CN113354736B

CN113354736B - Targeting human PD-L1 monoclonal antibody or antigen binding fragment thereof and application thereof

Info

Publication number: CN113354736B
Application number: CN202110758664.4A
Authority: CN
Inventors: 谢朋辉
Original assignee: Albertson Jiangsu Biotechnology Co ltd
Current assignee: Albertson Jiangsu Biotechnology Co ltd
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2022-05-20
Anticipated expiration: 2041-07-05
Also published as: CN113354736A

Abstract

The invention discloses a targeted human PD-L1 monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises CDR1, CDR2 and CDR3, the CDR1 of the light chain variable region is an amino acid sequence shown in SEQ ID NO.1, the CDR2 of the light chain variable region is an amino acid sequence shown in SEQ ID NO.2, and the CDR3 of the light chain variable region is an amino acid sequence shown in SEQ ID NO. 3; the heavy chain variable region comprises CDR1, CDR2 and CDR3, CDR1 of the heavy chain variable region is the amino acid sequence shown in SEQ ID No.5, CDR2 of the heavy chain variable region is the amino acid sequence shown in SEQ ID No.6, and CDR3 of the heavy chain variable region is the amino acid sequence shown in SEQ ID No. 7. The antibody of the invention can be applied to the development of immunotherapy targeted monoclonal drugs, and can also be modified into bispecific antibody drugs such as scFv single-chain antibody and the like.

Description

Targeting human PD-L1 monoclonal antibody or antigen binding fragment thereof and application thereof

Technical Field

The invention relates to the technical field of phage display monoclonal antibodies, in particular to a targeted human PD-L1 monoclonal antibody or an antigen binding fragment thereof and application thereof.

Background

Programmed death protein-1 (PD-1) and ligand (PD-L1) inhibitors thereof are important targets of immunotherapy monoclonal antibody medicines. PD-1 is mainly expressed in activated T cells and B cells, functions to inhibit the activation of cells, and is a normal self-stabilization mechanism of an immune system, and PD-1 is a physical protector of our human body because excessive T/B cell activation can cause autoimmune diseases. However, the tumor microenvironment can induce infiltrated T cells to highly express PD-1 molecules, and the tumor cells can highly express ligands PD-L1 and PD-L2 of PD-1, so that PD-1 channels in the tumor microenvironment are continuously activated, T cell functions are inhibited, and the tumor cells cannot be killed. Antibodies to PD-1 can block this pathway, partially restoring T cell function, enabling these cells to continue to kill tumor cells. PD-L1, which is up-regulated in many tumor cells, binds to PD-1 on T cells, inhibits T cell proliferation and activation, leaves T cells in an inactive state, and ultimately induces immune escape. The two inhibitors can block the combination of PD-1 and PD-L1, up-regulate the growth and proliferation of T cells, enhance the recognition of T cells to tumor cells, activate the attacking and killing functions of the T cells, and realize the anti-tumor effect by mobilizing the self immune function of a human body.

Two PD-1 monoclonal antibody drugs Opdivo and Keytruda approved by the FDA in the United states are clinically used for various tumors such as melanoma, non-small cell lung cancer, urinary epithelial cancer and the like. The Hengrui medicine, Baiji Shenzhou, Xindao and other medicine, namely the PD-1 monoclonal antibody medicine, are continuously on the market in this year. However, more preferred blocking antibodies against PD-1/PD-L1 for different indications still need to be developed in the art.

Disclosure of Invention

The present invention aims to provide a monoclonal antibody or an antigen-binding fragment thereof targeting human PD-L1 and uses thereof, which address one or more of the above-mentioned problems of the prior art.

In a first aspect, the present invention provides a targeted human PD-L1 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises CDR1, CDR2 and CDR3, CDR1 of the light chain variable region is the amino acid sequence shown in SEQ ID No.1, CDR2 of the light chain variable region is the amino acid sequence shown in SEQ ID No.2, and CDR3 of the light chain variable region is the amino acid sequence shown in SEQ ID No. 3;

the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 of the heavy chain variable region is an amino acid sequence shown in SEQ ID NO.5, the CDR2 of the heavy chain variable region is an amino acid sequence shown in SEQ ID NO.6, and the CDR3 of the heavy chain variable region is an amino acid sequence shown in SEQ ID NO. 7.

