Anti-PD-1 humanized antibody or antigen-binding fragment thereof and use thereof

Abstract

The present disclosure belongs to the field of biomedicine technology, and specifically provides an anti-PD-1 humanized antibody or antigen-binding fragment thereof and uses thereof, which can efficiently and specifically bind to PD-1 and effectively inhibit the binding of PD-1 to PD-L1 and PD-L2. Thus, the antibody or antigen-binding fragment thereof, as well as its associated nucleic acid, vector, cell or pharmaceutical composition, can be used to prepare a medicament for treating a PD-1-mediated disease or condition.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and more particularly to a humanized anti-PD-1 antibody or an antigen-binding fragment thereof and uses thereof. [Background technology]

[0002] Programmed death factor-1 (PD1) is a member of the CD28 family and is expressed in activated B cells, T cells, and myeloid cells. Human PD1 is encoded by the 9.6-kb gene Pdcd1, located at 2q37.3, and consists of five exons and four introns, with a 663-bp upstream promoter. PD1 is a 55-kDa type I transmembrane protein. Its molecular structure consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains one IgV domain of the immunoglobulin variable region, while the intracellular domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The amino acid sequence of the PD-1 extracellular domain shares 24% identity with CTLA-4 and 28% identity with CD28. Upon T cell activation, PD-1 recruits the tyrosine phospholipase SHP2, primarily via its ITIM, leading to the dephosphorylation of downstream effector molecules.

[0003] PD-1 has two ligands, PD-L1 and PD-L2, both of which are B7 homologs. The PD-L1 and PD-L2 genes are located on the human chromosome 9P24.2 locus, are 42 kb in size, and each molecule contains one immunoglobulin-like variable region domain, one constant region-like domain, one transmembrane domain, and a short cytoplasmic tail.

[0004] PD-1 can downregulate T cell activation after binding to PD-L1 and PD-L2. PD-L1 is expressed on the surface of many tumor cells, including lung cancer, gastric cancer, liver cancer, esophageal cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, skin cancer, colon cancer, bladder cancer, glioma, head and neck cancer, and oral squamous cell carcinoma. Large numbers of CD8+ T cells expressing PD-L1 have been found in the periphery of these cancers, and clinical results show that high levels of PD-L1 expression on tumor cells are associated with poor prognosis in cancer patients. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem to be solved by the present invention is to overcome the drawbacks and deficiencies of existing antibodies, such as their low affinity and specificity for binding to PD-1, and to provide an anti-PD-1 humanized antibody or antigen-binding fragment thereof, and uses thereof. [Means for solving the problem]

[0006] The present invention provides a humanized anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises a light chain CDR region and a heavy chain CDR region, wherein the heavy chain CDR region comprises HCDR1, HCDR2, and HCDR3, and the light chain CDR region comprises LCDR1, LCDR2, and LCDR3, and wherein HCDR1 The amino acid sequence of is as shown in SEQ ID NO: 8, HCDR3 amino acid sequence teeth Sequence number 10 The amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs: 11 to 13, respectively, and the amino acid sequence of the heavy chain variable region of the antibody is as shown in any one of SEQ ID NOs: 3 to 5.

[0007] The amino acid sequence of the light chain variable region of the antibody is as shown in any one of SEQ ID NOs: 6 to 7.

[0008] The present invention further relates to nucleic acids, vectors, cells or pharmaceutical compositions related to said antibodies or antigen-binding fragments thereof.

[0009] The invention further relates to the use of said antibodies or antigen-binding fragments thereof, and their related nucleic acids, vectors, cells or pharmaceutical compositions in the manufacture of a medicament for treating a PD-1 mediated disease or condition.

[0010] The present invention further relates to a method for treating a PD-1-mediated disease or condition, the method comprising administering to a subject an effective amount of the above-described antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, or pharmaceutical composition.

[0011] The present invention further relates to the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or pharmaceutical composition for use in therapy.

[0012] The present invention further relates to the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or pharmaceutical composition for treating a PD-1 mediated disease or condition. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the effect of different concentrations of PD-1-112-C2 on IL-2 / IFN-γ secretion. [Figure 2] This figure shows the effects of different concentrations of PD-1-112-C2 on T cell proliferation and T cell secretion of the cellular factor IL-2. [Figure 3] This figure shows the effects of different concentrations of PD-1-112-C2 on T cell proliferation and T cell IFN-γ secretion. [Figure 4] This shows the results of the effect of anti-PD-1 humanized antibody c53 on tumor volume. [Figure 5] This shows the effect of the anti-PD-1 humanized antibody c53 on mouse survival time. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be further described below with reference to specific examples, which are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment employed in the present invention are conventional reagents, methods, and equipment in the art.

[0015] Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0016] The present invention relates to a humanized anti-PD-1 antibody or an antigen-binding fragment thereof, the antibody comprising a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consisting of HCDR1, HCDR2, and HCDR3, the light chain CDR region consisting of LCDR1, LCDR2, and LCDR3, and HCDR1 The amino acid sequence of is as shown in SEQ ID NO: 8, The amino acid sequence of HCDR3 is Distribution Column number 10 The amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs: 11 to 13, respectively; the amino acid sequence of the heavy chain variable region of the antibody is as shown in any one of SEQ ID NOs: 3 to 5; and the amino acid sequence of the light chain variable region of the antibody is as shown in any one of SEQ ID NOs: 6 to 7.

