41BB agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor

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-4-1BBL bispecific antibody fusion protein could enhance the immune-mediated antitumoral effect of these treatments given in combination. Methods: Murine transplantable TS/A tumor cells line was used to investigate increases in FAP expression in tumors after irradiation by IHQ and RT-PCR. We treated bilateral tumor-bearing mice in which only one of the lesions was locally irradiated (2 × 6 Gy) given alone or in combination with a systemic administration of the FAP-4-1BBL 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. Antibody of interest was labeled and tracked its distribution by CT. Colorectal carcinoma samples were also validated. Results: Irradiation of TS/A+CAF tumors showed clear increases of FAP expression levels after local irradiation. The suboptimal radiotherapy regimen in combination with FAP targeted 4-1BBL worked to attain primary tumor control as well as partial abscopal effect. Immune landscape analysis showed an effector and proinflammatory phenotype with an increased infiltration of immune cells. The antitumoral mechanism relied on CD8+ T cells, IFN-I, IFN-γ and CD137 expression. Biodistribution studies of bispecific antibody performed indicated enhanced tumor targeting after tumor irradiation. Human sample results confirmed enhanced immune infiltration and FAP expression after radiotherapy treatment. Conclusion: Our data provides a proof- of- concept and mechanistic insights pertaining the therapeutic efficacy of bispecific FAP-41BBL combined with local radiotherapy.