In certain embodiments, the light chain variable region sequence consists of the amino acid sequence of SEQ ID No. 4.

In certain embodiments, the heavy chain variable region sequence consists of the amino acid sequence of SEQ ID No. 8.

In certain embodiments, the antibody is a whole antibody or an scFv antibody.

In certain embodiments, the antigen binding fragment is an antigen binding fragment in the Fab form or in the Fab2 form.

In certain embodiments, the antibody is a murine, chimeric, or humanized antibody.

In certain embodiments, a method of preparing an anti-human PD-L1 monoclonal antibody comprises the steps of:

expressing and purifying S1 and PD-L1 recombinant protein;

s2, preparing and immunizing animals, and collecting spleens after immunization is finished;

s3, extracting total RNA;

s4, RNA reverse transcription and antibody variable region PCR;

s5, constructing and transforming a phagemid vector;

s6, library amplification and concentration;

s7, antigen coating and cell preparation;

s8, screening and re-amplifying the library;

s9, library detection.

In certain embodiments, step S1 is specifically: cloning a protein fragment encoded by a human PD-L1 gene into a eukaryotic expression vector, adding a 6-his label at the C end of the protein, carrying out endotoxin-free medium-volume extraction on the vector after correct sequencing, transfecting HEK293F cells with the extracted endotoxin-free plasmid by using a PEI transfection reagent, carrying out fermentation culture after transfection, collecting a surface supernatant, carrying out affinity purification on the supernatant by using a Ni column to obtain a target protein, and dialyzing the target protein into PBS (the pH is 7.4).

In certain embodiments, step S3 is specifically: grinding the fresh spleen collected in the step S2 into powder, weighing a proper amount of the fresh spleen, adding the powder into Trizol, mixing and smashing, standing at room temperature, mixing 2 parts of each sample with chloroform, standing, taking supernatant, mixing the two obtained supernatants, continuously adding isopropanol with the same volume, mixing, standing, centrifuging, removing the supernatant, adding ethanol to wash the precipitate, drying at room temperature, adding DEPC water, and completely dissolving to obtain the total spleen RNA.

In certain embodiments, step S4 is specifically: after the total RNA is quantified by using a micro spectrophotometer, reverse transcription is carried out by a 50 mu L reverse transcription system according to the operation instruction of a reverse transcriptase kit, the PCR primer of the heavy chain variable region of the antibody is properly modified and optimized, and the upstream and the downstream of the primer are introduced with an enzyme cutting site and a linker primer.

In certain embodiments, step S5 is specifically: digesting the antibody heavy and light chain splicing fragment obtained by PCR and a pCom3 vector expression vector by enzyme digestion, and carrying out overnight connection according to a T4 ligase using instruction, wherein the method specifically comprises the following steps: thawing the competent bacteria XL1-blue at 4 ℃, simultaneously precooling an electric shock cup at 4 ℃, uniformly mixing the plasmid and the competent bacteria XL1-blue, adding the mixture into the electric shock cup for ice bath and electric shock, adding an SOC culture medium, taking out electric shock competent cells, combining and collecting the competent cells after repeated times, adding the culture medium after recovery, and culturing and recovering to obtain a bacterium solution.

In certain embodiments, step S6 is specifically: and (2) continuously culturing the bacterial liquid obtained in the step (S5) and a culture medium containing ampicillin, tetracycline and glucose, then adding auxiliary phage, continuously culturing for resuscitation after standing and co-incubation, centrifuging to remove the supernatant, adding the culture medium containing ampicillin, tetracycline and kanamycin for overnight culture and amplification, collecting the overnight culture supernatant, adding a mixed ice bath of PEG8000 and NaCl, centrifuging to remove the supernatant, adding a small-volume PBS sterile buffer solution for redissolving, filtering and sterilizing by using a 0.2um filter membrane to obtain a concentrated library, finally adding DMSO antifreeze solution for uniformly mixing, and freezing and storing at-80 ℃.

