Preclinical evaluation of homodimeric FAPI target modules for UniCAR-mediated targeting of FAP-expressing tumor and stromal cells

Hugo Boutier; Simona Mattiussi; Liliana R Loureiro; Nicole Berndt; Antonia Stammberger; Nathalia Jones-Cifuentes; Lydia Hoffmann; Emile Verhulst; Marc Schmitz; Ingrid De Meester; Umberto M Battisti; Claudia Arndt; Anja Feldmann; Frank Roesch; Matthias M Herth; Michael Bachmann

doi:10.1080/2162402X.2026.2717836

. 2026 Aug 21;15(1):2717836. doi: 10.1080/2162402X.2026.2717836

Preclinical evaluation of homodimeric FAPI target modules for UniCAR-mediated targeting of FAP-expressing tumor and stromal cells

Hugo Boutier ^a,¹, Simona Mattiussi ^b,¹, Liliana R Loureiro ^a, Nicole Berndt ^a, Antonia Stammberger ^c, Nathalia Jones-Cifuentes ^a, Lydia Hoffmann ^a, Emile Verhulst ^d, Marc Schmitz ^c,^e,^f, Ingrid De Meester ^d, Umberto M Battisti ^b, Claudia Arndt ^a,^g, Anja Feldmann ^a,^e,^f, Frank Roesch ^h, Matthias M Herth ^b,ⁱ, Michael Bachmann ^a,^e,^f,^j,^*

^a Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Dresden, Germany

^b Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

^c Institute of Immunology, Faculty of Medicine Carl Gustav Carus, TUD University of Technology, Dresden, Germany

^d Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Wilrijk-Antwerp, Belgium

^e German Cancer Consortium (DKTK), Partner Site Dresden, Dresden, Germany

^f National Center for Tumor Diseases (NCT), NCT/UCC Dresden, a partnership between DKFZ, Faculty of Medicine and Univ. Hosp. Carl Gustav Carus, TU Dresden & Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany

^g Mildred Scheel Early Career Center, Faculty of Medicine Carl Gustav Carus, TUD Dresden, University of Technology, Dresden, Germany

^h Department of Chemistry-TRIGA Site, Johannes Gutenberg University Mainz, Mainz, Germany

ⁱ Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet, Copenhagen, Denmark

^j Faculty of Medicine and University Hospital Carl Gustav Carus, TUD, Dresden, Germany

CONTACT Michael Bachmann profmiba@googlemail.com Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, 01328, Dresden, Germany

^¹

Hugo Boutier and Simona Mattiussi contributed equally to this work.

Roles

Hugo Boutier: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Simona Mattiussi: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Liliana R Loureiro: Formal analysis, Methodology, Writing – review & editing

Nicole Berndt: Formal analysis, Investigation, Methodology, Writing – review & editing

Antonia Stammberger: Formal analysis, Investigation, Methodology, Writing – review & editing

Nathalia Jones-Cifuentes: Formal analysis, Investigation, Methodology, Writing – review & editing

Lydia Hoffmann: Formal analysis, Investigation, Methodology, Writing – review & editing

Emile Verhulst: Formal analysis, Investigation, Methodology, Writing – review & editing

Marc Schmitz: Methodology, Supervision, Writing – review & editing

Ingrid De Meester: Methodology, Supervision, Writing – review & editing

Umberto M Battisti: Formal analysis, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing

Claudia Arndt: Formal analysis, Methodology, Writing – review & editing

Anja Feldmann: Conceptualization, Resources, Supervision, Writing – review & editing

Frank Roesch: Conceptualization, Writing – review & editing

Matthias M Herth: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing

Michael Bachmann: Conceptualization, Funding acquisition, Resources, Writing – original draft, Writing – review & editing

PMCID: PMC13502003 PMID: 42627862

Abstract

Due to its overexpression in the tumor microenvironment of most solid malignancies, fibroblast activation protein (FAP) has emerged as an ideal target for (immuno)theranostic applications. The clinically tested UniCAR system represents a promising adapter CAR T-cell approach, in which CAR T-cell activity is regulated through the administration of target modules (TMs). Here, we report the first homodimeric FAP inhibitor (FAPI)-based TMs that enable efficient adapter CAR T-cell immunotherapy of FAP-positive tumors. The novel TMs consist of two UAMC-1110 FAPI moieties, the E5B9 UniCAR epitope, a suitable polyethylene glycol spacer to ensure epitope accessibility, and a functional group that allows for further TM functionalization for diagnostic and radiotherapeutic applications. Following the synthesis of the novel FAPI TMs, we evaluated their functionality using both in vitro and in vivo models. The FAPI TMs successfully redirect UniCAR T-cells and mediate potent lysis of FAP-positive cells in vitro and in an immunodeficient mouse model. In addition, we established a 3D heterospheroid model consisting of tumor cells expressing prostate stem cell antigen (PSCA) alongside FAP-positive stromal fibroblasts. We showed that simultaneous dual-targeting leads to enhanced UniCAR-mediated cytotoxicity. Notably, fibroblast killing is mediated by the release of PSCA from dying tumor cells and its subsequent binding to the surface of neighboring PSCA-negative fibroblasts, thereby making them susceptible to PSCA-directed targeting. Overall, our work not only summarizes the successful development of novel dimeric FAPI adapter molecules for immunotherapeutic applications in solid tumors but also provides novel insights into the targeting of PSCA-positive tumors.

Introduction

To improve the efficacy of current therapies against solid malignancies, recent research has focused on targeting the tumor microenvironment (TME), which plays a critical role in cancer progression, invasion, metastasis, immune escape, and therapy resistance. ¹ ^, ² Within the TME, fibroblast activation protein (FAP) has emerged as one of the most promising stromal markers. ³ FAP expression has been reported in numerous tumor entities, making it a potential pan-tumor target. ⁴ Importantly, FAP is not only overexpressed in the tumor stroma of most epithelial malignancies, but has also been detected on the surface of tumor cells themselves. ⁵ ^, ⁶ Due to its absence or very low expression in healthy adult tissues, FAP has been extensively used as a target for radioligand-based drug delivery and immunotherapeutic applications such as chimeric antigen receptor (CAR) T-cell therapy. ^7-9

To date, the clinically approved CAR T-cell treatments have demonstrated remarkable efficacy in hematologic malignancies. ¹⁰ However, their broader clinical application remains limited by severe side effects and challenges in targeting solid tumors, such as antigen heterogeneity and the immunosuppressive TME. ¹¹ To address these issues, the UniCAR system was developed. ¹² In this platform, T-cells are engineered to express the UniCAR, which recognizes the non-immunogenic E5B9 epitope derived from the human nuclear La-SS/B protein. ¹³ ^, ¹⁴ UniCAR T-cells only interact with target cells through a target module (TM), which enables the activation of effector cells and the selective elimination of target cells. So far, the functionality of the UniCAR system has been demonstrated in numerous preclinical studies for a variety of antigens (e.g. FAP, CD123, PSCA) and has already been translated into phase I clinical trials (NCT04230265, NCT04633148). ¹² ^, ^15-19 We recently developed FAP-targeted immunotheranostic TMs based on the potent and selective FAP inhibitor (FAPI) UAMC-1110. ¹⁹ ^, ²⁰ These monomeric FAPI TMs induced significant UniCAR-mediated antitumor activity in vivo, and enabled non-invasive diagnostic imaging of FAP-positive tumors. ¹⁹

In the context of targeted radioligand therapy (TRT), monomeric FAPI tracers often exhibit relatively rapid pharmacokinetics and a short biological half-life, which can limit tumor retention and consequently reduce therapeutic efficacy. ²¹ To address this limitation, several groups have explored multimerization strategies to enhance FAP targeting. ^22-27 In particular, dimeric FAPIs have displayed enhanced binding avidity to FAP, improved tumor uptake, and increased retention times compared with their monomeric counterparts in both diagnostic and endoradiotherapeutic applications. ^22-27 These characteristics are particularly advantageous for TRT, where extended tumor residence can increase radiation dose delivery to the tumor.

