CN115466329A - anti-PD-1 humanized antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological medicines, and particularly relates to an anti-PD-1 humanized antibody and application thereof. The antibody can be efficiently and specifically combined with PD-1, and can effectively block the combination of PD-1 with PD-L1 and PD-L2, and the antibody or the antigen binding fragment thereof, and related nucleic acid, vector, cell or pharmaceutical composition thereof can be used for preparing a medicament for treating PD-1 mediated diseases or symptoms.

Description

A kind of anti-PD-1 humanized antibody and its application

technical field

The invention belongs to the technical field of biomedicine. More specifically, it relates to an anti-PD-1 humanized antibody and its application.

Background technique

Programmed death factor-1 (PD1) is a member of the CD28 family, expressed on activated B cells, T cells and myeloid cells. Human PD1 is encoded by the gene 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). The amino acid sequence of the extracellular domain of PD-1 has 24% homology with CTLA-4 and 28% homology with CD28. After T cells are activated, PD-1 mainly assembles tyrosine phospholipase SHP2 through ITIM, leading to dephosphorylation of downstream effector molecules.

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. Moreover, 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.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of existing antibodies that have low affinity and specificity for binding to PD-1 and cannot effectively block the binding of PD-1 to PD-L1 and PD-L2, and provide a highly efficient An anti-PD-1 humanized antibody that binds to PD-1 and can effectively block the binding of PD-1 to PD-L1 and PD-L2.

The purpose of the present invention is to provide an anti-PD-1 humanized antibody or its antigen-binding fragment, the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region is composed of HCDR1, HCDR2, HCDR3, the light chain The CDR region is composed of LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 9-11, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 12-14. The amino acid sequence of the heavy chain variable region of the antibody 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: 6-8 Show.

Another object of the present invention is to provide nucleic acid, vector, cell or pharmaceutical composition related to the antibody or antigen-binding fragment 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.

Description of drawings

Figure 1 is a graph showing the effect of different concentrations of PD-1-76-C2 on the secretion of IL-2/IFN-γ.

Figure 2 is a graph showing the effects of different concentrations of PD-1-76-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-1-76-C2 on the proliferation of T cells and the secretion of cytokine IFN-γ by T cells.

Figure 4 is a graph showing the effect of anti-PD-1 humanized antibodies h31, h61, and h43 on tumor volume.

Figure 5 is a graph showing the effects of anti-PD-1 humanized antibodies h31, h61, and h43 on the survival of mice.

detailed description

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. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

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 NOs: 9 to 11, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 12 to 14. The antibody The amino acid sequence of the heavy chain variable region of the antibody 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: 6-8.

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.

In the present invention, 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. Typically, 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.

In some embodiments, 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: 6, 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 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, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

In some embodiments, 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 κ chain or a λ chain.

In some embodiments, the species source of the heavy chain constant region and the light chain constant region is selected from human, mouse or monkey.

In some embodiments, the antibody is a chimeric antibody or a multispecific antibody (eg, a bispecific antibody).

In the present invention, the term "multispecific antibody" is an antigen binding protein or antibody that targets more than one antigen or epitope.

In the present invention, the term "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.

In some embodiments, the antigen-binding fragment is any one or more of F(ab') ₂ , Fab, scFv, Fv and single domain antibody.

In the present invention, 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.

In the present invention, the term "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.

In the present invention, the term "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)).

In the present invention, the term "Fv" comprises variable regions from heavy and light chains, but lacks constant regions.

In the present invention, the term "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.

The present invention also relates to a nucleic acid encoding the anti-PD-1 humanized antibody or an antigen-binding fragment thereof.

In a preferred embodiment, 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.

In the present invention, 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. For example, 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. In addition, 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.

In the present invention, the term "vector" is a nucleic acid delivery tool into which a polynucleotide can be inserted. When 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. 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).

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.

In the present invention, 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.

In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients.

In the present invention, 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.

In addition, 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.

In some embodiments, the pharmaceutical composition or medicament is in a form suitable for injection.