In certain embodiments, step S7 is specifically: diluting PD-L1 protein with carbonate buffer solution, adding into 96# enzyme label plate, standing overnight at 4 deg.C, taking out the next day, drying the liquid in the hole, adding PBST washing solution, washing and drying, adding BSA solution for room temperature sealing, washing with PBST, drying, and freezing at-20 deg.C; HCC827 cells are revived and cultured by using RPMI-1640 culture medium, and are subcultured and expanded for later use as required according to the number of cells required by the screening process.

In certain embodiments, step S8 is specifically: taking a concentrated library, diluting the concentrated library in a proper amount, adding skimmed milk powder, mixing, standing and incubating, adding the incubated library into an ELISA plate coated with PD-L1 protein, and incubating; after the library solution is dried, adding a PBST solution, standing, drying, and repeating the steps for a plurality of times; eluting and adjusting pH to be neutral, adding bacterial liquid for mixing, infecting, standing for incubation, centrifuging and removing supernatant after culture recovery, adding a culture medium containing ampicillin, tetracycline and kana antibiotics for overnight culture and amplification, collecting overnight culture supernatant the next day, adding PEG8000 and NaCl for mixing and ice-bath for 1h, centrifuging and removing supernatant at 4 ℃, adding PBS (phosphate buffer solution) with the total volume of 5 percent of the total volume before concentration for dissolving, finally adding DMSO for mixing, freezing and storing at-80 ℃, completing one round of library screening and amplification, and performing two rounds of library screening and amplification again to obtain a library screened and amplified for 3 times.

In a second aspect, the invention provides a use of the monoclonal antibody targeting human PD-L1 or an antigen binding fragment thereof in a medicament for treating lung cancer or melanoma.

Has the advantages that: the antibody provided by the invention can be applied to the development of immunotherapy targeting monoclonal drugs, and can also be transformed into bispecific antibody drugs such as scFv single-chain antibodies; compared with the existing PD-L1 monoclonal antibody, the antibody of the invention has different sequence numbers and structures, and is a brand new structure antibody different from the existing antibody drugs.

Drawings

FIG. 1 is an electrophoretogram of PD-L1, PD-1 purified protein of the present invention;

FIG. 2 is an electrophoretogram of a variable region fragment of a PCR antibody of the present invention;

FIG. 3 shows absorbance values at different dilution times under the conditions of coating PD-L1 protein according to the present invention;

FIG. 4 is a diagram showing the results of Elisa detection of the library of the present invention;

FIG. 5 is a diagram of the PD-1/PD-L1 competition assay of the present invention;

FIG. 6 is a diagram showing the results of immunofluorescence detection of HCC827 and R002 phages in accordance with the present invention;

FIG. 7 is a graph showing the results of competition between R002 of the present invention and a commercially available antibody.

Detailed Description

The present invention will be described in further detail below with reference to embodiments.

Example 1PD-L1 recombinant protein expression and purification

The Met 1-Thr 239 fragment of the protein encoded by the human PD-L1 gene (purchased from leadingbiology) is cloned into a pCDNA3.1 eukaryotic expression vector, the C end of the protein is added with a 6-his tag for purification, and the vector after correct sequencing is subjected to endotoxin-free medium-weight extraction of plasmids. The extracted endotoxin-free plasmids were transfected into HEK293F cells using PEI transfection reagent (purchased from Anlun) at the appropriate plasmid ratio, the transfected cells were cultured to a density of 2X 106 using 293-TII medium (purchased from Okinawa), and the supernatant was collected after 7 days of fermentation culture at 37 ℃ and 165RPM in 5% CO2 after transfection. The supernatant was affinity-purified using a Ni column, and the target protein was eluted in 50mM Tris-HCl, pH8.0,500mM NaCl, 500mM imidazole, dialyzed against PBS (pH 7.4), and the purity and yield of the purified protein were confirmed by SDS-PAGE. The results are shown in FIG. 1, and the final purity of the purified protein is 90% and the yield is 20 mg/L. In the same way, the protein Met 1-Gln 167 fragment encoded by human PD-1 gene (purchased from leadingbiology) was cloned into pCDNA3.1 eukaryotic expression vector, and 6-his tag was added to the C-terminus of the protein for purification. Finally, the purity of the purified protein is 90%, and the yield is 3 mg/L.

