WO2020192770A1 - 一种筛选pd-l1/pd-1检测点抑制剂的方法 - Google Patents

一种筛选pd-l1/pd-1检测点抑制剂的方法 Download PDF

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WO2020192770A1
WO2020192770A1 PCT/CN2020/081787 CN2020081787W WO2020192770A1 WO 2020192770 A1 WO2020192770 A1 WO 2020192770A1 CN 2020081787 W CN2020081787 W CN 2020081787W WO 2020192770 A1 WO2020192770 A1 WO 2020192770A1
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culture system
glycosylation
protein
inhibitor
level
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俞强
陈芳芳
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中国科学院上海药物研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a method for screening inhibitors of PD-L1/PD-1 checkpoints.
  • Immune escape is a major feature of tumors.
  • the body's immune system can reduce the incidence of tumors by identifying and removing cancerous cells in the early stages of tumorigenesis.
  • Tumor cells can achieve immune escape and avoid immune killing by adjusting the state of themselves or the tumor microenvironment.
  • Immune checkpoints play an important role in regulating the immune response and maintaining immune tolerance to self-antigens. Up-regulating the expression of immune checkpoint inhibitory molecules is a common mechanism for tumor cells to avoid immune killing. By antagonizing the immune checkpoint signaling pathway, tumor growth can be effectively inhibited. Immunotherapy is becoming an important method of tumor treatment.
  • PD-1 Programmed cell death protein 1, also known as CD279
  • CD279 is currently one of the most concerned immune checkpoint receptors. It is a type I transmembrane protein with 288 amino acids that is encoded by the PDCD1 gene and belongs to the CD28/CTLA-4 immunoglobulin superfamily. It is mainly expressed in activated mature T cells.
  • PD-L1 Programmed death-ligand 1, also known as CD274, B7-H1
  • CD274, B7-H1 is an immunoglobulin-like type I transmembrane protein with 290 amino acids encoded and expressed by the CD274 gene. It is the main ligand of PD-1.
  • PD-L1 and PD-1 can mediate the phosphorylation of the intracellular domain of the latter, thereby recruiting SHP-2 phosphatase in the cytoplasm to dephosphorylate adjacent TCR pathway molecules, thereby inhibiting the downstream signaling pathway of TCR. Lead to T cell exhaustion.
  • T cell effector functions such as ELIZA detecting IFN- ⁇ levels or CFSE method detecting T cell expansion
  • the purpose of the present invention is to provide a drug screening and evaluation system for conveniently, effectively and accurately evaluating candidate drugs for inhibiting PD-L1/PD-1 interaction and signal transduction.
  • a method for screening candidate compositions of PD-L1/PD-1 checkpoint inhibitors including the steps:
  • the candidate composition is a PD-L1/PD-1 checkpoint inhibitor
  • the candidate composition is a PD-L1/PD-1 checkpoint inhibitor.
  • the PD-L1/PD-1 checkpoint inhibitor is a membrane transfer inhibitor.
  • the PD-L1/PD-1 checkpoint inhibitor is a glycosylation inhibitor that inhibits the formation of 55kD PD-L1.
  • the cell line expressing PD-1 includes: Jurkat/PD-1, stimulated by cytokines (IL-2, etc.), ionomycin combined with phorbol ester or anti-CD3 antibody combined with anti-CD28 antibody Later, Jurkat cells, MOLT-4 and RPMI-8226 that upregulated PD-1.
  • cytokines IL-2, etc.
  • ionomycin combined with phorbol ester or anti-CD3 antibody combined with anti-CD28 antibody
  • RPMI-8226 that upregulated PD-1.
  • the cell strain expressing PD-L1 includes: PC-9/PD-L1, H1975, H460, H2228, Hcc827, MDA-MB-231, A2058, A375, SK-MEL-5, NCI-H226, EBC-1, HDLM-2, U-251MG, U-138MG, CAPAN-2, TIME, SK-BR-3 and A431.
  • the culture system is a co-culture system of Jurkat/PD-1 and PC-9/PD-L1.
  • the "significantly higher” means that the ratio of P1a/P0a is greater than 1, preferably ⁇ 2, more preferably ⁇ 4.
  • the "significantly higher” means that the ratio of P1a/P0a is 1.5-10, preferably 2-5.
  • step (b) adding a candidate composition (candidate drug or candidate compound) and observing the expression level of PD-1 and/or the glycosylation of PD-L1 in the test group
  • the interval time t is 2-36 hours, preferably 6-30 hours, more preferably 10-24 hours.
  • the change in glycosylation of PD-L1 includes a change in the distribution of western blot bands of the PD-L1 protein.
  • the "change in glycosylation of PD-L1 in the culture system of the test group” is selected from the group consisting of a decrease in the level of PD-L1 protein at 55kD, an increase in the level of PD-L1 protein at 43kD, or Its combination.
  • the glycosylation change of PD-L1 includes a decrease in the level of PD-L1 protein of 55 kD.
  • the change in glycosylation of PD-L1 includes an increase in the level of 43 kD PD-L1 protein.
  • glycosylation change of PD-L1 is expressed by G value, and the G value calculation formula is as follows:
  • E1 is the number or level of 43kD PD-L1 in the training system of the test group
  • E2 is the number or level of 55kD PD-L1 in the training system of the test group
  • R1 is the number or level of 43kD PD-L1 in the control culture system
  • R2 is the number or level of 55kD PD-L1 in the control culture system.
  • the G value is ⁇ 2, preferably ⁇ 3, and more preferably ⁇ 5.
  • the value of G is 2-100, preferably 5-20.
  • the PD-1 is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.
  • the PD-1 includes PD-1 protein, encoding nucleic acid, active fragment or derivative thereof.
  • the candidate composition is selected from the group consisting of small molecule compounds, proteins, nucleic acids, antibodies, or combinations thereof.
  • the PD-L1/PD-1 checkpoint inhibitor includes a PD-1 antibody or an inhibitor of PD-L1/PD-1 activity.
  • the PD-L1/PD-1 checkpoint inhibitor is selected from the following group: two small molecule compounds purchased from Selleck (PD-1/PD-L1 inhibitor 1 and BMS-202) And a cyclic peptide inhibitor (PD-1/PD-L1inhibitor 3) and its structural analogs.
  • the PD-L1/PD-1 checkpoint inhibitor inhibits the lysosomal degradation of PD-1, which is manifested as an increase in the expression of PD-1.
  • the PD-L1 inhibitor is selected from the group consisting of compound no. 1166 developed by Bristol-Myers Squibb (Example 1166 of US 2015/0291549 A1) and structural analogs thereof.