Here, we aimed to develop the first low-molecular-weight homodimeric FAPI TMs for application in UniCAR T-cell immunotherapy. The design aimed to combine the advantages of multivalent FAP targeting with the modular controllability of the UniCAR platform, while also providing chemical flexibility for future diagnostic imaging and radiotherapeutic applications. After successful synthesis of the novel homodimeric FAPI TMs, their functionality was demonstrated in the context of UniCAR T-cell immunotherapy using 2D and 3D in vitro models as well as an immunodeficient mouse model.

Materials and methods

Organic synthesis

We synthesized a homodimeric TM targeting FAP by linking two UAMC-1110 moieties as previously described. ²² The E5B9 UniCAR epitope was coupled to the FAPI homodimer via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). A polyethylene glycol (PEG) spacer of sixteen or twenty-four units was introduced between the FAPI dimer and UniCAR epitope. Detailed synthesis and analytical characterization of the novel dimeric FAPI molecules are presented in the supplementary material (Supplemental Figures 1-3).

Cell culture

The cell lines HT1080, 3T3, MRC-5 and A-549 were obtained from the American Type Culture Collection (ATCC). The HT1080 cell line was genetically modified to express the firefly luciferase (Luc) and overexpress human FAP (hFAP), and named HT1080 hFAP. ¹⁵ The lung cancer cell line A-549 was genetically modified via lentiviral transduction to overexpress PSCA, the resulting line was named A-549 PSCA. MRC-5 and A-549 PSCA cell lines were lentivirally transduced to express Luc. All cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO₂ and cultured in complete DMEM medium. ¹⁹ Routine testing for mycoplasma contamination was performed using PCR.

Heterospheroid generation

To differentiate the cell lines in the heterospheroids, MRC-5 and A-549 PSCA cells were first labeled with Cell Proliferation Dyes eFluor™ 450 and eFluor™ 670 (Thermo Fisher Scientific), respectively. Heterospheroids were generated in 96-well plates precoated with 1% agarose by seeding unstained or eFluor^TM-stained MRC-5 and A-549 PSCA cells at a fibroblast-to-tumor ratio of 5:1 in 100 µL of complete DMEM (10,000 total cells). The plate was centrifuged at 850x g for 3 min and incubated at 37 °C for 24 h to allow for spheroid formation.

Expression of PSCA-specific UniCAR TM

The αPSCA-scFv TM containing the E5B9 UniCAR epitope and the PSCA-specific single chain variable fragment (MB1), was cloned into the lentiviral vector p6NST50 as described previously. ¹⁶ This vector was used to transduce 3T3 cells for stable expression of the αPSCA-scFv TM. Secreted TM was purified by Ni-NTA affinity chromatography (Qiagen) followed by analysis of protein concentration and purity through SDS-PAGE and immunoblotting, as described elsewhere. ¹⁸

Binding assays using flow cytometry

FAP and PSCA expression at the surface of target cell lines was assessed using flow cytometry. In short, 1 × 10⁵ target cells or trypsinized heterospheroids (previously blocked with Fc blocking reagent (Miltenyi Biotec GmbH)) were stained for 30 min with anti-hFAP mAb (clone #427819, R&D systems) or anti-PSCA 7F5 mAb ²⁸ , and binding was detected using goat anti-mouse polyclonal IgG Abs conjugated with Pacific Blue^TM (PB) or Phycoerythrin^TM (PE) (Invitrogen). The binding properties of the FAPI TMs to target cell lines were assessed using the anti-La monoclonal antibody (mAb) 5B9 (in-house produced antibody ²⁹ ), as described previously. ¹⁹ Live and dead cells were distinguished by adding propidium iodide (PI) solution (Miltenyi Biotec GmbH). All stained cells were analyzed using MACSQuant 10 Analyzer, MACSQuantify software (Miltenyi Biotec) and FlowJo 10.10 Software (BD Life Sciences). Binding curves were obtained and K _D values were calculated using the nonlinear regression curve fit with GraphPad Prism 10.4.1.

T-cell Isolation and generation of UniCAR T-cells

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats of healthy donors (German Red Cross, Dresden) and Pan T-cells were purified as described elsewhere. ³⁰ Subsequent T-cell activation was performed using T-Cell TransAct (Miltenyi Biotec) and UniCAR T-cells were generated via lentiviral transduction as previously described. ³⁰ During expansion, T-cells were cultured in TexMACS medium (Miltenyi Biotec) supplemented with human IL-2, IL-7 and IL-15 (Miltenyi Biotec). UniCAR T-cells were maintained in complete RPMI 1640 medium without cytokines for 24 hours prior to experiments. ¹⁹

In vitro luciferase-based cytotoxicity assay

The cytotoxic potential of UniCAR T-cells was analyzed using the luciferase-expressing target cell lines according to previously published protocols. ¹⁵ Dose-response curves were plotted and half maximal effective concentration (EC₅₀) values were calculated using GraphPad Prism 10.4.1 (GraphPad Software).

Cytokine release assay

Levels of secreted interferon gamma (IFNγ), interleukin 2 (IL-2), tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor (GM-CSF) by target cells were determined using OptEIA Human IFNγ, IL-2, TNF, and GM-CSF ELIZA Sets (BD BioSciences) as previously described. ¹⁵

In vivo cytotoxicity assay

All animal experiments presented in this study were performed according to the guidelines of the German Regulations for Animal Welfare and adhered to the ARRIVE guidelines. To evaluate the cytotoxic potential of UniCAR T-cells in combination with the FAPI TMs in vivo, a co-injection experiment was performed following a well-established protocol (e.g. ¹⁹ ) and has been accepted by the legal authorities as prerequisite for a clinical application of the UniCAR system. The protocol was approved by the local Ethical Committee for Animal Experiments (DD25-5131/562/49, Landesdirektion Sachsen, Chemnitz, Germany). The co-injection experiment was performed using 8-week-old female NXG mice (NOD-Prkdc ^scid -IL2rg ^Tm1/Rj, Janvier Labs, Saint Berthevin, Cedex, France). Mice were kept under specific pathogen free (SPF) conditions, with a temperature of 22–26 °C, humidity of 40-70%, and a 12-hour light/dark cycle. Mice were randomly divided into 4 groups with each five individuals. Prior to subcutaneous injections and subsequent optical imaging, mice were temporarily anesthetized by administration of 4-5% sevoflurane in an oxygen-air mixture (40% oxygen) at a flow rate of 0.6 L/min. All anesthetized animals were kept warm, with a maximum anesthesia duration of 10 minutes per session. All the groups were subcutaneously injected in the right flank with 0.5 × 10⁶ of luciferase-expressing HT1080 hFAP target cells. Control groups 1 and 2 received target cells alone or in combination with UniCAR T-cells (E:T ratio 1:1), respectively. Treatment groups 3 and 4 received target cells, UniCAR T-cells, and 1 nmol of either (FAPI)₂-PEG₁₆-E5B9 or (FAPI)₂-PEG₂₄-E5B9. Bioluminescence imaging of the mice was performed at the day of injection (day 0) and monitored at 1, 2, 5, 7, 9 and 12 days post-injection to follow up tumor growth. No animal was excluded during the experiment. For each measurement, anesthetized mice were subcutaneously injected in the neck fold with 150 µl Luciferin solution (15 mg/ml D-Luciferin (IVISbrite D-Luciferin Potassium Salt Bioluminescent Substrate (XenoLight), Revvity) in sterile 1x Dulbecco's phosphate buffered saline, w/o Mg²⁺ and Ca²⁺) and bioluminescence imaging was performed 10 min post injection using IVIS Spectrum In Vivo Imaging System (PerkinElmer) and the Living Image Software (PerkinElmer). On the last day of measurement mice were euthanized via cervical dislocation according to the approved experimental application.