[0017] Although the present invention has labeled the CDR regions using the Kabat numbering system, CDR regions labeled in other ways are also within the scope of the present invention.

[0018] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO:3 As shown in The amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 6, or the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 4. As shown in The amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 7, or the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 5. As shown in The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:7.

[0019] In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, and the light chain constant region is a kappa chain or a lambda chain.

[0020] In some embodiments, the species of the heavy chain constant region and the light chain constant region is selected from human, murine, or simian.

[0021] In some embodiments, the antibody is a chimeric antibody or a multispecific antibody (eg, a bispecific antibody).

[0022] In the present invention, the term "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope.

[0023] As used herein, the term "bispecific antibody" refers to a multispecific antigen-binding protein or antibody, which can be produced by a variety of methods, including, but not limited to, hybridoma fusion or Fab' fragment ligation. See, for example, 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 bind to two different epitopes, which may reside on the same or different protein targets.

[0024] In the present invention, the term "specific binding" or similar expressions refers to the binding of an antibody or an antigen-binding fragment thereof to an epitope on a predetermined antigen. Generally, an antibody or an antigen-binding fragment thereof binds to an epitope on a predetermined antigen at a specific specificity of about 10 -6 Less than m, e.g., about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M or about 10 -10 Affinity (K DKD refers to the ratio of the dissociation rate to the association rate (koff / kon), and this quantity can be measured by methods well known to those skilled in the art.

[0025] In some embodiments, the antigen-binding fragment is any one or more of a F(ab')2, a Fab, a scFv, an Fv, and a single domain antibody.

[0026] In the present invention, the term "F(ab')2" refers to a fragment comprising two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. An F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0027] In the present invention, the term "Fab" refers to a molecule consisting of one light chain, CH1, and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0028] In the present invention, the term "scFv" refers to an Fv molecule in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain (which forms the antigen-binding region) (see, for example, Bird et al., Science. 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA. 90:5879-5883 (1988)).

[0029] In the present invention, the term "Fv" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0030] In the present invention, the term "single domain antibody" refers to a VHH that contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but does not readily adhere to each other or even aggregate like engineered single-chain antibodies (scFv). More importantly, the individually cloned and expressed VHH structure is the smallest known unit capable of binding to a target antigen, with structural stability and antigen-binding activity comparable to that of the original heavy chain antibody.

[0031] The present invention further relates to nucleic acids encoding the anti-PD-1 humanized antibodies or antigen-binding fragments thereof.

[0032] In a preferred embodiment, the nucleic acid comprises a first nucleic acid encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof, and / or a second nucleic acid encoding a light chain variable region of the antibody or antigen-binding fragment thereof.

[0033] In the present invention, nucleic acids are generally RNA or DNA, and nucleic acid molecules may be single-stranded or double-stranded. Ku Preferably, the nucleic acid is 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 it affects the transcription of the coding sequence. When it is connected to a vector, it is preferable to use DNA. Furthermore, because antibodies are membrane proteins, the nucleic acid generally comprises a signal peptide sequence.

[0034] The present invention further relates to a vector carrying said nucleic acid.

[0035] In the present invention, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The genetic material elements carried by the vector can be expressed in a host cell by introducing the vector into the host cell via transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), phages (e.g., lambda phage or M13 phage), animal viruses, and the like. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0036] The invention further relates to cells, said cells carrying said nucleic acid, containing said vector, or capable of expressing said antibody or antigen-binding fragment thereof.

[0037] The present invention further relates to a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, or the cell.

[0038] In the present invention, the term "pharmaceutical composition" refers to a composition in which the biological activity of the active ingredient is present in a form that allows it to be effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0039] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable vector and / or excipient.

[0040] In the present invention, the term "pharmaceutically acceptable vector" means ,rawAny and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physically compatible may be included.

[0041] Also included within the scope of the present invention is the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell, or the pharmaceutical composition in the manufacture of a medicament for treating a PD-1-mediated disease or condition.

[0042] In some embodiments, the pharmaceutical composition or medicament is in a form suitable for injection.

[0043] In a preferred embodiment, the pharmaceutical composition or medicament is in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.

[0044] [Effects of the invention]

[0045] The present invention has the following beneficial effects:

[0046] The anti-PD-1 humanized antibodies or antigen-binding fragments thereof provided in the present invention are capable of binding with high affinity to CHO-hPD1 cells, CHO-cyno cells, and activated PBMCs, with affinities significantly improved compared to positive controls, and are capable of binding to PD-1 efficiently and specifically. They can also effectively inhibit the binding of PD-L1 / PD-L2 ligands to CHO-hPD1, and in MLR, inhibit the binding of PD-1 to its ligands and suppress the PD-1 signaling pathway, thereby promoting T cell proliferation and the secretion of IL-2 and IFN-γ cytokines. Therefore, the antibodies or antigen-binding fragments thereof, and their related nucleic acids, vectors, cells, or pharmaceutical compositions have broad potential for use in the manufacture of medicaments for the treatment of PD-1-mediated diseases or conditions. [Example]

[0047] Example 1: Preparation of anti-PD-1 antibodies 1. Immunogen Human PD-1 sequence (NCBINP005009) was artificially synthesized, and the extracellular domain was amplified by PCR using the upstream primer: 5'-CCGCAAGCTTGCCGCCACCATG-3' (SEQ ID NO: 1) and the downstream primer: 5'-CCGGAATTCTCATTAATGGTGATGGTGATGATGCTGGAACTGGCCGGCAGGTC-3' (SEQ ID NO: 2). After double digestion with HindIII and EcoRI, the amplified extracellular domain was cloned into the pCDNA3.4A eukaryotic expression system. 293 cells were transfected with this plasmid, and the supernatant was collected and purified to obtain human PD-1 recombinant protein (hPD-1).

