Unicar: A Novel Modular Retargeting Platform Technology for CAR T Cells

Abstract

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.

3D model for CAR ‐mediated cytotoxicity using patient‐derived colorectal cancer organoids

“…Note that EGFRvIII-CAR NK-92 cells are also GFP + , but which does not perturb the analysis (see Fig EV4). active CAR dosage (Cartellieri et al, 2015) or the SynNotch technology that depends on combinatorial detection of multiple TAAs (Roybal et al, 2016). Testing in patient-matched normal and tumor organoids may greatly assist to further adapt these strategies.…”

A theranostic PSMA ligand for PET imaging and retargeting of T cells expressing the universal chimeric antigen receptor UniCAR

“…Moreover, with EC 50 values in a low nM range, killing efficiency of UniCAR T cells armed via the PSMA PLT-TM is comparable to all so far described combinations with Ab-based TMs. [44][45][46][47][48][49][50] Furthermore, we have shown for the first time that not only Ab fragments but also peptide ligands can be successfully fused to the UniCAR epitope E5B9. In context of solid tumor UniCAR T cell therapy, such small molecules are advantageous due to increased tumor permeability and short serum half-lifes.…”

“…We therefore established a modular platform technology termed UniCAR, which is a two-component system consisting of UniCAR T cells and target modules (TMs). [43][44][45][46][47][48][49][50] UniCARs are second-generation CARs, which target the La/SS-B-derived peptide epitope E5B9 (UniCAR epitope). 51,52 As this epitope is physiologically not accessible on the surface of intact living cells, adoptively transferred UniCAR T cells are in contrast to TAA-specific CAR T cells per se inactive.…”

“…Thus, UniCAR T cells are engaged for tumor cell killing only in the presence of TMs. [43][44][45][46][47][48][49][50] Based on rapid TM elimination, adverse effects of redirected UniCAR T cells should be easily controlled by stopping TM infusion. After recovery of the patient, TM supply and hence immunotherapy can be restarted.…”

“…Since the first presentation of the UniCAR idea in 2014, 43 several, so far only Ab-based TMs were described including TMs directed against the TAAs CD33, CD123, prostate stem cell antigen, PSMA, epidermal growth factor receptor, GD2, CD19, or STn. [43][44][45][46][47][48][49][50][51][52][53][54] The aim of the presented study was to develop a novel theranostic PSMA ligand for management of PCa patients that combines the fields of nuclear medicine and CAR T cell technology. Thus, we converted PSMA-11, one of the most commonly used PCa imaging agent, by fusion of the 10-amino acid peptide epitope E5B9 into a TM for the UniCAR system ( Figure 1b).…”