Chimeric antigen receptors containing the OX40 signalling domain enhance the persistence of T cells even under repeated stimulation with multiple myeloma target cells

Abstract

Persistence of CAR-T cell function is associated with relapse rate after CAR-T therapy, while co-stimulatory agents are highly concerned with the persistence of CAR-T cells. In this study, we designed and constructed a series of BCMA-targeting second-generation CAR constructs containing CD28, 41BB, and OX40 molecules, respectively, to identify the costimulatory domains most favorable for persistence. The results of routine in vitro studies showed that OX40-CAR-T and 41BB-CAR-T had similar antitumor effects and were superior to CD28-CAR-T in terms of proliferation and cytotoxicity. Although difficult to distinguish by conventional functional assays, OX40-CAR-T cells exhibited greater proliferation and enhanced immune memory than 41BB-CAR-T cells with the repeated stimulation assay by BCMA-expressing target cells. In vivo studies further demonstrated that OX40-CAR-T cells had stronger proliferative activity than 41BB-CAR-T cells, which was highly consistent with the in vitro antitumor activity and proliferation results. Our study provides for the first time a scientific basis for designing OX40-CAR-T cell therapy to improve relapse in patients with MM after CAR-T treatment.

Keywords: 41BB; CD28; Chimeric antigen receptor T cells; Costimulatory molecules; Multiple myeloma; OX40; Persistence.

Fig. 1

Characterizations of antitumour efficacy among CD28, OX40 and 41BB based CAR-T cells. A The structure of the BCMA-CAR consists of the same variable region of the BCMA single-chain antibody, the CD8 hinge and transmembrane regions, different costimulatory molecules (from 41BB, CD28, or OX40) and CD3ζ. B Coincubation of effector cells with target 8226 cells for 24 h at a ratio of 5:1 between D10 and D15 (different days were used in different donors) and the supernatant was collected. Cytokines were detected by a human Th1/Th2/Th17 kit using flow cytometry. The qualitative analysis of the expression of IFN-γ, TNF-α was performed with Phyton 3.7 using the Matplotlib package (https://matplotlib.org/) (n = 3 donors). C The expression of the exhaustion-related markers LAG-3, TIM-3, PD-1, and CTLA-4 on T cells expressing BCMA-CAR were measured on day 7 (D7) (n = 3 donors). Data were analyzed with FlowJo software, and graphs were plotted with Phyton 3.7 using the Matplotlib package (https://matplotlib.org/). D The Cell Trace TM CFSE Cell Proliferation Kit was used to detect cell proliferation. On D13, effector T cells were stained with CFSE (CFDA-SE) dye and incubated with target K562 (negative control) and 8226 cells at a ratio of 5:1. After 5 days of incubation, CFSE fluorescence intensity was detected by flow cytometry (K562 data not shown). E Effector T cells were incubated for 24 h with target K562 (negative control) and 8226 cells at E:T ratios of 10:1, 5:1, 2.5:1, and 1:1 on D13. Cytotoxicity was determined from the amount of released LDH in the culture supernatants using an LDH kit at a wavelength of 490 nm. The figure shows the result of effector T cells incubated with the 8226 target cells (n = 3, P < 0.001 and P < 0.01, error bars denote standard deviation)

Fig. 2

OX40-CAR-T cells are the least exhausted and have superior persistence. A Effector cells were subjected to three consecutive repeated stimulations with 8226 target cells (CAR⁺ cells and 8226 target cells at a ratio of 1:1; 3-day interval was used for each stimulation), changes in CAR⁺ cell subtypes were determined with flow cytometry (n = 3). B CAR⁺ cells from different experimental groups after repeated stimulation. As described in A, FlowJo was used for data analysis and presentation (n = 3). C The average copy number of the CAR gene in single cells was detected by qPCR technology on day 7 (D7), D14, and D21 after the cells were activated with anti-CD3/CD28 antibodies (n = 3, P < 0.001 and P < 0.01, respectively. error bars denote standard deviation). D Schematic outline of the mouse model experiment (n = 5). All the Methods and Materials were described in the Additional file 4. E Tumour progression was monitored by IVIS imaging. In order to scientifically show the small gap between different CAR-T, the scales are normalized for PBS and NC group with 1 × 10⁵ ~ 1 × 10⁶, and 4-1BB, CD28 and OX40 CAR-T group with 1 × 10⁴ ~ 1 × 10⁵. F Tumour progression was monitored by total flux of each mice. G The proportion of CAR-T in WBC (white blood cell) were detected from mice PB by flow cytometry on day 7. ***P ≤ 0.001, NS no significant. H Representative GSEA of DNA repair pathways and MSigDB hallmark gene set for two different BCMA-targeted CAR-T cells (OX40-CAR-T cells and 41BB-CAR-T cells). I Representative GSEA of the oxidative phosphorylation pathway and MSigDB hallmark gene sets for different BCMA-targeted CAR-T cells