CN111344305A

CN111344305A - Antibodies against PD-L1 and uses thereof

Info

Publication number: CN111344305A
Application number: CN201880055538.9A
Authority: CN
Inventors: 宋德勇; 董创创; 窦昌林; 刘秀; 成岩; 韩镇; 沙春洁; 韩静
Original assignee: Shandong Bioanty Biological Technology Co ltd
Current assignee: Shandong Bioanty Biological Technology Co ltd
Priority date: 2017-11-27
Filing date: 2018-11-26
Publication date: 2020-06-26
Anticipated expiration: 2038-11-26
Also published as: WO2019101196A1; CN111344305B

Abstract

Relates to an anti-PD-L1 antibody and application thereof. In particular to an anti-PD-L1 antibody and an antigen binding fragment thereof and application thereof in diagnosing and treating cancer and predicting prognosis.

Description

Antibodies against PD-L1 and uses thereof

Technical Field

The invention relates to an anti-PD-L1 antibody and application thereof. In particular, the present invention relates to an anti-PD-L1 antibody and antigen binding fragments thereof and their use for the diagnosis and treatment of inflammatory diseases, autoimmune diseases and cancer and for predicting prognosis.

Background

The immune system must strike a balance between an effective response to eliminate the pathogenic agent and maintaining tolerance to prevent autoimmune disease. T cells are critical to maintaining this balance and their proper regulation is primarily mediated by the B7-CD28 family of molecules. The interaction between the B7 family member (which functions as a ligand) and the CD28 family member (which functions as a receptor) not only provides a critical positive signal that initiates, enhances and maintains a T cell response, but also, where appropriate, provides a critical negative signal that facilitates the limiting, terminating and/or attenuating of a T cell response. One member of the CD28 family, known as PD-1 (also known as programmed cell death-1), is upregulated on activated T cells, B cells, and monocytes; the ligand in the B7 family, PD-L1 (also known as B7H1 or programmed cell death-1 ligand 1), interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses.

PD-L1(B7-H1) is a 40kDa cell surface glycoprotein belonging to the B7 family, having IgV and IgC-like regions, a transmembrane region and a cytoplasmic tail. The gene was first discovered and cloned in 1999 (Do. mu.g H et al, Nat Med 5: 1365-. PD-L1 is expressed in lower amounts in normal human tissues in addition to immune cells, but is expressed in higher amounts in some tumor cell lines, such as lung Cancer, ovarian Cancer, colon Cancer and melanoma (Iwai et al, PNAS 99: 12293-.

It has been shown that high expression of PD-L1 in tumor cells plays an important role in immune escape of tumors by increasing T cell apoptosis. For example, in a large number of samples of ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, and melanoma, expression of PD-L1 is associated with poor prognosis and short overall survival, regardless of subsequent treatment. In the PD-1 gene-knocked-out mice, the PD-L1/PD-1 pathway is blocked, and tumors cannot be formed after tumor cell inoculation (Do μ g H et al, Nat Med 8:793-800, 2002).

Given the important role of PD-L1 in cancer development and immune system regulation, there is a continuing need to detect the presence of PD-L1 and effective antibodies that block the PD-L1/PD-1 pathway.

Disclosure of Invention

The present invention relates to an anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the light chain region of the antibody comprises:

1) sequence KSSQX₁VLYSSNNKNYLX₂LCDR1 sequence shown by W (SEQ ID NO:14), wherein X₁Is S or N, and X₂Is A or P;

2) the sequence LCDR2 shown by sequence WASTRES (SEQ ID NO: 15); and

3) a LCDR3 sequence shown as sequence QQYYSTPLT (SEQ ID NO: 16); and is

Wherein the heavy chain region of the antibody comprises:

1) the sequence HCDR1 shown by the sequence GFTFSSY (SEQ ID NO: 17);

2) sequence SX₃The HCDR2 sequence shown by DGSNK (SEQ ID NO:18), wherein X₃Is F or Y; and

3) the sequence HCDR3 shown by sequence DRIYLDY (SEQ ID NO: 19).

In one aspect of the invention, the antibody is an anti-PD-L1 antibody.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 1 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 2.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 3 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 4.

In one aspect of the invention, the light chain region of the antibody has the sequence of SEQ ID NO. 5 and the heavy chain region of the antibody has the sequence of SEQ ID NO. 6.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 7 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 8.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO 9 and the sequence of the heavy chain region of the antibody is SEQ ID NO 10.

In one aspect of the invention, the light chain region of the antibody has the sequence of SEQ ID NO. 11 and the heavy chain region of the antibody has the sequence of SEQ ID NO. 12.

