Targeted Blockade Of PD-1/PD-L1 Pathway In NSCLC Immunotherapy

BackgroundImmune checkpoints are a class of immune system regulators involved in self-tolerance and prevent self-attacks and over-immunization.Immune checkpoints are classified into two types;active type and inhibitory type.Inhibitory immune checkpoints are often used by tumor cells in the tumor microenvironment,inhibiting immune cell activity,and achieving immune evasion.The inhibitory type commonly seen in activated T lymphocyte include PD-1(CD279),CTLA-4(CD152)),LAG3(CD223),TIM-3(CD366),killer cell immunoglobulin-like receptors(KIR)and so on.PD-1 may play an important role in the process of death induction.The binding of PD-1 to ligands up-regulates the E3-ubiquitin ligase CBL-b and c-CBL triggering downregulation of T cell receptor,so that binding of PD-L1 to its receptor inhibits T cell activation and cytokine release.PD-L1 is lowly expressed in activated T cells,dendritic cells,B cells,monocytes,keratinocytes.PD-L1 is also highly expressed in non-small cell lung cancer.It has been reported that PD-L1 can be used as a predictive biomarker for tumor immunotherapy.According to 2015 statistics,lung cancer is the most common cancer in China,and it is also a major cause of cancer-related deaths in China,followed by gastric cancer,esophageal cancer,and liver cancer.Among them,non-small cell lung cancer(NSCLC)accounts for 80-85%of lung cancer cases.Compared with small cell carcinoma,non-small cell lung cancer is insensitive to chemotherapy and is treated primarily by surgical resection.The primary treatment for stage Ⅰ-Ⅱ NSCLC is surgery.Most newly diagnosed cases of NSCLC are stage Ⅳ.Over the past two decades,great progress has been made in the pathology,pathogenesis,and treatment of tumor progression.Lung cancer is a molecularly heterogeneous disease.The treatment of lung cancer has shifted from cytotoxic treatment based on physicians’ experience preferences to personalized treatment based on individual characteristics,based on the state of patient genetic alterations and the presence of programmed death ligands(PD-L1).The situation is handled in a corresponding manner.Platinum-based dual therapy has become the standard treatment for patients with advanced NSCLC and has performed better.Strengthening the immune response and introducing antibodies targeting CTLA-4 and PD-1 have opened up new directions for the treatment of lung cancer.In 2015,the FDA approved the immune checkpoint blocker nivolumab for patients with disease progression during or after platinum therapy.Methods for restoring T cell mediated anti-tumor immunity are provided.Chimeric antigen receptors(CARs)are a type of fusion protein that contains at least three major domains.CAR molecules are expressed on the surface of T cells(CAR-T)and recognize tumor antigens.After being activated,CAR-T cells begin to lyse.target cells.As of August 2017,more than 200 clinical trials have achieved remarkable results and promoted the transition to solid tumor therapeutic applications.Therefore,finding safe and effective therapeutic targets quickly becomes an important task today.Clustered regularly interspaced short palindromic repeat(CRISPR)is a virus-eliminating immune mechanism developed by bacteria during the evolution process,which can be directed to find the target DNA sequence under the guidance of a RNA,and then the DNA sequence was excised,so that the gene could not be normally transcribed,so as to achieve an effective scavenging effect.It has been reported that PD-1 knocked out T cells have a higher tumor killing effect.From 2016,a series of clinical trials of tumor immunotherapy based on the CRSIPR/Cas9 knock-out technique are underway.Given the successful clinical application of monoclonal antibodies at early immune checkpoints,we have reason to believe that with the development of clinical trials,With the escalation of CRISPR/Cas9 technology,The knockout of immunological checkpoints may also bring positive results.The paper is divided into three aspects.The first part analyzes the molecular composition of PD-1 monoclonal antibody and the labeling with fluorescent molecules,detects the binding ability of fluorescent labeled monoclonal antibody with PBMCs,and determines optimizing conditions of PD-1 