The role of the different circulating regulatory T-cells (Treg) subsets, as well as their correlation with clinical outcome of non-small cell lung cancer (NSCLC) patients is poorly understood. Peripheral blood from 156 stage III/IV chemotherapy-naive NSCLC patients and 31 healthy donors (HD) was analyzed with flow cytometry for the presence and functionality of CD4+ Treg subsets (naive, effector and terminal effector). Their frequencies were correlated with the clinical outcome. All CD4+ Treg subsets exhibited highly suppressive activity by TGF-β and IL-10 production. The percentages of naive Treg were found elevated in NSCLC patients compared to HD and were associated with poor clinical outcome, whereas the percentage of terminal effector Treg was lower compared to HD and higher levels were correlated with improved clinical response. At baseline, normal levels of naive and effector Treg were associated with longer overall survival (OS) compared to high levels, while the high frequency of the terminal effector Treg was correlated with longer Progression-Free Survival and OS. It is demonstrated, for first time, that particular CD4+ Treg subtypes are elevated in NSCLC patients and their levels are associated to the clinical outcome. The blocking of their migration to the tumor site may be an effective therapeutic strategy.

Combination of a CAR and a chimeric costimulatory receptor augments cytotoxicity and durability of T cells and elimination of antigen-low tumors.

Purpose: The microenvironment of multiple myeloma (MM) can critically impair therapy outcome, including immunotherapies. In this context, we have earlier demonstrated that bone marrow mesenchymal stromal cells (BMMSC) protect MM cells against the lytic machinery of MM-reactive cytotoxic T cells (CTL) and daratumumab-redirected natural killer (NK) cells through the upregulation of antiapoptotic proteins Survivin and Mcl-1 in MM cells. Here, we investigated the significance of this mode of immune escape on T cells engineered to express chimeric antigen receptors (CAR T cells). Experimental Design: We tested the cytolytic ability of a panel of 10 BCMA-, CD38-, and CD138-specific CAR T cells with different affinities against a model MM cell line and against patient-derived MM cells in the presence versus absence of BMMSCs. Results: Although BMMSCs hardly protected MM cells from lysis by high-affinity, strongly lytic BCMA- and CD38-CAR T cells, they significantly protected against lower affinity, moderately lytic BCMA-, CD38-, and CD138-specific CAR T cells in a cell–cell contact-dependent manner. Overall, there was a remarkable inverse correlation between the protective ability of BMMSCs and the lytic activity of all CAR T cells, which was dependent on CAR affinity and type of costimulation. Furthermore, BMMSC-mediated resistance against CAR T cells was effectively modulated by FL118, an inhibitor of antiapoptotic proteins Survivin, Mcl-1, and XIAP. Conclusions: These results extend our findings on the negative impact of the microenvironment against immunotherapies and suggest that outcome of CAR T cell or conventional CTL therapies could benefit from inhibition of antiapoptotic proteins upregulated in MM cells through BMMSC interactions.

Due to the CD1d restricted recognition of altered glycolipids, Vα24-invariant natural killer T (iNKT) cells are excellent tools for cancer immunotherapy with a significantly reduced risk for graft-versus-host disease when applied as off-the shelf-therapeutics across Human Leukocyte Antigen (HLA) barriers. To maximally harness their therapeutic potential for multiple myeloma (MM) treatment, we here armed iNKT cells with chimeric antigen receptors (CAR) directed against the MM-associated antigen CD38 and the plasma cell specific B cell maturation antigen (BCMA). We demonstrate that both CD38- and BCMA-CAR iNKT cells effectively eliminated MM cells in a CAR-dependent manner, without losing their T cell receptor (TCR)-mediated cytotoxic activity. Importantly, iNKT cells expressing either BCMA-CARs or affinity-optimized CD38-CARs spared normal hematopoietic cells and displayed a Th1-like cytokine profile, indicating their therapeutic utility. While the costimulatory domain of CD38-CARs had no influence on the cytotoxic functions of iNKT cells, CARs containing the 4-1BB domain showed a better expansion capacity. Interestingly, when stimulated only via CD1d+ dendritic cells (DCs) loaded with α-galactosylceramide (α-GalCer), both CD38- and BCMA-CAR iNKT cells expanded well, without losing their CAR- or TCR-dependent cytotoxic activities. This suggests the possibility of developing an off-the-shelf therapy with CAR iNKT cells, which might even be boostable in vivo by administration α-GalCer pulsed DCs.

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In our previous study, high levels of one granulocytic (G-MDSC) and two monocytic (M-MDSCs) subpopulations of immunosuppressive MDSCs were found and their overexpression was correlated with worse prognosis in NSCLC patients. Using flow cytometry, the impact of chemotherapy on the percentages and functionality of M-MDSC (CD14+CD15-CD11b+CD33+HLA-DR-Lin- and CD14+CD15+CD11b+ CD33+HLA-DR-Lin-) and G-MDSC (CD14-CD15+CD11b+CD33+HLA-DR-Lin-) was evaluated in the peripheral blood of patients (n=141) prior to chemotherapy and after the 3rd and 6th cycle of treatment. The M-MDSC (CD14+CD15-CD11b+CD33+HLA-DR-Lin-) percentages were found significantly decreased post chemotherapy compared to baseline, whereas chemotherapy had no effect on CD14+CD15+CD11b+CD33+HLA-DR-Lin- M-MDSCs. In contrast, the production of iNOS by M-MDSCs was significantly increased compared to the baseline. Both G-MDSC levels and their ROS expression were not affected by chemotherapy. In addition, both subtypes of MDSCs, when co-cultured with CD3+ T cells, were able to significantly suppress IFN-γ secretion by T cells. Finally, the M-MDSC (CD14+CD15-CD11b+CD33+HLA-DR-Lin-) percentages were found significantly decreased in patients that did not develop progressive disease during or after treatment completion, compared to patients with disease progression. Chemotherapy seems to have an impact on MDSC expression and functionality, hence targeting these cells may lead to a better clinical outcome in NSCLC patients.

Background:Multiple Myeloma (MM), a disease characterized by a clonal expansion of plasma cells, still remains incurable with current treatment options. The genetic engineering of T lymphocytes with chimeric antigen receptors (CAR) has recently emerged as a promising therapeutic approach and BCMA has shown to be a good MM‐specific antigenic target. Despite its specificity, the intensity of BCMA is variable in MM patients and may not be uniform across different subclonal populations which might explain why first clinical trials of BCMA‐CAR‐T cells report lower complete remission rates as well as relapses of BCMA‐low clonal variants. Another MM‐related antigen, CD38, is uniformly highly expressed in all malignant MM cells.Aims:We hypothesized that targeting 2 MM‐related antigens would increase cytotoxic ability of CAR‐T cells and the incorporation of a dual costimulation design would confer increased persistence. Thus, we propose to improve CAR‐T cell therapy for MM by dual targeting of BCMA and CD38 and incorporating full costimulation with 4‐1BB and CD28 signaling by using the combination of a CAR and a chimeric costimulatory receptor (CCR).Methods:To this end, we designed first and second generation CAR constructs targeting BCMA (BCMA‐z and BCMA‐28z), CCR constructs targeting CD38 (CD38‐28BB and CD38‐BB) as well as a CD38‐engager construct lacking intracellular domain (CD38del). We then evaluated in vitro the anti‐MM cytotoxic potential, cytokine production, proliferative capacity and exhaustion of the double‐targeting CAR‐T cells (BCMA‐z/38‐28BB, BCMA‐28z/CD38‐BB or BCMA‐z/CD38del) compared to single‐targeting T cells carrying a conventional CAR (BCMA‐28z, BCMA‐BBz, BCMA‐z), or CCR (38‐28z or 38‐28BB).Results:We found that double‐targeting BCMA‐z/38‐28BB CAR‐T cells showed significantly increased cytotoxic capacity against BCMA+CD38+ MM cell lines and primary MM cells, even in low effector to target ratios, as compared to the single‐targeting BCMA‐CAR‐T cells. This effect was diminished when a BCMA+CD38‐ cell line was used as target, while cells carrying only a CCR had no cytotoxic potential. Interestingly, BCMA‐z/CD38del T cells, where CD38‐engagement does not mediate any costimulation signal, had similar cytotoxic ability as BCMA‐z/38‐28BB cells, therefore the increased killing capacity of double‐targeting CAR‐T cells is due to CD38 engagement and the consecutive increase in avidity. Finally, double‐targeting BCMA‐z/38‐28BB CAR‐T cells showed significantly higher cytokine secretion (IL‐2, IFN‐γ, TNF‐α), proliferative capacity and induction of PD‐1 expression than single‐targeting BCMA‐CAR‐T cells, which was not reproduced by BCMAz/CD38del T cells indicating that this effect is a result of combining both CD28 and 4‐1BB costimulation.Summary/Conclusion:In conclusion, we here show that double targeting MM with a BCMA‐CAR and a CD38‐CCR provides higher effector‐MM avidity and full costimulation. Engagement of the CD38‐CCR enhances the anti‐MM cytotoxicity and persistence and reduces the exhaustion of double‐targeting compared to conventional single‐targeting CAR T cells. Therefore double‐targeting of BCMA and CD38 is a powerful strategy to improve clinical outcomes of BCMA‐CAR T cell therapy.