In one aspect of the invention, the sequence of the light chain region of the antibody comprises one or more mutations selected from: a mutation of the amino acid at position 1 from E to D, a mutation of the amino acid at position 2 from L to I, a mutation of the amino acid at position 4 from L to M and a mutation of the amino acid at position 105 from D to E, and

the heavy chain region sequence of the antibody comprises one or more mutations selected from the group consisting of: a mutation of amino acid 1 from E to Q, a mutation of amino acid 6 from Q to E, and a mutation of amino acid 16 from K to R.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 20 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 21.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 22 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 23.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 24 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 25.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO 26 and the sequence of the heavy chain region of the antibody is SEQ ID NO 27.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO 28 and the sequence of the heavy chain region of the antibody is SEQ ID NO 29.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 30 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 31.

In one aspect of the invention, the sequence of the light chain region of the antibody is SEQ ID NO. 32 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 33.

In one aspect of the invention, the antigen binding fragment is a Fab, Fab ', F (ab')2, scFv or dsFv fragment.

In one aspect of the invention, the invention relates to a method of treating cancer using an antibody or antigen-binding fragment thereof of the invention, comprising administering an antibody or antigen-binding fragment thereof of the invention to a subject in need thereof. Preferably, the cancer is selected from lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.

In one aspect of the invention, the invention relates to the use of an antibody or antigen binding fragment thereof of the invention for the preparation of a medicament for the treatment of cancer, preferably selected from lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.

In one aspect of the invention, the invention relates to a method of diagnosing cancer using an antibody or antigen-binding fragment thereof of the invention, comprising contacting an antibody or antigen-binding fragment thereof of the invention with a sample from a subject, and determining the binding of the antibody to PD-L1, thereby determining the level of PD-L1 expression in the sample from the subject, thereby diagnosing cancer in the subject. Preferably, the cancer is selected from lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.

In one aspect of the invention, the invention relates to the use of an antibody or antigen binding fragment thereof of the invention for the preparation of an agent for the diagnosis of cancer, preferably selected from lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.

In one aspect of the invention, the invention relates to a method of using an antibody or antigen-binding fragment thereof of the invention for making a method of determining the prognosis or overall survival of a cancer patient, comprising contacting the antibody or antigen-binding fragment thereof of the invention with a sample from a subject, and determining the binding of the antibody to PD-L1, thereby diagnosing the level of PD-L1 expression in the subject sample, thereby determining the prognosis or overall survival of the cancer patient. Preferably, the cancer is selected from lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma. The results indicate that patients with the presence of PD-L1 expression have a better prognosis or longer overall survival when treated with a drug (e.g., treatment with the anti-cancer therapy of the invention) relative to patients without the presence of PD-L1 expression.

In one aspect of the invention, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention for the preparation of a reagent for determining the prognosis or overall survival of a patient with a cancer, preferably selected from the group consisting of lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.

The antibody or the antigen binding fragment thereof can effectively bind to PD-L1 and block a PD-L1/PD-1 pathway, thereby effectively diagnosing and treating cancer, and the effect of the antibody or the antigen binding fragment thereof is obviously superior to that of a commonly used anti-PD-L1 specific antibody known in the field.

In one aspect of the invention, the invention relates to a method of treating an inflammatory disease or an autoimmune disease using an antibody or antigen-binding fragment thereof of the invention, comprising administering the antibody or antigen-binding fragment thereof of the invention to a subject in need thereof. Preferably, the inflammatory or autoimmune disease is selected from the group consisting of viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, crohn's disease, multiple sclerosis, and inflammatory bowel disease.

In one aspect of the invention, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention for the preparation of a medicament for the treatment of an inflammatory or autoimmune disease, preferably selected from the group consisting of a viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, crohn's disease, multiple sclerosis, and inflammatory bowel disease.

In one aspect of the invention, the invention relates to a method of diagnosing an inflammatory disease or an autoimmune disease using an antibody or antigen-binding fragment thereof of the invention, comprising contacting the antibody or antigen-binding fragment thereof of the invention with a sample from a subject and determining the binding of the antibody to PD-L1, thereby determining the level of PD-L1 expression in the sample from the subject, thereby diagnosing the inflammatory disease or the autoimmune disease. Preferably, the inflammatory or autoimmune disease is selected from the group consisting of viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, crohn's disease, multiple sclerosis, and inflammatory bowel disease.

