CN110964111A - anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof - Google Patents
anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and particularly provides an anti-PD-L1 monoclonal antibody or an antigen binding fragment thereof, which comprises amino acid sequences of a heavy chain variable region and a light chain variable region, wherein the anti-PD-L1 monoclonal antibody has higher affinity and good biological activity, can well inhibit the combination of PD-L1 and PD-1 in vitro, ensures the normal proliferation and activation of T cells and the secretion of cytokines, further ensures the recognition capability and the killing capability of the T cells on tumor cells, avoids the occurrence of immune escape of the tumor cells, can be effectively used for treating cancer diseases, and has wide development prospect; cancer diseases include, but are not limited to, bladder cancer, non-small cell lung cancer, head and neck cancer, hodgkin's lymphoma or melanoma, and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a fully human anti-PD-L1 monoclonal antibody, an antigen binding fragment and application thereof.
Background
In recent years, immunotherapy has become the fourth treatment means of tumor after surgery, radiotherapy and chemotherapy, and especially the treatment aiming at immune checkpoint molecules obtains very positive treatment effect on the clinical treatment of tumor. Programmed cell death molecule 1 and its ligand protein PD-L1(Programmed cell death ligand 1) are important immune check point proteins with inhibitory action, and PD-1/PD-L1 medicine is a new kind of tumor therapy medicine which is currently spotlighted and is also the prime force in current immunotherapy. At present, there are 8 kinds of drugs on the market for monoclonal antibodies against these two proteins, and although the indications for the 8 kinds of monoclonal antibodies are different, they all play a very important role in the process of tumor treatment.
Programmed death receptor-1 (PD-1) is an inhibitory receptor on T cells, PD-1 can interact with ligand (PD-Ls) PD-L1 and PD-L2 to inhibit T cell proliferation, activation and cytokine secretion, therefore PD-1 is an important immune sentinel for regulating T cell response. In a normal organism, a PD-1 and PD-Ls signal channel plays an important role in maintaining the immune tolerance of the organism; in tumor occurrence, both the PD-1 and PD-Ls signal pathways can inhibit the immune response of T cells to promote the occurrence of tumor immune escape, so that the development of a PD-1 or PD-Ls blocking agent with remarkable curative effect and low adverse reaction by taking the PD-1 and PD-Ls signal pathways as targets becomes a great hotspot in the field of tumor immunotherapy in recent years.
Programmed cell death-Ligand 1 (PD-L1), a first type transmembrane protein of 40kDa in size, normally responds to foreign antigens accumulated in lymph nodes or spleen by the immune system, promotes the proliferation of antigen-specific T cells, the combination of PD-1 and PD-L1 can transmit inhibitory signals, reduce the proliferation of T cells, one way for tumor cells to escape T-cell destruction is by generating PD-L1 on its surface, thereby leading a large amount of PD-1 to be combined with PD-L1, leading T cells to be incapable of finding tumor cells, furthermore, the tumor cells can not send out attack signals, so that the PD-L1/PD-1 combination is blocked, and the method is a very potential direction in the field of tumor immunotherapy.
At present, the fully human antibody is the main direction of development of therapeutic antibodies, and the emergence of antibody library technology provides a good technical platform for preparation and screening of the fully human antibody. The antibody library technology bypasses the hybridoma process necessary in the previous monoclonal antibody development process, and can obtain various antibody genes and antibody molecular fragments even without an immunization process. Phage antibody libraries were the earliest and most widely used antibody libraries at present. The phage antibody library display technology is a technology firstly established by Smith, inserts a gene coding a foreign protein or polypeptide into a phage capsid protein gene, and leads the foreign protein or polypeptide and the phage capsid protein to be fused and expressed on the surface of a phage. The phage antibody library utilizes the principle to express antibodies with different specificities or functional fragments thereof (Fab, Fv and ScFv) on the surface of phage, and then carries out screening by using antigen. The phage antibody library is divided into immune library and non-immune library according to the source of antibody gene, and the non-immune library comprises natural library, semi-synthetic library and fully-synthetic library. The screening of the phage antibody library simulates the process of antibody affinity maturation, usually the antigen is coated on a solid phase medium, the phage antibody library to be screened is added, and a plurality of rounds of processes of 'adsorption-washing-elution-amplification' (namely panning) are carried out until the specific high-affinity antibody is screened.
Disclosure of Invention
In order to meet the requirements of domestic markets, the invention screens a monoclonal antibody resisting PD-L1 from a fully-synthesized phage antibody library, constructs a library for light chain CDR123 region mutation of the monoclonal antibody obtained by primary screening by a method for constructing a small-capacity synthesized phage antibody library, performs affinity maturation on a light chain, selects a monoclonal antibody with higher affinity through screening identification, performs affinity maturation on the heavy chain CDR123 region mutation library, and finally screens an anti-PD-L1 monoclonal antibody or an antigen binding fragment thereof with higher affinity and better activity.
The specific technical scheme of the invention is as follows:
the invention provides an anti-PD-L1 monoclonal antibody or an antigen binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of HCDR1 of the heavy chain variable region is selected from SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3; the amino acid sequence of HCDR2 of the heavy chain variable region is selected from SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6; the amino acid sequence of HCDR3 of the heavy chain variable region is selected from SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9; the amino acid sequence of LCDR1 of the light chain variable region is selected from SEQ ID NO. 10 or SEQ ID NO. 11; the amino acid sequence of LCDR2 of the light chain variable region is selected from SEQ ID NO. 12 or SEQ ID NO. 13; the amino acid sequence of LCDR3 of the light chain variable region is selected from SEQ ID NO. 14 or SEQ ID NO. 15.
Further, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18; the amino acid sequence of the light chain variable region is selected from SEQ ID NO 19 or SEQ ID NO 20.
Further, the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG 4;
preferably, the heavy chain constant region is human IgG 1.
Further, the monoclonal antibody or the antigen binding fragment thereof is a full-length antibody or an antibody fragment, and the antibody fragment comprises one or a combination of more of Fab, F (ab)2, Fv or ScFv.
The invention also provides a polypeptide or protein, wherein the polypeptide or the protein comprises the anti-PD-L1 monoclonal antibody or an antigen binding fragment thereof.
The invention also provides a polynucleotide sequence or a combination which codes the amino acid sequence of the anti-PD-L1 monoclonal antibody or the antigen binding fragment thereof.
The invention also provides a recombinant DNA expression vector which comprises the polynucleotide sequence or the combination.
The invention also provides a host cell for transfecting the recombinant DNA expression vector, wherein the host cell comprises a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell;
preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293E cell, a CHO cell, or a NS0 cell.
The invention also provides a medicament or a pharmaceutical composition, which comprises the anti-PD-L1 monoclonal antibody or an antigen-binding fragment thereof.
The invention also provides application of the anti-PD-L1 monoclonal antibody or the antigen binding fragment thereof in preparing a medicament for treating cancer diseases;
preferably, the cancer disease comprises bladder cancer, non-small cell lung cancer, head and neck cancer, hodgkin's lymphoma or melanoma.
The invention has the following beneficial effects: the anti-PD-L1 monoclonal antibody provided by the invention has higher affinity and good biological activity, can well inhibit the combination of PD-L1 and PD-1 in vitro, ensures the normal proliferation and activation of T cells and the secretion of cytokines, further ensures the recognition capability and killing capability of the T cells to tumor cells, avoids the occurrence of tumor cell immune escape, can be effectively used for treating cancer diseases, and has wide development prospect; cancer diseases include, but are not limited to, bladder cancer, non-small cell lung cancer, head and neck cancer, hodgkin's lymphoma or melanoma, and the like.
Drawings
FIG. 1 is a plasmid map of pScFvDisb-s according to example 1 of the present invention;
FIG. 2 is a line graph of the relative affinity of the gradient dilution phase ELISA for identifying phase-Abs in example 3 of the present invention;
FIG. 3 is a diagram of pTSE plasmid vector in example 4 of the present invention;
FIG. 4 is an SDS-PAGE electrophoresis of the whole antibody in example 4 of the present invention;
FIG. 5 is a graph comparing the binding capacity of the whole antibody and PD-L1 at molecular level in example 5 of the present invention;
FIG. 6 is a graph comparing the competitive inhibition ability of whole antibody and PD-1 in example 6 of the present invention;
FIG. 7 is a graph showing a comparison of the activities of the PD-L1 monoclonal antibody tested for Mixed Lymphocyte Reaction (MLR) in example 7 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples.
