The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide ‘proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo.
The adoptive transfer of T cells engineered with chimeric antigen receptors (CARs) is currently considered as a highly promising therapeutic option for treatment of otherwise incurable malignant diseases. CARs combine the cellular and humoral arm of the immune response by assembling a single-chain fragment variable (scFv) as binding moiety which provides the antigen-specificity and an activating immune receptor. It has been demonstrated both in vitro and in vivo, that CAR engrafted effector T cells mediate long-lasting anti-tumor responses. Despite encouraging clinical efficacy targeting CD19 in recent clinical trials, the appearance of potentially life-threatening adverse reactions and the lack of control mechanisms once initiated, prevent more widespread application of the CAR technology. To overcome limitations of conventional CAR T cells, a unique chimeric antigen receptor (UniCAR) technology was developed (Fig. 1) which allows precise control of CAR T cell reactivity, thus lowering the risk of side effects while preserving efficacy. Moreover, the UniCAR technology enables the retargeting of engrafted T cells against more than one antigen simultaneously or subsequently, thus reducing the risk for development of antigen-loss tumor variants under treatment. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CAR into two individual components. T cells are engineered to express a universal CAR (UniCAR), which has specificity for a short peptide motif of 10 amino acids derived from a human nuclear protein. Thus, T cells engineered to express UniCAR remain inactivated after re-infusion, since the UniCAR target is not available for binding under physiological conditions. The ultimate antigen-specificity of the system is provided separately by targeting modules (TMs) comprising a binding domain e.g., a tumor-antigen specific scFv, fused to the nuclear antigen motif recognized by the UniCAR binding domain. Here we provide first in vitro and in vivo prove of concept for this new approach. Antigen-specific redirection of T cells armed with the universal CAR in the presence of different targeting modules against various antigens (CD33, CD123, CD19, CD20, PSCA, PSMA,) was effective at femtomolar concentrations of the targeting module both. Taken together, the modular nature of UniCAR technology will allow retargeting of autologous, patient-derived T cells to several antigens under controlled pharmacological conditions and has the potential to become a highly effective treatment option for late stage cancer patients with reduced risks for side effects. Figure 1. Schematic representation of T cell recruitment with the modular UniCAR system. The UniCAR T cell recruitment system consists of two separated units. The first unit is the UniCAR expressed on T cells with a single-chain fragment variable (scFv) specific for a short 10 aa long peptide motif. The intracellular signalling domain of the UniCAR contains a costimulatory domain derived from CD28 and the T cell receptor z chain. The second unit is a targeting molecule (TM) which consists of a scFv fused to the peptide epitope. The cross-linkage of T cell and target cell is mediated by interaction between the UniCAR binding domain on T cells and target cell binding TM. Figure 1. Schematic representation of T cell recruitment with the modular UniCAR system. / The UniCAR T cell recruitment system consists of two separated units. The first unit is the UniCAR expressed on T cells with a single-chain fragment variable (scFv) specific for a short 10 aa long peptide motif. The intracellular signalling domain of the UniCAR contains a costimulatory domain derived from CD28 and the T cell receptor z chain. The second unit is a targeting molecule (TM) which consists of a scFv fused to the peptide epitope. The cross-linkage of T cell and target cell is mediated by interaction between the UniCAR binding domain on T cells and target cell binding TM. Disclosures Cartellieri: Cellex Patient Treatment GmbH: Employment. Loff:GEMoaB Monoclonals GmbH: Employment. Ehninger:GEMoaB Monoclonals GmbH: Employment, Patents & Royalties: related to the UniTARG system. Ehninger:GEMoaB Monoclonals GmbH: Equity Ownership; Cellex Patient Treatment GmbH: Equity Ownership. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system.
The universal modular chimeric antigen receptor (UniCAR) platform redirects CAR-T cells using a separated, soluble targeting module with a short half-life. This segregation allows precise controllability and flexibility. Herein we show that the UniCAR platform can be used to efficiently target solid cancers and using a pre-clinical prostate cancer model which overexpresses prostate stem cell antigen (PSCA). Short-term administration of the targeting module to tumor bearing immunocompromised mice engrafted with human UniCAR-T cells significantly delayed tumor growth and prolonged survival of recipient mice both in a low and high tumor burden model. In addition, we analyzed phenotypic and functional changes of cancer cells and UniCAR-T cells in association with the administration of the targeting module to reveal potential immunoevasive mechanisms. Most notably, UniCAR-T cell activation induced upregulation of immune-inhibitory molecules such as programmed death ligands. In conclusion, this work illustrates that the UniCAR platform mediates potent anti-tumor activity in a relevant and solid tumor model.
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