An anti-PD-1 humanized antibody and its application

Abstract

The present invention relates to the field of biomedicine, and more specifically, to a humanized anti-PD-1 antibody and its use. The antibody can efficiently and specifically bind to PD-1 and effectively block the binding of PD-1 to PD-L1 and PD-L2. The antibody or its antigen-binding fragment, as well as its related nucleic acid, vector, cell, or pharmaceutical composition, can be used to prepare a drug for treating PD-1-mediated diseases or conditions.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. More specifically, it relates to an anti-PD-1 humanized antibody and its application. Background Art

[0002] Programmed death factor 1 (PD1) is a member of the CD28 family and is expressed on activated B cells, T cells, and myeloid cells. Human PD1 is encoded by the gene Pdcd1, located at 2q37.3. It is 9.6 kb long and consists of five exons and four introns, with a 663-bp promoter upstream. PD1 is a 55 kDa type I transmembrane protein composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains an immunoglobulin variable region (IgV) domain, while the intracellular domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch interaction module (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 primarily recruits the tyrosine phospholipase SHP2 through the ITIM, leading to dephosphorylation of downstream effector molecules.

[0003] PD-1 has two ligands: PD-L1 and PD-L2. Both PD-L1 and PD-L2 are B7 homologs. The PDL gene is located at locus 24.2 on human chromosome 9 and is 42 kb in size. Its molecular structure consists of an immunoglobulin-like variable region, a constant region-like domain, a transmembrane region, and a short cytoplasmic tail.

[0004] PD-1 binds to PD-L1 and PD-L2, which can downregulate T cell activation. PD-L1 is expressed on the surface of various tumor cells, including lung, gastric, liver, esophageal, kidney, ovarian, cervical, breast, skin, colon, bladder, glioma, head and neck, and oral squamous cell carcinoma. Numerous CD8+ T cells expressing PD-L1 are found in the vicinity of these cancers. Clinical results show that high levels of PD-L1 expression on tumor cells are associated with a poor prognosis in cancer patients. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing antibodies, such as low affinity and specificity for binding to PD-1 and inability to effectively block the binding of PD-1 to PD-L1 and PD-L2, and to provide an anti-PD-1 humanized antibody that can efficiently bind to PD-1 and effectively block the binding of PD-1 to PD-L1 and PD-L2.

[0006] The object of the present invention is to provide an anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2, and HCDR3, and the light chain CDR region consists of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 12 to 14, respectively. The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs: 3 to 5; the amino acid sequence of the light chain variable region of the antibody is shown in any one of SEQ ID NOs: 6 to 8.

[0007] Another object of the present invention is to provide nucleic acids, vectors, cells or pharmaceutical compositions related to the antibodies or antigen-binding fragments thereof.

[0008] The present invention also relates to the use of the antibody or antigen-binding fragment thereof, and related nucleic acids, vectors, cells or pharmaceutical compositions in the preparation of drugs for treating PD-1-mediated diseases or conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a graph showing the effects of different concentrations of PD-1-76-C2 on IL-2 / IFN-γ secretion.

[0010] Figure 2 This is a graph showing the effects of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IL-2.

[0011] Figure 3 This is a graph showing the effects of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IFN-γ.

[0012] Figure 4 This is a graph showing the effects of anti-PD-1 humanized antibodies h31, h61, and h43 on tumor volume.

[0013] Figure 5 This is a graph showing the effects of anti-PD-1 humanized antibodies h31, h61, and h43 on mouse survival. DETAILED DESCRIPTION

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

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

[0016] The present invention relates to an anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2, and HCDR3, and the light chain CDR region consists of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 12 to 14, respectively. The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs: 3 to 5; the amino acid sequence of the light chain variable region of the antibody is shown in any one of SEQ ID NOs: 6 to 8.

[0017] The present invention uses the Kabat numbering system to identify CDR regions, but CDR regions identified by other methods also fall within the scope of protection of the present invention.

[0018] In the present invention, the term "specific binding" or similar expressions refers to the binding of an antibody or its antigen-binding fragment to a predetermined epitope on an antigen. -6 M, for example, less than approximately 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D KD refers to the ratio of the off-rate to the on-rate (koff / kon), which can be measured by methods familiar to those skilled in the art.

[0019] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region 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, and the amino acid sequence of the light chain variable region 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:5, and the amino acid sequence of the light chain variable region 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, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0020] In some embodiments, the antibody contains a heavy chain constant region and a light chain constant region, the heavy chain constant region is any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM; the light chain constant region is a κ chain or a λ chain.

[0021] In some embodiments, the species origin of the heavy chain constant region and the light chain constant region is selected from human, mouse or monkey.

[0022] In some embodiments, the antibody is a chimeric antibody or a multispecific antibody (eg, a bispecific antibody).

[0023] In the present invention, the term "multispecific antibody" refers to an antigen binding protein or antibody that targets more than one antigen or epitope.

[0024] As used herein, the term "bispecific antibody" refers to a multispecific antigen-binding protein or antibody and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. 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 will bind to two different epitopes, either on the same or different protein targets.

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

[0026] In the present invention, the term "F(ab')2" refers to two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.

