CAR'TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment.

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and 5-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR'TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro, particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR'TCR-T, CAR-T, and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR'TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies.

Combining CSPG4-CAR and CD20-CCR for treatment of metastatic melanoma.

The prognosis for patients with metastatic melanoma is poor and treatment options are limited. Genetically-engineered T cell therapy targeting chondroitin sulfate proteoglycan 4 (CSPG4), however, represents a promising treatment option, especially as both primary melanoma cells as well as metastases uniformly express CSPG4. Aiming to prevent off-tumor toxicity while maintaining a high cytolytic potential, we combined a chimeric co-stimulatory receptor (CCR) and a CSPG4-directed second-generation chimeric antigen receptor (CAR) with moderate potency. CCRs are artificial receptors similar to CARs, but lacking the CD3ζ activation element. Thus, T cells expressing solely a CCR, do not induce any cytolytic activity upon target cell binding, but are capable of boosting the CAR T cell response when both CAR and CCR engage their target antigens simultaneously. Here we demonstrate that co-expression of a CCR can significantly enhance the anti-tumor response of CSPG4-CAR T cells in vitro as well as in vivo. Importantly, this boosting effect was not dependent on co-expression of both CCR- and CAR-target on the very same tumor cell, but was also achieved upon trans activation. Finally, our data support the idea of using a CCR as a powerful tool to enhance the cytolytic potential of CAR T cells, which might open a novel therapeutic window for the treatment of metastatic melanoma.

Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells.

Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-ß. TGF-ß plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-ß within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-ß, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-ß and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-ß.

Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer.

Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications.