，通过采集小鼠尾血来监测免疫应答。通过FACS筛选(如下所述)，使用具有足够抗hPD-1免疫球蛋白滴度的小鼠来进行融合。用抗原进行腹腔加强免疫后3天，处死小鼠并取出脾进行细胞融合。
3.选择产生抗hPD-1抗体的BAL b/C小鼠
为了选择产生抗hPD-1抗体的BAL b/C小鼠，通过FACS对经免疫的小鼠血清进行测试。来自hPD-1重组蛋白免疫小鼠的血清稀释液与转染了hPD1的CHO细胞在4摄氏度孵育30分钟，以PBS洗涤3次后，加入0.4ug/ml的PE山羊抗小鼠IgG(Biolegend 405307)并在4摄氏度育孵30分钟。以PBS洗涤3次后，将样品放入Beckman Coulter公司流式细胞仪(CytoFLEX A00-1-1102)检测以验证其是否可以与转染了hPD1的CHO细胞结合，筛出产生抗hPD-1抗体的BAL b/C小鼠，然后进行细胞融合。
4.生成产生针对hPD-1的鼠单克隆抗体杂交瘤
将经免疫过BAL b/C小鼠的脾细胞与小鼠骨髓瘤细胞融合，然后对得到的杂交瘤进行抗原特异性抗体的筛选。用PEG 1500(Roche 10783641001)将来自经免疫过小鼠的脾细胞的单细胞悬浮液与五分之一数目且不分泌免疫球蛋白的小鼠骨髓瘤细胞(SP2/0，ATCC CRL1581)进行细胞融合。将融合细胞以约1×10 5个/ 孔铺在96孔细胞培养板中，放入培养箱(Panasonic MCO-18AIC)，培养条件为37℃，5％CO 2。随后在HAT选择性培养基中培养大约一周，所述培养基在1640培养基中含1X青链霉素双抗(Gibco 15140122)，1×HAT(Sigma CRLP-7185)和有20％胎牛血清(Royacel RY-F11-01)。1周后，再用HT培养基(1640培养基含1X青链霉素双抗(gibco 15140122)，1×HT(gibco 11067030)和20％胎牛血清(Royacel RY-F11-01))替换HAT的培养基进行培养，然后通过FACS检测融合板的细胞培养上清液，筛出分泌可结合hPD-1蛋白抗体的杂交瘤。对分泌可结合hPD-1蛋白抗体的杂交瘤重新铺板，再次进行筛选。对筛出的结合hPD-1蛋白抗体阳性的杂交瘤，通过有限释法亚克隆至少两次。然后体外培养稳定亚克隆并生成小量抗体用于进一步分析。选择杂交瘤克隆PD1-112-C2进行下一步的分析。
实施例2抗PD-1鼠单抗亲和力表征
根据常规方法，利用重组技术制备在细胞表面表达重组人PD-1的CHO(中国仓鼠卵巢细胞)细胞系(CHO-hPD1)，表达猴PD1(Uniprot：B0LAJ2)的CHO细胞系(CHO-cynoPD1)，表达小鼠PD1(Uniprot：Q02242)的CHO细胞系(CHO-mousePD1)，细胞系将用于流式细胞计量术(FCM)测定抗PD-1鼠单抗PD-1-112-C2的结合表征。
为了评估抗PD-1鼠单抗跟CHO-hPD1的结合，在96孔板里加入2×10 5CHO-hPD1细胞以及经浓度梯度稀释(初始浓度为10μg/ml，三倍梯稀释)的抗PD-1鼠单抗，4℃孵育30分钟，缓冲液(PBS含3％BSA)洗涤细胞一次后加入用PE标记的抗鼠IgG(Fc)Ab(Biolegend)荧光二抗，4℃孵育30分钟后缓冲液洗涤细胞一次后PBS重悬，而后细胞悬液经CytoFlex(贝克曼流式细胞仪)进行流式细胞分析，根据染色的平均荧光强度(MFI)来测量结合到细胞上的抗体量；同样的方法用于评估此抗PD-1鼠单抗与CHO-cyno细胞、CHO-mousePD1(在本发明中有时简写为“CHO-mPD1”)细胞的结合。
结果如表1所示，数据表明，抗PD-1鼠单抗PD-1-112-C2都能较高亲和力结合CHO-hPD1细胞以及CHO-cyno细胞；同时，鼠单抗都不结合CHO-mousePD1细胞。
