CN110964111B - 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
- Publication number
- CN110964111B CN110964111B CN201911352088.2A CN201911352088A CN110964111B CN 110964111 B CN110964111 B CN 110964111B CN 201911352088 A CN201911352088 A CN 201911352088A CN 110964111 B CN110964111 B CN 110964111B
- Authority
- CN
- China
- Prior art keywords
- ser
- monoclonal antibody
- seq
- binding fragment
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 high 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 tumor cell immune escape, can be effectively used for treating cancer diseases, and has a wide development prospect, and the cancer diseases comprise but are not limited to bladder cancer, non-small cell lung cancer, head and neck cancer, Hodgkin 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 thereof and application thereof.
Background
Programmed cell death molecule 1(Programmed cell death ligand 1) and ligand protein PD-L1 (Programmed cell death ligand 1) molecules are important immune checkpoint proteins with inhibitory effect, PD-1/PD-L1 drugs are a new class of currently-spotlighted tumor therapy drugs and are the main force in current immunotherapy, at present, monoclonal antibodies aiming at the two proteins are available on the market for 8 drugs, and the 8 monoclonal antibodies play a very important role in the tumor therapy process although the indications for the two monoclonal antibodies are different.
Programmed death receptor-1 (PD-1) is an inhibitory receptor on T cells, PD-1 can interact with ligand (Programmed death-1ligand, PD-L s) PD-L1 and PD-L2 to inhibit the proliferation, activation and cytokine secretion of T cells, therefore, PD-1 is an important immune sentinel for regulating T cell response, in normal organisms, PD-1 and PD-L s signal channels play an important role in maintaining the immune tolerance of organisms, and in tumorigenesis, PD-1 and PD-L s signal channels can inhibit the immune response of T cells to promote the occurrence of tumor immune escape, therefore, PD-1 and PD-L s signal channels are taken as targets, and the development of PD-1 or PD-L s blocking agents with remarkable curative effect and low adverse reaction becomes a hot spot in the field of tumor immunotherapy in recent years.
Programmed cell death-L igand 1 (PD-L1) is a first type transmembrane protein with the size of 40kDa, an immune system can react to foreign antigens gathered in lymph nodes or spleen under normal conditions to promote T cell proliferation with antigen specificity, and the binding of PD-1 and PD-L1 can transmit inhibitory signals to reduce the proliferation of T cells, so that one way for tumor cells to escape from T cell destruction is to generate PD-L1 on the surface of the tumor cells, so that a large amount of PD-1 and PD-L1 are bound, the T cells cannot find the tumor cells and cannot send attack signals to the tumor cells, and therefore, the blocking of the binding between PD-L1/PD-1 is a very potential direction in the field of tumor immunotherapy.
The phage display technology is a technology which is firstly established by Smith and inserts a gene coding foreign protein or polypeptide into phage capsid protein gene, so that the foreign protein or polypeptide and phage capsid protein are fused and expressed on the surface of phage, the phage display technology utilizes the principle to express antibodies with different specificities or functional fragments thereof (Fab, Fv and ScFv) on the surface of phage, then the antigen is used for screening, the phage antibody library is divided into an immune library and a non-immune library according to the source of the antibody gene, the non-immune library comprises a natural library, a semi-synthetic library and a fully synthetic library, the screening process of the antibody affinity is simulated, the mature antigen is screened on a solid phase, the antigen is screened, and the high-activity antibody screening medium is added into the phage display library, and the anti-human antibody library is selectively eluted through a high-affinity screening process, namely, a high-affinity screening process of the phage antibody, a high-affinity screening process of the anti-antibody is carried out through a high-affinity screening technology of the phage antibody, a high-affinity screening technology of the high-affinity antibody.
Disclosure of Invention
In order to meet the requirements of domestic markets, the anti-PD-L1 monoclonal antibody is screened from a fully-synthesized phage antibody library, then a small-capacity synthetic phage antibody library is constructed by mutating and banking the light chain CDR123 region of the monoclonal antibody obtained by primary screening, affinity maturation is carried out on the light chain, the monoclonal antibody with higher affinity is selected by screening and identifying, then the heavy chain CDR123 region mutation is constructed and the affinity maturation is carried out, and finally the anti-PD-L1 monoclonal antibody or the antigen binding fragment thereof with higher affinity and better activity is screened.
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 L CDR1 of the light chain variable region is selected from SEQ ID NO 10 or SEQ ID NO 11, the amino acid sequence of L CDR2 of the light chain variable region is selected from SEQ ID NO 12 or SEQ ID NO 13, and the amino acid sequence of L CDR3 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 the antigen binding fragment thereof.
