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

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

An anti-PD-L1 humanized monoclonal antibody and its application

technical field

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

Background technique

PD-L1, English full name programmed cell death-Ligand 1, also known as programmed cell death-ligand 1. The PD-L1 protein is the ligand of PD-1, which is related to the inhibition of the immune system and can transmit inhibitory signals. Once PD-1 and PD-L1 are combined, they will send a negative regulatory signal to T cells, induce T cells to enter a quiescent state, reduce the proliferation of CD8+ T cells in lymph nodes, make them unable to recognize cancer cells, and make T cells unable to recognize cancer cells. Self-proliferation is reduced or apoptosis is effectively relieved of the body's immune response, so cancer cells can grow recklessly. The anti-PD-L1 antibody provided by the present invention can be combined with PD-1 protein to activate T cells to recognize and kill cancer cells.

SUMMARY OF THE INVENTION

The present invention provides an anti-PD-L1 monoclonal antibody and a preparation method thereof. The anti-PD-L1 monoclonal antibody has a brand-new sequence and can specifically bind to PD-1 protein, so that immune cells can specifically recognize and kill cancer cells. achieve the purpose of cancer treatment.

The present invention is achieved through the following technical solutions:

An anti-PD-L1 humanized monoclonal antibody, the CDR-H1 of the heavy chain variable region of the antibody is the amino acid sequence shown in SEQ ID NO:1, and the CDR-H2 is shown in SEQ ID NO:2 Amino acid sequence, and CDR-H3 is the amino acid sequence shown in 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, and CDR-L2 is SEQ ID NO:4 The amino acid sequence shown in ID NO:5, and 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 CDR-L1 of the light chain variable region of the antibody is the nucleotide sequence shown in SEQ ID NO: 10, and CDR-L2 is SEQ ID NO : the nucleotide sequence shown in SEQ ID NO: 11, and CDR-L3 is the nucleotide sequence shown in SEQ ID NO: 12.

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

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

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

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

The present invention also protects a pharmaceutical composition comprising the above-mentioned antibody and a pharmaceutically acceptable carrier.

The present invention also protects the application of the above-mentioned antibodies in pharmacy.

The present invention also protects the application of the above-mentioned antibody in the preparation of a drug for treating cancer.

The beneficial effects of the present invention are:

The anti-PD-L1 monoclonal antibody provided by the present invention has a brand-new sequence, can specifically bind to human PD-L1 protein, has high affinity, enables immune cells to specifically recognize and kill cancer cells, and achieves the purpose of cancer treatment. The molecular and cellular level performance is not lower than the products currently on sale.

Description of drawings

Figure 1 shows the results of humanized antibody SA126-C9 inducing T cells to secrete IL2;

Figure 2 shows the results of humanized antibody SA126-C9 inducing T cells to secrete IFN-γ;

Figure 3 is a graph showing the binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to human PD-L1 antigen;

Figure 4 is a graph showing the binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to monkey PD-L1 antigen;

Fig. 5 is a graph showing the binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to mouse PD-L1 antigen.

Detailed ways

In order to better understand the present invention, the present invention will be further described below with reference to the embodiments and the accompanying drawings. The following embodiments are only to illustrate the present invention and not to limit it.

The materials, reagents, instruments and methods used in the following examples, unless otherwise specified, are conventional materials, reagents, instruments and methods in the art, and can be obtained through commercial channels.

Example 1: Preparation of PD-L1 antigen

Commercially available cDNA of human tumor PD-L1. A human IgG1 Fc tag was added to the synthetic extracellular region PD-L1 gene, and Xba I and Bam HI restriction sites were introduced at both ends to connect to the pTT5 expression plasmid, which was verified to be correct by sequencing. The sequenced plasmids were transfected into Trans10, single clones were picked, and inoculated into 1 liter of LB liquid medium. When the OD ₆₀₀ was 1, the cells were collected by centrifugation, and the plasmids were extracted with a plasmid extraction kit.

