Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The invention relates to an anti-PD-1 humanized 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 consists of HCDR1, HCDR2 and HCDR3, the light chain CDR region consists of LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are sequentially shown as SEQ ID NO 9-11, the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are sequentially shown as SEQ ID NO 12-14, the amino acid sequence of the heavy chain variable region of the antibody is shown as any one of SEQ ID NO 3-5, and the amino acid sequence of the light chain variable region of the antibody is shown as any one of SEQ ID NO 6-8.
The present invention uses the Kabat numbering system to designate CDR regions, but CDR regions designated by other methods are also within the scope of the present invention.
In the present invention, the term "specific binding" or the like refers to the binding of an antibody or antigen-binding fragment thereof to an epitope on a predetermined antigen. Typically, the antibody or antigen binding fragment thereof binds with an affinity (KD) of about less than 10-6 M, e.g., about less than 10-7M、10-8M、10-9 M or 10-10 M or less. KD refers to the ratio of off-rate to on-rate (koff/kon) and this amount can be measured by methods familiar to those skilled in the art.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6, or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 4, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6, or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7, or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.
In some embodiments, the antibody contains a heavy chain constant region that is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE, or IgM and a light chain constant region that is a kappa chain or a lambda chain.
In some embodiments, the species source of the heavy chain constant region and the light chain constant region is selected from human, murine, or monkey.
In some embodiments, the antibody is a chimeric antibody or a multispecific antibody (e.g., bispecific antibody).
In the present invention, the term "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope.
In the present invention, the term "bispecific antibody" is a multispecific antigen-binding protein or multispecific antibody, and may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or ligation 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 two different epitopes present on the same or different protein targets.
In some embodiments, the antigen binding fragment is any one or more of F (ab')2, fab, scFv, fv, and a single domain antibody.
In the present invention, the term "F (ab')2" contains two light chains and two heavy chains containing portions 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 thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
In the present invention, the term "Fab" consists of a variable region of one light chain and CH1 and one heavy chain. The heavy chain of a Fab molecule is unable to form disulfide bonds with another heavy chain molecule.
In the present invention, the term "scFv" is an Fv molecule in which the heavy and light chain variable regions are joined by a flexible linker to form a single polypeptide chain that forms an antigen binding region (see, e.g., bird et al, science 242:423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA.90:5879-5883 (1988)).
In the present invention, the term "Fv" comprises variable regions from the heavy and light chains, but lacks constant regions.
In the present invention, the term "single domain antibody" comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but is not as easily attached to each other or even aggregated into blocks as the artificial engineered single chain antibody (scFv). More importantly, the VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity to the antigen, the smallest unit known to bind the antigen of interest.
The invention also relates to a nucleic acid encoding the anti-PD-1 humanized antibody or antigen-binding fragment thereof.
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.
In the present invention, the nucleic acid is usually RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA is preferably used when it is incorporated into a vector. Furthermore, since antibodies are membrane proteins, nucleic acids typically carry signal peptide sequences.
The invention also relates to a vector carrying the nucleic acid.
In the present invention, the term "vector" is a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby 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), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40).
The invention also relates to a cell carrying said nucleic acid, containing said vector or being capable of expressing said antibody or antigen binding fragment thereof.
The invention also relates to a pharmaceutical composition comprising said antibody or antigen binding fragment thereof, said nucleic acid, said vector or said cell.
In the present invention, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not comprise additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
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, and the like that are physiologically compatible for extending the shelf life or efficacy of the antibodies.
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 manufacture of a medicament for the treatment of a PD-1 mediated disease or disorder is also within the scope of the present invention.
In some embodiments, the pharmaceutical composition or medicament is in a form suitable for injection.
In a preferred embodiment, the pharmaceutical composition or medicament is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
The invention has the following beneficial effects:
The invention provides an anti-PD-1 humanized antibody or an antigen binding fragment thereof, which has high affinity and strong specificity, can be combined with CHO-hPD cells, CHO-cyno cells and activated PBMC with high efficiency and specificity, can effectively block the combination of a ligand PD-L1/PD-L2 and CHO-hPD1, can block the combination of PD-1 and the ligand in MLR, and inhibit the PD-1 signal path so as to promote the proliferation of T cells and the secretion of IL-2 and IFN-gamma cytokines, thus the antibody or the antigen binding fragment thereof and related nucleic acid, vector, cell or pharmaceutical composition thereof have wide application prospect in preparing medicaments for treating PD-1 mediated diseases or symptoms.
Example 1 preparation of anti-PD-1 antibodies
1. Immunogens
Human PD-1 sequence (NCBI NP 005009) was synthesized manually, the upstream primer 5'-CCGCAAGCTTGCCGCCACCATG-3' (SEQ ID NO: 1) and the downstream primer 5'-CCGGAATTCTCATTAATGGTGATGGTGATGATGCTGGAACTGGCCGGCA GGTC-3' (SEQ ID NO: 2), the extracellular ends were amplified by PCR, cloned into a pCDNA3.4A eukaryotic expression system after double digestion with HindIII and EcoRI, transfected into 293 cells with this plasmid, and the supernatant was harvested and purified to obtain human PD-1 recombinant protein (hPD-1).
2. Immunization of animals
125Ug of hPD-1 recombinant protein at a concentration of 1.23mg/ml was used as antigen and mixed with the equivalent amount of the immunological adjuvant Freund's adjuvant (Sigma-Aldrich F5881), and 5 female BAL b/C mice 6 weeks old were subcutaneously immunized with 25ug of each mouse. After the primary immunization, the same dose of booster immunization was performed once a week. After a total of 5 immunizations, the immune response was monitored by collecting mouse tail blood. Mice with sufficient anti-hPD-1 immunoglobulin titers were used for fusion by FACS screening (described below). 3 days after intraperitoneal booster immunization with antigen, mice were sacrificed and spleens were removed for cell fusion.
3. Selection of BAL b/C mice producing anti-hPD-1 antibodies
To select BAL b/C mice producing anti-hPD-1 antibodies, the immunized mice serum was tested by FACS. Serum dilutions from hPD-1 recombinant protein immunized mice were incubated with hPD-transfected CHO cells for 30min at 4 degrees celsius, washed 3 times with PBS, added with 0.4ug/ml PE goat anti-mouse IgG (Biolegend 405307) and incubated for 30min at 4 degrees celsius. After washing 3 times with PBS, the samples were examined by Beckman Coulter flow cytometer (CytoFLEX A-1-1102) to verify that they could bind to hPD-transfected CHO cells, and BAL b/C mice producing anti-hPD-1 antibodies were screened out and then subjected to cell fusion.
4. Generation of a murine monoclonal antibody hybridoma against hPD-1
Spleen cells of immunized BAL b/C mice were fused with mouse myeloma cells, and the resulting hybridomas were screened for antigen-specific antibodies. Cell fusion was performed with PEG 1500 (Roche 10783641001) from a single cell suspension of splenocytes from immunized mice with one fifth the number of mouse myeloma cells (SP 2/0, atcc crl 1581) that did not secrete immunoglobulins. The fused cells were plated at about 1X 105 cells/well in 96-well cell culture plates and placed in an incubator (Panasonic MCO-18 AIC) at 37℃under 5% CO2. This was followed by approximately one week in HAT selective medium containing 1X green streptomycin diabody (Gibco 15140122), 1X HAT (SIGMA CRLP-7185) and 20% fetal bovine serum (Royacel RY-F11-01) in 1640 medium. After 1 week, the HAT medium was replaced with HT medium (1640 medium containing 1 XStreptomyces lividans (gibco 15140122), 1 XHT (gibco 11067030) and 20% fetal bovine serum (Royacel RY-F11-01)) and the cell culture supernatant of the fusion plate was examined by FACS and hybridomas secreting hPD-1 protein-binding antibodies were screened. Hybridomas secreting hPD-1 protein-binding antibodies were re-plated and screened again. The screened hybridomas positive for the binding hPD-1 protein antibody were subcloned at least twice by the limited release method. The stable subclones were then cultured in vitro and small amounts of antibodies were generated for further analysis. Hybridoma clone PD-76-C2 was selected for further analysis.
Example 2 characterization of anti-PD-1 murine monoclonal antibody affinity
According to conventional methods, a CHO (Chinese hamster ovary) cell line expressing recombinant human PD-1 on the cell surface (CHO-hPD 1), a CHO cell line expressing monkey PD1 (Uniprot: B0LAJ 2) (CHO-cynoPD), a CHO cell line expressing mouse PD1 (Uniprot: Q02242) (CHO-mousePD 1) was prepared using recombinant techniques and the cell line was used for Flow Cytometry (FCM) assay of binding characterization of anti-PD-1 murine monoclonal antibody PD-1-76-C2.