Example 2 animal preparation and immunization

The PD-L1 obtained by purification is taken as immunogen, and about 2Kg of healthy Japanese big ear white rabbit is selected for immunization. The first immunization is carried out by emulsifying PD-L1 with equivalent volume of Freund's complete adjuvant according to 500 mug/dose, and injecting the rabbit at multiple points on the back by subcutaneous injection; after the first immunization, every 14 days, after the PD-L150 mu g/dose is emulsified with equal volume of Freund incomplete adjuvant, rabbits are immunized by multipoint subcutaneous injection on the back for 4 times. The indirect ELISA is used for measuring the antibody titer of rabbit serum, and when the titer reaches 81000-fold dilution, the spleen is collected after the rabbit is injected with 500 mu g PD-L1 in the abdominal cavity for 4 days of strengthening immunity.

Example 3 Total RNA extraction

Collecting fresh spleen, adding liquid nitrogen, grinding in mortar until it is fine powder, adding appropriate amount of Trizol, rapidly mixing well, and crushing several times with homogenizer. After standing at room temperature for 5min, 1ml of each sample was added to 2 1.5ml EP tubes in turn. Add 200. mu.L of chloroform to 1.5ml of EP tube and cover the sample tube tightly. The tube was shaken vigorously by hand for 1min and then allowed to stand for 5 min. Approximately 350. mu.L of supernatant was taken from each tube and two tubes were combined to one tube, yielding approximately 700. mu.L of supernatant. Adding equal volume of isopropanol, mixing, and standing at room temperature for 20-30 min. The sample was centrifuged at 11000rpm at 4 ℃ for 10min and the supernatant was discarded. Add 700. mu.L of 75% ethanol and wash the precipitate twice. Centrifuge at 7000rpm for 5min at 4 ℃ and discard the supernatant. Air-drying at room temperature, adding appropriate amount of DEPC water, and dissolving completely to obtain spleen total RNA.

Example 4 reverse transcription of RNA and antibody variable region PCR

After the total RNA was quantified using a microspectrophotometer, reverse transcription was performed in a 50. mu.L reverse transcription system according to the reverse transcriptase kit protocol. The antibody heavy and light chain variable region PCR primer reference [ Shenbei Chengniannan Liu Min Bei, recombinant antibody ] is modified and optimized properly, and the upstream and downstream of the primer are introduced with enzyme cutting site and linker primer. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, at 94 ℃ for 30s, at 55 ℃ for 30s, at 72 ℃ for 5min, for 30 cycles. The antibody heavy and light chain splicing conditions were: pre-denaturation at 94 ℃ for 5min, at 98 ℃ for 15s, at 58 ℃ for 30s, and at 72 ℃ for 1min for 5 cycles; then, the temperature is 15s at 98 ℃, 1min at 68 ℃ and 5min at 72 ℃. The PCR electrophoresis results are shown in FIG. 2. As can be seen from FIG. 2, the PCR fragment was about 500bp in size, and the antibody variable region fragment was efficiently amplified in accordance with the theoretical size.

Example 5 phagemid vector construction and transformation

After digesting the antibody heavy and light chain splicing fragment obtained by PCR and the vector expression vector of pCom3 by enzyme digestion, the antibody heavy and light chain splicing fragment and the vector expression vector are connected overnight according to the instruction of T4 ligase. The competent bacteria XL1-blue were thawed at 4 ℃ and the cuvette was placed at 4 ℃ for 10 minutes. The voltage of the electric rotating instrument is adjusted to 1800V/mm, the mixture with the competent plasmid is added into an electric rotating cup, ice bath is carried out for half a minute, then electric shock is carried out for 5ms, SOC culture medium is rapidly added, and then electric shock competent cells are taken out. Repeating the steps for a plurality of times, combining and collecting competence, adding 2YT culture medium after recovery, and culturing at 37 ℃ and 250rpm for recovery for a plurality of minutes. And (5) taking 100uL of bacterial liquid, diluting and paving the plate, and calculating the storage capacity.

