WO2019101196A1 - 抗pd-l1的抗体及其用途 - Google Patents

抗pd-l1的抗体及其用途 Download PDF

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WO2019101196A1
WO2019101196A1 PCT/CN2018/117430 CN2018117430W WO2019101196A1 WO 2019101196 A1 WO2019101196 A1 WO 2019101196A1 CN 2018117430 W CN2018117430 W CN 2018117430W WO 2019101196 A1 WO2019101196 A1 WO 2019101196A1
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antibody
sequence
seq
chain region
cancer
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PCT/CN2018/117430
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French (fr)
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宋德勇
董创创
窦昌林
刘秀
成岩
韩镇
沙春洁
韩静
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山东博安生物技术有限公司
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Publication of WO2019101196A1 publication Critical patent/WO2019101196A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

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  • the present invention relates to an antibody against PD-L1 and uses thereof.
  • the present invention relates to an antibody against PD-L1 and antigen-binding fragments thereof and use thereof for diagnosing and treating inflammatory diseases, autoimmune diseases and cancers, and predicting prognosis.
  • T cells are critical for maintaining this balance, and their proper regulation is primarily through molecular coordination of the B7-CD28 family.
  • the interaction between members of the B7 family (which function as ligands) and members of the CD28 family (which function as receptors) not only provides a critical positive signal, can initiate, enhance and maintain T cell responses, but when appropriate
  • a key negative signal that promotes restriction, termination, and/or attenuation of T cell responses is also provided.
  • PD-1 also known as programmed cell death-1
  • B cells B cells
  • monocytes ligands in the B7 family
  • PD-L1 also known as B7H1 or programmed cell death-1 ligand 1
  • B7H1 or programmed cell death-1 ligand 1 interacts with receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses.
  • PD-L1 (B7-H1) is a 40 kDa cell surface glycoprotein belonging to the B7 family with IgV and IgC-like regions, transmembrane regions and cytoplasmic tails. This gene was first discovered and cloned in 1999 (Do ⁇ g H et al., Nat Med 5: 1365-1369, 1999), which interacts with receptor PD-1 on T cells in the negative regulation of immune response. Play an important role. In addition to expression on immune cells, PD-L1 is expressed in normal human tissues, but has high expression in some tumor cell lines, for example, lung cancer, ovarian cancer, colon cancer, and melanoma (Iwai). Et al., PNAS 99: 12293-7, 2002; and Ohigashi et al., Clin Cancer Res 11: 2947-53, 2005).
  • PD-L1 which is highly expressed in tumor cells, plays an important role in the immune escape of tumors by increasing the apoptosis of T cells.
  • the expression of PD-L1 is associated with poor prognosis and short overall survival, regardless of subsequent treatment.
  • mice that knocked out the PD-1 gene the PD-L1/PD-1 pathway was blocked, and tumors could not be formed after inoculation of tumor cells (Do ⁇ g H et al., Nat Med 8: 793-800, 2002).
  • the present invention relates to an antibody against PD-L1 or an antigen-binding fragment thereof, wherein the light chain region of the antibody comprises:
  • the heavy chain region of the antibody comprises:
  • the antibody is an antibody against PD-L1.
  • sequence of the light chain region of the antibody is SEQ ID NO: 1
  • sequence of the heavy chain region of the antibody is SEQ ID NO: 2.
  • 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.
  • sequence of the light chain region of the antibody is SEQ ID NO: 5
  • sequence of the heavy chain region of the antibody is SEQ ID NO: 6.
  • 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.
  • 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.
  • 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 sequence of the light chain region of the antibody comprises one or more mutations selected from the group consisting of a mutation of amino acid at position 1 from E to D, a mutation of amino acid at position 2 from L to I, Mutation of the amino acid at position 4 from L to M and mutation of 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 at position 1 from E to Q, a mutation of amino acid at position 6 from Q to E, and an amino acid at position 16 from K to R. mutation.
  • sequence of the light chain region of the antibody is SEQ ID NO: 20
  • sequence of the heavy chain region of the antibody is SEQ ID NO: 21.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 antigen binding fragment is a Fab, Fab', F(ab')2, scFv or dsFv fragment.
  • 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.
  • the cancer is selected from the group consisting of lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • 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 the group consisting of lung cancer, colon cancer, ovarian cancer, kidney cancer, colon Rectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • 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 antibody The binding condition to PD-L1, thereby determining the PD-L1 expression level of the subject sample, thereby diagnosing cancer in the subject.
  • the cancer is selected from the group consisting of lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention for the preparation of a medicament for diagnosing cancer, preferably selected from the group consisting of lung cancer, colon cancer, ovarian cancer, kidney cancer, colon Rectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • the invention relates to a method of using the antibody or antigen-binding fragment thereof of the invention for the preparation of a prognosis or overall survival of a cancer patient comprising administering an antibody or antigen-binding fragment thereof of the invention to a subject The sample is contacted and the binding of the antibody to PD-L1 is determined, thereby diagnosing the PD-L1 expression level of the subject sample to determine the prognosis or overall survival of the cancer patient.
  • the cancer is selected from the group consisting of lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • a drug for treatment for example, treatment of the anticancer therapy of the present 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 determining the prognosis or overall survival of a cancer patient, preferably selected from the group consisting of lung cancer, colon cancer, ovary Cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • the antibody or antigen-binding fragment thereof of the present invention can effectively bind to PD-L1 and block the PD-L1/PD-1 pathway, thereby effectively diagnosing and treating cancer, and the effect thereof is remarkably superior to the commonly used anti-PD known in the art.
  • -L1 specific antibody can effectively bind to PD-L1 and block the PD-L1/PD-1 pathway, thereby effectively diagnosing and treating cancer, and the effect thereof is remarkably superior to the commonly used anti-PD known in the art.
  • 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 an antibody or antigen-binding fragment thereof of the invention to a subject in need thereof By.
  • the inflammatory disease 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.
  • 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 disease or an autoimmune disease, preferably the inflammatory disease or an autoimmune disease It is selected from the group consisting of viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, Crohn's disease, multiple sclerosis, and inflammatory bowel disease.
  • 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 administering an antibody or antigen-binding fragment thereof of the invention to a sample from a subject Contacting and determining the binding of the antibody to PD-L1, thereby determining the PD-L1 expression level of the subject sample, thereby diagnosing an inflammatory disease or an autoimmune disease.
  • the inflammatory disease or autoimmune disease is selected from the group consisting of a viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, lupus erythematosus, Crohn's disease, multiple sclerosis, and inflammatory bowel disease.
  • the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention for the preparation of a medicament for diagnosing an inflammatory disease or an autoimmune disease, preferably an inflammatory disease or an autoimmune disease It is 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 antigen-binding fragment thereof of the present invention can effectively detect the presence of PD-L1 and block the PD-L1/PD-1 pathway, and the effect thereof is superior to the anti-PD-L1 antibody known in the prior art, for example, the city. Attuzumab sold.
  • Figure 1 shows the serum titers of antibodies against PD-L1 in 6 transgenic mice.
  • Figure 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 candidate antibodies and PD-L1 using an ELISA assay.
  • Figure 4A shows the results of detecting the binding kinetics of the candidate antibody PLH03-BT613-hIgG1 using BiAcore.