[0027] In the present invention, the term "Fab" is composed of a light chain and CH1 and the variable region of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

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

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

[0030] In this context, the term "single-domain antibody" refers to a single heavy chain variable region (VHH) and two conventional CH2 and CH3 domains. However, unlike engineered single-chain antibodies (scFvs), these antibodies are not prone to adhesion or even aggregation. More importantly, the individually cloned and expressed VHH structure possesses comparable structural stability and antigen-binding activity to the original heavy chain antibody, representing the smallest known unit capable of binding to the target antigen.

[0031] The present invention also relates to a nucleic acid encoding the anti-PD-1 humanized antibody or its antigen-binding fragment.

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

[0033] In the present invention, nucleic acids are typically RNA or DNA. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. When incorporated into a vector, DNA is preferably used. In addition, since antibodies are membrane proteins, nucleic acids typically carry a signal peptide sequence.

[0034] The present invention also relates to a vector, wherein the vector carries the nucleic acid.

[0035] In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0036] The present invention also relates to a cell, wherein the cell carries the nucleic acid, contains the vector, or is capable of expressing the antibody or the antigen-binding fragment thereof.

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

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

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

[0040] In the present invention, the term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible and serve to prolong the shelf life or effectiveness of the antibody.

[0041] In addition, the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell or the pharmaceutical composition in the preparation of a drug for treating a PD-1-mediated disease or condition should also be within the scope of protection of the present invention.

[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 injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0044] The present invention has the following beneficial effects:

[0045] The present invention provides an anti-PD-1 humanized antibody or an antigen-binding fragment thereof. The antibody has high affinity and strong specificity, can bind to CHO-hPD1 cells, CHO-cyno cells and activated PBMCs with high affinity, efficiently and specifically binds to PD-1, effectively blocks the binding of the ligands PD-L1 / PD-L2 to CHO-hPD1, and can block the binding of PD-1 to the ligand in the MLR, inhibit the PD-1 signaling pathway, thereby promoting T cell proliferation and the secretion of IL-2 and IFN-γ cytokines; therefore, the antibody or antigen-binding fragment thereof, and related nucleic acids, vectors, cells or pharmaceutical compositions have broad application prospects in the preparation of drugs for treating PD-1-mediated diseases or conditions.

[0046] Example 1 Preparation of anti-PD-1 antibodies

[0047] 1. Immunogen

[0048] The human PD-1 sequence (NCBI NP 005009) was artificially synthesized using the upstream primer: 5'-CCGCAAGCTTGCCGCCACCATG-3' (SEQ ID NO: 1) and the downstream primer: 5'-CCGGAATTCTCATTAATGGTGATGGTGATGATGCTGGAACTGGCCGGCA GGTC-3' (SEQ ID NO: 2). The extracellular end was amplified by PCR, digested with Hind III and EcoRI, and cloned into the pCDNA3.4A eukaryotic expression system. 293 cells were transfected with this plasmid, and the supernatant was harvested and purified to obtain human PD-1 recombinant protein (hPD-1).

[0049] 2. Immunization of Animals

[0050] 125 μg of hPD-1 recombinant protein at a concentration of 1.23 mg / ml was mixed as an antigen with an equal amount of Freund's adjuvant (Sigma-Aldrich F5881) and five six-week-old female BAL b / C mice were subcutaneously immunized with 25 μg of antigen per mouse. After the initial immunization, booster immunizations of the same dose were performed weekly. After a total of five immunizations, the immune response was monitored by collecting tail blood from the mice. Mice with sufficient anti-hPD-1 immunoglobulin titers were screened by FACS (as described below) and used for fusion. Three days after intraperitoneal booster immunization with antigen, the mice were sacrificed and the spleens were removed for cell fusion.

[0051] 3. Selection of BAL b / C mice producing anti-hPD-1 antibodies

[0052] To select BAL b / C mice that produce anti-hPD-1 antibodies, immunized mouse sera were tested by FACS. Sera dilutions from mice immunized with hPD-1 recombinant protein were incubated with hPD1-transfected CHO cells at 4 degrees Celsius for 30 minutes. After washing three times with PBS, 0.4 μg / ml of PE goat anti-mouse IgG (Biolegend 405307) was added and incubated at 4 degrees Celsius for 30 minutes. After washing three times with PBS, the samples were placed in a Beckman Coulter flow cytometer (CytoFLEXA00-1-1102) to verify whether they could bind to hPD1-transfected CHO cells. BAL b / C mice that produce anti-hPD-1 antibodies were screened and then cell fusion was performed.

[0053] 4. Generation of Hybridomas Producing Mouse Monoclonal Antibodies Targeting hPD-1

[0054] Splenocytes from immunized BAL b / C mice were fused with mouse myeloma cells, and the resulting hybridomas were screened for antigen-specific antibodies. Single-cell suspensions of splenocytes from immunized mice were fused with one-fifth the number of immunoglobulin-free mouse myeloma cells (SP2 / 0, ATCC CRL1581) using PEG 1500 (Roche 10783641001). The fused cells were plated at approximately 1×10 5 Cells were plated at 1000 cells / well in a 96-well cell culture plate and placed in an incubator (Panasonic MCO-18AIC) at 37°C with 5% CO2. They were then cultured for approximately one week in HAT selective medium (1640 medium containing 1X penicillin-streptomycin (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 (Gibco 15140122), 1X HT (Gibco 11067030), and 20% fetal bovine serum (Royacel RY-F11-01)). Cell culture supernatants from the confluent plates were then analyzed by FACS to screen for hybridomas that secrete antibodies that bind to the hPD-1 protein. Hybridomas secreting antibodies that bind to the hPD-1 protein were replated and screened again. Hybridomas positive for hPD-1 binding were subcloned at least twice by limited release. Stable subclones were then cultured in vitro to generate small amounts of antibody for further analysis. Hybridoma clone PD-76-C2 was selected for further analysis.

