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1348 Development of enLIGHTEN™ Alpha-201 herpes simplex viral vectors encoding payloads targeting the tumor microenvironment
  1. Anne R Diers,
  2. Qiuchen Guo,
  3. John D Christie,
  4. David Krisky,
  5. Karen Bouch,
  6. Paul P Tak and
  7. Francesca Barone
  1. Candel Therapeutics, Needham, MA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Failure to respond to conventional immunotherapies arises from heterogeneous mechanisms present in the tumor microenvironments (TME) that drive resistance in non-responding patients. Candel’s enLIGHTEN™ Discovery Platform applies advanced analytics to generate in silico prediction of multi-gene payload combinations with potential therapeutic benefit for specific solid tumor indications. Selected payloads, screened through ex vivo and in vivo multiplex assays, are then integrated into a viral chassis selected from Candel’s modified herpes simplex virus-1 (HSV-1) vector suite resulting in a multimodal, programmable treatment combined in a single therapeutic. We applied enLIGHTEN™ to immune checkpoint inhibitor (ICI)-treated patient datasets1–5 to identify potential gene payload combinations that may help overcome mechanisms underlying lack of response to ICI.

Methods Replication-defective HSV-1 vectors (Alpha-201 series) encoding payloads such as IFNγ and IL-12, selected in silico using enLIGHTEN™ advanced analytics, were tested in vitro in Hs578T cells (10 PFU/cell, 24–72 h) followed by RNASeq and gene set enrichment analysis. The ability of multiplexed vector combinations to induce immune activation and subsequent cancer cell killing was tested in an ex vivo cancer/peripheral blood mononuclear cell (PBMC) coculture system using flow cytometry-based readouts. Values are expressed as mean±SEM.

Results From a suite of modified HSV-1 vectors, we selected the Alpha-201 viral chassis for delivery of therapeutic payloads based on its ability to induce MYC and E2F targets, G2M cell cycle checkpoint, and allograft rejection responses (normalized enrichment scores = 2.4, 3.2, 3.0, 1.81, and 1.43 p<0.05), pathways associated with effective anti-tumor immune responses to ICI. Payload expression for Alpha-201-IFNγ was confirmed for up to 6 days post-infection (peak ~1 ng/mL/day; 2 days post-infection) and we observed a strong IFNγ response gene signature including upregulation of ISG15, CCL2, and CXCL10. Alpha-201 infection of cancer cells induced PBMC-mediated cell killing and infection- and payload-dependent alterations in lymphoid and myeloid cell populations. Alpha-201-IFNγ infection increased the number of CD11c+CD16+CD14-Ki67+MHCIIhigh dendritic cells (13.2-fold±3.58, p=0.019) and granzyme B+ NK cells (1.46-fold±0.14, p=0.013) compared to uninfected cocultures. Further, t-sne analysis demonstrated robust phenotypic changes in CD14+ monocyte populations upon infection with Alpha-201 vectors. Immune activation and PBMC-mediated cancer cell killing was further enhanced by selected payload combinations identified by in silico predictions (>60% compared to vector alone).

Conclusions These data validate enLIGHTEN™ in silico predictions of gene payload combinations and highlight the utility of the enLIGHTEN™ Discovery Platform to guide development of novel viral immunotherapeutics to modify the TME by design.


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