  • Figure 4B shows the results of detecting the binding kinetics of the candidate antibody PLH06-CA782-hIgG1 using BiAcore.
  • Figure 4C shows the results of detecting the binding kinetics of the candidate antibody PLH06-CA783-hIgG1 using BiAcore.
  • Figure 4D shows the results of detecting the binding kinetics of the candidate antibody PLH01-CA785-hIgG1 using BiAcore.
  • Figure 4E shows the results of detecting the binding kinetics of the candidate antibody PLH01-CA786-hIgG1 using BiAcore.
  • Figure 4F shows the results of detecting the binding kinetics of the candidate antibody PLH01-CA787-hIgG1 using BiAcore.
  • Figure 5 shows the results of different candidate antibodies blocking the binding between PD-L1 and PD-1.
  • Figure 6 shows the results of comparison of the binding affinities of antibody BT613 and antibody PL-GEN-IgG1 to PD-L1.
  • Figure 7 shows the results of comparison of the activity of antibody BT613 and antibody PL-GEN-IgG1 to block binding between PD-L1 and PD-1.
  • Figure 8 shows the results of comparison of the activity of the antibody BT613 and the antibody PL-GEN-IgG1 to block the binding between PD-L1 and B7-1.
  • Figure 9 shows the results of comparison of the binding affinities of antibody BT613 and antibody PL-GEN-IgG1 to mouse PD-L1.
  • Figure 10 shows the results of comparison of the binding affinities of the antibody BT613 and the antibody PL-GEN-IgG1 to the PD-L1 related protein.
  • Figure 11 shows that in GS-C2/PD-L1/GS-J2/PD-1 cell line, antibody BT613 and antibody PL-GEN-IgG1 (Atezolizumab) block the binding between PD-L1 and PD-1 Comparison of the activity of the results.
  • Figure 12 shows the effect of MLR in vitro detection antibody BT613 and antibody PL-GEN-IgG1 (Atezolizumab) on IL-2 production.
  • Figure 13 shows the pharmacokinetic profile of C57BL/6 mice after intravenous injection of 10.0 mg/kg antibody BT613 and antibody Atezolizumab.
  • Figure 14A shows the inhibition of tumor volume increase by 3.0 mg/kg antibody BT613 and altuzumab.
  • Figure 14B shows a photograph of a mouse tumor in which TS1503-A is antibody BT613 and TS1503-D is altuzumab.
  • Figure 15 shows tumor weight in the solvent, antibody BT613 and atetuzumab groups.
  • Figure 16 shows mouse body weight in the solvent, antibody BT613 and altuzumab groups.
  • Figure 17A shows the results of detecting the binding kinetics of BT613 mutant BT613 (HM ⁇ LM) using BiAcore.
  • Figure 17B shows the results of detecting the binding kinetics of BT613 mutant BT613 (H/LM) using BiAcore.
  • Figure 17C shows the results of detecting the binding kinetics of BT613 mutant BT613 (HM/L) using BiAcore.
  • Figure 17D shows the results of detecting the binding kinetics of BT613 mutant BT613 (HM/LM-1) using BiAcore.
  • Figure 17E shows the results of detecting the binding kinetics of BT613 mutant BT613 (HM/LM-2) using BiAcore.
  • Figure 17F shows the results of detecting the binding kinetics of BT613 mutant BT613 (HM/LM-3) using BiAcore.
  • Figure 17G shows the results of detecting the binding kinetics of BT613 mutant BT613 (HM/LM-4) using BiAcore.
  • Figure 18A shows the inhibition of tumor volume increase by 3.0 mg/kg antibody BT613 and altuzumab.
  • Figure 18B shows a photograph of a mouse tumor in which TS1503-1 is BT613 and TS1503-4 is altuzumab.
  • Figure 19 shows tumor weights in the solvent, antibody BT613 and atetuzumab groups.
  • Figure 20 shows mouse body weight in the solvent, antibody BT613 and altuzumab groups.
  • the PD-L1 protein PD-L1-His (Sinobiological, Cat. No. 10084-H08H) was used together with an adjuvant to immunize the full-body antibody transgenic mouse LuyeMab (the preparation method of transgenic mice is described in Chinese patent CN201210281415).
  • the adjuvant was complete Freund's adjuvant, and the adjuvants of Dim and Triad were incomplete freund's adjuvant (Sigma).
  • a total of 17 mice were co-immunized, and 6 mice with high antibody serum titer were boosted with adjuvant-free antigen after 3 immunizations (see Figure 1). After 4 days, the mice were sacrificed and the spleen was removed. Store frozen for later use.
  • RNA extraction was performed from the frozen mouse spleens by Trizol (Thermo Scientific, catalog No. 15596-026) according to a conventional protocol using the Roche Reverse Science Kit Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, catalog No. 4897030001) RNA was reverse transcribed into cDNA according to its instructions.
  • the procedure for establishing the phage library was carried out according to the method described in "Phage display" edited by Tim Clackson. First, the variable regions of the heavy and light chains were obtained from the cDNA by PCR, and the variable regions of the heavy and light chains were added.
  • ScFv was obtained by overlap extension PCR, SfiI was digested with SfiI at 50 ° C (for 5 h), the cleaved ScFv (single-chain Fv) was ligated to plasmid pCOMB3x, and the ligation product was electrotransfected to E. coli XL1- Blue competent cells.
  • the phage library PD-L1-H3-1 was established with the mouse numbered LYQ013, and the storage capacity was 4.2*10 8 ; the phage library PD-L1-H6-1 was established with the mouse numbered LYQ024, and the storage capacity was 4.0*10 8 .
  • Plate screening Plates were plated with 0.2 ⁇ g/well of PD-L1-His protein (high-adsorption microplate, Costar), placed at 4 ° C overnight, and the plate was blocked with 3% skim milk powder for 1 h the next day, and the phage library PD was added.
  • -L1-H3-1/PD-L1-H6-1 colony formation unit: 2*10 12
  • PBST PBST
  • the PD-L1-specifically bound phage was eluted with an elution buffer (pH 2.2, 0.1 M HCl-Gly).
  • the PD-L1-Fc protein was biotinylated according to Thermo's biotin labeling procedure (molar ratio of PD-L1 protein to biotin 1:2), and then magnetic beads of Thermo (Invitrogen Dynabeads) M-280 Streptavidin, 00355871), and then incubated with the phage library PD-L1-H3-1/PD-L1-H6-1 (colony forming unit: 2*10 12 ) to obtain PD-L1 specific binding clones .
  • the cloned PLH6-CA782 ⁇ CA783 ⁇ CA785 ⁇ CA786 ⁇ CA787 was obtained by plate screening, and the cloned PLH3-BT613 was obtained by magnetic bead screening.
  • the PLH3 represents the library PD-L1-H3-1 established by mouse LYO013, and the PLH6 represents the library established by mouse LYO024. PD-L1-H6-1.
  • a library positive for ELISA by phage enzyme-linked immunosorbent assay (ELISA) was coated with 2YT (Amp 100 ⁇ g/ml) plates, and the monoclonals were picked directly and induced to express using commercially available ZYM medium, and the purified products after expression were used for The blocking activity was examined and the results are shown in Figure 2.