[0055] Example 2 Affinity Characterization of Anti-PD-1 Mouse Monoclonal Antibodies

[0056] According to conventional methods, recombinant technology was used to prepare a CHO (Chinese Hamster Ovary) cell line (CHO-hPD1) expressing recombinant human PD-1 on the cell surface, a CHO cell line (CHO-cynoPD1) expressing monkey PD1 (Uniprot: B0LAJ2), and a CHO cell line (CHO-mousePD1) expressing mouse PD1 (Uniprot: Q02242). The cell lines will be used for flow cytometry (FCM) to determine the binding characterization of the anti-PD-1 mouse monoclonal antibody PD-1-76-C2.

[0057] To evaluate the binding of anti-PD-1 mouse monoclonal antibody to CHO-hPD1, 2 × 10 5CHO-hPD1 cells and a concentration gradient dilution (initial concentration of 10 μg / ml, three-fold dilution) of an anti-PD-1 mouse monoclonal antibody were incubated at 4°C for 30 minutes. The cells were washed once with buffer (PBS containing 3% BSA) and then a PE-labeled anti-mouse IgG (Fc) Ab (Biolegend) fluorescent secondary antibody was added. After incubation at 4°C for 30 minutes, the cells were washed once with buffer and resuspended in PBS. The cell suspension was then analyzed by flow cytometry using a CytoFlex (Beckman flow cytometer). The amount of antibody bound to the cells was measured based on 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 the present invention) cells.

[0058] The results are shown in Table 1. The data show that the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 can bind to CHO-hPD1 cells and CHO-cyno cells with high affinity; at the same time, the mouse monoclonal antibody does not bind to CHO-mousePD1 cells.

[0059] Example 3 Binding of anti-PD-1 antibodies to activated PBMCs

[0060] Fresh human peripheral blood mononuclear cells (PBMC) can be activated and proliferated under the stimulation of PHA (Sigma), and PD1 is expressed at the highest abundance on the third day. It can be used to conduct binding experiments between PD-1 antibodies and activated lymphocytes naturally expressing PD1.

[0061] Fresh human peripheral blood was centrifuged through lymphocyte separation gradient centrifugation to obtain PBMCs, and the density was adjusted to 1×10 6 1 μg / ml of PHA-L (Sigma) was added to stimulate lymphocyte proliferation. After standing in a 37°C, 5% CO2 incubator for 3 days, the cell suspension was removed, centrifuged to remove the supernatant, and resuspended in buffer (PBS containing 3% BSA) and the cells were plated at 2×10 5 Cells were added to a 96-well U-shaped plate, and then anti-PD-1 antibodies were added in a 3-fold gradient dilution starting from 30 μg / ml, with a total of 10 concentration gradients. After incubation at 4°C for 30 minutes, the cells were centrifuged at 300g for 5 minutes, and the cells were washed once with buffer. PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated at 4°C for 30 minutes. After washing the cells once by centrifugation, the cells were resuspended in PBS and analyzed by CytoFlex flow cytometer to detect the amount of antibody bound to PBMCs.

[0062] The results are shown in Table 1. Anti-PD1 antibodies can bind to activated lymphocytes with high affinity.

[0063] Table 1

[0064]

[0065] Example 4 Binding Specificity of Anti-PD-1 Mouse Monoclonal Antibodies

[0066] Anti-PD-1 mouse monoclonal antibodies were bound to four different CD28 family proteins to verify the specificity of the antibody binding to PD-1. Using a standard ELISA protocol, PD-1, CD28, CTLA-4, and ICOS (ACRO) were immobilized on an ELISA plate at a concentration of 1 μg / ml. Anti-human PD-1 mouse monoclonal antibodies were added at a concentration of 10 μg / ml, and peroxidase (HRP)-conjugated anti-mouse IgG (Sigma) was used as a secondary antibody. TMB was used for color development, and after termination, the OD450 value was read on a microplate reader.

[0067] The results are shown in Table 2. The anti-PD-1 mouse monoclonal antibody PD-1-76-C2 can specifically bind to PD-1 but not to other CD28 family members.

[0068] Table 2

[0069]

[0070] Example 5 Determination of affinity of anti-human PD-1 mouse monoclonal antibody by biolayer interferometry (BLI)

[0071] ForteBio (Octet Qke) affinity assay: HISIK biosensors were loaded with 5 μg / ml of PD-1-his (ACRO) recombinant protein for 120 seconds. The loaded sensors were then equilibrated in standard buffer (PBST, PBS + 0.02% Tuween 20) for 120 seconds. The sensors were then transferred to a dilution of anti-PD-1 mouse monoclonal antibody for 180 seconds to measure association rates, and then transferred to standard buffer for 20 minutes to measure dissociation rates. Kinetic modeling was used for analysis.

[0072] The data processing results are shown in Table 3.

