Anne R. Diers

Background: Patients with advanced non-small cell lung cancer who progress on immune checkpoint inhibitors (ICIs) face limited options and poor survival. Aglatimagene besadenovec (CAN-2409), a replication-defective adenoviral vector encoding herpes simplex virus-thymidine kinase, plus valacyclovir, has been shown to induce immunogenic cell death and systemic immune activation. Methods: In this open-label, phase 2a clinical trial, 76 patients were enrolled (intention-to-treat population) and 73 received at least one dose of aglatimagene besadenovec (safety population); 46 patients who received two intratumoral aglatimagene besadenovec injections, completed protocol-defined valacyclovir exposure, and underwent a 12-week imaging comprised the evaluable (per-protocol) population. Endpoints included clinical and immunological outcomes. Results: In the 73 patients included in the safety analysis (including patients who did not receive a second injection due to apparent pseudoprogression), median OS was 14.3 months. In the evaluable (per-protocol) population of 46 patients who completed protocol-defined treatment and week-12 imaging, median OS was 24.5 months (95% CI 16.0 to 33.8). Among the 41 evaluable patients in cohort 2, 15 (37%) were alive at ≥24 months and 5 (12%) at ≥40 months. In the 46-patient evaluable population, the objective response rate was 10.9% (5/46; 95% CI 4.7% to 23.0%) and the disease control rate was 71.7% (33/46; 95% CI 57.5% to 82.7%). In the 73 patients included in the safety analysis (including patients who did not receive a second injection due to apparent pseudoprogression) median overall survival was 14.3 months. Exploratory biomarkers demonstrated systemic immune reactivation, including cytotoxic T-cell expansion and regression of uninjected lesions, with stronger signals in non-squamous histology. No grade 4 treatment-related adverse events or treatment-related deaths were reported. Most treatment-related adverse events were grade 1–2; 10/73 patients (13.7%) experienced grade 3 treatment-related adverse events. Six patients discontinued because of adverse events, including two with events considered possibly related to treatment (including ongoing ICI). Conclusions: In this single-arm phase 2a study, aglatimagene besadenovec plus valacyclovir with ongoing ICI was associated with systemic immune remodeling and encouraging survival. Because comparisons with historical controls are descriptive and vulnerable to selection and other biases, efficacy requires confirmation in a randomized controlled clinical trial, which has been initiated. Trial registration number: NCT04495153 .

Supplemental Figure 2 shows the tumor growth inhibition rate 4 days after treatment and immune cell quantification in tumor tissue in Kaede mice

Supplemental Figure 1 shows individual tumor growth of animals treated with and without CAN-2409 and prodrug

Supplemental Figure 5 shows the flow cytometry gating scheme applied to tumor-draining lymph node samples

Supplemental Figure 3 shows the combined effect of CAN-2409 and anti-CTLA-4 Ab treatment on individual tumor growth curves and immune cell quantification in tumor tissue

Supplemental Figure 4 shows the flow cytometry gating scheme applied to tumor samples

CAN-2409 + prodrug induces expansion of immune cells and neoantigen-specific T cells retained in tumors. A, Summary of MC38 implanted subcutaneous on the flank of C57BL/6 Kaede mice and photoconverted by violet light exposure. B, Study timeline for experiments assessing the effect of CAN-2409 + prodrug on TME modulation in Kaede photoconvertible mice. C, Representative flow cytometry plots showing KR+ and KG+ expression in immune subpopulations within tumors. MC38 pentamers were used to identify antigen-specific CD8+ T cells. D, Quantification of newly entering (KG+) cells for each T-cell subset. N = 4–6 mice per group. Two-tailed t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Supplemental Figure 4 shows the flow cytometry gating scheme applied to tumor samples

Supplemental Figure 1 shows individual tumor growth of animals treated with and without CAN-2409 and prodrug

CAN-2409 is a replication-defective adenovirus that delivers the herpes simplex virus–thymidine kinase gene to infected cells. Intratumoral administration of CAN-2409, followed by prodrug, results in the formation of a toxic metabolite able to induce immunogenic cell death, exposure of tumor-associated antigens, and activation of local and systemic immune responses. We used a dynamic labeling model with MC38 tumor cells implanted in photoconvertible Kaede mice. Violet light was used to label the tumor microenvironment (TME), distinguishing retained versus newly entering cells and allowing real-time monitoring of immune compartment changes within tumors. Administration of CAN-2409 + prodrug led to control of tumor growth and significantly increased effector CD8+ T-cell responses. Photolabeling of the TME revealed that rather than enhancing recruitment of T cells to the tumor, CAN-2409 altered the TME whereby newly entering and retained CD8+ T cells were significantly more proliferative. CAN-2409 supported reinvigoration of tumor-associated antigen–specific CD8+ T cells and expansion of regulatory T cells of an altered phenotype. Moreover, the combination of CAN-2409 + prodrug and anti–CTLA-4 antibody treatment further improved control of tumor growth, in part by the enhanced CD8+ T-cell–mediated effector function and diminished regulatory T cell–mediated immunosuppression. Collectively, these data defined at least two temporally distinct pathways underpinning the mechanism of action of CAN-2409 that overcome cell exhaustion and decrease immune suppression. The results also support the rationale for future clinical trials of CAN-2409 treatment combined with anti–CTLA-4 antibody therapy.Significance:This study utilized a novel photoconvertible mouse tumor model to track immune cell trafficking upon treatment with an investigational viral immunotherapy (CAN-2409), revealing enhanced T-cell responses after viral immunotherapy associated with local proliferation of T cells within tumors that could further enhance antitumor efficacy in combination with immune checkpoint inhibitors. These findings define temporally and spatially distinct interactions of immune cells that could be harnessed by novel therapeutics.