CN110878122B - Recombinant anti-PD-L1 monoclonal antibodies - Google Patents

Abstract

A recombinant anti-PD-L1 monoclonal antibody relates to the technical field of biological medicine and is used for providing effective therapeutic drugs for patients with advanced or metastatic cancers, in particular to patients with ineffective or drug-resistant existing anti-PD-L1 drug treatment. The complementarity determining region of the antibody has the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 6. Compared with the existing anti-PD-L1 drugs, the antibody has unique binding epitopes, and has better affinity to human PD-L1 and better tumor inhibiting effect.

Description

Recombinant Anti-PD-L1 Monoclonal Antibody

technical field

The invention relates to the technical field of biomedicine, and more specifically, the invention discloses a monoclonal antibody suitable for the treatment or detection of cancer and immune diseases.

Background technique

PD-L1 (Programmed cell death 1 ligand 1, programmed cell death ligand 1) is also known as surface antigen differentiation cluster 274 (cluster of differentiation 274, CD274) or B7 homolog 1 (B7 homolog 1, B7-H1) , is a transmembrane protein with a size of 40kDa, one of the two ligands of PD-1 (Programmed Cell Death Protein 1), highly expressed in the heart, skeletal muscle, placenta, and lung, and in the thymus, spleen, kidney, and liver Low expression, also expressed in activated T cells, B cells, dendritic cells and other immune cells, and widely expressed on a variety of tumor cells.

The combination of PD-L1 and PD-1 can transmit immunosuppressive signals and reduce the proliferation of T cells. This "immune checkpoint" (Immune checkpoints) mechanism can be used by cancer cells to achieve immune escape. Therefore, PD-1 and PD- L1 has become a target for cancer immunotherapy, and multiple anti-PD-1 and anti-PD-L1 monoclonal antibodies have entered clinical trials. In May 2016, the recombinant anti-PD-L1 humanized monoclonal antibody Atezolizumab was approved by the US Food and Drug Administration (FDA) for the treatment of locally advanced or metastatic urothelial carcinoma, with the trade name Tecentriq, which is the first in the world A therapeutic antibody targeting PD-L1 that has been approved for marketing. In October of the same year, the indication of Tecentriq was added to metastatic non-small cell lung cancer, which was the first time in the world to approve the anti-PD-L1 antibody for the treatment of lung cancer.

In a 310-person clinical trial of Tecentriq in the treatment of locally advanced or metastatic urothelial carcinoma, the overall response rate (ORR) was 26.0% in patients with PD-L1 expression ≥ 5%, and the complete response rate (CR ) is only 12.0%. In a clinical trial of Tecentriq in the treatment of metastatic non-small cell lung cancer with 287 participants, the overall response rate was 15%, of which the complete response rate was only 0.7%, and the median survival period of patients was 12.6 months, which was higher than that of docetaxel. The control group of the race was extended by 2.9 months. Therefore, it can be seen that more than half of the patients have no obvious response to anti-PD-L1 treatment (this may be related to factors such as individual genetic diversity, tumor type and variability), and the prolongation of patient survival is also limited compared with chemotherapy drugs. The effectiveness and efficacy of anti-PD-L1 therapy need to be further improved. Discover and screen anti-PD-L1 monoclonal antibodies with new pharmacodynamic and pharmacokinetic characteristics, such as monoclonal antibodies with new binding epitopes, binding/dissociation kinetics, tissue permeability, and stability, It is a way to provide patients with diversified alternatives and improve the effectiveness of anti-PD-L1 therapy.

Antibody drugs, also known as therapeutic antibodies (Therapeutic Antibodies), are targeted drugs, generally IgG immunoglobulins. The basic structure of IgG type immunoglobulin is composed of four peptide chains, that is, two identical peptide chains with smaller molecular weight are called light chains (L chains) and two identical peptide chains with larger molecular weights are called heavy chains ( H chain). In most cases the H chain plays a more important role in binding to the antigen. The amino acid sequence of the amino terminal (N-terminal) of the H chain or L chain varies greatly, and this region is called the variable region (V), and the V regions of the L chain and the H chain are called VL and VH, respectively; The end) is relatively stable with little change, and this region is called the constant region (C), and the C regions of the L chain and H chain are called CL and CH, respectively.

The amino acid composition and sequence of certain local regions in VL and VH have a higher degree of variability, and these regions are called hypervariable regions (HVR). The amino acid group and arrangement of non-HVR parts in the V region are relatively conservative, which is called the framework region (Framework). X-ray crystallography analysis proves that the hypervariable region is indeed the position where the antibody binds to the antigen, so it is called the complementarity determining region (CDR). VL and VH each have three HVR regions, which are called HVR1, HVR2 and HVR3, respectively, and can also be called CDR1, CDR2 and CDR3, and CDR3 generally has a higher degree of hypervariability.

Genetic markers with partial allotypes on CL and CH. The CH2 region of IgG immunoglobulin has a complement binding point, which can activate the classical activation pathway of complement and induce CDC effect. The CH3 region has the function of binding to the Fc receptors of monocytes, macrophages, granulocytes, B cells and NK cells, and can induce ADCC effects. There is a conserved N-linked glycosylation site at the 297th aspartic acid (Asn297) in the constant region of the H chain. Different forms of glycosylation modification have CDC effect, ADCC effect, immunogenicity, half-life and other properties of the antibody have an important impact.