In the present invention, the CDR is a complementary-determining region; the ScFv is a single-chain antibody (single-chain fragment variable); the HEK293E cells were human embryonic kidney 293E cells (human embryo kidney 293E cells); the CHO cell is a Chinese hamster ovary cell (Chinese hamster ovary cell); NS0 cells were mouse NS0 thymoma cells.
The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from an antibody fragment containing one or more CDRs or any other fragment of an antibody that binds an antigen but does not have the structure of an intact native antibody. In certain embodiments, the antibodies described herein are antigen binding fragments.
An "Fab" fragment of an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of one light chain (comprising the variable and constant regions) and one heavy chain of the variable and first constant regions, joined by disulfide bonds. Fab may be obtained by papain digestion at residues near the N-terminus of the disulfide bond between the heavy chains of the antibody hinge region.
"F (ab) 2" refers to a dimer of Fab, which comprises two light chains and a portion of two heavy chains.
The "Fv" segment of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. The Fv fragment consists of the variable region of one light chain joined to the variable region of one heavy chain.
"scFv" refers to an engineered antibody comprising a light chain variable region linked directly to a heavy chain variable region or via a polypeptide linker sequence.
Example 1
The embodiment 1 of the invention provides an anti-PD-L1 monoclonal antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of HCDR1 of the heavy chain variable region is selected from SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; the amino acid sequence of HCDR2 of the heavy chain variable region is selected from SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6; the amino acid sequence of HCDR3 of the heavy chain variable region is selected from SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9; the amino acid sequence of LCDR1 of the light chain variable region is selected from SEQ ID NO. 10 or SEQ ID NO. 11; the amino acid sequence of LCDR2 of the light chain variable region is selected from SEQ ID NO. 12 or SEQ ID NO. 13; the amino acid sequence of LCDR3 in the light chain variable region is selected from SEQ ID NO. 14 or SEQ ID NO. 15.
Preferably, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18; the variable region in the light chain has an amino acid sequence selected from SEQ ID NO 19 or SEQ ID NO 20.
Specifically, the monoclonal antibody of anti-PD-L1 is screened out from a fully-synthesized phage antibody library by utilizing a phage antibody library display technology, then a light chain CDR123 region mutation library of the monoclonal antibody obtained by primary screening is built by a method for constructing a small-capacity synthesized phage antibody library, affinity maturation is carried out on a light chain, the monoclonal antibody with higher affinity is selected by screening and identifying, then the heavy chain CDR123 region mutation library thereof is subjected to affinity maturation, and finally the high-affinity monoclonal antibody of anti-PD-L1 is screened out. The method comprises the following steps:
the method comprises the following steps: a series of gene cloning methods are adopted to modify a vector pComb3 (purchased from China plasmid vector strain cell strain gene collection center) for construction and expression of a phage single-chain antibody library. The transformed vector is named pScFvDisb-s, the plasmid map of the transformed vector is shown in figure 1, and a fully synthetic phage antibody library is constructed on the basis of the vector.
Step two: PD-L1 is used as an antigen coating immune tube, the antigen coating amount is 5 mug/500 mug/tube, the immune tube and a total synthetic phage antibody library are respectively sealed by 4% skimmed milk powder/PBST at 4 ℃ for one night, and the immune tube and the total synthetic phage antibody library are respectively sealed for 1h at room temperature. Adding the sealed fully-synthetic phage antibody library into an immune tube for antigen-antibody binding, wherein the input amount of phage is about 109～1012After 1h reaction at room temperature, unbound phage were washed off using PBST-PBS and washed with 0.1M Glycine-HCl, pH2.2Finally, the eluted phage antibody solution was neutralized to pH7.0 with 1.5M Tris-HCl pH 8.8.