In one aspect of the invention, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention for the preparation of an agent for the diagnosis of an inflammatory or autoimmune disease, preferably selected from the group consisting of viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, crohn's disease, multiple sclerosis and inflammatory bowel disease.

The antibody or the antigen-binding fragment thereof can effectively detect the existence of PD-L1 and block a PD-L1/PD-1 pathway, and the effect is superior to that of an anti-PD-L1 antibody known in the prior art, such as commercial Abutizumab.

Drawings

Figure 1 shows the serum titers of antibodies against PD-L1 in 6 transgenic mice.

FIG. 2 shows the activity of monoclonal prokaryotic expression of ScFv to block the binding of PD-1 to PD-L1.

Figure 3 shows the sensitivity of detecting binding between a candidate antibody and PD-L1 using an ELISA assay.

FIG. 4A shows the results of detection of binding kinetics of candidate antibody PLH03-BT613-hIgG1 using BiAcore.

FIG. 4B shows the results of detection of the binding kinetics of candidate antibody PLH06-CA782-hIgG1 using BiAcore.

FIG. 4C shows the results of using BiAcore to detect the binding kinetics of candidate antibody PLH06-CA783-hIgG 1.

FIG. 4D shows the results of using BiAcore to detect the binding kinetics of candidate antibody PLH01-CA785-hIgG 1.

FIG. 4E shows the results of using BiAcore to detect the binding kinetics of candidate antibody PLH01-CA786-hIgG 1.

FIG. 4F shows the results of using BiAcore to detect the binding kinetics of candidate antibody PLH01-CA787-hIgG 1.

FIG. 5 shows the results of different candidate antibodies blocking the binding between PD-L1 and PD-1.

FIG. 6 shows the results of a comparison of the binding affinity of antibody BT613 and antibody PL-GEN-IgG1 to PD-L1.

FIG. 7 shows the results of a comparison of the activity of antibody BT613 and antibody PL-GEN-IgG1 in blocking the binding between PD-L1 and PD-1.

FIG. 8 shows the results of a comparison of the activity of antibody BT613 and antibody PL-GEN-IgG1 in blocking the binding between PD-L1 and B7-1.

FIG. 9 shows the results of a comparison of the binding affinity of antibody BT613 and antibody PL-GEN-IgG1 to mouse PD-L1.

FIG. 10 shows the results of a comparison of the binding affinities of antibody BT613 and antibody PL-GEN-IgG1 for a PD-L1-related protein.

FIG. 11 shows the results of a comparison of the activity of antibody BT613 and antibody PL-GEN-IgG1(Atezolizumab) to block the binding between PD-L1 and PD-1 in the GS-C2/PD-L1/GS-J2/PD-1 cell line.

FIG. 12 shows the effect of MLR in vitro detection of antibody BT613 and antibody PL-GEN-IgG1(Atezolizumab) on IL-2 production.

FIG. 13 shows the time course of administration of the antibody BT613, antibody Atezolizumab (Atezolizumab), 10.0mg/kg, intravenously injected into C57BL/6 mice.

Figure 14A shows the inhibitory effect of 3.0mg/kg antibody BT613 and atlizumab on tumor volume growth.

FIG. 14B shows a photograph of a mouse tumor, wherein TS1503-A is antibody BT613, and TS1503-D is atezumab.

Figure 15 shows tumor weights in the solvent, antibody BT613 and attentizumab groups.

Figure 16 shows mouse body weights in the solvent, antibody BT613 and atlizumab groups.

Figure 17A shows the results of detection of binding kinetics of BT613 mutant BT613(HM \ LM) using BiAcore.

FIG. 17B shows the results of testing the binding kinetics of BT613 mutant BT613(H/LM) using BiAcore.

FIG. 17C shows the results of measuring the binding kinetics of BT613 mutant BT613(HM/L) using BiAcore.

FIG. 17D shows the results of detecting the binding kinetics of BT613 mutant BT613(HM/LM-1) using BiAcore.

FIG. 17E shows the results of the detection of binding kinetics of BT613 mutant BT613(HM/LM-2) using BiAcore.

FIG. 17F shows the results of measuring the binding kinetics of BT613 mutant BT613(HM/LM-3) using BiAcore.

FIG. 17G shows the results of detecting the binding kinetics of BT613 mutant BT613(HM/LM-4) using BiAcore.

Figure 18A shows the inhibitory effect of 3.0mg/kg antibody BT613 and atlizumab on tumor volume growth.

FIG. 18B shows a photograph of a mouse tumor, wherein TS1503-1 is BT613 and TS1503-4 is atelizumab.