  • the clones PLH3-BT613, PLH6-CA782 ⁇ CA783 ⁇ CA785 ⁇ CA786 ⁇ CA787 were sent to Invitrogen Biotechnology Co., Ltd. for sequencing.
  • the amino acid sequence of each clone is described in Table 1 below.
  • the antibody gene was digested with 5' Xba I (NEB catalog #R0145S) and 3' EcoRV (NEB catalog #R3195S), and purified.
  • the antibody gene was ligated into the vector pCDNA3.4 (Life Technology) by T4 DNA ligase (NEB catalog #M0202S), and then transfected into HEK293 cells, DMEM medium at 37 ° C ⁇ 8% CO 2 ⁇ 125 rpm shaker After 6 days, the transient expression supernatant was purified by Protein A (GE Healthcare) according to a conventional affinity chromatography procedure to obtain an antibody against PD-L1, and the antibody concentration was determined by UV280 binding extinction coefficient.
  • control antibody The amino acid sequence of Roche PD-L1 antibody atetuzumab was determined by IMGT data and patent US 8217149B2. After whole gene synthesis and sequencing, the vector pCDNA3.4 was inserted and expressed in HEK293 cells, and the antibody produced was named PL- GEN-IgG1.
  • 96-well ELISA plates (BEVER, 40301) were used at different concentrations (0.4 ⁇ g/ml, 0.2 ⁇ g/ml, 0.1 ⁇ g/ml, 0.05 ⁇ g/ml, 0.025 ⁇ g/ml, 0.0125 ⁇ g/ml, 0.00625 ⁇ g/ml, 0 ⁇ g).
  • the antigen PD-L1-His was coated at 100 ⁇ l/well, incubated overnight at 4 ° C; blocked with 3% skim milk powder at 37 ° C for 1 h; each well was added with 2 ⁇ g / ml candidate antibody 100 ⁇ l, and incubated at 37 ° C for 1 h; Then, goat anti-human IgG/HRP (Zhongshan Jinqiao) was added, and the cells were incubated at 37 ° C for 1 h, and after color development for 10 min, OD450 was read on the microplate reader. The results are shown in Figure 3. The 6 strains of antibodies have similar binding sensitivities to the PD-L1 protein.
  • Antibody binding kinetics were measured using a BIAcore X100 instrument based on surface plasmon resonance (SRP) technology.
  • the anti-human IgG antibody amino group was coupled to a CM5 biosensor chip (GE, BR-1000-12) by a GE anti Human IgG FC amino coupling kit (GE, cat#BR-1008-39) to obtain approximately 1000 Response units (RU).
  • GE CM5 biosensor chip
  • GE GE anti Human IgG FC amino coupling kit
  • RU Response units
  • antibodies were serially diluted 2-fold with HBS-EP + 1 x (GE, cat #BR-1006-69) buffer, starting at 50 mM, diluting 4 concentration gradients 2 fold, and setting a concentration of 0.
  • the assay conditions were as follows: antibody: 1 ⁇ g/ml, injection time 60 s, flow rate 5 ⁇ l/min, stable for 5 s; PD-L1 protein (50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, 0 mM): combined with 60 s, flow rate 30 ⁇ l / Min, dissociation 300s; regeneration: regeneration with 3M MgCl 2 buffer for 30s, start 2 times. Binding constants (ka) and dissociation constants (kd) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2), and the equilibrium dissociation constant (kD) was calculated as the ratio kd/ka.
  • the ELISA plate was coated with PD-1-Fc at a concentration of 0.25 ⁇ g/ml at 100 ⁇ l/well, incubated overnight at 4 ° C; blocked with 3% skim milk powder for 1 h; and PD-L1-Fc-biotin (0.5) Gg/ml) (preparation step is the same as 1.3) and different concentrations (4 ⁇ g/mL, 2 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL, 0.25 ⁇ g/mL, 0.125 ⁇ g/mL, 0.0625 ⁇ g/mL, 0 ⁇ g/mL)
  • Candidate antibodies were incubated for 1 h at 37 ° C, then added to the blocked ELISA plate, incubated for another 1 h at 37 ° C; then added streptomycin / HRP, incubated at 37 ° C for 1 h; after 10 min of color development, in the microplate reader Read the OD450 on it. The results are shown in Figure 5.
  • the 96-well microtiter plate was coated with B7-1-Fc (purchased from Yishen Shenzhou) (concentration: 0.5 ⁇ g/ml) at 100 ⁇ l/well, coated at 4 degrees overnight, and blocked with 3% skim milk powder the next day.
  • B7-1-Fc purchased from Yishen Shenzhou
  • Binding of BT613 to PL-GEN-IgG1 and PD-L1 related proteins The results of ELISA assay showed that the binding of BT613 and PL-GEN-IgG1 to human PD-L1 ⁇ PDL2 ⁇ PDL3 protein was different. , showing that both are specifically binding to human PD-L1.
  • GS-C2/PD-L1 cells/GS-J2/PD-1 cells are functional cell lines established by Kingsray. In both cells, binding of PD-1 ⁇ PD-L1 inhibits the expression of luciferase in cells, and when PD-L1/PD-1 antibody is added to block this binding, luciferase expression is enhanced. The concentration of antibody added was positively correlated with the expression of luciferase. The results are shown in Figure 11. Both BT613 antibody and altuzumab (purchased from foreign countries according to the regular procedure) can block the binding of PD-1 to PD-L1 at the cellular level and enhance the expression of luciferase. Similar IC50 values.
  • Peripheral blood mononuclear cells are isolated from healthy humans, promote differentiation and maturation of DC cells by dendritic cell (DC) differentiation culture, and then CD4+ T cells purified from blood of another healthy person according to DC cells: CD4 +T cells were mixed at a ratio of 1:10.
  • RPMI 1640 complete medium was diluted with anti-PD-L1 antibody (8 ⁇ g/mL, 0.8 ⁇ g/mL, 0.08 ⁇ g/mL, 0.008 ⁇ g/mL, 0.0008 ⁇ g/mL).
  • BT613 has a functional activity superior to that of altuzumab, especially at 200 ⁇ g/mL and 2 ⁇ g/mL, BT613 better stimulates CD4+ T cell production.
  • MLR mixed lymphocyte reaction
  • mice Four C57BL/6 mice were administered intravenously to each antibody at a dose of 10.0 mg/kg, 0 h before administration, 0.5 h, 1 h, 8 h and 1 d, 2 d, 4 d, 7 d, 10 d, 12 d, 14 d. Serum detection antibody concentration was taken at 17d, 21d, and 28d. The specific results are shown in Figure 13 and Table 3. After administration, BT613 is more persistent in mouse serum.
  • MC38-hPD-L1 colon cancer was subcutaneously modeled, and MC38-hPD-L1 colon cancer cells 5 ⁇ 10 5 /0.1 mL were inoculated subcutaneously into the right anterior flank of female B-hPD-1 humanized mice.
  • the tumor volume was randomly grouped into 6 groups of 3 groups, respectively: solvent control group, PLH03-BT613-IgG1 (3 mg/kg, Q2Dx8) group, and Atelizumab. (3mg/kg, Q2Dx8) group.
  • the route of administration was intraperitoneal injection, and the experiment was terminated on the 21st day after the group administration.