Example 6 library amplification and concentration

Adding 2YT culture medium containing ampicillin, tetracycline and 2% glucose into the recovered bacterial liquid, and continuously culturing for 1 hour. Then, the helper phage was added, incubated at 37 ℃ and allowed to stand for co-incubation, and then incubated at 37 ℃ and 250rpm for resuscitation for 2 hours. Subsequently, the supernatant was removed by centrifugation, and 2YT medium containing ampicillin, tetracycline and kanamycin was added thereto, followed by amplification by overnight culture at 30 ℃ and 250 rpm. The overnight culture supernatant was collected, added with 4% PEG8000 (biologicals) and 3% NaCl, mixed well, ice-cooled for 1 hour, centrifuged at 13000g for 40 minutes at 4 ℃ after which the supernatant was removed, and dissolved in PBS sterile buffer solution of 5% of the total volume before concentration. Finally adding DMSO with the final volume of 7% to mix evenly, and freezing and storing at-80 ℃.

Example 7 antigen coating and cell preparation

The PD-L1 protein is diluted to 10 mu g/mL by carbonate buffer solution (pH9.6), 100 mu L of the solution is added into a detachable 96# enzyme label plate according to each hole, the plate is placed at 4 ℃ overnight, the plate is taken out the next day, the liquid in the hole is dried, PBST washing solution is added for washing twice and dried, then 2% BSA solution is added for sealing at room temperature for 1 hour, PBST washing solution is used for washing twice again and dried, and the solution is frozen and stored at-20 ℃ for standby. HCC827 cells are revived and cultured by using RPMI-1640 culture medium, and are subcultured and expanded for later use as required according to the number of cells required by the screening process.

Example 8 library screening and reamplification

Taking 1mL of the concentrated library, diluting the library by a proper amount, adding 3% skimmed milk powder, mixing the library and the skimmed milk powder uniformly, and standing and incubating the library for 30 minutes at 37 ℃. 100uL of the post-incubation library was added to each well of the PD-L1 protein-coated microplate and incubated at 37 ℃ and 60rpm for 1.5 hours. Draining the library solution, adding 330 mu L of PBST lotion into each hole, standing for 1 minute, and draining; then, 330. mu.L of PBST solution was added, left to stand for 1 minute, and then rinsed off, and the procedure was repeated 6 times. After the PBST washing solution is dried, 100 mu L of PD-1 recombinant protein is added into each hole, standing incubation is carried out for 40 minutes at 37 ℃, the eluent is sucked out and evenly mixed with XL1-blue bacterial liquid (OD is about 1.0), infection is carried out for 30 minutes at 37 ℃, then culture is carried out for 2 hours at 37 ℃ and 250rpm, and after standing co-incubation at 37 ℃, culture is carried out for 1.5 hours at 37 ℃ and 250 rpm. Subsequently, the supernatant was removed by centrifugation, and 2YT medium containing ampicillin, tetracycline and kanamycin was added thereto, followed by amplification by overnight culture at 30 ℃ and 250 rpm. The culture supernatant was collected overnight the next day, 4% PEG8000 (biologics) and 3% NaCl were added, mixed well and then subjected to ice-bath for 1 hour, after which time 13000g was centrifuged at 4 ℃ for 40 minutes to remove the supernatant, which was then dissolved in 5% PBS (sterile buffer) in total volume before concentration. Finally adding DMSO with the final volume of 7% to mix evenly, and freezing and storing at-80 ℃. Thus, completing a round of library screening and amplification, repeating the library screening and library screening process once again, and obtaining the library screened and amplified 2nd (2 times). Starting from the 3rd screening, one round of incubation and screening using HCC827 cells yielded a library that was able to bind to HCC827 cells, and the eluted library was infected with XL1-blue bacterial fluid and amplified according to the previous two rounds of amplification to yield a 3rd screening amplified library.