Figure 19 shows tumor weights in the solvent, antibody BT613 and attentizumab groups.

Figure 20 shows mouse body weights in the solvent, antibody BT613 and atlizumab groups.

Detailed Description

The invention will be better understood with reference to the following examples. However, it is to be understood that the following examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

Example 1 Generation of Single chain antibodies against PD-L1

1.1 immunization of mice

Whole human antibody transgenic mice LuyeMab prepared by immunizing green leaf drugs with PD-L1 protein PD-L1-His (Sinobiological, catalog No. 10084-H08H) and adjuvant (the preparation method of the transgenic mice is described in Chinese patent CN 201210281415), complete Freund's adjuvant is used as the adjuvant for priming, and incomplete Freund's adjuvant (Sigma) is used as the adjuvant for secondary and tertiary immunization. A total of 17 mice were immunized, 6 mice with higher antibody serum titers were selected after 3 immunizations and boosted with antigen without adjuvant (see FIG. 1), and the mice were sacrificed 4 days later and the spleens were removed and stored frozen for future use.

1.2 phage library creation

RNA extraction was performed from the frozen mouse spleen by Trizol (Thermo Scientific, catalog # 15596-026) according to a conventional protocol, and RNA was reverse-transcribed into cDNA using Roche Applied Science, catalog # 4897030001, according to the instructions thereof, using the Roche reverse transcription Kit Transcriptor First Strand cDNA Synthesis Kit. Step of establishing phage libraryReferring to the procedure described in Phage display, edited by Tim Clackson, the variable regions of heavy and light chains were first obtained from cDNA by PCR, then ScFv was obtained by overlap extension PCR, SfiI cleavage of ScFv was performed at 50 ℃ for 5 hours, the cleaved ScFv (single chain Fv) was ligated to plasmid pCOMB3x, and the ligation product was then electroporated into E.coli XL1-Blue competent cells. Phage library PD-L1-H3-1, library volume 4.2 x10, established with mouse numbered LYQ013⁸(ii) a Phage library PD-L1-H6-1, library volume 4.0 × 10, established with mouse number LYQ024⁸。

1.3 clonal selection

Screening a flat plate: plates (high adsorption microplates, Costar) were coated with PD-L1-His protein at 0.2. mu.g/well, left overnight at 4 ℃ the next day, plates were blocked with 3% skimmed milk powder for 1H, phage library PD-L1-H3-1/PD-L1-H6-1 (colony forming unit: 2 × 10)¹²) And incubated for 2h, after washing 4-10 times with PBST (0.01M, pH 7.4, 0.05% tween-20), PD-L1-specifically bound phage were eluted with elution buffer (pH 2.2, 0.1M HCl-Gly).

Screening magnetic beads: the PD-L1-Fc protein was biotinylated according to the procedure of the Thermo's Biotin labeling instructions (molar ratio of PD-L1 protein to biotin 1:2), bound to Thermo's magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, 00355871) and then bound to the phage library PD-L1-H3-1/PD-L1-H6-1 (colony-forming unit: 2: 10)¹²) And incubated to obtain a clone specifically bound by PD-L1.

The plate screening obtains clone PLH6-CA782\ CA783\ CA785\ CA786\ CA787, and the magnetic bead screening obtains clone PLH3-BT613, wherein PLH3 represents library PD-L1-H3-1 established by mouse LYO013, and PLH6 represents library PD-L1-H6-1 established by mouse LYO 024.

1.4 monoclonal prokaryotic expression of ScFv to block the binding of PD-1 and PD-L1

Libraries that were positive by phage enzyme-linked immunosorbent assay (ELISA) were coated with 2YT (Amp 100. mu.g/ml) plates, single clones were picked directly and induced to express using commercially available ZYM medium, and the purified products after expression were used to detect blocking activity, the results are shown in FIG. 2.

Example 2 molecular construction and production of anti-PD-L1 antibodies

Clones PLH3-BT613, PLH6-CA782\ CA783\ CA785\ CA786\ CA787 were sent to Invitrogen Biotechnology Inc. for sequencing. The amino acid sequence of each clone is set forth in Table 1 below.

TABLE 1 amino acid sequence of clones with blocking Activity

After fusing the antibody gene with the Fc sequence (N297A) SEQ NO ID:13, the antibody gene was cleaved with 5 'Xba I (NEB catalog # R0145S) and 3' EcoRV (NEB catalog # R3195S), and the purified antibody gene was ligated into vector pCDNA3.4(Life Technology) by T4 DNA ligase (NEB catalog # M0202S), transfected into HEK293 cells at 37 ℃ with 8% CO₂Culturing in DMEM medium at 125rpm, and after 6 days, purifying the transient expression supernatant by Protein A (GE Healthcare) according to conventional affinity chromatography to obtain anti-PD-L1 antibody, and determining the antibody concentration by UV280 binding extinction coefficient.

