Background Combination of antibody drug conjugates (ADC) and immunotherapy (IO) is emerging as a treatment paradigm with clinical efficacy in several tumor indications. Identifying translatable pre-clinical models to demonstrate the potential of novel ADC and IO combinations has been a challenge for the field. We describe the development of a humanized mouse model of NSCLC which demonstrates significant anti-tumor activity and tumor microenvironment (TME) regulation in response to the combination of ADC and a novel checkpoint immunotherapy.

Methods Newborn immunodeficient NOD-scid Il2rg^null NSG mice transgenic for the human cytokines SCF1, GM-CSF and IL-3 (SGM3) were engrafted with human cord blood CD34 positive stem cells. Twelve weeks post engraftment, mice were implanted with NCI-H358 human NSCLC cells, which were a partial HLA-ABC match to the CD34 positive stem cell donor. When implanted tumor volumes reached a mean of ~175 mm³ mice were randomized to receive either an unconjugated human IgG isotype, isotype IgG conjugated to the topoisomerase I inhibitor AZ’0132, PD1-TIM3 bispecific antibody sabestomig, a TROP2 targeting antibody (hRS7) conjugated to AZ’0132, or the combination of both agents. TME composition was assessed by immunohistochemistry, high-plex multi-color immunofluorescence (IF) imaging (Leica CellDive), spectral flow cytometry, and bulk RNA sequencing.

Results Monotherapy treatment with sabestomig or TROP2 ADC each resulted in significant reduction in tumor volume compared to controls. Combination treatment yielded tumor regression greater than 30% from baseline in all animals treated, for a RECIST response rate of 100%. Terminal assessment of the TME by IHC and high-plex IF showed the combination induced significant increases in total T cells and T cells expressing activation and effector molecules PD-1, CTLA-4 and Granzyme B. A follow on pharmacodynamic study evaluated the same treatments at 8 days post dosing initiation. Flow cytometry revealed significantly increased human immune cell infiltration into tumors in the combination group, driven by a significant increase in CD8 T cells. Bulk RNA sequencing of the same tumors showed that combination strongly upregulated genes in the Inflammatory, IFNγ and IFNα response pathways as well as the antigen presentation pathway suggesting immunogenic modulation of the TME.

Conclusions Our data suggests that a novel bispecific immunotherapy targeting PD-1 and TIM-3 combined with a topoisomerase ADC modulates the TME leading to increased tumor immune infiltration, activation and anti-tumor response.

Acknowledgements Authors would like to acknowledge Michael Stephens and Yang Song for support with the bulk RNA sequencing.

Ethics Approval The animals run in these in vivo studies were humanely treated and housed according to Institutional Animal Care and Use Committee approved protocols (AUP-22-17) in the Laboratory Animal Resources facility at AstraZeneca, an Association for Animal Accreditation of Laboratory Animal Care and USA Department of Agriculture-licensed facility.