In a preferred embodiment, 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 present invention has the following beneficial effects:

The present invention provides an anti-PD-1 humanized antibody or an antigen-binding fragment thereof. The antibody has high affinity and strong specificity, and can bind to CHO-hPD1 cells, CHO-cyno cells and activated PBMCs with high affinity. Specifically binds to PD-1 and effectively blocks the binding of ligand PD-L1/PD-L2 to CHO-hPD1. In MLR, it can block the binding of PD-1 and ligand, inhibit the PD-1 signaling pathway, and promote T cells proliferate and secrete IL-2, IFN-γ 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 diseases of the drug has broad application prospects.

Example 1 Preparation of anti-PD-1 antibody

1. Immunogen

Artificially synthesized human PD-1 sequence (NCBI NP 005009), upstream primer: 5'-CCGCAAGCTTGCCGCCACCATG-3'(SEQ ID NO:1), downstream primer: 5'-CCGGAATTCTCATTAATGGTGATGGTGATGATGCTGGAACTGGCCGGCA GGTC-3'(SEQ ID NO:2), The extracellular end was amplified by PCR, cloned into the pCDNA3.4A eukaryotic expression system after double digestion with Hind III and EcoRI, transfected with this plasmid into 293 cells, harvested the supernatant and purified to obtain human PD-1 recombinant protein (hPD-1 ).

2. Immunization of Animals

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 initial immunization, a booster immunization with 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.

3. Selection of BAL b/C mice that produce anti-hPD-1 antibodies

To select for BAL b/C mice producing anti-hPD-1 antibodies, immunized mouse sera 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. After washing 3 times with PBS, put the sample into the flow cytometer (CytoFLEXA00-1-1102) of Beckman Coulter Company to detect whether it can bind to the CHO cells transfected with hPD1, and screen out the cells that produce anti-hPD-1 antibody. BAL b/c mice were then subjected to cell fusion.

4. Generation of hybridomas producing mouse monoclonal antibodies against hPD-1

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 suspension of splenocytes from immunized mice was fused with one-fifth the number of non-immunoglobulin-secreting mouse myeloma cells (SP2/0, ATCCCRL1581) using PEG 1500 (Roche 10783641001) . 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 . Then cultivate about one week in HAT selective medium, and described medium contains 1X penicillin double antibody (Gibco 15140122) in 1640 medium, 1×HAT (SigmaCRLP-7185) and has 20% fetal calf serum ( Royacel RY-F11-01). After 1 week, replace HAT with HT medium (1640 medium containing 1X penicillin-streptomycin double antibody (gibco 15140122), 1X HT (gibco 11067030) and 20% fetal bovine serum (RoyacelRY-F11-01)). The 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. For 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 PD-76-C2 was selected for further analysis.

Example 2 Characterization of the affinity of anti-PD-1 mouse monoclonal antibody

According to conventional methods, 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-1-76-C2 by flow cytometry (FCM) characterization.

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 μg/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.

The results are shown in Table 1. The data show that the anti-PD-1 mouse monoclonal antibody PD-1-76-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 .

Example 3 Binding of Anti-PD-1 Antibodies to Activated PBMCs

Under the stimulation of PHA (Sigma), fresh human peripheral blood mononuclear cells (PBMC) can activate and proliferate lymphocytes, and express PD1 at the highest abundance on the third day, which can be used for natural expression of PD-1 antibodies and activated lymphocytes PD1 binding experiments.

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 μg/ml to stimulate lymphocyte proliferation. After standing in a 5% _CO2 incubator at 37°C for 3 days, take out the cell suspension, centrifuge to remove the supernatant, resuspend in buffer (PBS containing 3% BSA), add 2× ¹⁰ cells/well to 96-well U Add anti-PD1 antibody starting from 30μg/ml, 3-fold serial dilution, a total of 10 concentration gradients, incubate at 4°C for 30 minutes, then centrifuge at 300g for 5 minutes, wash the cells with buffer once, add PE-labeled goat Anti-human IgG fluorescent antibody (Biolegend), incubated at 4°C for 30 minutes; cells were washed by centrifugation once, resuspended in PBS, and analyzed by CytoFlex flow cytometry to detect the amount of antibody bound to PBMCs.

The results are shown in Table 1. The anti-PD1 antibody can bind to activated lymphocytes with high affinity.