Contents of the invention

In order to provide effective therapeutic drugs for patients with advanced or metastatic cancer, especially to provide new drug options for patients who are ineffective or resistant to Tecentriq treatment, the present invention provides recombinant anti-PD-L1 humanized monoclonal antibody and mouse anti-PD-L1 PD-L1 monoclonal antibody, the technical scheme is as follows:

For the recombinant anti-PD-L1 monoclonal antibody, the CDR1, CDR2, and CDR3 regions of the heavy chain contain the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

In the antibody, the heavy chain variable region contains the sequence of SEQ ID NO: 7 (humanized antibody), or contains the sequence of SEQ ID NO: 10 (murine antibody).

In the antibody, the light chain CDR1, CDR2, and CDR3 regions contain the sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In the antibody, the light chain variable region contains the sequence of SEQ ID NO: 8 or SEQ ID NO: 9 (humanized antibody), or contains the sequence of SEQ ID NO: 11 (murine antibody).

The humanized antibody is an antibody of human IgG1 subtype.

Use of the humanized antibody in the manufacture of therapeutic or preventive drugs for cancer and immune diseases.

The use of the murine antibody in the detection of cancer or immune diseases, and in the prediction of drug efficacy.

Specifically, the names and sequences of the humanized antibodies are as follows:

Table 1. Sequences of recombinant anti-PD-L1 humanized monoclonal antibodies T0004-BC and T0004-C

The names and sequences of the murine antibodies are as follows:

Table 2. Sequence of murine anti-PD-L1 monoclonal antibody T0004 Antibody characteristics amino acid (or nucleotide) sequence SEQ ID NO: heavy chain CDR1 SAYIWH 1 heavy chain CDR2 YIHYSTITKYNPSLKS 2 heavy chain CDR3 EGNDYGGFAY 3 heavy chain variable region DVQLQESGPDLVKPSQSLSLTCTVTGYSITSAYIWHWIRQFPGNKLEWMGYIHYSTITKYNPSLKSRFSITRDTSKNQFFLQLSSVTTEDTATYYCAREGNDYGGFAYWGQGTLVTVSA 10 light chain CDR1 KASQSVSNGVA 4 light chain CDR2 YASNRYT 5 light chain CDR3 QQDYSSPYT 6 light chain variable region SIVMTQTPKFLLVSAGDRVTITCKASQSVSNGVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTRLEIKR 11

Surface plasmon resonance detection shows that the mouse antibody T0004 has a very high affinity for human PD-L1, and the equilibrium dissociation constant (KD value) is less than 1 pM (the detection result is 0.817 pM, which exceeds the detection range of the instrument); humanization Antibodies T0004-BC (KD value 1.008E-11M), T0004-C (KD value 1.250E-11M) have higher human PD-L1 affinity than Tecentriq (KD value 2.720E-11M); and T0004-BC (Kd Value 4.452E-5), T0004-C (Kd value 4.852E-5) and human PD-L1 (Kd value 1.410E-4) are more stable than Tecentriq.

ELISA and flow cytometry detection showed that T0004-BC and T0004-C could compete with Tecentriq for binding to soluble or membrane surface human PD-L1, indicating that the binding epitopes of these three antibodies on human PD-L1 are relatively close , or have a steric blocking effect. However, T0004-BC and T0004-C can bind to the D49Y and V111E mutants of human PD-L1 on the cell membrane surface, while Tecentriq cannot bind to the mutants, indicating that their binding epitopes are different. T0004-BC, T0004- The binding epitope of C on human PD-L1 is unique compared with commercial anti-PD-L1 antibodies.

In animal experiments using mice as a model, T0004-BC and T0004-C can significantly inhibit the growth of tumors formed by MC38 colorectal cancer cells, and the tumor inhibitory effect is better than that of commercial anti-PD-L1 antibodies.

Since murine antibody T0004 has the same CDR region as humanized antibodies T0004-BC and T0004-C, it can compete with human PD-1 for binding to human PD-L1, so it can be speculated that they have the same human PD-L1 binding epitope . In addition, the murine antibody T0004 has a very high affinity for human PD-L1, and is expected to be used for cancer or immune disease detection and drug efficacy prediction.

In conclusion, the present invention provides a recombinant anti-PD-L1 humanized monoclonal antibody with better affinity to human PD-L1, unique binding epitope and better anti-tumor effect, as well as a humanized monoclonal antibody with extremely high affinity to human PD-L1 , Mouse-derived antibodies that can be used for cancer or immune disease detection and drug efficacy prediction.

Description of drawings

Figure 1: The binding ability of murine antibody T0004 antibody to PD-L1 on the cell membrane surface was detected by flow cytometry.

Figure 2: Detection of the binding ability of murine antibody T0004 antibody to soluble PD-L1 by enzyme-linked immunosorbent assay.

Figure 3: Surface plasmon resonance detection of the affinity of murine antibody T0004 to human PD-L1.

Figure 4: ELISA detection of murine antibody T0004 competing with PD-1 for binding to PD-L1.

Figure 5: ELISA detection of murine antibody T0004 competing with Tecentriq for binding to PD-L1.

Figure 6: Four humanization schemes of the heavy chain variable regions A, B, C, and D of the murine antibody T0004.

Figure 7: Four humanization schemes of the light chain variable regions A, B, C, and D of the murine antibody T0004.

Figure 8: Measurement of thermal stability of humanized antibody T0004-BC by differential scanning calorimetry.

