Laura M. Moser ^1,2,3,4,5* Catrin Heim ¹ Sebastian E. Koschade ^3,4,5,6,7 Philipp Wendel ^2,3,8,9 Süleyman Bozkurt ⁷ Sabine Harenkamp ¹⁰ Hermann Kreyenberg ^1,2 Michael Merker ^1,2 Christian Münch ^11,4,7 Elise Gradhand ¹² Meike Vogler ^13,3,4 Evelyn Ullrich ^2,3,4,5,8 Halvard Bönig ^10,14 Jan-Henning Klusmann ^15,3,4,5 Peter Bader ^1,2,3,4,5 Winfried S Wels ^16,3,4 Eva Rettinger ^1,2,3,4,5*

• ¹ Division for Stem Cell Transplantation and Immunology, Department of Pediatrics, Goethe University Frankfurt, University Hospital, Frankfurt, Germany
• ² Department of Pediatrics, Goethe University Frankfurt, University Hospital, Frankfurt, Germany
• ³ German Cancer Consortium (DKTK), partner site Frankfurt/Mainz, Frankfurt, Germany
• ⁴ Frankfurt Cancer Institute (FCI), Frankfurt, Hesse, Germany
• ⁵ Universitäres Centrum für Tumorerkrankungen (UCT), Frankfurt, Germany
• ⁶ Department of Medicine, Hematology/Oncology, Goethe University Frankfurt, University Hospital, Frankfurt, Germany
• ⁷ Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Frankfurt, Hesse, Germany
• ⁸ Experimental Immunology & Cell Therapy, Department of Pediatrics, Goethe University, Frankfurt, Germany
• ⁹ Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany
• ¹⁰ Department of Cellular Therapeutics/Cell Processing, Institute for Transfusion Medicine and Immunotherapy, Frankfurt, Germany
• ¹¹ Cardio-Pulmonary Institute, Frankfurt, Germany
• ¹² Department of Pediatric and Perinatal Pathology, Dr. Senckenberg Institute of Pathology, Frankfurt, Germany
• ¹³ Institute for Experimental Pediatric Hematology and Oncology, Frankfurt, Germany
• ¹⁴ Department of Medicine, Division of Hematology, University of Washington, Seattle, Washington, United States
• ¹⁵ Department of Pediatrics, Goethe University Frankfurt, Halle, Bavaria, Germany
• ¹⁶ Institute for Tumor Biology and Experimental Therapy, Georg Speyer Haus, Frankfurt, Hesse, Germany

Introduction: CAR-T cell therapy, though successful in hematologic malignancies, faces challenges in solid tumors due to limitations of autologous T cells. Cytokine-induced killer (CIK) cells can be given safely across allogeneic barriers and constitute alternative effector cells generated from healthy donors. CIK cells are a heterogenous population of predominantly T cells with a mixed natural killer (NK) phenotype and combine non-MHC-restricted cytotoxicity with potent anti-tumor capacity of the adaptive immune system. Here, we characterize and compare efficacy, phenotypic subpopulations and modes of action of CAR-CIK cells and conventional CAR-T cells from same-donor samples in ErbB2+ rhabdomyosarcoma (RMS). Methods: To benchmark CAR-CIK against conventional CAR-T cells, effector cells were generated from same-donor samples and lentivirally transduced with a second generation CD28-CD3ζ CAR. Effector subpopulations and their dynamics upon target cell exposure were phenotypically characterized by flow cytometry. Efficacy was assessed in human ErbB2+ RMS cancer cell lines and primary patient samples in vitro and ex vivo using cytotoxicity and spheroid co-incubation assays. Modes of action were assessed by comparing cytokine secretion profiles using bead-based multiplexed flow cytometry and by liquid chromatography mass spectrometry whole cell proteomics. Finally, we used an in vivo model of RMS mimicking minimal metastatic residual disease to compare anti-tumor potency of CAR-CIK vs. CAR-T cells and to assess their target organ infiltration. Results: In vitro assays demonstrated superior cytotoxicity of CAR-CIK cells against RMS cell lines and primary tumor samples. Long-term co-incubation with tumor spheroids led to expansion of CAR-CIK cells and enrichment of CD3+CD56+ TNK cells. CAR-CIK cell cytokine signature showed significantly increased secretion of effector molecules like interferon-γ, perforin and granulysin, and lower secretion of Th2 cytokines IL-2, IL-4 and IL-10. Whole cell proteomics showed corresponding upregulation of chemokine signaling and NK-cytotoxicity pathways in CAR-CIK cells. In NSG mice xenografted with ErbB2+ RMS, a single injection of either CAR-effector cells strongly impeded metastatic tumor development and significantly improved survival.Conclusion: Our results demonstrate that CAR-CIK cells are at least equipotent to CAR-T cells. Combined with their favorable safety profile and allogeneic applicability, these findings position CAR-CIK cells as promising immune effectors for solid tumors.