表1
Figure PCTCN2022097854-appb-000001
实施例3抗PD-1抗体与活化的PBMC的结合
新鲜人外周血单个核细胞(PBMC)在PHA(Sigma)的刺激下，淋巴细胞能活化增殖，并在第三天最高丰度表达PD1，可以用来进行PD-1抗体与活化的淋巴天然表达PD1的结合实验。
新鲜人的外周血通过淋巴分离液梯度离心方法得到PBMC后，调整密度为1×10 6细胞/ml接种到T75中，同时加入终浓度为1μg/ml PHA-L(Sigma)刺激淋巴细胞增殖，37℃，5％CO 2培养箱中静置3天后，取出细胞悬液，离心去掉上清，缓冲液(PBS含3％BSA)重悬，按照2×10 5个/孔加入到96孔U型板中，再加入从30μg/ml起始，3倍梯度稀释，共10个浓度梯度的抗PD1抗体，4℃孵育30分钟后300g离心5分钟，缓冲液洗涤细胞一次，加入PE标记的山羊抗人IgG荧光抗体(Biolegend)，4℃孵育30分钟；离心洗涤细胞一次后PBS重悬细胞，而后进行CytoFlex流式细胞仪分析，检测结合到PBMC上的抗体量。
结果如表1所示，抗PD1抗体能高亲和力的与活化的淋巴细胞结合。
实施例4抗PD-1鼠单抗结合特异性
将抗PD-1鼠单抗与四种不同CD28家族成员蛋白结合来验证抗体结合PD-1的特异性。利用标准ELISA方法，将浓度1μg/ml的PD-1，CD28，CTLA-4，ICOS(ACRO)固定在ELISA板上，加入浓度为10μg/ml的抗人PD-1鼠单抗，将偶联了过氧化物酶(HRP)的抗小鼠IgG作为二抗(Sigma)。TMB显色，终止后，酶标仪读OD450值。
结果如表2所示，抗PD-1鼠单抗PD-1-112-C2都能特异性结合PD-1，而不结合CD28其他家族成员。
表2
Figure PCTCN2022097854-appb-000002
实施例5生物层干涉(BLI)法测定抗人PD-1鼠单抗的亲和力
ForteBio(Octet Qke)亲和力测定：通过将浓度为5μg/ml的PD-1-his(ACRO) 重组蛋白装载在HISIK生物传感器上120秒，然后将装载好的传感器在标准缓冲液(PBST，PBS+0.02％Tuween20)中平衡120秒，之后传感器转移到抗PD-1鼠单抗稀释液中停留180秒测量结合速率，再转移至标准缓冲液中停留20分钟测量解离速率。最后利用动力学模型来进行分析。
数据处理结果如表3所示。
表3
待测抗体 kon(1/Ms) kdis(1/s) KD(M)
Opdivo(ABA0333) 1.38E+06 3.63E-06 2.62E-12
PD-1-112-C2 7.32E+05 3.18E-05 4.35E-11
实施例6抗PD-1鼠单抗阻断配体PD-L1/PD-L2与CHO-hPD1结合
通过流式细胞仪分析抗PD-1鼠单抗对于阻断配体与经转染CHO细胞表面稳定表达PD-1的结合能力。实验中用到的配体蛋白为重组的PD-L1/PD-L2胞外段连接上human IgG1Fc段融合蛋白：PD-L1-hFc(ACRO)，PD-L2-hFc(ACRO)。
CHO-PD1细胞用缓冲液(PBS含3％BSA)重悬，调整密度为2×10 6细胞/ml，100μl/孔细胞悬液加入到96孔U型板中，300g离心5分钟后，去掉上清。
后续过程可分为两种阻断模式进行：模式一，向细胞孔中加入浓度为3μg/ml的PD-L1-hFc/PD-L2-hFc，4℃孵育30分钟后，再加入从30μg/ml起始，3倍梯度稀释，共10个浓度梯度释的抗PD-1鼠单抗，4℃孵育30分钟；模式二，向细胞孔中加入从30μg/ml起始，3倍梯度稀释，共10个浓度梯度的抗PD-1鼠单抗，4℃孵育30分钟后，再加入浓度为3μg/ml的PD-L1-hFc/PD-L2-hFc蛋白，4℃孵育30分钟。