  • the method is a non-diagnostic and non-therapeutic method.
  • Figure 1A was detected by Western blot method and showed that PC-9/PD-L1 cells highly expressed PD-L1 compared with wild-type (WT) cells, and GAPDH was the internal reference;
  • Figure 1B was used to detect the cellular localization of PD-L1 by immunofluorescence method The situation indicated that the PD-L1 highly expressed in PC-9/PD-L1 cells was localized on the cell membrane.
  • Figure 2A shows that compared with wild-type (WT) cells, Jurkat/PD-1 cells express PD-1 with a Western blot method, and GAPDH is the internal reference;
  • Figure 2B uses flow cytometry to detect PD-1 expression on the cell surface , Indicating that the highly expressed PD-1 in Jurkat/PD-1 cells is located on the cell membrane.
  • NC is a negative control without antibody, and Alexa 488 PD-1 Ab is an antibody that can recognize PD-1 coupled to the Alexa 488 group.
  • Figure 3 detects the expression levels of PD-L1 and PD-1 after PC-9/PD-L1 and Jurkat/PD-1 cells are co-cultured for the indicated time, showing that the protein levels of PD-L1 and PD-1 are negative for the co-culture time Related. Among them, GAPDH or ⁇ -Tubulin was used as the internal control.
  • the line graph below is the quantitative result normalized by internal reference after scanning the gray level.
  • FIG. 4A While PC-9/PD-L1 and Jurkat/PD-1 cells were co-cultured, chloroquine (CQ, chloroquine) or bortezomib (BTZ, bortezomib) was added to inhibit lysosomal degradation pathway or proteasome degradation pathway, respectively The results show that CQ can inhibit the degradation of PD-1 caused by co-cultivation for 24 hours, indicating that co-cultivation promotes the degradation of PD-1 protein through lysosomes;
  • Figure 4B transfected with Lipo 2000 in PC-9/PD-L1 cells Negative control or PD-L1 interfering RNA (PD-L1 WT or PD-L1 KD) was treated for 24 hours and co-cultured with Jurkat/PD-1.
  • CQ chloroquine
  • BZ bortezomib
  • Figure 5A pre-treated PC-9/PD-L1 cells with the indicated concentrations of PD-L1 Ab for 1 hour, and then co-cultured with Jurkat/PD-1 cells for 0 or 17 hours.
  • the results showed that the PD-1 level can vary with PD-L1 The concentration of Ab increases; in Figure 5B, PC-9/PD-L1 cells were pretreated with the compound at the indicated concentration for 1 hour, and then Jurkat/PD-1 cells were added for co-cultivation for the indicated time. The results showed that all three inhibitors can be used. Significantly up-regulate the expression level of PD-1.
  • PC-9/PD-L1 cells were lysed after being treated with DMSO (NC) or 10 ⁇ M no.1166 overnight. Part of the cell lysate was used as input, and the rest was combined with ConA (Agarose bound ConA). ) Suspended overnight. The results showed that PD-L1 of about 43kD was easier to bind to Conavaline lectin compared with PD-L1 of about 55kD, indicating that 43kD PD-L1 may be a high-mannose glycoprotein; PC in Figure 8B -9/PD-L1 cells were treated with DMSO (NC) or 10 ⁇ M no.1166 overnight, and the cell lysate was collected and incubated with Endo H for 1 hour.
  • NC DMSO
  • 10 ⁇ M no.1166 10 ⁇ M no.1166
  • PC-9/PD-L1 cells were pre-treated with PD-L1 Ab or no.1166 at the indicated concentration for 1 hour, and then added with Jurkat/PD-1 for co-cultivation for 17 hours. Detection of PD-1 levels showed no.1166 Up-regulating the level of PD-1 is equivalent to that of PD-L1 Ab, indicating that the two have the same ability to inhibit the interaction of PD-L1/PD-1. Among them, only no.1166 will affect the distribution of PD-L1 bands.
  • the screening system consists of two artificially constructed cell lines-PC-9/PD-L1 and Jurkat/PD-1. The co-cultivation of these two cells in vitro causes the interaction of PD-L1 and PD-1 and leads to the degradation of PD-1 protein. Drug interference can inhibit the degradation of PD-1 protein.
  • the expression level of PD-1 in the co-culture system after treatment with the candidate drug can indicate the degree of blocking of the PD-L1/PD-1 detection point by the drug.
  • the terms “candidate composition”, “candidate drug”, and “candidate test substance” are used interchangeably, and refer to any substance that needs to be tested as a PD-L1/PD-1 checkpoint inhibitor. It can be a single substance. The compound can also be a mixture (such as an extract).
  • the term includes (but is not limited to): small molecule compounds, proteins, nucleic acids, antibodies, or combinations thereof.
  • the term includes substances with known structures as well as substances with unknown structures.
  • the co-culture screening method of the present invention is more in line with the physiological process; compared with the method of detecting T cell effects, it leads to The mechanism of T cell effect changes is complex and diverse. Blocking the binding of PD-L1/PD-1 is only one of them. The detection of PD-1 expression by co-culture method can more intuitively reflect the effect of the drug on the interaction of the two proteins, while PC- The glycosylation of PD-L1 in 9/PD-L1 cells can directly reflect the influence of PD-L1 protein membrane transfer.
  • the invention has the characteristics of simple operation, low cost, high efficiency and high accuracy of results.
  • the cell lines were cultured in an incubator containing 5% CO 2 at 37°C, and the experiment was performed when the cells were passed to 3-8 generations and in the logarithmic growth phase.
  • Ponceau staining Ponceau (Ponceau 4g, trichloroacetic acid 60g, sulfosalicylic acid 60g, add water to make the volume to 2L) dye for 5-7min and observe the transfer effect;
  • F. Incubation with primary antibody Dilute the corresponding antibody in a 5% bovine serum albumin (BSA) TBST solution at a ratio of 1:1000-1:4000.
  • BSA bovine serum albumin
  • the primary antibody solution covers the protein bands and incubate at room temperature on a vertical shaker for 1 hour, then move to 4°C and incubate overnight. After incubating for 1 hour at room temperature on the second day, remove the primary antibody solution, add TBST solution and place on a horizontal shaker to wash for 10 minutes, replace with a new TBST solution and repeat twice;
  • BSA bovine serum albumin
  • A. Cell climbing film Put a wash-free cover glass in a 24-well plate, spot plate with 50,000-80,000 cells to ⁇ 50% density;
  • DAPI solution (1:1000 dilution and PBS, 0.1g/ml), 300 ⁇ l/well, incubate at room temperature for 10min, wash with PBST solution three times;
  • Olympus FLUOVIEW FV1000 Confocal Laser Microscope takes pictures under a 60 ⁇ (1.42NA) PLAPON objective lens (oil lens), and FV10-ASW software processes the pictures.