The invention also provides a polynucleotide sequence or a combination, which encodes 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 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.
The anti-PD-L1 monoclonal antibody has the advantages that the anti-PD-L monoclonal antibody has high 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 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, and the cancer diseases comprise but are not limited to bladder cancer, non-small cell lung cancer, head and neck cancer, Hodgkin 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 gradient-diluted phase E L ISA of example 3 of the present invention identifying the relative affinity of phase-Abs;
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 ability of the whole antibody and PD-L1 at the 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 activities of the PD-L1 monoclonal antibody tested for mixed lymphocyte reaction (M L R) 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
Example 1 of the present invention provides an anti-PD-L1 monoclonal antibody or 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 L CDR1 of the light chain variable region is selected from SEQ ID No. 10 or SEQ ID No. 11, the amino acid sequence of L CDR2 of the light chain variable region is selected from SEQ ID No. 12 or SEQ ID No. 13, and the amino acid sequence of L CDR3 of 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 invention utilizes phage antibody library display technology to screen out monoclonal antibody of anti-PD-L1 from fully synthesized phage antibody library, then constructs method of small capacity synthetic phage antibody library to build library for light chain CDR123 region mutation of monoclonal antibody obtained by primary screening, carries out affinity maturation to light chain, selects monoclonal antibody with higher affinity through screening identification, then carries out affinity maturation to heavy chain CDR123 region mutation library, and finally screens out monoclonal antibody of anti-PD-L1 with high affinity, the biopanning 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, taking PD-L1 as an antigen coating immune tube, coating the antigen with the amount of 5 mug/500 mug/tube at 4 ℃ overnight, respectively sealing the immune tube and a fully synthetic phage antibody library by 4% skimmed milk powder/PBST, sealing the immune tube and the fully synthetic phage antibody library for 1h at room temperature, adding the sealed fully synthetic phage antibody library into the immune tube for antigen-antibody combination, wherein the input amount of phage is about 109～1012After 1 hour of reaction at room temperature, unbound phage was washed off with PBST-PBS, eluted with 0.1M Glycine-HCl (pH2.2), and the eluted phage antibody solution was neutralized to pH7.0 or so with 1.5M Tris-HCl (pH8.8).
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 the positive clone of the PD-L1 phage single-chain antibody, selecting well-separated monoclonal colony after three rounds of screening, inoculating the colony in a 96-hole deep-hole plate added with 2YTAG liquid culture medium, culturing the colony to the logarithmic growth phase under the conditions of 37 ℃ and 220rpm, and adding about 10 percent of monoclonal antibody into each hole10The helper phage M13KO7 is statically infected at 37 ℃ for 30min, centrifuged at 4000rpm for 15min, the supernatant is discarded, the phage is resuspended and precipitated with 2YTAK, cultured overnight at 28 ℃ and 220rpm, centrifuged at 4000rpm and 4 ℃ for 15min, the amplified phage supernatant is extracted for E L ISA identification, and the anti-PD-L1 single-chain antibody with high affinity is obtained by screening.
