CN113150155B

CN113150155B - anti-PD-L1 humanized monoclonal antibody and application thereof

Info

Publication number: CN113150155B
Application number: CN202110436333.9A
Authority: CN
Inventors: 汪国兴; 刘培培; 樊丽; 鲍习琛
Original assignee: Anhui Rubiox Vision Biotechnology Co ltd
Current assignee: Anhui Rubiox Vision Biotechnology Co ltd
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2022-04-08
Anticipated expiration: 2041-04-22
Also published as: CN113150155A

Abstract

The invention relates to the technical field of immunology and antibody engineering, in particular to an anti-PD-L1 humanized monoclonal antibody and application thereof. The CDR-H1 of the heavy chain variable region of the antibody is an amino acid sequence shown by SEQ ID NO. 1, the CDR-H2 is an amino acid sequence shown by SEQ ID NO. 2, and the CDR-H3 is an amino acid sequence shown by SEQ ID NO. 3; and the CDR-L1 of the light chain variable region of the antibody is the amino acid sequence shown in SEQ ID NO. 4, the CDR-L2 is the amino acid sequence shown in SEQ ID NO. 5, and the CDR-L3 is the amino acid sequence shown in SEQ ID NO. 6. The anti-PD-L1 monoclonal antibody provided by the invention has a brand new sequence, can be specifically combined with human PD-L1 protein, has high affinity, enables immune cells to specifically recognize and kill cancer cells, achieves the aim of treating cancer, and has molecular cell level expression not lower than that of the currently sold products.

Description

anti-PD-L1 humanized monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of immunology and antibody engineering, in particular to an anti-PD-L1 humanized monoclonal antibody and application thereof.

Background

PD-L1, its full name programmed cell death-Ligand 1, is also called as apoptosis-Ligand 1. The PD-L1 protein is a ligand of PD-1, is related to the inhibition of an immune system, and can transmit inhibitory signals. Once PD-1 and PD-L1 bind, they transmit a negative regulatory signal to T cells, inducing them to go to rest, decreasing the proliferation of lymph node CD8+ T cells, making them unable to recognize cancer cells, and decreasing or apoptosis of T cells themselves, effectively relieving the immune response of the body, so that cancer cells can grow erratically. The anti-PD-L1 antibody provided by the invention can be combined with PD-1 protein, activate T cell recognition and kill cancer cells.

Disclosure of Invention

The invention provides an anti-PD-L1 monoclonal antibody and a preparation method thereof, the anti-PD-L1 monoclonal antibody has a brand new sequence, can be specifically combined with PD-1 protein, enables immune cells to specifically recognize and kill cancer cells, and achieves the purpose of treating cancer.

The invention is realized by the following technical scheme:

an anti-PD-L1 humanized monoclonal antibody, wherein CDR-H1 of a heavy chain variable region of the antibody is an amino acid sequence shown as SEQ ID NO. 1, CDR-H2 is an amino acid sequence shown as SEQ ID NO. 2, and CDR-H3 is an amino acid sequence shown as SEQ ID NO. 3; and the CDR-L1 of the light chain variable region of the antibody is the amino acid sequence shown in SEQ ID NO. 4, the CDR-L2 is the amino acid sequence shown in SEQ ID NO. 5, and the CDR-L3 is the amino acid sequence shown in SEQ ID NO. 6.

Preferably, the CDR-H1 of the heavy chain variable region of the antibody is the nucleotide sequence shown in SEQ ID NO. 7, the CDR-H2 is the nucleotide sequence shown in SEQ ID NO. 8, and the CDR-H3 is the nucleotide sequence shown in SEQ ID NO. 9; and the CDR-L1 of the light chain variable region of the antibody is the nucleotide sequence shown in SEQ ID NO. 10, the CDR-L2 is the nucleotide sequence shown in SEQ ID NO. 11, and the CDR-L3 is the nucleotide sequence shown in SEQ ID NO. 12.

Preferably, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 13.

Preferably, the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 14.

Preferably, the heavy chain nucleotide sequence of the antibody is shown as SEQ ID NO. 15.