  • BT613 has a better inhibitory effect than atetuzumab at a dose of 3 mg/kg.
  • the results of mouse body weight are shown in Fig. 16. The results showed that the mice gained a slight increase in body weight after administration of BT613. Combined with the results of inhibition of tumor growth, the growth volume was roughly equivalent to the tumor volume, indicating that BT613 was safer.
  • BT613 has stronger tumor suppressing activity than altuzumab at a dose of 3 mg/kg, and the specific results are shown in Fig. 15.
  • CA782, CA783, CA785, CA786, and CA787 also obtained the results of the above experiments similar to BT613.
  • MC38-hPD-L1 colon cancer was subcutaneously modeled, and MC38-hPD-L1 colon cancer cells 5 ⁇ 10 5 /0.1 mL were inoculated subcutaneously into the right anterior flank of female B-hPD-1 humanized mice.
  • the tumors grew to about 111 mm 3
  • the tumor volume was randomly grouped into 6 groups of 3 groups, respectively: solvent control group, PLH03-BT613-IgG1 (3 mg/kg, Q2Dx8) group, and altuzumab (3mg/kg, Q2Dx8) group.
  • the route of administration was intraperitoneal injection, once every two days for a total of 8 administrations, and the experiment was terminated on the 17th day after the group administration.
  • BT613 has a better inhibitory effect than atetuzumab at a dose of 3 mg/kg.
  • the results of mouse body weight are shown in Fig. 20. The results showed that the weight of mice increased slightly after administration of BT613. Combined with the results of tumor growth inhibition, the growth volume was roughly equivalent to the tumor volume, indicating that BT613 was safe.
  • BT613 has stronger tumor suppressing activity than altuzumab at a dose of 3 mg/kg, and the specific results are shown in Fig. 19.
  • CA782, CA783, CA785, CA786, and CA787 also obtained the results of the above experiments similar to BT613.
  • Example 8 BT613 sequence continues to optimize germline genes
  • the non-germline amino acids at both ends of the antibody BT613 sequence are continually optimized to be closer to the germline, thereby reducing the risk of immunogenicity.
  • Table 4 below lists the specific amino acid mutation positions. The amino acid numbers are numbered according to the Kabat rule.
  • the affinity values of different mutants of BT613 are shown in Table 4.
  • the specific binding kinetic curves are shown in Figures 17A to 17G.
  • the amino acid sequences of different mutants of BT613 See Table 5.

Abstract

涉及一种抗PD-L1的抗体及其用途。具体而言，涉及一种抗PD-L1的抗体及其抗原结合片段以及其用于诊断和治疗癌症及预测预后的用途。

Description

抗PD-L1的抗体及其用途 技术领域
本发明涉及一种抗PD-L1的抗体及其用途。具体而言，本发明涉及一种抗PD-L1的抗体及其抗原结合片段以及其用于诊断和治疗炎性疾病、自身免疫性疾病和癌症及预测预后的用途。
背景技术
免疫系统必须在有效应答以清除致病体与保持耐受以防止自身免疫疾病之间实现平衡。T细胞对于保持这种平衡是关键的，它们的适当的调节主要是通过B7-CD28家族的分子协调进行。B7家族成员(其行使配体的功能)和CD28家族成员(其行使受体的功能)之间的相互作用不仅提供了关键的正信号，可发起、提高和维持T细胞应答，而且在适当时还提供促进限制、终止和/或消弱T细胞应答的关键负信号。CD28家族的一个成员，称为PD-1(也称为程序化细胞死亡-1)在活化的T细胞、B细胞和单核细胞上被增量调节；B7家族中的配体，PD-L1(也称为B7H1或程序化细胞死亡-1配体1)与T细胞上的受体PD-1相互作用，在免疫应答的负性调控方面发挥着重要作用。
PD-L1(B7-H1)是40kDa的细胞表面糖蛋白，属于B7家族，具有IgV和IgC样区、跨膜区及胞浆区尾部。该基因于1999年首次被发现并克隆(Doμg H et al.,Nat Med 5:1365-1369,1999)，它与T细胞上的受体PD-1相互作用，在免疫应答的负性调控方面发挥着重要作用。PD-L1除了在免疫细胞上表达外，在人类正常的组织中表达量较低，但是在一些肿瘤细胞系上却有着较高的表达，例如，肺癌、卵巢癌、结肠癌和黑色素瘤(Iwai et al.,PNAS 99:12293-7,2002；和Ohigashi et al.,Clin Cancer Res 11:2947-53,2005)。
已有的结果显示，肿瘤细胞中高表达的PD-L1是通过增加T细胞的凋亡从而在肿瘤的免疫逃逸中起着重要的作用。例如，在卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌和黑色素瘤的大量样品中，在不考虑后续治疗的情况下，PD-L1的表达与不良预后和总存活期短有关。敲除 PD-1基因的小鼠，因PD-L1/PD-1通路被阻断，则接种肿瘤细胞后不能形成肿瘤(Doμg H et al.,Nat Med 8:793-800,2002)。