SEQ ID NO:13

Production of control antibody: the amino acid sequence of the Roche PD-L1 antibody attlizumab is determined by IMGT data and the patent US 8217149B2, the complete gene is synthesized and sequenced, then the vector pCDNA3.4 is inserted and expressed by HEK293 cells, and the produced antibody is named PL-GEN-IgG 1.

Example 3 characterization of candidate antibodies

3.1 ELISA detection of binding of candidate antibodies to PD-L1 protein

96-well ELISA plates (BEVER, 40301) were coated with different concentrations (0.4. mu.g/ml, 0.2. mu.g/ml, 0.1. mu.g/ml, 0.05. mu.g/ml, 0.025. mu.g/ml, 0.0125. mu.g/ml, 0.00625. mu.g/ml, 0. mu.g/ml) of the antigen PD-L1-His at 100. mu.l/well and incubated overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100 μ l of each candidate antibody of 2 μ g/ml into each well, and incubating at 37 deg.C for 1 h; then adding goat anti-human IgG/HRP (China fir gold bridge), incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on an enzyme-linked immunosorbent assay. The results are shown in FIG. 3, and the 6-strain antibody has similar binding sensitivity to PD-L1 protein.

3.2 SPR detection of binding of candidate antibodies to PD-L1 protein

Antibody binding kinetics was measured using a BIAcoreX100 instrument based on surface plasmon resonance (SRP) technology by GE anti Human IgG FC amino coupling kit (GE, cat # BR-1008-39) anti Human IgG antibody amino was coupled to a CM5 biosensor chip (GE, BR-1000-12) to obtain approximately 1000 response units (response units, RU). for kinetic measurements, the antibody was serially diluted 2-fold with HBS-EP +1 × (GE, cat # BR-1006-69) buffer, starting with 50mM, 2-fold over 4 concentration gradients and set at 0 concentration.the assay conditions were antibody: 1. mu.g/ml, injection time 60s, flow rate 5. mu.l/min, stable 5s, PD-L1 protein (50mM, 25mM, 12.5mM, 6.25mM, 3.125mM, 0mM binding 60s, flow rate 30 min, dissociation flow rate 300. mu.l/min, regeneration with MgCl 3. mu.3M) and regeneration with MgCl 3. mu.25M₂Buffer regeneration 30s, start 2 times. The association constant (ka) and dissociation constant (kD) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2), with the equilibrium dissociation constant (kD) calculated as the ratio kD/ka.

Table 2 BiAcore assay candidate antibody binding kinetics

Antibody ID	Ka(1/Ms)	Kd(1/s)	KD(M)
PLH03-BT613-hIgG1	1.237E+6	8.303E-4	6.713E-10
PLH06-CA782-hIgG1	2.32E+06	0.0023	9.92E-10
PLH06-CA783-hIgG1	2.35E+06	2.23E-03	9.48E-10

PLH01-CA785-hIgG1	2.213E+6	1.395E-3	6.305E-10
PLH01-CA786-hIgG1	2.232E+6	1.270E-3	5.690E-10
PLH01-CA787-hIgG1	2.542E+6	1.341E-3	5.275E-10
PL-GEN-IgG1	1.185E+6	4.932E-4	4.163E-10

3.3 candidate antibodies block the binding of PD-L1 protein to PD-1 protein

ELISA plates were coated with PD-1-Fc at a concentration of 0.25. mu.g/ml at 100. mu.l/well and incubated overnight at 4 ℃; sealing with 3% skimmed milk powder for 1 hr; simultaneously co-incubating PD-L1-Fc-biotin (0.5. mu.g/mL) (prepared as in 1.3) and various concentrations (4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.0625. mu.g/mL, 0. mu.g/mL) of candidate antibody at 37 ℃ for 1h, then adding to the blocked ELISA plate and incubating at 37 ℃ for an additional 1 h; subsequently adding streptomycin/HRP, and incubating for 1h at 37 ℃; after development for 10min, OD450 was read on a microplate reader. The results are shown in FIG. 5, and the 6-strain antibody can effectively block the binding of PD-1 and PD-L1, and has similar blocking activity.