Step three: and infecting 10ml of TG1 bacterial solution growing to the logarithmic phase with the neutralized phage, standing in an incubator at 37 ℃ for 30min, taking out part of the bacterial solution, performing gradient dilution, and coating on a 2YTAG plate for calculating the output of the phage. The remaining bacterial solution was centrifuged and the supernatant discarded, and the pellet was resuspended in a small volume of medium, aspirated and plated on a 2YTAG large plate in preparation for the next round of screening.
Step four: scraping the infected and plated thallus from a large plate, inoculating the thallus to a 2YTAG liquid culture medium, shaking to a logarithmic phase, adding M13KO7 to assist the superinfection of the phage, culturing at 28 ℃, 220rpm overnight to prepare the phage, and settling and purifying the phage by PEG/NaCl for the next round of screening. Three rounds of phage library enrichment screening were performed.
Step five: screening of PD-L1 phage single-chain antibody positive clones: after three rounds of screening, well-separated monoclonal colonies were picked, inoculated into a 96-well deep-well plate containing 2YTAG liquid medium, cultured at 37 ℃ and 220rpm to its logarithmic growth phase, and added to about 10/well10The helper phage M13KO7 was quiescently infected at 37 ℃ for 30 min. Centrifugation was carried out at 4000rpm for 15min, the supernatant was discarded, and the mycelia were resuspended in 2YTAK and precipitated, and cultured overnight at 28 ℃ and 220 rpm. And centrifuging at 4000rpm and 4 ℃ for 15min, sucking amplified phage supernatant for ELISA identification, and screening to obtain the anti-PD-L1 single-chain antibody with high affinity.
Step six: performing in vitro affinity maturation on the anti-PD-L1 single-chain antibody obtained by primary screening in the step five, and specifically comprising the following steps: on the basis of the light chain of the anti-PD-L1 single-chain antibody obtained by primary screening, firstly synthesizing a light chain CDR123 mutant library, and carrying out screening identification according to the second step to the fifth step to obtain two clones with higher affinity; synthesizing the two light chains respectively, constructing a heavy chain CDR123 mutation library, screening and identifying according to the steps two to five, finally obtaining six monoclonal antibodies with higher affinity, respectively named as AH10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3 and BH2-E3, and determining the obtained monoclonal antibodies as correct antibody sequences by gene sequencing
After sequencing, the sequences of the 6 monoclonal antibodies screened by the method are as follows:
the amino acid sequence of the heavy chain variable region of AH10-B11 is SEQ ID NO:16, and the amino acid sequence of the light chain variable region is SEQ ID NO: 19;
the amino acid sequence of the heavy chain variable region of AC9-B11 is SEQ ID NO:17, and the amino acid sequence of the light chain variable region is SEQ ID NO: 19;
the amino acid sequence of the heavy chain variable region of BH2-B11 is SEQ ID NO. 18, and the amino acid sequence of the light chain variable region is SEQ ID NO. 19;
the amino acid sequence of the heavy chain variable region of AH10-E3 is SEQ ID NO:16, and the amino acid sequence of the light chain variable region is SEQ ID NO: 20;
the amino acid sequence of the heavy chain variable region of AC9-E3 is SEQ ID NO:17, and the amino acid sequence of the light chain variable region is SEQ ID NO: 20;
the amino acid sequence of the heavy chain variable region of BH2-E3 is SEQ ID NO 18, and the amino acid sequence of the light chain variable region is SEQ ID NO 20.
Specifically, SEQ ID NO 16:
EVQLVESGGGLVQPGGSLRLSCAASGWTLSDSLQHWVRQAPGKGLEWVGFQSAYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHYYTAWDYWGQGTLVTVSS；
SEQ ID NO:17：
EVQLVESGGGLVQPGGSLRLSCAASGCTWSDSWDHWVRQAPGKGLEWVDYESTYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWNATFDYWGQGTLVTVSS；
SEQ ID NO:18：
EVQLVESGGGLVQPGGSLRLSCAASGLTWSDSYKHWVRQAPGKGLEWVHWNSTYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHFASTWDYWGQGTLVTVSS；
SEQ ID NO:19：
DIQMTQSPSSLSASVGDRVTITCRASQNIHNSLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQERRVPWTFGQGTKVEIK；
SEQ ID NO:20：
DIQMTQSPSSLSASVGDRVTITCRASQSIHNYLAWYQQKPGKAPKLLIYSASNLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGLHTPPTFGQGTKVEIK。
further, the selected monoclonal antibody further comprises a heavy chain constant region, wherein the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG 4;
preferably, the heavy chain constant region is human IgG 1.
