Introduction

Despite the importance of LNs initiating immune responses for systemic immunity/tolerance, immune checkpoint inhibitor (ICI) immunotherapies are administered intravenously, often failing to reach the dLNs that are the sites of dysfunctional T-cell priming responsible for tolerance to tumor Ags (tAgs). When administered systemically, these therapies act in an Ag-indiscriminate and system-wide fashion often breaching central tolerance and leading to severe immune related adverse events (irAEs) or delayed immune related events (DIREs). We hypothesize that regional delivery of immunotherapies within the distinct lymphatic watersheds will enhance anti-tumor immunity while preserving central tolerance.

Methods

Foxp3^DTR mice received i.p. administration of 250 ng diptherion toxin (DT) on days 1 and 2 to transiently deplete Tregs, making the susceptible to normal tissue lymphocyte infiltration. These animals received 5 mg/kg of αCTLA-4 or α4-1BB, administered i.v. or i.d. at the right base of the tail on day 1, 3, and 5.  At day 1 and 7, a subset of animals received ipsilateral i.d. injection of 10 µg virus-like-particle (VLP) Qβ(1668) conjugated to B16F10 tumor neoantigen (tNeoAg) PMEL, as cancer vaccine. In a subset of WT or Foxp3-DTR animals dosed with PBS, B16F10 was implanted on day 8 and were treated with VLP and ICI as described above. Caliper measurements of tumor growth were made every other day on tumor bearing animals. At day 11, all animals were sacrificed and liver, lung, and colon tissues were collected and fixed for later evaluation for infiltrating lymphocytes and pathological scoring.

Results

Foxp3^DTR animals that received DT and ICI i.v., had significantly greater spleen weights and more infiltrating lymphocytes in the liver (p<0.005) than those dosed with PBS. Interestingly, when ICI was administered i.v. to Qβ- vaccinated Foxp3^DTR mice, lymphocyte infiltration was significantly reduced (p<0.005) in comparison to unvaccinated mice. Qβ-vaccinated and unvaccinated Foxp3^DTR animals that received DT and ICI i.d. did not experience significant normal tissue lymphocyte infiltration when compared to control mice. Pathology evaluation indicated greater inflammatory responses in the colon, lung, and liver tissues of animals dosed with ICI i.v compared to those dosed within a lymphatic watershed or control animals. However, tumor growth rates were significantly reduced when ICI was administered i.d. in vaccinated WT mice.

Conclusion

Given the recent trend to combine systemic doses of ICI with chemotherapy in order to improve response rates, the chances for worsening irAEs and DIREs increase. Currently, αCTLA-4 is associated with a greater rate of irAEs than αPD-1/-L1, and α4-1BB has not translated beyond Phase I trials due to severe irAEs. In this work, we have shown that by dosing αCTLA-4 and α4-1BB regionally to a regional LNs, improvements in anti-tumor responses occur in WT animals and reductions in normal tissue lymphocyte infiltration occur in the Foxp3^DTR mouse model of transient Treg depletion.

In addition, antigen-directed ICI (i.e. dosing drug with cancer vaccine to a non-tumor draining lymphatic watershed) may also help to reduce indiscriminate T-cell priming and normal tissue lymphocyte infiltration.

Supported in parts by CPRIT RP190019 and NIH R21CA267263.