Preferably, the light chain nucleotide sequence of the antibody is shown as SEQ ID NO 16.

The invention also provides a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.

The invention also protects the application of the antibody in pharmacy.

The invention also protects the application of the antibody in preparing a medicament for treating cancer.

The invention has the beneficial effects that:

the anti-PD-L1 monoclonal antibody provided by the invention has a brand new sequence, can be specifically combined with human PD-L1 protein, has high affinity, enables immune cells to specifically recognize and kill cancer cells, achieves the aim of treating cancer, and has molecular cell level expression not lower than that of the currently sold products.

Drawings

FIG. 1 shows the result that humanized antibody SA126-C9 induces T cells to secrete IL 2;

FIG. 2 shows the result of humanized antibody SA126-C9 inducing T cells to secrete IFN-gamma;

FIG. 3 is a graph showing the binding activity of SA126-C9-hIgG1, SA126-G7-hIgG1 antibody to human PD-L1 antigen;

FIG. 4 is a graph showing the binding activity of SA126-C9-hIgG1, SA126-G7-hIgG1 antibody to monkey PD-L1 antigen;

FIG. 5 is a graph showing the binding activity of SA126-C9-hIgG1, SA126-G7-hIgG1 antibody to murine PD-L1 antigen.

Detailed Description

For a better understanding of the present invention, the present invention will be further described with reference to the following examples and the accompanying drawings, which are illustrative of the present invention and are not to be construed as limiting thereof.

The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available.

Example 1: preparation of PD-L1 antigen

The cDNA of human tumor PD-L1 was purchased commercially. A human IgG1 Fc label is added after the synthesized extracellular region PD-L1 gene, two restriction enzyme sites of Xba I and Bam H I are introduced at two ends and are connected to a pTT5 expression plasmid, and the correctness is verified by sequencing. The sequenced plasmid was transfected into Trans10, and a single clone was picked and inoculated into 1 liter of LB liquid medium to OD₆₀₀When the number is 1, the thalli are collected by centrifugation, and plasmids are extracted by a plasmid macroextraction kit.

The sequencing is identified correctlyThe expression vector of (3) was transfected into 293F cells at 37 ℃ and 5% CO₂After culturing at 130rpm/min for 7 days, the supernatant was collected by centrifugation. Centrifuging the supernatant at 4000rpm for 10min, and filtering with 0.45 μm filter membrane; adding 400mM NaCl into the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2 μm filter, it was loaded onto a 5mL HiTrap Protein A column equilibrated with PBS; after the sample is completely loaded, the sample is washed by PBS, the flow rate is 5mL/min, and the ultraviolet monitoring is horizontal. 1M Glycine, pH 3.5 elution, flow rate 1mL/min, collection of the effluent peak with Tris neutralized to pH 7.5. And concentrating the elution peak by using an ultrafiltration concentration tube and changing the elution peak into PBS (phosphate buffer solution), thereby obtaining the PDL1-Fc antigen fusion protein. Enzyme-cutting PDL1-Fc with enterokinase, placing at 37 ℃ for 12 hours, passing the product after enzyme-cutting through a HiTrap Protein A column for adsorbing the cut Fc label, and collecting effluent, namely the PD-L1 extracellular region antigen.

Example 2: antigen-immunized mice and hybridoma screening

3 female BALB/c mice of 8 weeks old were selected for this experiment, and the mice were immunized by intraperitoneal injection with the PD-L1 extracellular region antigen prepared in example 1 mixed with Freund's complete adjuvant 1 time per week for 3 times in total. Measuring the serum titer of the mice one week after the last immunization, strengthening the immunization once after the condition titer is more than 8K is met, and the result shows that 3 mice completely meet the titer (the titer of the antibody is determined by the dilution value corresponding to the OD450 value which is more than 2 times of the negative control and more than 0.25, and the titer is more than or equal to 8K, so that the requirement is met), killing the mice after 3 days, taking the spleen of the mice, and grinding to obtain the splenocytes. B cells of anti-human tumor PD-L1 antibody were selected by flow cytometry (FACS), and placed in RPMI1640 medium, myeloma cells (SP2/0) were added and mixed, and cell fusion was performed with 50% PEG solution. The fused cells are diluted properly, cultured in multiple 96-well culture plates, HAT selective medium is added to kill unfused B cells and myeloma cells, and hybridoma cells are obtained. Collecting the cell culture supernatant of a 96-well plate after culturing for 2 weeks, combining the cell culture supernatant with a 96-well enzyme label plate paved with PD-L1 antigen for 1 hour, adding an anti-mouse/HRP secondary antibody for incubation for 1 hour, finally adding a TMB color reagent for reaction for 10 minutes, measuring the light absorption value at 450nm by using an enzyme label instrument, and selecting the hybridoma cells with the binding activity with PD-L1. And then screening by flow cytometry (FACS), selecting hybridoma cells with PD-1/PD-L1 blocking activity, and carrying out subcloning by a limiting dilution method to finally obtain the anti-PD-L1 murine monoclonal antibody.

Example 3: anti-PD-L1 murine antibody variable region Gene calling

Selecting the anti-PD-L1 murine monoclonal antibody prepared in example 2, extracting total RNA by Trizol method, using antibody subtype (Isotype) specific primer or general primer to make reverse transcription PCR, respectively amplifying antibody light chain variable region (VL) and heavy chain variable region (VH) genes, and then connecting to cloning vector to make DNA sequencing analysis. Finally, the complete DNA sequences of VL and VH are obtained and translated into the corresponding amino acid sequences.

Example 4: anti-PD-L1 murine monoclonal antibody gene humanization transformation and affinity maturation

Human germline genes (germline genes) with high homology to the VH gene of the mouse PD-L1 antibody were analyzed and relevant humanization modifications were performed.

Antibody mutant libraries were designed for 6 CDR regions of the anti-PD-L1 humanized antibody, respectively, with the mutation sites covering the non-conserved sites of all CDR regions. Obtaining single-chain antibody (scFv) genes by adopting SOE-PCR reaction, recovering DNA gel, carrying out enzyme digestion, connecting with a pCANTAB-5E phage display carrier after enzyme digestion, and electrically transforming TG1 competent bacteria to obtain 6 single-chain antibody libraries containing CDR mutation. Recombinant phages were prepared by infecting M13KO7 helper phage, and three rounds of panning were performed in total to retain and enrich for antibody mutants with strong binding capacity. And (3) respectively combining the recombinant phage with biotin-labeled recombinant human PD-L1 antigen for 2 hours in each round of panning, then adding streptavidin magnetic beads for combination for 30 minutes, washing with 2% TPBS, 1% TPBS and PBS for 5 times in sequence, each time for 5 minutes, and immediately infecting TG1 cells after panning for the preparation of the recombinant phage in the next round. Selecting the enriched TG1 monoclonals after three rounds of elutriation, preparing recombinant phage supernatant, combining with a 96-hole enzyme label plate paved with 1ug/mL PD-L1 antigen for 1 hour, adding M13/HRP secondary antibody for incubation for 1 hour, finally adding TMB for color reaction for 10 minutes, and measuring the light absorption value at 490nm by using an enzyme label analyzer. After analyzing the data, the relative affinities of the antibody-containing mutants are calculated, the results of the affinity determination are shown in table 1, and 1 clone (SA126-C9) with obviously improved affinity is obtained by screening to carry out the next step of research. The amino acid and nucleotide sequences of the heavy chain of the anti-PD-L1 humanized antibody with mature affinity are respectively SEQ ID NO 13 and SEQ ID NO 15; wherein, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are respectively SEQ ID NO. 1-3, and the nucleotide sequences are respectively SEQ ID NO. 7-9. The amino acid and nucleotide sequences of the affinity-matured light chain of the anti-PD-L1 humanized antibody are SEQ ID NOS: 14 and 16, respectively; wherein, the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region are respectively SEQ ID NO. 4-6, and the nucleotide sequences are respectively SEQ ID NO. 10-12.