300g离心5分钟，缓冲液洗涤细胞一次，加入PE标记的山羊抗人IgG荧光抗体(Biolegend)，4℃孵育30分钟。离心洗涤细胞一次后PBS重悬细胞，而后进行CytoFlex流式细胞仪分析，检测结合到细胞上的配体蛋白量，计算得到PD-1抗体结合阻断的IC 50值。
结果如表4所示，抗PD-1鼠单抗：PD-1-112-C2在两种模式中都能有效的阻断PD-L1/PD-L2与细胞CHO-PD1的结合。
表4
Figure PCTCN2022097854-appb-000003
Figure PCTCN2022097854-appb-000004
实施例7抗PD-1抗体对SEB刺激的PBMC细胞的细胞因子释放的影响
此实施例中，过夜培养的外周血单个核细胞(PBMC)通过加入超抗原金黄色葡萄球菌肠毒素B(SEB)刺激时，在抗PD-1抗体存在或缺失情况下，检测细胞因子分泌的影响。
新鲜的外周单个核细胞(PBMC)用含10％FBS的X-VIVO 15培养基(LONZA)重悬后，添加到T25培养瓶，37℃，5％CO 2静置过夜培养，次日，取悬浮细胞，离心后，新鲜X-VIVO(含10％FBS)培养基重悬，并添加终浓度为200ng/ml的SEB超抗原(Toxin technology)，然后按照每孔1×10 5细胞添加到96孔平板中，同时添加不同浓度的抗PD-1抗体，另外设置同型对照抗体(mIgG1同型对照抗体(Biolegend)；hIgG4同型对照抗体(Biolegend))，无抗体对照孔。3天后，样本孔中取样，使用IL2/IFN-γHuman Uncoated ELISA Kit(eBioscience)试剂盒测量IL-2/IFN-γ水平。
不同浓度的PD-1-112-C2对IL-2/IFN-γ分泌的影响结果如图1所示，抗PD-1抗体以浓度依赖性方式提高IL-2/IFN-γ的分泌。这些结果表明，在SEB超抗原刺激PBMC中，抗PD-1抗体：PD-1-112-C2能进一步促进T细胞分泌细胞因子。
实施例8抗PD-1抗体在混合淋巴细胞反应中的影响
在混合淋巴细胞反应(MLR)中，抗PD-1抗体的存在与否能证明PD1信号被阻断情形下的T细胞增殖情况和T细胞分泌细胞因子水平高低。
用CD14 MicroBeads，human(Miltenyi)从新鲜PBMC中分离得到CD14 +单核细胞(monocyte)，在GM-CSF/IL-4存在下，诱导6天后，添加TNF-α，3天后诱导DC的成熟；实验当天，用EasySep TM Human T Cell Enrichment Kit(StemCell)纯化PBMC中的T细胞，1×10 4的DC细胞与1×10 5的T细胞混合培养，并且向混合细胞中添加不同浓度梯度的抗PD-1抗体，另外设置同型对照抗体(mIgG1同型对照抗体和hIgG4同型对照抗体(Biolegend))，无抗体对照孔。混合培养3天后，取上清进行IL-2的检测，再培养2天后，取上清进行IFN-γ的检测。
不同浓度的PD-1-112-C2对T细胞增殖和T细胞分泌细胞因子IL-2的影响 结果如图2所示，不同浓度的PD-1-112-C2对T细胞增殖和T细胞分泌细胞因子IFN-γ的影响结果如图3所示，图2和图3的结果表明抗PD-1抗体：PD-1-112-C2在MLR实验中，能以抗体浓度依赖性方式的阻断PD1与配体的结合，抑制PD1信号通路，从而促进T细胞增殖，促进T细胞分泌IL-2，IFN-γ细胞因子。
实施例9抗PD-1鼠单抗人源化