Example 4 comparison of BT613 and Roche PD-L1 antibodies

4.1 comparison of PL-BT613-IgG1 (hereinafter or simply BT613) with Roche PD-L1 antibody. Binding affinity comparisons were first performed on a cell line with high expression of PD-L1, GS-C2/PD-L1 cells (purchased from Nanjing Kingsri) were adjusted to 5E5 cells, 100. mu.l/w, with FACS buffer (PBS containing 0.2% BSA), 100. mu.g of BT613 (sample A) or PL-GEN-IgG1 (sample D) antibody was added, mixed and allowed to stand at 4 ℃ for 1 h. After washing the cells 2 times with FACS buffer, the cells were resuspended in 100. mu.l FACS buffer, and 1. mu.g of goat anti-human IgG fluorescent secondary antibody (Life, A21445) was added thereto, mixed well, and allowed to stand at 4 ℃ for 1 hour. FAThe cells were washed 2 times with CS buffer, resuspended in 100. mu.l FACS buffer and flow-checked (BD Accuri)^TMC6) In that respect The BT613 antibody showed similar binding affinity to the roche PD-L1 antibody on GS-C2/PD-L1 cells (see fig. 6).

4.2 comparison of BT613 and PL-GEN-IgG1 on the activity of blocking the binding of PD-1 and PD-L1, the procedure of ELISA was the same as in 3.3, and the results are shown in FIG. 7, where BT613 was found to have a better activity on blocking the binding of PD-L1 and PD-1 than PL-GEN-IgG1 from Roche.

4.3 comparison of BT613 to PL-GEN-IgG1 for Activity in blocking the binding of B7-1 to PD-L1

Coating a 96-well enzyme label plate with B7-1-Fc (purchased from Yi Qiao Shen) (the concentration is 0.5 mu g/ml) at 100 mu l/well, coating at 4 ℃ overnight, and sealing with 3% skimmed milk powder for 1h the next day; simultaneously, PD-L1-Fc-organisms (0.8. mu.g/ml) were incubated with gradient-diluted PLH03-BT613-hIgG1 or PL-GEN-IgG1 (8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0. mu.g/ml) at 37 ℃ for 1h, then added to the microplate which had been coated with B7-1 and incubated at 37 ℃ for a further 1 h; then streptomycin/HRP is added, incubation is carried out for 1h at 37 ℃, and OD450 is read on a microplate reader after color development is carried out for 10 min. The results are shown in FIG. 8, BT613 and Roche PL-GEN-IgG1 have similar activity in blocking the binding of PD-L1 to B7-1.

4.4 binding of BT613 and PL-GEN-IgG1 to PD-L1 of different species: the results of the ELISA assay showed that BT613 and PL-GEN-IgG1 showed similar binding to both human and cynomolgus PD-L1 (results not shown), but different binding behavior on mouse PD-L1 protein (see FIG. 9).

4.5 binding of BT613 to PL-GEN-IgG1 to PD-L1-related protein: the results of detection by ELISA showed differences in binding of BT613 and PL-GEN-IgG1 to human PD-L1\ PDL2\ PDL3 proteins, and the results are shown in FIG. 10, showing that both bind specifically to human PD-L1.

4.6 comparison of BT613 and Roche PD-L1 antibodies in cell function in vitro

4.6.1 functional cell line-based Activity assays: GS-C2/PD-L1 cell/GS-J2/PD-1 cell (purchased from Kinsley of Nanjing) is a functional cell line established by Kinsley. In the two cells, the binding of PD-1\ PD-L1 can inhibit the expression of luciferase in the cells, when the binding is blocked by adding PD-L1/PD-1 antibody, the expression of the luciferase is enhanced, and the concentration of the added antibody is positively correlated with the expression of the luciferase. Results see figure 11, BT613 antibody and atlizumab (purchased overseas according to the regular protocol) both blocked PD-1 binding to PD-L1 at the cellular level and enhanced luciferase expression, both with similar IC50 values.

4.6.2 Mixed Lymph Reaction (MLR) assay for cellular Activity of antibodies

Peripheral blood mononuclear cells were isolated from a healthy person, and differentiation and maturation of Dendritic Cell (DC) cells were promoted by DC differentiation medium, and then compared with CD4+ T cells purified from blood of another healthy person according to DC cell: CD4+ T cells ═ 1: 10 in proportion. Meanwhile, RPMI 1640 complete medium was used to dilute anti-PD-L1 antibody (8. mu.g/mL, 0.8. mu.g/mL, 0.08. mu.g/mL, 0.008. mu.g/mL, 0.0008. mu.g/mL) in gradient. The mixed cells were added to a 96-well cell culture plate with diluted antibody, and placed at 37 ℃ and 5% CO₂After 5 days in the cell culture chamber of (1), 100. mu.l of cell supernatant was collected and the concentration of IL2 was determined. Referring to fig. 12, BT613 had a functional activity superior to that of astuzumab in the Mixed Lymphoid Response (MLR) assay, in particular at 200 μ g/mL and 2 μ g/mL, BT613 stimulated CD4+ T cells to produce IL2 better.

