Genetic engineering of T cells to express chimeric antigen receptors (CARs) is recognized as a promising new approach for relapsed/refractory acute lymphoblastic leukemia1. However, its activity in extramedullary leukemia has not been well-characterized2. The efforts are underway to find strategies that can increase its anti-tumor efficacy, which is highly dependent on the optimal molecular design of the CAR. The CARs are fusion proteins with well-defined functional domains, among which the costimulatory molecule domains are required for activation, expansion and survival of CAR-T cells. Several costimulatory molecules such as CD28, 4-1BB, ICOS, OX-40, CD27, TLR2 and so on have been tried to incorporated into CAR and can influence the function of CAR-T cells2,3. C3aR, the G protein-coupled receptor for the complement fragment C3a, plays a role in the induction and regulation of effector CD4 and CD8 T cells responses4. Previous studies found C3aR signaling direct CD4 T cells differentiation towards tumor-killing Th17 cells and against the regulatory effect on Treg commitment, highlighting the potential role of C3aR5,6. Here we introduced the C3aR domain to the 3’ end of CD3ζ, which was followed 4-1BB domain, to generate a new BB-ζ-C3aR CAR-T cell.
Then we would like to evaluate whether C3aR costimulation incorporation play a role in the efficacy of CAR-T cell therapy regardless of targeted antigen. Here the BB-ζ-C3aR CAR-T cell were established with targeting CD19-specific or BCMA-specific tumor antigen (Additional file 1: Fig. S1). The CAR constructs contain the CD19-scFv or BCMA-scFv and CD3ζ signal transduction domain with the 4-1BB and C3aR signaling domains in tandem (Additional file 1: Fig. S1a, c). All CAR vectors were efficiently transduced and the CAR-GFP were effectively expressed in transduced T cells (Additional file 1: Fig. S1b, d).
Initially, we detected the activity of the novel 19-BB-ζ-C3aR CAR-T cells in vitro. NALM6, Raji, and K562.CD19 cell lines were applied as tumor model in vitro. Incubation of CAR-T cells with these tumor cells resulted in significant potent cytotoxicity at indicated ratios of effector:target. Both 19-BB-ζ-C3aR and 19-BB-ζ CAR-T cells lysed CD19-expressing NALM6, Raji, and CD19-K562 compared with mocked T cells, with the cytotoxicity was significantly higher in 19-BB-ζ-C3aR CAR-T cells (Fig. 1a). Of note, we further found that C3aR incorporation improved the generation of IL-17-expressing Th17 cells while suppressed the differentiation of Foxp3 + CD25 + CD4 + Treg cells compared to the mock transduced T or 19-BB-ζ CAR-T cells (Fig. 1b, Additional file 2: Fig. S2a, b).
To test the in vivo activity of 19-BB-ζ-C3aR CAR-T cells, NCG (NOD-SCID-IL2rg−/−) mice were injected intravenously with NAML6-ffLuc and treated with twice i.v. injections of indicated CAR-T cells (1 × 105 via tail vein injection on Day 2 and Day 8). These mice were then followed with serial bioluminescence imaging (Fig. 1c). As expected, mice treated with mock transduced T cells succumbed quickly to disease, whereas mice treated with CAR-T cells showed significant reduction of leukemia. Of note, the 19-BB-ζ-C3aR CAR-T cells infusion got a more pronounced effect of tumor eradication than that of 19-BB-ζ control (Fig. 1e). Consistently, the mice received 19-BB-ζ-C3aR CAR-T cells exhibited a better survival advantage (Fig. 1d). Furthermore, 19-BB-ζ-C3aR CAR-T cells showed the most potent ability to eradicate the CD19 blasts in blood, whereas no difference on the accounts of GFP + CAR-T cells, CD4 + T, and CD8 + T cells in circulation were observed among groups (Fig. 1f-i).
As we known, extramedullary relapse is an aggressive type of leukemia and the conventional CAR-T cells had shown little evidence of antitumor activity against it7. It is important to validate the therapeutic potential of the novel CAR-T cells to eradicate extramedullary leukemia cells. We therefore established a subcutaneous leukemia model and examined whether 19-BB-ζ-C3aR CAR-T cells harbored cytolytic activity against extramedullary leukemia. The immunodeficient NCG mice received a subcutaneous injection of 2×105 NALM-6 cells that were tagged with GFP and luciferase (Fig. 1j). The Bioluminescence imaging (BLI) results revealed that tumors were significantly suppressed in mice treated with 19-BB-ζ-C3aR in comparison to 19-BB-ζ CAR-T cells (Fig. 1l). The tumor volume was examined at indicated time points and the extracted tumors were measured. Although there was no significant statistic difference of tumor volume and weight between 19-BB-ζ CAR-T and 19-BB-ζ-C3aR CAR-T cell group (Fig. 1k,m), more tumor-bearing mice after 19-BB-ζ-C3aR CAR-T cell infusion attained thorough removal (Fig. 1l) and less CD19-expressing tumor cells were detected (Fig. 1n), highlighting the potent efficacy of 19-BB-ζ-C3aR CAR-T cells against extramedullary leukemia. Taken together, this novel CAR with C3aR incorporation displayed enhanced anti-leukemia efficacy particularly in the subcutaneous leukemia-bearing xenografts.
