TW202320852A - Anti-PD-1 antibody and application thereof to preparation of medicine for treating cervical cancer patients - Google Patents

Abstract

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Description

Anti-PD-1 antibodies and their use in the preparation of medicaments for the treatment of cervical cancer patients

The present invention relates to the field of antibody drugs, in particular to anti-PD-1 antibodies related to cervical cancer.

Cervical Cancer (CC) is the second most common malignant tumor that seriously affects women's health. New cases of cervical cancer in my country every year account for about 1/4 of the world's total. Growing evidence from preclinical and clinical outcomes suggests that targeting immune checkpoints is emerging as the most promising approach to treating cancer patients. Programmed cell death molecule 1 is one of the immune checkpoint proteins, which plays a major role in limiting the activity of T cells. The T cells provide the main immune resistance mechanism. Through this restriction, tumor cells can escape immune surveillance. . The interaction of PD-1 expressed on activated T cells and PD-L1 expressed on tumor cells negatively regulates immune responses and attenuates anti-tumor immunity. Expression of PD-L1 on tumors is associated with reduced survival in esophageal, pancreatic and other cancer types, highlighting this pathway as a new and promising target for cancer immunotherapy. Pharmaceutical companies have developed a variety of drugs targeting the PD-1 pathway, such as Bristol-Myers Squibb (BMS), Merck & Co., Roche and GlaxoSmithKline (GSK). Data from clinical trials show early evidence of durable clinical activity and a favorable safety profile in patients across a variety of tumor types. Nivolumab is a PD-1 drug developed by BMS that is being put into the center stage of the next generation field. Now in six late-stage studies, the treatment prompted tumor shrinkage in three of the five cancer groups studied, including 18 percent of 72 lung cancer patients and nearly a third of 98 melanoma patients. One and 27% of 33 patients with kidney cancer. Lambrolizumab, developed by Merck, is a fully human monoclonal IgG4 antibody that targets PD-1 and captured the FDA's new breakthrough designation after impressive IB data against skin cancer. Results from the phase IB study showed objective anti-tumor responses in 51% of 85 cancer patients, with complete responses in 9% of patients. Roche's experimental MPDL3280A demonstrated that it shrank tumors in 29 (21%) of 140 patients with advanced cancer who had tumors of various sizes.

The prognosis of advanced cervical cancer is poor. The first-line standard treatment is platinum-based combination chemotherapy. The overall survival of cisplatin monotherapy is about 6 to 9 months; the overall survival of cisplatin-based combination chemotherapy is about 9 to 18 months. months, and the progression-free survival is about 4 months. For patients with recurrent or metastatic cervical cancer who fail first-line standard chemotherapy, more treatment options need to be provided. The U.S. FDA approved pembrolizumab for the treatment of patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors whose disease has progressed after treatment or for whom there is no satisfactory alternative therapy, and for the treatment of PD-L1-positive (CPS ≥1) Patients with recurrent or metastatic cervical cancer whose disease has progressed during or after chemotherapy. The 2018 NCCN Guidelines recommend pembrolizumab for the second-line treatment of PD-L1-positive (CPS ≥ 1) or MSI-H/dMMR recurrent or metastatic cervical cancer (category 2A evidence). Most patients with recurrent and metastatic cervical cancer have undergone multiple lines of platinum-containing chemotherapy. After platinum resistance develops, the options available are very limited and the treatment effect is poor. Therefore, there is still a great demand for treatment of patients at this stage. Continue to Supplement and replace the treatment plan for patients at this stage.

The present invention aims at the problem of poor clinical treatment effect of cervical cancer, and provides the use of anti-PD-1 antibodies or antigen-binding fragments thereof in the treatment of cervical cancer, especially in the treatment of recurrence or metastasis after first-line or above platinum-containing standard chemotherapy. Use in PD-L1 expression-positive cervical cancer.

A first aspect of the present invention provides the use of an anti-PD-1 antibody or an antigen-binding fragment thereof in preparing a medicament for treating cervical cancer patients, wherein the antibody or an antigen-binding fragment thereof includes: a) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 1, the CDR2 shown in SEQ ID NO: 3, and the CDR3 shown in SEQ ID NO: 5; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 7, CDR2 shown in SEQ ID NO: 9 and CDR3 shown in SEQ ID NO: 11; b) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 13, the CDR2 shown in SEQ ID NO: 15, and the CDR3 shown in SEQ ID NO: 5; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 7, CDR2 shown in SEQ ID NO: 17 and CDR3 shown in SEQ ID NO: 11; c) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 1, the CDR2 shown in SEQ ID NO: 15, and the CDR3 shown in SEQ ID NO: 5; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 7, CDR2 shown in SEQ ID NO: 17 and CDR3 shown in SEQ ID NO: 19; d) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 21, the CDR2 shown in SEQ ID NO: 23, and the CDR3 shown in SEQ ID NO: 25; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 27, CDR2 shown in SEQ ID NO: 29 and CDR3 shown in SEQ ID NO: 31; or e) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 33, the CDR2 shown in SEQ ID NO: 35, and the CDR3 shown in SEQ ID NO: 37; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 39, CDR2 shown in SEQ ID NO: 41 and CDR3 shown in SEQ ID NO: 43.

Further, the antibody or antigen-binding fragment thereof includes: a) the heavy chain variable region, which includes SEQ ID NO: 45; and the light chain variable region, which includes SEQ ID NO: 47; b) heavy chain variable region, which includes SEQ ID NO: 49; and light chain variable region, which includes SEQ ID NO: 51; c) heavy chain variable region, which includes SEQ ID NO: 53; and light chain variable region, which includes SEQ ID NO: 55; d) a heavy chain variable region comprising SEQ ID NO: 57; and a light chain variable region comprising SEQ ID NO: 59; or e) A heavy chain variable region, which includes SEQ ID NO: 61; and a light chain variable region, which includes SEQ ID NO: 63.

Further, the antibody or its antigen-binding fragment specifically binds to human PD-1 with a Kd value not exceeding 10 ^-8 M, and the Kd value is determined by a plasmon resonance binding method.

Further, the antibody or antigen-binding fragment thereof binds to monkey PD-1 with an EC ₅₀ of no more than 100 nM, or no more than 10 nM, and/or does not bind to mouse PD-1.