Example 9 library detection

The 3rd amplification library was diluted 1-fold, 10-fold, 100-fold, 1000-fold, 100 μ L per well was added to coated PD-L1 elisa plates and 2% BSA-blocked blanks, incubated for 1 hour at room temperature, and PBST wash was run twice and dried. anti-M13 HRP-labeled secondary antibody (mounting biology) was added, incubated at room temperature for 40 minutes, and the PBST wash was washed three times and the buttons were dried. TMB developing solution is added, after developing for 20 minutes at room temperature, 2M sulfuric acid is added to stop the development, and an enzyme-labeling instrument OD450nm reads the light absorption value, and the result is shown in figure 3. As can be seen in FIG. 3, the apparent absorbance values at different dilution times under the conditions of coating PD-L1 protein were higher than those of the control, indicating that antibodies against PD-L1 were present in the library.

EXAMPLE 10 monoclonal expression and Elisa test

After the positive library was diluted appropriately in the third round of test, it was infected with XL1-blue E.coli and plated. The next day, monoclonal colonies were picked from the plate and cultured. Reserving one part of the cultured bacterial liquid as a sequencing backup, incubating the remaining bacterial liquid by using auxiliary phage, adding 2YT culture medium containing ampicillin, tetracycline and kanamycin antibiotic, and carrying out overnight culture and amplification at 30 ℃ and 250 rpm. 50ng of 96# microplate was coated with PD-L1 per well for 1 plate in total, as control in 2% BSA blocked blank. After blocking, washing and patting dry, 10 times diluted expression samples were added, incubated at room temperature for 1 hour, and then washed twice with PBST wash solution and dried. anti-M13 HRP-labeled secondary antibody (purchased to leadingbiology) was added, incubated for 40 minutes at room temperature, and washed three times with PBST wash and dried. Adding TMB color development solution, developing for 20 minutes at room temperature, adding 2M sulfuric acid to stop color development, and reading a light absorption value by an enzyme-labeling instrument OD450 nm. The absorbance of clones detected as positive is shown in FIG. 4.

Example 11 PD-1/PD-L1 Competition assay

The clones with better positive detection are subjected to a PD-1/PD-L1 blocking detection experiment. Specifically, 50ng of the ELISA plate was coated with PD-L1 in each well, and the two wells were coated. Each sample was diluted 10 times and added to one coated well and an equal volume of PBS solution was added to the other coated well and incubated at room temperature for 1 hour before PBST wash was washed twice and dried. Then 50ng of biotin-labeled PD-1 was added to each well, and after incubation for 30 minutes at room temperature, the PBST wash was washed twice and dried. anti-Avidin HRP-labeled secondary antibody (purchased to leadingbiology) was added, incubated for 40 minutes at room temperature, and washed three times with PBST wash and drained. Adding TMB developing solution, developing for 20 minutes at room temperature, adding 2M sulfuric acid to stop developing, and reading the light absorption value by an enzyme-labeling instrument OD450 nm. The competition result of R002 is shown in FIG. 5, and the competition rate reaches 92.35%.

Example 12 immunofluorescence detection and sequence analysis

Competitive assay the most competitive sample was further analyzed for targeting HCC827 cell activity by immunofluorescence. Diluting the monoclonal sample, adding the diluted monoclonal sample into a 96# plate paved with HCC827 cells, incubating for 1 hour, washing the plate, adding a rabbit anti-M13 antibody, incubating for 1 hour, washing the plate, adding a goat anti-rabbit IgG-FITC labeled secondary antibody, continuing to incubate for half an hour, washing the plate to remove unbound secondary antibody, and taking a fluorescent photograph to read the cell binding result. In the experiment, rabbit anti-M13 antibody and goat anti-rabbit IgG-FITC were purchased to leadingbiology. Finally, R002 clone with a high affinity binding signal with HCC827 cells is obtained through immunofluorescence detection, and basically no signal is obtained with 293t cells as a negative control, and the result is shown in FIG. 6, and further sequencing analysis is carried out to obtain an antibody variable region sequence of the antibody, wherein the light chain variable region sequence is shown in SEQ ID NO.4, the CDR 1-containing sequence is shown in SEQ ID NO.1, the CDR 2-containing sequence is shown in SEQ ID NO.2, and the CDR 3-containing sequence is shown in SEQ ID NO. 3. The heavy chain variable region sequence is shown in SEQ ID NO.7, the CDR1 sequence is shown in SEQ ID NO.5, the CDR2 sequence is shown in SEQ ID NO.6, and the CDR3 sequence is shown in SEQ ID NO. 7.