Furthermore, the monoclonal antibody or the antigen binding fragment thereof is a full-length antibody or an antibody fragment, and the antibody fragment comprises one or a combination of more of Fab, F (ab)2, Fv or ScFv.
Example 2
The embodiment 2 of the invention also provides a polypeptide or protein based on the embodiment 1, and the polypeptide or protein comprises the anti-PD-L1 monoclonal antibody or the antigen binding fragment thereof in the embodiment 1.
The present invention also provides a polynucleotide sequence or combination encoding the amino acid sequence of the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of example 1.
The invention also provides a recombinant DNA expression vector which comprises the polynucleotide sequence or the combination.
The invention also provides a host cell of the transfected recombinant DNA expression vector, wherein the host cell comprises prokaryotic cells, yeast cells, insect cells or mammalian cells;
preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293E cell, a CHO cell, or a NS0 cell.
The present invention also provides a medicament or pharmaceutical composition comprising the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of example 1.
The invention also provides an application of the anti-PD-L1 monoclonal antibody or the antigen binding fragment thereof in preparing a medicament for treating cancer diseases;
preferably, the cancer disease comprises bladder cancer, non-small cell lung cancer, head and neck cancer, hodgkin's lymphoma or melanoma.
Example 3 gradient dilution ELISA comparison of the affinity of anti-PD-L1 phage monoclonal antibodies
The monoclonal antibodies of 6 strains (AH10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3 and BH2-E3) obtained in example 1 are subjected to monoclonal phage display and purification, then a phage gradient dilution ELISA experiment is carried out to identify the affinity, and the anti-PD-L1 monoclonal antibody provided by the core patent CN102245640A of the marketed product atezolizumab is selected as a positive control, and the specific method is as follows:
PD-L1 was coated with a carbonate buffer solution of pH9.6 at a concentration of 100 ng/well/100. mu.l, the solution was coated overnight at a temperature of 4 ℃ and washed three times with PBST, and the 6-strain phage monoclonal antibody selected in example 1 and the anti-PD-L1 phage monoclonal antibody provided in patent CN102245640A were each diluted with a PBST triple gradient, 100. mu.l of the diluted sample was added to each well, and the mixture was allowed to stand at room temperature for 1 hour. The ELISA plate was washed with PBST, and the HRP-anti-M13 monoclonal antibody diluted with PBST was added to the ELISA plate and left at room temperature for 1 h. Developing with TMB color development kit at room temperature for 10 min, and performing color development with 2M H2SO4After termination, readings were taken at 450nm/630nm and the corresponding EC50 values were calculated as follows:
| cloning | AH10-B11 | AC9-B11 | BH2-B11 | AH10-E3 | AC9-E3 | BH2-E3 | CN102245640A |
| EC50 | 8.134 | 5.133 | 2.008 | 9.191 | 9.28 | 5.604 | 35.46 |
Through the data and as shown in FIG. 2, the selected 6 different monoclonal antibodies can be combined with PD-L1, and compared with the anti-PD-L1 monoclonal antibody provided by patent CN102245640A, the monoclonal antibody provided by the invention has stronger binding ability with PD-L1 and higher affinity.
Example 4 preparation of anti-PD-L1 Total antibody
The heavy chain VH and light chain Vkappa genes of the 6 monoclonal antibodies selected in example 1 were cloned into vector pTSE (shown in FIG. 3) containing heavy chain and light chain constant region genes, respectively, the heavy chain constant region is human IgG1 constant region (amino acid sequence is shown in SEQ ID NO:21), the light chain constant region is kappa chain constant region (amino acid sequence is shown in SEQ ID NO:22), the pTSE vector structure is shown in FIG. 3, and the preparation process is described in page 3 [0019] of the CN103525868A specification.