To assess binding of anti-PD-1 murine mab to CHO-hPD1, 2X 105 CHO-hPD cells were added to 96-well plates and the amount of antibody bound to the cells was measured by concentration gradient dilution (initial concentration 10. Mu.g/ml, triplicate dilution) followed by 30min incubation with buffer (PBS containing 3% BSA) followed by one wash of the cells with PE-labeled anti-murine IgG (Fc) Ab (Biolegend) fluorescent secondary antibody, 30min incubation at 4℃followed by one wash of the cells with buffer followed by PBS re-suspension, and then the cell suspension was subjected to flow cytometry by CytoFlex (Beckmann flow cytometer), the amount of antibody bound to the cells was measured according to the mean fluorescence intensity of staining (MFI), the same method was used to assess binding of this anti-PD-1 murine mab to CHO-cyno cells, CHO-mousePD1 (sometimes abbreviated as "CHO-mPD1" cells in the present invention).
The results are shown in Table 1, and the data indicate that anti-PD-1 murine monoclonal antibody PD-1-76-C2 can bind to CHO-hPD cells and CHO-cyno cells with higher affinity, and at the same time, murine monoclonal antibody does not bind to CHO-mousePD1 cells.
EXAMPLE 3 binding of anti-PD-1 antibodies to activated PBMC
Fresh human Peripheral Blood Mononuclear Cells (PBMC) can activate and proliferate by stimulation of PHA (Sigma), and express PD1 at highest abundance on the third day, and can be used for carrying out the binding experiment of PD-1 antibodies and activated lymphocytes for naturally expressing PD 1.
After fresh human peripheral blood was subjected to a lymphocyte separation gradient centrifugation to obtain PBMC, cells were inoculated into T75 at a density of 1X 106 cells/ml, PHA-L (Sigma) was added at a final concentration of 1. Mu.g/ml to stimulate lymphocyte proliferation, the cells were removed from the culture in a 5% CO2 incubator for 3 days at 37℃after which the cell suspension was removed by centrifugation, the supernatant was resuspended in a buffer (PBS containing 3% BSA) and added to a 96-well U-plate at 2X 105 cells/well, followed by centrifugation at 300g for 5 minutes after incubation at 4℃for 30 minutes starting with 30. Mu.g/ml, 10 concentration gradients of anti-PD 1 antibody was added, the cells were washed once with buffer, goat anti-human IgG fluorescent antibody (Biolegend) was added at 4℃for 30 minutes, the cells were resuspended in PBS after centrifugation once, and then subjected to a CytoFlex flow cytometer analysis to detect the amount of antibody bound to PBMC.
The results are shown in Table 1, where anti-PD 1 antibodies bind to activated lymphocytes with high affinity.
TABLE 1
EXAMPLE 4 anti-PD-1 murine monoclonal antibody binding specificity
Anti-PD-1 murine mab was conjugated to four different CD28 family member proteins to verify the specificity of the antibody for binding to PD-1. PD-1, CD28, CTLA-4, ICOS (ACRO) was immobilized on ELISA plates at a concentration of 1. Mu.g/ml using standard ELISA methods, anti-human PD-1 murine monoclonal antibody was added at a concentration of 10. Mu.g/ml, and peroxidase (HRP) -conjugated anti-mouse IgG was used as secondary antibody (Sigma). After TMB development and termination, the OD450 values were read by the microplate reader.
The results are shown in Table 2, where anti-PD-1 murine monoclonal antibody PD-1-76-C2 specifically binds PD-1 and does not bind other family members of CD 28.
TABLE 2
Example 5 Biological Layer Interferometry (BLI) assay for affinity of anti-human PD-1 murine monoclonal antibody
ForteBio (Octet Qke) affinity assay the binding rate was measured by loading PD-1-his (ACRO) recombinant protein at a concentration of 5. Mu.g/ml onto HISIK biosensor for 120 seconds, then equilibration of the loaded sensor in standard buffer (PBST, PBS+0.02% Tuwen 20) for 120 seconds, after which the sensor was transferred to anti-PD-1 murine mab diluent for 180 seconds to measure the binding rate and then to standard buffer for 20 minutes to measure the dissociation rate. Finally, the analysis is performed by using a kinetic model.
The data processing results are shown in table 3.
TABLE 3 Table 3
| Antibody to be tested | kon(1/Ms) | kdis(1/s) | KD(M) |
| Opdivo(ABA0333) | 1.38E+06 | 3.63E-06 | 2.62E-12 |
| PD-1-76-C2 | 7.71E+05 | <1.0E-07 | <1.0E-12 |
EXAMPLE 6 binding of anti-PD-1 murine monoclonal antibody blocking ligand PD-L1/PD-L2 to CHO-hPD1
Anti-PD-1 murine mab was analyzed by flow cytometry for its ability to block ligand binding to stably expressed PD-1 on the surface of transfected CHO cells. The ligand protein used in the experiment is recombinant PD-L1/PD-L2 extracellular segment connected human IgG1 Fc segment fusion protein PD-L1-hFc (ACRO) and PD-L2-hFc (ACRO).