Further, the antibody or antigen-binding fragment thereof blocks the binding of human or monkey PD-1 to its ligand with an _IC50 of no more than 100 nM.

Further, the antibody or antigen-binding fragment thereof does not bind to CD28 or CTLA4.

Further, the antibody or antigen-binding fragment thereof does not mediate ADCC or CDC or neither.

Further, the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody.

Further, in the antibody or antigen-binding fragment thereof, the fully human monoclonal antibody is produced from transgenic rats.

Further, the antibody or antigen-binding fragment thereof blocks the binding of human PD-1 to its ligand, and thus provides at least one of the following activities: a) inducing the production of IL-2 in CD4 ⁺ T cells; b) Induces IFNγ production in CD4 ⁺ T cells; c) induces proliferation of CD4 ⁺ T cells; and d) reverses T reg suppressive function.

Further, the antibody or antigen-binding fragment thereof is a diabody, scFv, scFv dimer, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab' or F(ab' )2.

Further, in the antibody or antigen-binding fragment thereof, the diabody is a BsFv or ds diabody.

Further, the antibody or antigen-binding fragment thereof further includes an immunoglobulin constant region.

Further, the antibody or antigen-binding fragment thereof further includes a conjugate.

Further, the cervical cancer is a tumor related to immunosuppression caused by PD-1.

Further, the cervical cancer is recurrent or metastatic cervical cancer.

Further, the cervical cancer is recurrence or metastasis after first-line or above platinum-containing standard chemotherapy and/or PD-L1 expression-positive cervical cancer.

Further, the cervical cancer is recurrence or metastasis after first-line or above platinum-containing standard chemotherapy and/or PD-L1 expression-positive cervical cancer.

The preferred embodiments of the present invention are described below in conjunction with the drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention. Example 1 : Generation of antibody hybridomas

1.1 Generation of immunogen: Synthesize DNA encoding the ECD of PD-1 and PD-L1 or both full-length DNA and insert into the expression vector pcDNA3.3. Plastid DNA was prepared in large quantities and the inserted DNA sequence was verified by sequencing. The fusion protein of PD-1ECD and PD-L1ECD includes a variety of tags, including human Fc, mouse Fc and His tags. The fusion protein is prepared by transfecting the human PD-1ECD gene into CHO-S or HEK293 cells. After 5 days, the supernatants harvested from the transiently transfected cell cultures were used for protein purification. The fusion protein was purified and quantified for immunization and screening.

1.2 Establish stable cell lines. In order to obtain tools for antibody screening and validation, PD-1 and PD-L1 transfected cell lines were established. Briefly, CHO-K1, 293F or Ba/F3 cells were transfected with pCND3.3 expression vector containing full-length PD-1 or PD-L1 using Lipofectamine 2000 transfection kit according to the manufacturer's instructions. 48-72 hours after transfection, the transfected cells were cultured in medium containing Blasticidin or G418 for selection. After a period of time, cells with stably incorporated PD-1 or PD-L1 genes in their genomic DNA are selected. At the same time, verify whether the cells have expression of the target genes PD-1 and PD-L1. Once expression is verified, individual clones of interest are selected by limiting dilution and scaled up to large volumes. The established individual cell lines were then maintained in media containing low doses of the antibiotic blasticidin or G418.

1.3 Establishment of antibody hybridoma.

1.3.1 Immunization and cell fusion: 8-10 week old OMT-rats (obtained from Open Monoclonal Technology, Inc., Palo Alto, US) were used for primary challenge on the footpad with 10 μg of human PD-1 ECD protein in TiterMax. Immunization was repeated every 3 days with PD-1ECD configured with aluminum. Rats were bled every 2 weeks to collect serum and determine antibody titers by ELISA or FACS testing. When the antibody titer reaches a high enough level, the rats are given a final challenge without adjuvant (adding 100 μl 1XPBS instead), and cell fusion is performed as follows: Lymph nodes isolated from the immunized OMT-rats Cell fusion of B lymphocytes with myeloma cells (1:1 ratio). Wash and resuspend the cell mixture with 5-10 ml ECF solution. Add ECF solution to adjust the concentration to ^2x10 cells/ml. Immediately after cell electrofusion, the cell suspension in the fusion chamber was transferred into a sterile tube containing a larger volume of culture medium. After incubation at 37°C for over 24 hours, the cell suspension was mixed and pipetted into a 96-well plate ( ^0.5x10 cells/plate). Culture cells at 37°C, 5% _CO2 . When the clones are large enough, 100 μl of the supernatant is transferred from the 96-well plate for antibody screening testing.

1.3.2 First-round and confirmatory screening of hybridoma supernatants: Use ELISA testing as a first-round screening method to test the binding of hybridoma supernatants to PD-1 protein. Briefly, plates were coated with 1 μg/ml soluble protein of human PD-1 extracellular domain overnight at 4°C. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then washed and subsequently incubated with secondary antibodies goat anti-rat IgGl HRP (Bethyl) and goat anti-rat IgG2b HRP (Bethyl) for 45 minutes. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. Use a microplate reader (Molecular Device) to read the absorbance value at 450 nm. To confirm the natural binding of PD-1 antibodies to the conformational PD-1 molecules expressed on the cell membrane, FACS analysis was performed on PD-1-transfected CHO-S cell lines. Transfer PD-1-expressing CHO-S cells to a 96-well U-shaped bottom plate (BD) at a concentration of ^1x10 cells/ml. The hybridoma supernatant was then transferred to the plate and incubated at 4°C for 1 hour. After washing with 1XPBS/1% BSA, goat anti-rat FITC (Jackson Immunoresearch Lab) secondary antibody was added and incubated with the cells for 1 hour at 4°C in the dark. The cells were then washed and resuspended in 1XPBS/1% BSA or fixed in 4% formalin and analyzed by flow cytometry (BD). The same method was used to bind the antibody to the maternal CHO-S cell line.

