2855 - Combination of Radiotherapy and Complement-Mediated Immunotherapy

L. Mai¹, Y. Liang¹, M. Kirschfink¹, and P. E. Huber^2,3; ¹German Cancer Research Center (DKFZ), Molecular and Radiooncology, Heidelberg, Germany, ²University Hospital Heidelberg, RadioOncology and Radiotherapy, Heidelberg, Germany, ³German Cancer Research Center (dkfz) and University Hospital Center, Molecular and Radiation Oncology, Heidelberg, Germany

Purpose/Objective(s): Radiotherapy is a cornerstone of cancer treatment, and the integration of immunotherapy, such as checkpoint inhibition, is increasingly becoming part of cancer treatment strategies. While the complement system plays a crucial role in the human anti-cancer immune response, the full extent of its interaction with radiotherapy remains incompletely understood. Therefore, this study aims to investigate the rationale and interaction mechanisms of combining antibody-based complement-mediated immunotherapy with radiotherapy. Materials/

Methods: Human solid cancer (lung A549, prostate Du145, HCC HEPG2, breast MCF7), lymphoma (Raji, Ramos), and leukemia cells (Reh, K562) were irradiated using X-rays (0 to 8 Gy, up to 72h) and treated with polyclonal antibodies or anti-CD20 monoclonal antibodies, respectively. Chromium release assays measured cell lysis after complement activation. The expression of membrane complement regulatory proteins (mCRPs; CD59, CD55, CD46), which confer resistance against complement activation, apoptosis, cell cycle, and radiation-induced DNA double-strand breaks (DSB, via ?H2AX), were measured by FACS.
Results: Irradiation induced a marked and dose-dependent upregulation of mCRPs in all investigated solid and hematological cell types. Consequently, cytotoxicity assays demonstrated that Ab-induced complement-mediated cell lysis was markedly reduced by pretreatment with irradiation, aligning with the irradiation-induced up-regulation of mCRPs as negative regulators of complement activation. In contrast, clonogenic survival assays showed that intrinsic radiosensitivity increased when complement was activated before irradiation, but not after irradiation. Similarly, radiation-induced apoptosis rates increased with prior complement activation, while complement activation after irradiation did not increase apoptosis. Interestingly, irradiation also upregulated CD20 expression in hematological tumor cells, thus enhancing the targets of monoclonal antibody-based immunotherapy. Mechanistically, in the context of intrinsic radiosensitivity, we found that blocking mCRPs by a combination of Abs to CD59, CD55, CD46 did not change radiation-induced primary DSB induction but increased ?H2AX intensity after 24h and 48h in line with cell cycle changes, suggesting reduced DSB repair capacity by activated complement.
Conclusion: Complement regulatory proteins (mCRPs) play a role in radiation response in human solid and hematologic tumor cells, influencing DNA repair and radioresistance. Irradiation can reduce complement activation by increasing the expression of regulatory mCRPs, potentially impeding the efficiency of antibody therapy. In contrast, pretreatment with complement may increase radiosensitivity and anti-cancer effects, suggesting a role for complement activation in combination with radiotherapy for solid and hematological tumors.