  • AlexaFluor 488 anti-human PD-1 is directly added to the tube and incubated for 30 minutes, 2 ⁇ l/tube;
  • the PC-9/PD-L1 cell dot plate adheres to the wall, 90% density is appropriate;
  • the cell dot plate adheres to the wall, and when the density reaches 30%-50%, proceed to the next step;
  • RNA Dilute RNA in the same volume of serum-free medium, pipette several times;
  • the cells in the well are cultured with serum-free medium, and the mixture in D is added to the well;
  • the siRNA (5' to 3') used in the experiment was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.
  • the sequence is as follows:
  • Negative control sense chain-UUC UCC GAA CGU GUC ACG UTT (SEQ ID No.: 1); antisense chain-ACG UGA CAC GUU CGG AGA ATT (SEQ ID No.: 2);
  • PD-L1 human: sense chain-GAG GAA GAC CUG AAG GUU CAG CAU A (SEQ ID No.: 3); antisense chain-UAU GCU GAA CCU UCA GGU CUU CCU C (SEQ ID No.: 4);
  • PD-L1no.2 human: Sense Chain-CCU ACU GGC AUU UGC UGA ACG CAU U (SEQ ID No.: 5); Antisense Chain-AAU GCG UUC AGC AAA UGC CAG UAG G (SEQ ID No.: 6) );
  • PD-L1 no.3 human: Justice chain-UGA UAC ACA UUU GGA GGA GAC GUA A (SEQ ID No.: 7); Antisense chain-UUA CGU CUC CUC CAA AUG UGU AUC A (SEQ ID No.: 8 ).
  • Example 1 The expression level of PD-1 in the co-culture system can indicate the degree of blocking of the PD-L1/PD-1 detection point by the drug
  • the screening system of the present invention consists of two artificially constructed cell lines: PC-9/PD-L1 and Jurkat/PD-1.
  • PC-9/PD-L1 is a human lung adenocarcinoma transfected and highly expressing human PD-L1 Stable cell line ( Figure 1);
  • Jurkat/PD-1 is a leukemia T cell line that is transfected and highly expresses human PD-1 ( Figure 2).
  • the Jurkat/PD-1 and PC-9/PD-L1 cells were co-cultured to simulate the behavior of T cells infiltrating tumor tissues. It can be found that as the co-cultivation time increases, the protein content of PD-1 and PD-L1 appears time The trend of dependent reduction, the amount of protein decreased significantly after 12-24 hours of co-cultivation, and the change of PD-1 was particularly significant, as shown in Figure 3.
  • CQ chloroquine
  • BTZ bortezomib
  • the PC-9/PD-L1 cells were transfected with Lipo 2000 with negative control or PD-L1 interfering RNA (PD-L1 WT or PD-L1 KD) for 24 hours and then co-cultured with Jurkat/PD-1.
  • the results are shown in Figure 4B, indicating that down-regulation of PD-L1 can stabilize the expression of PD-1.
  • PC-9/PD-L1 cells were pretreated with PD-L1 Ab or blank solvent for 1 hour, and then Jurkat/PD-1 cells were added for co-cultivation for the indicated time.
  • the results are shown in Figure 4C.
  • PD-L1 Ab can significantly reduce PD-1 was down-regulated after 24 hours of culture.
  • PC-9/PD-L1 cells were pretreated with the indicated concentrations of PD-L1 Ab for 1 hour, and then co-cultured with Jurkat/PD-1 cells for 0 or 17 hours.
  • the results are shown in Figure 5A, indicating that the PC-9 In the co-culture system of 9/PD-L1 and Jurkat/PD-1, the PD-L1 antibody can gradually increase the protein level of PD-1 as the concentration increases.
  • PC-9/PD-L1 cells use Selleck's three commercialized PD-L1/PD-1 inhibitors-two small molecule compounds (PD-1/PD-L1inhibitor 1 and BMS-202) and one Cyclic peptide inhibitors (PD-1/PD-L1inhibitor 3) were pretreated for 1 hour, and then added to Jurkat/PD-1 cells for the time indicated by co-cultivation. The results are shown in Figure 5B. All three inhibitors can significantly upregulate PD -1 expression level.
  • Example 2 The change in glycosylation of PD-L1 in the co-culture system can characterize the degree of blocking of the PD-L1/PD-1 detection point by the drug
  • PD-L1 WT or PD-L1 KD transfect negative control with Lipo 2000 or PD-L1 interfering RNA (PD-L1 WT or PD-L1 KD) for 24 hours and detect the PD-L1 protein level, using GAPDH as an internal control.
  • PD-L1 is a type I membrane integrated glycoprotein, it is known that there are a large number of N-linked-glycosylation modifications at its four asparagine residues N35, N192, N200 and N219, and such glycosylation modifications
  • the PD-L1 protein showed multiple non-uniform bands on the detection results.
  • N-linked glycosylation is a post-translational modification of proteins. It is a key step in the maturation of transmembrane glycoproteins, starting from the synthesis of polypeptide chains in the endoplasmic reticulum cavity, and then further cleaved and processed into high mannose type by different enzymes in the endoplasmic reticulum-Golgi secretion pathway , Heterozygous or compound structure. According to the glycotype of the protein, the secretion stage after protein synthesis can be inferred. In PC-9/PD-L1 cells, the Western blot band distribution of PD-L1 protein can reflect the state of PD-L1 protein.
  • the compound no.1166 (US 2015/0291549 A1) developed by Bristol-Myers Squibb can promote the decrease of PD-L1 protein level of about 55kD and the increase of protein of about 43kD in a concentration and time-dependent manner.
  • PD-L1 of about 43kD is a high-mannose glycoprotein, which can be digested by Endo H (Endo H), belongs to the early stage of synthesis and secretion, and is mainly distributed in the endoplasmic reticulum; while 55kD
  • the left and right PD-L1 are glycoproteins connected with complex glycans, which can tolerate Endo H. They are mature glycoproteins and are mainly distributed on the cell membrane (Figure 8A-C).
  • the change of the Western blot band of PD-L1 protein by candidate drugs can characterize its effect on PD-L1 protein membrane transfer.
  • the candidate drug is a PD-L1/PD-1 checkpoint inhibitor that acts on PD-L1 protein membrane transfer, it will cause changes in the glycosylation of PD-L1 in the co-culture system, including 55kD PD -Decrease in L1 protein level, increase in 43kD PD-L1 protein level, or a combination thereof.