Example 5 study of pharmacokinetics of BT613 and atlizumab in mice

4 mice C57BL/6 were selected for each antibody and administered intravenously at a dose of 10.0mg/kg, and the antibody concentrations were measured at 0h before administration, 0.5h after administration, 1h, 8h and 1d, 2d, 4d, 7d, 10d, 12d, 14d, 17d, 21d, 28d from the blood serum, and the results were more durable in the mouse serum after administration, see FIG. 13 and Table 3.

TABLE 3 pharmacokinetic study of BT 613/atlizumab (10.0mg/kg) in mice

Example 6 pharmacodynamic study of BT 613/Attributab in B-hPD-1 humanized mouse MC38-hPD-L1 Colon cancer subcutaneous graft tumor model

A colon cancer tumor model is established by using B-hPD-1 mice with human PD-1 purchased from Beijing Baioeiosu chart company and MC38-hPD-L1 colon cancer cells with human PD-L1 to carry out functional research on the PD-L1 antibody.

Performing subcutaneous modeling on MC38-hPD-L1 colon cancer, and performing subcutaneous modeling on MC38-hPD-L1 colon cancer cells 5 × 10⁵0.1mL of the cells were inoculated subcutaneously into the right anterior flank of a female B-hPD-1 humanized mouse until the tumor grew to about 150mm³The tumor volumes were randomly grouped, 6 per group, for a total of 3 groups, which were: solvent control group, PLH03-BT613-IgG1(3mg/kg, Q2Dx8) group, and astuzumab (3mg/kg, Q2Dx8) group. The administration route was intraperitoneal injection, and the experiment was ended on day 21 after divided administration. Tumor volume and body weight were measured 2 times per week and mouse body weight and tumor volume were recorded. The inhibition results of the antibody on the tumor volume increase of the mice are shown in fig. 14A and 14B, and BT613 has a better inhibition effect than atuzumab at a dose of 3 mg/kg. The results of the body weight of the mice are shown in fig. 16, and the results show that the body weight of the mice slightly increases after the administration of BT613, and the increase volume is approximately equivalent to the tumor volume by combining the analysis of the results of the inhibition of tumor growth, which indicates that BT613 has better safety.

At the end of the experiment on day 21 post-group, animals were euthanized, tumor removed weighed, and the relative tumor weight Inhibition Rate (IRTW) was calculated. From the view of tumor weight, BT613 has stronger tumor inhibition activity than atlizumab at the dose of 3mg/kg, and the specific results are shown in fig. 15.

CA782, CA783, CA785, CA786 and CA787 also gave results of the above experiments similar to BT 613.

Example 7 pharmacodynamic study of BT 613/Attributab in B-hPD-1 humanized mouse MC38-hPD-L1 subcutaneous graft tumor model

Performing subcutaneous modeling on MC38-hPD-L1 colon cancer, and performing subcutaneous modeling on MC38-hPD-L1 colon cancer cells 5 × 10⁵0.1mL of the cells were inoculated subcutaneously into the right anterior flank of a female B-hPD-1 humanized mouse until the tumor grew to about 111mm³The tumor volumes were randomly grouped, 6 per group, for a total of 3 groups, which were: solvent control group, PLH03-BT613-IgG1(3mg/kg, Q2Dx8) group, and astuzumab (3mg/kg, Q2Dx8) group. The administration route is intraperitoneal injection, 1 time is given every two days, 8 times are given in total, and the experiment is finished on the 17 th day after grouped administration. Tumor volume and body weight were measured 2 times per week and mouse body weight and tumor volume were recorded. The results of the inhibition of the tumor volume growth in mice by the antibody are shown in fig. 18A and 18B, and BT613 has a better inhibitory effect than atuzumab at a dose of 3 mg/kg. The results of the body weight of the mice are shown in fig. 20, and the results show that the body weight of the mice slightly increases after the administration of BT613, and the increase volume is approximately equivalent to the tumor volume by combining the analysis of the results of the inhibition of tumor growth, which indicates that BT613 has better safety.

