Background Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has greatly improved treatment options for patients with recurrent/metastatic (R/M) melanoma. With current treatment options, complete response (CR) does not exceed 4% in R/M head and neck cancer (HNC). TIL therapy harnesses the patient‘s immune cells to combat cancer. Briefly, parts of the patient’s tumor are surgically resected to identify and expand tumor-specific TIL ex vivo for reinfusion into the patient. It is now widely accepted that radiation therapy (RT) can be immunostimulatory. Here we investigated the potential of RT to improve TIL therapy. This study aimed to achieve two objectives: (1) to enhance ex vivo TIL expansion by preconditioning tumors with RT, and (2), to enhance the anti-tumor effect by combining RT with ACT.

Materials and Methods (1) Experiments were conducted in a murine HPV+ HNC model. MEER-bearing mice received a local dose of 1x8 Gray (Gy) or 0Gy. Five days after radiation, tumors were harvested to extract RNA for RNA-sequencing, to characterize different immune subsets using flow cytometry, and to set up individual tumor fragments in media with 6000IU/ml IL-2 for ex vivo TIL expansion. Wells that showed >90% confluency of lymphocytes without the presence of tumors cells were expanded. After 5 weeks, ex vivo expanded TIL were harvested from both groups and reinfused into tumor bearing mice. (2) TC-1 bearing mice received 1x8 Gy before they were adoptively transferred with T cells specific to HPV E7 peptide (‘E7 T cells’). SJL mice were utilized to assess tumor infiltration of adoptively transferred T cells after radiation. SJL mice were injected with tumor in both flanks: The ‘primary tumors’ were irradiated with 1x8Gy, whereas the ‘abscopal tumors’ were lead-shielded.

Results (1) Pre-irradiating tumors significantly increased the proportion of fragments with ex vivoTIL expansion (p=0.0014). Tumors in mice that received ex vivo expanded TIL from pre-irradiated tumors showed increased tumor growth delay (p=0.02). RNA-sequencing revealed an increase of CCL21 5 days after radiation. We found that immune cells that express the receptor for CCL21 (CCR7) were increased in the tumor 5 days after radiation (p=0.002). (2) Mice treated with ACT+radiation had a median survival of 67 days after treatment, compared to a median survival of 39 days in the groups ACT or radiation alone. Mice that received either radiation or ACT did not achieve CR. 13 of 27 mice treated with both ACT+radiation achieved CR, of which 9 were durable CR. Combining ACT+radiation increased the tumor infiltration with adoptively transferred T cells in both primary and abscopal tumors.

Conclusions Our findings highlight the significant potential of radiation to enhance TIL therapy, not only by improving ex vivo TIL expansion but also by increasing the anti-tumor effect of adoptively transferred T cells. The role of CCR7 within the synergy of radiation and TIL therapy remains to be elucidated. In summary, the combination of TIL therapy and radiation offers a promising approach to enhance the likelihood of complete response in recurrent/metastatic solid tumors.

N. Obertopp: None. A. Thomas: None. J. Ali: None. J. Blauvelt: None. A. Hall: None. H. Enderling: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; NIH-NCI U01 CA244100-01. S. Pilon-Thomas: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Provectus Biopharmaceuticals, Celgene, Iovance Biotherapeutics, Intellia Therapeutics, Dyve Biosciences, and Turnstone Biologics. F. Consultant/Advisory Board; Significant; Seagen Inc. and KSQ Therapeutics.