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Justyna Jureczek,1– 3 Anja Feldmann,3 Ralf Bergmann,3,4 Claudia Arndt,3 Nicole Berndt,3 Stefanie Koristka,3 Liliana Rodrigues Loureiro,3,5 Nicola Mitwasi,3 Anja Hoffmann,3 Alexandra Kegler,1– 3 Tabea Bartsch,3 Michael Bachmann1– 3,5,6 1German Cancer Consortium (DKTK), Dresden, Germany; 2German Cancer Research Center (DKFZ), Heidelberg, Germany; 3Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany; 4Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary; 5National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden; Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany; 6Tumor Immunology, University Cancer Center (UCC), University Hospital Carl Gustav Carus Dresden, Technische Universität Dresden, Dresden, GermanyCorrespondence: Michael BachmannHelmholtz-Zentrum Dresden-Rossendorf e.V. Managing Director, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstraße 400, Dresden 01328, GermanyTel +49 351 260 3170Fax +49 351 260 3232Email m.bachmann@hzdr.deIntroduction: Since epithelial growth factor receptor (EGFR) overexpression is linked to a variety of malignancies, it is an attractive target for immune therapy including chimeric antigen receptor (CAR)-engineered T cells. Unfortunately, CAR T cell therapy harbors the risk of severe, even life-threatening side effects. Adaptor CAR T cell platforms such as the previously described UniCAR system might be able to overcome these problems. In contrast to conventional CARs, UniCAR T cells are per se inert. Their redirection towards target cells occurs only in the presence of a tumor-specific target molecule (TM). TMs are bifunctional molecules being able to recognize a tumor-associated antigen and to cross-link the CAR T cell via a peptide epitope recognized by the UniCAR domain.Materials and Methods: Here, we compare αEGFR TMs: a nanobody (nb)-based αEGFR TM derived from the camelid αEGFR antibody 7C12 with a murine and humanized single-chain fragment variable (scFv) based on the clinically used antibody Cetuximab®.Results: In principle, both the nb- and scFv-based TM formats are able to redirect UniCAR T cells to eliminate EGFR-expressing tumor cells in an antigen-specific and TM-dependent manner. However, the scFv-based αEGFR TM was significantly superior to the nb-based TM especially with respect to lysis of tumor cells.Discussion: Improved efficiency of the scFv-based TM allowed the redirection of UniCAR T cells towards tumor cells expressing high as well as low EGFR levels in comparison to nb-based αEGFR TMs.Keywords: EGFR, UniCAR, CAR T cells, adaptor CARs, solid tumors, immunotherapy

Induction or selection of radioresistant cancer (stem) cells following standard radiotherapy is presumably one of the major causes for recurrence of metastatic disease. One possibility to prevent tumor relapse is the application of targeted immunotherapies including, e.g., chimeric antigen receptor (CAR) T cells. In light of long-term remissions, it is highly relevant to clarify whether radioresistant cancer cells are susceptible to CAR T cell-mediated killing. To answer this question, we evaluated the anti-tumor activity of the switchable universal chimeric antigen receptor (UniCAR) system against highly radioresistant head and neck squamous cell carcinoma cells both in vitro and in vivo. Following specific UniCAR T cell engagement via EGFR or CD98 target modules, T cell effector mechanisms were induced including secretion of pro-inflammatory cytokines, up-regulation of granzyme B and perforin, as well as T cell proliferation. CD98- or EGFR-redirected UniCAR T cells further possess the capability to efficiently lyse radioresistant tumor cells. Observed anti-tumor effects were comparable to those against the radiosensitive parental cell lines. Finally, redirected UniCAR T cells significantly inhibited the growth of radioresistant cancer cells in immunodeficient mice. Taken together, our obtained data underline that the UniCAR system is able to overcome radioresistance. Thus, it represents an attractive technology for the development of combined radioimmunotherapeutic approaches that might improve the outcome of patients with metastatic radioresistant tumor diseases.

Digital, Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution., 1 ed.

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies.