And step six, performing in-vitro affinity maturation on the anti-PD-L1 single-chain antibody obtained by the primary screening in the step five, specifically, synthesizing a light chain CDR123 mutation library on the basis of the anti-PD-L1 single-chain antibody light chain obtained by the primary screening, performing screening identification according to the step two to the step five to obtain two clones with higher affinity, synthesizing the two light chains respectively, constructing a heavy chain CDR123 mutation library, performing screening identification according to the step two to the step five to finally obtain six monoclonal antibodies with higher affinity, which are named as AH10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3 and BH2-E3 respectively, and performing gene sequencing on the obtained monoclonal antibodies to determine the correct antibody sequence
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 of E L ISA comparison of 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 display and purification of monoclonal phages, then phage gradient dilution E L ISA experiments are carried out to identify the affinity, and the anti-PD-L1 monoclonal antibody provided by 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 carbonate buffer solution (pH 9.6) at 100 ng/well/100. mu.l overnight at 4 ℃, washed three times with PBST, and the 6 phage monoclonal antibodies obtained in example 1 were screenedAnd the anti-PD-L1 phage monoclonal antibody provided by patent CN102245640A were diluted with PBST three times of gradient, 100. mu.l of diluted sample was added to each well, and left to stand at room temperature for 1 hour, the E L ISA plate was washed with PBST, the HRP-anti-M13 monoclonal antibody diluted with PBST was added to the E L ISA plate, and left to stand at room temperature for 1 h.TMB color development kit for color development at room temperature for 10 minutes, and 2M H was used for color development2SO4After 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
Coating PD-L1 with carbonate buffer solution of pH9.6, coating 100 ng/well/100. mu.l, coating overnight at 4 deg.C, washing with 300. mu.l/well PBST five times, adding 1% BSA-PBS, blocking at 37 deg.C for 2h, adding total Anti-AH 10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3, BH2-E3 and Anti-PD-L1 total antibodies of patent CN102245640A at different dilution concentrations, starting maximum concentration of 7 total antibodies being 1. mu.g/ml, each total antibody being diluted 3 times, each being incubated with 8 gradients, washing at 37 deg.C for 1h, washing with 300. mu.l/well PBST five times, adding Anti-HuFc-HRP diluted with 1% BSA-PBS1:10000, incubating at 37 deg.C for 1h, developing color at room temperature, developing for 100. mu.l/well, developing color at 2M H min, developing at room temperature, developing for 100. mu.l/well, developing color at 678 min, and developing room temperature2SO4The 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 |
As shown in fig. 5, the selected full antibodies of 6 different monoclonal antibodies can bind to PD-L1, and the EC50 values of the full antibodies of the 6 different monoclonal antibodies provided by the present invention are significantly lower than the anti-PD-L1 full antibody in CN102245640A, which indicates that the monoclonal antibody provided by the present invention has higher binding affinity with PD-L1 and better activity, and in addition, as can be seen from fig. 5 and the above data, the full antibody of BH2-B11 in the 6 different monoclonal antibodies has the best activity and the EC50 value is the lowest, indicating that the full antibody has the best binding ability with PD-L1, the highest affinity and the best activity.
Example 6 full antibody Competition inhibits the binding of PD-L1 to PD-1
Coating PD-L1-Fc 200 ng/well/100 μ l with carbonate of pH9.6, coating overnight at 4 deg.C, washing five times with PBST, blocking for 2H at 37 deg.C in 1% BSA-PBS, diluting AH10-B11, AC9-B11, BH2-B11, AH10-E3, AC9-E3, BH2-E3 whole antibody and whole antibody provided by patent CN102245640A with PD-1-His of 5 μ g/ml, respectively, the initial highest concentration of all antibodies is 50 μ g/ml, after 3-fold gradient dilution, each whole antibody is subjected to 12 dilution gradients, incubating for 1H at 37 deg.C, washing five times with PBST, adding mouse anti-His antibody diluted with HRP-PBS of 1%, incubating for 1H at 37 deg.C, developing for 100H with TMB, developing for 100 min, developing for 10 μ l at room temperature, and developing for 10 μ l with H8H at room temperature2SO4Color 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 |
The data and results are shown in fig. 6, 7 kinds of full antibodies can inhibit the binding of PD-L1 and PD-1, but the full antibody inhibiting ability of the 6 strains selected in the present invention is significantly stronger than that of the full antibody in patent CN102245640A, and it can be found from fig. 6 and the data that BH2-B11 has the strongest inhibiting ability for the binding of PD-L1 and PD-1 in the 6 different monoclonal antibodies.
Example 7 Mixed lymphocyte reaction (M L R) testing of anti-PD-L1 monoclonal antibody Activity
Separating PBMC from fresh peripheral blood by density gradient centrifugation, and sorting CD14 with magnetic beads+Culturing CD14 in culture medium of 20ng/m L GM-CSF and 10ng/m L I L-4+The dendritic DC cells are induced to become mature DC cells by adding 25ng/m L TNF- α two days before the dendritic DC cells are used, collecting the mature DC cells, and preparing the mature DC cells with the cell density of 1x105Cell suspension of L/m CD4 was separated from fresh PBMC by magnetic bead separation+T cells were counted to make the cell density 1x106Cell suspension of L pieces/m 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-strain anti-PD-L1 whole antibody 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-fold gradients was set for each whole antibody, 50. mu.l of each was added to a 96-well plate, and after 5 days, cck8 tested 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-L full antibodies screened by the invention are all obviously lower than that of the anti-PD-L full antibody provided by patent CN102245640A, which indicates that the activity of the anti-PD-L1 full antibody provided by the invention is all higher than that of the anti-PD-L full antibody provided by patent CN102245640A, which indicates that the anti-PD-L monoclonal antibody provided by the invention has better activity and stronger affinity.