[0048] 2. Immunizing animals Five 6-week-old female BALb / C mice were subcutaneously immunized with 125 μg of 1.23 mg / ml hPD-1 recombinant protein, mixed with an equal volume of Freund's adjuvant (Sigma-Aldrich F5881). Each mouse received 25 μg of antigen. After the primary immunization, booster immunizations with the same amount were administered weekly. After a total of five immunizations, immune responses were monitored by tail bleeding. Mice with sufficient anti-hPD-1 immunoglobulin titers, as determined by FACS screening (described below), were used for cell fusion. Three days after the intraperitoneal booster immunization with antigen, the mice were sacrificed, and their spleens were removed for cell fusion.

[0049] 3. Selection of BALb / C mice producing anti-hPD-1 antibodies To select BALb / C mice producing anti-hPD-1 antibodies, immunized mouse sera were tested by FACS. Dilutions of serum from hPD-1 recombinant protein-immunized mice were incubated with hPD1-transfected CHO cells for 30 minutes at 4°C. After three washes with PBS, 0.4 μg / ml PE goat anti-mouse IgG (Biolegend 405307) was added and incubated for 30 minutes at 4°C. After three washes with PBS, the samples were detected using a Beckman Coulter flow cytometer (CytoFLEXA00-1-1102) to confirm the ability of hPD1 to bind to the transfected CHO cells. BALb / C mice producing anti-hPD-1 antibodies were then screened for, followed by cell fusion.

[0050] 4. Generation of mouse monoclonal antibody hybridomas against hPD-1 Spleen cells from immunized BALb / C mice were fused with mouse myeloma cells, and the resulting hybridomas were screened for antigen-specific antibodies. Single-cell suspensions of spleen cells from immunized mice were fused with one-fifth the number of non-immunoglobulin-secreting mouse myeloma cells (SP2 / 0, ATCC CRL1581) using PEG1500 (Roche 10783641001). The fused cells were plated in a 96-well cell culture plate at approximately 1 × 10 5The cells were plated at 100 cells / well and placed in an incubator (Panasonic MCO-18AIC) at 37°C and 5% CO. They were then cultured for approximately 1 week in HAT selection medium, which consisted of 1640 medium supplemented with 1x penicillin-streptomycin double antibiotic (Gibco 15140122), 1x HAT (Sigma CRLP-7185), and 20% fetal bovine serum (Royacel RY-F11-01). After one week, the HAT medium was replaced with HT medium (1640 medium containing 1x penicillin-streptomycin double antibiotic (Gibco 15140122), 1x HT (Gibco 11067030), and 20% fetal bovine serum (Royacel RY-F11-01)). The cell culture supernatants from the fusion plates were screened by FACS to identify hybridomas secreting antibodies capable of binding to hPD-1 protein. Hybridomas secreting antibodies capable of binding to hPD-1 protein were replated and screened again. Hybridomas that were positive for hPD-1 protein antibody binding were subcloned at least twice by limiting dilution. The subclones were then stabilized in vitro, and small amounts of antibody were generated for further analysis. Hybridoma clone PD1-112-C2 was selected for further analysis.

[0051] Example 2. Affinity characterization of anti-PD-1 mouse monoclonal antibodies Using conventional recombinant techniques, we produced three CHO (Chinese hamster ovary) cell lines expressing recombinant human PD-1 on the cell surface (CHO-hPD1), monkey PD1 (Uniprot: B0LAJ2) (CHO-cynoPD1), and mouse PD1 (Uniprot: Q02242) (CHO-mousePD1). These cell lines were used to measure the binding properties of the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 by flow cytometry (FCM).

[0052] To evaluate the binding of anti-PD-1 mouse monoclonal antibodies to CHO-hPD1, 2 × 10 cells were cultured in a 96-well plate. 5CHO-hPD1 cells were incubated with gradient-diluted anti-PD-1 mouse monoclonal antibody (initial concentration 10 μg / ml, diluted 3-fold) at 4°C for 30 minutes. The cells were washed once with buffer (PBS containing 3% BSA) and incubated with a PE-labeled anti-mouse IgG (Fc) Ab (Biolegend) fluorescent secondary antibody for 30 minutes at 4°C. The cells were then washed once with buffer and resuspended in PBS. The cell suspension was then subjected to flow cytometry analysis using a CytoFlex (Beckman flow cytometer) to measure the amount of antibody bound to the cells by measuring the mean fluorescence intensity (MFI) of the staining. The same method was used to evaluate the binding of this anti-PD-1 mouse monoclonal antibody to CHO-cyno cells and CHO-mousePD1 (sometimes abbreviated as "CHO-mPD1" in this specification) cells.

[0053] The results are shown in Table 1. The data indicate that the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 can bind to CHO-hPD1 cells and CHO-cyno cells with relatively high affinity, while none of the mouse monoclonal antibodies bind to CHO-mousePD1 cells.

[0054] [Table 1]

[0055] Example 3 Binding of anti-PD-1 antibodies to activated PBMCs Fresh human peripheral blood mononuclear cells (PBMCs) are stimulated with PHA (Sigma) to activate and proliferate lymphocytes, and on day 3 they express the most PD1, allowing for binding experiments between PD-1 antibodies and PD1 naturally expressed by activated lymphocytes.