对以上获得的抗PD-1鼠单抗PD-1-112-C2(其HCDR1、HCDR2、HCDR3的氨基酸序列依次如SEQ ID NO：8～10所示，LCDR1、LCDR2、LCDR3的氨基酸序列依次如SEQ ID NO：11～13所示，重链可变区序列如SEQ ID NO：14所示，轻链可变区序列如SEQ ID NO：15所示)进行人源化，具体方法为：
人工合成人PD-1序列(NCBI NP 005009)，克隆到PCDNA3.4A真核表达系统，以此质粒转染293细胞，收获上清后纯化获得人PD-1重组蛋白。将获得的人PD-1重组蛋白进行皮下免疫雌性BAL b/C小鼠，将经免疫过BAL b/C小鼠的脾细胞与小鼠骨髓瘤细胞融合，然后对得到的杂交瘤进行抗原特异性抗体的筛选。筛出的结合hPD-1蛋白抗体阳性的杂交瘤，通过有限稀释法亚克隆至少两次后，体外培养稳定亚克隆并生成小量抗体在进一步筛选后得到PD-1-112-C2克隆。
SEQ ID NO：8：TYYMY
SEQ ID NO：9：GINPSNGGTNFNEKFKS
SEQ ID NO：10：RDSNYDGGFDY
SEQ ID NO：11：RASKSVSTSGYSYMH
SEQ ID NO：12：LAYHLES
SEQ ID NO：13：QHSWELPIT
SEQ ID NO：14：
Figure PCTCN2022097854-appb-000005
SEQ ID NO：15：
Figure PCTCN2022097854-appb-000006
Figure PCTCN2022097854-appb-000007
参照SEQ ID NO：14和SEQ ID NO：15，在Germline数据库中选取与其非CDR区匹配最好的人源化模板。其中抗体重链的模板为IGHV1，抗体的轻链模板为IGKV1，在以不影响抗体结构稳定性，不影响抗体与抗原结合，不引入糖基化，磷酸化等蛋白修饰位点，不引入已被氧化氨基化等位点，增强结构稳定性的原则下，设计重链人源化序列为VH1-5，设计轻链人源化序列为VL1-3，同时轻链以同源性最高的IGKV7-3*01为另一组人源化设计模板，设计人源化序列VL-4。设计轻重链配对形式为IGHV1/IGKV2为常见配对形式，得到人源化抗体。
根据各人源化抗体轻链和重链氨基酸序列合成基因，用Hind III(NEB)和EcoRI(NEB)双酶切后，将基因片段采用T4DNA连接酶(TAKARA 2011A)通过Hind III(NEB)/EcoRI(NEB)酶切位点插入到PCDNA3.4A表达载体(Invitrogen)上。将表达载体和转染试剂PEI(Poly science，Inc.Cat.NO.23966)以1∶2的比例转染HEK293细胞(Life Technologics Cat.NO.11625019)，并置于CO 2培养箱培养5-7天。表达的抗体通过离心回收后按照常规方法进行抗体纯化，得到本发明的抗PD-1人源化抗体(c11、c21、c31、c41、c51、c12、c22、c32、c42、c52、c43、c53、c44、c54)；其中，3株抗PD-1人源化抗体(c22、c43、c53)的氨基酸序列如表5所示。
表5 3株抗PD-1人源化抗体(c22、c43、c53)的氨基酸序列
Figure PCTCN2022097854-appb-000008
SEQ ID NO：3：
Figure PCTCN2022097854-appb-000009
SEQ ID NO：4：
Figure PCTCN2022097854-appb-000010
Figure PCTCN2022097854-appb-000011
SEQ ID NO：5：
Figure PCTCN2022097854-appb-000012
SEQ ID NO：6：
Figure PCTCN2022097854-appb-000013
