Abstract Topoisomerase I inhibitors are used as standard-of-care chemotherapy in many types of cancer but are associated with significant toxicities. There is potential to improve their efficacy further by combining with inhibitors of the DNA damage response, such as the PARP inhibitor olaparib. However, while preclinical data highlight the improved efficacy of this combination, subsequent clinical trials have struggled due to dose limiting myelotoxicity. CRLX101 is an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin (the most potent topoisomerase I inhibitor known). This agent is preferentially targeted to tumours and demonstrated a favourable toxicity profile in the clinic. Here, we explored the molecular mechanism and therapeutic potential of combining CRLX101 with either olaparib or the WEE1 inhibitor AZD1775, by testing both efficacy and safety in preclinical models. In vitro studies using NCI-H417a SCLC cells demonstrated that combination with both olaparib and AZD1775 potentiated the efficacy of CRLX101 although by different mechanisms. Cellular analyses revealed that CRLX101 treatment alone predominantly activated ATM-mediated DNA damage response and resulted in late S/G2 cell cycle arrest. Combination with a PARP inhibitor further enhanced the CRLX101-induced DNA damage response and prolonged cell cycle arrest in late S/G2 phase. In contrast, WEE1 inhibition abrogated late S/G2 cell cycle arrest induced by CRLX101, resulting in aberrant mitotic entry and enhanced cell death. Our in vivo studies using wild type Wistar rat model showed that CRLX101, olaparib and AZD1775, are well tolerated as single agents. However, concurrent combination of CRLX101 with either olaparib or AZD1775 resulted in a dose-dependent decrease in haematological parameters. We investigated sequenced schedules and demonstrated that at a 24h delay between the CRLX101 and olaparib mitigates much of the combined bone marrow toxicity, while improving the efficacy above CRLX101 alone in xenograft tumours from NCI-H417a cells. Collectively, these preclinical data demonstrate increased anti-tumour efficacy of CRLX101 when combined with DDR inhibitors. The combination schedule for CRLX101 and olaparib identified in our preclinical models as providing an increased therapeutic index has been used to develop protocols to test this combination in a relapsed (2nd line) SCLC human clinical trial (in collaboration with NCI). Citation Format: Lenka Oplustil O’Connor, Anderson T. Wang, David R. Jones, Rajesh Odedra, Michael Spreadborough, Joanne Wilson, Aaron Smith, Peter Cotton, Jaimini Reens, Jen Barnes, Victoria Sheridan, Scott Eliasof, Andres Tellez, Alan Lau, Claire Sadler, Mark J. O’Connor. A camptothecin-containing nanoparticle-drug conjugate combination with DDR agents provides a novel approach to increasing therapeutic index. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3721.