[0056] PBMCs were obtained from fresh human peripheral blood by gradient centrifugation using lymphoid isolation fluid, and 1 × 10 6The density was adjusted to 10 cells / ml and inoculated into T75 wells. At the same time, PHA-L (Sigma) was added at a final concentration of 1 μg / ml to stimulate lymphocyte proliferation. After leaving the cells in an incubator at 37°C and 5% CO for 3 days, the cell suspension was taken, the supernatant was removed by centrifugation, and the cells were resuspended in buffer (PBS containing 3% BSA) and plated at 2 × 10 cells / ml in a 96-well U-plate. 5 PBMCs were added at 1000 μg / well, followed by a 3-fold gradient dilution of anti-PD1 antibody (10 concentrations total) starting from 30 μg / ml. After incubation at 4°C for 30 minutes, the cells were centrifuged at 300 g for 5 minutes. The cells were washed once with buffer, and a PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated at 4°C for 30 minutes. The cells were washed once by centrifugation, resuspended in PBS, and analyzed by CytoFlex flow cytometry to detect the amount of antibody bound to PBMCs.

[0057] As shown in Table 1, the results showed that the anti-PD1 antibody bound to activated lymphocytes with high affinity.

[0058] Example 4 Binding specificity of anti-PD-1 mouse monoclonal antibodies To confirm the specificity of antibody binding to PD-1, the anti-PD-1 mouse monoclonal antibody was conjugated to four different CD28 family member proteins. Using standard ELISA techniques, PD-1, CD28, CTLA-4, and ICOS (ACRO) were immobilized on an ELISA plate at a concentration of 1 μg / ml, and anti-human PD-1 mouse monoclonal antibody was added at a concentration of 10 μg / ml. Anti-mouse IgG conjugated with peroxidase (HRP) was used as the secondary antibody (Sigma). After development with TMB and quenching, the OD450 values ​​were read using a microplate reader.

[0059] The results are shown in Table 2. The anti-PD-1 mouse monoclonal antibody PD-1-112-C2 specifically binds to PD-1, but does not bind to other members of the CD28 family.

[0060] [Table 2]

[0061] Example 5: Measurement of affinity of anti-human PD-1 mouse monoclonal antibody using biolayer interferometry (BLI) ForteBio (OctetQke) affinity assay: PD-1-his(ACRO) recombinant protein at a concentration of 5 μg / ml was loaded onto a HISIK biosensor for 120 seconds. The loaded sensor was then equilibrated with standard buffer (PBST, PBS + 0.02% Tuween 20) for 120 seconds. The sensor was then transferred to a dilution of anti-PD-1 mouse monoclonal antibody for 180 seconds to measure the binding rate, and then transferred to standard buffer for 20 minutes to measure the dissociation rate. Final analysis was performed using a kinetic model.

[0062] The results of the data processing are shown in Table 3.

[0063] [Table 3]

[0064] Example 6: Inhibition of binding of anti-PD-1 mouse monoclonal antibodies to ligands PD-L1 / PD-L2 and CHO-hPD1 Flow cytometry was used to analyze the ability of anti-PD-1 mouse monoclonal antibodies to block the binding of ligands to PD-1 stably expressed on the surface of transfected CHO cells. The ligand proteins used in the experiments were recombinant PD-L1 / PD-L2 extracellular segments fused to a human IgG1 Fc segment: PD-L1-hFc(ACRO) and PD-L2-hFc(ACRO).

[0065] CHO-PD1 cells were resuspended in buffer (PBS containing 3% BSA) to a density of 2 × 10 6 The cell suspension was adjusted to cells / ml, and 100 μl / well of the cell suspension was added to a 96-well U-plate, centrifuged at 300 g for 5 minutes, and the supernatant was removed.

[0066] The subsequent process could be performed in two inhibition modes: In mode 1, PD-L1-hFc / PD-L2-hFc was added to the cell wells at a concentration of 3μg / ml and incubated at 4℃ for 30 minutes, followed by the addition of a total of 10 concentrations of anti-PD-1 mouse monoclonal antibodies diluted 3-fold starting from 30μg / ml and incubation at 4℃ for 30 minutes; in mode 2, 10 concentrations of anti-PD-1 mouse monoclonal antibodies diluted 3-fold starting from 30μg / ml and incubation at 4℃ for 30 minutes, followed by the addition of PD-L1-hFc / PD-L2-hFc protein at a concentration of 3μg / ml and incubation at 4℃ for 30 minutes.

[0067] The cells were centrifuged at 300 g for 5 min and washed once with buffer, followed by the addition of PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) and incubation for 30 min at 4°C. The cells were washed once by centrifugation and resuspended in PBS. The amount of ligand protein bound to the cells was detected by CytoFlex flow cytometry analysis, and the IC of PD-1 antibody binding inhibition was calculated. 50 The value was calculated.

[0068] The results, as shown in Table 4, show that the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 can effectively inhibit the binding of PD-L1 / PD-L2 to CHO-PD1 cells in both modes.

[0069] [Table 4]

[0070] Example 7 Effect of anti-PD-1 antibody on cytokine release from PBMC cells stimulated with SEB In this example, we detected the effect of overnight culture of peripheral blood mononuclear cells (PBMCs) on cytokine secretion when stimulated with the superantigen Staphylococcus aureus enterotoxin B (SEB) in the presence or absence of anti-PD-1 antibodies.

