Combination Of Targeted Drugs To Improve The Safty And Efficacy Of CAR T Cell Therapy

Background and Aim:Chimeric antigen receptor(CAR)T cell therapy has emerged as a promising treatment for human cancers,such as hematological malignancies,hepatocellular carcinoma,gastric cancer,and pancreatic cancer.However,its safety during treatment and effectiveness against solid tumors still need to be further improved.For example,the administration of CAR T cells against acute lymphoblastic leukemia has a high response rate,but has also been associated with significant adverse events,such as cytokine-release syndrome and neurotoxicity,so how to enhance its safety becomes very urgent.Our previous clinical study about GPC3-targeted CAR T cell therapy showed a high safety profile in patients with hepatocellular carcinoma(HCC).However,the clinical response ratio of GPC3-CAR T cells still needs to be further increased.In this study we tried to apply antibody-drug conjugate CH12-MMAF and a new safety switch to increase the safety of CAR-T cells.We also tried to increase the antitumor activities of GPC3-directed CAR T cells by the combined usasge of sorafenib,an FDA-approved tyrosine kinase inhibitor.Methods:Antibody-drug conjugate CH12-MMAF was applied to eliminate CAR T cells for improved security in the case of adverse events.We engineered CAR T cells with a fusion receptor(FR806),which can be selectively targeted by CH12-MMAF and mediate its internalization.The expression of FR806 and CAR was detected by flow cytometry.Cytotoxicity of CH12-MMAF to FR806-expressing CAR T cells was evaluated both in vitro and NOD/SCID mice.To enhace the anti-tumor efficacy of GPC3-CAR T cell therapy,we combined GPC3-CAR T cells with a multikinase inhibitor,sorafenib.First,we engineered mouse splenic T cells with GPC3-targeted CAR and applied it in a transplantable mouse HCC model established with a murine hepatocarcinoma cell line Hepa1-6-ch GPC3 in immune-competent mice.Then,we investigated the impact of sorafenib on the activities of mouse GPC3-CAR T cells in vitro and tested the combined effect of mouse GPC3-CAR T cells and sorafenib against murine HCC xenografts in vivo.The interaction between sorafenib-treated macrophages and mouse GPC3-CAR T cells was further observed to study their synergy mechanism.At last,we evaluated the impact of sorafenib on the function of human peripheral blood mononuclear cell(PBMC)-derived CAR T cells in vitro,and tested the combined effect of sorafenib and human CAR T cells in an established human HCC xenografts in immune-compromised mice.The synergistic mechanism in immunodeficient mice was further explored by immunohistochemical staining.Results:In the results about increasing the safety of CAR T cells,we found that the expression of FR806 allows CAR T cells to be targeted with CH12-MMAF,an antibody-drug conjugate recognizing the 806 epitope but not wild-type EGFR in healthy tissues.Antibody-drug conjugate CH12-MMAF could be efficiently internalized into the FR806-expressing T cells and potently lyse these cells.In immuno-compromised mice,CH12-MMAF eliminated most of the transferred targeted CAR T cells expressing FR806.In the results about GPC3-CAR T cells,we found that mouse GPC3-CAR T cells induced regression of small-sized tumors(about 130 mm~3 in tumor volume),whereas had no effect on large established tumors(about 400 mm~3 in tumor volume)in an immune-competent mice model.We further showed that pharmacologic doses of sorafenib(10?M in vitro;30 mg/kg in vivo)strongly impaired the functions of mouse GPC3-CAR T cells in vitro and vivo,while sub-pharmacologic doses of sorafenib(1-5?M in vitro;7.5 mg/kg in vivo)augmented the anti-tumor effects of mouse GPC3-CAR T cells,partly because of the promotion of IL-12secretion in tumor-associated macrophages(TAMs).At last,we found that both sub-pharmacologic and pharmacologic doses of sorafenib had limited impact on the function of human GPC3-CAR T cells in vitro,and showed synergistic effects with human GPC3-CAR T cells in vivo in immune-compromised mice model.Immunohistochemical staining analysis indicated a synergistic promotion of apoptosis between sorafenib and CAR T cells.Conclusions:This study proved that antibody-drug conjugate CH12-MMAF can be applied to eliminate CAR T cells expressing FR806.The high specificity of CH12-MMAF to 806 epitope and the internalization capacity of FR?make CH12-MMAF&FR806 system as a useful tool to increase the safety of CAR T cell therapy.Additionally,sorafenib can be applied to enhance the anti-HCC activity of GPC3-CAR T cells and demonstrated the potentiality of a clinical combination of sorafenib with GPC3-CAR T cells against hepatocellular carcinoma.