Multiplex immunohistochemistry staining of heterospheroids

Unstained heterospheroids were fixed in 4% neutral buffered formalin (NBF) for 2 h at room temperature, and subsequently stained with hematoxylin. Thereafter, spheroids were embedded in histogel (Fisher Scientific) and fixed overnight in 4% NBF at 4 °C, followed by dehydration. After paraffin embedding, 2.5 µm thin sections were cut using a microtome and mounted on adhesive slides (Agilient Dako). Multiplex immunohistochemistry was performed using OPAL technology (Akoya Biosciences) on the Ventana Discovery Ultra platform (Ventana medical Systems) was performed as previously described. ³¹ Briefly, formalin-fixed, paraffin-embedded (FFPE) spheroids were deparaffinized and subjected to heat-mediated antigen retrieval. Sections were then incubated with a primary antibody, followed by application of a horseradish peroxidase (HRP)-coupled secondary antibody. Upon addition of tyramide signal amplification (TSA)-fluorophores (Akoya Biosciences), HRP converted the TSA molecules to reactive TSA radicals, which covalently bound to tyrosine residues on the epitope. Subsequent heat-mediated stripping removed antibody complexes, allowing the next staining cycle to begin. This staining cycle was performed three times in total using three different primary antibodies and TSA fluorophores with distinct emission wavelengths. Details on the antibodies, their concentrations, and suppliers are listed in Supplemental Table 1. Staining was completed by counterstaining with DAPI (Sigma Aldrich) and slides were mounted with Fluoromount-G® medium (SouthernBiotech). Stained slides were stored at 4 °C in the dark until imaging. Multispectral images (MSIs) of spheroids were acquired at 200x magnification using the PhenoImager HT 2.0 fluorescence microscope (Akoya Biosciences). MSIs were subsequently spectrally unmixed in inForm software (Akoya Biosciences) using a previously generated spectral library and exported as multichannel TIFF files. Representative staining images were processed using ImageJ software. ³²

Flow cytometry-based killing assay

Heterospheroids were co-cultured with UniCAR T-cells at an E:T ratio of 2:1 in the absence or presence of 2 nM (FAPI)₂-PEG₂₄-E5B9 TM and/or 1 nM αPSCA-scFv TM diluted in complete RPMI. ¹⁹ After 16 hours, heterospheroids were either imaged with the Keyence microscope BZ X710 or dissociated for flow cytometry analysis. For each condition, 3 spheroids were pooled and washed once with PBS and trypsinized at 37 °C for 10 min. Cells were washed with complete DMEM and resuspended in 50 μL medium. All stained cells were analyzed using MACSQuant 10 Analyzer and MACSQuantify software (Miltenyi Biotec).

Statistical evaluation

All statistical analyzes were performed using GraphPad Prism 10.4.1. Statistical significance was determined using one-tailed or two-tailed paired T-test or two-way ANOVA with a Greenhouse-Geisser correction and a Tukey’s multiple comparisons test. Significance was set at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Results

Design and synthesis of dimeric FAPI TMs

Given the potential of multimeric FAP inhibitors as endoradiotherapeutic agents, ³³ we generated two homodimeric FAPI target modules enabling UniCAR T-cell immunotherapy. As summarized in Figure 1a,b, the novel dimeric FAPI TMs consist of two UAMC-1110 moieties, ²⁰ ^, ²² a PEG spacer of varying length, and the E5B9 peptide. Our previous work on monomeric FAPI TMs demonstrated that the length of the spacer between the E5B9 epitope and the FAPI moiety is critical for UniCAR T-cell redirection. ¹⁹ Since substantial functional differences were observed between the PEG₁₂- and PEG₂₄-based monomeric FAPI TMs in favor of the longer version, PEG spacers of 16 or 24 units were selected for the novel homodimeric FAPI TMs. To form the homodimeric structure, two UAMC-1110 moieties were connected by incorporating a glutamic acid linker between the 2 monomers, as already described for the DOTAGA.Glu.(FAPi)₂ tracer applied in patients. ²² A cysteine residue was employed to connect the FAPI homodimer to the PEG spacer and the E5B9 peptide. The fast and selective CuAAC reaction was used to conjugate the UniCAR epitope to the FAPI homodimer. In the future, the functional thiol group of the cysteine may be further substituted with various chemical entities, such as a chelator for radioligand delivery (Figure 1b). Detailed synthesis is described in the supplemental section (Supplemental Figures 1-3).

A 2-panel diagram shows UniCAR T-cell system and FAP-specific target modules. Panels display molecular structures and sizes. The two-panel diagram presents the UniCAR T-cell system and FAP-specific target modules. Panel A shows the UniCAR T-cell with an extracellular scFv binding domain, CD28 transmembrane, and CD3 zeta signaling domains. The presence of a target module containing the E5B9 UniCAR epitope and a FAP-specific domain is required to redirect UniCAR T-cells to FAP-positive cells. Panel B displays chemical structures of dimeric FAPI target modules consisting of the E5B9 epitope, a PEG spacer, and two UAMC-1110 FAPI moieties. The panels also show molecular weights of small molecules, monomers, dimers, scFv, and IgG derivatives. — Design of dimeric FAPI TMs for UniCAR T-cell immunotherapy. (a) Schematic representation of the UniCAR system and FAP-specific TMs based on small molecules (monomeric/dimeric FAPI) or antibody derivatives (αFAP-scFv/αFAP-IgG4 TMs). The UniCAR is a second-generation CAR composed of an extracellular scFv binding domain targeting the E5B9 epitope, the CD28 transmembrane and intracellular signaling domains, and the CD3 zeta signaling domain. The presence of a TM, containing the E5B9 UniCAR epitope (orange) and a FAP-specific domain (blue), is required to redirect UniCAR T-cells to FAP-positive cells and trigger effector cell functions. (b) Chemical structures of the dimeric FAPI TMs, consisting of the E5B9 UniCAR epitope (orange), a PEG spacer of 16 or 24 units (gray) and two UAMC-1110 FAPI moieties (blue).

Dimeric FAPI TMs promote UniCAR-mediated lysis of HT1080 hFAP in vitro

The initial functional analysis of the novel dimeric FAPI TMs was performed using the well-established FAP-overexpressing HT1080 hFAP model. ¹⁵ ^, ¹⁸ ^, ¹⁹ As confirmed by flow cytometry, over 99% of HT1080 hFAP cells express FAP (Figure 2a). The specific binding of (FAPI)₂-PEG₁₆-E5B9 and (FAPI)₂-PEG₂₄-E5B9 TMs to HT1080 hFAP cells was successfully detected using the anti-La mAb 5B9 and PB-conjugated anti-mouse IgG Abs (Figure 2a). Both dimeric FAPI TMs could bind similarly and with high affinity to the target (Figure 2b). The apparent K_D value of (FAPI)₂-PEG₁₆-E5B9 and (FAPI)₂-PEG₂₄-E5B9 TMs were calculated as 124 and 68.9 nM, respectively. Importantly, both molecules were able to redirect UniCAR T-cells in a dose-dependent manner and promote the specific lysis of HT1080 hFAP cells (Figure 2c). The dimeric FAPI TMs showed no substantial differences, exhibiting EC₅₀ values of 641 and 179 pM for (FAPI)₂-PEG₁₆-E5B9 TM and (FAPI)₂-PEG₂₄-E5B9 TM, respectively. When UniCAR T-cells were incubated with wild-type (FAP-negative) HT1080 cells in the presence of either FAPI TM, no lysis was observed (Supplemental Figure 4). Furthermore, consistent with their cytotoxic potential, the UniCAR T-cells secreted IFNγ, IL-2, TNF, and GM-CSF only in the presence of each FAPI TM (Figure 2d). Taken together, these results demonstrate the in vitro functionality of the novel dimeric FAPI TMs.

