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1134 Dynamic analyses of tumor microenvironment modulation by CAN-2409 treatment using kaede photoconvertible transgenic mice
  1. David Withers1,
  2. Anne Diers2,
  3. Zhi Li1,
  4. Suaad Idris1,
  5. Claire Willis1,
  6. Paul Peter Tak2 and
  7. Francesca Barone2
  1. 1University of Birmingham, Birmingham, UK
  2. 2Candel Therapeutics, Needham, MA, USA


Background CAN-2409 is a replication-defective adenovirus that delivers the HSV-thymidine kinase gene. 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. Use of state-of-the-art preclinical tools for dynamic assessment of the lymphocyte response in vivo will enable assessment of the evolution of the anti-tumor immune response induced by CAN-2409 and provide the immunological rationale for potential therapeutic combinatory approaches.

Methods We utilized a dynamic labeling model1 where MC38 tumor cells were implanted subcutaneously into photoconvertible Kaede mice; violet light was used to simultaneously label the entire tumor microenvironment (TME), enabling the discrimination of cells retained within the tumor (Kaede Red+) versus newly entering cells (Kaede Green+) and the ability to assess real-time changes occurring in the immune compartment of the TME. CAN-2409 was administered intratumorally on day 10 after tumor inoculation followed by prodrug (ganciclovir, 50mg/kg IP QD on days 11 to 14). Photoconversion occurred on day 12, and analysis was performed 48h later. Dynamic of MC38-tumor specific T cell clones was evaluated by flow cytometry. We also explored the effect of CAN-2409 in combination with immune checkpoint inhibition (ICI).

Results Administration of CAN-2409 led to control of tumor growth and a significantly increased effector CD8 T cell response. Photolabeling of the TME revealed that rather than enhancing recruitment of T cells to the tumor, CAN-2409 altered the TME such that newly entering, but also retained CD8 T cell were significantly more proliferative. Amongst the retained population, terminally exhausted neoantigen-specific CD8 T cells showed evidence of reinvigoration, adopting a CX3CR1+ GZMB+ phenotype. There was also enhanced proliferation within the PD-1+ stem-like compartment of newly recruited cells and we observed expansion of a population of non-activated, likely less suppressive, Tregs. The combination of CAN-2409 and anti-CTLA-4 (clone 9H10) treatment further improved control of tumor growth and remodeled the Treg compartment to further skew the Treg:CD8 ratio in favor of the effector response.

Conclusions CAN-2409 alters the TME such that newly entering CD8 T cells expand and retain key effector functions while the exhausted CD8 compartment is reinvigorated, likely reducing Treg-mediated suppression. Collectively, these data suggest at least two temporally distinct pathways underpinning CAN-2409 action that overcome cell exhaustion and decreased immune suppression, supporting the rationale for the use of CAN-2409 either as monotherapy or in combination.


  1. Zhi Li, Zewen K Tuong, Isaac Dean etal. In vivo labeling reveals continuous trafficking of TCF-1+ T cells between tumor and lymphoid tissue. J Exp Med 2022;219(6):e20210749. doi: 10.1084/jem.20210749

Ethics Approval All in vivo experiments were conducted in accordance to UK HO guidelines on an approved PPL

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