[0073] Table 3

[0074] Antibodies to be tested kon(1 / Ms) kdis(1 / s) KD(M) Opdivo (ABA0333) 1.38E+06 3.63E-06 2.62E-12 PD-1-76-C2 7.71E+05 <1.0E-07 <1.0E-12

[0075] Example 6 Anti-PD-1 Mouse Monoclonal Antibody Blocks Binding of Ligands PD-L1 / PD-L2 to CHO-hPD1

[0076] Flow cytometry was used to analyze the ability of anti-PD-1 mouse monoclonal antibodies to block ligand binding to transfected CHO cells stably expressing PD-1. The ligand proteins used in the experiments were recombinant PD-L1 / PD-L2 extracellular domains linked to the human IgG1 Fc domain: PD-L1-hFc (ACRO) and PD-L2-hFc (ACRO).

[0077] CHO-PD1 cells were resuspended in buffer (PBS containing 3% BSA) and the density was adjusted to 2×10 6 cells / ml, 100 μl / well cell suspension was added to a 96-well U-shaped plate, centrifuged at 300 g for 5 minutes, and the supernatant was removed.

[0078] The subsequent process can be divided into two blocking modes: Mode 1, add PD-L1-hFc / PD-L2-hFc at a concentration of 3 μg / ml to the cell wells, incubate at 4°C for 30 minutes, and then add anti-PD-1 mouse monoclonal antibody starting from 30 μg / ml, 3-fold gradient dilution, a total of 10 concentration gradients, and incubate at 4°C for 30 minutes; Mode 2, add anti-PD-1 mouse monoclonal antibody starting from 30 μg / ml, 3-fold gradient dilution, a total of 10 concentration gradients, and incubate at 4°C for 30 minutes, and then add PD-L1-hFc / PD-L2-hFc protein at a concentration of 3 μg / ml and incubate at 4°C for 30 minutes.

[0079] The cells were centrifuged at 300 g for 5 minutes, washed once with buffer, and PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated at 4°C for 30 minutes. After centrifugation and washing once, the cells were resuspended in PBS and analyzed by CytoFlex flow cytometry to detect the amount of ligand protein bound to the cells and calculate the IC value of PD-1 antibody binding blocking. 50 value.

[0080] The results are shown in Table 4. The anti-PD-1 mouse monoclonal antibody: PD-1-76-C2 can effectively block the binding of PD-L1 / PD-L2 to cell CHO-PD1 in both modes.

[0081] Table 4

[0082]

[0083] Example 7 Effect of anti-PD-1 antibodies on cytokine release from SEB-stimulated PBMC cells

[0084] In this example, the effects of cytokine secretion on peripheral blood mononuclear cells (PBMCs) cultured overnight were examined by stimulation with the superantigen Staphylococcus aureus enterotoxin B (SEB) in the presence or absence of anti-PD-1 antibodies.

[0085] Fresh peripheral mononuclear cells (PBMCs) were resuspended in X-VIVO 15 medium (LONZA) containing 10% FBS, added to T25 culture flasks, and cultured overnight at 37°C in 5% CO2. The next day, the suspended cells were collected, centrifuged, and resuspended in fresh X-VIVO medium (containing 10% FBS). SEB superantigen (Toxin technology) was added at a final concentration of 200 ng / ml, and then 1×10 cells were plated per well. 5 Cells were plated in a 96-well plate and supplemented with various concentrations of anti-PD-1 antibodies, along with isotype control antibodies (mIgG1 isotype control antibody (Biolegend) and hIgG4 isotype control antibody (Biolegend)), and no-antibody control wells. Three days later, samples were collected from the sample wells and IL-2 / IFN-γ levels were measured using the IL2 / IFN-γ Human Uncoated ELISA Kit (eBioscience).

[0086] The effects of different concentrations of PD-1-76-C2 on IL-2 / IFN-γ secretion are shown in the following table. Figure 1 As shown, anti-PD-1 antibodies increased IL-2 / IFN-γ secretion in a concentration-dependent manner. The results showed that in PBMCs stimulated with SEB superantigen, anti-PD-1 antibody: PD-1-76-C2 can further promote T cell cytokine secretion.

[0087] Example 8 Effect of anti-PD-1 antibodies in mixed lymphocyte reaction

[0088] In mixed lymphocyte reaction (MLR), the presence or absence of anti-PD-1 antibodies can demonstrate the T cell proliferation and the level of cytokine secretion by T cells when PD1 signaling is blocked.

[0089] CD14 was isolated from fresh PBMC using CD14 MicroBeads, human (Miltenyi) + Monocytes were induced in the presence of GM-CSF / IL-4 for 6 days, and TNF-α was added to induce DC maturation 3 days later. On the day of the experiment, EasySep TM Human T Cell Enrichment Kit (StemCell) was used to purify T cells from PBMC, 1×10 4 DC cells with 1×10 5T cells were mixed and cultured, and anti-PD-1 antibodies were added to the mixed cells at varying concentrations. Isotype control antibodies (mIgG1 and hIgG4 (Biolegend)) were also added, along with no-antibody control wells. After three days of mixed culture, the supernatant was collected for IL-2 detection. After another two days of culture, the supernatant was collected for IFN-γ detection.