TABLE 1 results of affinity assay

Example 5: expression and purification of anti-PD-L1 humanized antibody

The cDNA of the light chain of the antibody was artificially synthesized, the cDNA of the heavy chain of the antibody was artificially synthesized, and the synthesized cdnas were cloned into the pTT5 plasmid, respectively, and confirmed by sequencing that the plasmid construction was correct. The correct heavy and light chain expression vectors (1:1) were identified by sequencing and CO-transfected into 293F cells at 37 ℃ with 5% CO₂After culturing at 130rpm/min for 7 days, the supernatant was collected by centrifugation. Centrifuging the supernatant at 4000rpm for 10min, filtering with 0.45 μm filter membrane, and collecting filtrate; adding 400mM NaCl into the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2 μm filter, it was loaded onto a 5mL HiTrap Protein A column equilibrated with PBS; after the sample is completely loaded, the sample is washed by PBS, the flow rate is 5mL/min, and the ultraviolet monitoring is horizontal. 1M Glycine, pH 3.5 elution, flow rate 1mL/min, collection of the effluent peak with Tris neutralized to pH 7.5. The eluted peak was concentrated by an ultrafiltration concentration tube, and the solution was exchanged into PBS by a desalting column, whereby an antibody protein was obtained.

Example 6: humanized antibody induces T cell to secrete IL2 in vitro

Monocytes were isolated from PBMC using CD14+ microspheres (Miltenyi), CD4+ T cells were isolated from PBMC using CD4+ T cell isolation kit (Miltenyi), and the cells were cryopreserved in liquid nitrogen. Monocytes were thawed and cultured in RPMI1640 medium for 6 days to produce iDCs. Half of the medium was replaced every 2 or 3 days and fresh medium was supplemented with 1000U/mL rhGM CSF and 500U/mL rhIL-4. CD4+ T cells were thawed and incubated at 37 ℃ for 2-3 hours. CD4+ T cells and iDCs were added to a 96-well plate at a rate of 50. mu.L/well, antibody dilutions (at concentrations of 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 0.08. mu.g/mL, 0.016. mu.g/mL, 0.003. mu.g/mL, 0. mu.g/mL, respectively) were added to 100. mu.L/well, and hIgG1 at a concentration of 10. mu.g/mL was added as a negative control. After 3 days of culture at 37 ℃, the supernatant was collected and the secretion level of IL2 of the supernatant was measured using a Luminex apparatus (purchased from life technology) and a cytokine IL2 detection kit (purchased from BD Biosciences). As shown in FIG. 1, the antibodies are all effective in stimulating T cell function, secreting IL2, and are related to antibody concentration. In the MLR assay, SA126-C9 was slightly superior to Atezolizumab in its ability to induce IL-2 secretion.

Example 7: humanized antibody induces T cell to secrete IFN-gamma in vitro

Monocytes were isolated from PBMC using CD14+ microspheres (Miltenyi), CD4+ T cells were isolated from PBMC using CD4+ T cell isolation kit (Miltenyi), and the cells were cryopreserved in liquid nitrogen. Monocytes were thawed and cultured in RPMI1640 medium for 6 days to produce iDCs. Half of the medium was replaced every 2 or 3 days and fresh medium was supplemented with 1000U/mL rhGM CSF and 500U/mL rhIL-4. CD4+ T cells were thawed and incubated at 37 ℃ for 2-3 hours. CD4+ T cells and iDCs were added to a 96-well plate at a rate of 50. mu.L/well, antibody dilutions (at concentrations of 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 0.08. mu.g/mL, 0.016. mu.g/mL, 0.003. mu.g/mL, 0. mu.g/mL, respectively) were added to 100. mu.L/well, and hIgG1 at a concentration of 10. mu.g/mL was added as a negative control. The cells were cultured at 37 ℃ for 5 days. After 5 days, 100. mu.L of medium was removed from each culture for cytokine IFN-. gamma.measurement. The level of IFN-. gamma.was determined using the OptEIA ELISA kit (from BD Biosciences). As shown in FIG. 2, the antibodies are all effective in stimulating the functional secretion of the cytokine IFN-. gamma.by T cells, and are concentration dependent. In the MLR test, the SA126-C9 has better capability of inducing IFN-gamma secretion under high concentration condition than Atezolizumab.