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Abstract

一种筛选PD-L1/PD-1检测点抑制剂的方法，包括步骤：(a)提供一个表达PD-1的细胞株和表达PD-L1的细胞株的共培养体系；(b)分别在测试组和对照组中，向共培养体系中添加候选组合物，并观察测试组和对照组中PD-1的表达量和/或PD-L1的糖基化情况。该方法操作简便、成本低、高效率，能直观地反映药物对PD-L1/PD-1蛋白相互作用或PD-L1蛋白膜转移的影响。

Description

一种筛选PD-L1/PD-1检测点抑制剂的方法 技术领域
本发明属于生物技术领域，具体涉及一种筛选PD-L1/PD-1检测点抑制剂的方法。
背景技术
“免疫逃逸”是肿瘤的一大特征。机体的免疫系统在肿瘤发生初期可通过识别和清除癌变细胞来降低肿瘤发生率。而肿瘤细胞可通过调整自身或肿瘤微环境的状态来实现免疫逃逸，规避免疫杀伤。免疫检测点在调控免疫应答、维持对自身抗原的免疫耐受过程中担任着重要的角色。上调免疫检测点抑制分子的表达，是肿瘤细胞规避免疫杀伤的一种常见机制。通过拮抗免疫检测点信号通路可有效地抑制肿瘤生长。免疫治疗正在成为肿瘤治疗的重要方法。
PD-1(Programmed cell death protein 1，又称CD279)，是目前最受关注的免疫检测点受体之一。它是由PDCD1基因编码表达含288个氨基酸的I型跨膜蛋白，属于CD28/CTLA-4免疫球蛋白超级家族，主要表达在活化的成熟T细胞中。PD-L1(Programmed death-ligand 1，又称CD274，B7-H1)，是由CD274基因编码表达的含290个氨基酸的免疫球蛋白样I型跨膜蛋白，是PD-1的主要配体，目前已发现过表达于多种恶性肿瘤细胞中，包括淋巴瘤、黑色素瘤、肺癌、乳腺癌、恶性胶质瘤、卵巢癌、肾癌和膀胱癌等。PD-L1与PD-1的结合可介导后者胞内域发生磷酸化，以此招募胞质中的SHP-2磷酸酶对邻近的TCR通路分子去磷酸化从而抑制TCR下游信号通路，最终导致T细胞功能衰竭(T cell exhaustion)。
自2013年起，靶向PD-L1/PD-1通路的药物研发逐渐成为肿瘤免疫治疗的热点，主要的策略是通过设计抗体阻断PD-L1与PD-1的结合，解除PD-1通路对T细胞的抑制。截止至2018年6月，已有5个PD-L1/PD-1抗体或FDA批准上市，它们在临床上取得了突破性的治疗效果，尤其是针对晚期或转移性癌症的治疗。但同时，抗体类药物本身存在许多限制，比如其局限于静脉注射的单一用药方式、免疫原性、弱组织渗透性、低稳定性和高成本等。因此，其它类型尤其是小分子药物的开发显得日趋重要。尽管在目前而言，小分子化合物 的开发远远滞后于抗体类药物，但随着PD-L1/PD-1复合物结构的细节被揭露，开发可有效阻断PD-L1/PD-1相互作用的小分子抑制剂已成为可能。
与开发PD-L1/PD-1抑制剂的巨大需求相对应的是目前尚不健全的筛选方法。目前已有多种结构的小分子化合物出现在已发表的专利或文献中，但鲜少有进一步的报道。它们在临床前研究中的失败大部分归咎于所使用的筛选方法。它们大多数都是基于检测体外的同源蛋白结合的筛选原理，比如最常用的HTRF法(结合时间分辨荧光分析法(TRF,time-resolved fluoroimmunoassay)和荧光能量共振转移(FRET)的一种方法)和体外蛋白竞争实验，这些体外检测方法虽然具备极高的敏感性，但与生理状况相去甚远，容易得到假阳性的结果。另外，通过检测T细胞效应功能(如ELIZA检测IFN-γ水平或CFSE法检测T细胞扩增)来筛选也是常见的手段，但影响T细胞效应改变的机理复杂多样，这些方法并不能直观地反映药物对PD-L1/PD-1检测点的影响。
由此可见，如何能更有效、准确地评估候选抗体或化合物针对PD-L1/PD-1相互作用的方法，成为一个亟待解决的问题。
因此，本领域迫切需要开发一种便捷、有效、准确地筛选PD-L1/PD-1检测点抑制剂的方法。
发明内容
本发明的目的就是提供一种便捷、有效、准确地评估候选药物抑制PD-L1/PD-1相互作用和信号转导的药物筛选和评价体系。
在本发明的第一方面，提供了一种筛选PD-L1/PD-1检测点抑制剂的候选组合物的方法，包括步骤：
(a)提供一培养体系，所述培养体系为表达PD-1的细胞株和表达PD-L1的细胞株的共培养体系；
(b)在测试组中，向所述培养体系中添加候选组合物，并观察所述测试组PD-1的表达量和/或PD-L1的糖基化情况；在对照组中，在相同培养体系中不添加所述的候选组合物，并观察所述对照组中PD-1的表达量和/或PD-L1的糖基化情况；
其中，如果测试组培养体系中PD-1的表达量P1a显著高于对照组P0a，则说明所述候选组合物为PD-L1/PD-1检测点抑制剂；
如果测试组培养体系中PD-L1的糖基化情况改变，则说明所述候选组合物为PD-L1/PD-1检测点抑制剂。
在另一优选例中，所述PD-L1/PD-1检测点抑制剂为膜转移抑制剂。
在另一优选例中，所述PD-L1/PD-1检测点抑制剂为抑制形成55kD PD-L1的糖基化抑制剂。
在另一优选例中，所述表达PD-1的细胞株包括：Jurkat/PD-1、经细胞因子(IL-2等)、离子霉素联合佛波酯或抗CD3抗体联合抗CD28抗体刺激后上调PD-1的Jurkat细胞、MOLT-4和RPMI-8226。
在另一优选例中，所述表达PD-L1的细胞株包括：PC-9/PD-L1、H1975、H460、H2228、Hcc827、MDA-MB-231、A2058、A375、SK-MEL-5、NCI-H226、EBC-1、HDLM-2、U-251MG、U-138MG、CAPAN-2、TIME、SK-BR-3和A431。
在另一优选例中，所述培养体系为Jurkat/PD-1与PC-9/PD-L1的共培养体系。
在另一优选例中，所述“显著高于”指P1a/P0a的比值大于1，较佳地≥2，更佳地≥4。
在另一优选例中，所述“显著高于”指P1a/P0a的比值为1.5-10，较佳地2-5。
在另一优选例中，在步骤(b)中，在添加候选组合物(候选药物、或候选化合物)和观察所述测试组PD-1的表达量和/或PD-L1的糖基化情况之间，间隔时间t为2-36小时，较佳地6-30小时，更佳地10-24小时。