[0071] Fresh peripheral blood mononuclear cells (PBMCs) were resuspended in X-VIVO15 medium (LONZA) containing 10% FBS, added to a T25 flask, and cultured overnight at 37°C and 5% CO. The next day, the suspended cells were harvested, centrifuged, and resuspended in fresh X-VIVO medium (containing 10% FBS). SEB superantigen (Toxin Technology) was added to a final concentration of 200 ng / ml, and then plated at 1 x 10 cells per well in a 96-well flat plate. 5 Cells were added simultaneously with varying concentrations of anti-PD-1 antibodies. Isotype control antibodies (mIgG1 isotype control antibody (Biolegend) and hIgG4 isotype control antibody (Biolegend)) were also added; no antibody control wells were used. After 3 days, samples were collected from the sample wells, and IL-2 / IFN-γ levels were measured using the IL-2 / IFN-γ Human Uncoated ELISA Kit (eBioscience).

[0072] The effects of different concentrations of PD-112-C2 on IL-2 / IFN-γ secretion are shown in Figure 1. Anti-PD-1 antibody increased IL-2 / IFN-γ secretion in a concentration-dependent manner. These results indicate that the anti-PD-1 antibody PD-1-112-C2 further enhanced cytokine secretion by T cells in PBMCs stimulated with SEB superantigen.

[0073] Example 8: Effect of anti-PD-1 antibody on mixed lymphocyte reaction In mixed lymphocyte reaction (MLR), the presence or absence of anti-PD-1 antibodies can demonstrate the proliferation status of T cells and the level of cytokine secretion by T cells in the presence of PD1 signaling inhibition.

[0074] CD14 MicroBeads, human (Miltenyi) were used to detect CD14 from fresh PBMCs. +Monocytes were isolated and induced in the presence of GM-CSF / IL-4 for 6 days, followed by the addition of TNF-α for 3 days to induce DC maturation. On the day of the experiment, T cells were purified from PBMCs using the EasySep™ Human T Cell Enrichment Kit (StemCell) and 1 × 10 4 DC cells and 1 x 10 5 T cells were cultured in a mixed culture, and anti-PD-1 antibodies at varying concentrations were added to the mixed cells. Isotype control antibodies (mIgG1 isotype control antibody and hIgG4 isotype control antibody (Biolegend)) were also added to wells without antibody controls. After 3 days of mixed culture, the supernatant was collected and assayed for IL-2. After another 2 days of culture, the supernatant was collected and assayed for IFN-γ.

[0075] The effects of different concentrations of PD-1-112-C2 on T cell proliferation and T cell secretion of the cytokine IL-2 are shown in Figure 2, and the effects of different concentrations of PD-1-112-C2 on T cell proliferation and T cell secretion of the cytokine IFN-γ are shown in Figure 3. The results in Figures 2 and 3 demonstrate that the anti-PD-1 antibody PD-1-112-C2 can inhibit PD1 ligand binding and inhibit the PD1 signaling pathway in an antibody concentration-dependent manner in MLR experiments, thereby promoting T cell proliferation and T cell secretion of the cytokines IL-2 and IFN-γ.

[0076] Example 9 Humanization of anti-PD-1 mouse monoclonal antibody The anti-PD-1 mouse monoclonal antibody PD-1-112-C2 obtained above (the amino acid sequences of its HCDR1, HCDR2, and HCDR3 are set forth in SEQ ID NOs: 8 to 10, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are set forth in SEQ ID NOs: 11 to 13, respectively; the heavy chain variable region sequence is set forth in SEQ ID NO: 14; and the light chain variable region sequence is set forth in SEQ ID NO: 15) was humanized by the following specific method.

[0077] Human PD-1 sequence (NCBINP005009) was artificially synthesized and cloned into the PCDNA3.4A eukaryotic expression system. This plasmid was transfected into 293 cells, and the supernatant was collected and purified to obtain human PD-1 recombinant protein. The resulting human PD-1 recombinant protein was subcutaneously immunized into female BALb / C mice. Splenocytes from the immunized BALb / C mice were fused with mouse myeloma cells, and the resulting hybridomas were then screened for antigen-specific antibodies. Hybridomas that were screened for positive hPD-1 protein antibody binding were subcloned at least twice by limiting dilution, and stable subclones were cultured in vitro to generate small amounts of antibody. After further screening, the PD-1-112-C2 clone was obtained. SEQ ID NO: 8:TYYMY SEQ ID NO: 9: GINPSNGGTNFNEKFKS SEQ ID NO: 10: RDSNYDGGFDY SEQ ID NO: 11: RASKSVSTSGYSYMH SEQ ID NO: 12: LAYHLES SEQ ID NO: 13: QHSWELPIT SEQ ID NO: 14: QVQLQQPGAELVKPGASVKLSCKASGYTFTTYYMYWVKQRPGQGLEWIGGINPSNGGTNFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRRDSNYDGGFDYWGQGTTLTVSS SEQ ID NO: 15: DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLAYHLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSWELPITFGSGTKLEIKR

[0078] Based on SEQ ID NO: 14 and SEQ ID NO: 15, the humanized template with the best fit to the non-CDR region was selected from the Germline database. The antibody heavy chain template was IGHV1, and the antibody light chain template was IGKV1. Based on the principles 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 or phosphorylation, and not introducing sites such as oxidation or amination, thereby enhancing structural stability, the heavy chain humanized sequence was designed as VH1-5, and the light chain humanized sequence was designed as VL1-3. At the same time, the light chain was designed using the most identical IGKV7-3*01 as a separate group of humanized design templates, and the humanized sequence VL-4 was designed. The light and heavy chain pairing format was designed with the commonly seen IGHV1 / IGKV2 pairing format, resulting in a humanized antibody.