CHO-PD1 cells were resuspended in buffer (PBS containing 3% bsa) at a density of 2x 106 cells/ml, 100 μl/well of cell suspension was added to a 96 well U-plate and after centrifugation at 300g for 5 min the supernatant was removed.
The subsequent procedure can be divided into two blocking modes, mode one, in which PD-L1-hFc/PD-L2-hFc was added to the cell wells at a concentration of 3. Mu.g/ml, incubated at 4℃for 30 minutes, followed by addition of PD-L1-hFc/PD-L2-hFc protein starting at 30. Mu.g/ml, 3-fold gradient dilution, followed by addition of 10 concentration gradient released anti-PD-1 murine monoclonal antibody, incubated at 4℃for 30 minutes, and mode two, in which cell wells were added with 10 concentration gradient anti-PD-1 murine monoclonal antibody starting at 30. Mu.g/ml, incubated at 4℃for 30 minutes, followed by addition of PD-L1-hFc/PD-L2-hFc protein at a concentration of 3. Mu.g/ml, followed by incubation at 4℃for 30 minutes.
300G was centrifuged for 5 min, the cells were washed once with buffer, PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated for 30 min at 4 ℃. After washing the cells once by centrifugation, the cells were resuspended in PBS and then analyzed by CytoFlex flow cytometry to detect the amount of ligand protein bound to the cells, and the IC50 value for blocking PD-1 antibody binding was calculated.
The results are shown in Table 4, and the anti-PD-1 murine monoclonal antibody PD-1-76-C2 can effectively block the binding of PD-L1/PD-L2 to cell CHO-PD1 in both modes.
TABLE 4 Table 4
Example 7 Effect of anti-PD-1 antibodies on cytokine Release from SEB stimulated PBMC cells
In this example, overnight cultured Peripheral Blood Mononuclear Cells (PBMCs) were stimulated by the addition of the superantigen staphylococcus aureus enterotoxin B (SEB) in the presence or absence of anti-PD-1 antibodies to detect the effect of cytokine secretion.
Fresh peripheral mononuclear cells (PBMC) were resuspended in X-VIVO 15 medium (LONZA) containing 10% FBS, added to T25 flasks, incubated at 37℃with 5% CO2 overnight, the next day, the suspension cells were taken, centrifuged, resuspended in fresh X-VIVO (10% FBS) medium, and added to SEB super antigen (Toxin technology) at a final concentration of 200ng/ml, then 1X 105 cells per well were added to 96-well plates, while varying concentrations of anti-PD-1 antibody were added, and isotype control antibodies (mIgG 1 isotype control antibody (Biolegend); hIgG4 isotype control antibody (Biolegend)) were additionally provided without antibody control wells. After 3 days, samples were taken from the sample wells and IL-2/IFN- γ levels were measured using the IL2/IFN- γ Human Uncoated ELISA Kit (eBioscience) kit.
The effect of different concentrations of PD-1-76-C2 on IL-2/IFN-gamma secretion results are shown in FIG. 1, where anti-PD-1 antibodies increased IL-2/IFN-gamma secretion in a concentration-dependent manner. The result shows that the anti-PD-1 antibody PD-1-76-C2 can further promote the secretion of cytokines by T cells in PBMC stimulated by the SEB superantigen.
Example 8 Effect of anti-PD-1 antibodies in Mixed lymphocyte reaction
In Mixed Lymphocyte Reaction (MLR), the presence or absence of anti-PD-1 antibodies can demonstrate T cell proliferation and levels of T cell secreting cytokines in cases where PD1 signaling is blocked.
CD14+ monocytes (monocyte) were isolated from fresh PBMC using CD14 microblades, human (Miltenyi), induced for 6 days after TNF- α addition and 3 days after DC maturation in the presence of GM-CSF/IL-4, on the day of the experiment, T cells from PBMC were purified using EasSepTM Human T CELL ENRICHMENT KIT (StemCell), 1X 104 DC cells were mixed with 1X 105 T cells and different concentration gradients of anti-PD-1 antibody were added to the mixed cells, and isotype control antibodies (mIgG 1 isotype control antibody and hIgG4 isotype control antibody (Biolegend)) were additionally provided without antibody control wells. After 3 days of mixed culture, the supernatant was assayed for IL-2, and after 2 days of culture, the supernatant was assayed for IFN-gamma.