A diagram shows FAPI TM binding to hFAP cells, binding affinity, and UniCAR T-cell cytotoxicity and cytokine release. The four-panel diagram presents in vitro evaluation of the cytotoxicity and immunostimulatory cytokine production by UniCAR T-cells redirected by dimeric FAPI TMs against HT1080 hFAP cells. Panel A shows surface expression of FAP and specific binding of dimeric FAPI TMs to HT1080 hFAP cells assessed by flow cytometry. Panel B displays the binding affinity of the FAPI TMs to HT1080 hFAP cells investigated by flow cytometry using increasing TM concentrations. Panel C shows in vitro elimination of HT1080 hFAP cells by UniCAR T-cells redirected by the FAPI TMs. Panel D quantifies cytokines released by UniCAR T-cells cultured with target cells and FAPI TMs. — In vitro evaluation of the cytotoxicity and immunostimulatory cytokine production by UniCAR T-cells redirected by dimeric FAPI TMs against HT1080 hFAP cells. (a) Surface expression of FAP and specific binding of dimeric FAPI TMs to HT1080 hFAP cells assessed by flow cytometry. FAP expression (light blue) was detected using anti-FAP mAb and goat anti-mouse IgG Pacific Blue^TM. The binding of 1000 nM of (FAPI)₂-PEG₁₆-E5B9 (dark blue) and (FAPI)₂-PEG₂₄-E5B9 (purple) TMs was detected using anti-La mAb 5B9 and goat anti-mouse IgG Pacific Blue^TM. Data is shown for one representative experiment out of three independent experiments with similar results (n = 3). (b) The binding affinity of (FAPI)₂-PEG₁₆-E5B9 (dark blue) and (FAPI)₂-PEG₂₄-E5B9 (purple) TMs to HT1080 hFAP was further investigated by flow cytometry using increasing concentrations of the respective TMs, anti-La mAb 5B9 and goat anti-mouse IgG Pacific Blue^TM. Titration curves are plotted as normalized mean fluorescence intensity (MFI) ± SD from three independent experiments (n = 3). (c) In vitro elimination of HT1080 hFAP cells by UniCAR T-cells redirected by dimeric FAPI TMs. Target cell lysis was measured using luciferase-based assays. UniCAR T-cells were cultured with HT1080 hFAP for 16 h at an E:T ratio of 5:1 in the absence or presence of (FAPI)₂-PEG₁₆-E5B9 (dark blue) or (FAPI)₂-PEG₂₄-E5B9 (purple) TM. The dotted line indicates the level of target cell lysis in the absence of TM. (d) Cytokines released by UniCAR T-cells cultured with target cells and 10 nM of FAPI TMs were quantified by ELISA. All results are plotted as mean $\pm$ SD from three independent donors and experiments (n = 3), each performed with three technical replicates. Statistical analysis was performed using one-tailed paired T-test in GraphPad Prism 10.4.1. Stars indicate statistical significance; p-values close to the cutoff (0.05) are shown on the graphs. K _D: equilibrium dissociation constant; EC₅₀: half maximal effective concentration.

UniCAR T-cells combined with dimeric FAPI TMs promote significant tumor killing in vivo

We next evaluated whether the promising in vitro findings could be recapitulated in vivo using a well-established co-injection model. ¹⁵ ^, ¹⁹ For this purpose, NXG mice were subcutaneously injected with either luciferase-expressing HT1080 hFAP cells alone or HT1080 hFAP cells together with UniCAR T-cells in the absence or presence of either (FAPI)₂-PEG₁₆-E5B9 TM or (FAPI)₂-PEG₂₄-E5B9 TM (Figure 3a, b). Tumor growth was monitored using bioluminescence imaging for up to 12 days. The tumor growth in the control group injected with HT1080 hFAP cells alone gradually increased over time (Figure 3b, black curve). Tumor size within the second control group injected with HT1080 hFAP cells and UniCAR T-cells in the absence of TM, was stable up to 2 days post-injection before growing significantly up to day 9 (Figure 3b, gray curve). Interestingly, a substantial reduction of the tumor signal until day 2 was observed for the groups treated with UniCAR T-cells and (FAPI)₂-PEG₁₆-E5B9 or (FAPI)₂-PEG₂₄-E5B9 TM (Figure 3b, blue and purple curves). Between days 2 and 9, comparable tumor regrowth was observed for both treatments, which is most likely attributed to the limited in vivo half-life of the small-molecule TMs. Nonetheless, the overall tumor burden in both treatment groups was significantly lower than both control groups between days 2 and 9. Taken together, these results demonstrate that both dimeric FAPI TMs combined with UniCAR T-cells exert significant inhibition of tumor growth in vivo.

A 2-panel diagram shows in vivo killing of UniCAR T-cells by homodimeric FAPI TMs. The two-panel diagram presents an assessment of in vivo killing of UniCAR T-cells redirected by homodimeric FAPI TMs. Panel A shows the bioluminescence signal of HT1080 hFAP cells across all groups over 9 days. Panel B displays a quantitative analysis of the bioluminescence plotted as mean plus or minus standard deviation from 5 mice per group. Panel A and Panel B show the UniCAR T-cells combined with either (FAPI) 2 -PEG 16 -E5B9 or (FAPI) 2 -PEG 24 -E5B9 resulted in a significant reduction in tumor growth compared to UniCAR T-cells alone. — Assessment of in vivo killing of UniCAR T-cells redirected by homodimeric FAPI TMs. Female NXG mice were injected with either HT1080 hFAP cells (black), HT1080 hFAP and UniCAR T-cells (gray), or HT1080 hFAP and UniCAR T-cells at E:T ratio of 1:1 in combination with 1 nmol of either (FAPI)₂-PEG₁₆-E5B9 (blue) or (FAPI)₂-PEG₂₄-E5B9 (purple). (a) The bioluminescence signal of HT1080 hFAP cells across all groups is shown for days 0, 1, 2, 5, 7 and 9 days after injection. (b) Quantitative analysis of the bioluminescence was conducted, and results were plotted as mean ± SD from five mice per group (n = 5). Statistical significance was assessed in GraphPad Prism 10.4.1 using two-way ANOVA with Greenhouse-Geisser correction and with Tukey´s multiple comparison test with respect to the control group injected with only HT1080 hFAP cells and UniCAR T-cells. Stars indicate statistical significance.

In vitro functional evaluation of PSCA- and FAP-targeted UniCAR T-cells against tumor-stroma heterospheroids

We next aimed to progress toward a 3D in vitro model to further investigate tumor-stroma interactions in the context of FAP-targeted CAR T-cell immunotherapy. Given the critical role of fibroblasts in modulating tumor growth, architecture, and therapeutic response, we selected a three-dimensional heterospheroid co-culture system combining tumor cells with fibroblasts to better reflect the cellular complexity of the TME. The MRC-5 lung fibroblast cell line, which natively expresses FAP, and the A-549 PSCA lung cancer cell line, which was genetically modified to express PSCA, were chosen to represent the stromal and malignant compartments, respectively. Prior to heterospheroid establishment, each cell line was characterized independently in 2D models to establish the functional properties relevant to UniCAR T-cell immunotherapy.

On the one hand, we first confirmed that the wild-type MRC-5 cell line natively expresses FAP and does not express PSCA using flow cytometry (Figure 4a). The homodimeric adapter (FAPI)₂-PEG₂₄-E5B9 was selected to target FAP-positive fibroblasts. The successful binding of the PEG₂₄-based TM to MRC-5 cells was confirmed by flow cytometry (Figure 4a), resulting in an apparent K_D of 45.3 nM (Supplemental Figure 5a). On the other hand, we confirmed that the recombinant A-549 PSCA cell line expresses PSCA, and does not natively express FAP (Figure 4b). To redirect UniCAR T-cells against this cell line, the already established scFv-based anti-PSCA TM (αPSCA-scFv TM), was used in this study. ¹⁶ The binding of the αPSCA-scFv TM to A-549 PSCA cells was subsequently confirmed by flow cytometry (Figure 4b), yielding an apparent K_D constant of 171 nM (Supplemental Figure 5b).