在另一优选例中，所述PD-L1的糖基化情况改变包括PD-L1蛋白的western blot条带分布的改变。
在另一优选例中，所述的“测试组培养体系中PD-L1的糖基化情况改变”选自下组：55kD的PD-L1蛋白水平下降、43kD的PD-L1蛋白水平增加、或其组合。
在另一优选例中，所述PD-L1的糖基化情况改变包括55kD的PD-L1蛋白水平下降。
在另一优选例中，所述PD-L1的糖基化情况改变包括43kD的PD-L1蛋白水平增加。
在另一优选例中，所述的PD-L1的糖基化情况改变用G值表示，G值计算公式如下：
Figure PCTCN2020081787-appb-000001
式中，
E1是测试组培养体系中43kD PD-L1的数量或水平；
E2是测试组培养体系中55kD PD-L1的数量或水平；
R1是对照组培养体系中43kD PD-L1的数量或水平；
R2是对照组培养体系中55kD PD-L1的数量或水平。
在另一优选例中，G值≥2，较佳地≥3，更佳地≥5。
在另一优选例中，G值为2-100，较佳地5-20。
在另一优选例中，所述PD-1来源于哺乳动物；优选地，来源于人、小鼠、大鼠、或兔；更优选地，来源于人。
在另一优选例中，所述PD-1包括PD-1的蛋白、编码核酸、活性片段或其衍生物。
在另一优选例中，所述的候选组合物选自下组：小分子化合物、蛋白质、核酸、抗体、或其组合。
在另一优选例中，所述PD-L1/PD-1检测点抑制剂包括PD-1抗体、或PD-L1/PD-1的活性抑制剂。
在另一优选例中，所述PD-L1/PD-1检测点抑制剂选自下组：购自Selleck公司的两个小分子化合物(PD-1/PD-L1 inhibitor 1和BMS-202)和一个环肽类抑制剂(PD-1/PD-L1inhibitor 3)及其结构类似物。
在另一优选例中，所述PD-L1/PD-1检测点抑制剂抑制PD-1的溶酶体降解，表现为PD-1的表达量增加。
在另一优选例中，所述PD-L1的抑制剂选自下组：Bristol-Myers Squibb开发的化合物no.1166(US 2015/0291549 A1的实施例1166)及其结构类似物。
在另一优选例中，所述方法是非诊断性和非治疗性方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1A用Western blot方法检测表明与野生型(WT)细胞相比，PC-9/PD-L1细 胞高表达PD-L1，其中GAPDH为内参；图1B用免疫荧光方法检测PD-L1的细胞定位情况，表明PC-9/PD-L1细胞中高表达的PD-L1定位在细胞膜上。
图2A用Western blot方法检测表明与野生型(WT)细胞相比，Jurkat/PD-1细胞高表达PD-1，其中GAPDH为内参；图2B用流式细胞仪检测细胞表面PD-1表达情况，表明Jurkat/PD-1细胞中高表达的PD-1定位在细胞膜上。NC为无抗体阴性对照，Alexa 488 PD-1 Ab为耦合Alexa 488基团的可识别PD-1的抗体。
图3检测了PC-9/PD-L1与Jurkat/PD-1细胞共培养所示时间后PD-L1和PD-1的表达水平，显示PD-L1和PD-1的蛋白水平与共培养时间负相关。其中以GAPDH或α-Tubulin为内参。下方的折线图为扫描灰度后经内参标准化的定量结果。
图4A在PC-9/PD-L1与Jurkat/PD-1细胞共培养的同时加入氯喹(CQ,chloroquine)或硼替佐米(BTZ,bortezomib)来分别抑制溶酶体降解通路或蛋白酶体降解通路，结果显示CQ可抑制共培养24小时后引起的PD-1降解，表明共培养促进了PD-1蛋白通过溶酶体降解；图4B在PC-9/PD-L1细胞中用Lipo 2000转染negative control或PD-L1干扰RNA(PD-L1 WT或PD-L1 KD)处理24小时后与Jurkat/PD-1共培养，结果表明下调PD-L1可稳定PD-1的表达；图4C中PC-9/PD-L1细胞用PD-L1 Ab或空白溶剂预处理1小时，再加入Jurkat/PD-1细胞共培养所示时间，结果显示PD-L1 Ab可显著减少共培养24小时后PD-1的下调。图4B和C中下方柱状图为三次重复实验的定量结果。
图5A分别用所示浓度PD-L1 Ab预处理PC-9/PD-L1细胞1小时，再与Jurkat/PD-1细胞共培养0或17小时，结果表明PD-1水平可随PD-L1 Ab浓度增加而增加；图5B中PC-9/PD-L1细胞分别用所示浓度化合物预处理1小时，再加入Jurkat/PD-1细胞共培养所示时间，结果显示三种抑制剂都可以显著上调PD-1的表达水平。
图6在PC-9/PD-L1细胞中用Lipo 2000转染negative control或PD-L1干扰RNA(PD-L1 WT或PD-L1 KD)处理24小时后检测PD-L1蛋白水平，以GAPDH为内参。
图7中用所示浓度的no.1166化合物处理PC-9/PD-L1细胞过夜(A)或用10μM no.1166处理PC-9/PD-L1细胞所示时间(B)，结果显示no.1166可改变PD-L1蛋白的Western blot条带分布。
图8A中PC-9/PD-L1细胞经DMSO(NC)或10μM no.1166处理过夜后被裂解，其中部分细胞裂解液作为input，余下部分与结合刀豆凝集素的琼脂糖(Agarose  bound ConA)混悬过夜，结果表明与55kD左右的PD-L1相比，43kD左右的PD-L1更易与刀豆凝集素结合，表明43kD PD-L1可能是高甘露糖型的糖蛋白；图8B中PC-9/PD-L1细胞经DMSO(NC)或10μM no.1166处理过夜，收集细胞裂解液同Endo H培养1小时，结果显示与55kD左右的PD-L1相比，43kD左右的PD-L1不耐受Endo H，说明43kD PD-L1属于分泌晚期的产物；图8C用免疫荧光方法检测PD-L1(Green)与内质网(ER-tracker Red)的共定位情况，结果显示43kD PD-L1分布在内质网中，而55kD PD-L1分布在细胞膜上。