1.3.3 Hybridoma subpopulation lines: Once specific binding and blocking have been verified through the first round and confirmation screening, the positive hybridoma cell lines can be used for subpopulation. Briefly, for each hybridoma cell line, cells were counted and diluted in selection medium to 5 cells/well, 1 cell/well, and 0.5 cells/well. Plate 200 μl/well into 96-well plates, one plate at 5 cells/well, one plate at 1 cell/well and four plates at 0.5 cells/well. Place all plates at 37°C, 5% _CO2 . Incubate until all cell lines can be checked by ELISA test. Example 2 : Antibody hybridoma cell sequencing and fully human antibody characterization

2.1 Antibody hybridoma cell sequencing: Use Trizol reagent to isolate RNA from single hybridoma cells. The VH and VL of the PD-1 antibody were amplified using the following protocol: Briefly, RNA was first reverse transcribed into cDNA using reverse transcriptase reaction system (20 μl) as described in this application:

Take 10 μl of the PCR reaction product for ligation reaction with pMD18-T vector. Use 10 μl of ligation product to transform Top10 competent cells and transfer the mixture to 2-YT+Cab plates preheated according to standard protocols and incubate overnight. Positive selections were checked by PCR using M13-48 and M13-47 primers, followed by sequencing.

2.2 Construction of fully human antibody molecules: Amplify the VH and VL of the PD-1 antibody as described above. The PCR reaction product was purified by PCR clean-up kit and the VL and pCI vectors were digested with restriction enzymes Pme I and BssH II at 37°C for 2 hours. The reaction product was electrophoresed in a 1% agarose gel and gel extracted according to the manufacturer's instructions. Ligate the digested VL and pCI vectors using the following steps:

Incubate the mixture at 16°C for 30 minutes. Use 10 μl of the reaction product for transformation and growth of selected strains. Use the confirmed selection strain to extract plastid pCI-VL DNA. The pCI-VL vector and VH fragment were then digested with Xbal and Sal I and the purified digested VH and vector were ligated using T4 DNA ligase at 16°C for 30 minutes. Once the sequences of the inserted VL and VH were verified by sequencing, an expression vector containing the entire IgG of the fully human PD-1 antibody was used for transient transfection and establishment of stable cell lines. Example 3 : Characterization of fully human antibodies

3.1 Full kinetic binding affinity determined by surface plasmon resonance (SPR): The affinity and binding kinetics of the antibody to PD-1 were characterized by the SPR method using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on the GLM sensor chip (Bio-Rad) via amine coupling. Purified antibodies are flowed over the sensor wafer and captured by Protein A. The wafer was rotated 90° and washed with running buffer (1XPBS/0.01% Tween20, Bio-Rad) until the baseline was stable. Five concentrations of human PD-1 and electrophoresis buffer were flowed through the antibody flow unit at a flow rate of 100 μL/min, first for 240 s in the binding phase, and then 600 s in the dissociation phase. The wafers were regenerated with _H3PO4 pH _1.7 after each run. Binding and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software.

3.2 The binding affinity of PD-1 antibodies to cell surface PD-1 molecules measured by flow cytometry (FACS): The binding affinity of antibodies to cell surface PD-1 was tested by FACS analysis. Transfer PD-1-expressing CHO-S cells to a 96-well U-shaped bottom plate (BD) at a concentration of ^5x10 cells/ml. Antibodies to be tested were serially diluted 1:2 in wash buffer (1XPBS/1%BSA) and incubated at 4°C for 1 hour. Secondary antibody goat anti-human IgG Fc FITC (3.0 moles of FITC per mole of IgG, (Jackson Immunoresearch Lab)) was added and incubated for 1 hour at 4°C in the dark. Cells were then washed once and resuspended in 1XPBS/1% BSA and analyzed by flow cytometry (BD). Fluorescence intensity was converted to molecules bound per cell based on the beads being quantified (QuantumTM MESF Kit (Bangs Laboratories, Inc.). _KD was calculated using Graphpad Prism5.

3.3 Effect of human PD-1 antibody on T cell proliferation. Test the effect of PD-1 antibodies on T lymphocyte proliferation using alloreactivity. Primary dendritic cell (DC)-stimulated MLR was performed in 96-well U-bottomed tissue culture plates in 200 μl RPMI 1640 containing 10% FCS and antibiotics. DCs were mixed with ^1X10 of allogeneic total CD4 ⁺ T cells at a DC:T cell ratio of 1:10 and 1:100. Cultures were performed in the presence or absence of neutralizing mAb: human PD-1 antibody and reference antibodies A and B were used at a concentration of 10 μg/ml. The test was incubated for 5 days, with 1 uCi/well of [ ^3H ]thymidine added during the last 16 hours. [ ³ H]thymidine incorporation was determined by scintillation counting, and the proliferative response was expressed as average [ ³ H]thymidine incorporation (counts per minute) in triplicate wells. Counts for DC only are typically <1000cpm. Results shown are representative examples of a minimum of 5 trials.

Human dendritic cells (DC) and CD4 ⁺ T, CD8 ⁺ T and total cells used in the above allogeneic MLR were generated from PBMC by using the human monocyte enrichment cocktail kit according to Purification of human monocytes from PBMC by negative selection according to the manufacturer's (StemCell Meylan) instructions. Briefly, PBMCs were isolated from healthy donor blood using Ficoll-Paque gradients. Cells were washed twice with PBS, subsequently resuspended in isolation buffer at ^1X10 cells/ml and incubated with monocyte concentrated Ab mixture for 30 minutes at 4°C. Unlabeled monocytes passing through the MACS column were collected. To generate iDCs, monocytes were cultured with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml) in RPMI 1640 medium containing 10% FCS and antibiotics. The cell concentration is 2X10 ⁶ cells/ml. Half of the medium was replaced daily with medium containing GM-CSF and IL-4. iDCs were stimulated with LPS (026:B6; Sigma-Aldrich, St. Louis, MO; 1 μg/ml) for an additional 24 h on day 5 to generate mature DCs. CD4 ⁺ T, CD8 ⁺ T and total T cells were purified by negative selection ^by incubating PBMC with a concentrated mixture of human CD4 + T, CD8 ⁺ T and total T cells and magnetic colloids according to the manufacturer's instructions (Stemsep).

Human CD4 ⁺ T cells were stimulated with allogeneic DC in the presence or absence of PD-1 antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb. CD4 ⁺ T cell proliferation was assessed by [ ^3H ]thymidine incorporation. 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15 hAb and 1.153.7hAb enhanced concentration-dependent T cell proliferation.

