Background
Ionizing radiation (IR) is a double-edged sword for immunotherapy as it may have both immunosuppressive and immunostimulatory effects. The biological effects of IR on the tumor microenvironment (TME) are a key factor for this balance. Fibroblast activation protein (FAP) is expressed on the surface of cancer associated fibroblasts (CAF) in many cancer types and its abundance is associated with poor immune response to immune-checkpoint-blockade in patients. We hypothesized that IR increases FAP expression in CAFs, therefore the combination of IR with targeted immunomodulators such as an agonistic anti-FAP-4-1BBL fusion protein could enhance the immune-mediated antitumoral effects of these treatments.
Methods
The murine transplantable TS/A tumor-cell-line co-engrafted with CAFs was used to investigate increases in FAP expression in tumors following irradiation using immunohistochemistry, RT-PCR and multiplex tissue immunofluorescence. One lesion of bilateral tumor-bearing mice was only locally irradiated or combined with weekly injections of the bispecific muFAP-4-1BBL fusion protein (a mouse surrogate for RG7826). Tumor sizes were followed over time and the cellular composition of the TME was assessed by immunochemistry and multiplex tissue immunofluorescence. Selective mAb-mediated depletions of immune cell populations, neutralizing IFNAR and IFN mAbs and gene-modified mice (CD137-/-) were used to delineate the immune cell subsets and mechanisms required for efficacy. 67Ga labeled muFAP-4-1BBL tracked by SPECT-CT was used to study biodistribution. In human colorectal carcinoma samples the inducibility of FAP expression following RT was explored by immunohistochemistry.
Results
Irradiation of TS/A+CAF tumors in mice showed an increase of FAP levels after local irradiation. A suboptimal radiotherapy regimen in combination with muFAP-4-1BBL attained primary tumor control and measurable abscopal effects. Immune TME landscape analyses showed post-treatment increased infiltration of activated immune cells associated with the combined radioimmunotherapy treatment. Efficacy depended on CD8+ T-cells, Type I IFN, IFN- and abililty to express CD137. Biodistribution studies of muFAP-4-1BBL indicated enriched tumor targeting to irradiated tumors. Human colorectal cancer samples pre and post irradiation showed enhanced FAP expression after radiotherapy.
Conclusions
Increased FAP expression in the TME as a result of radiotherapy can be exploited to target agonist 4-1BB immunotherapy to malignant tumor lesions using a FAP-4-1BBL antibody fusion protein.
Ethics Approval
The experimental protocols were approved by the Ethics Committee of the University of Navarra (CEEA037-20 and CEAAE27-23) in accordance with European Council Guidelines All patients signed informed consent forms for their tissues to be used in this study.