SEQ ID NO：7：
Figure PCTCN2022097854-appb-000014
实施例10抗PD-1人源化抗体亲和力表征
1、实验方法
利用重组技术在细胞表面表达重组人PD-1的CHO(中国仓鼠卵巢细胞)细胞系(CHO-hPD1)，表达猴PD1的CHO细胞系(CHO-cynoPD1)，这两种细胞系将用于流式细胞计量术(FCM)测定抗PD-1人源化候选单抗(c22、c43、c53)的结合表征。具体方法为：
为了评估抗人源化抗体跟CHO-hPD1的结合，在96孔板里加入2×10 5CHO-hPD1细胞以及经浓度梯度稀释(初始浓度为30μg/ml，三倍梯稀释)的人源化抗体，4℃孵育30分钟，缓冲液(PBS含3％BSA)洗涤细胞一次后加入用PE标记的抗人IgG(Fc)Ab(Biolegend)荧光二抗，4℃孵育30分钟后缓冲液洗涤细胞一次后PBS重悬，而后细胞悬液经CytoFlex(贝克曼流式细胞仪)进行流式细胞分析，根据染色的平均荧光强度(MFI)来测量结合到细胞上的抗体量；同样的方法用于评估此抗人源化抗体与CHO-cyno细胞。
2、实验结果
抗PD-1人源化抗体亲和力表征如表6所示，结果显示本发明抗PD-1人源化抗体都能较高亲和力结合CHO-hPD1细胞以及CHO-cynoPD1细胞。
表6抗PD-1人源化抗体亲和力表征
Figure PCTCN2022097854-appb-000015
实施例11抗PD-1人源化抗体与活化的PBMC的结合
1、实验方法
新鲜人外周血单个核细胞(PBMC)在PHA(Sigma)的刺激下，淋巴细胞能活化增殖，并在第三天最高丰度表达PD1，可以用来进行抗PD-1人源化抗体(c22、c43、c53)与活化的淋巴天然表达PD1的结合实验。具体方法为：
新鲜人的外周血通过淋巴分离液梯度离心方法得到PBMC后，调整密度为1×10 6cells/ml接种到T75中，同时加入终浓度为1μg/ml PHA-L(Sigma)刺激淋巴细胞增殖，37℃，5％CO 2培养箱中静置3天后，取出细胞悬液，离心去掉上清，缓冲液(PBS含3％BSA)重悬，按照2E5/孔加入到96孔U型板中，再加入不同浓度梯度的人源化抗体，4℃孵育30分钟后300g离心5分钟，缓冲液洗涤细胞一次，加入PE标记的山羊抗人IgG荧光抗体(Biolegend)，4℃孵育30分钟；离心洗涤细胞一次后PBS重悬细胞，而后进行CytoFlex流式细胞仪分析，检测结合到PBMC上的抗体量。
2、实验结果
抗PD-1人源化抗体与活化的PBMC的结合能力测定结果如表6所示，结果显示本发明抗PD-1人源化抗体能高亲和力的与活化的淋巴细胞结合。
与实施例2和3中的抗PD-1鼠单抗PD-1-112-C2(表1)相比，本发明抗PD-1人源化抗体(c22、c43、c53)与CHO-hPD1细胞、CHO-cyno细胞和活化的PBMC的结合能力与其相当。
实施例12抗PD-1人源化抗体结合特异性
1、实验方法
将本发明抗PD-1人源化抗体(c22、c43、c53)与四种不同CD28家族成员蛋白结合来验证抗PD-1人源化抗体结合PD-1的特异性。利用标准ELISA方法，将浓度1μg/ml的PD-1(Acro)，CD28(Acro)，CTLA-4(Acro)，ICOS固定在ELISA板上，加入浓度为10μg/ml的人源化抗体，将偶联了过氧化物酶(HRP)的抗人IgG(Fab)作为二抗。TMB显色，终止后，酶标仪读OD450值。
2、实验结果
抗PD-1人源化抗体结合特异性结果如表7所示，结果显示本发明抗PD-1人源化抗体都能特异性结合PD-1，而不结合CD28其他家族成员。
表7抗PD-1人源化抗体结合特异性结果
Figure PCTCN2022097854-appb-000016
