| BackgroundCancer is a major public health problem.The prevalence of multiple myeloma in China is about 3/100,000.The application of targeted drug therapy,radiotherapy and chemotherapy has improved its remission rate,but the 5-year survival rate is still less than 25%.Furthermore,the solid tumors including the most common types of lung and liver cancers are mainly treated by surgery and 70%of these patients are in the advanced stages of cancers when they are first diagnosed;therefore,they lost the opportunity of radical radiotherapy.CAR-T cell therapy is to genetically engineer T cells to recognize and specifically kill tumor cells in a major histocompatibility complex-independent manner.At present,four generations of CAR-T therapies have been developed based on the structures and numbers of costimulatory signals contained.At present,the FDA only approved the second-generation CAR-T therapy,which complete remission rate of current CAR-T therapy for R/R MM is 20%-65%and the recurrence rate within one year after remission is 30-50%.For advanced solid tumors,the challenge is ineffective initial treatment and an urgent need to develop new therapies.Research purpose and significance(1)To develop a safe,efficient and durable immune cell therapy,we designed and constructed the fourth generation CAR-T cells targeting multiple myeloma cells BCMA namely BCMA-7.21 CAR-T cells,based on the fourth-generation CAR-T technology that secretes the third T cell costimulatory signal including interleukin 7(IL-7)and chemokine 21(CCL21).IL-7 promotes T cell proliferation,inhibits apoptosis/functional exhaustion and CCL21 recruits dendritic cells/peripheral T cells to clear MM cells,resulting in improved immune cell infiltration.(2)The fourth-generation CAR-T cells(secreting IL-7 and CCL19)that target solid tumor cell adhesion protein 4(Nectin4)and cancer-associated fibroblasts FAP in the tumor stroma namely Nectin4-7.19 CAR-T and FAP-7.19 CAR-T cells,are used in intravenous infusion adjuvant therapy for advanced solid tumors with positive Nectin4 and FAP immunohistochemical tests.It can combat and remove solid tumor cells and their cancer-associated fibroblasts(CAFs)in the tumor microenvironment,and synergistically or complementarily improve the tumor immunosuppressive microenvironment,thereby activating the host immune system to produce intrinsic active anti-tumor immune effects,thereby improving the efficacy.Research contents and methods(1)To design and construct BCMA-7.21 CAR-T cells targeting myeloma cells,and quantified the secretion,proliferation and chemotactic functions of IL-7 and CCL21.The cytotoxic effect of BCMA-7.21 CAR-T cells to three tumor cells in vitro was conducted by luciferase-based bioluminescence imaging and flow cytometry.A xenogeneic MM model was constructed to demonstrate the in vivo anti-tumor effect of the animal model(2)The Simon two-stage design was selected as the clinical trial method to decide early invalidation termination.Setting parameters:The expected complete remission rate,the control group complete remission rate,with a and β,using for calculate the sample size and evaluation indicators for each stage.The research endpoint was determined based on the 2016 International Myeloma Task Force Response Evaluation Criteria.he 2010 WHO Response Evaluation Criteria were used for Dual Diameter Measurement of Solid Tumors.Results1.Preclinical studies of fourth-generation BCMA-7.21 CAR-T cells for R/R MM(1)Preparation of fourth-generation BCMA-7.21 CAR-T cells and second-generation BCMA CAR-TLentivirus transduced T cells to construct the second-generation BCMA-7.21 CAR-T cells and the fourth-generation BCMA-7.21 CAR-T cells.The expression rates of CAR were 52.7%and 51.1%,respectively.Construction three cell lines which expressing BCMA and luciferase simultaneously.(2)Cell subtypesBCMA-7.21 CAR-T cells showed higher stem-cell like memory T cell(Tscm)ratio by flow cytometry,this may be related to delayed terminal differentiation.No significant difference in the proportions of CD4 and CD8.(3)IL-7 and CCL21 functionELISA quantification assays shown the BCMA-7.21 CAR-T cells secreted high levels IL-7 and CCL21 in the supernatant.Through microscopic observation,absolute number recording and CFSE labeled the fourth-generation BCMA-7.21 CAR-T cells were more obviously clustered and grew faster(P=0.0043).Migration assay demonstrated that CCL21 could promote T cell migration(P<0.0001).(4)In vitro cytotoxicity functionCAR-T cells were co-cultured with three target cells to quantify their in vitro cytotoxicity functions,data shown that BCMA-7.21 CAR-T can specifically killed tumors at different target ratios,especially at low effect-target ratio,the cytotoxicity of BCMA-7.21 CAR-T was better than the second-generation BCMA CAR-T cells(70%vs 53%,P=0.0001).No significant difference in the expression of IL-2,IFN-y and TNF-α.The level of CD 107 α was higher and the proportion of late apoptotic cells was lower in BCMA-7.21 CAR-T cells,while the expressions of CD69,PD1.(5)Efficacy of BCMA-7.21 CAR-T cells on myeloma xenograft modelMyeloma cell MM1S-luc2 which expressing both BCMA antigens and luciferase proteins,was injected intravenously into NSG mice to generate a xenogeneic model with MM.After intravenous infusion of CAR-T cells,optical imaging results showed that the fourth generation BCMA-7.21 cells have better efficacy and longer lasting effect than the second generation BCMA CAR-T cells on eliminate multiple myeloma.2.Prospective clinical trial of fourth-generation BCMA-7.21 CAR-T for R/R MM(1)Simon’s two-stage minimum and maximum designThe complete remssion rate of R/R MM in three tertiary hospitals was retrospectively analyzed at 5.2%,and the phase Ⅲ R/R MM clinical trials were Metaanalyzed to evaluate the remission rate in the past five years.Set the expected CR of the experimental group to be 0.45 and the CR of the control group to be 0.1,α=0.05,β=0.10,and the SIMON SAS macro program(n1,n,r1,r)is(5,14,0,3).(2)Patient information and cell preparationAll enrolled patients were ineffective or relapsed after receiving multiple lines of therapy,and eventually developed R/R MM.The median age was 63.5 years(43-75 years),the median treatment line was 3.5(2-6 times),and the ECOG score was 0-2.After lymph node dissection,patients were infused with 2×106/kg of BCMA-7.21 CAR-T cells.(3)Safety and adverse eventsThe incidence of cytokine release syndrome(CRS)was 50%,fever≥ 38℃ was the most common symptom,the median number of days between onset of fever was 1 day(0-5 days),and immune effector cell-related neurotoxicity(ICANS)was 7.1%.The cytokine with the largest median increase after treatment was IL-6.All adverse reactions ranged in severity were elevated from grade 1 to grade 3,and self-limiting,reversible,and could be relieved without specific treatment.(4)Clinical response and persistence analysisThe median follow-up time was 10 months(3-35 months),the complete response rate was 71.4%,and the objective response rate was 100%.Two patients with extramedullary recurrence had significantly reduced tumor volume.Ten patients had marked reductions in bone marrow plasma cells,decreased serum BCMA,and sustained complete remission for up to 35 months after treatment,and were MRD(minimal residual disease)negative.The 1-year PFS(progression-free survival)probability was 70.1%,the 1-year survival probability was 85.1.According to Simon’s two-stage design method,4 patients in the first stage achieved complete remission,and 6 patients in the second stage achieved complete remission,so this therapy is worthy of further clinical trial research.3.A single-arm,prospective clinical trial of fourth-generation CAR-T cells targeting Nectin4/FAP in the treatment of advanced solid tumors(1)Simon’s two-stage minimum and maximum designThe objective response rate(ORR)of patients with solid tumors after treatment was retrospectively studied.The expected ORR of the experimental group was 0.4,and the ORR of the control group was 0.1,α=0.05,β=0.20,calculated by the SIMON SAS macro program(n1,n,r1,r)is(5,15,0,3).(2)Patient information and cell preparationThe tumor targets were screened by histology and showed different degrees of expression of Nectin4 and FAP antigens.The median age was 56 years(32-75 years),the median treatment line was 5.5(2-9),and all received more than 3 treatment lines.Through single cell collection,lentiviral transfection,in vitro expansion and reinfusion of clinical grade Nectin4-7.19 CAR-T(2 × 106/Kg)and FAP-7.19 CAR-T cells(1×106/Kg).(3)Safety and adverse eventsThe incidence of CRS was 73.3%,and 1 patient experienced grade 3 CRS.The incidence of ICANS was 20%,which returned to normal after tocilizumab and symptomatic treatment.The median number of days between onset of fever was 1 day(0-2 days).The largest median fold increase were IL-6 and IL-10,which were related to the grade of CRS(P=0.031,P=0.0207).The peak absolute number of CAR-T cells was not related to the grade of CRS and the efficacy.(4)Clinical responseThe ORR for objective response was 40%,and 6 patients with cell therapy showed significant tumor shrinkage by imaging.The median follow-up period was 7 months(2-15 months),the 1-year PFS probability was 43.6%,the 1-year survival probability was 60%.According to Simon’s two-stage design method,2 patients achieved objective remission in the first stage,and 4 patients achieved objective remission in the second stage,so this therapy is worthy of further clinical trial research.ConclusionCAR-T therapy has changed and improved the therapeutic methods for cancer treatment.We successfully designed and constructed the fourth-generation CAR-T(BCMA-7.21 CAR-T)cells and verified BCMA-7.21 CAR-T cells have stronger T lymphocyte recruitment,proliferation capabilities,and can significantly enhance stem memory T cells in vitro.We developed and validated the effectiveness of BCMA 7.21 CAR-T cells against MM cells in vitro and in vivo studies.Then,we develop the firstin-human clinical trial of single-arm prospective BCMA-7.21 CAR-T cell therapy for R/R MM.The study initially showed excellent safety and efficacy.Moreover,a singlearm prospective clinical trial of advanced solid tumors was carried out using Nectin47.19 CAR-T targeting tumor cells combined with FAP-7.19 CAR-T cells targeting CAF,achieving an objective response rate of 30.7%.According to Simon’s two-stage design,our research showed the fourth-generation CAR-T cells are expected to provide a safe and effective novel therapy for R/R MM and advanced solid tumors. |
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