The effect of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IL-2 is shown in FIG. 2, the effect of different concentrations of PD-1-76-C2 on T cell proliferation and T cell secretion of cytokine IFN-gamma is shown in FIG. 3, and the results of FIG. 2 and FIG. 3 show that the anti-PD-1 antibody, PD-1-76-C2, can block the binding of PD1 and ligand in an antibody concentration dependent manner in MLR experiments, inhibit PD1 signal path, thereby promoting T cell proliferation and promoting T cell secretion of IL-2, IFN-gamma cytokine.
EXAMPLE 9 anti-PD-1 murine monoclonal antibody humanization
The anti-PD-1 murine monoclonal antibody PD-1-76-C2 obtained in the above way (the amino acid sequences of HCDR1, HCDR2 and HCDR3 are sequentially shown as SEQ ID NO: 9-11, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are sequentially shown as SEQ ID NO: 12-14, the heavy chain variable region sequence is shown as SEQ ID NO:15 and the light chain variable region sequence is shown as SEQ ID NO: 16) is humanized by the specific method:
Human PD-1 sequence (NCBI NP 005009) is synthesized manually, cloned into PCDNA3.4A eukaryotic expression system, transfected 293 cells with the plasmid, and the supernatant is harvested and purified to obtain human PD-1 recombinant protein. The obtained human PD-1 recombinant protein is subjected to subcutaneous immunization of female BAL b/C mice, spleen cells immunized with the BAL b/C mice are fused with mouse myeloma cells, and then the obtained hybridomas are subjected to antigen-specific antibody screening. The screened hybridoma positive to the hPD-1 protein antibody is subcloned by a limiting dilution method at least twice, and then stable subclones are cultured in vitro to generate a small amount of antibody, and PD-1-76-C2 clones are obtained after further screening.
SEQ ID NO:9:NYGMN
SEQ ID NO:10:WINTHTGEPTYADDFKG
SEQ ID NO:11:EGEGIGFAY
SEQ ID NO:12:RSSQSIVYSNGKTYLE
SEQ ID NO:13:KVSNRFS
SEQ ID NO:14:FQGSHVPNT
SEQ ID NO:15:
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQIKNLKNEDVATYFCTKEGEGIGFAYWGQGTLVTVSA
SEQ ID NO:16:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPNTFGGGTKLEIKR
Referring to SEQ ID NO. 15 and SEQ ID NO. 16, a humanized template which is best matched with a non-CDR region is selected from Germline database, wherein the template of an antibody heavy chain is IGHV1/7, the template of an antibody light chain is IGKV2, and under the principle that the structural stability of the antibody is not influenced, the combination of the antibody and antigen is not influenced, protein modification sites such as glycosylation, phosphorylation and the like are not introduced, oxidized amination and the like are not introduced, the structural stability is enhanced, the humanized sequence of the heavy chain is designed to be VH1-6, the humanized sequence of the light chain is designed to be VL1-3, and the pairing form of the light chain and the heavy chain is designed to be IGHV1/IGKV2, so that the humanized antibody is obtained.
Based on the amino acid sequences of the light and heavy chains of each humanized antibody, the gene fragment was inserted into PCDNA3.4A expression vector (Invitrogen) by HindIII (NEB)/EcoRI (NEB) cleavage site using T4 DNA ligase (TAKARA 2011A) after double cleavage with HindIII (NEB) and EcoRI (NEB). HEK293 cells (Life Technologics Cat. No. 11625019) were transfected with the expression vector and the transfection reagent PEI (Poly science, inc. Cat. No. 23966) at a ratio of 1:2 and incubated in a CO2 incubator for 5-7 days. The expressed antibodies were collected by centrifugation and then purified by a conventional method to obtain anti-PD-1 humanized antibodies (h 11, h21, h31, h41, h51, h61, h12, h42, h13, h23, h33, h43, h53, h 63), wherein the amino acid sequences of the 4 anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) are shown in Table 5.