[0079] Genes were synthesized based on the amino acid sequences of the light and heavy chains of each humanized antibody. After double digestion with HindIII (NEB) and EcoRI (NEB), the gene fragments were inserted into the pCDNA3.4A expression vector (Invitrogen) via the HindIII (NEB) / EcoRI (NEB) digestion sites using T4 DNA ligase (TAKARA2011A). HEK293 cells (LifeTechnologics Cat. No. 11625019) were transfected with the expression vector and transfection reagent PEI (Polyscience, Inc. Cat. No. 23966) at a 1:2 ratio and incubated in a CO2 incubator for 5–7 days. The expressed antibodies were collected by centrifugation and purified according to standard methods to obtain the anti-PD-1 humanized antibodies of the present invention (c11, c21, c31, c41, c51, c12, c22, c32, c42, c52, c43, c53, c44, and c54). The amino acid sequences of the three anti-PD-1 humanized antibodies (c22, c43, and c53) are shown in Table 5.

[0080] [Table 5]

[0081] SEQ ID NO:3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKSRVTMTVDKSTSTAYMELSSLRSEDTAVYYCTRRDSNYDGGFDYWGQGTTVTVSS

[0082] SEQ ID NO:4: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMYWVRQAPGQGLEWIGGINPSNGGTNYAEKFKGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRRDSNYDGGFDYWGQGTTVTVSS

[0083] SEQ ID NO:5: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMYWVRQAPGQGLEWMGGINPSNGGTNYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCTRRDSNYDGGFDYWGQGTTVTVSS

[0084] SEQ ID NO:6: DIQLTQSPSSLSASVGDRATITCRASKSVSTSGYSYMHWYQQKPGKAPKLLIYLAYHLESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQHSWELPITFGQGTKLEIKR

[0085] SEQ ID NO:7: DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKAPKLLIYLAYHLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSWELPITFGQGTKLEIKR

[0086] Example 10. Affinity characterization of anti-PD-1 humanized antibodies 1. Experimental method Two cell lines, a Chinese hamster ovary (CHO) cell line expressing recombinant human PD-1 on its cell surface (CHO-hPD1) and a CHO cell line expressing monkey PD-1 (CHO-cynoPD1), were used to evaluate the binding characteristics of anti-PD-1 humanized candidate monoclonal antibodies (c22, c43, and c53) by flow cytometry (FCM). The specific method is as follows:

[0087] To evaluate the binding of anti-humanized antibodies to CHO-hPD1, 2 x 10 cells were placed in a 96-well plate. 5 CHO-hPD1 cells were incubated with gradient-diluted humanized antibody (initial concentration 30 μg / ml, 3-fold gradient dilution) at 4°C for 30 minutes. After washing the cells once with buffer (PBS containing 3% BSA), a PE-labeled fluorescent anti-human IgG (Fc) Ab (Biolegend) secondary antibody was added and incubated at 4°C for 30 minutes. The cells were then washed once with buffer and resuspended in PBS. The cell suspension was then subjected to flow cytometry analysis using a CytoFlex (Beckman flow cytometer) to measure the amount of antibody bound to the cells by the mean fluorescence intensity (MFI) of the staining. The same method was used to evaluate this anti-humanized antibody and CHO-cyno cells.

[0088] 2. Experimental results The affinity characterization of the anti-PD-1 humanized antibodies is shown in Table 6. The results demonstrate that all of the anti-PD-1 humanized antibodies of the present invention bind with high affinity to CHO-hPD1 cells and CHO-cynoPD1 cells.

[0089] [Table 6]

[0090] Example 11 Binding of humanized anti-PD-1 antibodies to activated PBMCs 1. Experimental method Fresh human peripheral blood mononuclear cells (PBMCs) were stimulated with PHA (Sigma) to activate and proliferate lymphocytes. On day 3, PD1 expression was at its highest. This allowed for binding experiments between anti-PD-1 humanized antibodies (c22, c43, and c53) and the naturally expressed PD1 of activated lymphocytes. The specific method is as follows:

[0091] PBMCs were obtained from fresh human peripheral blood by gradient centrifugation with lymphoid isolation fluid, and 1 × 10 6 The cells were adjusted to a density of 1000 cells / ml and inoculated into T75 cells. At the same time, PHA-L (Sigma) was added at a final concentration of 1 μg / ml to stimulate lymphocyte proliferation. After 3 days of incubation in an incubator at 37°C with 5% CO2, the cell suspension was removed, the supernatant was removed by centrifugation, and the cells were resuspended in buffer (PBS containing 3% BSA) and plated at 2E5 / well into a 96-well U-plate. A gradient of humanized antibodies was then added and incubated at 4°C for 30 minutes, followed by centrifugation at 300 g for 5 minutes. The cells were washed once with buffer, and a PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated at 4°C for 30 minutes. The cells were then washed once by centrifugation, resuspended in PBS, and analyzed by CytoFlex flow cytometry to detect the amount of antibody bound to PBMCs.

[0092] 2. Experimental results The measurement results of the binding ability of the anti-PD-1 humanized antibodies to activated PBMCs are shown in Table 6. The results demonstrate that the anti-PD-1 humanized antibodies of the present invention bind to activated lymphocytes with high affinity.