21 (constant region sequence of human IgG 1):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG；
22 (light chain constant region sequence of kappa chain):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
HEK293E cells are transiently transfected, whole antibody expression is carried out, whole antibody protein of 6 monoclonal antibodies is obtained by protein A affinity column purification through an AKTA instrument, protein concentration determination is carried out through a BCA kit, protein size is identified through SDS-PAGE, and as a result, as shown in figure 4, anti-PD-L1 monoclonal antibodies provided by protein molecular weight Marker, AH10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3, BH2-E3 and core patent CN102245640A of atezolizumab which is a commercial product are arranged from left side to right side in sequence, and the molecular weight of each band is consistent with theory.
Example 5 binding experiment of Whole antibody to PD-L1
PD-L1 was coated with carbonate buffer pH9.6 at 100 ng/well/100. mu.l overnight at a temperature of 4 ℃. Washing with 300 μ L/hole PBST five times, adding 1% BSA-PBS, blocking for 2h at 37 ℃, adding different diluted concentrations of full-antibody AH10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3, BH2-E3 and anti-PD-L1 full-antibody in patent CN102245640A, wherein the initial highest concentration of 7 full-antibodies is 1 μ g/ml, respectively diluting by 3 times, making 8 gradients for each full-antibody, and incubating for 1h at 37 ℃. The membrane was washed five times with 300. mu.l/well PBST, and Anti-Human Fc-HRP diluted with 1% BSA-PBS1:10000 was added thereto, followed by incubation at 37 ℃ for 1 h. Developing with TMB color development kit at 100 μ l/well for 8min at room temperature, and then developing with 2M H2SO4The color development was terminated. Readings were taken at 450nm/630nm and the corresponding EC50 values were calculated as follows:
| cloning | AH10-B11 | AC9-B11 | BH2-B11 | AH10-E3 | AC9-E3 | BH2-E3 | CN102245640A |
| EC50(ng/ml) | 1.425 | 0.5744 | 0.5351 | 1.909 | 2.174 | 1.228 | 12.26 |
The data and experimental results are shown in fig. 5, the whole antibodies of the 6 different monoclonal antibodies screened out can be combined with PD-L1, the EC50 values of the whole antibodies of the 6 different monoclonal antibodies provided by the present invention are obviously lower than that of the anti-PD-L1 whole antibody in patent CN102245640A, which indicates that the monoclonal antibody provided by the present invention has high binding affinity with PD-L1 and good activity, and in addition, it can be obtained from fig. 5 and the data above, the whole antibody of BH2-B11 in the 6 different monoclonal antibodies has the best activity and the EC50 value is the lowest, which indicates that the monoclonal antibody has the best binding ability with PD-L1, the highest affinity and the best activity.
Example 6 full antibody Competition inhibits PD-L1 binding to PD-1
PD-L1-Fc 200 ng/well/100 μ L was coated with carbonate pH9.6, coated overnight at 4 deg.C, washed five times with PBST, blocked in 1% BSA-PBS at 37 deg.C for 2h, and diluted with 5 μ g/ml PD-1-His respectively to obtain AH10-B11, AC9-B11, BH2-B11, AH10-E3, AH 10-B3,The total antibodies of AC9-E3, BH2-E3 and the total antibodies provided by patent CN102245640A are all 50 mu g/ml in initial highest concentration, after 3-fold gradient dilution, each total antibody is subjected to 12 dilution gradients, is incubated for 1H at the temperature of 37 ℃, is washed for five times by PBST, is added with HRP-labeled mouse anti-His antibody diluted by 1% BSA-PBS, is incubated for 1H at the temperature of 37 ℃, is subjected to color development by a TMB color development kit, is 100 mu l/hole, is subjected to color development for 8min at room temperature, and is finally subjected to color development by 10% H2SO4Color development was stopped and read at 450nm/630nm with the following data:
| cloning | AH10-B11 | AC9-B11 | BH2-B11 | AH10-E3 | AC9-E3 | BH2-E3 | CN102245640A |
| IC50(ng/ml) | 108.2 | 82.04 | 61.18 | 136 | 138 | 92.73 | 510 |
As shown in FIG. 6, the 7 kinds of total antibodies can inhibit the binding of PD-L1 and PD-1, but the inhibition ability of the 6 strains of total antibodies selected in the invention is significantly stronger than that of the total antibody in the patent CN102245640A, and it can be found from FIG. 6 and the above data that BH2-B11 has the strongest inhibition ability to the binding of PD-L1 and PD-1 in the 6 different monoclonal antibodies.