Figure 9: Thermal stability of humanized antibody T0004-C measured by differential scanning calorimetry.

Figure 10: Flow cytometry detection results of humanized antibody T0004-BC binding to membrane PD-L1.

Figure 11: Flow cytometry detection results of humanized antibody T0004-C binding to membrane PD-L1.

Figure 12: Flow cytometry detection results of commercial antibody Tecentriq (control) binding to membrane PD-L1.

Figure 13: Detection of the binding ability of T0004-BC, T0004-C and soluble PD-L1 by ELISA.

Figure 14: Surface plasmon resonance detection of the affinity of humanized antibody T0004-BC to human PD-L1.

Figure 15: Surface plasmon resonance detection of the affinity of humanized antibody T0004-C to human PD-L1.

Figure 16: Surface plasmon resonance detection of the affinity of the commercial antibody Tecentriq (control) to human PD-L1.

Figure 17: Flow cytometry detection of the ability of T0004-BC to compete with Tecentriq to bind to PD-L1 on the cell membrane surface.

Figure 18: Flow cytometry detection of the ability of T0004-C to compete with Tecentriq to bind to PD-L1 on the cell membrane surface.

Figure 19: Flow cytometry detection of the ability of Tecentriq (control) to compete with Tecentriq to bind to PD-L1 on the cell membrane surface.

Figure 20: ELISA detection of the ability of T0004-BC, T0004-C to compete with Tecentriq to bind to soluble PD-L1.

Figure 21: Flow cytometry detection of the ability of T0004-BC to compete with PD-1 to bind to PD-L1 on the cell membrane surface.

Figure 22: Flow cytometry detection of the ability of T0004-C to compete with PD-1 to bind to PD-L1 on the cell membrane surface.

Figure 23: Flow cytometry detection of the ability of Tecentriq (control) to compete with PD-1 to bind to PD-L1 on the cell membrane surface.

Figure 24: ELISA detection of the ability of T0004-BC, T0004-C to compete with PD-1 for binding to soluble PD-L1.

Figure 25: The biological activity of humanized antibody T0004-BC to activate TCR was detected by reporter gene method.

Figure 26: The biological activity of humanized antibody T0004-C to activate TCR was detected by reporter gene method.

Figure 27: The reporter gene method detects the biological activity of Tecentriq (control) to activate TCR.

Figure 28: The ability of humanized antibody T0004-BC to induce ADCC effect was detected by reporter gene method.

Figure 29: The ability of humanized antibody T0004-C to induce ADCC effect was detected by reporter gene method.

Figure 30: Reporter gene method to detect the ability of Tecentriq (control) to induce ADCC effect.

Figure 31: Detection of CDC effect of humanized antibodies T0004-BC and T0004-C by lactate dehydrogenase method.

Figure 32: The binding ability of T0004-BC and T0004-C to human PD-L1 mutants detected by flow cytometry.

Figure 33: Study on the tumor inhibitory ability of humanized antibodies T0004-BC and T0004-C in animals.

Detailed ways

Embodiment 1, construction and screening of hybridoma cells

1. Immunized animals

Use the recombinantly expressed PD-L1-Fc fusion protein (fused from the extracellular region of human PD-L1 to the Fc segment of the antibody, the amino acid sequence is the sequence numbered Q9NZQ7-1 on the UniProt website) with a dose of 100-500 μg/mL intraperitoneally Immunize Balb/c mice for 3 times. 3-7 days before cell fusion, boost immunization with PD-L1-Fc fusion protein 50-100 μg/mL tail vein.

2. Cell Fusion

The mouse myeloma cell line NS-1 was fused with the splenocytes of Balb/c mice after immunization, and then placed in a 96 empty cell culture plate, screened with a complete medium containing HAT, half of the medium was changed in 3-5 days, 2 Colony formation can be seen in about a week.

3. Identification of culture supernatant

The hybridoma cell culture supernatant was detected by ELISA technique. Coat multi-well plates with recombinant PD-L1-Fc fusion protein and irrelevant antibody Fc fragment respectively, add hybridoma cell culture supernatant, and then add horseradish peroxidase (HRP)-labeled anti-mouse antibody to select PD-L1 -Fc hole single positive clone (PD-L1-Fc hole and Fc hole double positive clone is anti-Fc antibody), that is, hybridoma cells secreting mouse anti-human PD-L1 antibody, cultured with complete medium containing HT, and Perform subcloning.

4. Subtype detection

The mouse anti-human PD-L1 monoclonal antibody secreted by each hybridoma cell line was detected with a commercially available mouse subtype detection kit to determine the light and heavy chain subtypes.

5. Research results

A hybridoma cell strain secreting mouse anti-human PD-L1 monoclonal antibody was obtained through screening, numbered T0004, and the subtype of the heavy chain was identified as IgG1, and the subtype of the light chain was identified as κ.

Example 2. Flow cytometry detection of the binding ability of T0004 antibody to PD-L1 on the cell membrane surface

The expression vector plasmid containing the full-length coding sequence of human PD-L1 was stably transfected into CHO-K1 host cells, and CHO-K1 engineered cells stably expressing PD-L1 on the membrane surface were obtained by pressurized screening (denoted as CHO-K1/hmPD- L1). The T0004 antibody was diluted to 15 concentrations starting from 10 μg/mL, mixed with CHO-K1/hmPD-L1 cells in the logarithmic growth phase, and kept on ice for 45 minutes to allow it to interact with the surface of CHO-K1/hmPD-L1 cells PD-L1 binding. Wash 2 times with PBS containing 1% fetal bovine serum, add FITC fluorescently labeled rabbit anti-mouse IgG (H+L) secondary antibody, ice bath for 45min, wash 2 times with PBS containing 1% fetal bovine serum. The pellet was resuspended in PBS containing 1% fetal bovine serum, and the fluorescence intensity was analyzed by flow cytometry.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the mean fluorescence intensity (MFI). See Figure 1. The EC50 ( half maximum effect concentration) was 274.7112 ng/mL.