Example 8: species cross reaction

The antibodies SA126-C9 and SA126-G7 replace humanized IgG1 subtype, so that the affinity is improved, and the species cross reaction is detected. Atezolizumab as a control antibody, cPD-L1, mPD-L1, hPD-L2 antigens, commercially available proteins were purchased. 0.1mg of cPD-L1 (cynomolgus monkey antigen, manufactured by R & D), 0.1mg of mPD-L1 (mouse antigen, manufactured by BioLegend), and 0.1mg of hPD-L2 (human antigen, manufactured by BioLegend) were purchased for use.

The binding activity of the antibodies to be tested SA126-C9-hIgG1 and SA126-G7-hIgG1 and three different species of PD-L1 antigens is identified by an ELISA method, and the IC50 value is respectively calculated and compared with a control antibody to judge the species cross-reaction specificity. The results are shown in FIGS. 3-5:

the binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to human PD-L1 antigen (FIG. 3) can be seen: the affinity of the antibody SA126-C9-hIgG and Atezolizumab to be detected is approximately same, and the affinity is higher than that of SA126-G7-hIgG 1.

② the binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibody and monkey PD-L1 antigen (FIG. 4) can be seen: the tested antibody SA126-C9-hIgG and SA126-G7-hIgG1 have equivalent affinities and are weaker than the affinity of Atezolizumab.

③ SA126-C9-hIgG1, SA126-G7-hIgG1 antibody with murine PD-L1 antigen (FIG. 5) it can be seen that: atezolizumab has stronger affinity with mouse PD-L1, and the antibodies SA126-C9-hIgG1 and SA126-G7-hIgG1 to be detected are not combined with mouse PD-L1 antigen, so that no cross reaction exists.

The comprehensive experiment result shows that SA126-C9 has better affinity with human PD-L1 antigen and has no cross reaction with mouse PD-L1 antigen, and can be used as a candidate antibody for the next experiment.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Han Hai Boxing Biotechnology Limited in Anhui

<120> anti-PD-L1 humanized monoclonal antibody and application thereof

<130> 2021

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<170> PatentIn version 3.3

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Claims

1. An anti-PD-L1 humanized monoclonal antibody characterized by: the amino acid sequence of CDR-H1, the amino acid sequence of CDR-H2 and the amino acid sequence of CDR-H3 of the heavy chain variable region of the antibody are respectively shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; and the amino acid sequence of CDR-L1, the amino acid sequence of CDR-L2 and the amino acid sequence of CDR-L3 of the light chain variable region of the antibody are respectively shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

2. The anti-PD-L1 humanized monoclonal antibody according to claim 1, characterized in that: the nucleotide sequence of CDR-H1 of the heavy chain variable region of the antibody is shown as SEQ ID NO. 7, the nucleotide sequence of CDR-H2 is shown as SEQ ID NO. 8, and the nucleotide sequence of CDR-H3 is shown as SEQ ID NO. 9; and the nucleotide sequence of CDR-L1 of the light chain variable region of the antibody is shown as SEQ ID NO. 10, the nucleotide sequence of CDR-L2 is shown as SEQ ID NO. 11, and the nucleotide sequence of CDR-L3 is shown as SEQ ID NO. 12.

3. The anti-PD-L1 humanized monoclonal antibody according to claim 1, characterized in that: the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO 13.

4. The anti-PD-L1 humanized monoclonal antibody according to claim 1, characterized in that: the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO. 14.

5. The anti-PD-L1 humanized monoclonal antibody according to claim 2, characterized in that: the nucleotide sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 15.

6. The anti-PD-L1 humanized monoclonal antibody according to claim 2, characterized in that: the nucleotide sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO: 16.

7. A pharmaceutical composition characterized by: the composition comprises the antibody of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. Use of the antibody of any one of claims 1 to 6 in the manufacture of a medicament for the treatment of cancer mediated by PD-L1.