实施例13抗PD-1人源化抗体的亲和力测定
1、实验方法
ForteBio(Octet Qke)亲和力测定：通过将浓度为5μg/ml的PD-1-his重组蛋白装载在HISIK生物传感器上120秒，然后将装载好的传感器在标准缓冲液(PBST，PBS+0.02％Tuween20)中平衡120秒，之后传感器转移到抗PD-1人源化抗体(c22、c43、c53)稀释液中停留180秒测量结合速率，再转移至标准缓冲液中停留20分钟测量解离速率。最后利用动力学模型来进行分析，数据处理。以Opdivo(ABA0333)为阳性对照。
2、实验结果
抗PD-1人源化抗体的亲和力测定结果如表8所示，结果显示本发明的抗PD-1人源化抗体均能高亲和力结合PD-1。
与实施例5中的抗PD-1鼠单抗PD-1-112-C2(表3)相比，本发明抗PD-1人源化抗体的亲和力与其相当；和阳性对照Opdivo相比(表3)，本发明抗PD-1人源化抗体的亲和力显著提高。
表8抗PD-1人源化抗体的亲和力测定结果
待测抗体 kon(1/Ms) kdis(1/s) KD(M)
c22 9.85E+05 ＜1.0E-07 ＜1.0E-12
c43 8.84E+05 ＜1.0E-07 ＜1.0E-12
c53 9.79E+05 ＜1.0E-07 ＜1.0E-12
实施例14抗PD-1人源化抗体阻断配体PD-L1/PD-L2与CHO-hPD1结合
1、实验方法
通过流式细胞仪分析抗PD-1人源化抗体对于阻断配体与经转染CHO细胞表面稳定表达PD-1的结合能力。实验中用到的配体蛋白为重组的PD-L1/PD-L2胞外段连接上mouse IgG1Fc段融合蛋白：PD-L1-mFc，PD-L2-mFc。
CHO-PD1细胞用缓冲液(PBS含3％BSA)重悬，调整密度为2×10 6cells/ml，100μl/孔细胞悬液加入到96孔U型板中，300g离心5分钟后，去掉上清。向细胞孔中加入浓度为0.2μg/ml的PD-L1-mFc/PD-L2-mFc，4℃孵育30分钟后，再加入浓度梯度稀释的抗PD-1人源化抗体(c22、c43、c53)，4℃孵育30分钟。
300g离心5分钟，缓冲液洗涤细胞一次，加入PE标记的山羊抗鼠IgG荧光抗体(Biolegend)，4℃孵育30分钟。离心洗涤细胞一次后PBS重悬细胞，而后进行CytoFlex流式细胞仪分析，检测结合到细胞上的配体蛋白量，计算得到PD-1抗体结合阻断的IC 50值。
2、实验结果
抗PD-1人源化抗体的亲和力测定结果如表9所示，结果显示本发明抗PD-1人源化抗体都能有效的阻断PD-L1/PD-L2与细胞CHO-PD1的结合。
表9抗PD-1人源化抗体的亲和力测定结果
Figure PCTCN2022097854-appb-000017
Figure PCTCN2022097854-appb-000018
实施例15抗PD-1人源化抗体在混合淋巴细胞反应中的影响
1、实验方法
在混合淋巴细胞反应(MLR)中，抗PD-1人源化抗体的存在与否能证明PD1信号被阻断情形下的T细胞增殖情况和T细胞分泌细胞因子水平高低。具体方法为：
用CD14MicroBeads，human(Miltenyi)从新鲜PBMC中分离得到CD14 +单核细胞(monocyte)，在GM-CSF/IL-4存在下，诱导6天后，添加TNF-α，3天后诱导DC的成熟；实验当天，用EasySep TM Human T Cell Enrichment Kit(StemCell)纯化PBMC中的T细胞，1×10 4cells/孔的DC细胞与1×10 5cells/孔的T细胞混合培养，并且向混合细胞中添加不同浓度梯度的抗PD-1人源化抗体(c22、c43、c53)，另外设置同型对照抗体，无抗体对照孔。混合培养3天后，取上清进行IL-2的检测，再培养2天后，取上清进行IFN-γ的检测。