[0093] The anti-PD-1 humanized antibodies (c22, c43, c53) of the present invention have comparable binding abilities to CHO-hPD1 cells, CHO-cyno cells, and activated PBMCs compared to the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 of Examples 2 and 3 (Table 1).

[0094] Example 12 Binding specificity of anti-PD-1 humanized antibodies 1. Experimental method The anti-PD-1 humanized antibodies of the present invention (c22, c43, and c53) were conjugated to four different CD28 family member proteins to confirm the binding specificity of the anti-PD-1 humanized antibodies to PD-1. Using standard ELISA techniques, PD-1 (Acro), CD28 (Acro), CTLA-4 (Acro), and ICOS were immobilized on an ELISA plate at a concentration of 1 μg / ml, and the humanized antibodies were added at a concentration of 10 μg / ml. Anti-human IgG (Fab) conjugated with peroxidase (HRP) served as the secondary antibody. After color development with TMB and quenching, the OD450 values ​​were read using a microplate reader.

[0095] 2. Experimental results The binding specificity results of the anti-PD-1 humanized antibodies are shown in Table 7. The results demonstrate that all of the anti-PD-1 humanized antibodies of the present invention specifically bind to PD-1, but do not bind to other members of the CD28 family.

[0096] [Table 7]

[0097] Example 13: Affinity measurement of anti-PD-1 humanized antibodies 1. Experimental method ForteBio (OctetQke) affinity assay: PD-1-his recombinant protein at a concentration of 5 μg / ml was loaded onto a HISIK biosensor for 120 seconds. The loaded sensor was then equilibrated with standard buffer (PBST, PBS + 0.02% Tuween 20) for 120 seconds. The sensor was then transferred to a diluted solution of anti-PD-1 humanized antibodies (c22, c43, c53) for 180 seconds to measure the binding rate, and then transferred to standard buffer for 20 minutes to measure the dissociation rate. Final analysis and data processing were performed using a kinetic model. Opdivo (ABA0333) was used as a positive control.

[0098] 2. Experimental results The affinity measurement results of the anti-PD-1 humanized antibodies are shown in Table 8. The results demonstrate that all of the anti-PD-1 humanized antibodies of the present invention bind to PD-1 with high affinity.

[0099] The anti-PD-1 humanized antibodies of the present invention have affinity comparable to that of the anti-PD-1 mouse monoclonal antibody PD-1-112-C2 (Table 3) in Example 5. The anti-PD-1 humanized antibodies of the present invention have significantly improved affinity compared to the positive control Opdivo (Table 3).

[0100] [Table 8]

[0101] Example 14: Inhibition of binding of anti-PD-1 humanized antibodies to ligands PD-L1 / PD-L2 and CHO-hPD1 1. Experimental method Flow cytometry was used to analyze the ability of anti-PD-1 humanized antibodies to block the binding of ligands to PD-1 stably expressed on the surface of transfected CHO cells. The ligand proteins used in the experiments were recombinant PD-L1 / PD-L2 extracellular segments fused to a mouse IgG1 Fc segment: PD-L1-mFc and PD-L2-mFc.

[0102] CHO-PD1 cells were resuspended in buffer (PBS containing 3% BSA) to a density of 2 × 10 6 The cell suspension was adjusted to cells / ml, and 100μl / well of the cell suspension was added to a 96-well U-plate. After centrifugation at 300g for 5 minutes, the supernatant was removed. PD-L1-mFc / PD-L2-mFc was added to the cell wells at a concentration of 0.2μg / ml and incubated at 4℃ for 30 minutes. Gradient-diluted anti-PD-1 humanized antibodies (c22, c43, c53) were then added and incubated at 4℃ for 30 minutes.

[0103] The cells were centrifuged at 300 g for 5 min and washed once with buffer, followed by the addition of PE-labeled goat anti-mouse IgG fluorescent antibody (Biolegend) and incubation for 30 min at 4°C. The cells were washed once by centrifugation and resuspended in PBS. The amount of ligand protein bound to the cells was detected by CytoFlex flow cytometry analysis, and the IC of PD-1 antibody binding inhibition was calculated. 50 The value was calculated.

[0104] 2. Experimental results The affinity assay results for the anti-PD-1 humanized antibodies are shown in Table 9. The results demonstrate that all of the anti-PD-1 humanized antibodies of the present invention can effectively inhibit the binding of PD-L1 / PD-L2 to CHO-PD1 cells.

[0105] [Table 9]

[0106] Example 15: Effect of anti-PD-1 humanized antibody on mixed lymphocyte reaction 1. Experimental method In mixed lymphocyte reaction (MLR), the presence or absence of anti-PD-1 humanized antibodies can be used to demonstrate T cell proliferation and cytokine secretion levels in the absence of PD1 signaling.

[0107] CD14 was isolated from fresh PBMCs using CD14 MicroBeads, human (Miltenyi). + Monocytes were isolated and induced in the presence of GM-CSF / IL-4 for 6 days, followed by the addition of TNF-α for 3 days to induce DC maturation. On the day of the experiment, T cells were purified from PBMCs using the EasySep™ Human T Cell Enrichment Kit (StemCell) and 1 × 10 4 cells / well DC cells and 1 x 10 5T cells were cultured at 1000 cells / well, and gradient concentrations of anti-PD-1 humanized antibodies (c22, c43, c53) were added to the culture. Isotype control antibodies were also added. After 3 days of culture, supernatants were collected for IL-2 assay. After another 2 days of culture, supernatants were collected for IFN-γ assay.