Example 3. Enzyme-linked immunoassay detection of binding ability of T0004 antibody to soluble PD-L1

Dilute the human PD-L1-Fc fusion protein to 10 μg/mL with coating solution, add 100 μL/well to the microtiter plate, and coat at 37°C for 1-2 hours. Discard the coating solution, add blocking solution, 350 μL/well, block overnight at 2-8°C, and wash 7 times with a plate washer. Dilute the T0004 antibody with PBS, starting from 50000ng/mL, dilute 15 concentrations at a 4-fold ratio, 100μL/well, react at 37°C for 1-2 hours, discard the liquid in the microplate plate, and wash 7 times with a plate washer. Add horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) diluted 1:10000, 100 μL/well, react at 37°C for 1 hour, discard the liquid in the microtiter plate, and wash 7 times with a plate washer . Add TMB substrate solution to the ELISA plate, 50 μL/well, develop color in the dark for 1-10 minutes, add 50 μL/well stop solution, and mix quickly. Read OD450nm with a microplate reader, and use OD570nm as the reference wavelength.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the OD value. See Figure 2. The EC50 (half maximum effect concentration) of T0004 binding to soluble PD-L1 was calculated. was 1.471 ng/mL.

Example 4. Detection of the affinity between T0004 and human PD-L1 by surface plasmon resonance

The human PD-L1-Fc fusion protein was labeled with biotin, and then coupled to the detection chip of the surface plasmon resonance instrument BIACORE. The T0004 antibody was diluted 10 times starting from 1 μg/mL, and added to the BIACORE equipment. Detect and record the optical signal changes caused by the binding-dissociation process between the T0004 antibody and the human PD-L1-Fc fusion protein, and draw the curves on the horizontal and vertical coordinates with time and optical response values respectively. The obtained map is shown in Figure 3. T0004 antibody The affinity dissociation constant (KD value) with human PD-L1-Fc fusion protein is 8.170×10 ^-13 M.

Example 5. Enzyme-linked immunoassay detection of T0004 competing with PD-1 for binding to PD-L1

Dilute the recombinant human PD-1-Fc fusion protein to 10 μg/mL with coating solution, add 100 μL/well to the microtiter plate, and coat at 37°C for 2 hours. Discard the coating solution, add blocking solution, 350 μL/well, block overnight at 2-8°C, and wash 7 times with a plate washer. Mix T0004 antibody with biotin-labeled human PD-L1-Fc fusion protein in different ratios, dilute 15 concentration gradients at a 2-fold ratio starting from a final concentration of T0004 antibody of 500 μg/mL, add 100 μL/well to the microtiter plate, 37 °C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Add 1:1000 diluted Avidin (avidin)-HRP (horseradish peroxidase) at 100 μL/well, and react at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Add TMB chromogenic substrate solution to the microtiter plate, 50 μL/well, and develop color in the dark for 15 minutes. Add the stop solution to the microtiter plate, 50 μL/well, and mix quickly. The microplate reader reads OD450nm, and 570nm is the reference wavelength.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the OD value. See Figure 4. The calculated T0004 antibody inhibits the binding of PD-1 to its ligand PD-L1-Fc The EC50 (half-maximal effect concentration) is 6.298 μg/mL.

Example 6. ELISA detection of T0004 competing with Tecentriq for PD-L1 binding

Dilute the recombinant human PD-L1-Fc fusion protein to 10 μg/mL with coating solution, add 100 μL/well to the microtiter plate, and coat at 37°C for 2 hours. Discard the coating solution, add blocking solution, 350 μL/well, block overnight at 2-8°C, and wash 7 times with a plate washer. The T0004 antibody was mixed with the biotin-labeled Tecentriq antibody in different ratios. The final concentration of the T0004 antibody was diluted 15 concentration gradients at a 2-fold ratio starting from 500 μg/mL, and 100 μL/well was added to the microtiter plate, and reacted at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Add 1:1000 diluted Avidin (avidin)-HRP (horseradish peroxidase) at 100 μL/well, and react at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Add TMB chromogenic substrate solution to the microtiter plate, 50 μL/well, and develop color in the dark for 15 minutes. Add the stop solution to the microtiter plate, 50 μL/well, and mix quickly. The microplate reader reads OD450 nm, and 570 nm is the reference wavelength.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the OD value. See Figure 5. The EC50 (half-maximal effect) of the T0004 antibody competing with Tecentriq for binding to PD-L1 was calculated. concentration) was 4.917 μg/mL.

Example 7, T0004 Antibody Sequencing and Humanization Transformation

The outsourced service company was commissioned to use mouse-derived primers to determine the cDNA coding sequence of the T0004 antibody secreted by the T0004 hybridoma cell line and translate it into an amino acid sequence. The results showed that the heavy and light chain variable region sequences of the T0004 antibody were SEQID NO: 10, Sequence of SEQ ID NO: 11.