3、实验结果
抗PD-1人源化抗体在混合淋巴细胞反应中的影响结果如表10所示，结果显示本发明抗PD-1人源化抗体在MLR实验中，能阻断PD1与配体的结合，抑制PD1信号通路，从而促进T细胞增殖，促进T细胞分泌IL-2、IFN-γ细胞因子。
表10抗PD-1人源化抗体在混合淋巴细胞反应中的影响结果
Figure PCTCN2022097854-appb-000019
实施例16抗PD-1人源化抗体对小鼠结肠癌细胞的体内抗肿瘤药效评价
1、实验方法
实验目的：测定抗PD-1人源化抗体(c53)对小鼠结肠癌细胞(MC38细胞) 的体内抗肿瘤活性，同时设置同型对照组(Isotype)、阳性对照组(Sintilimab)。
实验材料：hPD1 Knock in小鼠，雌性，6-8周(C57BL/6背景，来源：北京维通达生物技术有限公司)；MC38细胞(国家实验细胞共享资源平台)；FBS(Gibco，10091-148)，0.25％胰蛋白酶-EDTA(Gibco，25200056)，DMSO(Sigma，D2650)，DPBS(Hyclone，SH30028.02)，青霉素-链霉素(Gibco，15140122)，DMEM高糖培养基(Gibco，11965084)。胎牛血清(Gibco)，谷氨酰胺(Gibco)，
仪器设备：电子天平(上海舜宇恒平科学仪器有限公司，JA12002)，游标卡尺(上海美耐特实业有限公司，MNT-150T)，显微镜(重庆奥特光学仪器有限公司，BDS200)，医用离心机(湖南湘仪实验室开发有限公司，L530R)，数显恒温水浴锅(普瑞斯机械有限公司，HH-S)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)，双人垂直型超净台(无锡易净化设备有限公司，SW-CJ-VS2)。
实验步骤：
细胞培养：将MC38细胞培养在含有10％胎牛血清、1％谷氨酰胺与1％青霉素-链霉素(1 ∶1)的DMEM高糖培养基中。
接种：收集对数生长期的MC38细胞，调节细胞浓度为3×10 6/mL。取雌性hPD1小鼠40只，皮下接种MC38细胞，接种体积为0.1mL/小鼠，即3×10 5/小鼠。
给药：接种当天记为第0天(D0)，第7天，将小鼠按瘤体积随机分为3组，每组8只，开始给药(MC38肿瘤模型给药剂量、方式以及频率如表11所示)。
Figure PCTCN2022097854-appb-000020
记录：D7开始测量肿瘤体积并记录，之后每周2次用游标卡尺测量肿瘤长径和短径。以公式：(1/2)×长径×(短径) 2计算肿瘤体积。每只小鼠达到实验终点时(肿瘤体积超过2000mm 3达到仁慈终点)，颈椎脱臼法处死小鼠，记录生存曲线。
2、实验结果
抗PD-1人源化抗体对肿瘤体积的影响结果如表12和图4所示，可以看出，与Isotype组相比，抗PD-1人源化抗体(c53)对MC38肿瘤模型的肿瘤生长有显著的抑瘤作用(TGI＝104.44％，肿瘤全消小鼠7只)，与Sintilimab组抗肿瘤作用相当(TGI＝105.8l％，肿瘤全消小鼠7只)。
抗PD-1人源化抗体对小鼠生存期的影响结果如图5所示，可以看出，与Isotype组相比，抗PD-1人源化抗体(c53)能够明显延长小鼠生存期。
表12抗PD-1人源化抗体对肿瘤体积的影响结果(mm 3)
Figure PCTCN2022097854-appb-000021
以上结果表明：本发明提供的抗PD-1人源化抗体(c53)能够显著抑制MC38细胞的生长，有效延长小鼠生存期，对治疗小鼠结肠癌具有显著疗效。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (13)

