Antibody Drug Conjugate Targeted Radio-Immunosensitization

Purpose/Objective(s)

Concurrent chemo-radiotherapy is the basis of organ sparing therapy for locally advanced cancers. However, non-targeted chemotherapies cause toxicities. Antibody drug conjugates (ADCs) spatially target drug delivery for biomarker selected cancer patients. The potent anti-tubulin monomethyl auristatin E (MMAE) is the most common ADC drug warhead. In prior studies, MMAE radiosensitized and improved irradiated tumor control of human tumor xenografts in immune-deficient mice. No studies to date have examined MMAE radiosensitization in the tumor immune microenvironment context. We hypothesized MMAE-ADCs achieve spatially precise radiosensitized tumor kill that engages tumor immune responses.

Materials/Methods

ADCs are optimized to bind human receptors. To synthesize a MMAE-ADC recognizing murine isoforms, we conjugated MMAE to anti-HER3 antibody CDX3379 creating CDX3379-MMAE and labeled it with Cy5 for non-invasive tracking. An advantage of CDX3379 is it recognizes both human & murine HER3. MMAE radiosensitization of murine cancer cell lines (B16, LL2, MC38) was tested by clonogenic survival. In vivo tumor targeting and efficacy studies were done in HER3+ murine syngeneic tumors grown subcutaneously (B16, MOC1) or orthotopically in tongues (MOC2) in C57BL/6 mice. CDX3379-MMAE (2.5 nmoles) was given i.v. followed by focal fractionated ionizing radiation (IR) of 5 Gy x 3. Anti-PD-1 antibody was given i.p. (200 ug, BioXCell) with start of CDX3379-MMAE for 3 doses. For CD8 depletion, mice were i.p. injected with anti-CD8 antibody (10 mg/kg, BioXCell) prior to tumor cell implant and then weekly. Statistical analysis was done by ANOVA and log-rank (Mantel-Cox). Fluorescent mouse imaging for Cy5 labeled ADC evaluated tumor targeting. Efficacy was determined by tumor volume and mouse survival. Tumor immune infiltration was assayed by flow cytometry.

Results

In cell culture, MMAE arrested all murine cancer cell lines tested in G₂/M and radiosensitized in the 1-3 nmolar range, P<0.01. In syngeneic models, CDX3379-MMAE accumulated in HER3+ but not HER3 negative tumors by Cy5 imaging. Combining CDX3379-MMAE with IR increased tumor growth delay and mouse survival, P<0.01. Temporal analysis of tumor immune infiltration demonstrated CDX3379-MMAE + IR therapy increased CD8+ T cell tumor immune infiltration, P<0.01. Mechanistically, tumor control by CDX3379-MMAE + IR was attenuated with CD8 T cell depletion. Finally, anti-PD-1 immune checkpoint inhibition potentiated CDX3379-MMAE + IR durable tumor control, P<0.0001.

Conclusion

These studies provide the first demonstration MMAE-ADC radiosensitization produces durable tumor control in a CD8 T cell dependent manner that is potentiated by immune checkpoint inhibition. Our studies have exciting translational implications by providing a foundation for irradiated cancer patients to be concurrently treated with spatially precise ADC radio-immunosensitization.