blocking with monoclonal antibody.In the second part,PD-L1 CAR-T cells were constructed to determine the killing effect of PD-L1 targeting CAR-T cells on PD-L1 high expression tumor cell lines.In the third part,we use the CRISPR/Cas9 technology to screen the high efficient knockout targets of PD-1 and other immune checkpoints at the cellular level.MethodsTo label monoclonal antibody drug with fluorescent kit and use SDS-PAGE,protein gel fluorescence scanning to analysis composition of nivolumab monoclonal antibody molecules;Detection of the binding of fluorescent antibody and PBMC by flow cytometry to determine the best blocking conditions in vitro,the tumor killing experiment was used to assess the in vitro blocking effect;PD-L1 chimeric antigen receptor lentiviral vector was constructed,and CAR-T cells were obtained through virus infection of PBMCs.The specific killing effect of CAR-T on PD-L1 highly expressing cell lines was determined by apoptosis assay.The construction of stable cell lines with expression of PD-1 or other immune checkpoint,and knocking out targets screening of PD-1 and other immune checkpoint based on CRISPR/Cas9 technology.ResultsPart Ⅰ PD-1 blocking with the monoclonal antibodyTwo subunits of PD-1 monoclonal antibody could be effectively labeled with fluorescent dyes.The fluorescent labeled monoclonal antibody against PD-1 could effectively block PBMC;PD-1 monoclonal antibody could bind with PBMC in a buffer system of pH 5.8.The binding constant is the lowest,and the binding is stable.When NCI-H358 is not stimulated by cytokines,PD-L1 is highly expressed.After stimulation with cytokines,the expression of PD-L1 is increased.When met NCI-H358 with highly expression PD-L1,the binding of monoclonal antibody with PBMC were unaffected and the PD-1 monoclonal antibody was stable;It is the best temperature of binding at 37℃for 1 h(the binding constant was the lowest);Both of 0.9%NaCl and OKM200+5%FBS could serve as a blocking medium;The killing effects of NCI-H358 and J82 were not significantly decreased for the blocking in vitro,and the killing activity was significantly higher than that of the non-blocked group at some certain effect-target ratio.Part II Killing effect of PD-L1 CAR-T cells on PD-L1 high expression tumor cell linesThe scFv,scFv-28Bz,and Fab of PD-L1 were successfully constructed in lentiviral vectors,respectively;The extracellular scFv expression of pLVX-anti-PD-L1-scFv and pLVX-anti-PD-L1-scFv-28Bz were able to be detected;Lentivirus packaged with plasmids pLVX-anti-PD-L1-scFv and pLVX-anti-PD-L1-scFv-28B could effectively infect PBMCs.The infection rate is more than 30%,and cell surface scFv could be effective detected,the transmembrane domain and signal domain did not affect the expression of CAR molecules;With three culture conditions,CD3+ cells were stable above 90%after 7 days of culture,and the proportion of NKT in mixed cells gradually increased to about 40%.Magnetic bead activation and X-VIVO 15 culture medium were selected and the OKM-200 medium was properly supplemented after 14 days of culture,the PD-L1 CAR expressing PBMC could be effectively expanded by about 200 times.PD-L1 CAR-T cells lysed non-small cell lung cancer with PD-L1 high expression but had no significant killing effect on lung cancer cells with PD-L1 low exression.CAR-T cells were able to release IFN-y at a high level when co-cultured with NCI-H358 cells,but not when co-cultured with A549 cells,which demonstrated a PD-L1-specific interaction with scFv.PartⅢI Immune checkpoint knockout target screening based on CRISPR/Cas9 methodThe expression of immune checkpoints in tumor patients is diverse.The level of PD-1 expression varies from person to person.The expression of other immune checkpoints varies from patient to patient;293TN cells does not express PD-1,CD 152,CD160,CD223,CD244,CD272,CD366,VISTA,stable cell lines were constructed to express immune checkpoints in 293TN cells;CRISPR/Cas9 based PD-1 knockout vectors were successfully constructed and transfected to 293TN-PD-1 to verify the efficiency of knockout.Four high efficient knock-out targets were obtained.Two efficient knock-out vectors were randomly selected for lentivirus packaging and PBMCs infection.The PD-1 knockout effectiveness was significant.