Abstract Antibody-Drug Conjugates (ADCs) are a growing class of therapeutic agents for the treatment of cancer, with thirteen approved and over 100 others in clinical development. The pyridinobenzodiazepine (PDD) platform is a toolbox of sequence-selective, DNA Guanine mono-alkylating ADC payloads with varying potencies and biophysical properties, enabling a payload-based approach to targeting solid tumours and haematological malignancies. We recently reported the development of a lower potency DNA mono-alkylator, FGX20-75, with substantially superior in vivo properties to other DNA alkylating agents and an efficacy/tolerability profile on-par with a topoisomerase inhibitor-based ADC. This molecule is active in the low nanomolar range (i.e., median potency of 6 nM across a 15 cell-line screen), but is sufficiently hydrophilic to allow efficient conjugation to IgG antibodies with Drug Antobody Ratios (DARs) of up to 7. A maleimide-linked analogue of FGX20-75 has been conjugated to a number of antibodies with a DAR of 4. The ADCs produced exhibit significant in vivo efficacy in human tumour xenograft models. For example, in one case, an ADC targeted to an undisclosed antigen produced complete tumour regressions at 10 mg/kg dosing (Q7dx2) in patient-derived xenograft model of osteosarcoma. A substantially increased tolerability profile compared to other DNA-interactive payloads has been observed (e.g., single dose MED of < 1 mg/kg and MTD in mice of up to 80 mg/kg for a DAR 4 construct). Furthermore, in cynomolgus toxicity studies, a HNSTD has yet to be established at 8 mg/kg. Thus, the increase in loading compared to other DNA-interactive payloads compensates for the drop in potency of FGX20-75, resulting in a potent ADC with substantially increased tolerability and potent efficacy in solid tumours. The favourable hydrophobicity profile of the low potency alkylator FGX20-75, and its ease of conjugation to antibodies, along with the significant in vivo efficacy and tolerability of the ADCs produced from it, suggest that this low potency DNA-alkylating payload represents a promising new approach in the treatment of solid tumours. Citation Format: Paul J. Jackson, Paolo Andriollo, Nicolas Veillard, George Procopiou, David E. Thurston. In vivo efficacy and toxicity of a reduced potency DNA guanine mono-alkylating ADC payload suitable for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6334.