[0090] The effects of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IL-2 are shown in the following table. Figure 2 The effects of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IFN-γ are shown in Figure 3 As shown, Figure 2 and Figure 3 The results showed that the anti-PD-1 antibody: PD-1-76-C2 can block the binding of PD1 to the ligand in an antibody concentration-dependent manner in the MLR experiment, inhibit the PD1 signaling pathway, thereby promoting T cell proliferation and promoting T cell secretion of IL-2 and IFN-γ cytokines.

[0091] Example 9 Humanization of anti-PD-1 mouse monoclonal antibody

[0092] The anti-PD-1 mouse monoclonal antibody PD-1-76-C2 obtained above (the amino acid sequences of its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 9 to 11, respectively, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 12 to 14, respectively, the heavy chain variable region sequence is shown in SEQ ID NO: 15, and the light chain variable region sequence is shown in SEQ ID NO: 16) was humanized by the following method:

[0093] The human PD-1 sequence (NCBI NP 005009) was artificially synthesized and cloned into the PCDNA3.4A eukaryotic expression system. This plasmid was transfected into 293 cells, and the supernatant was harvested and purified to obtain recombinant human PD-1 protein. The resulting recombinant human PD-1 protein was subcutaneously immunized in female BAL b / C mice. Splenocytes from these immunized BAL b / C mice were fused with mouse myeloma cells, and the resulting hybridomas were screened for antigen-specific antibodies. Hybridomas positive for antibodies binding to the hPD-1 protein were subcloned at least twice by limiting dilution. Stable subclones were cultured in vitro to produce small amounts of antibodies, and further screening was performed to obtain the PD-1-76-C2 clone.

[0094] SEQ ID NO:9:NYGMN

[0095] SEQ ID NO:10:WINTHTGEPTYADDFKG

[0096] SEQ ID NO:11:EGEGIGFAY

[0097] SEQ ID NO:12:RSSQSIVYSNGKTYLE

[0098] SEQ ID NO:13:KVSNRFS

[0099] SEQ ID NO:14:FQGSHVPNT

[0100] SEQ ID NO: 15:

[0101] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQIKNLKNEDVATYFCTKEGEGIGFAYWGQGTLVTVSA

[0102] SEQ ID NO: 16:

[0103] DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPNTFGGGTKLEIKR

[0104] With reference to SEQ ID NO: 15 and SEQ ID NO: 16, the humanized template that best matched their non-CDR regions was selected from the Germline database, wherein the template for the antibody heavy chain was IGHV1 / 7 and the template for the antibody light chain was IGKV2; under the principle of not affecting the structural stability of the antibody, not affecting the binding of the antibody to the antigen, not introducing protein modification sites such as glycosylation and phosphorylation, not introducing sites that have been oxidized and aminoated, and enhancing structural stability, the heavy chain humanized sequence was designed to be VH1-6, the light chain humanized sequence was designed to be VL1-3, and the light and heavy chain pairing form was designed to be IGHV1 / IGKV2 as the common pairing form to obtain a humanized antibody.

[0105] Genes were synthesized based on the amino acid sequences of the light and heavy chains of each humanized antibody. After double digestion with Hind III (NEB) and EcoRI (NEB), the gene fragments were inserted into the pCDNA3.4A expression vector (Invitrogen) using T4 DNA ligase (TAKARA 2011A) via the Hind III (NEB) / EcoRI (NEB) restriction sites. HEK293 cells (Life Technologies Cat. No. 11625019) were transfected with the expression vector and transfection reagent PEI (Poly Science, Inc. Cat. No. 23966) at a ratio of 1:2 and cultured in a CO2 incubator for 5-7 days. The expressed antibodies were recovered by centrifugation and purified according to conventional methods to obtain anti-PD-1 humanized antibodies (h11, h21, h31, h41, h51, h61, h12, h42, h13, h23, h33, h43, h53, and h63). The amino acid sequences of four anti-PD-1 humanized antibodies (h31, h61, h42, and h43) are shown in Table 5.

[0106] Table 5 Amino acid sequences of four humanized anti-PD-1 antibodies (h31, h61, h42, h43)

[0107]

[0108] SEQ ID NO: 3:

[0109] QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTKEGEGIGFAYWGQGTTVTVSS

[0110] SEQ ID NO: 4:

[0111] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQKFQGRVTMTLDTSISTAYMELSRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS

[0112] SEQ ID NO: 5:

[0113] QIQLVQSGAEVKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYADDFKGRFTFTLDTSISTAYLEISRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS

[0114] SEQ ID NO: 6:

[0115] DVVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR

[0116] SEQ ID NO: 7:

[0117] DIVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR

[0118] SEQ ID NO:8:

[0119] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGKTYLEWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR

[0120] Example 10 Determination of the binding ability of anti-PD-1 humanized antibodies to CHO-hPD1 cells and CHO-cyno cells

[0121] 1. Experimental methods

[0122] Using recombinant technology, a CHO (Chinese Hamster Ovary) cell line expressing recombinant human PD-1 on the cell surface (CHO-hPD1) and a CHO cell line expressing monkey PD1 (CHO-cynoPD1) will be used for flow cytometry (FCM) to determine the binding characteristics of the anti-PD-1 humanized candidate monoclonal antibody. The specific method is as follows:

[0123] To evaluate the binding of anti-PD-1 humanized antibodies to CHO-hPD1 cells, 2 × 10 5CHO-hPD1 cells and humanized antibodies diluted in a concentration gradient (initial concentration 30 μg / ml, three-fold dilution) were incubated at 4°C for 30 minutes. The cells were washed once with buffer (PBS containing 3% BSA) and then a PE-labeled anti-human IgG (Fc) Ab (Biolegend) fluorescent secondary antibody was added. After incubation at 4°C for 30 minutes, the cells were washed once with buffer and resuspended in PBS. The cell suspension was then analyzed by flow cytometry using a CytoFlex (Beckman flow cytometer). The amount of antibody bound to the cells was measured based on the mean fluorescence intensity (MFI) of the staining. The binding of anti-PD-1 humanized antibodies to CHO-cyno cells was evaluated using the same method.