Select a human antibody sequence with high homology to the light and heavy chains of the T0004 antibody as a template; determine the amino acids involved within 5 Å of the CDR region according to the crystal structure of the mouse antibody; combine the mouse antibody and human template within 5 Å of the CDR region Different amino acids were back-mutated to determine the final sequence of the humanized antibody, and four different humanization schemes, A, B, C, and D, were established for the heavy chain variable region and light chain variable region of the T0004 antibody. , see Figure 6 and Figure 7, the related sequences are SEQ ID NO: 7-9, 12-16.

Select the humanization scheme of B and C of the variable region of the heavy chain and the variable region of the light chain, and obtain four different sequence combinations of the variable region of the light chain and the heavy chain, namely T0004-B (light chain B+heavy chain B) , T0004-BC (light chain B + heavy chain C), T0004-C (light chain C + heavy chain C), T0004-CB (light chain C + heavy chain B). The variable regions of these four antibodies were fused with the constant regions of human IgG1κ, and the codons preferred by CHO (Chinese hamster ovary) cells were selected according to the amino acid sequences of the light and heavy chains for reverse translation, and the encoded cDNA sequences were synthesized and inserted into the pcDNA3.1 vector. Transfected CHO cells for expression. Harvest the culture supernatant of CHO cells, perform immunoblotting (Western Blot) with recombinant human PD-L1-Fc fusion protein, and initially identify the affinity, the results show that T0004-BC, T0004-C are fused with recombinant human PD-L1-Fc Proteins have affinity. T0004-BC and T0004-C antibody proteins in the cell culture supernatant were separated and purified by recombinant protein A affinity chromatography for subsequent detection.

Example 8. Measurement of thermal stability of T0004-BC and T0004-C antibody proteins by differential scanning calorimetry

Differential scanning calorimetry was used to measure the thermal stability of T0004-BC and T0004-C antibody proteins. The results are shown in Figure 8 and Figure 9. The Tm values of T0004-BC and T0004-C antibody proteins were 78.60 °C and 78.56 °C, respectively. ℃, indicating that the T0004-BC and T0004-C antibodies have good thermal stability.

Example 9, flow cytometry detection T0004-BC, T0004-C and membrane PD-L1 binding ability

Dilute T0004-BC (test product), T0004-C (test product), and Tecentriq (reference product) to 15 concentrations from 10 μg/mL, respectively, and CHO-K1/hmPD in the logarithmic growth phase - L1 cells (see Example 2 for the preparation method) were mixed and kept on ice for 45 min to allow them to bind to PD-L1 on the surface of CHO-K1/hmPD-L1 cells. Wash 2 times with PBS containing 1% fetal bovine serum, add FITC fluorescently labeled goat anti-human IgG (H+L) secondary antibody, ice bath for 45min, wash 2 times with PBS containing 1% fetal bovine serum. The pellet was resuspended in PBS containing 1% fetal bovine serum, and the fluorescence intensity was analyzed by flow cytometry.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the mean fluorescence intensity (MFI). See Figures 10-12. The EC50 (half maximum effect concentration) of Tecentriq binding to PD-L1 on the cell membrane surface is 336.20383 ng/mL, 324.08248 ng/mL, and 318.10122 ng/mL, respectively.

Example 10. Enzyme-linked immunoassay detection of binding ability of T0004-BC, T0004-C and soluble PD-L1

Dilute the human PD-L1-Fc fusion protein to 10 μg/mL with coating solution, add 100 μL/well to the microtiter plate, and coat at 37°C for 1-2 hours. Discard the coating solution, add blocking solution, 350 μL/well, block overnight at 2-8°C, and wash 7 times with a plate washer. Dilute T0004-BC (test product), T0004-C (test product) and Tecentriq (reference product) antibodies with PBS respectively, starting from 50000ng/mL and diluting 15 concentrations by 4 times, 100μL/well, 37℃ for 1 -2 hours, discard the liquid in the ELISA plate, and wash it 7 times with a plate washer. Add horseradish peroxidase (HRP)-labeled goat anti-human κ chain antibody diluted 1:10000, 100 μL/well, react at 37 °C for 1 hour, discard the liquid in the microplate, and wash 7 times with a plate washer. Add TMB substrate solution to the ELISA plate, 50 μL/well, develop color in the dark for 1-10 minutes, add 50 μL/well stop solution, and mix quickly. Read OD450nm with a microplate reader, and use OD570nm as the reference wavelength.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the OD value. See Figure 13. The calculated combination of T0004-BC, T0004-C, Tecentriq and soluble PD-L1 The EC50 (half-maximum effect concentration) were 38.17 ng/mL, 54.65 ng/mL, and 56.53 ng/mL, respectively.

Example 11. Surface plasmon resonance detection of affinity between T0004-BC, T0004-C and human PD-L1

Human PD-L1-Fc fusion protein was labeled with biotin, and then coupled to the detection chip of the surface plasmon resonance instrument BIACORE, T0004-BC (test product), T0004-C (test product), Tecentriq (reference product) Antibodies were diluted 10 times starting from 1 μg/mL, and added to BIACORE equipment, respectively, to detect and record the optical signal changes caused by the binding-dissociation process of each antibody and human PD-L1-Fc fusion protein. Curves were drawn on the horizontal and vertical coordinates with time and optical response values. See Figures 14 to 16 for the spectra, and Table 3 for the obtained parameter values.