TABLE 54 amino acid sequences of anti-PD-1 humanized antibodies (h 31, h61, h42, h 43)
SEQ ID NO:3:
QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTKEGEGIGFAYWGQGTTVTVSS
SEQ ID NO:4:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQKFQGRVTMTLDTSISTAYMELSRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS
SEQ ID NO:5:
QIQLVQSGAEVKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYADDFKGRFTFTLDTSISTAYLEISRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS
SEQ ID NO:6:
DVVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR
SEQ ID NO:7:
DIVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR
SEQ ID NO:8:
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGKTYLEWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR
EXAMPLE 10 determination of binding Capacity of anti-PD-1 humanized antibody to CHO-hPD1 cells and CHO-cyno cells
1. Experimental method
CHO (chinese hamster ovary) cell line expressing recombinant human PD-1 on the cell surface (CHO-hPD 1), CHO cell line expressing monkey PD1 (CHO-cynoPD 1) using recombinant technology, both cell lines will be used for the binding characterization of anti-PD-1 humanized candidate monoclonal antibodies in Flow Cytometry (FCM) assays. The specific method comprises the following steps:
To assess binding of anti-PD-1 humanized antibodies to CHO-hPD1 cells, 2 x105 CHO-hPD1 cells were added to 96-well plates and the humanized antibodies were diluted by concentration gradients (initial concentration 30 μg/ml, triplicate dilution), incubated for 30min at 4 ℃, buffer (PBS containing 3% bsa) was added to wash the cells once, PE-labeled anti-human IgG (Fc) Ab (Biolegend) fluorescent secondary antibody was added, buffer was washed once after 30min incubation at 4 ℃, PBS resuspended after 30min, and the cell suspension was subjected to flow cytometry analysis by CytoFlex (beckman flow cytometer) to measure the amount of antibody bound to the cells according to the Mean Fluorescence Intensity (MFI) of the staining. The binding of anti-PD-1 humanized antibodies to CHO-cyno cells was evaluated in the same manner.
2. Experimental results
The binding capacity of the anti-PD-1 humanized antibody to CHO-hPD cells and CHO-cyno cells was measured as shown in Table 6, and the results show that the anti-PD-1 humanized antibody of the present invention can bind to both CHO-hPD1 cells and CHO-cyno cells with high affinity.
EXAMPLE 11 determination of binding Capacity of anti-PD-1 humanized antibody to activated PBMC
1. Experimental method
Fresh human Peripheral Blood Mononuclear Cells (PBMC) can activate and proliferate by stimulation of PHA (Sigma), and express PD-1 in highest abundance on the third day, and can be used for measuring the binding capacity of the anti-PD-1 humanized antibody and the activated lymphocyte to naturally express PD-1. The specific method comprises the following steps:
After fresh human peripheral blood was subjected to a lymphocyte separation gradient centrifugation to obtain PBMC, the density was adjusted to 1X 106 cells/ml, inoculated into T75, PHA-L (Sigma) was added at a final concentration of 1. Mu.g/ml to stimulate lymphocyte proliferation, the cell suspension was removed after standing for 3 days in a 37℃5% CO2 incubator, the supernatant was centrifuged off, buffer (PBS containing 3% BSA) was resuspended, 2E 5/well was added to a 96-well U-plate, anti-PD-1 humanized antibodies were added at different gradients, 300g was centrifuged for 5 minutes after incubation at 4℃for 30 minutes, the cells were washed once with buffer, PE-labeled anti-human IgG fluorescent antibody (Biolegend) was added, 4℃was incubated for 30 minutes, PBS was resuspended after centrifugation, and then CytoFlex flow cytometry analysis was performed to detect the amount of antibodies bound to PBMC.
2. Experimental results
The binding capacity of the anti-PD-1 humanized antibodies to activated PBMC is determined as shown in Table 6, which shows that the anti-PD-1 humanized antibodies of the invention are capable of binding to activated PBMC with high affinity.
In comparison with the anti-PD-1 murine monoclonal antibodies of examples 2 and 3, PD-1-76-C2 (Table 1), the anti-PD-1 humanized antibodies of the invention bind to CHO-hPD cells, CHO-cyno cells and activated PBMC in an amount comparable thereto.
TABLE 6 determination of binding Capacity of anti-PD-1 humanized antibodies to CHO-hPD1 cells, CHO-cyno cells and activated PBMC
Example 12 binding specificity of anti-PD-1 humanized antibodies
1. Experimental method
The anti-PD-1 humanized antibodies of the invention were conjugated to four different CD28 family member proteins to verify the specificity of the anti-PD-1 humanized antibodies for binding to PD-1. PD-1, CD28, CTLA-4, ICOS was immobilized on ELISA plates at a concentration of 1. Mu.g/ml, anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) at a concentration of 10. Mu.g/ml were added, and peroxidase (HRP) -conjugated anti-human IgG (Fab) was used as secondary antibody, using standard ELISA methods. After TMB development and termination, the OD450 values were read by the microplate reader.
2. Experimental results
The binding specificity results of the anti-PD-1 humanized antibodies are shown in Table 7, and 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, thus indicating that the anti-PD-1 humanized antibodies have high binding specificity with PD-1.