Since epithelial growth factor receptor (EGFR) mutations or overexpression is linked with variety of malignancies, including lung, breast, stomach, colorectal, head and neck, and pancreatic carcinomas as well as glioblastomas it is an attractive target for tailored treatment of solid cancer. Thus over the last twenty years many strategies targeting EGFR were developed and even clinically approved, including disrupting intracellular signalling involving tyrosine kinase inhibitors (TKIs) or the inhibition of ligand binding using therapeutic monoclonal antibodies like e.g. Cetuximab, Panitumumab or Necitumumab. Unfortunately, cancers treated with these targeted drugs commonly become resistant to them. These limitations justify the need of more efficient therapy options. As chimeric antigen receptor (CAR) engineered T cells highly effectively eliminate hematological malignancies already in the clinics, one idea is to redirect CAR T cells also against EGFR expressing solid cancers. However, CAR T cell therapy can lead to severe even life-threatening side effects and its effectiveness against solid tumors is still limited. Particular worrying is that EGFR is a widespread antigen commonly expressed also on healthy tissues bearing a high risk of severe on-target/off-tumor side effects due to EGFR-targeted therapies, which cannot be controlled in patients. In order to overcome these challenges our UniCAR technology might be an appropriate answer combining high anti-tumor effectiveness, tumor specificity, flexibility, and safety control mechanisms. In contrast to conventional CARs, UniCAR T cells are per se inert because UniCARs are directed against a small peptide epitope, which is not present on living cells. The redirection of UniCAR T cells to tumor cells occurs only in the presence of a tumor specific targeting module (TM). TMs, on one hand carry the specificity for a certain tumor antigen and on the other hand contain the UniCAR peptide epitope recognized by UniCARs mediating the cross-linkage of UniCAR T cells and antigen presenting tumor cells. As TMs have a very short half-life in vivo they can be used as a switch to control UniCAR T cell activity on demand in patients. In detail, UniCAR T cells are only switched on in the presence of antigen specific TMs realized by permanent TM infusion, but could be rapidly switched off when the application of the TM is stopped and the TM is eliminated. Meanwhile we successfully generated a series of different TMs against different tumor antigens and entities. Interestingly, TMs can be made of different molecules showing various structures and can flexible be exchanged in order to target any tumor antigen and overcome tumor escape variants. Commonly our TMs consist of a humanized single-chain variable fragment (scFv) derived from the variable heavy and light chain domains of a murine monoclonal antibody. In addition, we successfully generated TMs based on different monovalent and bivalent antibody derivatives, nanobodies derived from one variable camelid antibody domain, affibodies and even small peptide molecules. Recently we demonstrated proof-of-concept for the redirection of UniCAR T cells to EGFR expressing tumor cells by a nanobody based αEGFR TM derived from the camelid αEGFR antibody 7C12. Considering that the affinity and anti-tumor efficiency of the eucaryotically expressed αEGFR nanobody based TM was limited, we therefore asked the question, whether we could further improve the therapeutic effect against EGFR positive tumor cells using the UniCAR technology. In order to answer this question, we generated a novel TM based on a scFv derived from the clinically used chimeric monoclonal antibody Cetuximab (IMC C-225). In detail, we designed a murine and humanized αEGFR scFv TM, successfully expressed them in mammalian cell lines and compared their functionality with the eucaryotic αEGFR nanobody-based TM in vitro and in vivo. In principle, we observed that both TM formats, the αEGFR nanobody as well as the scFv-based TM, are able to redirect UniCAR T cells eliminate EGFR-expressing tumor cells in an antigen-specific and TM-dependent manner. As both the murine and humanized scFv TM variants worked equally well, obviously humanization of the αEGFR scFv does not affect its functionality. However most interestingly, the tumor killing efficiency of the αEGFR scFv TM was significantly superior in comparison to the αEGFR nanobody based TM. Here, the half maximal effective TM concentration (EC50) value of scFv based TM was improved 1000-fold, from nM to pM range. Consequently, UniCAR T cells in combination with the scFv based TM efficiently eliminate also target cells expressing a low EGFR density level, while UniCAR T cells redirected by the nb based TM clearly attack only highly EGFR expressing tumor cells. Furthermore, the high anti-tumor efficacy of the αEGFR scFv TM over nb TM was manifested in experimental mice. In summary, we successfully established different αEGFR TM formats that are able to redirect UniCAR T cells to eliminate EGFR-positive tumor cells. However, the analysed αEGFR TM formats differ with respect to their anti-tumor efficiency, which might decide whether UniCAR T cells attack target cells showing different EGFR density levels.