图9中PC-9/PD-L1细胞预先被所示浓度的PD-L1 Ab或no.1166处理1小时，然后加入Jurkat/PD-1共培养17小时，检测PD-1水平表明no.1166上调PD-1的水平与PD-L1 Ab相当，表明两者抑制PD-L1/PD-1相互作用能力相当。其中只有no.1166会影响PD-L1条带分布。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种便捷、有效、准确地评估候选药物抑制PD-L1和PD-1相互作用和信号转导的药物筛选和评价体系。该筛选体系由两种人工构建的细胞株组成——PC-9/PD-L1和Jurkat/PD-1。此两种细胞在体外的共培养造成PD-L1和PD-1的相互作用并导致PD-1蛋白降解。药物干扰能抑制PD-1蛋白的降解，经候选药物处理后共培养体系中PD-1的表达水平可以表征该药物对PD-L1/PD-1检测点的阻断程度。另一方面，由于PD-L1的糖蛋白性质，作用于PD-L1的候选药物也会影响PD-L1的糖基化修饰从而改变PD-L1蛋白在Western blot上的分布模式。因此在PC-9/PD-L1细胞中，PD-L1蛋白在Western blot上条带的改变可以用来表征化合物对PD-L1蛋白膜转移的影响。在此基础上完成了本发明。
术语
如本文所用，术语“候选组合物”、“候选药物”、“候选测试物”可互换使用，指需要测试其是否为PD-L1/PD-1检测点抑制剂的任何物质，可以是单一化合物，也可以是混合物(如提取物)。此外，该术语包括(但并不限于)：小分子化合物、蛋白质、核酸、抗体、或其组合。此外，该术语包括结构已知的物质，也包括结构未知的物质。
本发明的主要优点包括：
1)本发明所述的共培养筛选方法与现有的体外筛选方法(如均相时间分辨荧光技术和体外蛋白竞争实验)比较，更符合生理过程；与检测T细胞效应的方法相比，导致T细胞效应改变的机理复杂多样，阻断PD-L1/PD-1结合只是其中之一，通过共培养方法检测PD-1表达能更直观地反映药物对两蛋白相互作用的影响，而PC-9/PD-L1细胞的PD-L1糖基化可直观反映PD-L1蛋白的膜转移的影响。
2)本发明具有操作简便，成本低，高效率，结果准确度高的特点。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
通用材料
1.药品与试剂
Figure PCTCN2020081787-appb-000002
Figure PCTCN2020081787-appb-000003
2.细胞与细胞培养基
Figure PCTCN2020081787-appb-000004
细胞株均置于37℃含5％CO 2的培养箱中培养，待细胞传至3-8代，处于对数生长期时进行实验。
通用方法
1.免疫印迹法(Western blot)分析
A.收集蛋白。加入1×sample buffer裂解细胞，收集细胞裂解液，100℃加热煮沸7-10min，使蛋白充分变性；
B.分离蛋白。12％-8％SDS-聚丙烯酰胺(SDS-PAGE)凝胶电泳分离蛋白(Tris-甘氨酸电泳液由Tris 3.02g，Glycine 18.8g和SDS 1g定容至1L)。浓缩 胶电压维持80v，分离胶电压为120-140v；
C.半干法转膜。将分离的蛋白从胶中转移至硝酸纤维素膜上(转膜液由Glycine 2.9g，Tris 5.8g和SDS 0.37g溶于800ml去离子水中，使用前加入200ml甲醇)，转膜电压为9V，转膜时间视蛋白大小而定(34-55kD PD-L1和PD-1蛋白转膜时间为25min)；
D.丽春红染色。丽春红(丽春红4g，三氯乙酸60g，磺基水杨酸60g，加水定容至2L)染色5-7min后观察转膜效果；
E.牛奶封闭。5％脱脂奶粉TBST(Tris 2.42g，氯化钠8g，加水定容至1L，调节pH为7.5，使用前加入1ml Tween 20)溶液覆盖蛋白条带于垂直摇床室温孵育1h；
F.一抗孵育。在5％牛血清白蛋白(BSA)TBST溶液中按1:1000-1:4000比例稀释相应抗体，一抗溶液覆盖蛋白条带于垂直摇床室温孵育1h，再移至4℃孵育过夜，第二日于室温孵育1h后去除一抗溶液，加入TBST溶液放置于水平摇床洗10min，更换新的TBST溶液重复两遍；
G.二抗孵育。在5％牛血清白蛋白(BSA)TBST溶液中按1:5000比例稀释相应抗体，二抗溶液覆盖蛋白条带于垂直摇床室温孵育1h，去除上清加入TBST溶液放置于水平摇床洗10min，更换新的TBST溶液重复两遍；
H.化学发光显影。将化学发光底物(Millipore)按1:1混合，孵育在蛋白膜上，2-3min后在AZURE c300中显影拍照。
2.免疫荧光检测蛋白定位
A.细胞爬片。24孔板中放入免洗盖玻片，50,000-80,000细胞点板至～50％密度；
B.固定。移除上清，PBS溶液(氯化钠8g，氯化钾0.2g，十二水磷酸氢二钠3.49g，磷酸二氢钾0.2g，加水定容至1L，pH 7.4)洗三遍；4％多聚甲醛(PFA)溶液(PFA 40g/L，加入相应体积所需PBS所需的化合物，加入超纯水至80％，温育使PFA溶解，待溶液冷却后加入氢氧化钠调pH为7.0，定容)按300μl/孔加入，室温固定15min，PBS洗两遍，每遍5min；
C.打孔(如只检测细胞表面的PD-L1等蛋白表达，省去这一步)。0.1％Triton X-100PBS溶液室温孵育15min，PBS洗两遍，每遍5min；
D.封闭。0.1％PBST溶液(PBS+0.1％Tween 20)+10％马血清封闭液37℃ 孵育30min，
E.一抗孵育。将封闭后的盖玻片转移至疏水板中，在封闭液中按1:200比例稀释相应抗体，每盖玻片加上50μl一抗溶液，4℃孵育过夜，PBST溶液洗三遍；
F.二抗孵育。在封闭液中按1:1000比例稀释相应的荧光二抗，37℃孵育1.5h，PBST溶液洗三遍；
G.细胞核染色。DAPI溶液(1:1000稀释与PBS中，0.1g/ml)，300μl/孔，室温孵育10min，PBST溶液洗三遍；
H.封片。盖玻片反扣在滴有DAKO抗淬灭剂的载玻片上，注意赶走大的气泡；
I.激光共聚焦显微镜拍照。Olympus FLUOVIEW FV1000激光共聚焦显微镜在60×(1.42NA)PLAPON物镜(油镜)下拍照，FV10-ASW软件处理图片。
3.流式细胞仪检测Jurkat细胞表面PD-1表达
A.将孔内细胞(6孔板，100万细胞/孔)移至离心管中离心，500g×5min，弃上清，加入PBS枪头吹打数下洗一遍；
B.封闭。10％马血清PBS溶液封闭15min，200μl/管；
C.抗体孵育。AlexaFluor 488 anti-human PD-1直接加入管中孵育30min，2μl/管；