3.5.2 Effect of human PD-1 antibody on cytokine IFNγ secretion in vitro: In order to evaluate the blocking effect of human PD-1 antibody on cytokine IFNγ production, we performed IFNγ production in allogeneic-MLR. Experiment. Briefly, human CD4 ⁺ T cells were purified from PBMCs by negative selection ^using the CD4 ⁺ T cell enrichment cocktail kit according to the manufacturer's instructions. Immature DCs were generated from monocytes cultured in GM-CSF and IL-4 for 5 days and stimulated with LPS at 1 μg/ml overnight to differentiate into mature DCs. CD4 ⁺ T cells and iDC/mDC were mixed at T:DC ratios of 10:1 and 100:1. Cultures were performed in the presence or absence of human PD-1 antibodies and baseline antibodies. After 5 days, the supernatant from each culture was collected and assayed for the cytokine IFNγ. IFNγ levels in the supernatant were determined by ELISA test. Briefly, Maxisorp plates were coated with anti-human IFNγ mAb diluted in coating buffer (0.75 μg/ml; i.e., diluted 1/1360) at 50 μl/well (i.e., 3.7 μl was added to a full 96-well plate). of antibody to 5 ml of coating buffer) and incubate overnight at 4°C. Add 200 μl/well of blocking buffer for 2 hours to block excess protein binding ability. Prepare a dilution of recombinant IFNγ as a standard solution, using complete medium for two-fold dilution from 8000pg/ml to 125pg/ml, plus the case of complete medium only. Wash the plate, add standard solution and test supernatant (100 μl/well), and incubate for 2-4 hours. Biotinylated anti-IFNγ mAb (1/1333) in blocking buffer was added, followed by additional avidin peroxidase. The reaction was carried out by adding TMB substrate and quenched with 2M HCl. The absorbance value was measured at 450 nm.

Results show stimulation of human CD4 ⁺ T cells with allogeneic DC in the presence or absence of 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb antibodies. IFNγ levels were determined by ELISA. The results showed that the fully human PD-1 antibody increased the secretion of IFNγ in a dose-dependent manner.

3.5.3 Effect of human PD-1 on interleukin 2 (IL-2) production in vitro: CD4 ⁺ T cells and iDC/mDC were mixed at T:DC ratios of 10:1 and 100:1. Cultures were performed in the presence or absence of human PD-1 antibodies and baseline antibodies. After 5 days, the supernatant from each culture was collected and assayed for cytokines. IL-2 levels in the supernatant were determined by ELISA test.

Results show stimulation of human CD4 ⁺ T cells with allogeneic DC in the presence or absence of antibodies of the present application or control antibodies. IL-2 levels were measured using ELISA. The results showed that the fully human PD-1 antibody increased the secretion of IFNγ in a dose-dependent manner. The results show that anti-PD-1 antibodies increase IL-2 secretion in a dose-dependent manner.

3.5.4 Effects of human PD-1 antibodies on cell proliferation and cytokine production through autologous antigen-specific immune responses: In this assay, T cells and DC cells were derived from the same donor. Briefly, CD4 ⁺ T cells were purified from PBMC and cultured in CMV pp65 peptide and low dose IL2 (20 U/ml), while PBMC from the same donor were cultured in GM-CSF and IL-4. DCs are generated from monocytes. After 5 days, CD4 ⁺ T cells treated with CMV pp65 peptide were co-cultured with DCs pulsed with pp65 peptide in the presence or absence of human PD-1 antibody and a reference antibody as a control. .

On day 5, 100 μl of supernatant from each culture was used for determination of the cytokines IFNγ and IL-2. Levels of IFNγ and IL-2 production were measured by ELISA test. Specific T cell proliferation in response to DC pulsed with CMV pp65 peptide was determined by incorporation of [ ^3H ]thymidine.

The results showed that PD-1 antibody enhanced concentration-dependent proliferation of CMV ⁺ -CD4 ⁺ T cells stimulated by autologous DC loaded with CMV pp65 peptide.

3.5.5 The effect of human PD-1 antibodies on the inhibitory function of regulatory T cells (Tregs): Tregs are a subset of T cells, which are key immune regulators and play a key role in maintaining autotolerance. .

CD4 ⁺ CD25 ⁺ regulatory T cells are relevant to tumors, as increased numbers of Tregs are found in patients with a variety of cancers and are associated with poor prognosis. To directly assess the effect of human PD-1 antibodies on immunosuppressive responses, we performed Tregs experiments. CD4 ⁺ CD25 ⁺ and CD4 ⁺ CD25 ⁻ T cells were isolated using specific anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) and positive or negative selection, respectively. Initially, CD4 ⁺ T cells were purified by negative selection by incubating PBMC with a human CD4 ⁺ T cell concentrate mixture and magnetic colloid according to the manufacturer's instructions (Stemsep). CD4 ⁺ T cells were then resuspended in MACS buffer, incubated with CD25 ⁺ beads on ice for 30 min, washed and loaded onto the column. CD4 ⁺ CD25 ^- T cells that do not bind to the column are collected from the effluent solution and washed before use. CD4 ⁺ CD25 ⁺ T cells were subsequently recovered from the column and washed before use. Tregs were cultured with CD4 ⁺ CD25 ⁻ T cells and DC (Treg:Teff ratio 1:1) in the presence or absence of human PD-1 antibody at a concentration of 10 μg/ml. Do not use antibodies or use isotype antibodies as negative controls. The supernatant of the culture was taken on day 5 for ELISA detection of cytokines, and cell proliferation was detected by adding [ ³ H]thymidine at a concentration of 1 uCi/well and further culturing for 18 hours. [ ^3H ]Thymidine incorporation was counted by scintillation. The results showed that the PD-1 antibody abrogated Treg suppressive function and restored responsive T cell proliferation and IFNγ secretion.

3.6 ADCC/CDC assay: To minimize unwanted toxicity to healthy PD-1 ⁺ cells, selected anti-PD-1 fully human antibodies were confirmed to be free of ADCC and CDC functions.

3.6.1ADCC: Use activated T cells expressing high levels of cell surface PD-1 as target cells and pre-incubate with different concentrations of fully human antibodies in a 96-well plate for 30 minutes, followed by addition at an effector/target cell ratio of 50:1 IL-2 activated PBMC (used as a source of natural killer (NK) cells, i.e. effector cells). [Incubate the plate for 6 hours at 37°C in a 5% _CO2 incubator. Target cell lysis was determined by a cytotoxicity assay kit (Roche). Optical density was measured by a Molecular Devices SpectraMax M5e microplate reader. The results showed that the fully human anti-PD-1 antibodies tested did not mediate ADCC.