[0124] 2. Experimental results

[0125] The results of the binding ability test of the anti-PD-1 humanized antibodies to CHO-hPD1 cells and CHO-cyno cells are shown in Table 6. The results show that the anti-PD-1 humanized antibodies of the present invention can bind to CHO-hPD1 cells and CHO-cyno cells with high affinity.

[0126] Example 11 Determination of the binding ability of anti-PD-1 humanized antibodies to activated PBMCs

[0127] 1. Experimental methods

[0128] Fresh human peripheral blood mononuclear cells (PBMC) are stimulated by PHA (Sigma), which activates and proliferates lymphocytes and expresses PD-1 at the highest abundance on the third day. This can be used to determine the binding ability of anti-PD-1 humanized antibodies to PD-1 naturally expressed by activated lymphocytes. The specific method is as follows:

[0129] Fresh human peripheral blood was centrifuged through lymphocyte separation gradient centrifugation to obtain PBMCs, and the density was adjusted to 1×10 6 cells / ml were inoculated into T75 cells, and PHA-L (Sigma) was added at a final concentration of 1 μg / ml to stimulate lymphocyte proliferation. After standing in a 37°C, 5% CO2 incubator for 3 days, the cell suspension was removed, the supernatant was removed by centrifugation, and the cells were resuspended in buffer (PBS containing 3% BSA) and added to a 96-well U-shaped plate at a rate of 2E5 / well. Then, different concentration gradients of anti-PD-1 humanized antibodies were added, incubated at 4°C for 30 minutes, and then centrifuged at 300g for 5 minutes. The cells were washed once with buffer, and PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated at 4°C for 30 minutes. After washing the cells once by centrifugation, the cells were resuspended in PBS and analyzed by CytoFlex flow cytometer to detect the amount of antibody bound to PBMC.

[0130] 2. Experimental results

[0131] The results of the binding ability test of the anti-PD-1 humanized antibody to activated PBMCs are shown in Table 6. The results show that the anti-PD-1 humanized antibody of the present invention can bind to activated PBMCs with high affinity.

[0132] Compared with the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 in Examples 2 and 3 (Table 1), the anti-PD-1 humanized antibody of the present invention has comparable binding ability to CHO-hPD1 cells, CHO-cyno cells and activated PBMCs.

[0133] Table 6 Results of the binding ability assay of anti-PD-1 humanized antibodies to CHO-hPD1 cells, CHO-cyno cells and activated PBMCs

[0134]

[0135]

[0136] Example 12 Binding Specificity of Anti-PD-1 Humanized Antibodies

[0137] 1. Experimental methods

[0138] The humanized anti-PD-1 antibody of the present invention was bound to four different CD28 family members to verify the specificity of the anti-PD-1 humanized antibody in binding to PD-1. Using a standard ELISA method, PD-1, CD28, CTLA-4, and ICOS at a concentration of 1 μg / ml were immobilized on an ELISA plate. Humanized anti-PD-1 antibodies (h31, h61, h42, and h43) were added at a concentration of 10 μg / ml, and an anti-human IgG (Fab) conjugated to peroxidase (HRP) was used as a secondary antibody. TMB was used for color development, and after termination, the OD450 value was read on a microplate reader.

[0139] 2. Experimental results

[0140] The binding specificity results of the anti-PD-1 humanized antibodies are shown in Table 7. The results show that the anti-PD-1 humanized antibodies can specifically bind to PD-1, but not to other proteins of the CD28 family members, indicating that the anti-PD-1 humanized antibodies of the present invention have high binding specificity to PD-1.

[0141] Table 7 Binding specificity results of anti-PD-1 humanized antibodies

[0142]

[0143] Example 13 Affinity determination of anti-PD-1 humanized antibodies

[0144] 1. Experimental methods

[0145] Based on biofilm interferometry (BLI) technology, using Fortebio The detection instrument measures the antibody affinity. The specific method is as follows:

[0146] The HISIK biosensor was loaded with 5 μg / ml of PD-1-his recombinant protein for 120 seconds. The loaded sensor was then equilibrated in standard buffer (PBST, PBS + 0.02% Tuween20) for 120 seconds. The sensor was then transferred to a dilution of anti-PD-1 humanized antibodies (h31, h61, h42, and h43) for 180 seconds to measure association rates, and then transferred to standard buffer for 20 minutes to measure dissociation rates. Kinetic modeling was used for analysis and data processing. Opdivo (ABA0333) was used as a positive control.

[0147] 2. Experimental results

[0148] The affinity measurement results of the anti-PD-1 humanized antibodies are shown in Table 8, which show that the anti-PD-1 humanized antibodies of the present invention can bind to PD-1 with high affinity.