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
<110> Beijing Oriental Baitai Biotechnology Ltd
<120> anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof
<160>22
<170>SIPOSequenceListing 1.0
<210>1
<211>10
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>1
Gly Trp Thr Leu Ser Asp Ser Leu Gln His
1 5 10
<210>2
<211>10
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>2
Gly Cys Thr Trp Ser Asp Ser Trp Asp His
1 5 10
<210>3
<211>10
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>3
Gly Leu Thr Trp Ser Asp Ser Tyr Lys His
1 5 10
<210>4
<211>10
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>4
Gly Phe Gln Ser Ala Tyr Gly Gly Ser Thr
1 5 10
<210>5
<211>10
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>5
Asp Tyr Glu Ser Thr Tyr Gly Gly Ser Thr
1 5 10
<210>6
<211>10
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>6
His Trp Asn Ser Thr Tyr Gly Gly Ser Thr
1 5 10
<210>7
<211>11
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>7
Ala Arg Arg His Tyr Tyr Thr Ala Trp Asp Tyr
1 5 10
<210>8
<211>11
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>8
Ala Arg Arg His Trp Asn Ala Thr Phe Asp Tyr
1 5 10
<210>9
<211>11
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>9
Ala Arg Arg His Phe Ala Ser Thr Trp Asp Tyr
1 5 10
<210>10
<211>7
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>10
Gln Asn Ile His Asn Ser Leu
1 5
<210>11
<211>7
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>11
Gln Ser Ile His Asn Tyr Leu
1 5
<210>12
<211>6
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>12
Gly Ala Ser Ser Leu Glu
1 5
<210>13
<211>6
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>13
Ser Ala Ser Asn Leu Pro
1 5
<210>14
<211>9
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>14
Gln Gln Glu Arg Arg Val Pro Trp Thr
1 5
<210>15
<211>9
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>15
Gln Gln Gly Leu His Thr Pro Pro Thr
1 5
<210>16
<211>118
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Trp Thr Leu Ser Asp Ser
20 25 30
Leu Gln His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Gln Ser Ala Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Tyr Tyr Thr Ala Trp Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>17
<211>118
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Cys Thr Trp Ser Asp Ser
20 25 30
Trp Asp His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Asp Tyr Glu Ser Thr Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Asn Ala Thr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>18
<211>118
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Trp Ser Asp Ser
20 25 30
Tyr Lys His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
His Trp Asn Ser Thr Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Phe Ala Ser Thr Trp Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>19
<211>107
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile His Asn Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Glu Arg Arg Val Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210>20
<211>107
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Asn Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Leu His Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210>21
<211>329
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>21
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210>22
<211>107
<212>PRT
<213> Artificial sequence (SIPOS sequence L sting 1.0)
<400>22
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
Claims (11)
1. 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 the heavy chain variable region is selected from SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO 19 or SEQ ID NO 20.
2. The anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of claim 1, further comprising a heavy chain constant region that is one of human IgG1, IgG2, IgG3, IgG 4.
3. The anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain constant region is human IgG 1.
4. The anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof of claim 2, 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 protein which is the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.
6. A polynucleotide encoding the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.
7. A recombinant DNA expression vector comprising the polynucleotide 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.
9. The host cell of claim 8, wherein the host cell is a mammalian cell that is a HEK293E cell, a CHO cell, or a NS0 cell.