3.6.2CDC: Mix target cells (activated T cells), diluted human serum complement (Quidel-A112) and different concentrations of fully human PD-1 antibodies in a 96-well plate. Incubate the plate for 4 hours at 37°C in a 5% _CO2 incubator. Target cell lysis was determined by CellTiter glo (Promega-G7573). Rituxan (Roche) and human B lymphocyte cell Raji (CD20 positive) were used as positive controls. Data show that PD-1 antibodies do not mediate CDC. Example 4 : Clinical trial results of recombinant fully human anti -PD-1 monoclonal antibody in the treatment of cervical cancer

Use the recombinant fully human anti-PD-1 monoclonal antibody injection of the present invention (such as GLS-010 injection) to study the recurrence or metastasis of GLS-010 after first-line or above platinum-containing standard chemotherapy and positive PD-L1 expression ( Anti-tumor efficacy in the treatment of cervical cancer patients with CPS ≥ 1), safety and tolerability of GLS-010 in patients with recurrence or metastasis and PD-L1 expression-positive cervical cancer that has progressed after first-line or above platinum-containing standard chemotherapy, study Pharmacokinetic (PK) characteristics and exposure-response relationship of GLS-010; observe the immunogenicity of recombinant fully human anti-PD-1 monoclonal antibody injection and initially evaluate microsatellite instability (MSI)/mismatch repair Correlation of defect (dMMR) and/or tumor mutation matching (TMB) with efficacy, as assessed by companion diagnostics (PD-L1).

1. Administration method:

All subjects were intravenously infused with GLS-010 at a dose of 240 mg/time, once every two weeks until confirmed disease progression or any of the following conditions (whichever occurs first): 1) The end of the treatment period, 2) Death, 3) Intolerable toxic side effects occur, 4) The subject is pregnant, 5) The researcher determines that the study should be terminated based on the best interests of the subject, 6) The subject or his/her guardian requests to withdraw from the study, 7) Subjects are lost to follow-up, 8) Subjects have poor compliance and are unable to follow the research protocol, etc.

The maximum treatment period is 2 years. After taking the drug for 2 years, if the researcher assesses that the subject can continue to benefit and there are no intolerable adverse reactions, based on the subject's wishes and with the approval of the sponsor, the subject can enter the extended treatment period of this study. (The extended treatment period is defined as the continued medication period after 2 years of medication), and continue to receive study drug treatment.

Each infusion time should be no less than 45 minutes. If the subject experiences mild allergic reactions such as dizziness, chills, rash, etc. during the infusion process, the infusion speed can be appropriately reduced (judged by the researcher), and the infusion time can be extended accordingly (judged by the researcher). judgment).

2. Experimental design:

The selected population for this study were patients with recurrent or metastatic cervical cancer who had progressed after first-line or above platinum-containing standard chemotherapy and had positive PD-L1 expression.

Phase I clinical studies show that the anti-PD-1 monoclonal antibody GLS-010 has good safety and tolerability in patients with advanced tumors, and shows preliminary anti-tumor efficacy. The results of a single-arm clinical study (KEYNOTE-158) of a similar drug, Pembrolizumab, in the treatment of advanced cervical cancer showed a certain proportion of objective response rate (ORR 14.3%), and 91% of patients responded for more than 6 months.

The U.S. FDA approved pembrolizumab for the treatment of patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors whose disease has progressed after treatment or for whom there is no satisfactory alternative therapy, and for the treatment of PD-L1-positive (CPS ≥1) Patients with recurrent or metastatic cervical cancer whose disease has progressed during or after chemotherapy. The 2018 NCCN Guidelines recommend pembrolizumab for the second-line treatment of PD-L1-positive (CPS ≥ 1) or MSI-H/dMMR recurrent or metastatic cervical cancer (category 2A evidence). Considering that most patients with recurrent and metastatic cervical cancer have undergone multiple lines of platinum-containing chemotherapy, and when platinum resistance develops, the options available are very limited and the treatment effect is poor. The similar anti-PD-1 monoclonal antibody drug Pembrolizumab is in clinical trials A certain efficacy has been achieved; the GLS-010 phase Ia study also found clinical benefits for patients with advanced cervical cancer. It is expected that the subjects may benefit from this study, but there may be ethical issues with using placebo as a control. Weighing scientific and ethical considerations, a single-arm study may serve the purposes of this invention.

3. The conditions for selected subjects are as follows: 1) Voluntarily participate in clinical research; fully understand and informed about this study and sign the informed consent form; willing to follow and have the ability to complete all trial procedures; 2) Female, aged 18 to 75 years old (inclusive) and 75 years old); 3) Cervical cancer patients with histologically confirmed PD-L1 positive expression (CPS ≥ 1); 4) Recurrent or metastatic cervical cancer confirmed by imaging after first-line or above platinum-containing standard chemotherapy. Cancer patients; 5) There is at least one measurable lesion according to RECIST 1.1 criteria, that is, the long diameter of non-lymph node lesions ≥ 10 mm based on CT cross-sectional images or MRI, or the short diameter of lymph node lesions ≥ 15 mm; 6) Eastern Cooperative Oncology Group (ECOG) performance status score is 0 to 1; 7) The expected survival period is not less than 12 weeks; 8) Organ function and hematopoietic function must meet the following requirements: Hemoglobin (HGB) ≥ 90 g/L; White blood cell count (WBC) ≥ 3×10 ⁹ /L; absolute neutrophil count (ANC) ≥ 1.5×10 ⁹ /L; platelet count (PLT) ≥ 100×10 ⁹ /L; total bilirubin (TBIL) ≤ 1.5×normal value Upper limit (ULN); aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5×ULN; if abnormal liver function is caused by tumor liver metastasis, AST and ALT ≤ 5×ULN ; Serum creatinine (Cr) ≤ 1.5×ULN; or creatinine clearance (CrCl) ≥ 50 mL/min international normalized ratio (INR) or plasma prothrombin time (PT) ≤ 1.5×ULN. 9) Female subjects of childbearing age must agree to take effective contraceptive measures after signing the informed consent form, during the study and within 5 months after the last dose of GLS-010. 10) Subjects must agree to provide sufficient tumor tissue samples for PD-L1 expression detection. Include archived tumor samples (paraffin blocks or unstained sections in quantities that meet the testing requirements specified in this study); if there are no archived tumor tissue samples, the subject agrees to undergo re-biopsy of the tumor lesions.