A six-panel diagram shows in vitro functional characterization of FAPI and PSCA TMs in MRC-5 and A-549 PSCA 2D models. The six-panel diagram presents the in vitro functional characterization of (FAPI) 2 -PEG 24 -E5B9 and αPSCA-scFv TMs in MRC-5 and A-549 PSCA monolayer models. Panel A shows FAP and PSCA expression on MRC-5 cells. Panel B displays FAP and PSCA expression on A-549 PSCA cells. Panel C demonstrates redirection of UniCAR T-cells by the (FAPI) 2 -PEG 24 -E5B9 TM against MRC-5 cells. Panel D shows redirection of UniCAR T-cells by the PSCA-specific TM against A-549 PSCA cells. Expected absence of redirection is shown in Panel E for PSCA-specific TM against MRC-5 cells and in Panel F for (FAPI) 2 -PEG 24 -E5B9 TM against A-549 PSCA cells. — In vitro functional characterization of (FAPI)₂-PEG₂₄-E5B9 and αPSCA-scFv TMs in MRC-5 and A-549 PSCA monolayer models. Surface expression of FAP and PSCA antigens and specific binding of UniCAR TMs to (a) MRC-5 cells and (b) A-549 PSCA cells assessed by flow cytometry. FAP (light blue) and PSCA (light red) expression was detected using anti-FAP mAb or anti-PSCA mAb, respectively, and goat anti-mouse IgG Pacific Blue^TM. The binding of (FAPI)₂-PEG₂₄-E5B9 TM (purple, 1000 nM) TM and αPSCA-scFv TM (red, 2000 nM) was detected using anti-La mAb 5B9 and goat anti-mouse IgG Pacific Blue^TM. Data is shown for one representative experiment out of three independent experiments with similar results (n = 3). Redirection of UniCAR T-cells (c) by (FAPI)₂-PEG₂₄-E5B9 TM against MRC-5 cells and (d) by αPSCA-scFv TM against A-549 PSCA cells. Expected absence of redirection of UniCAR T-cells (e) by αPSCA-scFv TM against MRC-5 cells and (f) by (FAPI)₂-PEG₂₄-E5B9 TM against A-549 PSCA cells. UniCAR T-cells were cultured with FAP⁺ or PSCA⁺ cells for 16 h in the absence or presence of (FAPI)₂-PEG₂₄-E5B9 or αPSCA-scFv TM at an E:T ratio of 2:1. Results are plotted as mean $\pm$ SD from three independent donors and experiments (n = 3), each performed with three technical replicates. Statistical analysis was performed using two-tailed paired T-test in GraphPad Prism 10.4.1 EC₅₀: half maximal effective concentration; ns: not-significant.

We subsequently investigated whether each TM could redirect UniCAR T-cells against both MRC-5 and A-549 PSCA cell lines. To this end, each cell line was co-cultured with UniCAR T-cells in the absence or presence of (FAPI)₂-PEG₂₄-E5B9 TM or αPSCA-scFv TM at an E:T ratio of 2:1. The FAPI and the PSCA-specific TMs were able to redirect UniCAR T-cells in a dose-dependent manner and promote the specific lysis of FAP⁺ PSCA^- MRC-5 and FAP^- PSCA⁺ A-549 PSCA cells, respectively (Figure 4c,d). Specific UniCAR T-cell activation was further corroborated by elevated secretion of IFN-γ, IL-2, TNF, and GM-CSF, which was observed exclusively in the presence of the respective UniCAR TM across each cell model (Supplemental Figure 6a, b). When UniCAR T-cells were incubated with wild-type (FAP^- PSCA^-) A-549 cells and αPSCA-scFv TM, no cell lysis was observed (Supplemental Figure 7). Importantly, no target cell death was observed when (1) FAP⁺ PSCA^- MRC-5 cells were co-cultured with UniCAR T-cells and αPSCA-scFv TM (Figure 4e), and (2) FAP^- PSCA⁺ A-549 PSCA cells were incubated with UniCAR T-cells and (FAPI)₂-PEG₂₄-E5B9 TM (Figure 4f). Taken together, these results demonstrate the prerequisites to establish a robust heterospheroid model based on FAP⁺ PSCA^- MRC-5 and FAP^- PSCA⁺ A-549 PSCA cell lines.

To differentiate the stromal and tumor cell lines in our 3D heterospheroid model, FAP⁺ PSCA^- MRC-5 and FAP^- PSCA⁺ A-549 PSCA cells were labeled with eFluor^TM 450 and eFluor^TM 670, respectively. As described in Figure 5a, heterospheroids were generated by seeding eFluor-stained MRC-5 and A-549 PSCA cells at a fibroblast-to-tumor ratio of 5:1. After 24 hours, UniCAR T-cells and (FAPI)₂-PEG₂₄-E5B9 TM or/and αPSCA-scFv TM were co-cultured with heterospheroids for 16 h (Figure 5a). As observed in Figure 5b (brightfield), the fibroblasts and tumor cells formed uniform, round, and compact heterospheroids, with an average diameter of 375-400 µm. Fibroblasts and tumor cells were heterogeneously distributed throughout the heterospheroids, with no clearly defined separation between the tumoral (Figure 5b, eFluor^TM 670) and the stromal compartments (Figure 5b, eFluor^TM 450). When incubated with UniCAR T-cells, the heterospheroids were surrounded by the effectors (Figure 5b, brightfield). Interestingly, the EGFP reporter signal of the UniCAR T-cells was detected exclusively in the presence of either TM and both TMs, and was predominantly localized around the spheroid (Figure 5b, GFP). Importantly, when both FAP- and PSCA-specific TMs were present, the EGFP signal was significantly increased compared to controls, suggesting increased effector recruitment (Figure 5b, GFP).

Two-panel diagram shows co-culture of fibroblasts and tumor cells, UniCAR T-cell recruitment, microscopy and flow cytometry. The two-panel diagram presents the process of co-culturing MRC-5 fibroblasts and A-549 PSCA tumor cells to form heterospheroids, followed by the recruitment of UniCAR T-cells and analysis using fluorescence microscopy, flow cytometry, and multiplex immunohistochemistry. Panel A shows the cell seeding and co-culture steps. Panel B displays brightfield and fluorescence images of the heterospheroids with MRC-5 cells in blue, A-549 PSCA cells in red, and UniCAR T-cells in green. — UniCAR T-cell recruitment to MRC-5/A-549 PSCA heterospheroids in the presence of dimeric FAPI and PSCA-specific TMs. (a) As schematically shown, eFluor^TM 450⁺ MRC-5 and eFluor^TM 670⁺ A-549 PSCA cells were seeded at a fibroblast-to-tumor ratio of 5:1 and co-cultured for 24 h to allow for heterospheroid formation. UniCAR T-cells were subsequently added at an E:T ratio of 2:1 in the absence or presence of 2 nM (FAPI)₂-PEG₂₄-E5B9 TM and/or 1 nM αPSCA-scFv TM. After a 16-hour incubation, heterospheroids were analyzed by fluorescence microscopy, flow cytometry, or multiplex immunohistochemistry (*figure created with BioRender.com*). (b) Heterospheroids were imaged with the Keyence microscope BZ X710 (scale bar: 100 µm). Brightfield images show heterospheroid morphology; eFluor^TM 450 and eFluor^TM 670 fluorescence indicate the spatial distribution of MRC-5 fibroblasts and A-549 PSCA tumor cells, respectively; and GFP signal indicates the presence and engagement of UniCAR T-cells.

We then further analyzed the specific killing of the fibroblast and tumor cell lines using flow cytometry (Figure 6a). A very low killing was observed in the presence of effector cells and in the absence of TM. Interestingly, in the presence of αPSCA-scFv TM, UniCAR T-cells promoted a similar lysis of both fibroblast and tumor cells (40%). In the presence of (FAPI)₂-PEG₂₄-E5B9 TM, UniCAR T-cells promoted the specific killing of FAP⁺ PSCA^- MRC-5 cells and no substantial lysis of the FAP^- PSCA⁺ A-549 PSCA cells. Importantly, when incubated with both (FAPI)₂-PEG₂₄-E5B9 and αPSCA-scFv TMs, UniCAR T-cells promoted a significantly increased lysis of both fibroblast and tumor cell lines (MRC-5: 60%; A-549 PSCA: 47%), suggesting a synergistic effect of the TMs.

