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Coexpression of IL-7 and CCL21 Increases Efficacy of CAR-T Cells in Solid 2 Tumors Without Requiring Preconditioned Lymphodepletion

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Coexpression of IL-7 and CCL21 Increases Efficacy of CAR-T Cells in Solid 2 Tumors Without Requiring Preconditioned Lymphodepletion Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/1078-0432.CCR-20-0777 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: Coexpression of IL-7 and CCL21 increases efficacy of CAR-T cells in solid 2 tumors without requiring preconditioned lymphodepletion 3 Running title: IL-7 and CCL21 enhance antitumor efficacy of CAR-T cells 4 Hong Luo1†, Jingwen Su 2†, Ruixin Sun2†, Yansha Sun2, Yi Wang1, Yiwei Dong2, Bizhi 5 Shi2, Hua Jiang2*, Zonghai Li1,2,3* 6 1. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 7 Renji Hospital, School of biomedical Engineering, Shanghai Jiao Tong University, 8 Shanghai, China. 9 2. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 10 Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 11 3. CARsgen Therapeutics, Shanghai, China. 12 †These authors contributed equally to this work. 13 14 Key words: CAR T, Immune cell therapy, IL-7, CCL21, Solid tumor 15 16 Additional information: 17 Funding: This work was supported by the Supporting Programs of the National 18 Natural 19 Science Foundation of China (No. 81871918, 81872483, 31800659), the Grant from 20 the State Key Laboratory of Oncogenes and Related Genes (ZZ-20-11RCPY). 21 *Corresponding Author: Zonghai Li and Hua Jiang, State Key Laboratory of 22 Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai 23 Jiaotong University School of Medicine, No.25/Ln2200, XieTu Road, Shanghai 24 200032, China. 25 E-mail addresses: [email protected]; [email protected] 26 Conflict of Interest: Author Dr. Zonghai Li has ownership interests of CAR-T cells 27 coexpressing with IL-7 and CCL21. All other authors declare no competing interests. 28 Other notes: word count: 4995; the total number of figures and tables: 6 29 30 Translational Relevance 31 There is limited success in using chimeric antigen receptor (CAR) T cells to treat 32 solid tumors. One of the major reasons is the suppressive tumor microenvironment 33 that impairs the infiltration, expansion and persistence of CAR-T cells in tumor 34 tissues. In this study, we engineered CLDN18.2 specific CAR-T cells to co-express 35 cytokines IL-7 and CCL21 (7×21 CAR-T). Our studies indicated that IL-7 and 36 CCL21 can enhance the survival of CAR-T cells and infiltration of T cells and 37 dendritic cells in tumor tissues. 7×21 CAR-T cells can efficiently destroy different 38 solid tumors with or without preconditioned lymphodepletion chemotherapy and 39 suppress the tumor growth with heterogenous antigen expression in vitro and in vivo. 40 These findings suggest that 7×21 could boost CAR-T cells antitumor activity and may 41 be served as a potential therapy strategy for solid tumors. 42 43 Abstract 1 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/1078-0432.CCR-20-0777 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 44 Purpose: T cell recruitment, survival and proliferation are the important limitations to 45 chimeric antigen receptor (CAR) T cells therapy in the treatment of solid tumors. In 46 this study we engineered CAR-T cells to co-express cytokines IL-7 and CCL21 (7×21 47 CAR-T), a cytokine combination in order to improve proliferation and chemotaxis of 48 CAR-T cells. 49 Experimental design: CLDN18.2 specific second-generation CAR-T cells 50 coexpressing cytokines were prepared using retroviral vector transduction. The 51 proliferation and migration of genetically engineered CAR-T cells were evaluated in 52 vitro. The antitumor activities of genetically engineered CAR-T cells were evaluated 53 against multiple solid tumors in C57BL/6 mice in vivo. 54 Results: In vitro, the proliferation and chemotaxis of 7×21 CAR-T cells are 55 significantly improved when compared to those of the conventional CAR-T cells. In 56 vivo, 7×21 CAR-T cells revealed superior therapeutic effects to either conventional 57 CAR-T cells or 7×19 CAR-T cells which co-expressing IL-7 and CCL19 as previous 58 reported in three different solid tumors without cyclophosphamide (CPA) 59 precondition. Interestingly, 7×21 CAR-T cells could also suppress the tumor growth 60 with heterogenous antigen expression and even induce tumor complete remission. 61 Mechanistically, IL-7 and CCL21 significantly improved survival and infiltration of 62 CAR-T cells and DCs in tumor. Additionally, CCL21 also inhibited the tumor 63 angiogenesis as proved by immunohistochemistry. 64 Conclusion: Coexpression of IL-7 and CCL21 could boost CAR-T cells antitumor 65 activity and 7×21 CAR-T cells may be served as a promising therapy strategy for 66 solid tumors. 