20 n（%） 19（42.2%） 38（36.2%） 57（38.0%） 表2 受試者既往腫瘤治療史 項目 第一階段（ N=45 ） n （ % ） 第二階段（ N=105 ） n （ % ） 總計（ N=150 ） n （ % ） 具有任意治療史受試者人數       抗腫瘤藥物治療 45（100.0%） 105（100.0%） 150（100.0%） 放療 42（93.3%） 100（95.2%） 142（94.7%） 腫瘤手術治療 37（82.2%） 68（64.8%） 105（70.0%） 其他抗腫瘤治療史 1（2.2%） 21（20.0%） 22（14.7%） 既往治療類型       化療 45（100.0%） 105（100.0%） 150（100.0%） 單株抗體治療 0 0 0 靶向治療 4（8.9%） 14（13.3%） 18（12.0%） 免疫治療 0 0 0 其他 1（2.2%） 1（1.0%） 2（1.3%） 治療線數       1線 8（17.8%） 104（99.0%） 112（74.7%） 2線 2（4.4%） 40（38.1%） 42（28.0%）

3線 1（2.2%） 10（9.5%） 11（7.3%） 抗腫瘤藥物治療史       化療 45（100.0%） 105（100.0%） 150（100.0%） 順鉑 33（73.3%） 81（77.1%） 114（76.0%） 紫杉醇 37（82.2%） 50（47.6%） 87（58.0%） 多西他賽 13（28.9%） 49（46.7%） 62（41.3%） 卡鉑 18（40.0%） 42（40.0%） 60（40.0%） 奈達鉑 15（33.3%） 40（38.1%） 55（36.7%） 白蛋白紫杉醇 3（6.7%） 32（30.5%） 35（23.3%） 貝伐珠單抗 5（11.1%） 28（26.7%） 33（22.0%） N為各階段全分析集人數。 百分比以各階段全分析集人數為分母並僅在分子不為0時計算。 用WHODrug 201909和MedDRA編碼字典（V 24.0）進行編碼。 表3 受試者研究者評估的客觀緩解率*（FAS）   第一階段（N=45） n（%） 第二階段（N=105） n（%） 總計（N=150） n（%） 最佳總體療效       完全緩解 3（6.7%） 3（2.9%） 6（4.0%） 部分緩解 8（17.8%） 19（18.1%） 27（18.0%） 疾病穩定 12（26.7%） 38（36.2%） 50（33.3%） 疾病進展 21（46.7%） 38（36.2%） 59（39.3%） 無法評估 0 0 0 客觀緩解率（ORR） 11（24.4%） 22（21.0%） 33（22.0%） 95%置信區間** （12.88%, 39.54%） （13.62%, 29.99%） （15.65%, 29.49%） P值*** 0.0008 ＜.0001 ＜.0001 *客觀緩解率是指根據RECIST1.1標準，總體療效達到完全緩解（CR）或部分緩解（PR）的受試者百分比。 **採用Clopper-Pearson精確法估計95%置信區間。 ***二項精確法檢驗。 N為各階段全分析集人數。 百分比以各階段全分析集人數為分母並僅在分子不為0時計算。 表4 受試者總生存期*（FAS）   第一階段（N=45） n（%） 第二階段（N=105） n（%） 總計（N=150） n（%） 評估結局       死亡（任何原因），n（%） 23（51.1%） 29（27.6%） 52（34.7%） 刪失，n（%） 22（48.9%） 76（72.4%） 98（65.3%）         中位總生存期（OS）**（月） 17.314 11.959 19.581 最小值，最大值 （1.643, 26.251） （0.756, 13.503） （0.756, 26.251） 95%置信區間** （7.195,

） （9.692,

） （9.889,

） *總生存期定義為自首次給藥開始到任何原因引起死亡的時間。 **中位總生存期由Kaplan-Meier方法計算得出，其雙側95%置信區間採用Brookmeyer and Crowley法計算。 N為各階段全分析集人數。 百分比以各階段全分析集人數為分母並僅在分子不為0時計算。 表5 受試者研究者評估的中位至緩解時間   第一階段（N=45） n（%） 第二階段（N=105） n（%） 總計（N=150） n（%） 中位至緩解時間（TTR）**（月） 1.906 1.873 1.873 最小值，最大值 （1.708, 5.520） （1.741, 5.421） （1.708, 5.520） 95%置信區間** （1.774, 3.680） （1.807, 1.938） （1.840, 1.906） *至緩解時間定義為從第一天用藥開始至第一次評估為完全緩解或部分緩解之間的時間，以先出現者為準。只適用於取得完全緩解或部分緩解的受試者。 **中位至緩解時間由Kaplan-Meier方法計算得出，其雙側95%置信區間採用Brookmeyer and Crowley法計算。 N為各階段全分析集人數。 表6 受試者安全性和耐受性評價（受試者治療中出現的不良事件（TEAE）概况（SS））   第一階段（N=45） 第二階段（N=105） 總計（N=150）   例次 例數n（%） 例次 例數n（%） 例次 例數n（%） 治療期不良事件（TEAE） 452 43（95.6%） 855 101（96.2%） 1307 144（96.0%） 與研究藥物相關的TEAE 224 36（80.0%） 413 78（74.3%） 637 114（76.0%） 免疫相關的TEAE 18 12（26.7%） 101 44（41.9%） 119 56（37.3%） 治療期的SAE 32 20（44.4%） 54 32（30.5%） 86 52（34.7%） 與研究藥物相關的SAE 7 5（11.1%） 17 12（11.4%） 24 17（11.3%） 3級及以上的TEAE 69 27（60.0%） 87 43（41.0%） 156 70（46.7%） 3級及以上免疫相關的TEAE 1 1（2.2%） 10 8（7.6%） 11 9（6.0%） 3級及以上與研究藥物相關的TEAE 21 12（26.7%） 31 19（18.1%） 52 31（20.7%） 導致治療終止的TEAE 3 3（6.7%） 11 11（10.5%） 14 14（9.3%） 導致退出研究的TEAE 11 11（24.4%） 15 15（14.3%） 26 26（17.3%） 導致死亡的TEAE的受試者 11 11（24.4%） 17 17（16.2%） 28 28（18.7%） N為各階段安全集人數。 百分比以各階段安全集人數為分母並僅在分子不為0時計算。 例次為不良事件發生的次數。 除了AE分析例次，其他每行中對於同一例受試者只計算一次。 SAE：以據研究者評估為準。 使用MedDRA編碼字典（V 24.0）進行編碼。 4. The test results are shown in the following Tables 1-6 and Figure 2. The recombinant fully human anti-PD-1 monoclonal antibody injection of the present invention can prevent recurrence or metastasis and PD-L1 expression after first-line or above platinum-containing standard chemotherapy. The anti-tumor efficacy in the treatment of patients with positive (CPS ≥ 1) cervical cancer is significant, the safety and tolerability are good, and the objective response rate and overall survival evaluated by subject researchers are better than other existing similar drugs. Table 1 PD-L1 expression in subjects indicator Statistics Stage 1 ( N=45 ) Second stage ( N=105 ) Total( N=150 ) PD-L1 expression CPS 1-<20 n(%) 26 (57.8%) 65 (61.9%) 91 (60.7%)