D.加入500μl PBST溶液，离心，弃上清；
E.加入500μl PBS溶液，离心，弃上清，最后加入500μl PBS；
F.流式细胞仪检测FL1-H通道。
4.共培养系统筛选PD-L1抑制剂
A.PC-9/PD-L1细胞点板贴壁，90％密度为宜；
B.加药预先处理PC-9/PD-L1细胞1h后，按PC-9/PD-L1的4倍数目加入Jurkat/PD-1细胞进行共培养；
C.17-24h后，收集细胞裂解液。
5.RNA干扰实验
A.细胞点板贴壁，待密度达到30％-50％进行下一步骤；
B.按产品说明书在无血清培养基中稀释lipofectamine 2000，枪头吹打数下，室温静置5min；
C.在同样体积的无血清培养基中稀释RNA，枪头吹打数下；
D.将B和C混匀，室温静置20min；
E.孔内细胞换用无血清培养基培养，将D中混合物加到孔内；
F.24h后进入下一处理。
实验中所用siRNA(5'至3')由上海吉玛制药技术有限公司合成，序列如下：
阴性对照：正义链-UUC UCC GAA CGU GUC ACG UTT(SEQ ID No.:1)；反义链-ACG UGA CAC GUU CGG AGA ATT(SEQ ID No.:2)；
PD-L1(人)：正义链-GAG GAA GAC CUG AAG GUU CAG CAU A(SEQ ID No.:3)；反义链-UAU GCU GAA CCU UCA GGU CUU CCU C(SEQ ID No.:4)；
PD-L1no.2(人)：正义链-CCU ACU GGC AUU UGC UGA ACG CAU U(SEQ ID No.:5)；反义链-AAU GCG UUC AGC AAA UGC CAG UAG G(SEQ ID No.:6)；
PD-L1no.3(人)：正义链-UGA UAC ACA UUU GGA GGA GAC GUA A(SEQ ID No.:7)；反义链-UUA CGU CUC CUC CAA AUG UGU AUC A(SEQ ID No.:8)。
6.Endo H酶消化实验
取已用1×sample buffer收集的蛋白样品10μl，按照产品说明书加入2μl10×GlycoBuffer 3，2μl Endo H，6μl水，37℃温育1h。
7.ER-tracker red染色
细胞加药处理后，移除培养基，加入ER-tracker工作液(ER-tracker red:稀释液＝1:1000-3000)300μl/孔，37℃孵育15-30min后移除，培养基洗涤1-2次，预冷的PBS快速洗一遍，4％PFA室温避光固定。
实施例1共培养体系中PD-1的表达水平高低可以表征该药物对PD-L1/PD-1检测点的阻断程度
本发明筛选体系由两种人工构建的细胞株组成：PC-9/PD-L1和 Jurkat/PD-1，PC-9/PD-L1为转染并高表达人PD-L1的人肺腺癌稳转细胞株(图1)；Jurkat/PD-1是转染并高表达人PD-1的白血病T细胞稳转细胞株(图2)。
将Jurkat/PD-1与PC-9/PD-L1细胞进行共培养以模拟T细胞浸润肿瘤组织的行为，可以发现随着共培养时间的延长，PD-1和PD-L1的蛋白量呈现时间依赖性减少的趋势，在共培养12-24小时后蛋白量明显降低，其中PD-1的变化尤为显著，如图3所示。
在PC-9/PD-L1与Jurkat/PD-1细胞共培养的同时加入氯喹(CQ,chloroquine)或硼替佐米(BTZ,bortezomib)来分别抑制溶酶体降解通路或蛋白酶体降解通路。结果如图4A所示，CQ可抑制共培养24小时后引起的PD-1降解，表明共培养促进了PD-1蛋白通过溶酶体降解。
在PC-9/PD-L1细胞中用Lipo 2000转染negative control或PD-L1干扰RNA(PD-L1 WT或PD-L1 KD)处理24小时后与Jurkat/PD-1共培养。结果如图4B所示，表明下调PD-L1可稳定PD-1的表达。
PC-9/PD-L1细胞用PD-L1 Ab或空白溶剂预处理1小时，再加入Jurkat/PD-1细胞共培养所示时间，结果如图4C所示，PD-L1 Ab可显著减少共培养24小时后PD-1的下调。
上述结果表明，通过RNA干扰的手段降低肿瘤细胞中PD-L1的表达，或使用阻断性抗体(PD-L1 Ab)削弱PD-L1/PD-1相互作用都可以阻止PD-1的降解。
此外，分别用所示浓度PD-L1 Ab预处理PC-9/PD-L1细胞1小时，再与Jurkat/PD-1细胞共培养0或17小时，结果如图5A所示，表明在PC-9/PD-L1和Jurkat/PD-1的共培养体系中，PD-L1抗体可以随浓度增加逐步增加PD-1的蛋白水平。
PC-9/PD-L1细胞分别用Selleck公司三种已商业化的PD-L1/PD-1抑制剂——两个小分子化合物(PD-1/PD-L1inhibitor 1和BMS-202)和一个环肽类抑制剂(PD-1/PD-L1inhibitor 3)预处理1小时，再加入Jurkat/PD-1细胞共培养所示时间，结果如图5B所示，三种抑制剂都可以显著上调PD-1的表达水平。
因此，这些结果说明，通过检测经候选药物处理后共培养体系中PD-1的表达水平高低可以表征该药物对PD-L1/PD-1检测点的阻断程度。
实施例2共培养体系中PD-L1的糖基化情况改变可以表征该药物对 PD-L1/PD-1检测点的阻断程度
在PC-9/PD-L1细胞中用Lipo 2000转染negative control或PD-L1干扰RNA(PD-L1 WT或PD-L1 KD)处理24小时后检测PD-L1蛋白水平，以GAPDH为内参，由于PD-L1属于I型膜整合糖蛋白，已知在其N35、N192、N200和N219四个天冬酰胺残基位点上存在大量N连接-糖基化修饰，而这样的糖基化修饰使得PD-L1蛋白在检测结果上呈现出多个非均匀的条带。
结果如图6所示，PC-9/PD-L1细胞株中PD-L1蛋白在43-55kD间存在多个特异性条带。
用不同浓度的no.1166化合物处理PC-9/PD-L1细胞过夜或用10μM no.1166处理PC-9/PD-L1细胞不同时间，结果如图7所示，no.1166可改变PD-L1蛋白的Western blot条带分布。