[0108] 3. Experimental results The effects of the anti-PD-1 humanized antibodies in the mixed lymphocyte reaction are shown in Table 10. The results show that the anti-PD-1 humanized antibodies of the present invention can inhibit the binding of PD1 to its ligand and inhibit the PD1 signaling pathway in the MLR experiment, thereby promoting T cell proliferation and T cell secretion of the cytokines IL-2 and IFN-γ.

[0109] [Table 10]

[0110] Example 16: Evaluation of the in vivo antitumor effect of anti-PD-1 humanized antibodies against mouse colon cancer cells 1. Experimental method Experimental objective: To measure the in vivo antitumor activity of anti-PD-1 humanized antibody (c53) against mouse colon cancer cells (MC38 cells), and also to establish an isotype control group (Isotype) and a positive control group (Sintilimab).

[0111] 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 Resources Platform); FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02), penicillin-streptomycin (Gibco, 15140122), DMEM high-sugar medium (Gibco, 11965084), fetal bovine serum (Gibco), glutamine (Gibco).

[0112] Equipment: Electronic balance (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd., JA12002), Vernier caliper (Shanghai Meitai Industrial Co., Ltd., MNT-150T), Microscope (Chongqing Akutaku Instruments Co., Ltd., BDS200), Medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L53) 0R), digital display constant temperature water tank (Prusis Machinery Co., Ltd., HH-S), carbon dioxide gas incubator (Japan Matsushita Health Medical Equipment Co., Ltd., MCO-18AC), 2-person vertical ultra clean table (Wuxi Yi Purification Equipment Co., Ltd., SW-CJ-VS2).

[0113] Testing Procedure: Cell culture: MC38 cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 1% glutamine, and 1% penicillin-streptomycin (1:1).

[0114] Inoculation: Collect MC38 cells in the logarithmic growth phase and inoculate them to a cell concentration of 3 × 10 6 MC38 cells were administered to 40 female hPD1 mice at 0.1 mL / mouse, i.e., 3 × 10 5 The mice were subcutaneously inoculated with the vaccine.

[0115] Administration: The day of inoculation was recorded as day 0 (D0), and on day 7, mice were randomly divided into three groups of eight mice each according to tumor volume, and administration began (the dosage, mode, and frequency of administration for the MC38 tumor model are shown in Table 11). [Table 11]

[0116] Recording: From D7 onwards, the tumor volume was measured and recorded. Subsequently, the long and short diameters of the tumor were measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: (1 / 2) × long diameter × (short diameter). 2 When each mouse reached the end point of the experiment (tumor volume of 2000 mm), 3 The benign endpoint was reached when the serotonin level exceeded 100 mg / kg / day. Mice were sacrificed by cervical dislocation and survival curves were recorded.

[0117] 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. Compared to the isotype group, the anti-PD-1 humanized antibody (c53) had a significant tumor-inhibitory effect on tumor growth in the MC38 tumor model (TGI = 104.44%, tumors completely disappeared in 7 mice), which was comparable to the anti-tumor effect of the sintilimab group (TGI = 105.81%, tumors completely disappeared in 7 mice).

[0118] The effects of anti-PD-1 humanized antibodies on mouse survival are shown in Figure 5. It can be seen that anti-PD-1 humanized antibody (c53) significantly extended mouse survival compared to the isotype group.

[0119] [Table 12]

[0120] These results demonstrate that the anti-PD-1 humanized antibody (c53) provided by the present invention significantly inhibits the growth of MC38 cells and effectively extends the survival time of mice, demonstrating its significant efficacy in treating mouse colon cancer.

[0121] The above examples are preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention are all considered equivalent substitutions and are included in the scope of the claims of the present invention.

Claims

1. an anti-PD-1 humanized antibody or an antigen-binding fragment thereof, comprising a light chain CDR region and a heavy chain CDR region, wherein the heavy chain CDR region consists of HCDR1, HCDR2, and HCDR3, and the light chain CDR region consists of LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are set forth in SEQ ID NOs: 11 to 13, respectively; An anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in any one of SEQ ID NOs: 3 to 5.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the amino acid sequence of the light chain variable region of the antibody is set forth in any one of SEQ ID NOs: 6 to 7.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 3 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 6, or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 4 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 7, or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 5 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

7.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, and the light chain constant region being a κ chain or a λ chain.

5. The antibody is a chimeric or multispecific antibody, and the antigen-binding fragment is F(ab'). ２ 2. The antibody or antigen-binding fragment thereof according to claim 1, which is any one or more of Fab, scFv, Fv and single domain antibodies.

6. A nucleic acid encoding the anti-PD-1 humanized antibody or antigen-binding fragment thereof according to claim 1.

7. 7. The nucleic acid according to claim 6, wherein the nucleic acid comprises a first nucleic acid encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof, and a second nucleic acid encoding a light chain variable region of the antibody or antigen-binding fragment thereof.

8. A vector comprising the nucleic acid according to claim 6.

9. A cell comprising the nucleic acid of claim 6, the vector of claim 8, or capable of expressing the antibody or antigen-binding fragment thereof of claim 1.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid according to claim 6, or the vector according to claim 8.

11. Use of the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid according to claim 6, or the vector according to claim 8 in the manufacture of a drug for treating a PD-1-mediated disease or condition.

12. Use of the pharmaceutical composition of claim 10 in the manufacture of a drug for treating a PD-1 mediated disease or condition.

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