The experimental results show that T0004-BC (KD value 1.008E-11M) and T0004-C (KD value 1.250E-11M) have higher human PD-L1 affinity than Tecentriq (KD value 2.720E-11M), and the KD value ( Equilibrium dissociation constant) is less than half of Tecentriq; and the kd value of T0004-BC, T0004-C and human PD-L1 is also much smaller than Tecentriq, indicating that T0004-BC, T0004-C binds to human PD-L1 Stability is higher than Tecentriq.

Table 3. Surface plasmon resonance detection of the affinity of each antibody to human PD-L1 Antibody name ka (1/Ms) kd (1/s) KD (M) Rmax (RU) Chi² (RU²) U-value T0004-BC 4.415E+6 4.452E-5 1.008E-11 18.30 0.377 7 T0004-C 3.881E+6 4.852E-5 1.250E-11 21.28 0.265 5 Tecentriq (control) 5.183E+6 1.410E-4 2.720E-11 20.75 0.894 4

Example 12. Flow cytometry detection of the ability of T0004-BC, T0004-C and Tecentriq to compete with PD-L1 on the cell membrane surface

CHO-K1/hmPD-L1 cells in the logarithmic growth phase were collected (see Example 2 for the preparation method), and different concentrations of competing antibodies T0004-BC, T0004-C, Tecentriq (control) and subsaturation concentrations of FITC fluorescently labeled Tecentriq Mix and dilute 15 concentration gradients at a 2-fold ratio from 500 μg/ml to the final concentration of the competing antibody. The mixture is ice-bathed for 45 min and washed twice with PBS containing 1% fetal bovine serum. The pellet was resuspended in PBS with 1% fetal bovine serum, and the competition was analyzed by flow cytometry.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the mean fluorescence intensity (MFI). See Figures 17 to 19. T0004-BC, T0004-C and Tecentriq competes to bind to the half effective dose (EC50) of PD-L1 on the cell membrane surface, and the results are shown in Table 4.

Table 4. T0004-BC, T0004-C compete with Tecentriq for binding to cell membrane surface

PD-L1 ability test results clone name EC50(μg/ml) Whether to compete T0004-BC 1.23916 compete T0004-C 1.20865 compete Tecentriq (control) 1.16779 compete

Example 13. Detection of the ability of T0004-BC, T0004-C and Tecentriq to compete with soluble PD-L1 by ELISA

Dilute the recombinant human PD-L1-Fc fusion protein to 10 μg/mL with coating solution, add 100 μL/well to the microtiter plate, and coat at 37°C for 1-2 hours. Discard the coating solution, add blocking solution, 350 μL/well, block overnight at 2-8°C. Plate washer washed 7 times. Mix the competing antibodies T0004-BC, T0004-C, and Tecentriq (control) with biotin-labeled Tecentriq in different proportions, add 100 μL/well to the microtiter plate, and the final concentration of the competing antibody in each well starts from 195 μg/mL to Dilute 14 concentration gradients by 2 times and react at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Dilute Avidin-HRP 1:1000, add 100 μL/well, and react at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Add TMB substrate solution to the microtiter plate, 50 μL/well, and develop color in the dark for 15 minutes. Add the stop solution to the microtiter plate, 50 μL/well, and mix quickly. The microplate reader reads OD450nm, and 570nm is the reference wavelength.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the OD value. See Figure 20. The calculated T0004-BC, T0004-C, Tecentriq and Tecentriq compete with PD- The EC50 (half maximum effect concentration) of L1 were 1.489 μg/mL, 1.429 μg/mL, and 1.494 μg/mL, respectively (Table 5).

Table 5, T0004-BC, T0004-C compete with Tecentriq for binding solubility

PD-L1 ability test results clone name EC50 (μg/mL) Whether to compete T0004-BC 1.489 compete T0004-C 1.429 compete Tecentriq 1.494 compete

Example 14, flow cytometry detection of T0004-BC, T0004-C and PD-1 competitive binding ability of PD-L1 on the cell membrane surface

CHO-K1/hmPD-L1 cells in the logarithmic growth phase were collected (see Example 2 for the preparation method), and the competing antibodies T0004-BC, T0004-C, Tecentriq (control) and subsaturating concentrations of biotin-labeled human PD- 1 Proteins were mixed in different proportions, and the final concentration of the competing antibody in the mixture was diluted 15 concentration gradients by 2 times starting from 10 μg/mL, and the mixture was kept on ice for 45 minutes. Wash 2 times with PBS solution containing 1% fetal bovine serum, add Avidin-PE fluorescently labeled secondary antibody, ice bath for 45min, and wash 2 times with PBS solution containing 1% fetal bovine serum. The pellet was resuspended in PBS solution containing 1% fetal bovine serum, and analyzed by flow cytometry.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the mean fluorescence intensity (MFI). See Figures 21 to 23. PD-1 competes to bind to the half effective dose (EC50) of PD-L1 on the cell membrane surface. The results are shown in Table 6.