10. A medicament comprising the anti-PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.
11. 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, including bladder cancer, non-small cell lung cancer, head and neck cancer, hodgkin's lymphoma or melanoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911352088.2A CN110964111B (en) | 2019-12-25 | 2019-12-25 | anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911352088.2A CN110964111B (en) | 2019-12-25 | 2019-12-25 | anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110964111A CN110964111A (en) | 2020-04-07 |
| CN110964111B true CN110964111B (en) | 2020-07-24 |
Family
ID=70036309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911352088.2A Active CN110964111B (en) | 2019-12-25 | 2019-12-25 | anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110964111B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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 |
| CN117106084B (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 |
| CN115925937A (en) * | 2022-08-05 | 2023-04-07 | 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106008714A (en) * | 2016-05-24 | 2016-10-12 | 瑞阳(苏州)生物科技有限公司 | Anti-human pd-1 humanized monoclonal antibody and application thereof |
| CN110194798A (en) * | 2019-05-31 | 2019-09-03 | 杭州科兴生物科技有限公司 | Anti- PD-L1 monoclonal antibody, segment and its medical usage |
| CN110392694A (en) * | 2016-11-02 | 2019-10-29 | 震动疗法股份有限公司 | For the antibody and application thereof of PD-1 |
| CN110404066A (en) * | 2018-04-28 | 2019-11-05 | 齐鲁制药有限公司 | A kind of monoclonal antibody formulation of anti-human PD-1, combination medicine and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR093984A1 (en) * | 2012-12-21 | 2015-07-01 | Merck Sharp & Dohme | ANTIBODIES THAT JOIN LEGEND 1 OF SCHEDULED DEATH (PD-L1) HUMAN |
-
2019
- 2019-12-25 CN CN201911352088.2A patent/CN110964111B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106008714A (en) * | 2016-05-24 | 2016-10-12 | 瑞阳(苏州)生物科技有限公司 | Anti-human pd-1 humanized monoclonal antibody and application thereof |
| CN110392694A (en) * | 2016-11-02 | 2019-10-29 | 震动疗法股份有限公司 | For the antibody and application thereof of PD-1 |
| CN110404066A (en) * | 2018-04-28 | 2019-11-05 | 齐鲁制药有限公司 | A kind of monoclonal antibody formulation of anti-human PD-1, combination medicine and application thereof |
| CN110194798A (en) * | 2019-05-31 | 2019-09-03 | 杭州科兴生物科技有限公司 | Anti- PD-L1 monoclonal antibody, segment and its medical usage |
Non-Patent Citations (2)
| Title |
|---|
| Structural basis of anti-PD-L1 monoclonal antibody avelumab for tumor therapy;Liu, Kefang等;《Cell Res.》;PMC;20160830;第27卷(第1期);第151-153页 * |
| 鼠抗人PD-L1单克隆抗体的制备及鉴定;陈娇等;《四川大学学报(医学版)》;CNKI;20110115;第42卷(第1期);第5-9页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110964111A (en) | 2020-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110964111B (en) | anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof | |
| JP7262597B2 (en) | Bispecific antibodies and methods of making and using the same | |
| CN107488229B (en) | PD-L1 antibodies and uses thereof | |
| CN114621345B (en) | anti-LAG-3 monoclonal antibody, antigen binding fragment thereof and application thereof | |
| CN111848800B (en) | PD-L1 single-domain antibody and application thereof | |
| CN109776678A (en) | A kind of humanization PD-L1 monoclonal antibody, preparation method and application | |
| CN110655579B (en) | Novel anti-CT L A-4 monoclonal antibody and application thereof | |
| CN112500485A (en) | anti-B7-H3 antibody and application thereof | |
| CN110563849A (en) | anti-VEGF-anti-PD 1 bispecific antibody with brand new sequence | |
| CN104829725A (en) | Construction and application of bispecific antibody CD133*CD3 | |
| CN112745391A (en) | PD-L1 binding molecules | |
| CN105504062B (en) | The detection antibody of anti-CD 6 monoclonal antibody T1h a kind of and application | |
| WO2022247804A1 (en) | Anti-gprc5d antibody, preparation method therefor, and use thereof | |
| WO2023273595A1 (en) | Antibody binding to trop2, bispecific antibody targeting trop2 and cd3, preparation methods therefor and uses thereof | |
| CN117586397A (en) | Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof | |
| CN104558181A (en) | Humanized single-chain variable fragments of targeted B lymphoma cells | |
| CN115505043A (en) | Antibodies specifically binding glycosylated CEACAM5 | |
| CN112521499B (en) | anti-CXCL 13 antibodies and uses thereof | |
| CN116199779B (en) | anti-LILRB 4 monoclonal antibody, antigen binding fragment thereof and application thereof | |
| CN116284393A (en) | LILRB 4-targeted monoclonal antibody | |
| CN116178550A (en) | anti-LILRB 4 monoclonal antibody | |
| CN115947850A (en) | Monoclonal antibody targeting LILRB4 | |
| CN116143922A (en) | anti-LILRB 4 monoclonal antibody | |
| CN115536746A (en) | anti-CRTAM antibodies and uses thereof | |
| CN113527491A (en) | Humanized antibody, chimeric antigen receptor, nucleic acid, vector, cell and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 406, room 1, building 2, 100176 Jingdong street, Beijing economic and Technological Development Zone, Beijing, Daxing District Patentee after: Beijing Dongfang Baitai Biotechnology Co., Ltd Address before: 406, room 1, building 2, 100176 Jingdong street, Beijing economic and Technological Development Zone, Beijing, Daxing District Patentee before: Beijing Dongfang Biotech Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder |