C3aR costimulation enhances the antitumor efficacy of CAR-T cell therapy through Th17 expansion and memory T cell induction.

Although chimeric antigen receptor (CAR)-modified adoptive T cell therapy is a promising immunotherapy for hematological malignancies, the efficacy improvement in relapsed/refractory acute lymphoblastic leukemia (ALL) with extramedullary infiltration and in multiple myeloma (MM) is still warranted. Since C3aR activation can promote the expansion of tumor-killing Th17 cells, we hypothesized that incorporating C3aR as a costimulatory domain would augment the antitumor activity of CAR-T. In this study, we introduced the C3aR domain into a CAR and generated BB-ζ-C3aR CAR-T targeting CD19 or BCMA. These new CAR-T exhibited a potent cytolytic ability to eradicate tumor cells expressing CD19 or BCMA in vitro. When administered intravenously to ALL or MM xenograft mouse models, BB-ζ-C3aR CAR-T reduced the tumor burden and improved the survival rate. Of note, these CAR-T could effectively eradicate subcutaneous CD19+ tumor cells, highlighting the therapeutic potential in extramedullary leukemia. Mechanistically, BB-ζ-C3aR CAR-T tended to exhibit a Th17 phenotype favoring tumor killing and suppressed Tregs. In addition, the induction of memory T cell in the BB-ζ-C3aR CAR-T cells indicated their long-term effects. Together, our findings suggest that the application of C3aR costimulation boosts the ability of CAR-T to eradicate aggressive tumor cells via Th17 expansion and memory T cell induction.

To the Editor:

The genetic engineering of T cell to express chimeric antigen receptors (CAR) is recognized as a promising approach for hematological malignancies, but the effects of CAR-T cell therapy on relapsed/refractory acute lymphoblastic leukemia (ALL) with extramedullary infiltration and on multiple myeloma (MM) are limited and need to be improved [1-3]. The costimulatory molecule domains in CAR are required for the activation, expansion, and survival of CAR-T. Currently, the optimal costimulatory molecules are still under investigation [4, 5]. C3aR, the receptor that recognizes the complement fragment C3a, not only mediates innate immune responses but also participates in the induction of T cell responses [6-8]. Thus, we introduced the C3aR domain (Additional file 1: Fig. S1a) to the 3’ end of CD3ζ, which followed the 4-1BB domain, to generate a novel type of BB-ζ-C3aR CAR.

To evaluate the efficacy of T cell bearing this new CAR in ALL or MM, an anti-CD19 scFv or anti-BCMA scFv was included in the CAR (Additional file 1: Fig. S1b-e). Initially, we detected the activity of 19-BB-ζ-C3aR CAR-T in vitro. They showed stronger cytotoxicity to tumor cells than did 19-BB-ζ controls (Fig. 1a). In vivo, NOD-SCID-IL2rg−/− (NCG) mice received an intravenous injection of NAML6-Luc cells, followed by treatment with CAR-T (2 × 106 cells intravenously administered on D2 and D8). Then, the mice were examined by serial bioluminescence imaging (BLI) (Fig. 1c). As expected, the effect of 19-BB-ζ-C3aR CAR-T was more pronounced in tumor eradication, with a better survival rate achieved than in those mice treated with the 19-BB-ζ control (Fig. 1d–f). Furthermore, lowest expression of PD-1 and fewest CD19+ blasts were found in the 19-BB-ζ-C3aR CAR-T cell group, whereas no differences were observed in the numbers of GFP+ CAR-T cell, CD4+ and CD8+ T cell among three groups (Additional file 2: Fig. S2; Fig. 1g–j).

Fig. 1

19-BB-ζ-C3aR CAR-T cells displayed potent anti-leukemia activity in vitro and in vivo, particularly in the xenografts extramedullary leukemia model. a The 19-BB-ζ-C3aR CAR-T cells showed significantly increased ability to lyse CD19-expressing tumor cells compared to 19-BB-ζ CAR-T cells. The cytotoxicity assay was performed at least three independent experiments. b Flow-cytometry results revealed enhanced expansion of Th17 cells and reduced Tregs in the 19-BB-ζ-C3aR CAR-T cells compared to 19-BB-ζ and mock-transduced T cells. c To establish the ALL model, 5 × 105 NAML6-Luc cells were administered intravenously into NCG mice, which were randomized to the treatment with 2 × 106 indicated T cell on Day 2 and Day 8. NAML6 tumor growth was then monitored by Xenogen imaging. d Bioluminescence images of NCG mice at Days 7, 14, 19, 26, and 33 are depicted for each group. e The curve of flux on indicated time points. f Kaplan–Meier survival analysis for ALL mice. Log-rank tests were used to perform statistical analyses of survival between groups. g The 19-BB-ζ-C3aR CAR-T group showed significantly fewer blast counts than Mock and 19-BB-ζ CAR-T groups. h The detectable GFP-positive T cells were similar in three groups. i, j There were no differences in CD4+ and CD8+ T cells between the indicated T cell populations. k Xenograft extramedullary leukemic model was established by subcutaneous injection of 5 × 105 NALM-6 cells. The indicated CAR-T with 2 × 106 dose were intravenously injected on Day 3 and Day 8, respectively. l NAML6 subcutaneous tumor growth was monitored by Xenogen imaging. m The curve of flux on indicated time points. n, o The tumor mass and weight were measured and recorded. p The 19-BB-ζ-C3aR CAR-T group showed the lowest CD19+ ALL blast counts on Day 22. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05, n.s. no significant

Notably, to validate the potential of 19-BB-ζ-C3aR CAR-T in eradicating extramedullary leukemic cells, a subcutaneous leukemia mouse model was established by subcutaneously injecting 5 × 105 NALM-6 cells (Fig. 1k). Strikingly, 19-BB-ζ-C3aR CAR-T significantly suppressed the subcutaneous tumor growth (Fig. 1i–m). Although no significant differences were found in tumor volume or weight between both CAR-T cell groups (Fig. 1m, o), more mice treated with 19-BB-ζ-C3aR CAR-T cells achieved complete tumor regression (Fig. 1l) and had fewer CD19-expressing tumor cells (Fig. 1p), highlighting their potent efficacy in extramedullary leukemia.

Similarly, BB-ζ-C3aR CAR-T targeting the BCMA antigen displayed better activity and efficacy than BCMA-BB-ζ CAR-T in vitro (Additional file 3: Fig. S3a). In vivo, MM-bearing mice receiving BCMA-BB-ζ-C3aR CAR-T showed the lowest tumor burden (Fig. 2a–c) and the longest survival time (Fig. 2d) with the fewest BCMA-expressing tumor cells (Fig. 2e). Thus, BB-ζ-C3aR CAR-T cells targeting BCMA possessed potent antitumor activity against MM.

Fig. 2

The BB-ζ-C3aR CAR-T cells significantly eradicated BCMA-expressing tumor cells through favoring Th17 cells expansion and memory T cells induction. a IM9-Luc cells with 5 × 105 dose were administered intravenously into NCG mice to establish the MM model. These mice were randomized to the treatment of 2 × 106 indicated T cell on Day 8 and Day 12. IM9 tumor growth was then monitored by Xenogen imaging. b Bioluminescence images of MM mice on Days 7, 14, 21, 28, and 35 are depicted for each group. c The curve of flux on indicated time points. d Kaplan–Meier survival analysis showed the longest time of survival in BCMA-BB-ζ-C3aR CAR-T cells group. e Hardly any BCMA+ tumor cells were detected in peripheral blood from mice treated with BCMA-BB-ζ-C3aR CAR-T cells. f In the xenograft MM mice, the BCMA-BB-ζ-C3aR CAR-T cells promoted the generation of IL-17-expressing Th17 cells and reduced the Tregs compared to the BCMA-BB-ζ CAR-T cells group. g A fraction of CD4+ or CD8+ cells exhibited the features of central memory cells (Tcm) with notably high expression of CCR7 and CD45RO. Tcm cells in CD8+ compartment were increased in the BCMA-BB-ζ-C3aR CAR-T-treated mice compared to BCMA-BB-ζ controls. In addition, the percentage of CD45RO+CCR7− effector memory cells (Tem) was significantly increased in both CD4+ and CD8+ compartments in the BCMA-BB-ζ-C3aR CAR-T cells. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05, n.s. no significant

Mechanistically, we found that C3aR incorporation improved the generation of Th17 cells while suppressing the differentiation of Tregs (Fig. 1b, Additional file 4: Fig. S4a, b). Consistently, the BB-ζ-C3aR CAR-T produced high level of IL-17, IL-22, GM-CSF, and IP-10 (Additional file 5: Fig. S5). Importantly, IL-17A blockade by secukinumab could abolish the cytotoxicity of 19-BB-ζ-C3aR CAR-T cells, indicating that IL-17A/Th17 was required for the tumor eradication process (Additional file 6: Fig. S6). In vivo, both the ALL and MM models exhibited an increase in Th17 cell and a decrease in Tregs after BB-ζ-C3aR CAR-T cell administration (Additional file 4: Fig. S4c, Fig. 2f), indicating that C3aR incorporation induced CAR-T to adopt the Th17 phenotype instead of differentiating into Tregs. In addition, we assessed memory T cell subsets to evaluate the persistence of CAR-T. In both the CD4+ and CD8+ compartments, enrichment of T central memory cells (Tcm) was observed in the 19-BB-ζ-C3aR group (Additional file 4: Fig. S4d). BCMA-BB-ζ-C3aR CAR-T cell treatment also presented increases in T central memory cells (Tem) and Tcm cells (Fig. 2g), suggesting that C3aR incorporation promoted the memory function of CAR-T.

In summary, we reported that C3aR, a novel costimulatory domain, significantly enhanced the antitumor ability of CAR-T and specifically improved therapeutic efficacy in extramedullary leukemia. BB-ζ-C3aR CAR-T promoted tumor eradication with long-term effects through Th17 expansion and memory T cell induction. These results not only highlight the importance of optimizing CAR engineering but also provide evidence that BB-ζ-C3aR CAR-T cells may be effective in treating refractory tumors, such as extramedullary leukemia and solid tumors.

Additional file 1: Fig. S1. Generation of the BB-ζ-C3aR CAR-T cells targeting CD19 or BCMA. a The amino acid sequence of incorporated C3aR domain. b Schematic representation of chimeric antigen receptors that contain C3aR as a costimulation signal associated with 4-1BB in the intracellular domains, CD19-scFv, and the CD3ζ signal transduction domain. c Transduction efficiency of T cell was determined by flow cytometry. Representative results are from at least three independent experiments. d Schematic representation of chimeric antigen receptors that contain the C3aR as a costimulation signal associated with 4-1BB in the intracellular domain, BCMA-scFv, and the CD3ζ signal transduction domain. e Transduction efficiency of T cell was determined by flow cytometry.

Additional file 2: Fig. S2. The expression of PD-1 was reduced in the 19-BB-ζ-C3aR CAR-T cells. The expression of PD-1 on T cell was determined by flow cytometry. The results showed that BB-ζ-C3aR CAR-T cells presented lower expression of PD-1 compared with mock T or BB-ζ CAR-T cells. *p ≤ 0.05.

Additional file 3: Fig. S3. BCMA-BB-ζ-C3aR CAR-T cells exhibited potent anti-tumor activity in vitro. a The results of cytotoxicity assay showed that BCMA-BB-ζ-C3aR CAR-T cells have improved the ability to lyse BCMA+ MM cells compared to BCMA-BB-ζ CAR-T cells. b Flow cytometry and the statistics revealed that BCMA-BB-ζ-C3aR CAR-T cells treatment group showed more IL-17-expressing Th17 cells and less CD4+CD25+FoxP3+ Tregs compared to BCMA-BB-ζ and mock-transduced T cells group. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

Additional file 4: Fig. S4. C3aR incorporation induced CAR-T cells to display phenotypes of Th17 and memory T cells in vivo. The statistics of IL-17-expressing Th17 cells (a) and CD4+CD25+FoxP3+ Tregs (b) in the co-culture system of CAR-T and CD19 expressing tumors. c In the xenograft leukemic mice, the 19-BB-ζ-C3aR CAR-T cells exhibited elevated expansion of Th17 cell phenotype. d In the 19-BB-ζ-C3aR CAR-T group, Tcm cells were highly induced in CD4+ and CD8+ T cells compared to those from 19-BB-ζ CAR-T group. No difference was observed in the percentage of Tem cells between both groups. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

Additional file 5: Fig. S5. 19-BB-ζ-C3aR CAR-T presented with an expression elevation of some cytokines. In vitro, increased expressions of IL-17, IL-22, GS-CSF, and IP-10 were observed in the 19-BB-ζ-C3aR CAR-T, whereas no differences in TNF-a and IFN-r were found between 19-BB-ζ-C3aR CAR-T and 19-BB-ζ CAR-T. **p ≤ 0.01, *p ≤ 0.05, n.s. no significant.

Additional file 6: Fig. S6. IL-17A blockade by secukinumab impaired the tumor eradication effect of 19-BB-ζ-C3aR CAR-T. In vitro, secukinumab, a human IgG1κ monoclonal antibody that binds to the IL-17A, suppressed the cytotoxicity of 19-BB-ζ-C3aR CAR-T on CD19-expressing NALM6 cells. ***p ≤ 0.001, **p ≤ 0.01.

Additional file 7. Materials and Methods.