112 Tumor-specific reactivity and effector function of chimeric antigen receptor engineered macrophages targeting MUC1

Background Chimeric antigen receptors (CAR) have demonstrated remarkable efficacy in licensing T cells for antitumor responses against hematopoietic malignancies but had limited success solid tumors. Macrophages, both archetypic phagocytes and professional presenting cells, may exert profound effector functions which complement adaptive cellular immunity. 1 Recently, it was shown that human macrophages engineered to express CARs (CAR-Ms) antigen-specific phagocytosis, inhibited xenograph tumors, induced an inflammatory tumor microenvironment boosting cell responses. 2 Kimura et al. previously completed the first prophylactic cancer vaccine trial based on a non-viral antigen, tumor-associated hypoglycosylated Mucin (MUC1). 3 A panel of fully-human affinity-matured MUC1-specific antibodies raised healthy subjects following immunization identified from these patients. 4 Using scFv domains CAR generation, we now MUC1-targeting CAR-Ms potentially possess reduced off-target specificities. Methods Lentiviral expression vectors containing three unique or CD20-specific antibody, CD3zeta signaling domain, CD28 OX40 co-stimulatory were constructed. The monocyte/macrophage U937, SC, THP-1 lines stably transduced flow-sort purified generate MUC1- CAR-Ms. differentiated into via 48 hour PMA treatment, subsequently evaluated function and/or CD20-expressing K562, ZR-75-1, Raji isolated lung tumors malignant pleural effusions. CAR-M phenotype by flow cytometry vitro differentiation polarization with conventional ‘M1’ ‘M2’ stimuli. Phagocytosis lysosomal processing phagocytosed cargo fluorescence microscopy GFP/CellTrace labeled targets detection pH-sensitive pHrodo co-culture, respectively. Antigen-specific cytokine production determined cytometric bead array co-culture 100mer MUC1 peptide. Results Differentiated possessed expressing IL-8 CD86 further enhanced IFNgamma LPS treatment resistant exhibited phagocytosis subsequent manner, minimal reactivity absence corresponding CD20 antigen. stimulated peptide MUC1+ secreted robust levels pro-inflammatory IL-8, TNFa, IL-1beta, not immunosuppressive IL-10. Conclusions potent tumor-restricted provide novel strategy either alone potential synergistic combination cell-mediated immunotherapies. Acknowledgements authors would like thank Dr. Olivera J. Finn generously providing reagents guidance Michael T. Lotze his mentorship. This study supported funding University Pittsburgh’s Department Cardiothoracic Surgery ACS RD. References Williams CB, Yeh ES, Soloff AC. Tumor-associated macrophages: unwitting accomplices breast malignancy. Npj Breast Cancer [Internet]. Research Foundation/Macmillan Publishers Limited; 2016; :15025. Available from: http://dx.doi.org/10.1038/npjbcancer.2015.25 Klichinsky M, Ruella Shestova O, Lu XM, Best A, Zeeman Human chimeric receptor immunotherapy. Nat Biotechnol 2020; 38 :947–53. T, McKolanis JR, Dzubinski LA, Islam K, Potter DM, Salazar AM, Vaccine Individuals Advanced Adenoma Colon: Immunoprevention Feasibility Study. Prev Res [Internet] 2013; 6 :18–26. http://cancerpreventionresearch.aacrjournals.org/content/6/1/18.abstract Lohmueller JJ, Sato S, Popova L, Chu IM, Tucker MA, Barberena R, Antibodies elicited show tumor-specificity immunotherapeutic potential. Sci Rep :31740. Ethics Approval approved Institutional Review Board approval number CR19120172-005.