  1. 抗PD-1人源化抗体或其抗原结合片段，所述抗体包含轻链CDR区和重链CDR区，重链CDR区由HCDR1、HCDR2、HCDR3组成，轻链CDR区由LCDR1、LCDR2、LCDR3组成，HCDR1、HCDR2、HCDR3的氨基酸序列依次如SEQ ID NO：8～10所示，LCDR1、LCDR2、LCDR3的氨基酸序列依次如SEQ ID NO：11～13所示，其特征在于，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3～5任一项所示。
  2. 根据权利要求1所述抗体或其抗原结合片段，其特征在于，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：6～7任一项所示。
  3. 根据权利要求2所述抗体或其抗原结合片段，其特征在于，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3、轻链可变区的氨基酸序列如SEQ ID NO：6所示，或所述抗体的重链可变区的氨基酸序列如SEQ ID NO：4、轻链可变区的氨基酸序列如SEQ ID NO：7所示，或所述抗体的重链可变区的氨基酸序列如SEQ ID NO：5、轻链可变区的氨基酸序列如SEQ ID NO：7所示。
  4. 根据权利要求1～3任一所述抗体或其抗原结合片段，其特征在于，所述抗体含有重链恒定区和轻链恒定区，所述重链恒定区为IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种；所述轻链恒定区为κ链或λ链。
  5. 根据权利要求1～4任一所述抗体或其抗原结合片段，其特征在于，所述抗体为嵌合抗体或多特异性抗体；所述抗原结合片段为F(ab’) 2、Fab、scFv、Fv及单域抗体中的任一种或几种。
  6. 一种核酸，其特征在于，编码权利要求1～5任一所述抗PD-1人源化抗体或其抗原结合片段；
    优选地，所述核酸包括：编码所述抗体或其抗原结合片段的重链可变区的第一核酸，和/或，编码所述抗体或其抗原结合片段的轻链可变区的第二核酸。
  7. 一种载体，其特征在于，所述载体携带权利要求6所述核酸。
  8. 一种细胞，其特征在于，所述细胞携带权利要求6所述核酸、含有权利要求7所述载体或能够表达权利要求1～5任一所述抗体或其抗原结合片段。
  9. 一种药物组合物，其特征在于，所述组合物含有权利要求1～5任一所述抗体或其抗原结合片段、权利要求6所述核酸、权利要求7所述载体或权利要求8所述细胞。
  10. 权利要求1～5任一所述抗体或其抗原结合片段、权利要求6所述核酸、权利要求7所述载体、权利要求8所述细胞或权利要求9所述药物组合物在制备用于治疗PD-1介导的疾病或病症的药物中的用途。
  11. 一种治疗PD-1介导的疾病或病症的方法，其特征在于，所述方法包括：
    给受试者摄入有效量的权利要求1～5任一所述抗体或其抗原结合片段、权利要求6所述核酸、权利要求7所述载体、权利要求8所述细胞或权利要求9所述药物组合物。
  12. 用于治疗的权利要求1～5任一所述抗体或其抗原结合片段、权利要求6所述核酸、权利要求7所述载体、权利要求8所述细胞或权利要求9所述药物组合物。
  13. 用于治疗PD-1介导的疾病或病症的权利要求1～5任一所述抗体或其抗原结合片段、权利要求6所述核酸、权利要求7所述载体、权利要求8所述细胞或权利要求9所述药物组合物。
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