The BCMA-Targeted Fourth-Generation CAR-T Cells Secreting IL-7 and CCL19 for Therapy of Refractory/Recurrent Multiple Myeloma

Abstract

Chimeric antigen receptor (CAR) technology has revolutionized cancer treatment, particularly in malignant hematological tumors. Currently, the BCMA-targeted second-generation CAR-T cells have showed impressive efficacy in the treatment of refractory/relapsed multiple myeloma (R/R MM), but up to 50% relapse remains to be addressed urgently. Here we constructed the BCMA-targeted fourth-generation CAR-T cells expressing IL-7 and CCL19 (i.e., BCMA-7 × 19 CAR-T cells), and demonstrated that BCMA-7 × 19 CAR-T cells exhibited superior expansion, differentiation, migration and cytotoxicity. Furthermore, we have been carrying out the first-in-human clinical trial for therapy of R/R MM by use of BCMA-7 × 19 CAR-T cells (ClinicalTrials.gov Identifier: NCT03778346), which preliminarily showed promising safety and efficacy in first two enrolled patients. The two patients achieved a CR and VGPR with Grade 1 cytokine release syndrome only 1 month after one dose of CAR-T cell infusion, and the responses lasted more than 12-month. Taken together, BCMA-7 × 19 CAR-T cells were safe and effective against refractory/relapsed multiple myeloma and thus warranted further clinical study.

Keywords: BCMA; CAR-T; CCL19; IL-7; multiple myeloma.

Figure 1

Efficient expression of CAR on lentiviral-transduced T cells and functional verification of IL-7 and CCL19. (A) Schematic representation of BCMA-hBBz CAR and BCMA-7x19 CAR. (B) CAR expressions of CAR-T cells were analyzed by flow cytometry. The numbers in scatter plots represent the percentages of positively stained cells. (C) The expression rates of BCMA CAR and BCMA-7x19 CAR. Data are means± SD obtained from seven donors. MOI = 80. P-value was calculated by two-tailed student t-test. ns, not statistically significant (P > 0.05). (D) Quantitative detection of IL-7 (left) and CCL19 (right) secretion by ELISA. (E) Number fold change of BCMA-hBBz and 7 × 19 CAR-T cells at the indicated time-points. ****P < 0.0001 by two-way ANOVA. (F) The count of migrating T cells (N = 3). ****P < 0.0001 was calculated by two-tailed student t-test. ***P < 0.001.

Figure 2

Phenotype of CAR-T cell. (A) Expression of CD45RA and CD45RO on CAR-T cells subsets in CD62L⁺ cells by flow cytometry. Tcm (CD62L⁺CD45RO⁺CD45RA⁻), Tscm (CD62L⁺CD45RO⁻CD45RA⁺). (B) Statistical chart of subset distribution. (C) CD4/CD8 ratios of CAR-T cells were analyzed by flow cytometry. (D) Change of Tscm ratio at the indicated time-points. P-value was calculated by two-way ANOVA. *P < 0.05, ****P < 0.0001.

Figure 3

Cytotoxicity analysis of BCMA-hBBz and 7 × 19 CAR-T cells in vitro and in vivo. (A) MM1S-Luc-GFP, U266-Luc-GFP and BCMA-K562 cell lines stably expressing BCMA and luciferase. (B) CAR-T cells and target tumor cells were co-incubated for 4 h at the indicated E:T ratios. Cytotoxicity assay with BCMA-K562 (left), MM1S-Luc-GFP (middle) and U266-Luc-GFP cells as targets (right). Differences between groups were determined using two-way ANOVA. Mean ± SD, ****p < 0.0001. (C) Cytokine release by CAR-T cells in response to multiple myeloma cell lines. CAR-T or mock-T cells were incubated with MM1S-Luc-GFP cells at 1:1 for 24 h, IL2 (left), IFN-γ (middle) and GM-CSF (right) were analyzed by intracellular staining or ELISA. P-value was calculated by two-tailed student t-test. ***P < 0.001. ns, not statistically significant (P > 0.05). (D) Flow chart of animal experimentation. (E). On day 0, NSG mice were injected intravenously with 4 × 10⁶ BCMA-K562 cells. On day 7, mice received 6 × 10⁶ BCMA-7 × 19 CAR-T cells (n = 3), BCMA-hBBz CAR-T cells (n = 3) or mock-T cells (n = 3). Luciferase bioluminescent imaging analysis on days 7, 10, 17, and 24. (F) Average bioluminescent signal for each group in different days [mean radiance (p/s/cm²/sr)] ±SD.

Figure 4

Clinical efficacy after BCMA-7 × 19 CAR-T infusion. (A) Duration of response and survival after BCMA-7X19 CAR-T cell infusion. (B) Scheme for CAR-T preparation and treatment. (C) Patient 1, serum immunoglobulin IgG, IgA, IgM are in the normal range, both before and after infusion. (D) Patient 1, representative CT scans at different baselines (baseline1: First extramedullary recurrence of multiple myeloma; 1 year after Len and DXM induction therapy shown in baseline 2: progression of extramedullary recurrence; baseline 3: 1 month before BCMA-7X19 CAR-T infusion); and CT scans post-CAR-T cell infusion 1, 4, and 10 months. Scale bars, 1 cm. (E) Patient 2, monitoring of urine light chain protein and (F) Serum immunoglobulin IgG, Ig A, Ig M levels before and after treatment. (G) Serum kinetics of a panel of cytokines in patients who received infusions with high doses of CAR-T cells, as determined by Luminex multiplex assay (R&D Systems) Horizontal lines denote mean values.