Specific Targeting of Multiple Myeloma by Dual Split-signaling Chimeric Antigen Receptor T cells Directed against CD38 and CD138.

PURPOSE: The success of B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells illustrates the potential of this novel therapy for multiple myeloma. Nonetheless, broadening CAR T-cell therapy beyond BCMA requires inventive strategies as there are only a few multiple myeloma- or plasma cell-specific target antigens. We investigated the feasibility of achieving multiple myeloma specificity by dual-split CD38/CD138 CAR targeting, whereby the stimulatory and costimulatory signals for T-cell activation are split into two separate stimulatory (sCAR) and costimulatory CARs (cCAR). EXPERIMENTAL DESIGN: Using various combinations of CD38 and CD138 sCARs and cCARs with different affinities, we generated several dual-split CAR T cells and analyzed them for multiple myeloma-specific effector functions in vitro. The best-functioning CAR T cells were tested in vivo in a murine xenograft model. RESULTS: We found optimal designs of both CD38sCAR/CD138cCAR and CD138sCAR/CD38cCAR combinations, that effectively lysed multiple myeloma cells but spared single CD38- or CD138-positive healthy hematopoietic cells. While the CD38sCAR/CD138cCAR T cells achieved multiple myeloma-specific activity solely due to the low affinity of the CD38sCARs, the multiple myeloma-specific cytotoxicity, cytokine release, and proliferation of CD138sCAR/CD38cCAR T cells were established through a true combinatorial stimulatory and costimulatory effect. The most optimal combination comprised a low-affinity CD138sCAR combined with a high-affinity CD38cCAR. These CD138sCAR/CD38cCAR T cells also showed dual-antigen specific anti-multiple myeloma effects in vivo. Importantly, they were also effective against multiple myeloma cells from daratumumab pretreated patients with decreased CD38 expression levels. CONCLUSIONS: We demonstrate the possibility to specifically target multiple myeloma cells, even after CD38 targeted therapy, with carefully-designed dual-split CARs directed against CD38 and CD138.

Directing CAR T cells towards the tumor vasculature for the treatment of solid tumors.

For successful application of chimeric antigen receptor (CAR) T cell therapy in solid tumors, major hurdles have to be overcome. CAR T cells have to cross the vascular barrier, which is hampered by the anergic state of the tumor vasculature, characterized by suppressed levels of leukocyte adhesion molecules on the endothelium. Additional immunosuppressive mechanisms in the solid tumor microenvironment can affect infiltration, activity and persistence of CAR T cells. Redirecting CAR T cells towards the tumor vasculature poses a possible solution, as molecular targets of tumor endothelial cells can be directly engaged from within the blood. In this review, we discuss recent advances in CAR T cell therapy against solid tumors, with a focus on targeting the tumor vasculature. Furthermore, we discuss opportunities to overcome challenges and barriers through engineering of CAR T cells to enhance trafficking, safety and efficacy.

Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence.

Despite the high remission rates achieved using T cells bearing a chimeric antigen receptor (CAR) against hematogical malignancies, there is still a considerable proportion of patients who eventually experience tumor relapse. Clinical studies have established that mechanisms of treatment failure include the down-regulation of target antigen expression and the limited persistence of effective CAR T cells. We hypothesized that dual targeting mediated by a CAR and a chimeric costimulatory receptor (CCR) could simultaneously enhance T cell cytotoxicity and improve durability. Concomitant high-affinity engagement of a CD38-binding CCR enhanced the cytotoxicity of BCMA-CAR and CD19-CAR T cells by increasing their functional binding avidity. In comparison to second-generation BCMA-CAR or CD19-CAR T cells, double-targeted CAR + CD38-CCR T cells exhibited increased sensitivity to recognize and lyse tumor variants of multiple myeloma and acute lymphoblastic leukemia with low antigen density in vitro. In addition, complimentary costimulation by 4-1BB and CD28 endodomains provided by the CAR and CCR combination conferred increased cytokine secretion and expansion and improved persistence in vivo. The cumulatively improved properties of CAR + CCR T cells enabled the in vivo eradication of antigen-low tumor clones, which were otherwise resistant to treatment with conventional CAR T cells. Therefore, multiplexing targeting and costimulation through the combination of a CAR and a CCR is a powerful strategy to improve the clinical outcomes of CAR T cells by enhancing cytotoxic efficacy and persistence, thus preventing relapses of tumor clones with low target antigen density.

Bone Marrow Mesenchymal Stromal Cells Can Render Multiple Myeloma Cells Resistant to Cytotoxic Machinery of CAR T Cells through Inhibition of Apoptosis.

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.

Preclinical Evaluation of Invariant Natural Killer T Cells Modified with CD38 or BCMA Chimeric Antigen Receptors for Multiple Myeloma.

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.

Prognostic value of circulating regulatory T cell subsets in untreated non-small cell lung cancer patients.

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-ß 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.