Chimeric antigen receptors (CARs) are highly efficient tools for T‐cell‐based cancer immunotherapy. Nonetheless, this approach is associated with mild to severe toxicities including cytokine release syndrome and on‐target/off‐tumor effects. To increase clinical safety while maintaining the efficacy of CAR T‐cell therapy, we developed a novel modular universal CAR (UniCAR) platform. UniCAR T‐cells are exclusively activated via a target module (TM) that establishes the cross‐link between UniCAR T‐cells and cancer cells. Given the small size of such molecules, they are rapidly eliminated and thus, have to be administrated via continuous infusion. Consequently, activation and possible side effects of UniCAR T‐cells can be easily controlled by TM dosing. Regulation of CAR T‐cell activity is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. Therefore, TMs with extended half‐life may improve eradication of residual tumor cells in late phase of treatment and further expedite clinical application. In this line of thought, we developed both short‐lived and longer lasting TMs directed against several tumor‐associated antigens. Short‐lived TMs are composed of a tumor‐specific binding moiety and the E5B9 peptide epitope which is recognized by UniCAR T‐cells. In order to generate extended half‐life TMs, these two components are fused to the human IgG4 Fc domain. Both short‐lived and longer lasting TMs efficiently redirect UniCAR T‐cells to cancer cells in a highly target‐specific manner, thereby promoting the secretion of pro‐inflammatory cytokines and tumor cell lysis in vitro and in vivo. As demonstrated by PET‐imaging, all TMs specifically enriched at the tumor site presenting either short or prolonged serum half‐lives. Taken together, combination of different short‐lived and extended half‐life TMs provides a highly promising and customized tool for retargeting of UniCAR T‐cells in a flexible, individualized and safe manner at different phases of tumor therapy.

Hematological malignancies are successfully treated with chimeric antigen receptor (CAR) armed T cells. Despite the clinical success, CAR T cell therapy struggles still with some problems including the selection of tumor escape variants and on-target, off-tumor side reactions as well as massive cytokine release and uncontrollability of CAR T cell activity in the patients. In order to enable controllability of CAR T cells and to avoid unspecific side effects, we established a novel switchable, split and adaptable CAR platform technology, termed RevCAR system. The novel RevCARs lack the single chain variable fragment (scFv) commonly used as extracellular domain in conventional CARs. Instead of the scFv, RevCARs contain only a small peptide epitope as extracellular portion. This design reduces the CAR size, avoids unspecific antigen binding and prevents antigen independent tonic signaling caused by scFv dimerization. As RevCAR T cells do not recognize anything, they are per se inert. Only in the presence of a corresponding bispecific antibody based target module (RevTM) they can be specifically redirected to tumor cells. Therefor RevTMs consist of two scFvs. One recognizes the RevCAR peptide epitope and the other one simultaneously binds to a tumor associated antigen (TAA). By dosing of the RevTM, which has a very short half-life, the reactivity of RevCAR T cells can be switched on and off reversibly. Another advantage is that the RevCAR system can be flexibly adapted to any tumor antigen simply by exchanging the RevTM. Furthermore, the small RevCAR size is favorable for inserting more than one RevCAR in the same T cell thus facilitating the mode of gated targeting which is a highly attractive approach to minimize the risk for on-target, off-tumor toxicities against healthy tissues and to increase tumor specificity of conventional CAR T cells. For 'AND' gate targeting via the RevCAR system, two different RevCARs were constructed and expressed simultaneously in the same T cell. The two RevCARs differed with respect to the extracellular peptide epitope and the intracellular signaling domain. Moreover, the respective transmembrane domain was selected to isolate the respective RevCAR signal. The first RevCAR is designed to transmit the activation signal, the second RevCAR to deliver a costimulatory signal. For efficient RevCAR T cell activation, both RevCARs must be engaged via their respective RevTM which on the one hand binds to one of the two RevCAR epitopes and on the other hand to one of two TAAs expressed on the same target cell. Here, we present two RevCAR/RevTM systems for retargeting of AML cells as well as solid tumor cells including via gated targeting. In summary, we show proof of concept for a novel switchable RevCAR system that can be used for retargeting of AML cells as well as solid tumors. The novel modular RevCAR platform is characterized by small size, lacks unwanted tonic signaling effects, allows the control of RevCAR T cell activity, enables gated targeting strategies, and can be adapted to any tumor antigen and tumor type.

Since epithelial growth factor receptor (EGFR) mutations or overexpression is linked with variety of malignancies, including lung, breast, stomach, colorectal, head and neck carcinomas as well as glioblastomas, it is an attractive target for tailored treatment. As chimeric antigen receptor (CAR) engineered T cells highly effectively eliminate hematological malignancies already in the clinics, one idea is to redirect CAR T cells also against EGFR expressing solid cancers. However, CAR T cell therapy can lead to severe, even life‐threatening side effects with high risk of on‐target/off‐tumor activity. To overcome these challenges our UniCAR technology might be an appropriate answer combining high anti‐tumor effectiveness, tumor specificity, flexibility, and safety control mechanisms. In contrast to conventional CARs, UniCAR T cells are per se inert because UniCARs are directed against a small peptide epitope, which is not present on living cells. The redirection of UniCAR T cells toward target cells occurs only in the presence of a tumor specific targeting molecule (TM) – bifunctional moieties carrying the specificity for a certain tumor antigen and contain the UniCAR peptide epitope recognized by UniCARs. TMs can be made of different molecules showing various structures. Here we are presenting the comparison in functionality, in vitro and in vivo, of two TM formats: nanobody based αEGFR TM derived from the camelid αEGFR antibody 7C12 and scFv derived TM from the clinically used chimeric monoclonal antibody Cetuximab (IMC C‐225). In principle, we observed that both TM formats are able to redirect UniCAR T cells to eliminate EGFR‐expressing tumor cells in an antigen‐specific and TM‐dependent manner. Most interestingly, the tumor killing efficiency of the αEGFR scFv TM was significantly superior in comparison to the nanobody based TM, what might decide whether UniCAR T cells attack target cells showing different EGFR density level.

Background: Chimeric antigen receptor (CAR) T cell therapy has demonstrated impressive clinical efficiency, but can also cause moderate to severe adverse effects that might be even fatal. Thus, preventing or managing CAR T cell toxicity is still an important issue for successful treatment of tumor patients. In order to provide a novel CAR technology platform with an improved safety profile, we established the switchable UniCAR system. This platform consists of (I) universal CAR (UniCAR) T cells that are per se inactive. Their anti-tumor activity can be specifically and repeatedly turned on/off in dependence of soluble tumor-binding target modules (TM).e.g.1-4 TMs are constructed by fusing an antigen-specific binding moiety with the E5B9 peptide epitope recognized by UniCARs. As these molecules are rapidly eliminated, UniCAR T cells can be easily controlled by TM dosing. Aims: As the risk for CAR T cell-related toxicities will also decrease with reduction of tumor burden, we intended to develop TMs with prolonged half-life that might ease clinical application and improve elimination of residual tumor cells in late phase of tumor therapy. Methods: We constructed a set of novel, longer lasting TMs by fusion of different tumor-specific single-chain fragment variables (scFv) and the E5B9 peptide epitope to the Fc domain of human IgG4 antibodies. The resulting IgG4-based TMs were functionally compared with smaller, scFv-based TMs in vitro and characterized for their pharmacokinetic properties in experimental mice. Results: The novel IgG4-based TMs are able to efficiently activate UniCAR T cells for killing of various tumor cell lines. In comparison to short-lived TMs, they are characterized by a comparable or increased efficiency at low TM concentrations. Pharmacokinetic studies in tumor-bearing mice further revealed that IgG4-based TM have a prolonged plasma half-life and enhanced bioavailability. Summary: Our data demonstrate that IgG4-based TMs in combination with smaller TMs are highly promising tools for redirection of UniCAR T cells to various cancer cells. Once the tumor burden is reduced, UniCAR T cells can be combined with IgG4-based TMs instead of small TMs. This is more convenient for patients as IgG4-based TM have not to be continuously infused due to their prolonged serum half-lives. Overall, the combination of UniCAR T cells with TMs of different size and specificity should allow a more convenient, individualized and safe treatment regimen of cancer patients.