The correct expression vector identified by sequencing was transfected into 293F cells, cultured at 37° C., 5% CO ₂ , 130 rpm/min for 7 days, and the supernatant was collected by centrifugation. The supernatant was centrifuged at 4000 rpm for 10 min, and then filtered through a 0.45 μm filter; 400 mM NaCl was added to the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2 μm filter, the sample was loaded onto a 5 mL HiTrap Protein A column that had been equilibrated with PBS; after the sample was loaded, it was rinsed with PBS at a flow rate of 5 mL/min, and the level was monitored by UV. 1 M glycine, pH 3.5 elution, flow rate 1 mL/min, elution peaks were collected and neutralized to pH 7.5 with Tris. The eluted peak was concentrated with an ultrafiltration concentrator and exchanged into PBS to obtain a PDL1-Fc antigen fusion protein. The PDL1-Fc was digested with enterokinase and placed at 37°C for 12 hours. After the digestion, the product was passed through a HiTrap Protein A column for adsorption of the excised Fc tag, and the effluent was collected, which was the PD-L1 extracellular region antigen.

Example 2: Antigen immunized mice and hybridoma screening

In this experiment, three 8-week-old female BALB/c mice were selected, and the mice were immunized with the PD-L1 extracellular domain antigen prepared in Example 1 mixed with Freund's complete adjuvant by intraperitoneal injection, once a week for a total of 3 times. The serum titer of the mice was measured one week after the last immunization, and the titer was more than 8K after the condition was met, and the immunization was boosted once. The results showed that all 3 mice met the titer (2 times greater than the negative control and greater than the dilution value corresponding to the OD450 value of 0.25). That is, the titer of the antibody, and the titer is greater than or equal to 8K to meet the requirements), and the mice were sacrificed after 3 days, and the spleen of the mice was taken, and the spleen cell group was obtained after grinding. B cells with anti-human tumor PD-L1 antibody were screened by flow cytometry (FACS), placed in RPMI1640 medium, myeloma cells (SP2/0) were added and mixed, and 50% PEG solution was used for cell fusion. The fused cells are appropriately diluted and cultured in multiple 96-well culture plates, and HAT selective medium is added to make unfused B cells and myeloma cells die to obtain hybridoma cells. After 2 weeks of culture, collect the cell culture supernatant from the 96-well plate, combine with the 96-well microtiter plate plated with PD-L1 antigen for 1 hour, add anti-mouse/HRP secondary antibody and incubate for 1 hour, and finally add TMB chromogenic reagent to react for 10 minutes , and the light absorption value at 450nm was measured with a microplate reader, and the hybridoma cells with binding activity to PD-L1 were selected. Then, flow cytometry (FACS) screening was performed to select hybridoma cells with PD-1/PD-L1 blocking activity, and subcloning by limiting dilution method to finally obtain an anti-PD-L1 mouse monoclonal antibody .

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

The anti-PD-L1 mouse-derived monoclonal antibody prepared in Example 2 was selected, the total RNA was extracted by the Trizol method, and reverse transcription PCR was performed using antibody isotype-specific primers or universal primers to amplify the antibody light chain respectively. The variable region (VL) and heavy chain variable region (VH) genes were then ligated into cloning vectors for DNA sequencing analysis. The complete VL and VH DNA sequences were finally obtained and translated into the corresponding amino acid sequences.

Example 4: Humanization and Affinity Maturation of Anti-PD-L1 Murine Monoclonal Antibody Gene

The human germline gene with high homology to the VH gene of mouse PD-L1 antibody was analyzed, and related humanization was carried out.