67 68 Introduction 69 The successful antitumor activities of second-generation (conventional) chimeric 70 antigen receptor (CAR) T cells have been proved for the treatment of B-cell leukemia 71 and lymphoma(1). However, limited success has been made in using CAR-T cells to 72 treat solid tumors. The hypoxic and extracellular matrix-rich tumor microenvironment 73 prevents T cells from infiltrating the tumor tissue. Furthermore, inhibitory surface 74 proteins, cytokines or soluble products of disrupted cell metabolism within the tumor 75 can impair the activation and persistence of T cells(2). Recently, in order to improve 76 the infiltration, accumulation and survival of CAR-T cells in solid tumor, CAR-T cells 77 engineered to express IL-7 and CCL19 (7×19 CAR ) have been developed to increase 78 their antitumor activities through enhancing the CAR-T cell survival and T/DC cell 79 infiltration(3). The combination showed remarkable efficacy in cancer 80 immunotherapy, but whether other combinations of the cytokines could be equally or 81 more effective remains to be explored. 82 Chemokines provide specific signaling to leukocytes for extravasation from the 83 blood and direct locomotion as well as microenvironmental homing of leukocytes 84 within tissues(4). In addition to CCL19, CCL21 is another homeostatic chemokine 85 ligand for chemokine (C-C motif) receptor 7 (CCR7), expressed constitutively in 86 endothelial cells of high endothelial venules and in stromal cells within the T-cell 87 zones of secondary lymphoid organs and strongly attract naïve T cells and 2 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/1078-0432.CCR-20-0777 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 88 antigen-presentation cells (APC), such as dendritic cell(5,6). Structurally, CCL21 89 differ from CCL19 in that it has an extra 32 amino acid C-terminus of basic amino 90 acids that may mediate the distinct binding of CCL21 to other molecules (7,8). 91 CCL21 can also bind to the receptor CXCR3 that possess the angiostatic activity to 92 inhibit tumor growth independent of leukocyte recruitment(9,10). 93 Previous studies have shown that CCL21/CCR7 plays a central role in 94 coordinating the meeting between mature DCs and naïve T-cells to initiate a pathogen 95 or tumor antigen-specific T-cell mediated immune response(9,11,12). It was recently 96 shown that the presence of CCL21 in tumor induces infiltration of dendritic cell and 97 CD8+ T cell and leads to immune-mediated inhibition of melanoma(12), lung(11), and 98 colon(13) carcinomas in experimental animals. The chemo-attractive properties of 99 CCL21 were also used in the development of the dendritic cell–based vaccines(14). 100 Treatment of tumor-bearing mice with these genetically altered dendritic cells resulted 101 in tumor growth inhibition and remission in some of the experimental tumors(12,15). 102 In the previous study, the cyclophosphamide (CPA) preconditional 103 chemotherapy was applied to potently increase the 7×19 CAR-T cells function in vivo 104 owing to differences in the susceptibility of tumors to CAR-T cells and/or in the 105 tumor growth rate. However, some patients are intolerant of lymphodepletion 106 chemotherapy and this study had also shown that the potent antitumor ability of 7×19 107 CAR-T cells were enhanced by the recipient T cells infiltration and the interaction of 108 T cells and DCs in tumor tissues. These results suggest that no prior preconditional 109 lymphodepletion is better for CAR-T cells interacted with immune cells in tumor. 110 CCL21-mediated antitumor responses were lymphocyte dependent as evidenced by 111 the fact that intratumoral injection of recombinant CCL21 did not alter tumor growth 112 in severe combined immunodeficiency (SCID) mice but mediate tumor regression in 113 immunocompetent mice(11). What’s more, mice deficient in CCL21 showed defects 114 in lymphocyte homing and dendritic cell localization(16), and mice lacking the CCR7 115 receptor also had defects in lymph node architecture(17). Collectively, these data 116 suggest that CCL21 may play an important role in immune response. Therefore, in the 117 absence of CPA, CAR-T cells armored with CCL21 may recruit effector cells into 118 tumor tissues and have the potential to enhance the antitumor responses by 119 cooperating with the natural immune system. 120 In mice, CCL21 includes three distinct genes of CCL21a, CCL21b and CCL21c. 121 These genes encode two CCL21 proteins distinguished from each other by one amino 122 acid at position 65 (either a serine or leucine residue) (Supplementary Fig. 123 S1a)(18,19). CCL21Ser (CCL21a) and CCL21Leu (CCL21b) proteins are 124 functionally equivalent in attracting CCR7-expressing cells(19) but the former is 125 predominantly detectable in immune organs while the latter is predominantly 126 expressed in nonimmune organs(18).
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