Table 6. T0004-BC, T0004-C compete with PD-1 for binding to cell membrane surface

PD-L1 ability test results clone name EC50(μg/ml) Whether to compete T0004-BC 0.16444 compete T0004-C 0.19668 compete Tecentriq (control) 0.16056 compete

Example 15. Detection of the ability of T0004-BC and T0004-C to compete with PD-1 for binding to soluble PD-L1 by enzyme-linked immunosorbent assay

Dilute the recombinant human PD-1-Fc fusion protein to 10 μg/mL with coating solution, add 100 μL/well to the microtiter plate, and coat at 37°C for 1-2 hours. Discard the coating solution, add blocking solution, 350 μL/well, block overnight at 2-8°C. Plate washer washed 7 times. Mix the competing antibodies T0004-BC, T0004-C, and Tecentriq (control) with biotin-labeled recombinant human PD-L1-Fc fusion protein in different proportions, add 100 μL/well to the microtiter plate, in which the competing antibodies are in each well The final concentration in the solution is 11 concentrations starting from 8800ng/mL, and reacted at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Dilute Avidin-HRP (avidin-coupled horseradish peroxidase) 1:1000, 100 μL/well, and react at 37°C for 1 hour. Discard the supernatant and wash 7 times with a plate washer. Add TMB substrate solution to the microtiter plate, 50 μL/well, and develop color in the dark for 15 minutes. Add the stop solution to the microtiter plate, 50 μL/well, and mix quickly. The microplate reader reads OD450nm, and 570nm is the reference wavelength.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the OD value. See Figure 24. The calculated T0004-BC, T0004-C, Tecentriq and Tecentriq compete with PD- See Table 7 for the EC50 (half maximum effect concentration) of L1.

Table 7, T0004-BC, T0004-C compete with human PD-1 for solubility

PD-L1 ability test results clone name EC50 (ng/mL) Whether to compete T0004-BC 3598 compete T0004-C 3294 compete Tecentriq 2503 compete

Example 16, reporter gene method to detect the biological activity of T0004-BC, T0004-C antibody activated TCR

Bright-Glo ^TM Luciferase Assay System kit (Promega, Cat. No. E2610) was used to use the human T lymphoma cell line Jurkat stably expressing PD-1 and NFAT-Luciferase (luciferase) reporter gene promoters on the cell membrane surface as effector cells , the target cell is the CHO cell (CHO-K1/hmPD-L1) that expresses human PD-L1 in the membrane prepared according to the method of Example 2.

Detection principle: In the absence of recombinant anti-PD-L1 humanized monoclonal antibody, after PD-1 on the surface of effector cells binds to PD-L1 on the surface of target cells, a negative regulation will be produced, which will not cause TCR (T cell receptor) Activation of NFAT promoter-induced luciferase reporter gene expression will not occur; when anti-PD-L1 antibody is present, anti-PD-L1 antibody can compete with effector cell surface PD-1 to bind to target cell surface PD-L1, When target cells are co-incubated with effector cells, the TCR complex on the surface of Jurkat effector cells will be activated to varying degrees with the concentration of anti-PD-L1 antibody (the higher the concentration, the higher the degree of activation), thereby triggering the NFAT promoter-induced The expression intensity of the luciferase reporter gene is different, the higher the antibody concentration, the stronger the expression of the luciferase reporter gene (the higher the RLU).

Detection method: Add 100 μL/well of the target cell culture solution with a concentration of 4×10 ⁵ cells/mL into a 96-well cell culture plate (the number is 4×10 ⁴ cells/well), and culture overnight in a 5% CO2 incubator at 37°C . The culture medium in the 96-well plate was discarded, and 40 μL of serially diluted T0004-BC, T0004-C or Tecentriq (control) antibody was added to each well (the antibody concentration was 1.5-fold dilution from 800 ng/mL to 9 concentrations), And 40 μL of effector cells with a concentration of 1.25×10 ⁶ cells/mL (the number is 5×10 ⁴ cells/well), and set up single target cell control wells, single effect cell control wells and target cell plus effector cell control wells. After the 96-well cell culture plate was placed in a 5% CO2 incubator at 37°C for 6 hours, 80 μL/well of the luminescent substrate was added and read with a GloMAX luminometer.

The analysis software Origin software was used to fit the standard curve, with the concentration (logarithm) of the sample as the abscissa, and the luminescence value as the ordinate, see Figures 25 to 27, and calculate the activation of T0004-BC, T0004-C, and Tecentriq (control) The EC50 (half maximum effect concentration) of the biological activity of TCR was 55.22212 ng/mL, 56.86219 ng/mL, and 53.09773 ng/mL, respectively.

Example 17. Detection of the ability of T0004-BC and T0004-C antibodies to induce ADCC (antibody-dependent cell-mediated cytotoxicity) effect by reporter gene method

The ADCC Effect Reporter Gene Detection Kit (Product No. G7014) from Promega was used, the effector cells were Jurkat NFAT-Re/CD16a(158V)-FcRγ/pcDNA3.1(hygro) cells, and the target cells were prepared according to the method in Example 2 CHO cells membrane expressing human PD-L1 (CHO-K1/hmPD-L1).

Add 30 μL/well of target cells at a concentration of 1×10 ⁶ cells/mL to a 96-well cell culture plate, and serially dilute T0004-BC, T0004-C or Tecentriq (control) antibodies 3-fold from 12,000 ng/mL11 Each concentration, 60 μL/well was added to a 96-well cell culture plate, and incubated at 37°C, 8% CO ₂ incubator for 60 minutes. Add 30 μL/well of logarithmic phase effector cells at a concentration of 1×10 ⁶ cells/mL to a 96-well cell culture plate, and act for 5 hours in a 37°C, 8% CO ₂ cell incubator. Add 120 μL/well of luminescent substrate and read with GloMAX luminometer.