Similarly, the activity and efficacy of the BB-ζ-C3aR CAR-T cells targeting BCMA antigen were evaluated in vitro and in vivo. The IM9, MM1S, K562, and K562.BCMA cell lines were utilized as BCMA-expressing tumor model in vitro. Strikingly, the BCMA-BB-ζ-C3aR CAR-T cells exhibited the most potent cytotoxicity, particularly in the MM1S culture system (Additional file 3: Fig. S3a). These BCMA-BB-ζ-C3aR CAR-T cells preferred to present with Th17 phenotype and away from tumor-tolerated Treg phenotype (Additional file 3: Fig. S3b). In vivo, the NCG mice was utilized to establish the multiple myeloma model by intravenous injection of IM9-ffLuc. At the day of 8 and 12, these mice received twice corresponding T cell therapies and monitored by serial BLI (Fig. 2a). As expected, the mice receiving BCMA-BB-ζ-C3aR CAR-T cells showed a lowest tumor burden (Fig. 2b) and a longest survival time (Fig. 2c) without any BCMA-expressing tumor cells (Fig. 2d). Thus, these data suggested that BB-ζ-C3aR CAR-T cells targeting BCMA possessed potent anti-tumor activity.
Finally, to evaluate the underlying mechanism of C3aR incorporation-mediated anti-tumor activity, the Th17 and Treg phenotypes were examined in the peripheral blood from NCG tumor mice after individual CAR-T cell therapy. A significant elevation of Th17 cells was observed in the 19-BB-ζ-C3aR CAR-T cell-treated mice, whereas the 19-BB-ζ signaling did not favor the expansion of Th17 cells compared to the mock control (Additional file 4: Fig. S4a). In contrast, the 19-BB-ζ-C3aR CAR-T cells infusion resulted in lowest percentage of Treg cells (Additional file 4: Fig. S4a), which facilitated tumor progression. Similarly, the BCMA-BB-ζ-C3aR CAR-T cell also promoted Th17 cell percentage and suppressed Treg percentage in tumor-bearing mice (Fig. 2e).
Considering the persistence of CAR-T cells is dependent on the establishment of CAR + memory T subsets8,9. We therefore assessed the expressions of CD45RO and CCR7 in the CD4 and CD8 T subsets to detect the CD45RO + CCR7 + T central memory cells (Tcm) and CD45RO + CCR7- T effector memory cells (Tem) in the blood from CAR-T cells-infused mice. In the CD4 + compartment, the enrichment of Tcm cells was higher in the 19-BB-ζ-C3aR CAR group in comparison to the 19-BB-ζ group (Additional file 4: Fig. S4b). Consistently, 19-BB-ζ-C3aR signaling improved the establishment of the Tcm population in the CD8 + compartment (Additional file 4: Fig. S4b). In contrast, both groups yielded a similar proportion of Tem phenotype, indicating that Tcm was predominantly required in the anti-tumor effect of 19-BB-ζ-C3aR CAR-T cells. Interestingly, the BCMA-BB-ζ-C3aR CAR-T cells induced a Tem phenotype in both CD4 and CD8 compartments, and a significant elevation of Tcm phenotype was only observed in CD8 cells (Fig. 2f). Together, these data suggested that C3aR incorporation promoted the memory feature of CAR-T cells.
In summary, we provide the unexpected observation that C3aR incorporation, a novel costimulatory domain for T cells function, had significantly enhanced the potency of CAR-T anti-tumor abilities in vitro and in vivo, particularly for treating extramedullary leukemia. The BB-ζ-C3aR CAR-T cells enhanced the tumor eradication activity through Th17 expansion and induced memory T phenotype for the long-term effect. The results not only highlight the importance of optimizing CAR engineering but also provide evidences that BB-ζ-C3aR CAR-T cells may be efficient for refractory tumors such as extramedullary leukemia and solid tumor.