TABLE 7 binding specificity results of anti-PD-1 humanized antibodies
Example 13 affinity assay for anti-PD-1 humanized antibodies
1. Experimental method
According to the biological membrane interference technique (BLI), fortebio is usedThe detection instrument is used for detecting the affinity of the antibody, and the specific method comprises the following steps:
The binding rate was measured by loading PD-1-his recombinant protein at a concentration of 5. Mu.g/ml on HISIK biosensors for 120 seconds, then equilibration of the loaded sensors in standard buffer (PBST, PBS+0.02% Tuwen 20) for 120 seconds, after which the sensors were transferred to dilutions of anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) for 180 seconds and then to standard buffer for 20 minutes for dissociation rates. And finally, analyzing by using a dynamic model, and processing data. The positive control was Opdivo (ABA 0333).
2. Experimental results
The affinity measurements of the anti-PD-1 humanized antibodies are shown in Table 8, which shows that the anti-PD-1 humanized antibodies of the invention can bind to PD-1 with high affinity.
Compared with the anti-PD-1 murine monoclonal antibody PD-1-76-C2 in example 5 (Table 3), the affinity of the anti-PD-1 humanized antibody of the invention is comparable to that of the anti-PD-1 humanized antibody, and compared with the positive control OPdivo (Table 3), the affinity of the anti-PD-1 humanized antibody of the invention is remarkably improved.
TABLE 8 affinity assay results for anti-PD-1 humanized antibodies
| Antibody to be tested | kon(1/Ms) | kdis(1/s) | KD(M) |
| h31 | 8.53E+05 | <1.0E-07 | <1.0E-12 |
| h61 | 8.92E+05 | <1.0E-07 | <1.0E-12 |
| h42 | 8.85E+05 | <1.0E-07 | <1.0E-12 |
| h43 | 8.20E+05 | <1.0E-07 | <1.0E-12 |
EXAMPLE 14 anti-PD-1 humanized antibody blocking ligand PD-L1/PD-L2 binding to CHO-hPD1
1. Experimental method
Anti-PD-1 humanized antibodies were analyzed by flow cytometry for their ability to block ligand binding to stably expressed PD-1 on the surface of transfected CHO cells. The ligand protein is recombinant PD-L1/PD-L2 extracellular domain connected mouse IgG1 Fc domain fusion protein PD-L1-mFc and PD-L2-mFc.
CHO-PD1 cells were resuspended in buffer (PBS containing 3% bsa) at a density of 2×106 cells/ml,100 μl/well of cell suspension was added to a 96-well U-plate and after centrifugation at 300g for 5min the supernatant was removed. PD-L1-mFc/PD-L2-mFc was added to the cell wells at a concentration of 0.2. Mu.g/ml, incubated at 4℃for 30min, and then anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) were added at a concentration gradient, and incubated at 4℃for 30 min.
300G was centrifuged for 5min, the cells were washed once with buffer, PE-labeled goat anti-mouse IgG fluorescent antibody (Biolegend) was added and incubated for 30 min at 4 ℃. After washing the cells once by centrifugation, the cells were resuspended in PBS and then analyzed by CytoFlex flow cytometry to detect the amount of ligand protein bound to the cells, and the IC50 value for blocking binding of anti-PD-1 humanized antibodies was calculated.
2. Experimental results
The anti-PD-1 humanized antibody blocks the binding capacity of the ligand PD-L1/PD-L2 and CHO-hPD1, and the results are shown in Table 9, and the result shows that the anti-PD-1 humanized antibody can effectively block the ligand PD-L1/PD-L2 from binding with CHO-hPD.
TABLE 9 determination of the ability of anti-PD-1 humanized antibodies to block ligand PD-L1/PD-L2 from binding to CHO-hPD1
Example 15 Effect of anti-PD-1 humanized antibodies in Mixed lymphocyte reaction
1. Experimental method
In Mixed Lymphocyte Reaction (MLR), the presence or absence of anti-PD-1 humanized antibodies can prove the proliferation of T cells and the level of cytokines secreted by T cells in the case of blocked PD-1 signals. The specific method comprises the following steps:
CD14+ monocytes (monocyte) were isolated from fresh PBMC using CD14 microblades, human (Miltenyi), induced 6 days after TNF- α addition and 3 days after DC maturation in the presence of GM-CSF/IL-4, on the day of the experiment, T cells from PBMC were purified using EaseSepTM Human T CELL ENRICHMENT KIT (StemCell), DC cells from 1X 104 cells/well were mixed with T cells from 1X 105 cells/well, and anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) were added to the mixture at different concentration gradients, and isotype control antibodies were additionally provided without antibody control wells. After 3 days of mixed culture, the supernatant was assayed for IL-2, and after 2 days of culture, the supernatant was assayed for IFN-gamma.
