Immunotheranostic target modules for imaging and navigation of UniCAR T-cells to strike FAP-expressing cells and the tumor microenvironment

(2023) 42:341 Loureiro et al. J Exp Clin Cancer Res https://doi.org/10.1186/s13046-023-02912-w Journal of Experimental & Clinical Cancer Research Open Access RESEARCH Immunotheranostic target modules for imaging and navigation of UniCAR T‑cells to strike FAP‑expressing cells and the tumor microenvironment Liliana R. Loureiro1*†, Lydia Hoffmann1†, Christin Neuber1, Luise Rupp2, Claudia Arndt1,3, Alexandra Kegler1, Manja Kubeil1, Christoph E. Hagemeyer4, Holger Stephan1, Marc Schmitz2,5,6,7, Anja Feldmann1,5,6,7* and Michael Bachmann1,5,6,7*    Abstract Background Chimeric antigen receptor (CAR) T-cells are a promising approach in cancer immunotherapy, particularly for treating hematologic malignancies. Yet, their effectiveness is limited when tackling solid tumors, where immune cell infiltration and immunosuppressive tumor microenvironments (TME) are major hurdles. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) and various tumor cells, playing an important role in tumor growth and immunosuppression. Aiming to modulate the TME with increased clinical safety and effectiveness, we developed novel small and size-extended immunotheranostic UniCAR target modules (TMs) targeting FAP. Methods The specific binding and functionality of the αFAP-scFv TM and the size-extended αFAP-IgG4 TM were assessed using 2D and 3D in vitro models as well as in vivo. Their specific tumor accumulation and diagnostic potential were evaluated using PET studies after functionalization with a chelator and suitable radionuclide. Results The αFAP-scFv and -IgG4 TMs effectively and specifically redirected UniCAR T-cells using 2D, 3D, and in vivo models. Moreover, a remarkably high and specific accumulation of radiolabeled FAP-targeting TMs at the tumor site of xenograft mouse models was observed. Conclusions These findings demonstrate that the novel αFAP TMs are promising immunotheranostic tools to foster cancer imaging and treatment, paving the way for a more convenient, individualized, and safer treatment of cancer patients. Keywords Cancer immunotherapy, UniCAR T-cells, Fibroblast activation protein (FAP), Tumor microenvironment (TME), 3D in vitro models, Immunotheranostic Target Modules (TMs) † Liliana R. Loureiro and Lydia Hoffmann contributed equally to this work. *Correspondence: Liliana R. Loureiro Anja Feldmann Michael Bachmann Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Loureiro et al. J Exp Clin Cancer Res (2023) 42:341 Page 2 of 17 Graphical Abstract Background Immunotherapeutic approaches, including CAR T-cell therapy, have revolutionized cancer treatment by leveraging immune cells to target cancerogenic cells [1]. Even though CAR T-cell therapy has demonstrated particularly remarkable success in the treatment of hematological malignancies, its efficacy when applied to solid tumors has been hampered by several hurdles, which include, for example, the immunosuppressive tumor microenvironment (TME) and antigen heterogeneity [1, 2]. Thus, it is crucial to find and target appropriate antigens as well as develop alternative and optimized CAR T-cell approaches. An example of such an alternative target antigen is the fibroblast activation protein (FAP), a cell surface protein upregulated in many cancers (over 90% of human epithelial carcinomas) and particularly highly expressed in stromal cells of the tumor microenvironment, like cancer-associated fibroblasts (CAFs) [3, 4]. FAP expression promotes tumor growth and invasion, emerging as an excellent candidate for diagnostic and therapeutic applications alongside modulation of the TME [5, 6]. Cancer therapies specifically targeting FAP are still in their early phases, with several strategies being explored, such as FAP-targeted antibodies and small molecule inhibitors, vaccine therapy, and CAR T-cell therapy (NCT03932565) [7–12]. As the TME is a complex and dynamic environment that influences the efficacy of CAR T-cell therapies, strategies to improve the outcome of such therapies may include the targeting of suitable TME-associated targets (e.g. FAP) and the optimization of CAR T-cell approaches. Alternatives to conventional CAR T-cells include the development of adapter CAR therapies, which provide greater flexibility and control in targeting cancer cells [13, 14]. The UniCAR system developed by our group is one of such modular approaches wherein an adapter molecule called target module (TM) is required and responsible for the specific bridging of UniCAR T-cells to tumor cells [15–21]. The findings from clinical studies using this approach meet the expectations related to high efficiency, safety, and controllability, aiming for its straightforward application in the treatment of both hematological and solid tumors (NCT04230265, NCT04633148) [22]. In detail, given that UniCAR T-cells express a CAR that does not recognize any surface antigen, in the absence of a TM these engineered T-cells are inert and harmless to patients. These only get activated and promote cell killing in the presence of a TM composed of a UniCAR peptide epitope (E5B9) linked to a binding moiety that specifically recognizes the target cells. Such TMs are highly versatile molecules that can be easily constructed in various formats and sizes to redirect UniCAR T-cells towards virtually any antigen [18, 23, 24]. Hence, they additionally hold great potential for diagnostic imaging applications when combined with appropriate radionuclides. Given all the above, here we have developed novel immunotheranostic TMs with different formats and sizes for diagnostic imaging and UniCAR T-cell therapy specifically targeting human FAP. Their functionality was extensively assessed using 2D, 3D, and in vivo models, envisioning a novel combined approach to help tackle the immunosuppressive tumor microenvironment commonly found in solid cance (...truncated)