At the end of the experiment on day 17 post-cohort, animals were euthanized, tumor removed weighed, and the relative tumor weight Inhibition Rate (IRTW) was calculated. From the tumor weight, BT613 had stronger tumor inhibitory activity than atlizumab at the dose of 3mg/kg, and the specific results are shown in fig. 19.

Example 8 BT613 sequence continues to optimize for germline genes

Non-germline amino acids at both ends of the antibody BT613 sequence were further optimized to be closer to germline (germline), thereby reducing the risk of immunogenicity. Table 4 below lists the specific amino acid mutation positions, the amino acid numbering is performed according to the Kabat rule, the affinity detection values of the different mutants of BT613 are shown in table 4, the specific binding kinetics curves are shown in fig. 17A to 17G, and the amino acid sequences of the different mutants of BT613 are shown in table 5.

TABLE 4 BT613 different mutant amino acid changes and corresponding affinities

TABLE 5 BT613 variant amino acid sequences

Claims

An anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the light chain region of the antibody comprises:

1) sequence KSSQX₁VLYSSNNKNYLX₂LCDR1 sequence shown by W (SEQ ID NO:14), wherein X₁Is S or N, and X₂Is A or P;

2) the sequence LCDR2 shown by sequence WASTRES (SEQ ID NO: 15); and

3) a LCDR3 sequence shown as sequence QQYYSTPLT (SEQ ID NO: 16); and is

Wherein the heavy chain region of the antibody comprises:

1) the sequence HCDR1 shown by the sequence GFTFSSY (SEQ ID NO: 17);

2) sequence SX₃The HCDR2 sequence shown by DGSNK (SEQ ID NO:18), wherein X₃Is F or Y; and

3) the sequence HCDR3 shown by sequence DRIYLDY (SEQ ID NO: 19).
The antibody or antigen-binding fragment thereof of claim 1, wherein the sequence of the light chain region of the antibody is SEQ ID No. 1 and the sequence of the heavy chain region of the antibody is SEQ ID No. 2; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 3 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 4; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 5 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 6; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 7 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 8; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO 9 and the sequence of the heavy chain region of the antibody is SEQ ID NO 10; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 11 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 12.
The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain region of the antibody comprises one or more mutations selected from the group consisting of: a mutation of the amino acid at position 1 from E to D, a mutation of the amino acid at position 2 from L to I, a mutation of the amino acid at position 4 from L to M and a mutation of the amino acid at position 105 from D to E, and

the heavy chain region sequence of the antibody comprises one or more mutations selected from the group consisting of: a mutation of amino acid 1 from E to Q, a mutation of amino acid 6 from Q to E, and a mutation of amino acid 16 from K to R.
The antibody or antigen-binding fragment thereof of claim 3, wherein the light chain region of the antibody has the sequence of SEQ ID NO 20 and the heavy chain region of the antibody has the sequence of SEQ ID NO 21; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 22 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 23; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 24 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 25; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO 26 and the sequence of the heavy chain region of the antibody is SEQ ID NO 27; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO 28 and the sequence of the heavy chain region of the antibody is SEQ ID NO 29; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO. 30 and the sequence of the heavy chain region of the antibody is SEQ ID NO. 31; or

Wherein the sequence of the light chain region of the antibody is SEQ ID NO:32 and the sequence of the heavy chain region of the antibody is SEQ ID NO: 33.
The antibody or antigen-binding fragment thereof of any one of claims 2-4, wherein the antigen-binding fragment is a Fab, Fab ', F (ab')2, scFv, or dsFv fragment.
Use of the antibody or antigen-binding fragment thereof of any one of claims 2-5 for the preparation of a medicament for the treatment of cancer, preferably, the cancer is selected from the group consisting of lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
Use of the antibody or antigen-binding fragment thereof of any one of claims 2-5 for the preparation of an agent for the diagnosis of cancer, preferably the cancer is selected from lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
Use of the antibody or antigen-binding fragment thereof of any one of claims 2-5 for the preparation of an agent for determining the prognosis or overall survival of a patient with a cancer, preferably, the cancer is selected from the group consisting of lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
Use of an antibody or antigen-binding fragment thereof according to any one of claims 2 to 5 for the manufacture of a medicament for the treatment of an inflammatory or autoimmune disease, preferably selected from the group consisting of a viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, crohn's disease, multiple sclerosis and inflammatory bowel disease.
Use of an antibody or antigen-binding fragment thereof according to any one of claims 2 to 5 for the preparation of an agent for the diagnosis of an inflammatory or autoimmune disease, preferably selected from the group consisting of a viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, crohn's disease, multiple sclerosis and inflammatory bowel disease.