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.

New treatment options especially of solid tumors including for metastasized prostate cancer (PCa) are urgently needed. Recent treatments of leukemias with chimeric antigen receptors (CARs) underline their impressive therapeutic potential. However CARs currently applied in the clinics cannot be repeatedly turned on and off potentially leading to severe life threatening side effects. To overcome these problems, we recently described a modular CAR technology termed UniCAR: UniCAR T cells are inert but can be turned on by application of one or multiple target modules (TMs). Here we present preclinical data summarizing the retargeting of UniCAR T cells to PCa cells using TMs directed to prostate stem cell- (PSCA) or/and prostate specific membrane antigen (PSMA). In the presence of the respective TM(s), we see a highly efficient target-specific and target-dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and PCa cell lysis both in vitro and experimental mice.

Based on compelling evidence from a vast number of in vitro and in vivostudies, Tregs have become an attractive cell population to treat or even prevent auto- and alloimmunity including Graft-versus-Host disease (GvHD). However, several safety concerns still exist as for example the risk of global immunosuppression using polyclonal Tregs. In fact, experiments in mice showed that adoptive transfer or induction of antigen-specific Tregs is more potent regarding suppression of pathogenic immune responses when compared to polyclonal Treg populations. Unfortunately, the isolation and expansion of naturally occurring antigen-specific Tregs is technically difficult, labour-intensive, and time-consuming. An attractive way to overcome these limitations and to endow polyclonal Treg populations with a desired antigen-specificity is their engraftment with chimeric antigen receptors (CARs). In this context, CAR-modification represents a promising approach to redirect polyclonal Tregs in an antigen-specific manner to suppress ongoing self-destructive immune responses at the site of inflammation. Nevertheless, until now redirection of CAR-engineered T cells is limited to a single target antigen, restricting this approach to an unflexible monospecific therapy. Therefore, we developed a more flexible universal CAR (UCAR) platform that allows redirection of T cells to an in principal unrestricted number of surface antigens. T cells are engrafted with UCARs that bind to a small peptide epitope derived from a human nuclear protein. Cross-linkage to target cells is mediated by independent target modules that provide antigen-specificity and comprise the peptide epitope recognized by the UCAR. In order to target different tissue antigens, the target modules can easily be exchanged. Thereby, once established, the treatment strategy can easily be applied to various auto- and alloimmune diseases. At present, the CD45RA+ population is the Treg subset of choice for a clinical application as these cells have the highest capacity to maintain phenotypic and functional Treg properties upon prolonged ex vivo expansion. Here we show that highly pure, sorted CD4+CD25+CD127lowCD45RA+ Tregs can be genetically manipulated using lentiviral gene transfer, resulting in approximately 70 % of UCAR-expressing Treg cells. The transduction procedure itself did not affect the phenotype of UCAR-engineered Tregs as it was similar to non-transduced wildtype cells. Both Treg populations presevered FOXP3 expression even after prolonged in vitro cultivation (> 95 % FOXP3+). Upon incubation with antigen-positive target cells and a respective target module UCAR-engineered Tregs upregulate the activation markers CD69 and LAP demonstrating that the cells can be restimulated antigen-specifically. Most importantly, UCAR-engrafted Tregs were functionally activated upon antigen encounter, demonstrated by suppression of proliferation and expansion of cocultured autologous T effector cells. Taken together, our results pave the way towards an application of UCAR technology for a site-specific recruitment of CAR-modified Tregs into inflamed tissues aiming at re-establishing immune homeostasis. Due to its high flexibility UCAR-engrafted Tregs can easily and universally be used for treatment of various autoimmune diseases or GvHD just by exchanging the tissue-specific target modules. Disclosures Cartellieri: Cellex Patient Treatment GmbH: Employment. Ehninger:GEMoaB GmbH: Employment, Patents & Royalties. Ehninger:GEMoaB GmbH: Consultancy, Patents & Royalties. Bachmann:GEMoaB GmbH: Consultancy, Patents & Royalties.

Chimeric antigen receptor T cells (CAR-T) targeting CD19 or B cell maturation antigen (BCMA) are highly effective against B cell malignancies. However, application of CAR-T to less differentially expressed targets remains a challenge due to lack of tumor-specific antigens and CAR-T controllability. CD123, a highly promising leukemia target, is expressed not only by leukemic and leukemia-initiating cells, but also by myeloid, hematopoietic progenitor, and certain endothelial cells. Thus, CAR-T lacking fine-tuned control mechanisms pose a high toxicity risk. To extend the CAR-T target landscape and widen the therapeutic window, we adapted our rapidly switchable universal CAR-T platform (UniCAR) to target CD123. UniCAR-T efficiently eradicated CD123 + leukemia in vitro and in vivo. Activation, cytolytic response, and cytokine release were strictly dependent on the presence of the CD123specific targeting module (TM123) with comparable efficacy to CD123-specific CAR-T in vitro. We further demonstrated a pre-clinical proof of concept for the safety-switch mechanism using a hematotoxicity mouse model wherein TM123-redirected UniCAR-T showed reversible toxicity toward hematopoietic cells compared to CD123 CAR-T. In conclusion, UniCAR-T maintain full anti-leukemic efficacy, while ensuring rapid controllability to improve safety and versatility of CD123-directed immunotherapy. The safety and efficacy of UniCAR-T in combination with TM123 will now be assessed in a phase I clinical trial (ClinicalTrials.gov: NCT04230265).

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.

Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced in vivo from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an in vivo TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells.

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.

Chimeric antigen receptor T cells (CAR-T) targeting CD19 have achieved significant success in patients with B cell malignancies. To date, implementation of CAR-T in other indications remains challenging due to the lack of truly tumor-specific antigens as well as control of CAR-T activity in patients. CD123 is highly expressed in acute myeloid leukemia (AML) blasts including leukemia-initiating cells making it an attractive immunotherapeutic target. However, CD123 expression in normal hematopoietic progenitor cells and endothelia bears the risk of severe toxicities and may limit CAR-T applications lacking fine-tuned control mechanisms. Therefore, we recently developed a rapidly switchable universal CAR-T platform (UniCAR), in which CAR-T activity depends on the presence of a soluble adapter called targeting module (TM), and confirmed clinical proof-of-concept for targeting CD123 in AML with improved safety. As costimulation via 4-1BB ligand (4-1BBL) can enhance CAR-T expansion, persistence, and effector functions, a novel CD123-specific TM variant (TM123-4-1BBL) comprising trimeric single-chain 4-1BBL was developed for transient costimulation of UniCAR-T cells (UniCAR-T) at the leukemic site in trans. TM123-4-1BBL-directed UniCAR-T efficiently eradicated CD123-positive AML cells in vitro and in a CDX in vivo model. Moreover, additional costimulation via TM123-4-1BBL enabled enhanced expansion and persistence with a modulated UniCAR-T phenotype. In addition, the increased hydrodynamic volume of TM123-4-1BBL prolonged terminal plasma half-life and ensured a high total drug exposure in vivo. In conclusion, expanding the soluble adapter optionality for CD123-directed UniCAR-T maintains the platforms high anti-leukemic efficacy and immediate control mechanism for a flexible, safe, and individualized CAR-T therapy of AML patients.