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Introduction: In experimental animal models, biological sex-differences in the manifestation and severity of normal tissue radiation injury have been well-documented. Previously we demonstrated male and female rats have differential and highly reproducible responses to high-dose partial body irradiation (PBI) with male rats having greater susceptibility to both gastrointestinal acute radiation syndrome (GI-ARS) and radiation pneumonitis than female rats.Methods: In the current study, we have investigated whether differential expression of the renin-angiotensin system (RAS) enzymes angiotensin converting enzyme (ACE) and ACE2 contribute to the observed sex-related differences in radiation response.Results: During the period of symptomatic pneumonitis, the relative ratio of ACE to ACE2 (ACE/ACE2) protein in the whole lung was significantly increased by radiation in male rats alone. Systemic treatment with small molecule ACE2 agonist diminazene aceturate (DIZE) increased lung ACE2 activity and reduced morbidity during radiation pneumonitis in both sexes. Notably DIZE treatment also abrogated morbidity in male rats during GI-ARS. We then evaluated the contribution of the irradiated bone marrow (BM) compartment on lung immune cell infiltration and ACE imbalance during pneumonitis. Transplantation of bone marrow from irradiated donors increased both ACE-expressing myeloid cell infiltration and immune ACE activity in the lung during pneumonitis compared to non-irradiated donors.Discussion: Together, these data demonstrate radiation induces a sex-dependent imbalance in the renin-angiotensin system enzymes ACE and ACE2. Additionally, these data suggest a role for ACE-expressing myeloid cells in the pathogenesis of radiation pneumonitis. Finally, the observed sex-differences underscore the need for consideration of sex as a biological variable in the development of medical countermeasures for radiation exposure.

Radiation therapy is received by over half of all cancer patients. However, radiation doses may be constricted due to normal tissue side effects. In thoracic cancers, including breast and lung cancers, cardiac radiation is a major concern in treatment planning. There are currently no biomarkers of radiation-induced cardiotoxicity. Complex genetic modifiers can contribute to the risk of radiation-induced cardiotoxicities, yet these modifiers are largely unknown and poorly understood. We have previously reported the SS (Dahl salt-sensitive/Mcwi) rat strain is a highly sensitized model of radiation-induced cardiotoxicity compared to the more resistant Brown Norway (BN) rat strain. When rat chromosome 3 from the resistant BN rat strain is substituted into the SS background (SS.BN3 consomic), it significantly attenuates radiation-induced cardiotoxicity, demonstrating inherited genetic variants on rat chromosome 3 modify radiation sensitivity. Genes involved with mitochondrial function were differentially expressed in the hearts of SS and SS.BN3 rats 1 week after radiation. Here we further assessed differences in mitochondria-related genes between the sensitive SS and resistant SS.BN3 rats. We found mitochondrial-related gene expression differed in untreated hearts, while no differences in mitochondrial morphology were seen 1 week after localized heart radiation. At 12 weeks after localized cardiac radiation, differences in mitochondrial complex protein expression in the left ventricles were seen between the SS and SS.BN3 rats. These studies suggest that differences in mitochondrial gene expression caused by inherited genetic variants may contribute to differences in sensitivity to cardiac radiation.

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.