Using the analysis software Origin software to fit the standard curve, the concentration (logarithm) of the sample is taken as the abscissa, and the luminescence value is the ordinate, as shown in Figures 28 to 30, and the EC50 ( half maximum effect concentration) were 6.39946 ng/mL and 7.00327 ng/mL respectively, and Tecentriq (control) had no ability to induce ADCC effect.

Example 18. Lactate dehydrogenase method to detect the ability of T0004-BC and T0004-C antibodies to induce CDC (complement-dependent cytotoxicity) effect

The Cytotoxicity Detection Kit (LDH) from Sigma-Aldrich Company (Product No. 11644793001) was used, and the target cells were CHO cells (CHO-K1/hmPD-L1) prepared by the method in Example 2 expressing human PD-L1 in the membrane.

Add human serum at 80 μL/mL to the target cells at a concentration of 3×10 ⁵ cells/mL as complement, add 50 μL/well to a 96-well cell culture plate, and add T0004-BC, T0004-C or Tecentriq (control) antibody Starting from 12,000 ng/mL, 3-fold serial dilutions were made to 11 concentrations, 50 μL/well was added to a 96-well cell culture plate, and the cells were incubated at 37°C and 8% CO ₂ for 4 hours. Add 100 μl/well of the mixed chromogenic substrate, and develop the color for 10-30 minutes in the dark, and detect its OD490 with a microplate reader, and OD630 is the reference wavelength.

Use the analysis software Origin software to fit the sample concentration (abscissa, logarithmic value) to the OD value (ordinate), see Figure 31, and calculate the EC50 (half maximum effect concentration ) were 734 ng/mL and 716 ng/mL respectively, and had weak CDC effect at high concentrations, while Tecentriq (control) had no ability to induce CDC effect.

Example 19. Detection of the binding ability of T0004-BC and T0004-C to human PD-L1 mutant strains by flow cytometry

Construct the D49Y and V111E mutants of human PD-L1 (referred to as hPD-L1-mu), that is, on the basis of the sequence number Q9NZQ7-1 on the UniProt website, the 49th aspartic acid and the 111th valine are mutated to Tyrosine, glutamic acid, design coding DNA sequence, insert into pcDNA3.1 expression vector, stably transfect CHO-K1 host cells, and obtain CHO-K1 engineered cells stably expressing hPD-L1-mu on the membrane surface through pressurized screening ( denoted as CHO-K1/hPD-L1-mu). Dilute the recombinant human PD-1-Fc fusion protein, T0004-BC, T0004-C, and Tecentriq antibody to 15 concentrations starting from 10 μg/mL, and CHO-K1/hPD-L1- The mu cells were mixed and kept on ice for 45 min to fully combine with hPD-L1-mu on the cell surface. Wash 2 times with PBS containing 1% fetal bovine serum, add FITC fluorescently labeled rabbit anti-mouse IgG (H+L) secondary antibody, ice bath for 45min, wash 2 times with PBS containing 1% fetal bovine serum. The pellet was resuspended in PBS containing 1% fetal bovine serum, and the fluorescence intensity was analyzed by flow cytometry.

The experimental data was analyzed using the analysis software Origin to fit the standard curve. The abscissa is the concentration (logarithm) of the sample, and the ordinate is the mean fluorescence intensity (MFI). See Figure 32. The results show that the recombinant human PD-1-Fc fusion protein and T0004 -BC, T0004-C antibodies bind to CHO-K1/hPD-L1-mu in a certain range in a dose-dependent manner, while Tecentriq does not bind to CHO-K1/hPD-L1-mu.

Example 20, T0004-BC, T0004-C animal tumor suppression ability research

BALB/c mice were subcutaneously inoculated with MC38 cells (mouse colon cancer cells, PD-L1 positive), and when the tumor grew to ^100mm3 , the tumor-bearing mice were divided into groups, and each group was injected into the tail vein at a dose of 10 mg/kg T0004-BC, T0004-C, Tecentriq or normal saline control, injected once every 3 days, treated continuously for 21 days, observed the changes of body weight and tumor size of mice in each group every 6 days, and compared the measured tumor volume with the first time The days after administration were plotted, and the results are shown in Figure 33, indicating that T0004-BC, T0004-C, and Tecentriq all had inhibitory effects on the tumors formed by MC38 cells in mice, and T0004-BC, T0004-C had obvious tumor inhibitory effects better.

Claims (8)

1. A recombinant anti-PD-L1 monoclonal antibody is characterized in that,

the sequences of the heavy chain CDR1, CDR2 and CDR3 regions are SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively; the sequences of the light chain CDR1, CDR2 and CDR3 regions are SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.

2. The antibody according to claim 1, wherein,

the sequence of the heavy chain variable region of the antibody is SEQ ID NO. 7.

3. The antibody according to claim 1, wherein,

the sequence of the antibody light chain variable region is SEQ ID NO. 8 or SEQ ID NO. 9.

4. The antibody according to claim 1, 2 or 3, wherein,

the antibodies are of the human IgG1 kappa subtype.

5. The antibody according to claim 1, wherein,

the sequence of the heavy chain variable region of the antibody is SEQ ID NO. 10.

6. The antibody according to claim 1, wherein,

the sequence of the antibody light chain variable region is SEQ ID NO. 11.

7. The antibody according to claim 1, 5 or 6, characterized in that,

the antibodies are of the murine IgG1 kappa subtype.

8. Use of the antibody of claim 1, 2 or 3 in the manufacture of a medicament for the treatment or prevention of cancer, immune disease.