BackgroundRadioimmunotherapy combines irradiation of tumor lesions with immunotherapy to achieve local and abscopal control of cancer. Most immunotherapy agents are given systemically, but strategies for delivering immunotherapy locally are under clinical scrutiny to maximize efficacy and avoid toxicity. Local immunotherapy, by injecting various pathogen-associated molecular patterns, has shown efficacy both preclinically and clinically. BO-112 is a viral mimetic based on nanoplexed double-stranded RNA (poly I:C) which exerts immune-mediated antitumor effects in mice and humans on intratumoral delivery. BO-112 and focal irradiation were used to make the proof-of-concept for local immunotherapy plus radiation therapy combinations.MethodsMurine transplantable tumor cell lines (TS/A, MC38 and B16-OVA) were used to show increased immunogenic features under irradiation, as well as in bilateral tumor models in which only one of the lesions was irradiated or/and injected with BO-112. Flow cytometry and multiplex tissue immunofluorescence were used to determine the effects on antitumor immunity. Depletions of immune cell populations and knockout mice for the IFNAR and BATF-3 genes were used to delineate the immune system requirements for efficacy.ResultsIn cultures of TS/A breast cancer cells, the combination of irradiation and BO-112 showed more prominent features of immunogenic tumor cell death in terms of calreticulin exposure. Injection of BO-112 into the tumor lesion receiving radiation achieved excellent control of the treated tumor and modest delays in contralateral tumor progression. Local effects were associated with more prominent infiltrates of antitumor cytotoxic tumor lymphocytes (CTLs). Importantly, local irradiation plus BO-112 in one of the tumor lesions that enhanced the therapeutic effects of radiotherapy on distant irradiated lesions that were not injected with BO-112. Hence, this beneficial effect of local irradiation plus BO-112 on a tumor lesion enhanced the therapeutic response to radiotherapy on distant non-injected lesions.ConclusionThis study demonstrates that local BO-112 immunotherapy and focal irradiation may act in synergy to achieve local tumor control. Irradiation plus BO-112 in one of the tumor lesions enhanced the therapeutic effects on distant irradiated lesions that were not injected with BO-112, suggesting strategies to treat oligometastatic patients with lesions susceptible to radiotherapy and with at least one tumor accessible for repeated BO-112 intratumoral injections.

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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.