Table 1

Example 4 Anti-PD-1 mouse monoclonal antibody binding specificity

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. Using the standard ELISA method, immobilize PD-1, CD28, CTLA-4, ICOS (ACRO) at a concentration of 1 μg/ml on the ELISA plate, add anti-human PD-1 mouse monoclonal antibody at a concentration of 10 μg/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-1-76-C2 can specifically bind to PD-1, but not to other family members of CD28.

Table 2

Example 5 Determination of the affinity of anti-human PD-1 mouse monoclonal antibody by biolayer interferometry (BLI)

ForteBio (Octet Qke) affinity determination: by loading PD-1-his (ACRO) recombinant protein with a concentration of 5 μg/ml on the HISIK biosensor for 120 seconds, and then loading the loaded sensor in standard buffer solution (PBST, PBS+ After equilibrating in 0.02% Tuween20) for 120 seconds, the sensor was transferred to the anti-PD-1 mouse monoclonal antibody dilution and stayed for 180 seconds to measure the association rate, and then transferred to the standard buffer solution for 20 minutes to measure the dissociation rate. Finally, the kinetic model is used for analysis.

The results of data processing are shown in Table 3.

table 3

Antibody to be tested kon(1/Ms) kdis(1/s) KD(M) Opdivo (ABA0333) 1.38E+06 3.63E-06 2.62E-12 PD-1-76-C2 7.71E+05 <1.0E-07 <1.0E-12

Example 6 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 μl/well of the cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, and removed supernatant.

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 μg/ml to the cell well, incubate at 4°C for 30 minutes, then add 30 μg/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 μg/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 μg/ml was added, and incubated at 4°C for 30 minutes.

Centrifuge at 300g for 5 minutes, wash the cells once with buffer, add PE-labeled goat anti-human IgG fluorescent antibody (Biolegend), and incubate at 4°C for 30 minutes. After the cells were centrifuged and washed once, the cells were resuspended in PBS, and then analyzed by CytoFlex flow cytometry to detect the amount of ligand protein bound to the cells, and the IC ₅₀ value of PD-1 antibody binding blocking was calculated.

The results are shown in Table 4. Anti-PD-1 murine monoclonal antibody: PD-1-76-C2 can effectively block the combination of PD-L1/PD-L2 and CHO-PD1 in both modes.

Table 4

Example 7 Effect of Anti-PD-1 Antibody on Cytokine Release of PBMC Cells Stimulated by SEB

In this example, overnight cultured peripheral blood mononuclear cells (PBMCs) were stimulated by the addition of the superantigen Staphylococcus aureus enterotoxin B (SEB), in the presence or absence of anti-PD-1 antibody, and the secretion of cytokines was detected. influences.

Fresh peripheral mononuclear cells (PBMCs) were resuspended in X-VIVO 15 medium (LONZA) containing 10% FBS, added to T25 culture flasks, cultured overnight at 37°C, 5% CO ₂ , and taken the next day Suspend cells, after centrifugation, resuspend in fresh X-VIVO (containing 10% FBS) medium, and add SEB superantigen (Toxin technology) at a final concentration of 200ng/ml, and then add ¹ ×105 cells per well to 96 In the well plate, different concentrations of anti-PD-1 antibodies were added at the same time, and isotype control antibodies (mIgG1 isotype control antibody (Biolegend); hIgG4 isotype control antibody (Biolegend)) were additionally set, and no antibody control wells were set. After 3 days, samples were taken from the sample wells, and IL-2/IFN-γ levels were measured using the IL2/IFN-γ Human Uncoated ELISA Kit (eBioscience) kit.

The results of different concentrations of PD-1-76-C2 on the secretion of IL-2/IFN-γ are shown in Figure 1. Anti-PD-1 antibodies increase the secretion of IL-2/IFN-γ in a concentration-dependent manner. The results showed that in SEB superantigen-stimulated PBMCs, anti-PD-1 antibody: PD-1-76-C2 could further promote T cells to secrete cytokines.

Example 8 Effect of Anti-PD-1 Antibody in Mixed Lymphocyte Reaction

In a mixed lymphocyte reaction (MLR), 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.

