Introduction: Adoptive T-cell therapy of gene modified to express CD19 chimeric antigen receptor (CAR) unveiled promising clinical outcomes, particularly in B-cell hematologic malignancies. Among several studies, CAR T-cell persistence is considered to be a surrogate marker of long-term clinical efficacy of CAR T-cell therapy. Consequently, various CD19 CAR T-cell costimulatory domains have been developed to improve T-cell signal, ultimately enhanced T-cell function and persistence. However, in the context of T-cell activation signaling, most of costimulatory molecules have already investigated, and no novel signaling domains have been reported to be useful recently. Here, we adopted B-cell signal moiety, CD79A, and dendritic cell signal moiety, CD40, and generated a composite costimulatory domain CD79A/CD40 to synergize T-cell signaling in order to improve T cell proliferation and persistence.
Methods: The CD79A/CD40 costimulatory domain was developed and inserted in the middle of anti-CD19-CD28TM-CD3z-tEGFR construct. Then the novel CD19.79a40z CAR gene was subsequently cloned into retroviral vector and transduced into human CD3+ cells. The CAR expression was assessed and enriched by anti-EGFR mAb, which resulted in achieving more than 90% purity. Then we expanded CD19 CAR T-cell by coculture with irradiated EBV-LCL. These expanded cells were further used for downstream experiments including 51Cr release assay, coculture assay, intracellular cytokine staining assay (IC-CS), T cell proliferation assay, and T cell phenotype, in order to compare with conventional CD19.28z CAR T-cells. In addition, CD19 CAR-transduced Jurkat-Dual luciferase reporter cells were also used to assess NF-κB pathway activity.
Results: After one course of viral transduction and LCL expansion, we obtained similar number of CAR-positive cells between CD19.79a40z and CD19.28z. The CD19.79a40z exhibited similar cytotoxic activities to CD19.28z, against K562-CD19 and two primary B-ALL samples (51Cr release assay). Also with coculture assay, both CD19.79a40z and CD19.28z showed robust cytotoxicity against K562-CD19 at E:T ratio of 1:1 and 1:8 after coculture for 120 hours without IL-2 supplementation. Interestingly, after extended coculture time to 168 hours, a tendency of target cell regrowth in CD19.28z was observed, whereas such regrowth was not seen in CD19.79a40z.
Regarding IC-CS assay against K562-CD19, the proportions of IFN-γ+ and IL-2+ responders were comparable between CD19.79a40z and CD19.28z. Similarly IC-CS assay, ELISA revealed that CD19.79a40z and CD19.28z evenly produced IFN-γ, IL-2, and TNF-α upon exposure to K562-CD19.
Furthermore, we also assessed the ability of CD19 CAR T-cell proliferation by coculture with K562-CD19, without IL-2 supplementation. Surprisingly, the novel CD19.79a40z unveiled robust T-cell proliferation and persistence throughout 2 weeks of culture when compared to CD19.28z (Figure).
We next determined the plausible factors owing to T-cell survival advantage. Firstly, we assessed the surface markers of T-cell subset as well as exhaustion phenotypes, after coculture with K562-CD19 for 10 days. Again, CD19.79a40z showed similar T-cell subset composition and exhaustion markers to CD19.28z. Secondly, we examined the NF-κB activity of CD19 CAR after exposure to target antigens that the novel CD19.79a40z exhibited significantly higher NF-κB activity upon exposure to K562-CD19 and Raji when compared to CD19.28z. [Luciferase activity (units) of CD19.28z vs. CD19.79a.40z: K562-CD19 11306.65±747.63 vs. 21291.93±924.06; Raji 5524.93±321.68 vs. 7537.96±336.16, respectively (p= 0.0001)].
Conclusions: The novel CD19.79a40z was successfully transduced into CD3+ cells with exhibited comparable cytotoxicity, cytokine secretion activities, T-cell subset composition, as well as T-cell exhaustion phenotypes to conventional CD19.28z CAR T-cells. On the contrary, the composite signaling domain, CD79A/CD40, astonishingly enhanced CD19 CAR T-cell proliferation and survival comparing to CD19.28z CAR T-cells. In addition, our construct presented higher NF-κB activity upon antigen exposure, which possibly synergized to T-cell signal and led to a proliferation advantage. Collectively, the greater CAR T-cell expansion might improve CAR T-cell persistence in vivo and ultimately improve the efficacy of adoptive CAR T-cell therapy.
Kiyoi: FUJIFILM Corporation: Patents & Royalties, Research Funding; MSD K.K.: Research Funding; Eisai Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis Pharma K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.
Asterisk with author names denotes non-ASH members.