考虑到PD-L1在癌症发展和免疫系统调节中的重要作用，持续需要检测PD-L1的存在情况以及阻断PD-L1/PD-1通路的有效抗体。
发明内容
本发明涉及一种抗PD-L1的抗体或其抗原结合片段，其中所述抗体的轻链区包含：
1)序列KSSQX 1VLYSSNNKNYLX 2W(SEQ ID NO:14)所示的LCDR1序列，其中X 1为S或N，并且X 2为A或P；
2)序列WASTRES(SEQ ID NO:15)所示的LCDR2序列；和
3)序列QQYYSTPLT(SEQ ID NO:16)所示的LCDR3序列；并且
其中所述抗体的重链区包含：
1)序列GFTFSSY(SEQ ID NO:17)所示的HCDR1序列；
2)序列SX 3DGSNK(SEQ ID NO:18)所示的HCDR2序列，其中X 3为F或Y；和
3)序列DRIYLDY(SEQ ID NO:19)所示的HCDR3序列。
在本发明的一个方面中，所述抗体为抗PD-L1的抗体。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:1，并且所述抗体的重链区的序列为SEQ ID NO:2。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:3，并且所述抗体的重链区的序列为SEQ ID NO:4。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:5，并且所述抗体的重链区的序列为SEQ ID NO:6。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:7，并且所述抗体的重链区的序列为SEQ ID NO:8。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:9，并且所述抗体的重链区的序列为SEQ ID NO:10。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:11，并且所述抗体的重链区的序列为SEQ ID NO:12。
在本发明的一个方面中，所述抗体的轻链区的序列包含一个或多个选自以下的突变：第1位氨基酸从E至D的突变、第2位氨基酸从L至I的突变、第4位氨基酸从L至M的突变和第105位氨基酸从D至E的突变，并且
所述抗体的重链区序列包含一个或多个选自以下的突变：第1位氨基酸从E至Q的突变、第6位氨基酸从Q至E的突变和第16位氨基酸从K至R的突变。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:20，并且所述抗体的重链区的序列为SEQ ID NO:21。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:22，并且所述抗体的重链区的序列为SEQ ID NO:23。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:24，并且所述抗体的重链区的序列为SEQ ID NO:25。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:26，并且所述抗体的重链区的序列为SEQ ID NO:27。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:28，并且所述抗体的重链区的序列为SEQ ID NO:29。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:30，并且所述抗体的重链区的序列为SEQ ID NO:31。
在本发明的一个方面中，所述抗体的轻链区的序列为SEQ ID NO:32，并且所述抗体的重链区的序列为SEQ ID NO:33。
在本发明的一个方面中，所述抗原结合片段为Fab、Fab'、F(ab')2、scFv或dsFv片段。
在本发明的一个方面中，本发明涉及使用本发明的抗体或其抗原结合片段治疗癌症的方法，包括将本发明的抗体或其抗原结合片段给予有需要的受试者。优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
在本发明的一个方面中，本发明涉及本发明的抗体或其抗原结合片段用于制备治疗癌症的药剂的用途，优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、 骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
在本发明的一个方面中，本发明涉及使用本发明的抗体或其抗原结合片段诊断癌症的方法，包括将本发明的抗体或其抗原结合片段与来自受试者样品接触，并且确定所述抗体与PD-L1的结合情况，由此确定受试者样品的PD-L1表达水平，从而诊断受试者中的癌症。优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
在本发明的一个方面中，本发明涉及本发明的抗体或其抗原结合片段用于制备诊断癌症的试剂的用途，优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
在本发明的一个方面中，本发明涉及使用本发明的抗体或其抗原结合片段用于制备确定癌症患者预后或总存活期的方法，包括将本发明的抗体或其抗原结合片段与来自受试者样品接触，并且确定所述抗体与PD-L1的结合情况，由此诊断受试者样品的PD-L1表达水平，从而确定癌症患者预后或总存活期。优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。结果表明，相对于不具有PD-L1表达的存在的患者，在使用药物进行治疗(例如本发明的抗癌疗法的治疗)时，具有PD-L1表达的存在的患者具有更好的预后或更长的总存活期。
在本发明的一个方面中，本发明涉及本发明的抗体或其抗原结合片段用于制备确定癌症患者预后或总存活期的试剂的用途，优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
本发明的抗体或其抗原结合片段可以有效地结合PD-L1并阻断PD-L1/PD-1通路，从而有效地诊断和治疗癌症，其效果明显优于本领域中已知的常用抗PD-L1的特异性抗体。
在本发明的一个方面中，本发明涉及使用本发明的抗体或其抗原结合片段治疗炎性疾病或自身免疫性疾病的方法，包括将本发明的抗体或其抗原结合片段给予有需要的受试者。优选地，所述炎性疾病或自身免疫性疾病选自病毒感染、类风湿性关节炎、骨关节炎、银屑病、红斑狼 疮、克罗恩病、多发性硬化症和炎症性肠病。
在本发明的一个方面中，本发明涉及本发明的抗体或其抗原结合片段用于制备治疗炎性疾病或自身免疫性疾病的药剂的用途，优选地，所述炎性疾病或自身免疫性疾病选自病毒感染、类风湿性关节炎、骨关节炎、银屑病、红斑狼疮、克罗恩病、多发性硬化症和炎症性肠病。
在本发明的一个方面中，本发明涉及使用本发明的抗体或其抗原结合片段诊断炎性疾病或自身免疫性疾病的方法，包括将本发明的抗体或其抗原结合片段与来自受试者样品接触，并且确定所述抗体与PD-L1的结合情况，由此确定受试者样品的PD-L1表达水平，从而诊断炎性疾病或自身免疫性疾病。优选地，所述炎性疾病或自身免疫性疾病选自病毒感染、类风湿性关节炎、骨关节炎、银屑病、红斑狼疮、克罗恩病、多发性硬化症和炎症性肠病。
在本发明的一个方面中，本发明涉及本发明的抗体或其抗原结合片段用于制备诊断炎性疾病或自身免疫性疾病的试剂的用途，优选地，所述炎性疾病或自身免疫性疾病选自病毒感染、类风湿性关节炎、骨关节炎、银屑病、红斑狼疮、克罗恩病、多发性硬化症和炎症性肠病。
本发明的抗体或其抗原结合片段可以有效地检测PD-L1的存在情况以及阻断PD-L1/PD-1通路，其效果优于现有技术中已知的抗PD-L1抗体，例如市售的阿特珠单抗。
附图说明
图1示出了6只转基因小鼠中抗PD-L1的抗体的血清滴度。
图2示出了单克隆原核表达ScFv阻断PD-1与PD-L1的结合的活性。
图3示出了利用ELISA测定检测候选抗体与PD-L1之间的结合的灵敏度。
图4A示出了利用BiAcore检测候选抗体PLH03-BT613-hIgG1的结合动力学的结果。