20 n(%) 19 (42.2%) 38 (36.2%) 57 (38.0%) Table 2 Subjects’ previous tumor treatment history Project First stage ( N=45 ) n ( % ) Second stage ( N=105 ) n ( % ) Total ( N=150 ) n ( % ) Number of subjects with any treatment history Anti-tumor drug treatment 45 (100.0%) 105 (100.0%) 150 (100.0%) radiotherapy 42 (93.3%) 100 (95.2%) 142 (94.7%) tumor surgical treatment 37 (82.2%) 68 (64.8%) 105 (70.0%) History of other anti-tumor treatments 1(2.2%) 21 (20.0%) 22 (14.7%) Type of previous treatment Chemotherapy 45 (100.0%) 105 (100.0%) 150 (100.0%) monoclonal antibody therapy 0 0 0 targeted therapy 4(8.9%) 14 (13.3%) 18 (12.0%) Immunotherapy 0 0 0 other 1(2.2%) 1 (1.0%) 2(1.3%) Number of treatment lines 1 line 8(17.8%) 104 (99.0%) 112 (74.7%) 2 lines 2(4.4%) 40 (38.1%) 42 (28.0%)

3 lines 1(2.2%) 10 (9.5%) 11 (7.3%) History of antineoplastic drug treatment Chemotherapy 45 (100.0%) 105 (100.0%) 150 (100.0%) Cisplatin 33 (73.3%) 81 (77.1%) 114 (76.0%) Paclitaxel 37 (82.2%) 50 (47.6%) 87 (58.0%) Docetaxel 13 (28.9%) 49 (46.7%) 62 (41.3%) carboplatin 18 (40.0%) 42 (40.0%) 60 (40.0%) nedaplatin 15 (33.3%) 40 (38.1%) 55 (36.7%) albumin paclitaxel 3(6.7%) 32 (30.5%) 35 (23.3%) Bevacizumab 5(11.1%) 28 (26.7%) 33 (22.0%) N is the number of people in the full analysis set at each stage. Percentages are calculated with the number of people in the full analysis set at each stage as the denominator and are calculated only when the numerator is not zero. Coding was performed with WHODrug 201909 and MedDRA coding dictionary (V 24.0). Table 3 Subject investigator-assessed objective response rate* (FAS) The first stage (N=45) n (%) Second stage (N=105) n (%) Total (N=150) n (%) best overall efficacy complete remission 3(6.7%) 3 (2.9%) 6(4.0%) partial relief 8(17.8%) 19 (18.1%) 27 (18.0%) disease stable 12 (26.7%) 38 (36.2%) 50 (33.3%) disease progression 21 (46.7%) 38 (36.2%) 59 (39.3%) Unable to evaluate 0 0 0 Objective response rate (ORR) 11 (24.4%) 22 (21.0%) 33 (22.0%) 95% confidence interval** (12.88%, 39.54%) (13.62%, 29.99%) (15.65%, 29.49%) P value*** 0.0008 <.0001 <.0001 *Objective response rate refers to the percentage of subjects whose overall response reaches complete response (CR) or partial response (PR) according to RECIST1.1 criteria. **95% confidence intervals were estimated using the Clopper-Pearson exact method. ***Binomial exact test. N is the number of people in the full analysis set at each stage. Percentages are calculated with the number of people in the full analysis set at each stage as the denominator and are calculated only when the numerator is not zero. Table 4 Subject overall survival* (FAS) The first stage (N=45) n (%) Second stage (N=105) n (%) Total (N=150) n (%) Assess outcomes Death (any cause), n (%) 23 (51.1%) 29 (27.6%) 52 (34.7%) Censored, n (%) 22 (48.9%) 76 (72.4%) 98 (65.3%) Median overall survival (OS)** (months) 17.314 11.959 19.581 minimum value, maximum value (1.643, 26.251) (0.756, 13.503) (0.756, 26.251) 95% confidence interval** (7.195,