A 2-panel diagram shows target cell killing and spatial organization of heterospheroids after UniCAR T-cell redirection. The two-panel diagram presents target cell-specific killing and spatial distribution of UniCAR T-cells within heterospheroids. Panel A shows specific lysis of MRC-5 and A-549 PSCA cells in the absence or presence of FAPI and PSCA target modules. Panel B displays the spatial organization of PSCA-positive cells, FAP-positive fibroblasts, and CD3-positive T-cells within heterospheroid cross-sections. It shows the heterospheroid architecture and UniCAR T-cell distribution under various treatment conditions. — Target cell-specific killing and spatial organization of MRC-5/A-549 PSCA heterospheroids following UniCAR T-cell redirection. Heterospheroids were co-cultured with UniCAR T-cells at an E:T ratio of 2:1 in the absence or presence of 2 nM (FAPI)₂-PEG₂₄-E5B9 TM and/or 1 nM αPSCA-scFv TM for 16 h. (a) Efluor^TM-labeled heterospheroids were subsequently dissociated and analyzed by flow cytometry to assess target cell-specific killing. Specific lysis of MRC-5 (FAP⁺ PSCA^-) and A-549 PSCA (FAP^- PSCA⁺) cells is shown in the absence of TM or in the presence of αPSCA-scFv TM, (FAPI)₂-PEG₂₄-E5B9 TM, or both TMs combined. Results are plotted as mean $\pm$ SD from three independent donors and experiments (n = 3). Statistical analysis was performed using one-tailed paired T-test in GraphPad Prism 10.4.1. Stars indicate statistical significance; p-values close to the cutoff (0.05) are shown on the graphs. (b) To further characterize the three-dimensional architecture of the heterospheroids and the spatial distribution of UniCAR T-cells within them, cross-sectioned, unstained heterospheroids were analyzed by multiplex immunohistochemistry staining for DAPI, PSCA, FAP, and CD3. Multispectral images were acquired at 200x magnification using the PhenoImager HT 2.0 fluorescence microscope (Akoya Biosciences) and spectrally unmixed in inForm software (Akoya Biosciences). Images show the spatial distribution of PSCA⁺ cells, FAP⁺ fibroblasts, and CD3⁺ UniCAR T-cells within representative heterospheroid cross-sections (scale bar: 100 µm) under multiple conditions: spheroids only; UniCAR: spheroid + UniCAR T-cells; UniCAR + FAPI: spheroid + UniCAR T-cells + FAPI TM; UniCAR + PSCA TM: spheroid + UniCAR T-cells + αPSCA-scFv TM; UniCAR + FAPI + PSCA TM: spheroid + UniCAR T-cells + FAPI TM + αPSCA-scFv TM.

To further characterize the three-dimensional architecture of the heterospheroids and the spatial distribution of UniCAR T-cells within them, we performed multiplex immunohistochemistry staining of cross-sectioned heterospheroids for DAPI, PSCA, FAP, and CD3 (Figure 6b). Consistent with the eFluor labeling shown in Figure 5b, PSCA⁺ tumor cells and FAP⁺ fibroblasts were heterogeneously distributed throughout the heterospheroid sections, without clear distinction between stromal and tumor compartments. CD3⁺ T-cells were found around, throughout and within the heterospheroids, indicating that UniCAR T-cell infiltration is not limited to the outer layers of the spheroids. This distribution may contribute to the effective killing of both fibroblast and tumor cell populations observed across TM conditions (Figure 6a).

To investigate the unexpected killing of fibroblasts in the presence of the PSCA TM (Figure 6a), dissociated heterospheroids were analyzed by flow cytometry using anti-PSCA and anti-FAP mAbs. Tumor cells remained negative for FAP and positive for PSCA across all conditions (Supplemental Figure 8a). While fibroblasts were FAP-positive in all conditions, a substantial PSCA signal was detected within the fibroblast compartment upon co-culture with tumor cells (Supplemental Figure 8b). This positivity was not observed in fibroblasts when cultured in absence of tumor cells (Figure 4a) and was enhanced in the presence of UniCAR T-cells and UniCAR TM (Supplemental Figure 8b). These findings suggest that PSCA positivity on fibroblasts may contribute to their susceptibility to PSCA-TM mediated UniCAR T-cell cytotoxicity.

To investigate whether PSCA positivity on the surface of MRC-5 fibroblasts resulted from de novo expression or material transfer from A-549 PSCA tumor cells, heterospheroids were generated using eFluor-labeled FAP^- PSCA^- A-549 wild-type cells and FAP⁺ PSCA^- MRC-5 fibroblasts. Following incubation with UniCAR T-cells and FAP- and/or PSCA-specific TMs, significant fibroblast killing was observed only in the presence of the FAP-specific TM, whereas no lysis of wild-type tumor cells occurred with the PSCA-specific TM (Supplemental Figure 9a). Importantly, the tumor compartment remained both PSCA- and FAP-negative across all conditions (Supplemental Figure 9b), and the fibroblast compartment remained FAP-positive and PSCA-negative (Supplemental Figure 9c). These findings suggest that the unexpected killing of MRC-5 fibroblasts when co-cultured with A-549 PSCA, UniCAR T-cells and UniCAR TM (Figure 6a) most likely arise from PSCA transfer to the fibroblast surface, explaining MRC-5 induced susceptibility to PSCA-directed killing.

Overall, fluorescence microscopy, flow cytometry and immunohistochemistry staining results yielded consistent results and suggested enhanced recruitment, expansion and cytotoxic activity of UniCAR T-cells against MRC-5/A-549 PSCA heterospheroids in the presence of both tumor- and stroma-specific TMs.

Discussion

Over the last years, FAP has been extensively used as target in diagnostic imaging, endoradiotherapy ⁸ ^, ^34-37 and immunotherapy. ³ ^, ⁹ ^, ³⁸ The use of small molecule (immuno)theranostic agents has gained much interest due to their small size associated with fast penetration into the tumor mass; ability to target intracellular molecules; relatively low production costs compared to antibodies; and predictable pharmacokinetics and pharmacodynamics. ^39-41 The development of FAPIs has led to the generation of numerous innovative radiotracers for nuclear imaging and TRT, ⁴ ^, ²⁶ ^, ³⁵ ^, ^42-52 as well as for adapter CAR T-cell immunotherapy. ¹⁹ ^, ⁵³ ^, ⁵⁴ To date, three monomeric adapter molecules based on FAPIs have been developed for the redirection of adapter CAR T-cells toward FAP-expressing cells. ¹⁹ ^, ⁵³ ^, ⁵⁴ Among these, our group reported the first monomeric FAPI-based adapter molecules enabling simultaneous nuclear imaging and elimination of FAP⁺ cells by UniCAR T-cells with a single molecule. ¹⁹ As monovalent FAPI radiotracers, the monomeric FAPI TMs exhibit rapid systemic clearance. ¹⁹ In parallel, dimeric FAPIs have displayed enhanced binding to FAP, improved tumor uptake, and increased retention times in radioligand delivery applications. ^22-27 These properties are particularly advantageous for TRT, where extended tumor residence is associated with increased radiation delivery. Based on these insights, we aimed to develop the first dimeric FAPI TMs allowing to combine enhanced tumor retention and pharmacokinetics properties with UniCAR-based immunotherapy and potential immunotheranostic applications.