Example 7 Mixed Lymphocyte Reaction (MLR) assay for anti-PD-L1 monoclonal antibody Activity
Separating PBMC from fresh peripheral blood by density gradient centrifugation, and sorting CD14 with magnetic beads+A T cell; culture of CD14 with medium of 20ng/mL GM-CSF and 10ng/mL IL-4+And (3) changing the solution every 2 days, and inducing the dendritic DC cells after 7-10 days, adding 25ng/mL TNF- α to induce the dendritic DC cells into mature DC cells two days before the dendritic DC cells are used, collecting the mature DC cells, and preparing the mature DC cells into the dendritic DC cells with the cell density of 1x105Cell suspension/mL. Magnetic bead separation of CD4 from fresh PBMC+T cells were counted to make the cell density 1x106Cell suspension/mL. Will CD4+Mu.l of each of the T cells and DC cells was added to a 96-well plate at a ratio of 5: 1.
The 6 anti-PD-L1 whole antibodies prepared in example 4, the anti-PD-L1 whole antibody provided in patent CN102245640A as a positive control and human IgG (hIgG) as an isotype control were diluted in 4-fold gradients, each of the 6 whole antibodies was subjected to 6-fold gradient dilution, 50. mu.l of each of the 6 whole antibodies was added to a 96-well plate, and after 5 days, cck8 was used to test CD4+T cells were propagated, read at 450nm, and corresponding EC50 values were calculated as follows:
| cloning | AH10-B11 | AC9-B11 | BH2-B11 | AH10-E3 | AC9-E3 | BH2-E3 | CN102245640A | hlgG |
| EC50(ng/ml) | 9.876 | 7.576 | 6.728 | 11.37 | 11.87 | 7.706 | 22.57 | - |
Through the data and as shown in fig. 7, the EC50 values of the 6 different anti-PD-L1 holoantibodies screened by the invention are all significantly lower than that of the anti-PD-L1 holoantibody provided by patent CN102245640A, which indicates that the activity of the anti-PD-L1 holoantibody provided by the invention is all higher than that of the anti-PD-L1 holoantibody provided by patent CN 102245640A. The monoclonal antibody of anti-PD-L1 provided by the invention has better activity and stronger affinity. In addition, as can be seen from FIG. 7 and the above data, the 6 monoclonal antibodies selected by the present invention had the highest BH2-B11 activity.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
Sequence listing
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Claims (10)
1. An anti-PD-L1 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of HCDR1 of the heavy chain variable region is selected from SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3; the amino acid sequence of HCDR2 of the heavy chain variable region is selected from SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6; the amino acid sequence of HCDR3 of the heavy chain variable region is selected from SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9; the amino acid sequence of LCDR1 of the light chain variable region is selected from SEQ ID NO. 10 or SEQ ID NO. 11; the amino acid sequence of LCDR2 of the light chain variable region is selected from SEQ ID NO. 12 or SEQ ID NO. 13; the amino acid sequence of LCDR3 of the light chain variable region is selected from SEQ ID NO. 14 or SEQ ID NO. 15.
2. The anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18; the amino acid sequence of the light chain variable region is selected from SEQ ID NO 19 or SEQ ID NO 20.
3. The anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of claim 2, further comprising a heavy chain constant region that is one of human IgG1, IgG2, IgG3, IgG 4;
preferably, the heavy chain constant region is human IgG 1.
4. The anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the monoclonal antibody or antigen-binding fragment thereof is a full-length antibody or antibody fragment comprising one or a combination of Fab, F (ab)2, Fv or ScFv.
5. A polypeptide or protein comprising the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.
6. A polynucleotide sequence or combination encoding the amino acid sequence of the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4.
7. A recombinant DNA expression vector comprising the polynucleotide sequence or combination of claim 6.
8. A host cell transfected with the recombinant DNA expression vector of claim 7, wherein said host cell comprises a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell;
preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293E cell, a CHO cell, or a NS0 cell.