Example 13 comparison of competitive effects on the level of intact antibody

To further verify the blocking effect at the antibody level, the sequence shown in SEQ ID NO.4 and the human light chain kappa constant region are fused and constructed to the pCDNA3.1 vector, and the sequence shown in SEQ ID NO.8 and the human heavy chain IgG1 constant region are fused and constructed to the pCDNA3.1 vector, and after the two plasmids are co-transfected into HEK293F cells and fermented for 6 days, the R002 recombinant antibody with the purity of more than 90 percent is obtained by purification through a proteinG column. Under the same condition, the heavy-light chain variable regions of a PD-L1 antibody which is already on the market are respectively recombined and expressed by the same method, and finally the recombinant antibody with the purity of more than 90 percent is obtained. The content of ELISA plate coated PD-L1 is further reduced through optimization of antibody blocking effect detection, and finally coating is carried out according to 20ng per well, and 3 parts of coating are carried out. Adding 100uL of a dilution containing 0.5ug/mL of PD-1 biotin labeled protein into the first part, adding 0.5ug/mL of PD-1 biotin labeled protein and 1ug/mL of R002 recombinant antibody into the second part, adding 0.5ug/mL of PD-1 biotin labeled protein and 1ug/mL of marketed recombinant expression antibody into the third part, incubating at room temperature for 30 minutes, and washing with PBST (Poly-p-phenylene benzobisoxazole) washing solution twice to remove the impurities. anti-Avidin HRP labeled secondary antibody was added, incubated at room temperature for 30 minutes, and rinsed three times with PBST wash and drained. Adding TMB color development solution, developing for 20 minutes at room temperature, adding 2M sulfuric acid to stop color development, and reading a light absorption value by an enzyme-labeling instrument OD450 nm. The competition result of R002 is shown in FIG. 7, the competition rate is as high as 95.74%, and the detection result shows that the competition rate is higher than that of the antibodies on the market.

In conclusion: the specific high-affinity PD-L1 monoclonal antibody can be applied to development of immunotherapy targeting monoclonal drugs and can be transformed into bispecific antibody drugs such as scFv single-chain antibodies. The results of a plurality of data show that the compound has excellent effect of blocking PD-1/PD-L1 and has larger drug development potential.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these should be considered as within the scope of the present invention.

Sequence listing

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Claims

1. A targeted human PD-L1 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises CDR1, CDR2 and CDR3, CDR1 of the light chain variable region is the amino acid sequence shown in SEQ ID No.1, CDR2 of the light chain variable region is the amino acid sequence shown in SEQ ID No.2, and CDR3 of the light chain variable region is the amino acid sequence shown in SEQ ID No. 3;

2. The targeted human PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region sequence consists of amino acid sequence SEQ ID No.4 and the heavy chain variable region sequence consists of amino acid sequence SEQ ID No. 8.

3. The monoclonal antibody or antigen-binding fragment thereof targeting human PD-L1 according to claim 1, characterized in that said antibody is a whole antibody or scFv antibody.

4. The human targeted PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein said antigen-binding fragment is an antigen-binding fragment in Fab or Fab2 form.

5. The targeted human PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a murine, chimeric or humanized antibody.

6. The use of the monoclonal antibody or antigen-binding fragment thereof targeting human PD-L1 according to any one of claims 1 to 5 in a medicament for the treatment of lung cancer or melanoma.