The clinical efficacy of CAR T cell therapies has been widely recognized, particularly in the treatment of hematologic malignancies. Nevertheless, CAR T cells also have the capability to elicit undesired effects such as on-target/off-tumor recognition and cytokine release syndrome. To increase clinical safety of CAR T cell therapy, a novel modular universal CAR platform termed UniCAR was developed by our group. In the UniCAR system, antigen-binding specificity and signaling features are two distinct moieties, in which the antigen specificity is provided by targeting modules (TMs) to redirect UniCAR T cells in an individualized time- and target-dependent manner. In this way, UniCAR T-cells acquire killing potential only in the presence of a tumour-specific TM. Given the reduced size of such molecules, they are rapidly eliminated and therefore, need to be continuously infused. Thus, possible side effects and activation of UniCAR T cells can be easily monitored and controlled by TM dosing. During the onset of therapy, tumor burden and the risk for severe side effects are high and regulation of CAR T cell activity is particularly important at this stage. For this reason, TMs with extended half-life may play an important role by improving eradication of residual tumor cells in late phases of treatment and further expedite clinical application. In this line of thought, a set of novel short-lived and longer-lasting TMs directed against several tumor-associated antigens was developed. Short-lived TMs are composed of a tumor-specific binding moiety fused to the La peptide epitope (E5B9) which is recognized by UniCAR T cells. In order to generate extended half-life TMs, these two components are fused via an Fc domain derived from the human IgG4 molecule. In vitro and in vivo assays have shown that both short-lived and longer-lasting TMs efficiently redirect UniCAR T cells to cancer cells in a highly target-specific manner, thereby promoting the secretion of pro-inflammatory cytokines and tumor cell lysis. Further assays using PET-imaging, demonstrated that all TM formats specifically enriched at the tumor site presenting either short or prolonged serum half-lives. From a clinical point of view, after the initial reduction of tumor burden promoted by the small TMs, IgG4-based TMs could be subsequently administrated allowing a more convenient and personalized treatment of the patients avoiding the continuous infusion of the short-lived TMs. Furthermore, the specific accumulation of such IgG4-based TMs at the tumor site sets these molecules as attractive candidates for in vivo imaging and endoradiotherapy. Taken together, combination of switchable UniCAR T cells and TMs with different sizes, specificities and half-lives represent a flexible and individualized approach at different stages of cancer treatment.

Introduction: Radiation is a common therapy for solid tumors. Unfortunately there is a high risk for the outgrowth of radioresistant tumor cells against which only limited treatment options exist. We challenged the idea whether or not chimeric antigen receptor (CAR) engineered T lymphocytes could be used as an adjuvant immunotherapy in combination with radiotherapy. Recently, we have established switchable universal CARs (UniCARs) that bind to a short peptide epitope (E5B9) which does not exist on the surface of living cells. UniCAR T cells are exclusively redirected to tumor cells in the presence of a target module (TM) that exhibits the E5B9 epitope and binds to a tumor associated antigen (TAA) on the tumor cell surface. Materials and Methods: We used different radioresistant sublines of the head and neck cancer cell line Cal33. Gene expression data for certain TAAs were confirmed by flow cytometry. TMs against potential targets were created from the variable domains of monoclonal antibodies, cloned in lentiviral vectors and purified from supernatants of permanently TM producing 3T3 cells. UniCAR T cells were generated by lentiviral transduction. Results: Radioresistant Cal33 cell lines expressed PSCA, EGFR and CD98. UniCAR TMs were created against these TAAs. Armed with these TMs UniCAR T cells efficiently killed radioresistant Cal33 cells in vitro and in vivo. Conclusions: Radioresistant tumor cells can efficiently be killed by redirecting UniCAR T cells against PSCA, CD98 and EGFR and thus resistance to radiotherapy can be overcome by immunotherapy based on the UniCAR technology to these targets.

Radiation therapy represents a commonly applied treatment regimen for solid tumors. Unfortunately, it is often accompanied by a high risk for the outgrowth of radioresistant cancer cells against which treatment options are limited. We challenged the idea whether or not chimeric antigen receptor (CAR)-modified T cells could be exploited as an adjuvant immunotherapy in combination with standard radiotherapy. Over the past several years, we have established switchable universal CAR constructs (UniCARs) that recognize a short peptide epitope (E5B9) which does not exist on the surface of living cells. UniCAR T cells are redirected to malignant cells exclusively in the presence of a target module (TM) that contains the epitope E5B9 and specifically binds to a tumor-associated antigen (TAA) on the tumor cell surface. For providing a rationale for the combination of CAR and radiation therapy, we used different radioresistant sublines of the head and neck cancer cell line Cal33. Expression of various TAAs including of PSCA, EGFR and CD98 was confirmed by flow cytometry analysis. Subsequently, TMs recognizing these potential targets were generated from the variable domains of monoclonal antibodies, cloned into lentiviral vectors and purified from cell culture supernatants of TM-producing stable cell lines. In parallel, T cells isolated from healthy donors were engrafted with UniCARs by lentiviral transduction. Armed with our anti-TAA TMs, UniCAR T cells efficiently lysed radioresistant Cal33 tumor cells both in vitro and in vivo. Taken together, we could demonstrate that radioresistant cancer cells can effectively be killed by retargeting UniCAR T cells against PSCA, CD98 and EGFR. Thus, resistance to standard of care radiotherapy can be overcome by concomitant or subsequent immunotherapy using the flexible UniCAR technology.