Use CD14 MicroBeads, human (Miltenyi) to isolate CD14 ⁺ monocytes from fresh PBMCs. In the presence of GM-CSF/IL-4, after 6 days of induction, add TNF-α, and induce DC maturation after 3 days; On the day of the experiment, 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-γ.

The results of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IL-2 are shown in Figure 2. Different concentrations of PD-1-76-C2 have effects on T cell proliferation and T cell secretion The effect of cytokine IFN-γ is shown in Figure 3. The results of Figure 2 and Figure 3 show that the anti-PD-1 antibody: PD-1-76-C2 can block in an antibody concentration-dependent manner in the MLR experiment The combination of PD1 and ligand inhibits the PD1 signaling pathway, thereby promoting the proliferation of T cells and promoting the secretion of IL-2 and IFN-γ cytokines by T cells.

Example 9 Humanization of anti-PD-1 mouse monoclonal antibody

For the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 obtained above (the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 9-11 in sequence, 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: 12-14, the sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the sequence of the light chain variable region is shown in SEQ ID NO: 16) 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. Subcutaneously immunize female BAL b/C mice with the obtained human PD-1 recombinant protein, fuse the splenocytes of the immunized BAL b/C mice with mouse myeloma cells, and then carry out antigen specificity on the obtained hybridoma Antibody screening. The screened hybridomas that were positive for binding hPD-1 protein antibodies were subcloned at least twice by the limiting dilution method, and the stable subclones were cultured in vitro to generate a small amount of antibodies. After further screening, the PD-1-76-C2 clone was obtained.

SEQ ID NO:9:NYGMN

SEQ ID NO:10:WINTHTGEPTYADDFKG

SEQ ID NO:11:EGEGIGFAY

SEQ ID NO:12:RSSQSIVYSNGKTYLE

SEQ ID NO:13:KVSNRFS

SEQ ID NO:14:FQGSHVPNT

SEQ ID NO: 15:

QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQIKNLKNEDVATYFCTKEGEGIGFAYWGQGTLVTVSA

SEQ ID NO: 16:

DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPNTFGGGTKLEIKR

Referring to SEQ ID NO:15 and SEQ ID NO:16, select the humanized template that best matches its non-CDR region in the Germline database, wherein the template for the heavy chain of the antibody is IGHV1/7, and the template for the light chain of the antibody is IGKV2; Under the principle of not affecting the structural stability of the antibody, not affecting the binding of the antibody to the antigen, not introducing protein modification sites such as glycosylation and phosphorylation, and not introducing sites that have been oxidized and aminated, and enhancing the structural stability, the design The heavy chain humanized sequence is VH1-6, the light chain humanized sequence is designed to be VL1-3, and the paired form of the light and heavy chains is designed to be IGHV1/IGKV2 as a common paired form to obtain a humanized antibody.

According to the amino acid sequences of the light chain and heavy chain of each humanized antibody, the gene was synthesized, after double digestion with Hind III (NEB) and EcoRI (NEB), the gene fragment was passed through Hind III (NEB) using T4 DNA ligase (TAKARA 2011A) The /EcoRI (NEB) restriction site was inserted into the pDNA3.4A expression vector (Invitrogen). Transfect HEK293 cells (Life Technologies Cat. NO. 11625019) with the expression vector and transfection reagent PEI (Poly science, Inc. Cat. NO. 23966) at a ratio of 1:2, and culture in a CO ₂ incubator for 5-7 sky. The expressed antibodies were recovered by centrifugation and purified according to conventional methods to obtain humanized anti-PD-1 antibodies (h11, h21, h31, h41, h51, h61, h12, h42, h13, h23, h33, h43, h53, h63); wherein, the amino acid sequences of the four anti-PD-1 humanized antibodies (h31, h61, h42, h43) are shown in Table 5.

Table 5 Amino acid sequences of 4 anti-PD-1 humanized antibodies (h31, h61, h42, h43)

SEQ ID NO: 3:

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTKEGEGIGFAYWGQGTTVTVSS

SEQ ID NO: 4:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQKFQGRVTMTLDTSISTAYMELSRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS

SEQ ID NO: 5:

QIQLVQSGAEVKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYADDFKGRFTFTLDTSISTAYLEISRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS

SEQ ID NO: 6:

DVVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR

SEQ ID NO: 7:

DIVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR

SEQ ID NO: 8:

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGKTYLEWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR

Example 10 Determination of the binding ability of anti-PD-1 humanized antibody to CHO-hPD1 cells and CHO-cyno cells

1. Experimental method

A CHO (Chinese hamster ovary cell) cell line (CHO-hPD1) expressing recombinant human PD-1 on the cell surface using recombinant technology, and a CHO cell line expressing monkey PD1 (CHO-cynoPD1), these two cell lines will be used in the flow Binding characterization of anti-PD-1 humanized candidate mAbs was determined by formula cytometry (FCM). The specific method is:

In order to evaluate the binding of anti-PD-1 humanized antibody to CHO-hPD1 cells, add 2×10 ⁵ CHO-hPD1 cells in a 96-well plate and dilute by concentration gradient (initial concentration is 30μg/ml, three-fold serial dilution) Humanized antibody, incubate at 4°C for 30 minutes, wash cells once with buffer (PBS containing 3% BSA), add PE-labeled anti-human IgG (Fc) Ab (Biolegend) fluorescent secondary antibody, incubate at 4°C for 30 minutes After the cells were washed once with buffer solution, they were resuspended in PBS, and then 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. Binding of anti-PD-1 humanized antibodies to CHO-cyno cells was evaluated in the same way.

2. Experimental results

The results of the determination of the binding ability of the anti-PD-1 humanized antibody to CHO-hPD1 cells and CHO-cyno cells are shown in Table 6, and the results show that the anti-PD-1 humanized antibody of the present invention can bind to CHO-hPD1 with high affinity cells and CHO-cyno cells.

Example 11 Determination of binding ability of anti-PD-1 humanized antibody to activated PBMC

1. Experimental method

Under the stimulation of PHA (Sigma), fresh human peripheral blood mononuclear cells (PBMC) can activate and proliferate lymphocytes, and express PD-1 at the highest abundance on the third day, which can be used for anti-PD-1 humanization Determination of the binding ability of antibodies to activated lymphocytes naturally expressing PD-1. The specific method is:

PBMCs were obtained from fresh human peripheral blood by gradient centrifugation of lymphatic separation fluid, 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 μg/ml to stimulate lymphocyte proliferation , 37°C, 5% CO ₂ incubator for 3 days, take out the cell suspension, centrifuge to remove the supernatant, resuspend in buffer (PBS containing 3% BSA), add to 96-well U-shaped plate according to 2E5/well , then add anti-PD-1 humanized antibodies with different concentration gradients, incubate at 4°C for 30 minutes, then centrifuge at 300g for 5 minutes, wash the cells once with buffer, add PE-labeled goat anti-human IgG fluorescent antibody (Biolegend), and incubate at 4°C 30 minutes; wash the cells by centrifugation once, resuspend the cells in PBS, and then perform CytoFlex flow cytometry analysis to detect the amount of antibody bound to PBMCs.

2. Experimental results

The results of the determination of the binding ability of the anti-PD-1 humanized antibody to activated PBMC are shown in Table 6, and the results show that the anti-PD-1 humanized antibody of the present invention can bind to activated PBMC with high affinity.

Compared with the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 (Table 1) in Examples 2 and 3, the anti-PD-1 humanized antibody of the present invention is compatible with CHO-hPD1 cells, CHO-cyno cells and The binding ability of activated PBMC is comparable to it.

Table 6 The results of the determination of the binding ability of anti-PD-1 humanized antibodies to CHO-hPD1 cells, CHO-cyno cells and activated PBMC

Example 12 Binding specificity of anti-PD-1 humanized antibody

1. Experimental method

The anti-PD-1 humanized antibody 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. Using the standard ELISA method, fix PD-1, CD28, CTLA-4, and ICOS at a concentration of 1 μg/ml on the ELISA plate, and add anti-PD-1 humanized antibodies (h31, h61, h42, h43), anti-human IgG (Fab) conjugated with peroxidase (HRP) was used as the secondary antibody. TMB color development, after termination, read the OD450 value with a microplate reader.

2. Experimental results

The binding specificity results of anti-PD-1 humanized antibodies are shown in Table 7, the results show that anti-PD-1 humanized antibodies can specifically bind to PD-1, but not to other proteins of CD28 family members; The invented anti-PD-1 humanized antibody has high binding specificity to PD-1.