) (9.692,

) (9.889,

) *Overall survival is defined as the time from the first dose to death from any cause. **Median overall survival was calculated by the Kaplan-Meier method, and its two-sided 95% confidence interval was calculated by the Brookmeyer and Crowley method. N is the number of people in the full analysis set at each stage. Percentages are calculated with the number of people in the full analysis set at each stage as the denominator and are calculated only when the numerator is not zero. Table 5 Median time to response as assessed by subject investigators The first stage (N=45) n (%) Second stage (N=105) n (%) Total (N=150) n (%) Median time to response (TTR)** (months) 1.906 1.873 1.873 minimum value, maximum value (1.708, 5.520) (1.741, 5.421) (1.708, 5.520) 95% confidence interval** (1.774, 3.680) (1.807, 1.938) (1.840, 1.906) *Time to response is defined as the time from the first day of dosing to the first assessment of complete or partial response, whichever occurs first. Applicable only to subjects who achieve complete remission or partial remission. **The median time to response was calculated by the Kaplan-Meier method, and its two-sided 95% confidence interval was calculated by the Brookmeyer and Crowley method. N is the number of people in the full analysis set at each stage. Table 6 Subject Safety and Tolerability Evaluation (Subject Treatment Emergent Adverse Events (TEAE) Profile (SS)) Phase 1 (N=45) Stage 2 (N=105) Total(N=150) Examples Number of examples n (%) Examples Number of examples n (%) Examples Number of examples n (%) Treatment Adverse Events (TEAEs) 452 43 (95.6%) 855 101 (96.2%) 1307 144 (96.0%) TEAEs related to study drug 224 36 (80.0%) 413 78 (74.3%) 637 114 (76.0%) Immune-related TEAEs 18 12 (26.7%) 101 44 (41.9%) 119 56 (37.3%) SAEs during treatment 32 20 (44.4%) 54 32 (30.5%) 86 52 (34.7%) SAEs related to study drug 7 5 (11.1%) 17 12 (11.4%) twenty four 17 (11.3%) TEAE Level 3 and above 69 27 (60.0%) 87 43 (41.0%) 156 70 (46.7%) Grade 3 and above immune-related TEAEs 1 1(2.2%) 10 8(7.6%) 11 9(6.0%) Grade 3 or higher TEAEs related to study drug twenty one 12 (26.7%) 31 19 (18.1%) 52 31 (20.7%) TEAEs leading to treatment discontinuation 3 3(6.7%) 11 11 (10.5%) 14 14 (9.3%) TEAEs leading to study withdrawal 11 11 (24.4%) 15 15 (14.3%) 26 26 (17.3%) Subjects with TEAEs resulting in death 11 11 (24.4%) 17 17 (16.2%) 28 28 (18.7%) N is the number of people safely gathered at each stage. The percentage is calculated with the number of people safely gathering at each stage as the denominator and is calculated only when the numerator is not zero. Examples are the number of times an adverse event occurs. Except for AE analysis cases, the same subject is only calculated once in each row. SAE: based on the researcher's assessment. Encoding was performed using the MedDRA encoding dictionary (V 24.0).

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the patent scope of the present invention and the scope of equivalent technologies, the present invention is also intended to include these modifications and variations.

without

Figure 1 is a flowchart of the study design for the phase II cervical cancer study. Figure 2 shows the investigator-assessed Kaplan-Meier survival curves* (FAS) for progression-free survival (stage 1 and stage 2 survival curves, respectively).

TW202320852A_111139115_SEQL.xml

Claims (18)

The use of an anti-PD-1 antibody or an antigen-binding fragment thereof in preparing a medicament for treating cervical cancer patients, wherein the antibody or an antigen-binding fragment thereof includes: a) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 1, the CDR2 shown in SEQ ID NO: 3, and the CDR3 shown in SEQ ID NO: 5; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 7, CDR2 shown in SEQ ID NO: 9 and CDR3 shown in SEQ ID NO: 11; b) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 13, the CDR2 shown in SEQ ID NO: 15, and the CDR3 shown in SEQ ID NO: 5; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 7, CDR2 shown in SEQ ID NO: 17 and CDR3 shown in SEQ ID NO: 11; c) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 1, the CDR2 shown in SEQ ID NO: 15, and the CDR3 shown in SEQ ID NO: 5; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 7, CDR2 shown in SEQ ID NO: 17 and CDR3 shown in SEQ ID NO: 19; d) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 21, the CDR2 shown in SEQ ID NO: 23, and the CDR3 shown in SEQ ID NO: 25; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 27, CDR2 shown in SEQ ID NO: 29 and CDR3 shown in SEQ ID NO: 31; or e) The heavy chain variable region, which includes CDR1 shown in SEQ ID NO: 33, the CDR2 shown in SEQ ID NO: 35, and the CDR3 shown in SEQ ID NO: 37; and the light chain variable region, which includes SEQ CDR1 shown in ID NO: 39, CDR2 shown in SEQ ID NO: 41 and CDR3 shown in SEQ ID NO: 43. The use as described in claim 1, wherein the antibody or antigen-binding fragment thereof includes: a) the heavy chain variable region, which includes SEQ ID NO: 45; and the light chain variable region, which includes SEQ ID NO: 47; b) heavy chain variable region, which includes SEQ ID NO: 49; and light chain variable region, which includes SEQ ID NO: 51; c) heavy chain variable region, which includes SEQ ID NO: 53; and light chain variable region, which includes SEQ ID NO: 55; d) a heavy chain variable region comprising SEQ ID NO: 57; and a light chain variable region comprising SEQ ID NO: 59; or e) A heavy chain variable region, which includes SEQ ID NO: 61; and a light chain variable region, which includes SEQ ID NO: 63. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds to human PD-1 with a Kd value not exceeding 10 ^-8 M, and the Kd value is determined by a plasma resonance binding method. Determination. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to monkey PD-1 with an EC ₅₀ of no more than 100 nM, or no more than 10 nM, and/or does not bind to mouse PD-1 combine. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof blocks the binding of human or monkey PD-1 to its ligand with an IC ₅₀ not exceeding 100 nM. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof does not bind to CD28 or CTLA4. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof does not mediate ADCC or CDC or both. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody. The use as claimed in claim 8, wherein in the antibody or antigen-binding fragment thereof, the fully human monoclonal antibody is produced by transgenic rats. The use according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof blocks the binding of human PD-1 to its ligand, and therefore provides at least one of the following activities: a) in CD4 ⁺ T cells inducing IL-2 production in; b) inducing IFNγ production in CD4 ⁺ T cells; c) inducing proliferation of CD4 ⁺ T cells; and d) reversing T reg suppressive function. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a diabody, scFv, scFv dimer, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment , Fab, Fab' or F(ab')2. The use as described in claim 11, wherein in the antibody or antigen-binding fragment thereof, the diabody is a BsFv or ds diabody. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof further includes an immunoglobulin constant region. The use as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof further includes a conjugate. The use as described in claim 1 or 2, wherein the cervical cancer is an immunosuppression-related tumor caused by PD-1. The use as described in claim 1 or 2, wherein the cervical cancer is recurrent or metastatic cervical cancer. The use as described in claim 1 or 2, wherein the cervical cancer is recurrence or metastasis and/or PD-L1 expression-positive cervical cancer after first-line or above platinum-containing standard chemotherapy. The use as described in claim 1 or 2, wherein the cervical cancer is recurrence or metastasis and/or PD-L1 expression-positive cervical cancer after first-line or above platinum-containing standard chemotherapy.

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