Antibodies (Abs) and their recombinant derivatives play an increasing role in tumor therapy and diagnostic imaging. The theranostic target modules (TMs) recently developed by us can be used for individualized and precise cancer immunotherapy and at the same time as radiotracers for tumor imaging. The TMs consist of an antigen binding moiety that binds to the prostate stem cell antigen (PSCA) on the tumor cell surface and present the peptide epitope E5B9. Via E5B9 the TMs can retarget human T cells that are genetically modified with universal anti-E5B9 chimeric antigen receptors (UniCARs) towards the tumor cells. The cross-linkage of UniCAR T cells and tumor cells via the TM results in T cell activation and tumor cell killing. This immunotherapeutic approach was shown as an inducible CAR platform technology that can be used for the treatment of leukemic as well as solid cancers. This technic has an intrinsic emergency brake: The short half-life of the TMs in vivo allows a fast switch off of the UniCAR system. In detail, UniCAR T cells per se are inactive because their UniCARs do not recognize an antigen on the cell surface. Only in the presence of tumor-bound TMs UniCAR T cells can be redirected to tumor cells and turned on. But they are switched off when the application of TMs is stopped and their concentration is too low to activate UniCAR T cells what also becomes particularly relevant in case of side effects occur in the patients. TMs cannot only be used as a therapeutic drug but also as a diagnostic tool for tumor imaging. For this purpose TMs can be modified with a bifunctional chelator. After conjugation they can be labeled with radiometals like 64Cu2+ or 68Ga3+ and used as an imaging tracer for PET analysis. Usually our TMs contain a tag of six histidine residues (His6-tag) at the C-terminus. The His6-tag is used for convenient purification by immobilized metal ion affinity chromatography and detection of the TMs. However the His6-tag may affect PK and biodistribution of the TMs. In particular with respect to clinical application of the TMs the His6-tag may induce immunologic reactions. Furthermore the purification of His6-tagged TMs by immobilized metal ion affinity chromatography might cause metal ions in the TMs which have influence on their radiolabeling and can be critical for patients’ health. To address these concerns we have generated an anti-PSCA TM without His6-tag (PSCA TM) and compared its theranostic properties to the corresponding TM with the His6-tag (PSCA-His TM). The PSCA TMs were successfully prepared by digestion with TEV protease recognizing a strict seven amino acid cleavage recognition site which was introduced upstream from the His6-tag in the PSCA-TEV-His TM construct. Both TMs with or without His6-tag showed similar high binding affinities to PSCA on PC3-PSCA prostate cancer cells whereas the Kd values were in the nM range. As shown by chromium release assays both target modules were able to redirect UniCAR T cells to efficiently kill PSCA-presenting tumor cells in a strictly target-dependent and target-specific manner. In vitro tumor cell killing of PSCA TM was less efficient than PSCA-His TM but still in pM range. Most importantly in NMRI-FOX1nu/nu mice the PSCA TM redirected UniCAR T cells to eliminate luciferase/PSCA-double positive tumor cells as efficient as the PSCA-His TM. PET analysis of the 64Cu-labeled TMs in these mice showed higher accumulation of the PSCA TM in the PSCA-positive tumors and less nonspecific binding in normal tissues (like blood and heart) after 2 h. At the same time the blood clearance of PSCA TM was distinctly faster than of the PSCA-His TM resulting in better contrast of the tumor for the PSCA TM in the PET images after 2 h. On the other hand, the PSCA-His TMs was slower cleared from the blood resulting in a higher PSCA-His TM concentration in the tumor later on after one day. To summarize we have established two PSCA TMs one with and the other without a C-terminal His6-tag which differ in their theranostic properties. Both TMs efficiently redirect UniCAR T cells to kill tumor cells. The PSCA TMs have a higher tumor accumulation and lower nonspecific binding in the normal tissues and blood resulting in a very short half-life that may allow an even faster emergency shut down of the UniCAR system if necessary and excellent short time imaging. The PSCA-His TMs are characterized by a larger nonspecific binding, slower blood-clearance and are apparently better suited for long-term imaging. Both TMs are applicable for the UniCAR based immunotherapy and imaging, complement each other and so they are suitable for the individualized, precise human theranostic.

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.