Table 7 Binding specificity results of anti-PD-1 humanized antibodies

Example 13 Affinity Determination of Anti-PD-1 Humanized Antibody

1. Experimental method

检测仪器对抗体亲和力进行测定，具体方法为：According to biofilm interferometry (BLI), using Fortebio

The detection instrument measures the affinity of the antibody, and the specific method is as follows:

Load the PD-1-his recombinant protein with a concentration of 5 μg/ml on the HISIK biosensor for 120 seconds, then equilibrate the loaded sensor in standard buffer (PBST, PBS+0.02%Tuween20) for 120 seconds, and then the sensor Transfer to the anti-PD-1 humanized antibody (h31, h61, h42, h43) dilution and stay for 180 seconds to measure the association rate, then transfer to the standard buffer solution for 20 minutes to measure the dissociation rate. Finally, the kinetic model is used for analysis and data processing. Opdivo (ABA0333) was used as a positive control.

2. Experimental results

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.

Compared with the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 (Table 3) in Example 5, 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.

Table 8 Affinity determination results of anti-PD-1 humanized antibodies

Antibody to be tested kon(1/Ms) kdis(1/s) KD(M) h31 8.53E+05 <1.0E-07 <1.0E-12 h61 8.92E+05 <1.0E-07 <1.0E-12 h42 8.85E+05 <1.0E-07 <1.0E-12 h43 8.20E+05 <1.0E-07 <1.0E-12

Example 14 Determination of the ability of anti-PD-1 humanized antibodies to block the binding of ligand PD-L1/PD-L2 to CHO-hPD1

1. Experimental method

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 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 μl/well of the cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, and removed supernatant. Add PD-L1-mFc/PD-L2-mFc at a concentration of 0.2 μg/ml to the cell wells, incubate at 4°C for 30 minutes, and then add anti-PD-1 humanized antibodies (h31, h61, h42, h43), incubate at 4°C for 30 minutes.

Centrifuge at 300g for 5 minutes, wash the cells once with buffer, add PE-labeled goat anti-mouse IgG fluorescent antibody (Biolegend), and incubate at 4°C for 30 minutes. After the cells were centrifuged and washed once, the cells were resuspended in PBS, and then analyzed by CytoFlex flow cytometry to detect the amount of ligand protein bound to the cells, and the IC ₅₀ value of anti-PD-1 humanized antibody binding blocking was calculated.

2. Experimental results

The results of the determination of the binding ability of the anti-PD-1 humanized antibody to block the ligand PD-L1/PD-L2 and CHO-hPD1 are shown in Table 9. The results show that the anti-PD-1 humanized antibody of the present invention can effectively block Ligands PD-L1/PD-L2 bind to CHO-hPD1.

Table 9 The results of the determination of the ability of anti-PD-1 humanized antibodies to block the binding of ligand PD-L1/PD-L2 to CHO-hPD1

Example 15 Effect of anti-PD-1 humanized antibody in mixed lymphocyte reaction

1. Experimental method

In a mixed lymphocyte reaction (MLR), the presence or absence of anti-PD-1 humanized antibodies can demonstrate T cell proliferation and the level of T cell secretion of cytokines in the case of PD-1 signaling blockade. The specific method is:

Use 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-α, and induce DC maturation after 3 days; On the day of the experiment, 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 to the mixed cells Anti-PD-1 humanized antibodies (h31, h61, h42, h43) with different concentration gradients were added, and isotype control antibodies were also set, and no antibody control wells were set. 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-γ.

2. Experimental results

The effect of anti-PD-1 humanized antibody in mixed lymphocyte reaction is shown in Table 10. The results show that anti-PD-1 humanized antibody can block the binding of PD-1 and ligand in MLR, inhibit PD-1 signaling pathway, thereby promoting the proliferation of T cells and promoting the secretion of IL-2 and IFN-γ cytokines by T cells.