The newly designed homodimeric FAPI-based adapter molecules incorporate two FAP-binding moieties, the E5B9 UniCAR epitope and a PEG₁₆ or PEG₂₄ spacer. After chemical synthesis, the specific binding of both TMs to HT1080 hFAP recombinant model could be detected using the anti-La mAb 5B9. In accordance with the binding data, (FAPI)₂-PEG₁₆-E5B9 and (FAPI)₂-PEG₂₄-E5B9 TMs effectively redirected UniCAR T-cells against FAP-expressing HT1080 cells. Besides cytotoxicity, it was also relevant to quantify the effector-mediated secretion of immunostimulatory cytokines as they mediate cytotoxicity and contribute to CAR T-cell proliferation and effector function. ^55-58 In a clinical context, these proinflammatory molecules also promote immune cell recruitment and may contribute to treatment-related side effects. ⁵⁹ In line with the cytotoxicity data, UniCAR T-cells released similar amounts of cytokines in the presence of either homodimeric FAPI TM. Importantly, cytokine secretion did not occur in absence of FAPI TM, indicating the robustness of the UniCAR system. We then assessed the immunotherapeutic potential of the novel TMs in an immunodeficient mouse model. When combined with either dimeric FAPI TM, UniCAR T-cells promoted significant, although transient, reduction of tumor growth. Tumor regrowth was not unexpected, as similar results have been observed for monomeric FAPI and antibody-derived FAP-specific adapter molecules using the same model. ¹⁵ ^, ¹⁹ This phenomenon may be explained by several factors, including exhaustion and/or limited persistence of UniCAR T-cells, FAP expression heterogeneity or downregulation within target cells following initial response (potentially allowing outgrowth of FAP-low or FAP-negative cell subpopulations), and the pharmacokinetic properties of the homodimeric FAPI TMs. Their small size may indeed lead to rapid systemic clearance, resulting in reduced UniCAR T-cell activity and subsequent tumor regrowth. In this context, such FAPI TMs may require frequent, if not continuous, administration to maintain tumor control. Overall, both PEG₁₆- and PEG₂₄-based dimeric TMs exhibited comparable in vitro and in vivo functionality, indicating that a PEG spacer of 16 units is sufficient to enable optimal and effective recognition of the E5B9 UniCAR epitope when the FAPI TM is already bound to FAP. ¹⁹

Despite promising efficacy in several preclinical studies, safety and potential on-target/off-tumor toxicity are a relevant consideration for the development and clinical translation of FAP-directed CAR T-cell therapies. While several groups have reported encouraging efficacy with no or very limited side effects, ³⁸ others have reported significant CAR-associated toxicities in mouse models, including cachexia and bone marrow toxicity. ⁶⁰ ^, ⁶¹ Although the reasons underlying the heterogeneous toxicity profiles observed across FAP-CAR preclinical studies remain incompletely understood, this variability underscores the importance of careful safety consideration for FAP-directed therapies. In this context, a valuable advantage of the UniCAR platform over conventional FAP CAR T-cells is its switchability. Since UniCAR T-cells require continuous presence of a TM to remain active, effector activity can be effectively terminated by stopping TM administration. This safety switch mechanism has recently been validated clinically in a first-in-human phase I trial evaluating the safety profile of UniCAR T-cells and a CD123-specific TM in relapsed/refractory acute myeloid leukemia (AML) patients (NCT04230265). In this trial, treatment-related toxicities could be rapidly and consistently resolved upon interruption of TM administration, ⁶² ^, ⁶³ providing clinical proof-of-concept for the on/off-switch mechanism in the context of CD123-positive AML. Although not yet tested in patients with FAP-positive solid tumors, these clinical results support the plausibility that the same safety switch could mitigate on-target/off-tumor toxicity in the context of FAP-directed UniCAR T-cells. Future safety and tolerability studies are required, ideally using immunocompetent models expressing physiological FAP levels in normal tissue.

As antigen density was shown to play a role on CAR T-cell efficacy, potential tumor escape and off-tumor toxicity, ¹¹ ^, ⁶⁴ ^, ⁶⁵ the potency of the (FAPI)₂-PEG₂₄-E5B9 TM was assessed using two monolayer cell models with varying FAP expression levels. As described in recent publications, while HT1080 hFAP recombinant cells used in this work express over 100,000 antigens per cell, MRC-5 cells natively express approximately 10,000 FAP molecules per cell. ¹⁵ ^, ¹⁹ Comparable cytotoxicity properties were observed for the (FAPI)₂-PEG₂₄-E5B9 TM in both MRC-5 or HT1080 hFAP models, with EC₅₀ values in the picomolar range. In contrast, higher amounts of pro-inflammatory cytokines were secreted by UniCAR T-cells against high-expressing HT1080 hFAP compared to MRC-5 cells. These results are consistent with other studies performed with small molecule- and antibody-derived anti-FAP UniCAR TMs. ¹⁵ ^, ¹⁹ In addition, recent reports indicate that CAR T-cell-mediated cytotoxicity and cytokine production require distinct antigen density thresholds, with cytotoxicity generally occurring at lower levels of antigen expression than cytokine release. ^65-69

Compared to our previously published monovalent PEG₂₄-based FAPI TM, ¹⁹ the novel homodimeric PEG₂₄-based FAPI TM exhibits comparable in vitro functional properties (Supplemental Table 2). Both molecules display nanomolar affinity for hFAP and mediate potent lysis of MRC-5 and HT1080 hFAP models, with half-maximal effective concentrations in the picomolar range (Supplemental Table 2). Both monomeric and dimeric FAPI TMs also enable substantial UniCAR-mediated tumor growth inhibition in immunodeficient mice (Supplemental Table 2). Under the experimental conditions applied, no clear functional superiority of the dimeric version was observed. However, these in vitro and in vivo assays do not capture potential differences in pharmacokinetics, which are known to be favorably influenced by dimerization (or multimerization) in the context of nuclear imaging and endoradiotherapy. ^22-27 ^, ⁷⁰ ^, ⁷¹ The homodimeric FAPI TMs may offer further advantages for in vivo theranostic applications, particularly with respect to tumor accumulation and residence time. Consequently, the here described homodimeric molecules provide a promising platform for future development, such as chelator conjugation for radiolabeling, allowing the combination of UniCAR T-cell immunotherapy and selective delivery of radioisotopes.

After demonstrating the functionality of our FAPI TMs, we established a physiologically relevant 3D heterospheroid co-culture system. This model reflects and recapitulates the key characteristics of the TME, including tumor-stroma interactions, cellular and architectural complexity, and infiltration limitations for immune cells and therapeutic molecules. ^72-78 As PSCA is a well-established and highly promising potential tumor-associated antigen (TAA), ¹⁶ ^, ²⁸ ^, ⁷⁹ ^, ⁸⁰ the FAP⁺ PSCA^- MRC-5 fibroblasts and FAP^- PSCA⁺ A-549 PSCA tumor cell lines were selected and independently characterized, first in 2D assays and subsequently in the context of a 3D heterospheroid model. Specific eradication of the FAP⁺ fibroblasts occurred only in the presence of (FAPI)₂-PEG₂₄-E5B9 TM, while elimination of PSCA⁺ tumor cells required the PSCA-specific UniCAR TM. Importantly, no UniCAR-mediated cytotoxicity was observed in the absence of TM or when target-mismatched TMs were used, confirming the controllability and the selectivity of the UniCAR system.

The combination of MRC-5 fibroblasts and A-549 PSCA tumor cells led to the formation of heterospheroids with heterogenous cell distribution. Interestingly, a partial elimination of FAP-positive fibroblasts was observed in the presence of UniCAR T-cells and PSCA-specific TM alone. To investigate these results, fibroblasts and tumor compartments were stained with FAP- and PSCA-specific mAbs. Detection of PSCA on fibroblasts occurred in the presence of UniCAR T-cells and TM. As fibroblasts did not express PSCA when cultured alone or when co-cultured with PSCA-negative tumor cells within heterospheroids, these observations argue against intrinsic PSCA expression in fibroblasts. These results rather suggest that PSCA positivity on fibroblasts may arise from tumor-stromal interactions within the heterospheroid system, potentially enabling acquisition of tumor-derived antigen by neighboring stromal cells. However, the underlying antigen transfer mechanism needs further clarification. It may occur through contact-dependent and/or contact-independent mechanisms such as protease-mediated PSCA cleavage, exchange of tumor-cell fragments, membrane debris or extracellular vesicles, or simply by passive release of PSCA from dying tumor cells. ⁸¹ ^, ⁸² Notably, morphological characterization of the heterospheroids revealed an heterogenous distribution of FAP-positive fibroblasts and PSCA-positive tumor cells, without a distinct core-shell compartmentalization (Figure 6b). This distribution supports close spatial proximity between the two cell populations and may facilitate contact-dependent transfer mechanisms such as trogocytosis or direct membrane exchange. Alternatively, the PSCA containing material may be released by dead cells and adsorbed to the neighboring cells.