图4B示出了利用BiAcore检测候选抗体PLH06-CA782-hIgG1的结合动力学的结果。
图4C示出了利用BiAcore检测候选抗体PLH06-CA783-hIgG1的 结合动力学的结果。
图4D示出了利用BiAcore检测候选抗体PLH01-CA785-hIgG1的结合动力学的结果。
图4E示出了利用BiAcore检测候选抗体PLH01-CA786-hIgG1的结合动力学的结果。
图4F示出了利用BiAcore检测候选抗体PLH01-CA787-hIgG1的结合动力学的结果。
图5示出了不同候选抗体阻断PD-L1与PD-1之间的结合的结果。
图6示出了抗体BT613和抗体PL-GEN-IgG1对PD-L1的结合亲和力的比较结果。
图7示出了抗体BT613和抗体PL-GEN-IgG1阻断PD-L1与PD-1之间的结合的活性的比较结果。
图8示出了抗体BT613和抗体PL-GEN-IgG1阻断PD-L1与B7-1之间的结合的活性的比较结果。
图9示出了抗体BT613和抗体PL-GEN-IgG1对小鼠PD-L1的结合亲和力的比较结果。
图10示出了抗体BT613和抗体PL-GEN-IgG1对PD-L1相关蛋白的结合亲和力的比较结果。
图11示出了在GS-C2/PD-L1/GS-J2/PD-1细胞系中，抗体BT613和抗体PL-GEN-IgG1(Atezolizumab)阻断PD-L1与PD-1之间的结合的活性的比较结果。
图12示出了MLR体外检测抗体BT613和抗体PL-GEN-IgG1(Atezolizumab)对IL-2产生的作用。
图13示出了C57BL/6小鼠静脉注射10.0mg/kg抗体BT613和抗体阿特珠单抗(Atezolizumab)后的药时曲线。
图14A示出了3.0mg/kg抗体BT613和阿特珠单抗对肿瘤体积增长的抑制作用。
图14B示出了小鼠肿瘤的照片，其中TS1503-A为抗体BT613，且TS1503-D为阿特珠单抗。
图15示出了溶剂、抗体BT613和阿特珠单抗组中的肿瘤重量。
图16示出了溶剂、抗体BT613和阿特珠单抗组中的小鼠体重。
图17A示出了利用BiAcore检测BT613突变体BT613(HM\LM)的结合动力学的结果。
图17B示出了利用BiAcore检测BT613突变体BT613(H/LM)的结合动力学的结果。
图17C示出了利用BiAcore检测BT613突变体BT613(HM/L)的结合动力学的结果。
图17D示出了利用BiAcore检测BT613突变体BT613(HM/LM-1)的结合动力学的结果。
图17E示出了利用BiAcore检测BT613突变体BT613(HM/LM-2)的结合动力学的结果。
图17F示出了利用BiAcore检测BT613突变体BT613(HM/LM-3)的结合动力学的结果。
图17G示出了利用BiAcore检测BT613突变体BT613(HM/LM-4)的结合动力学的结果。
图18A示出了3.0mg/kg抗体BT613和阿特珠单抗对肿瘤体积增长的抑制作用。
图18B示出了小鼠肿瘤的照片，其中TS1503-1为BT613，且TS1503-4为阿特珠单抗。
图19示出了溶剂、抗体BT613和阿特珠单抗组中的肿瘤重量。
图20示出了溶剂、抗体BT613和阿特珠单抗组中的小鼠体重。
具体实施方式
参照以下实施例可以更好地理解本发明。但是，应理解，以下实施例仅用于举例说明目的，而不应被理解为以任何方式限制本发明的保护范围。
实施例1.抗PD-L1的单链抗体的产生
1.1小鼠免疫
用PD-L1蛋白PD-L1-His(Sinobiological,目录号10084-H08H)与佐剂一起免疫绿叶制药的全人抗体转基因小鼠LuyeMab(转基因小鼠制备方法记载于中国专利CN201210281415中)，首免的佐剂为完全弗氏 佐剂，二免和三免的佐剂为非完全弗氏佐剂(incomplete freund’s adjuvant,Sigma)。共免疫17只小鼠，3次免疫后选取抗体血清滴度较高的6只小鼠用不含佐剂的抗原进行加强免疫(参见图1)，4天后杀死小鼠并取出脾脏，并冷冻保存备用。
1.2噬菌体库的建立
通过Trizol(Thermo Scientific，目录货号15596-026)从冻存的小鼠脾脏中按照常规的方案进行RNA提取，使用罗氏反转录试剂盒Transcriptor First Strand cDNA Synthesis Kit(Roche Applied Science，目录货号4897030001)按照其说明书将RNA反转录成cDNA。噬菌体库的建立步骤参照Tim Clackson编辑的《Phage display》中记载的方法进行，首先用PCR的方法从cDNA中获得重链和轻链的可变区，再将重链和轻链的可变区通过重叠延伸PCR的方法获得ScFv，对ScFv进行SfiI酶切50℃(持续5h)，将酶切后的ScFv(单链Fv)与质粒pCOMB3x连接，然后将连接产物电转染至大肠杆菌XL1-Blue感受态细胞中。以编号为LYQ013的小鼠建立的噬菌体库PD-L1-H3-1，库容4.2*10 8；以编号为LYQ024的小鼠建立的噬菌体库PD-L1-H6-1，库容4.0*10 8。
1.3克隆筛选
平板筛选：用PD-L1-His蛋白以0.2μg/孔包被平板(高吸附微孔板，Costar)，4℃下放置过夜，第二天通过3％脱脂奶粉封闭平板1h，加入噬菌体库PD-L1-H3-1/PD-L1-H6-1(集落形成单位:2*10 12)并孵育2h，用PBST(0.01M，pH 7.4，0.05％tween-20)洗涤4-10次后用洗脱缓冲液(pH 2.2，0.1M HCl-Gly)洗脱PD-L1特异性结合的噬菌体。
磁珠筛选：将PD-L1-Fc蛋白按照Thermo的生物素标记说明书步骤进行生物素化(投入的PD-L1蛋白与生物素的摩尔比1:2)，再与Thermo的磁珠(Invitrogen Dynabeads M-280 Streptavidin，00355871)结合，然后与噬菌体库PD-L1-H3-1/PD-L1-H6-1(集落形成单位:2*10 12)孵育，从而获得PD-L1特异性结合的克隆。
平板筛选获得克隆PLH6-CA782\CA783\CA785\CA786\CA787，磁珠筛选获得克隆PLH3-BT613，其中PLH3代表小鼠LYO013建立的库PD-L1-H3-1，PLH6代表小鼠LYO024建立的库PD-L1-H6-1。
1.4单克隆原核表达ScFv阻断PD-1与PD-L1的结合
将通过噬菌体酶联免疫(ELISA)法检测为阳性的库涂布2YT(Amp 100μg/ml)平板，挑取单克隆直接并用商业可获得的ZYM培养基进行诱导表达，表达后的纯化产物用于检测阻断活性，结果如图2中所示。
实施例2.抗PD-L1的抗体的分子构建与生产
将克隆PLH3-BT613、PLH6-CA782\CA783\CA785\CA786\CA787送至Invitrogen生物技术有限公司测序。每个克隆的氨基酸序列记载于下表1中。
表1.具有阻断活性的克隆的氨基酸序列
Figure PCTCN2018117430-appb-000001
Figure PCTCN2018117430-appb-000002
将抗体基因与Fc序列(N297A)SEQ NO ID:13融合后，抗体基因通过5’的Xba I(NEB目录货号#R0145S)与3’的EcoRV(NEB目录货号#R3195S)酶切，纯化后的抗体基因通过T4 DNA连接酶(NEB目录货号#M0202S)连接入载体pCDNA3.4(Life Technology)，然后转染进入HEK293细胞中，在37℃\8％CO 2\125rpm的摇床中DMEM培养基培养，6天后，取瞬时表达上清通过Protein A(GE Healthcare)按照常规亲和层析流程纯化，获得抗PD-L1的抗体，并通过UV280结合消光系数确定抗体浓度。
SEQ ID NO:13
Figure PCTCN2018117430-appb-000003
对照抗体的生产：通过IMGT数据及专利US 8217149B2确定罗氏 PD-L1抗体阿特珠单抗的氨基酸序列，全基因合成并测序后插入载体pCDNA3.4通过HEK293细胞表达，生产的抗体命名为PL-GEN-IgG1。
实施例3.候选抗体的表征
3.1 ELISA检测候选抗体与PD-L1蛋白的结合