[0149] Compared with the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 in Example 5 (Table 3), the affinity of the anti-PD-1 humanized antibody of the present invention is comparable thereto; compared with the positive control Opdivo (Table 3), the affinity of the anti-PD-1 humanized antibody of the present invention is significantly improved.

[0150] Table 8 Affinity determination results of anti-PD-1 humanized antibodies

[0151] Antibodies to be tested kon(1 / Ms) kdis(1 / s) KD(M) h31 8.53E+05 <1.0E-07 <1.0E-12 h61 8.92E+05 <1.0E-07 <1.0E-12 h42 8.85E+05 <1.0E-07 <1.0E-12 h43 8.20E+05 <1.0E-07 <1.0E-12

[0152] Example 14 Determination of the ability of anti-PD-1 humanized antibodies to block the binding of ligands PD-L1 / PD-L2 to CHO-hPD1

[0153] 1. Experimental methods

[0154] Flow cytometry was used to analyze the ability of humanized anti-PD-1 antibodies to block ligand binding to PD-1 stably expressed on the surface of transfected CHO cells. The ligand proteins used were recombinant PD-L1 / PD-L2 extracellular domains linked to the Fc domain of mouse IgG1: PD-L1-mFc and PD-L2-mFc.

[0155] CHO-PD1 cells were resuspended in buffer (PBS containing 3% BSA) and the density was adjusted to 2×10 6cells / ml, 100 μl / well cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, and the supernatant was removed. PD-L1-mFc / PD-L2-mFc was added to the cell wells at a concentration of 0.2 μg / ml, incubated at 4°C for 30 minutes, and then anti-PD-1 humanized antibodies (h31, h61, h42, h43) were added in a concentration gradient dilution and incubated at 4°C for 30 minutes.

[0156] The cells were centrifuged at 300 g for 5 minutes, washed once with buffer, and PE-labeled goat anti-mouse IgG fluorescent antibody (Biolegend) was added and incubated at 4°C for 30 minutes. After washing once with centrifugation, the cells were resuspended in PBS and analyzed by CytoFlex flow cytometry to detect the amount of ligand protein bound to the cells and calculate the IC50 of the anti-PD-1 humanized antibody binding blockade. 50 value.

[0157] 2. Experimental results

[0158] The results of the anti-PD-1 humanized antibody blocking the binding of the ligand PD-L1 / PD-L2 to CHO-hPD1 are shown in Table 9. The results show that the anti-PD-1 humanized antibody of the present invention can effectively block the binding of the ligand PD-L1 / PD-L2 to CHO-hPD1.

[0159] Table 9 Results of the ability determination of anti-PD-1 humanized antibodies to block the binding of ligands PD-L1 / PD-L2 to CHO-hPD1

[0160]

[0161] Example 15 Effect of humanized anti-PD-1 antibodies in mixed lymphocyte reaction

[0162] 1. Experimental methods

[0163] In mixed lymphocyte reaction (MLR), the presence or absence of anti-PD-1 humanized antibodies can demonstrate T cell proliferation and the level of cytokine secretion by T cells when PD-1 signaling is blocked. The specific method is:

[0164] CD14 was isolated from fresh PBMC using CD14 MicroBeads, human (Miltenyi) + Monocytes were induced in the presence of GM-CSF / IL-4 for 6 days, and TNF-α was added to induce DC maturation 3 days later. On the day of the experiment, EasySep TM Human T Cell Enrichment Kit (StemCell) was used to purify T cells from PBMC, 1×104 cells / well DC cells and 1×10 5 T cells were mixed and cultured at 100 cells / well. Different concentrations of humanized anti-PD-1 antibodies (h31, h61, h42, and h43) were added to the mixed cells. An isotype control antibody and a no-antibody control well were also set up. After 3 days of mixed culture, the supernatant was collected for IL-2 detection. After another 2 days of culture, the supernatant was collected for IFN-γ detection.

[0165] 2. Experimental results

[0166] The results of the effects of anti-PD-1 humanized antibodies in mixed lymphocyte reaction are shown in Table 10. The results show that anti-PD-1 humanized antibodies can block the binding of PD-1 to ligands and inhibit the PD-1 signaling pathway in MLR, thereby promoting T cell proliferation and promoting T cell secretion of IL-2 and IFN-γ cytokines.

[0167] Table 10 Effect of humanized anti-PD-1 antibodies on mixed lymphocyte reaction

[0168]

[0169]

[0170] Example 16 In vivo evaluation of the anti-tumor efficacy of humanized anti-PD-1 antibodies against mouse colon cancer cells 1. Experimental methods

[0171] Experimental purpose: To determine the in vivo anti-tumor activity of humanized anti-PD-1 antibodies (h31, h61, h43) against mouse colon cancer cells (MC38 cells), and to set up an isotype control group (Isotype group).

[0172] Experimental Materials: hPD1 knock-in female mice, 6-8 weeks old (C57BL / 6 background, source: Beijing Weitongda Biotechnology Co., Ltd.); MC38 cells (National Laboratory Cell Sharing Resource Platform); FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02), fetal bovine serum (Gibco), glutamine (Gibco), penicillin-streptomycin (Gibco, 15140122), DMEM high glucose medium (Gibco, 11965084).