From a translational perspective, this phenomenon could be associated with both potential risks and benefits, which needs to be carefully weighed. On the one hand, antigen acquisition by neighboring PSCA-negative stromal or healthy cells could narrow the therapeutic window of PSCA-directed immunotherapies by making non-malignant cells susceptible to PSCA-directed killing. On the other hand, the same phenomenon could in principle be advantageous by extending cytotoxic activity to PSCA-negative cell subpopulations within a heterogeneous tumor and TME. Determining how long transferred PSCA remains accessible on neighboring cells will be an important question for future studies aiming to optimize treatment protocols. An attractive hypothesis is that PSCA may represent a potential cell death target for combination with conventional tumor treatment strategies such as chemotherapy or radiation-based therapies, as previously described for the La autoantigen, ^83-86 although this will require confirmation of the underlying antigen transfer mechanism.

Besides the unexpected data, an important aspect of our work presented here is that the combination of both TMs resulted not only in enhanced UniCAR-mediated heterospheroid eradication but also in increased effector T-cell recruitment. All in all, the presented results underline the benefits of simultaneously targeting both the tumor and its stroma, which is known to promote immune evasion and therapy resistance in solid cancers. From a translational point of view, the ability to combine different TMs to target both the tumor and its microenvironment is a major advantage of adapter CAR T-cells like the UniCAR system over conventional CAR T-cell approaches, which are usually restricted to a single target. Although this novel 3D system provides a relevant and valuable platform to evaluate the effect of combinatorial targeting strategies on both tumor and stromal compartments under controlled conditions, future in vitro integrating patient-derived cancer cells and cancer-associated fibroblasts, together with in vivo validation in stroma-rich tumor models (e.g. syngeneic models), would enhance translational relevance by maintaining the phenotypic heterogeneity and stromal crosstalk of the native tumor microenvironment.

In conclusion, we have synthesized and characterized the first homodimeric FAPI adapter molecules that can be used in both preclinical in vitro and in vivo models for adapter CAR T-cell immunotherapy. Furthermore, we demonstrate that homodimeric FAPI TMs can be used in combination with a TAA-specific adapter molecule to promote synergistic killing within tumor-fibroblast 3D heterospheroids. Notably, these results suggest that PSCA may represent an attractive bystander cell death target.

Supplementary Material

ARRIVE_checklist_HB_dimeric.pdf

Supplementary File

KONI_A_2717836_SM8931.pdf^{(1.7MB, pdf)}

20260717_Dimeric_supp.docx

KONI_A_2717836_SM7153.docx^{(27MB, docx)}

Acknowledgments

We would like to acknowledge Annegret Riedel for her excellent technical support. We also warmly thank Eugenia Crespo, Cansu Daglar, Rutuja Gupte, Fabienne Weinberg, Eswari Chockalingam, and Lamice Shurafa for the shared routine laboratory work. We are grateful to Prof. Dr. Torsten Tonn (German Red Cross Blood Donation, Dresden) for providing buffy coats of healthy donors.

Funding Statement

HB and SM were supported by the European Union’s Horizon Europe Research and Innovation program under the Marie Skłodowska-Curie Action OncoProTools N°101073231 granted to MB, IDM, and MMH. CA is a fellow of the Mildred Scheel Early Career Center Dresden P² funded by the German Cancer Aid (Deutsche Krebshilfe). In addition, this research was funded by the Helmholtz Initiative and Networking Fund (Radio-Immunotheranostics (MHELTHERA), project ID: InterLabs-0031) granted to MB by the Helmholtz International Lab MHELTHERA. None of the funders was involved in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

Data confirming the results of this study are presented in the manuscript and are available from the corresponding authors upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed at https://doi.org/10.1080/2162402X.2026.2717836.

Complying with ethics of experimentation

The research conducted with human T-cells was approved by the local ethics committee of the Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Germany (EK138042014). All studies have complied with all relevant ethical regulations including the Declaration of Helsinki. All animal procedures were conducted in accordance with the guidelines of the German Regulations for Animal Welfare. The protocols were approved by the local Ethical Committee for Animal Experiments (DD25-5131/562/49, Landesdirektion Sachsen, Chemnitz, Germany).

Informed consent

Human samples were obtained from healthy blood donors who provided written informed consent (German Red Cross, Dresden, Germany).

Consent for publication

Consent for publication was obtained from all authors.

Abbreviations

AML: Acute myeloid leukemia
CAR: Chimeric antigen receptor
CuAAC: Copper(I)-catalyzed azide-alkyne cycloaddition
DMEM: Dubelcco’s modified Eagle’s medium
E:T: Effector-to-target
EC₅₀: Half maximal effective concentration
ELIZA: Enzyme-Linked Immunosorbent Assay
FAP: Fibroblast Activation Protein
FAPI: FAP inhibitor
GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor
IFNγ: Interferon gamma
IgG4: Immunoglobulin G4
IL-2: Interleukin 2
K _D: Equilibrium dissociation constant
mAb: Monoclonal antibody
MFI: Median fluorescence intensity
MS: Mass spectrometry
PBMCs: Peripheral blood mononuclear cells
PBS: Phosphate buffer saline
PEG: Polyethylene glycol
PI: Propidium iodide
RPMI: Roswell Park Memorial Institute medium
scFv: Single chain variable fragment
SD: Standard deviation
SDS: Sodium dodecyl sulfate
SPF: Specific pathogen-free
TAA: Tumor-associated antigen
TM: Target Module
TME: Tumor microenvironment
TNF: Tumor Necrosis Factor
TRT: Targeted radioligand delivery
UniCAR: Universal CAR
Luc: Luciferase

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ARRIVE_checklist_HB_dimeric.pdf

Supplementary File

KONI_A_2717836_SM8931.pdf^{(1.7MB, pdf)}

20260717_Dimeric_supp.docx

KONI_A_2717836_SM7153.docx^{(27MB, docx)}

Data Availability Statement

Data confirming the results of this study are presented in the manuscript and are available from the corresponding authors upon reasonable request.

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PERMALINK

Preclinical evaluation of homodimeric FAPI target modules for UniCAR-mediated targeting of FAP-expressing tumor and stromal cells

Hugo Boutier

Simona Mattiussi

Liliana R Loureiro

Nicole Berndt

Antonia Stammberger

Nathalia Jones-Cifuentes

Lydia Hoffmann

Emile Verhulst

Marc Schmitz

Ingrid De Meester

Umberto M Battisti

Claudia Arndt

Anja Feldmann

Frank Roesch

Matthias M Herth

Michael Bachmann

Roles

Abstract

Introduction

Materials and methods

Organic synthesis

Cell culture

Heterospheroid generation

Expression of PSCA-specific UniCAR TM

Binding assays using flow cytometry

T-cell Isolation and generation of UniCAR T-cells

In vitro luciferase-based cytotoxicity assay

Cytokine release assay

In vivo cytotoxicity assay

Multiplex immunohistochemistry staining of heterospheroids

Flow cytometry-based killing assay

Statistical evaluation

Results

Design and synthesis of dimeric FAPI TMs

Figure 1.

Dimeric FAPI TMs promote UniCAR-mediated lysis of HT1080 hFAP in vitro

Figure 2.

UniCAR T-cells combined with dimeric FAPI TMs promote significant tumor killing in vivo

Figure 3.

In vitro functional evaluation of PSCA- and FAP-targeted UniCAR T-cells against tumor-stroma heterospheroids

Figure 4.

Figure 5.

Figure 6.

Discussion

Supplementary Material

Acknowledgments

Funding Statement

Disclosure statement

Data availability statement

Supplementary material

Complying with ethics of experimentation

Informed consent

Consent for publication

Abbreviations

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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