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The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.

Digital, Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics., 1 ed.

As the expression of a tumor associated antigen (TAA) is commonly not restricted to tumor cells, adoptively transferred T cells modified to express a conventional chimeric antigen receptor (CAR) might not only destroy the tumor cells but also attack target-positive healthy tissues. Furthermore, CAR T cells in patients with large tumor bulks will unpredictably proliferate and put the patients at high risk of adverse side effects including cytokine storms and tumor lysis syndrome. To overcome these problems, we previously established a modular CAR technology termed UniCAR: UniCAR T cells can repeatedly be turned on and off via dosing of a target module (TM). TMs are bispecific molecules which cross-link UniCAR T cells with target cells. After elimination of the respective TM, UniCAR T cells automatically turn off. Here we describe novel TMs against the disialoganglioside GD2 which is overexpressed in neuroectodermal but also many other tumors. In the presence of GD2-specific TMs, we see a highly efficient target-specific and -dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and tumor cell lysis both in vitro and experimental mice. According to PET-imaging, anti-GD2 TM enrich at the tumor site and are rapidly eliminated thus fulfilling all prerequisites of a UniCAR TM.

Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.

We have recently described a novel modular targeting platform for T cell recruitment that not only efficiently replaces but also is superior to conventional T cell-engaging bispecific antibodies as it allows for the flexible targeting of several antigens and the delivery of co-stimulatory ligands to malignant lesions, thereby enhancing the antitumor potential of redirected T cells.

Regulatory T cells (Tregs) play an inevitable role in immune homeostasis by maintaining self-tolerance as well as regulating the magnitude of immune responses against foreign antigens. Over the last few years, the enormous potential of adoptive Treg transfer for treatment of auto- and alloimmunity including Graft-versus-Host disease (GvHD) has been validated in a vast number of in vitro and in vivo studies. For their clinical application, all modes of action should be well understood. Regarding their cytotoxic potential, only few and conflicting data exist. On the one hand, it is assumed that Tregs are capable of inducing apoptosis of effector T cells (Teff) utilizing granzyme/perforin or FasL expression. Others claim that Tregs are not capable of suppressing Teff via programmed cell death pathways but rather induce apoptosis by cytokine deprivation. However, it is of importance to clarify whether Tregs possess a cytotoxic potential particularly when activating the cells antigen-specifically using bispecific antibodies (bsAb). In recent years, bsAb have emerged as promising tools for an antigen-specific immunotherapy of malignant diseases. Their tremendous potential for tumor therapy has been verified in a plethora of in vitro and in vivo studies as well as in first clinical trials. So far, our group was able to demonstrate that not only Teff but also Tregs can be redirected by CD3-engaging bsAb (Koristka et al., J Immunol. 2012; J Autoimmun. 2013). According to a recent presentation (Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research, 2012, abstract nr 4841), bsAb-redirected Tregs can act as killer cells and efficiently mediate cancer cell lysis. In order to shed light onto this controversial issue, we decided to analyze this question in more detail. According to our investigations tumor cell elimination of bsAb-engaged Tregs is largely dependent on the purity of isolated Treg fractions. Tregs isolated on the basis of CD25 expression exhibited a remarkable killing capacity which is most probably due to contaminating CD25+FOXP3- Teff, as highly pure (> 99 %), FACS-isolated CD4+CD25+CD127low Tregs did not display any considerable cytotoxic effect upon cross-linkage to tumor cells via bsAb. The same applies for CD45RA-sorted, expanded Tregs. In comparison to autologous, expanded Teff, tumor cell lysis was negligible. Moreover, the lack of cytotoxicity was independent of the chosen target antigen, as redirecting Tregs with two different bsAb did not result in tumor cell eradication. Besides, upon polyclonal stimulation with conventional aCD3/CD28-coated beads Tregs were not capable of eliminating target cells. Furthermore, as opposed to autologous Teff, Tregs showed only a marginal upregulation of the degranulation marker CD107a when being activated either antigen-specifically via bsAb or polyclonally via beads. Taken together, our findings clearly demonstrate that Tregs bear no considerable cytotoxic potential and hence do not contribute to cancer cell lysis, as recently claimed. On the other hand, the results show that Tregs can be activated by bsAb without the risk of cytotoxic effects against the recognized target cells. This provides the basis for the application of bsAb for a site-specific recruitment of Tregs aiming at attenuating Teff-mediated proinflammatory immune responses and tissue destruction in order to treat auto- and alloimmune diseases including GvHD. Disclosures: No relevant conflicts of interest to declare.