将96孔ELISA板(BEVER，40301)用不同浓度(0.4μg/ml、0.2μg/ml、0.1μg/ml、0.05μg/ml、0.025μg/ml、0.0125μg/ml、0.00625μg/ml、0μg/ml)的抗原PD-L1-His以100μl/孔包被，4℃下孵育过夜；用3％脱脂奶粉37℃封闭1h；每孔加入2μg/ml候选抗体各100μl，37℃下孵育1h；然后加入山羊抗人IgG/HRP(中杉金桥)，37℃孵育1h，显色10min后，酶标仪上读取OD450。结果见图3,6株抗体具有相似的对PD-L1蛋白的结合灵敏度。
3.2 SPR检测候选抗体与PD-L1蛋白的结合
抗体结合动力学使用基于表面等离子共振(surface plasmon resonance，SRP)技术的BIAcoreX100仪器测量。通过GE anti Human IgG FC氨基偶联试剂盒(GE，cat#BR-1008-39)，将抗人IgG抗体氨基偶联到CM5生物传感器芯片(GE，BR-1000-12)上以获得大约1000应答单位(response units，RU)。对于动力学测量，将抗体用HBS-EP+1×(GE，cat#BR-1006-69)缓冲液2倍连续稀释，从50mM起始，2倍稀释4个浓度梯度，并设置0浓度。测定条件如下：抗体：1μg/ml，进样时间60s，流速5μl/min，稳定5s；PD-L1蛋白(50mM，25mM，12.5mM，6.25mM，3.125mM，0mM)：结合60s，流速30μl/min，解离300s；再生：用3M MgCl 2缓冲液再生30s，启动2次。使用简单一对一Languir结合模型(BIAcore Evaluation Software version 3.2)计算结合常数(ka)和解离常数(kd)，平衡解离常数(kD)以比率kd/ka计算。
表2 BiAcore检测候选抗体结合动力学
抗体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候选抗体阻断PD-L1蛋白与PD-1蛋白的结合
将ELISA板用浓度为0.25μg/ml的PD-1-Fc以100μl/孔包被，在4℃下孵育过夜；用3％脱脂奶粉封闭1h；同时将PD-L1-Fc-生物素(0.5μg/ml)(制备步骤同1.3)和不同浓度(4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL、0.0625μg/mL、0μg/mL)候选抗体在37℃共孵育1h，然后加入到封闭过的ELISA板上，在37℃再孵育1h；继而加入链霉素/HRP，在37℃下孵育1h；显色10min后，在酶标仪上读取OD450。结果见图5，6株抗体都能够有效阻断PD-1与PD-L1的结合，且具有相似的阻断活性。
实施例4 BT613与罗氏PD-L1抗体的比较
4.1 PL-BT613-IgG1(以下或简称为BT613)与罗氏PD-L1抗体的比较实验。首先在高表达PD-L1的细胞系上进行结合亲和力比较，将GS-C2/PD-L1细胞(购自南京金斯瑞)用FACS缓冲液(PBS，含0.2％BSA)调节细胞数至5E5个，100μl/w，加入100μg BT613(样品A)或PL-GEN-IgG1(样品D)抗体，混匀后在4℃下静置1h。用FACS缓冲液洗涤细胞2次后，用100μl FACS缓冲液重悬细胞，加入1μg羊抗人IgG荧光二抗(Life，A21445)混匀后在4℃下静置1h。FACS缓冲液洗涤细胞2次，用100μl FACS缓冲液重悬后进行流式检测(BD Accuri TMC6)。BT613抗体显示在GS-C2/PD-L1细胞上与罗氏PD-L1抗体有相似的结合亲和力(参见图6)。
4.2 BT613与PL-GEN-IgG1在阻断PD-1与PD-L1结合的活性上的比较，ELISA的操作步骤与3.3中相同，结果见图7，可见BT613相对于罗氏的PL-GEN-IgG1在阻断PD-L1与PD-1的结合上具有更好的活性。
4.3 BT613与PL-GEN-IgG1在阻断B7-1与PD-L1的结合的活性上 的比较
将96孔酶标板用B7-1-Fc(购自义翘神州)(浓度为0.5μg/ml)以100μl/孔包被，4度下包被过夜，第二天用3％脱脂奶粉封闭1h；同时将PD-L1-Fc-生物(0.8μg/ml)与梯度稀释的PLH03-BT613-hIgG1或PL-GEN-IgG1(8μg/ml、4μg/ml、2μg/ml、1μg/ml、0.5μg/ml、0.25μg/ml、0.125μg/ml、0μg/ml)在37℃下共孵育1h，然后加入到已经包被B7-1的酶标板上，在37℃下再孵育1h；再加入链霉素/HRP，37℃下孵育1h，显色10min后，酶标仪上读取OD450。结果见图8，BT613和罗氏的PL-GEN-IgG1在阻断PD-L1与B7-1的结合上具有相似的活性。
4.4 BT613与PL-GEN-IgG1与不同种属的PD-L1的结合：通过ELISA法检测的结果表明BT613和PL-GEN-IgG1与人和食蟹猴PD-L1都表现出相似的结合(结果未列出)，而在小鼠PD-L1蛋白上具有不一样的结合表现(参见图9)。
4.5 BT613与PL-GEN-IgG1与PD-L1相关蛋白的结合：通过ELISA法检测的结果表明BT613和PL-GEN-IgG1与人PD-L1\PDL2\PDL3蛋白的结合的差异，结果见图10，显示两者都特异性与人PD-L1结合。
4.6 BT613与罗氏PD-L1抗体在体外细胞功能上的比较
4.6.1基于功能细胞系的活性检测：GS-C2/PD-L1细胞/GS-J2/PD-1细胞(购自南京金斯瑞)为金斯瑞公司建立的功能细胞系。在这两种细胞中，PD-1\PD-L1的结合会抑制细胞内荧光素酶的表达，当加入PD-L1/PD-1抗体阻断这种结合时，会增强荧光素酶的表达，抗体加入的浓度与荧光素酶的表达正相关。结果见图11，BT613抗体和阿特珠单抗(从国外按照正规流程购买)都可以在细胞水平阻断PD-1与PD-L1的结合，并增强萤光素酶的表达，两者具有相似的IC50值。
4.6.2混合淋巴反应(MLR)检测抗体的细胞活性
从健康人中分离外周血单核细胞,通过树突状细胞(DC)分化培养液促进DC细胞的分化和成熟，然后与从另一健康人血液中纯化的CD4+T细胞按照DC细胞：CD4+T细胞＝1：10的比例混合。同时，RPMI 1640完全培养基梯度稀释抗PD-L1抗体(8μg/mL,0.8μg/mL,0.08μg/mL,0.008μg/mL,0.0008μg/mL)。将上述混合好的细胞与稀释 的抗体一起加入到96孔细胞培养板中，放于37℃和5％CO 2的细胞培养箱中培养5天后，收集100μl细胞上清液，测定IL2的浓度。具体结果参见图12，在混合淋巴反应(MLR)测定中，BT613具有优于阿特珠单抗的功能活性，尤其在200μg/mL和2μg/mL下，BT613更好的刺激CD4+T细胞产生IL2。
实施例5 BT613与阿特珠单抗在小鼠中的药代动力学的研究
每个抗体选4只C57BL/6小鼠静脉给药，剂量为10.0mg/kg，给药前0h、给药后0.5h、1h、8h及1d、2d、4d、7d、10d、12d、14d、17d、21d、28d时采血清检测抗体浓度，具体结果参见图13及表3，给药后BT613在小鼠血清中更加持久。
表3 BT613/阿特珠单抗(10.0mg/kg)在小鼠中的药代研究
Figure PCTCN2018117430-appb-000004
实施例6 BT613/阿特珠单抗在B-hPD-1人源化小鼠MC38-hPD-L1结肠癌皮下移植瘤模型中的药效学研究
利用购自北京百奥赛图公司的具有人源PD-1的B-hPD-1小鼠与具有人源PD-L1的MC38-hPD-L1结肠癌细胞建立结肠癌肿瘤模型进行PD-L1抗体的功能研究。
MC38-hPD-L1结肠癌进行皮下建模，将MC38-hPD-L1结肠癌细胞5×10 5个/0.1mL接种于雌性B-hPD-1人源化小鼠右侧前胁肋部皮下，待肿瘤生长到约150mm 3时按肿瘤体积随机分组，每组6只，共3组，分别为：溶剂对照组、PLH03-BT613-IgG1(3mg/kg,Q2Dx8)组，以及阿特珠单抗(3mg/kg,Q2Dx8)组。给药途径为腹腔注射，分组给药后第21天结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。抗体对小鼠肿瘤体积增长的抑制结果参见图14A和14B，BT613在3mg/kg的剂量上具有比阿特珠单抗更好的抑制效果。小鼠体重的结果参见图16，结果表明施用BT613后小鼠体重略有增长，结合肿瘤增长抑制的结果分析，增长体积大致相当于肿瘤体积，说明BT613安全性较好。
分组后第21天实验结束时，将动物安乐死，移除肿瘤称重，计算相对瘤重抑制率(IRTW)。从肿瘤重量上看，在3mg/kg剂量下，BT613具有比阿特珠单抗更强的肿瘤抑制活性，具体结果参见图15。
CA782、CA783、CA785、CA786和CA787也得到了与BT613相类似的上述实验的结果。
实施例7 BT613/阿特珠单抗在B-hPD-1人源化小鼠MC38-hPD-L1皮下移植瘤模型中的药效学研究
利用购自北京百奥赛图公司的具有人源PD-1的B-hPD-1小鼠与具有人源PD-L1的MC38-hPD-L1结肠癌细胞建立结肠癌肿瘤模型进行PD-L1抗体的功能研究。
MC38-hPD-L1结肠癌进行皮下建模，将MC38-hPD-L1结肠癌细胞5×10 5个/0.1mL接种于雌性B-hPD-1人源化小鼠右侧前胁肋部皮下，待肿瘤生长到约111mm 3时按肿瘤体积随机分组，每组6只，共3组，分别为：溶剂对照组、PLH03-BT613-IgG1(3mg/kg,Q2Dx8)组，以及阿特珠单抗(3mg/kg,Q2Dx8)组。给药途径为腹腔注射，每两天给药1次，共给药8次，分组给药后第17天结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。抗体对小鼠肿瘤体积增长的抑制结果参见图18A和18B，BT613在3mg/kg的剂量上具有比阿特珠单抗更好的抑制效果。小鼠体重的结果参见图20，结果表明施用BT613后小鼠体重略有增长，结合肿瘤增长抑制的结果分析，增长体积大致相当于肿瘤体积，说明BT613安全性较好。
分组后第17天实验结束时，将动物安乐死，移除肿瘤称重，计算相对瘤重抑制率(IRTW)。从肿瘤重量上看，在3mg/kg剂量下，BT613具有比阿特珠单抗更强的肿瘤抑制活性，具体结果参见图19。
CA782、CA783、CA785、CA786和CA787也得到了与BT613相类似的上述实验的结果。
实施例8 BT613序列继续向胚系基因优化
对抗体BT613序列两端的非胚系氨基酸继续进行优化，使之更接近种系(germline)，从而降低免疫原性风险。下表4列出了具体的氨基酸 突变位置，氨基酸编号按照Kabat规则进行编号，BT613不同突变体的亲和力检测值见表4，具体结合动力学曲线见图17A至17G，BT613不同突变体的氨基酸序列见表5。
表4 BT613不同突变体氨基酸变化及相应亲和力
Figure PCTCN2018117430-appb-000005
表5 BT613不同变体氨基酸序列
Figure PCTCN2018117430-appb-000006
Figure PCTCN2018117430-appb-000007

Claims (10)

  1. 一种抗PD-L1的抗体或其抗原结合片段，其中所述抗体的轻链区包含：
    1)序列KSSQX 1VLYSSNNKNYLX 2W(SEQ ID NO:14)所示的LCDR1序列，其中X 1为S或N，并且X 2为A或P；
    2)序列WASTRES(SEQ ID NO:15)所示的LCDR2序列；和
    3)序列QQYYSTPLT(SEQ ID NO:16)所示的LCDR3序列；并且
    其中所述抗体的重链区包含：
    1)序列GFTFSSY(SEQ ID NO:17)所示的HCDR1序列；
    2)序列SX 3DGSNK(SEQ ID NO:18)所示的HCDR2序列，其中X 3为F或Y；和
    3)序列DRIYLDY(SEQ ID NO:19)所示的HCDR3序列。
  2. 权利要求1的抗体或其抗原结合片段，其中所述抗体的轻链区的序列为SEQ ID NO:1，并且所述抗体的重链区的序列为SEQ ID NO:2；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:3，并且所述抗体的重链区的序列为SEQ ID NO:4；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:5，并且所述抗体的重链区的序列为SEQ ID NO:6；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:7，并且所述抗体的重链区的序列为SEQ ID NO:8；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:9，并且所述抗体的重链区的序列为SEQ ID NO:10；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:11，并且所述抗体的重链区的序列为SEQ ID NO:12。
  3. 权利要求2的抗体或其抗原结合片段，其中所述抗体的轻链区的序列包含一个或多个选自以下的突变：第1位氨基酸从E至D的突变、 第2位氨基酸从L至I的突变、第4位氨基酸从L至M的突变和第105位氨基酸从D至E的突变，并且
    所述抗体的重链区序列包含一个或多个选自以下的突变：第1位氨基酸从E至Q的突变、第6位氨基酸从Q至E的突变和第16位氨基酸从K至R的突变。
  4. 权利要求3的抗体或其抗原结合片段，其中所述抗体的轻链区的序列为SEQ ID NO:20，并且所述抗体的重链区的序列为SEQ ID NO:21；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:22，并且所述抗体的重链区的序列为SEQ ID NO:23；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:24，并且所述抗体的重链区的序列为SEQ ID NO:25；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:26，并且所述抗体的重链区的序列为SEQ ID NO:27；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:28，并且所述抗体的重链区的序列为SEQ ID NO:29；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:30，并且所述抗体的重链区的序列为SEQ ID NO:31；或者
    其中所述抗体的轻链区的序列为SEQ ID NO:32，并且所述抗体的重链区的序列为SEQ ID NO:33。
  5. 权利要求2-4中任一项的抗体或其抗原结合片段，其中所述抗原结合片段为Fab、Fab'、F(ab')2、scFv或dsFv片段。
  6. 权利要求2-5中任一项的抗体或其抗原结合片段用于制备治疗癌症的药剂的用途，优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
  7. 权利要求2-5中任一项的抗体或其抗原结合片段用于制备诊断癌 症的试剂的用途，优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
  8. 权利要求2-5中任一项的抗体或其抗原结合片段用于制备确定癌症患者预后或总存活期的试剂的用途，优选地，所述癌症选自肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
  9. 权利要求2-5中任一项的抗体或其抗原结合片段用于制备治疗炎性疾病或自身免疫性疾病的药剂的用途，优选地，所述炎性疾病或自身免疫性疾病选自病毒感染、类风湿性关节炎、骨关节炎、银屑病、红斑狼疮、克罗恩病、多发性硬化症和炎症性肠病。
  10. 权利要求2-5中任一项的抗体或其抗原结合片段用于制备诊断炎性疾病或自身免疫性疾病的试剂的用途，优选地，所述炎性疾病或自身免疫性疾病选自病毒感染、类风湿性关节炎、骨关节炎、银屑病、红斑狼疮、克罗恩病、多发性硬化症和炎症性肠病。
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