Abstract 1100: 4-1BBL agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor
Authors: Garate-Soraluze Eneko , Serrano-Mendioroz Irantzu , de Andrea Carlos E , Rullan Toni , Claus Christina , Umana Pablo , klein Christian , Melero Ignacio , Rodriguez-Ruiz Maria E. 
Published: April 2023

Abstract
Background. Ionizing radiation (IR) is a double-edge sword for immunotherapy as it may cause both immunosuppressive and immunostimulatory effects. The interactions of IR with the tumor microenvironment (TME) is a key factor for this balance. Fibroblast activation protein (FAP) is expressed on the surface of CAFs in many cancer types and its presence is associated with poor immune response to immune checkpoint blockade in patients. We hypothesize that IR increases FAP expression in CAFs, therefore the combination of IR with targeted immunomodulators such as an agonistic FAP-41BBL bispecific antibody-like fusion protein could enhance the immune-mediated antitumoral effect of these treatments given in combination.
Methods: Murine transplantable tumor cells lines (TSA and MC38) were used to investigate increases in FAP expression on CAFs in tumors under irradiation using IHQ and qPCR. Established orthotopic transplanted models were used. We treated bilateral tumor-bearing mice in which only one of the lesions was locally irradiated (2×6Gy) given alone or in combination with a systemic administration of the FAP-41BBL agonistic bispecific construct. Tumor sizes were followed over time and in the cellular composition microenvironment (TME) was assessed by immunochemistry and multiplex tissue immunofluorescence. Selective depletions of immune cell populations were used to delineate the immune system requirements for efficacy.We measured the changes in FAP expression following radiotherapy in engrafted syngeneic TSA and MC38 models using immunohistochemistry (IHC) and qPCR. TSA cells and CAFs were coinjected orthotopically creating bilateral tumor-bearing models, which were irradiated (2x6Gy) only inone of the lesions, either as a single treatment intervention or in combination with systemic administrations of FAP-41BBL. Treatment efficacy was evaluated measuring tumor volume and changes in the TME. Anti -D8 and a neutralizing anti-type-I IFN mAbs were used to define requirements for the efficacy of the radioimmunotherapy combination.
Results. Irradiation of TSA-breast cancer tumors showed clear increases of FAP expression levels after local irradiation. The suboptimal radiotherapy regimen chosen failed to control TSA-derived tumors in in which TSA-breast cancer cells had been co-engrafted with CAF. The combination of irradiation and FAP-41BBL resulted in more prominent increases of FAP expression after local irradiation and, importantly, induced durable complete responses in more than 50% of the mice treated with the combination. A role in the therapeutic effect for CD8+ T cells and type-I IFN was uncovered. Robust immune memory was observed in re-challenge experiments.
Conclusion. Our data provides a proof-of-concept and mechanistic insights pertaining the therapeutic efficacy of the bispecific FAP-41BBL fusion protein combined with local radiotherapy.
Citation Format: Eneko Garate-Soraluze, Irantzu Serrano-Mendioroz, Carlos E de Andrea, Toni Rullan, Christina Claus, Pablo Umana, Christian klein, Ignacio Melero, Maria E. Rodriguez-Ruiz. 4-1BBL agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1100.