An antibody mutant library was designed for the six CDR regions of the anti-PD-L1 humanized antibody, and the mutation sites covered the non-conserved sites of all CDR regions. The single-chain antibody (scFv) gene was obtained by SOE-PCR reaction. After DNA gel recovery and enzyme digestion, it was connected with the digested pCANTAB-5E phage display vector, and TG1 competent bacteria were electrotransformed to obtain 6 CDR mutations. single-chain antibody library. Recombinant phages were made by infecting M13KO7 helper phage, and three rounds of panning were performed to retain and enrich antibody mutants with strong binding ability. In each round of panning, the recombinant phage was combined with biotin-labeled recombinant human PD-L1 antigen for 2 hours, and then streptavidin magnetic beads were added for 30 minutes, and washed with 2% TPBS, 1% TPBS and PBS for 5 times. For 5 minutes each, TG1 cells were infected immediately after panning for the next round of recombinant phage preparation. The TG1 monoclone enriched after three rounds of panning was selected, and the recombinant phage supernatant was prepared, which was combined with a 96-well microtiter plate plated with 1ug/mL PD-L1 antigen for 1 hour, and the M13/HRP secondary antibody was added to incubate for 1 hour. TMB was added to carry out color reaction for 10 minutes, and the light absorption value at 490 nm was measured with a microplate reader. After analyzing the data, the relative affinity of the antibody mutants was calculated. The affinity determination results are shown in Table 1. One clone (SA126-C9) with significantly improved affinity was screened for further research. The heavy chain amino acid and nucleotide sequences of the affinity matured anti-PD-L1 humanized antibody are SEQ ID NOs: 13 and 15, respectively; wherein, the CDR-H1, CDR-H2 and CDR-H3 amino acids of the heavy chain variable region The sequences are SEQ ID NOs: 1-3, respectively, and the nucleotide sequences are SEQ ID NOs: 7-9, respectively. The light chain amino acid and nucleotide sequences of the affinity-matured anti-PD-L1 humanized antibody are SEQ ID NOs: 14 and 16, respectively; wherein, the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the light chain variable region They are SEQ ID NOs: 4-6, respectively, and the nucleotide sequences are SEQ ID NOs: 10-12, respectively.

Table 1 Affinity determination results

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

The cDNA of the light chain of the antibody was artificially synthesized, and the cDNA of the heavy chain of the antibody was artificially synthesized, and the synthesized cDNAs were cloned into the pTT5 plasmid respectively, and the correct construction of the plasmid was confirmed by sequencing. The correct heavy chain expression vector and light chain expression vector (1:1) identified by sequencing were co-transfected into 293F cells, cultured at 37° C., 5% CO ₂ , 130 rpm/min for 7 days, and the supernatant was collected by centrifugation. The supernatant was centrifuged at 4000 rpm for 10 min, and filtered with a 0.45 μm membrane filter to collect the filtrate; 400 mM NaCl was added to the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2 μm filter, the sample was loaded onto a 5 mL HiTrap Protein A column that had been equilibrated with PBS; after the sample was loaded, it was rinsed with PBS at a flow rate of 5 mL/min, and the UV level was monitored. 1 M glycine, pH 3.5 elution, flow rate 1 mL/min, elution peaks were collected and neutralized to pH 7.5 with Tris. The eluted peak was concentrated with an ultrafiltration concentrator, and the solution was exchanged with PBS with a desalting column to obtain antibody protein.

Example 6: Humanized antibody induces T cells to secrete IL2 in vitro

Monocytes were isolated from PBMCs using CD14+ microspheres (Miltenyi), CD4+ T cells were isolated from PBMCs using the CD4+ T cell isolation kit (Miltenyi), and cells were cryopreserved in liquid nitrogen. Monocytes were thawed and cultured in RPMI1640 medium for 6 days to generate iDCs. Half of the medium was replaced every 2 or 3 days, and 1000 U/mL rhGM CSF and 500 U/mL rhIL-4 were added to the fresh medium. Thaw CD4+ T cells and incubate at 37°C for 2-3 hours. CD4+ T cells and iDCs were added to the 96-well plate at a rate of 50 μL/well, respectively, and antibody dilution was added at 100 μL/well (concentrations were 10 μg/mL, 2 μg/mL, 0.4 μg/mL, 0.08 μg/mL, respectively. , 0.016 μg/mL, 0.003 μg/mL, 0 μg/mL), and 100 μL/well of hIgG1 with a concentration of 10 μg/mL was added as a negative control. After culturing at 37°C for 3 days, the supernatant was collected, and the secretion level of IL2 in the supernatant was detected with a Luminex instrument (purchased from LifeTechnology) and a cytokine IL2 detection kit (purchased from BD Biosciences). Figure 1 shows that both antibodies can effectively stimulate the function of T cells, secrete IL2, and are related to antibody concentration. SA126-C9 was slightly better than Atezolizumab in its ability to induce IL-2 secretion in the MLR assay.

Example 7: Humanized antibody induces T cells to secrete IFN-γ in vitro

Monocytes were isolated from PBMCs using CD14+ microspheres (Miltenyi), CD4+ T cells were isolated from PBMCs using the CD4+ T cell isolation kit (Miltenyi), and cells were cryopreserved in liquid nitrogen. Monocytes were thawed and cultured in RPMI1640 medium for 6 days to generate iDCs. Half of the medium was replaced every 2 or 3 days, and 1000 U/mL rhGM CSF and 500 U/mL rhIL-4 were added to the fresh medium. Thaw CD4+ T cells and incubate at 37°C for 2-3 hours. CD4+ T cells and iDCs were added to the 96-well plate at a rate of 50 μL/well, respectively, and antibody dilution was added at 100 μL/well (concentrations were 10 μg/mL, 2 μg/mL, 0.4 μg/mL, 0.08 μg/mL, respectively. , 0.016 μg/mL, 0.003 μg/mL, 0 μg/mL), and 100 μL/well of hIgG1 with a concentration of 10 μg/mL was added as a negative control. Cells were cultured at 37°C for 5 days. After 5 days, 100 μL of medium was removed from each culture for cytokine IFN-γ measurement. The level of IFN-γ was measured using OptEIA ELISA kit (purchased from BD Biosciences). Figure 2 shows that the antibodies can effectively stimulate the function of T cells to secrete cytokine IFN-γ, and it is related to the concentration. In the MLR assay, SA126-C9 was superior to Atezolizumab in its ability to induce IFN-γ secretion at high concentrations.

Example 8: Species Cross Reaction

The antibodies SA126-C9 and SA126-G7 were replaced with human IgG1 subtype to improve the affinity and detect species cross-reaction. Atezolizumab is used as a control antibody, and commercially available proteins can be purchased for cPD-L1, mPD-L1, and hPD-L2 antigens. 0.1 mg cPD-L1 (cynomolgus monkey antigen, produced by R&D company), 0.1 mg mPD-L1 (mouse antigen, produced by BioLegend company), 0.1 mghPD-L2 (human antigen, produced by BioLegend company) were purchased for use.

The binding activities of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to three different species of PD-L1 antigens were identified by ELISA, IC50 values were calculated respectively and compared with the control antibodies to determine their species cross-reactivity specificity . The result is shown in Figure 3-5:

①The binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to human PD-L1 antigen (Fig. 3) can be seen: the tested antibodies SA126-C9-hIgG and Atezolizumab have roughly the same affinity, and both are higher than SA126-G7- hIgG1 affinity is higher.

②The binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to monkey PD-L1 antigen (Figure 4) can be seen: the tested antibodies SA126-C9-hIgG and SA126-G7-hIgG1 have similar affinities, and both have higher affinity than Atezolizumab weak.

③The binding activity of SA126-C9-hIgG1 and SA126-G7-hIgG1 antibodies to mouse PD-L1 antigen (Fig. 5) shows that Atezolizumab has a strong affinity with mouse PD-L1, while the tested antibody SA126-C9-hIgG1 and SA126-G7-hIgG1 did not bind to the mouse PD-L1 antigen and had no cross-reactivity.

Comprehensive experimental results show that SA126-C9 has good affinity with human PD-L1 antigen and no cross-reaction with mouse PD-L1 antigen, and can be used as a candidate antibody for further experiments.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, , the protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Anhui Hanhai Boxing Biotechnology Co., Ltd.

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

<130> 2021

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Claims (8)

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.

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