N连接-糖基化是一种蛋白翻译后修饰。它是跨膜糖蛋白成熟的一个关键步骤，起始于内质网腔中多肽链的合成过程，随后在内质网-高尔基体分泌通路中被不同酶类进一步切割、加工为高甘露糖型、杂合型或复合型等不同结构。根据蛋白连接的糖型可以推断蛋白合成后的分泌阶段。在PC-9/PD-L1细胞中，PD-L1蛋白的Western blot条带分布可以反映PD-L1蛋白的状态。由Bristol-Myers Squibb开发的化合物no.1166(US 2015/0291549 A1)可随浓度和时间依赖性地促进55kD左右的PD-L1蛋白水平下降和43kD左右的蛋白增加。
本发明证实43kD左右的PD-L1为高甘露糖型的糖蛋白，可被糖苷内切酶H(Endo H)消化，属于合成-分泌早期的糖蛋白，主要分布在内质网中；而55kD左右的PD-L1为连接复合型聚糖的糖蛋白，可耐受Endo H，属于成熟糖蛋白，主要分布在细胞膜上(图8A-C)。
因此，通过候选药物对PD-L1蛋白的Western blot条带的改变可以表征其对PD-L1蛋白膜转移的影响。当候选药物是通过对PD-L1蛋白膜转移发挥作用的PD-L1/PD-1检测点抑制剂时，则会导致共培养体系中PD-L1的糖基化情况改变，其中包括55kD的PD-L1蛋白水平下降、43kD的PD-L1蛋白水平增加、或其组合。
PD-L1蛋白膜转移被抑制则必然会阻断PD-L1/PD-1的相互作用和信号转导。该结果可以通过上述共培养体系验证，如图9所示。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种筛选PD-L1/PD-1检测点抑制剂的候选组合物的方法，其特征在于，包括步骤：
    (a)提供一培养体系，所述培养体系为表达PD-1的细胞株和表达PD-L1的细胞株的共培养体系；
    (b)在测试组中，向所述培养体系中添加候选组合物，并观察所述测试组PD-1的表达量和/或PD-L1的糖基化情况；在对照组中，在相同培养体系中不添加所述的候选组合物，并观察所述对照组中PD-1的表达量和/或PD-L1的糖基化情况；
    其中，如果测试组培养体系中PD-1的表达量P1a显著高于对照组P0a，则说明所述候选组合物为PD-L1/PD-1检测点抑制剂；
    如果测试组培养体系中PD-L1的糖基化情况改变，则说明所述候选组合物为PD-L1/PD-1检测点抑制剂。
  2. 如权利要求1所述的方法，其特征在于，所述PD-L1/PD-1检测点抑制剂为PD-L1糖基化抑制剂。
  3. 如权利要求1所述的方法，其特征在于，所述表达PD-1的细胞株包括：Jurkat/PD-1、经细胞因子、离子霉素联合佛波酯或抗CD3抗体联合抗CD28抗体刺激后上调PD-1的Jurkat细胞、MOLT-4和RPMI-8226。
  4. 如权利要求1所述的方法，其特征在于，所述表达PD-L1的细胞株包括：PC-9/PD-L1、H1975、H460、H2228、Hcc827、MDA-MB-231、A2058、A375、SK-MEL-5、NCI-H226、EBC-1、HDLM-2、U-251 MG、U-138 MG、CAPAN-2、TIME、SK-BR-3和A431。
  5. 如权利要求1所述的方法，其特征在于，所述培养体系为Jurkat/PD-1与PC-9/PD-L1的共培养体系。
  6. 如权利要求1所述的方法，其特征在于，所述“显著高于”指P1a/P0a的比值大于1。
  7. 如权利要求1所述的方法，其特征在于，所述PD-L1的糖基化情况改变包括PD-L1蛋白的western blot条带分布的改变。
  8. 如权利要求1所述的方法，其特征在于，所述PD-L1/PD-1检测点抑制剂包括PD-1抗体、或PD-L1/PD-1的活性抑制剂。
  9. 如权利要求1所述的方法，其特征在于，所述PD-L1/PD-1检测点抑制剂选自下组：PD-1/PD-L1 inhibitor 1、BMS-202、PD-1/PD-L1 inhibitor 3及其结构类似物。
  10. 如权利要求1所述的方法，其特征在于，所述PD-L1的抑制剂选自下组：化合物no.1166及其结构类似物。
  11. 如权利要求1所述的方法，其特征在于，所述的“测试组培养体系中PD-L1的糖基化情况改变”选自下组：55kD的PD-L1蛋白水平下降、43kD的PD-L1蛋白水平增加、或其组合。
  12. 如权利要求2所述的方法，其特征在于，所述PD-L1/PD-1检测点抑制剂为抑制形成55kD PD-L1的糖基化抑制剂。
  13. 如权利要求11所述的方法，其特征在于，所述的PD-L1的糖基化情况改变用G值表示，G值计算公式如下：
    Figure PCTCN2020081787-appb-100001
    式中，
    E1是测试组培养体系中43kD PD-L1的数量或水平；
    E2是测试组培养体系中55kD PD-L1的数量或水平；
    R1是对照组培养体系中43kD PD-L1的数量或水平；
    R2是对照组培养体系中55kD PD-L1的数量或水平。
  14. 如权利要求13所述的方法，其特征在于，G值为2-100。
  15. 如权利要求1所述的方法，其特征在于，所述的候选组合物选自下组：小分子化合物、蛋白质、核酸、抗体、或其组合。
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SHAO, BIN AND LI CHIA-WEI, LIM SEUNG-OE, SUN LINLIN, LAI YUN-JU, HOU JUNWEI, LIU CHUNXIAO, CHANG CHIUNG-WEN, QIU YUFAN, HSU JUNG-M: "Deglycosylation of PD-L1 by 2-Deoxyglucose Reverses PARP Inhibitor- Induced Immunosuppression in Triple-Negative Breast Cancer", AMERICAN JOURNAL OF CANCER RESEARCH, vol. 8, no. 9, 1 September 2018 (2018-09-01), pages 1837 - 1846, XP055744670 *

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