[0173] Instruments and equipment: electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Menite Industrial Co., Ltd., MNT-150T), microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L530R), digital display constant temperature water bath (Prius Machinery Co., Ltd., HH-S), carbon dioxide incubator (Panasonic Healthcare Medical Devices Co., Ltd., Japan, MCO-18AC), double vertical clean bench (Wuxi Easy Purification Equipment Co., Ltd., SW-CJ-VS2).

[0174] Experimental steps:

[0175] Cell culture: MC38 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1% glutamine and 1% penicillin-streptomycin (1:1).

[0176] Inoculation: Collect MC38 cells in the logarithmic growth phase and adjust the cell concentration to 3×10 6 / mL. 40 female hPD1 mice were subcutaneously inoculated with MC38 cells at a volume of 0.1 mL / mouse, i.e., 3×10 5 / mice.

[0177] Administration: The day of inoculation was designated as day 0 (D0). On day 7, the mice were randomly divided into 4 groups according to tumor volume, with 8 mice in each group, and administration began (the dosage, method and frequency of administration for the MC38 tumor model are shown in Table 11).

[0178] Record: Tumor volume was measured and recorded starting from D7, and then the long and short diameters of the tumor were measured with a vernier caliper twice a week. The formula was: (1 / 2) × long diameter × (short diameter) 2 Calculate the tumor volume. When each mouse reaches the experimental endpoint (tumor volume exceeds 2000mm 3 The mice were killed by cervical dislocation and the survival curves were recorded.

[0179] Table 11 Dosage, method and frequency of administration in MC38 tumor model

[0180]

[0181]

[0182] 2. Experimental results

[0183] The results of the effect of anti-PD-1 humanized antibody on tumor volume are shown in Table 12 and Figure 4As shown, it can be seen that compared with the isotype group, the anti-PD-1 humanized antibodies (h31, h61, h43) have a significant inhibitory effect on the tumor growth of the MC38 tumor model (TGI is 100.85%, 94.77%, 99.05% respectively; the number of mice with complete tumor elimination is 6, 5, and 7 respectively).

[0184] The results of the effect of anti-PD-1 humanized antibody on the survival of mice are as follows Figure 5 As shown, it can be seen that compared with the Isotype group, anti-PD-1 humanized antibodies (h31, h61, h43) can significantly prolong the survival of mice.

[0185] Table 12 Effect of anti-PD-1 humanized antibodies on tumor volume (mm 3 )

[0186]

[0187] The above results show that the anti-PD-1 humanized antibodies (h31, h61, h43) provided by the present invention can significantly inhibit the growth of MC38 cells, effectively prolong the survival of mice, and have a significant therapeutic effect on the treatment of mouse colon cancer.

[0188] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention. SEQUENCE LISTING <110> Guangdong Feipeng Pharmaceutical Co., Ltd. <120> An anti-PD-1 humanized antibody and its application <130> 2021 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <400> 1 ccgcaagctt gccgccacca tg 22 <210> 2 <211> 53 <212> DNA <213> Artificial sequence <400> 2 ccggaattct cattaatggt gatggtgatg atgctggaac tggccggcag gtc 53 <210> 3 <211> 118 <212> PRT <213> Artificial sequence <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Glu Gly Glu Gly Ile Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 4[[ID=​​​​<213> Artificial Sequence <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Glu Gly Glu Gly Ile Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <400> 5 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Glu Ile Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Glu Gly Glu Gly Ile Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 6 ​​​​​​​​​​​​​​​​Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 7 <211> 113 <212> PRT <213> Artificial Sequence <400> 7 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <400> 8 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 9 <211> 5 <212> PRT <213> Artificial sequence <400> 9 Asn Tyr Gly Met Asn 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial sequence <400> 10 Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> 11 <211> 9 <212> PRT <213> Artificial sequence <400> 11 Glu Gly Glu Gly Ile Gly Phe Ala Tyr 1 5 <210> 12 <211> 16 <212> PRT <213> Artificial sequence <400> 12 Arg Ser Ser Gln Ser Ile Val Tyr Ser Asn Gly Lys Thr Tyr Leu Glu 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Artificial sequence <400> 13 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <400> 14 Phe Gln Gly Ser His Val Pro Asn Thr 1 5 <210> 15 <211> 118 <212> PRT <213> Artificial sequence <400> 15 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Lys Asn Leu Lys Asn Glu Asp Val Ala Thr Tyr Phe Cys 85 90 95 Thr Lys Glu Gly Glu Gly Ile Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 16 <211> 113 <212> PRT <213> artificial sequence <400> 16 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Asn Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Angry

Claims

1. An anti-PD-1 humanized antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

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

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody is a chimeric antibody or a multispecific antibody; the antigen-binding fragment is any one or more of F(ab')2, Fab, scFv and Fv.

4. A nucleic acid, characterized in that Encodes the anti-PD-1 humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that The nucleic acid comprises: a first nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof, and / or a second nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof.

6. A carrier, characterized in that The vector carries the nucleic acid according to claim 4 or 5.

7. A cell, characterized in that The cell carries the nucleic acid of claim 4 or 5, contains the vector of claim 6, or is capable of expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.

8. A pharmaceutical composition, characterized in that The composition contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, or the cell according to claim 7.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, the cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating colon cancer.

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