Real World Efficacy And Resistance Mechanism Of Osimertinib And CAR-T Cell Therapy For EGFR Mutant NSCLC Patients

Background and PurposeFor non small cell lung cancer(NSCLC),mutated epidermal growth factor receptor (EGFR)is the most common tumor-driving gene and therapeutic target.Patients with EGFR-mutated NSCLC can benefit from tyrosine kinase inhibitor(TKI)therapy.Osimertinib,as a third-generation TKI,has been approved for clinical treatment and recommanded by clinicians.However,studies have shown that therapeutic outcomes of osimertinib is limited in Chinese patients.Even in first-line osimertinib therapy,the progression-free survival(PFS)of chinese patients is worse than global data and the median survival(OS)is only 33.1 months which is less than three years.So the problems about osimertinib efficacy and resistance need to be sovled to prolong the survival of EGFR-mutant NSCLC patients.The preliminary study of resistance mechanism suggested that regardless of first-line and second-line therapy,the resistance mechanism of osimertinib is"fragmented",and there is a lack of"dominant target"and"accessible drug"for subsequent treatment.Immune checkpoint inhibitor therapy,as the most common immunotherapy strategy,has disappoiting efficacy in patients with EGFR-mutant NSCLC.Chemotherapy is still the standard subsequent treatment for these patients.But the therapeutic response rate is only 20%-30%.Additionally,previous study of our group indicated the sensitivity of chemotherapy will be reduced for the exposure of EGFR inhibitors,which even results in chemotherapy resistance.Patietns with EGFR-mutant NSCLC are hard to benefit from subsequent therapies.In general,conventional treatments for NSCLC,such as chemotherapy,targeted and immunotherapy,have limited efficacy in patients with the resistance to osimertinib.Therefore,based on the situation that different populations may have different responses to osimertinib treatment and the lack of effective rescue methods after osimertinib resistance,study on the real world data of osimertinib in the treatment of EGFR-mutant NSCLC in the Chinese population,and exploration of new strategies for the treatment are extremely urgent.Chimeric antigen receptor T cell(CAR-T cell)therapy is a cutting-edge immunotherapy strategy,and has potential application value in NSCLC patients,especially in those with EGFR mutations.After extracting and reenginerring,CAR-T cells have a major histocompatibility complex(MHC)-independent tumor recognition function relied on antigen binding structure.After activation in vitro,CAR-T cells can quickly recognize and kill tumor cells through chimeric antigen receptors.CAR-T cell therapy has entered clinical trials in solid tumors,but there is still a lack of reports in EGFR-mutant NSCLC.Application of CAR-T cell therapy in treatment of solid tumors has entered stage of clinical trial,but there is still a lack of study in EGFR-mutant NSCLC.For EGFR-mutant NSCLC,EGFR is commonly expressed in tumor tissues.Since mutations rarely occur in the extracellular sequence of EGFR,it can ensure that EGFR-mutant NSCLC can be effectively recognized by EGFR CAR-T cell.Theoretically,EGFR,as the core protein,is still highly expressed in EGFR-mutant NSCLC with resistance to osimertinib.The preliminary experiments of our group also found that long-term exposure of EGFR TKI will increase the expression of EGFR in cells.Thses fingdings ensure that EGFR CAR-T cell therapy will be suitable for the vast majority of osimertinib-resistant patients.In fact,almost all treatments for NSCLC patients with EGFR mutations,including targeted therapy after EGFR TKI resistance,are based on anti-EGFR treatment,such as combining strategy of EGFR antibody.As a result,EGFR CAR-T cell therapy is expected to become a new treatment strategy for EGFR-mutant NSCLC patients,especially the patients with EGFR-TKI resistant NSCLC.Improving the safety of CAR-T cell therapy on the basis of ensuring the efficacy is the key to the clinical application of CAR-T cells.Previous experiment by our team showed that cells may upregulated the expression of wild-type EGFR protein when exposed to EGFR TKI,which increasing the risk of the recognition of normal tissues and toxic effects of EGFR CAR-T cell.So the CAR-T treatment strategy needs to be further optimized to improve the safety.Receptor-tyrosine-kinase-like orphan receptor1(ROR1)is a receptor protein rarely expressed in normal tissues of adult.But,as a highly specific tumor-associated antigen,ROR1 is overexpressed in many malignant tumors,including lung cancer,breast cancer,ovarian cancer and so on.Previous studies of our group have verified the expression of ROR1 in lung adenocarcinoma patients which is nearly 94%,and the safety of ROR1 CAR-T cell in tumor therapy.In addition,ROR1 protein is involved in the regulation of cell apoptosis and proliferation mediated by EGFR signaling pathway in lung adenocarcinoma,indicating that ROR1has a tight correlation with the expression of EGFR.Therefore,we will try to optimize the CAR-T cell which is esigned for EGFR-mutant NSCLC relied on"chimeric costimulatory receptor plus chimeric antigen receptor"(CCR+CAR)structure.Considering above aspects,this study will review the real-world data of osimertinib in the treatment of patients with EGFR-mutant NSCLC,and clarify the resistance mechanisms as well as the distribution of EGFR mutations.Based on these characteristics of patients resistant to osimertinib,We take the lead in designing EGFR CAR-T cells for EGFR-mutant NSCLC patients and verifying the anti-tumor effects in vivo and in vitro.EGFR CAR-T cells and verify the anti-tumor effect in vivo and in vitro.Further,based on the correlation between EGFR and ROR1,we try the first attempt in world to construct"EGFR CCR+ROR1 CAR"T cells to improve the efficacy and safety of CAR-T cell therapy for EGFR-mutant NSCLC.Our research is expected to provide new methods and strategies for the treatment of patients with EGFR-mutant NSCLC and EGFR-TKI resistant NSCLC.Materials and MethodsWe selected and enrolled patients diagnosed with EGFR mutated NSCLC and treated with osimertinib at West China Hospital of Sichuan University between January 1,2018 and June 30,2020.Baseline clinical data and therapeutic benefits of the patients were collected to statistically analyzed the efficacy of osimertinib in the first-line and second-line treatment.Patients were grouped according to clinical baseline characteristics,and PFS and OS curves were plotted by Kaplan-Meier method.Cox multivariate analysis was used to explore possible independent factors,and imaging data were used to analyze the pattern of disease progression.The reports of next generation sequencing(NGS)were collected to analyze the genetic mechanisms of osimertinib resistance.In order to describe the treatment options and efficacy after osimertinib resistance occurred in the real world,all patients were followed up for scheme of subsequent treatment,clinical treatment outcomes and deaths of patients.The median PFS was calculated by Kaplan Meier method.At the same time,immunohistochemistry and flow cytometry were used to detect the expression of EGFR and ROR1 in clinical lung adenocarcinoma specimens and EGFR TKI-resistant cell lines.We also calculated the expression consistency of EGFR and ROR1expression by TIMER 2.0,which aimed to provide a basis for the design of subsequent CAR-T cells.Based on the results of retrospective study,sequence of human EGFR CAR was designed.Relied on molecular cloning technology and lentivirus system with the helper plasmid,we produced the virus containing the target CAR sequence.EGFR-CAR T cells were eventually produced by infecting healthy peripheral T cells with concentrated virus solution.Later,we validated the antitumor activity of EGFR CAR T cell in vitro.The target tumor cells were screened by flow cytometry.Positive and negative target cells were cultured with CAR T cells to detected the proliferation activity of T cells.And use the microscope to observed the clone masses of T cells after co-culture of tumor cells and CAR-T cells.Supernatant after co-culture was collected for LDH release experiment and enzyme-linked immunosorbent assay,which were used to monitor the specific cytotoxicity of CAR T cell and the release of interleukin 2(IL-2)and interferonγ(IFN-γ).Subsequently,we established a subcutaneous metastasis model of human EGFR-mutated lung adenocarcinoma in immunodeficient mice,and discussed the anti-tumor effect and potential toxicity risks of CAR T cell in vivo by monitoring the tumor volume and body weight of the mice.Based on the expression consistency of the above proteins,the single target car was modified to construct the structural sequence of"EGFR CCR+ROR1 CAR".ROR1CAR sequence was also designed as a control.Virus solution was prepared by molecular cloning and lentivirus system,and T cells stably expressing CAR were prepared.The antitumor effects of dual target CAR-T cells in vivo and in vitro were tested by the above verification experiments of proliferation and cytotoxic function,and compared with CAR-T cells with single target.ResultA total of 440 patients were enrolled,including 338 patients treated with second-line osimertinib and 102 patients treated with first-line osimertinib in this study.The objective response rate(ORR)of first-line and second-line treatment were 59.8%and43.9%,respectively.The median progression-free survival(m PFS)were 17.1 months(95%CI:14.074-20.126)and 14.1 months(95%CI:12.654-15.546).The median OS of patient received the second line osimertinib was 