Background Dysfunctional antigen presentation mediated by MHC downregulation in tumor cells and low infiltration of functional professional antigen presenting cells (APCs) are critical immune evasion mechanisms underlying intrinsic resistance to immunotherapies. Among APCs, conventional type 1 dendritic cells (cDC1s) in tumors are key drivers of efficient anti-tumor immunity and associated with better prognosis.¹ We have recently demonstrated that overexpression of transcriptional factors PU.1, IRF8 and BATF3 (PIB)^{2 3} drives cell fate reprogramming of tumor cells into antigen-presenting cDC1s, which upon in-vivo transfer mount efficient anti-tumor immunity in immune-checkpoint inhibitors (ICI)-resistant models.⁴ To circumvent ex-vivo cell manipulation challenges, Asgard Therapeutics is developing AT-108, a novel immunotherapy based on de-novo recreation of cDC1s’ functional properties in tumor cells by in-vivo direct reprogramming, to restore tumor immunogenicity and overcome immune evasion.

Methods We validated in-vivo cDC1 reprogramming and anti-tumor efficacy upon subcutaneous implantation of ex-vivo transduced cancer cells in xenografts and ICI-resistant cDC1-dependent mouse syngeneic models: T-cell^lowMHC^low B16, T-cell^HIGH YUMM1.7 and BRAF^V600ECOX2^KO (cDC1-deficient host). We used tumor spheroids generated with cancer-associated fibroblasts and anti-inflammatory cytokines to investigate the impact of immunosuppressive microenvironment in cDC1 reprogramming. To select an optimal platform to express PIB within tumors, we compared transduction and reprogramming of PIB-encoding lentiviral vectors, replication-deficient adenoviral (AdV) and adeno-associated viral (AAV) vectors.

Results In-vivo induced cDC1s drove tumor regression in YUMM1.7 (100% complete response, CR) and BRAF^V600ECOX2^KO (50% CR) models, and synergized with ICIs leading to tumor regression in B16 model (40% CR), expansion of tumor-reactive T-cells and abscopal effect, indicating that reprogramming reverses MHC downregulation and replenishes cDC1s in tumors promoting anti-tumor immunity. PIB-transduced human cancer cells acquired cDC1 (CLEC9A, XCR1) and antigen-presentation markers (MHC-I/II, CD40/80) in-vivo with similar efficiency and kinetics as in-vitro, and the cDC1 reprogramming process was not hindered by immunosuppressive environment. Comparison of delivery platforms revealed that AdV-PIB induced cDC1 phenotype with similar efficiency as LV-PIB but showed superior infiltration of spheroids and in-vivo transduction of tumors, supporting selection of AdVs for in-vivo PIB delivery. Remarkably, intra-tumoral injection of AT-108 allowed in-situ phenotypic reprogramming and resulted in 50% CR in B16 model up to 100 days, with long-term memory after B16 rechallenge.

Conclusions Our study demonstrates cDC1 reprogramming successfully overcomes immune evasion and revives anti-tumor immunity in ICI-resistant tumors, as monotherapy or in combination. We provide proof-of-concept for developing an AdV-based, off-the-shelf immunotherapy based on in-situ cDC1 reprogramming for patients with ICI primary resistance.

References

1. Zhang S, Chopin M, Nutt SL. Type 1 conventional dendritic cells: ontogeny, function, and emerging roles in cancer immunotherapy. Trends Immunol. 2021;42:1113–27.

2. Rosa FF, Pires CF, Kurochkin I, Ferreira AG, Gomes AM, Palma LG, et al. Direct reprogramming of fibroblasts into antigen-presenting dendritic cells. Sci Immunol. 2018;3:1–16.

3. Rosa FF, Pires CF, Kurochkin I, Halitzki E, Zahan T, Arh N, et al. Single-cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells. Sci Immunol. 2022;7:5539.

4. Zimmermannova O, Ferreira AG, Ascic E, Velasco M, Kurochkin I, Hansen M, et al. Restoring Tumor Immunogenicity with Dendritic Cell Reprogramming. Sci Immunol. 2023. In Press.

Ethics Approval Ethical approval for the experimental procedures was granted by Malmö – Lund Animal Research Ethics Committee on 2018/02/28 (Dnr 5.8.18–19343/2017).