2. Experimental results
The effect results of the anti-PD-1 humanized antibody in the mixed lymphocyte reaction are shown in Table 10, and the results show that the anti-PD-1 humanized antibody can block the combination of PD-1 and a ligand in MLR and inhibit the PD-1 signal path, thereby promoting T cell proliferation and promoting T cells to secrete IL-2 and IFN-gamma cytokines.
TABLE 10 Effect of anti-PD-1 humanized antibodies on Mixed lymphocyte reaction results
EXAMPLE 16 evaluation of in vivo anti-tumor efficacy of anti-PD-1 humanized antibody on mouse colon cancer cells 1, experimental method
The experimental purpose is to measure the in vivo anti-tumor activity of anti-PD-1 humanized antibodies (h 31, h61, h 43) on mouse colon cancer cells (MC 38 cells), and to set a isotype control group (Isotype groups).
Experimental materials hPD Knock in mice, females, 6-8 weeks (C57 BL/6 background, source: beijing Vietnam Biotechnology Co., ltd.), MC38 cells (national Experimental cell sharing resource platform), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), DMSO (Sigma, D2650), DPBS (Hyclone, SH 30028.02), fetal bovine serum (Gibco), glutamine (Gibco), penicillin-streptomycin (Gibco, 15140122), DMEM high sugar medium (Gibco, 11965084).
The instrument equipment comprises an electronic balance (Shanghai Zhenping scientific instruments Co., ltd., JA 12002), a vernier caliper (Shanghai Meinaite practical Co., ltd., MNT-150T), a microscope (Chongqing Ornithoid optical instruments Co., BDS 200), a medical centrifuge (Hunan Instrument laboratory developing Co., ltd., L530R), a digital display constant temperature water bath (Pris mechanical Co., ltd., HH-S), a carbon dioxide incubator (Japanese Songxia health medical instruments Co., ltd., MCO-18 AC), and a double vertical ultra clean bench (tin-free easy-purifying equipment Co., SW-CJ-VS 2).
The experimental steps are as follows:
cell culture MC38 cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 1% glutamine and 1% penicillin-streptomycin (1:1).
Inoculation, namely, collecting MC38 cells in logarithmic growth phase, and adjusting the cell concentration to be 3X 106/mL. 40 female hPD mice were inoculated subcutaneously with MC38 cells at a volume of 0.1 mL/mouse, i.e., 3X 105/mouse.
Administration on day 0 (D0) and day 7, mice were randomized into 4 groups of 8 animals each based on tumor volume and administration was initiated (MC 38 tumor model dosing, pattern and frequency are shown in Table 11).
Recording D7 tumor volume was measured and recorded, after which tumor long and short diameters were measured 2 times a week with vernier calipers. Tumor volume was calculated as (1/2) x major diameter x (minor diameter)2. When each mouse reached the end of the experiment (tumor volume exceeded 2000mm3 reached the end of the kernel-day), the mice were sacrificed by cervical dislocation and survival curves were recorded.
TABLE 11 dosing, modes and frequency of MC38 tumor model
2. Experimental results
The results of the effect of the anti-PD-1 humanized antibody on tumor volume are shown in Table 12 and FIG. 4, and it can be seen that the anti-PD-1 humanized antibody (h 31, h61, h 43) has a significant tumor inhibition effect on tumor growth of MC38 tumor model (TGI: 100.85%,94.77%,99.05% in order, and 6, 5, 7 tumor total-elimination mice, respectively) compared with Isotype groups.
The effect of anti-PD-1 humanized antibodies on the survival of mice as shown in FIG. 5, it can be seen that the anti-PD-1 humanized antibodies (h 31, h61, h 43) significantly prolonged the survival of mice compared to Isotype groups.
TABLE 12 results of the effect of anti-PD-1 humanized antibodies on tumor volume (mm3)
The results show that the anti-PD-1 humanized antibody (h 31, h61, h 43) provided by the invention can obviously inhibit the growth of MC38 cells, effectively prolong the survival period of mice and has obvious curative effect on treating colon cancer of mice.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Phepoaching pharmaceutical Co., ltd
<120> An anti-PD-1 humanized antibody and use thereof
<130> 2021
<160> 16
<170> PatentIn version 3.5
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Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
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Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Gln Gly Phe
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Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Gln Lys Phe
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Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr
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Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
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Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
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Lys Gly Arg Phe Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr
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Leu Glu Ile Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
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Thr Lys Glu Gly Glu Gly Ile Gly Phe Ala Tyr Trp Gly Gln Gly Thr
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
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Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
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Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
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Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
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Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
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Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
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Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
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Ser His Val Pro Asn Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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