38.1m(95%CI:26.892-49.308),while the m OS could not be evaluated temporarily for patients treated with first-line osimertinib.The progressions after osimertinib therapy commonly occurred in lung,brain,bone and pleura.The resistance mechanisms mainly include the loss of T790M mutation(only for patients treated with second-line osimertinib),EGFR point mutations,bypass activation,downstream signal activation,loss the alternation of driver gene,small cell lung cancer transformation,and unknown mechanisms.After osimertinib resistance occurred,the proportion of patients still carrying EGFR driver mutations reaches to 61%-86%.After the progression occurred,57%-59%of patients received subsequent chemotherapy.The median PFS was 4.1months to 4.2 months.In clinical samples of lung adenocarcinoma,the average expression ratio of EGFR was 77.50%.The expression rates of EGFR in EGFR-mutant NSCLC cell lines such as H1975 and HCC827 were 99.13%and 82.70%respectively.The EGFR expression rates of H1975 osimeritinib resistant strain(H1975OR),H1975 erlotinib resistant strain(H1975ER)and HCC827 gefitinib resistant strain(HCC827GR)were 57.80%,66.96%and 33.87%,respectively.In the same batch of clinical samples,the average expression ratio of ROR1 was 78.21%.All tumor samples expressed EGFR and ROR1at the same time.The expression of ROR1 was detected in EGFR-mutant NSCLC cell lines such as H1975,HCC827.As for the resistant cell lines,H1975OR,H1975ER and HCC827GR had the expression of ROR1.Timer 2.0 was used to test the expression of EGFR and ROR1 in lung adenocarcinoma in TCGA database,and the correlation coefficient was 0.456(P<0.001).Based on these findings,we constructed a second-generation car targeting EGFR.Based on clinical characteristics of these resistant patients,we constructed a second-generation CAR targeting human EGFR protein.CAR-T cell was prepared using peripheral T cells from healthy volunteers.Flow cytometry showed that the expression rate of EGFR CAR was 85.41%.We screened H1975 cell and H1975 OR cell as positive target cells,MCF-7 cells as negative target cells according to the results of flow cytometry.EGFR-CAR T cell can be effectively activated after co-culture with positive target cells.We observed the process of cell proliferation,release of intracellular vesicles containing CD107 and a large number of cytokines like IL-2,IFN-γ,which mediated the specific and efficient killing of cancer cells.We then validated the antitumor effect of EGFR-CAR T cell in vivo by mouse.Change of subcutaneous tumor volume in mice suggested that CAR T cell suppressed the increase of tumor.We also prepared the"EGFR CCR+ROR1 CAR"T cell.EGFR CCR and ROR1CAR were highly expressed in EGFR-ROR1 CAR-T cells,the expression rate were47.18%and 64.19%respectively.We screened H1975 cells and H1975OR cells as positive target cells,MCF-7 cells as negative target cells,H446 and HCTt15 as EGFR~+ROR1~-target cells by flow cytometry.EGFR-ROR1 CAR T cell can effectively proliferate and achieve anti-tumor effect after co-culture with positive target cells.EGFR-ROR1 CAR T cell released more cytokines than EGFR-CAR T cell,but there was no statistical difference with release level of ROR1 CAR T cell.Using microscope,we observed that EGFR ROR1 CAR T cells can aggregate to form distinct T cell clones when cultured with H1975OR cell.As for H446 cells and HCT15 cells,only EGFR-ROR1 CAR T cell and EGFR-CAR T cell could recognize and proliferate effectively.In vivo,mice treated with EGFR-ROR1 CAR T cells had the smallest tumor volume and growth rate,with the longest duration of tumor volume reduction.ConclusionFor patients with EGFR-mutant NSCLC,clinical outcomes of first-line and second-line osimertinib were similar to those in the prospective study.The resistance mechanism of osimertinib is scattered,but EGFR signaling is still the main pathway that tumor cells rely on after resistance.Chemotherapy is the main subsequent therapy of osimertinib-resistant patients.But the benefits outcomes are limited.So,it is necessary to explore new treatment strategies for patients with osimertinib resistance.Both EGFR-mutant and TKI-resistant NSCLC will highly express EGFR.And the expressions of EGFR and ROR1 are consistent.EGFR CAR-T cells and ROR1 CAR-T cells had specific antitumor activity towards EGFR-mutant NSCLC in vivo and in vitro.Dual-targeting EGFR-ROR1 CAR-T cell designed based on the"CCR+CAR"structure also have good anti-tumor activity.In vivo,CAR-T cell designed based on the"CCR+CAR"structure have better anti-tumor effects.Consideing with the data of in vitro research,dual-targeting EGFR-ROR1 CAR-T cells may have better safety.In summary,our research provides a research basis for clinical trials of CAR-T cells in the treatment of patients with EGFR mutations or EGFR TKI resistance.