9. A medicament or pharmaceutical composition comprising the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.
10. Use of the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4 in the manufacture of a medicament for the treatment of a cancer disease;
preferably, the cancer disease comprises bladder cancer, non-small cell lung cancer, head and neck cancer, hodgkin's lymphoma or melanoma.
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| CN110974958A (en) * | 2019-12-25 | 2020-04-10 | 北京东方百泰生物科技有限公司 | Injection preparation of anti-PD-L1 monoclonal antibody |
| CN112500486A (en) * | 2020-12-02 | 2021-03-16 | 杭州百凌生物科技有限公司 | anti-PD-L1 antibody and application thereof |
| CN114106175A (en) * | 2020-12-30 | 2022-03-01 | 江苏普若维生物技术有限责任公司 | PD-L1 monoclonal antibody, kit, preparation method and application thereof |
| CN114380913A (en) * | 2022-03-24 | 2022-04-22 | 上海济煜医药科技有限公司 | Fully human anti-PD-L1 antibody and application thereof |
| CN116789833A (en) * | 2022-08-05 | 2023-09-22 | 赛灵药业科技集团股份有限公司北京分公司 | Antibody or fragment thereof and pharmaceutical application thereof |
| CN117209603A (en) * | 2021-12-02 | 2023-12-12 | 北京东方百泰生物科技股份有限公司 | anti-TSLP monoclonal antibody, antigen binding fragment thereof and application thereof |
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| CN110974958B (en) * | 2019-12-25 | 2020-08-21 | 北京东方百泰生物科技有限公司 | Injection preparation of anti-PD-L1 monoclonal antibody |
| CN110974958A (en) * | 2019-12-25 | 2020-04-10 | 北京东方百泰生物科技有限公司 | Injection preparation of anti-PD-L1 monoclonal antibody |
| CN112500486B (en) * | 2020-12-02 | 2023-05-05 | 杭州百凌生物科技有限公司 | anti-PD-L1 antibody and application thereof |
| CN112500486A (en) * | 2020-12-02 | 2021-03-16 | 杭州百凌生物科技有限公司 | anti-PD-L1 antibody and application thereof |
| CN114106175A (en) * | 2020-12-30 | 2022-03-01 | 江苏普若维生物技术有限责任公司 | PD-L1 monoclonal antibody, kit, preparation method and application thereof |
| CN114106175B (en) * | 2020-12-30 | 2023-06-23 | 江苏普若维生物技术有限责任公司 | PD-L1 monoclonal antibody, kit, preparation method and application thereof |
| CN117209603A (en) * | 2021-12-02 | 2023-12-12 | 北京东方百泰生物科技股份有限公司 | anti-TSLP monoclonal antibody, antigen binding fragment thereof and application thereof |
| CN117209604A (en) * | 2021-12-02 | 2023-12-12 | 北京东方百泰生物科技股份有限公司 | anti-TSLP monoclonal antibody, antigen binding fragment thereof and application thereof |
| CN117209603B (en) * | 2021-12-02 | 2024-02-27 | 北京东方百泰生物科技股份有限公司 | anti-TSLP monoclonal antibody, antigen binding fragment thereof and application thereof |
| CN117209604B (en) * | 2021-12-02 | 2024-03-22 | 北京东方百泰生物科技股份有限公司 | anti-TSLP monoclonal antibody, antigen binding fragment thereof and application thereof |
| CN114380913B (en) * | 2022-03-24 | 2022-06-21 | 上海济煜医药科技有限公司 | Fully human anti-PD-L1 antibody and application thereof |
| CN114380913A (en) * | 2022-03-24 | 2022-04-22 | 上海济煜医药科技有限公司 | Fully human anti-PD-L1 antibody and application thereof |
| CN116789833A (en) * | 2022-08-05 | 2023-09-22 | 赛灵药业科技集团股份有限公司北京分公司 | Antibody or fragment thereof and pharmaceutical application thereof |
| CN116789833B (en) * | 2022-08-05 | 2023-12-12 | 赛灵药业科技集团股份有限公司北京分公司 | Antibody or fragment thereof and pharmaceutical application thereof |
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