Table 10 Effect of anti-PD-1 humanized antibody in mixed lymphocyte reaction

Example 16 In vivo anti-tumor efficacy evaluation of anti-PD-1 humanized antibody on mouse colon cancer cells 1. Experimental method

Experimental purpose: To determine the in vivo anti-tumor activity of anti-PD-1 humanized antibodies (h31, h61, h43) on mouse colon cancer cells (MC38 cells), and set up an isotype control group (Isotype group).

Experimental materials: 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), fetal bovine serum (Gibco), glutamine (Gibco), penicillin-streptomycin ( Gibco, 15140122), DMEM high glucose medium (Gibco, 11965084).

Equipment: electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Minate Industrial Co., Ltd., MNT-150T), microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), medical centrifuge ( Hunan Xiangyi Laboratory Development Co., Ltd., L530R), digital display constant temperature water bath (Price Machinery Co., Ltd., HH-S), carbon dioxide incubator (Matsushita Health and Medical Instrument Co., Ltd., Japan, MCO-18AC), double vertical type Ultra-clean bench (Wuxi Easy Purification Equipment Co., Ltd., SW-CJ-VS2).

Experimental steps:

Cell culture: 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 MC38 cells were inoculated subcutaneously with an inoculation volume of 0.1 mL/mouse, that is, 3×10 ⁵ /mouse.

Dosing: The day of inoculation was recorded as day 0 (D0). On the 7th day, the mice were randomly divided into 4 groups according to the tumor volume, with 8 mice in each group, and the administration was started (the dosage, method and frequency of the MC38 tumor model were as follows: shown in Table 11).

Recording: On D7, 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 . When each mouse reached the end point of the experiment (the tumor volume exceeded 2000 mm ³ to reach the benevolent end point), the mice were sacrificed by cervical dislocation, and the survival curve was recorded.

Table 11 Dosage, mode and frequency of administration in MC38 tumor model

2. Experimental results

The results of the effect of anti-PD-1 humanized antibodies on tumor volume are shown in Table 12 and Figure 4. It can be seen that compared with the Isotype group, anti-PD-1 humanized antibodies (h31, h61, h43) have a greater effect on MC38 The tumor growth of the tumor model has a significant tumor-inhibiting effect (TGI is 100.85%, 94.77%, 99.05% in turn; tumor-eliminated mice are 6, 5, and 7 respectively).

The effect of anti-PD-1 humanized antibodies on the survival of mice is shown in Figure 5. It can be seen that compared with the Isotype group, anti-PD-1 humanized antibodies (h31, h61, h43) can significantly prolong mouse survival.

Table 12 Effect of anti-PD-1 humanized antibody on tumor volume (mm ³ )

The above results show that: the anti-PD-1 humanized antibodies (h31, h61, h43) provided by the present invention can significantly inhibit the growth of MC38 cells, effectively prolong the survival period of mice, and have a significant effect on the treatment of colon cancer in mice.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangdong Feipeng Pharmaceutical Co., Ltd.

<120> An anti-PD-1 humanized antibody and its application

<130> 2021

<160> 16

<170> PatentIn version 3.5

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Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Gln Gly Phe

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Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

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Claims (10)

1. An anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein 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, the light chain CDR region consists of LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO 9-11 in sequence, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO 12-14 in sequence, and the antibody is characterized in that the amino acid sequence of the heavy chain variable region of the antibody is shown as any one of SEQ ID NO 3-5.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the variable region in the light chain of the antibody has an amino acid sequence as set forth in any one of SEQ ID NOS 6 to 8.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 3 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 6, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 4 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 6, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 5 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 7, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 5 and the amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID NO. 8.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a heavy chain constant region and a light chain constant region, wherein 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 kappa chain or a lambda chain.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody or a multispecific antibody; the antigen binding fragment is F (ab') ₂ Any one or more of Fab, scFv, fv and single domain antibody.

6. A nucleic acid encoding the anti-PD-1 humanized antibody or an antigen-binding fragment thereof;

preferably, the nucleic acid comprises: a first nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof, and/or, a second nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof.

7. A vector carrying the nucleic acid of claim 6.

8. A cell carrying the nucleic acid of claim 6, comprising the vector of claim 7, or capable of expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid of claim 6, the vector of claim 7, or the cell of claim 8.

10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid of claim 6, the vector of claim 7, the cell of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for treating a PD-1 mediated disease or disorder.

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