Abstract LB263: A first-in-class multimodal immunotherapy for induction of tertiary lymphoid structures as a novel therapeutic strategy for solid tumors

Abstract Immune checkpoint inhibitors (ICI) have transformed cancer treatment. However, long term disease control is often not achieved and resistance frequently occurs. Formation of organized intratumoral aggregates of lymphocytes (tertiary lymphoid structures, TLS) has been associated with response to ICI suggesting therapeutics able to induce intratumoral TLS could enhance the response to ICI treatment. TLS formation requires antigen presentation, stromal activation and immune stimulation - diverse events difficult to obtain with a single therapeutic. The enLIGHTEN™ Discovery Platform aims to create multimodal therapeutics by leveraging human biology and advanced analytics to select multigene payloads delivered by herpes simplex virus (HSV) vectors to the tumor microenvironment. Optimal payload combinations are predicted based on in silico experiments and then tested in proprietary multiplex assays used to evaluate the contribution of payload components and model the therapeutic biological effect of predicted payload combinations. Alpha-201, a replication-defective oncolytic virus, was selected from a suite of modified HSV vectors as the biological chassis due to its ability to release tumor antigens and activate pathways associated with response to ICI (e.g., MYC targets, E2F targets, G2M checkpoint; normalized enrichment scores of 2.4, 3.0, and 3.2, respectively, p<0.001). A library of single gene encoding Alpha-201 vectors was constructed, each expressing one in silico predicted payload component (e.g., IL-7, CXCL13, LTB or CCL19). The top two scoring combinations of these viral gene constructs (termed Multiplex A and Multiplex B) were next tested for their effects on TLS induction and anti-tumor efficacy in complementary in vivo models. First, the ability of HSV gene construct combinations to induce TLS was tested in murine salivary glands, a tissue highly permissive to TLS formation. As predicted, delivery of both multiplexes increased the number, size, organization and maturity of virally induced TLS. Of note, TLS were characterized by the presence of mature fibroblastic reticular cells and CCL21 expression, effects not observed with biological chassis administration alone. Second, anti-tumor efficacy was tested in EMT6 tumor-bearing mice. Both multiplexes induced tumor growth inhibition compared to vehicle control mice (Multiplex A: 39.8±10.5%, p=0.004, Multiplex B: 38.6%±12.1, p=0.011, n=8 per group). In combination with anti-PD-1 antibody therapy, multiplex treatment also increased the number of long-term survivors (>40 days) as compared to control (2/11 vs. 0/8). Characterization of intratumoral TLS induction is ongoing. In conclusion, we have demonstrated the ability of enLIGHTEN™ to design multimodal specific therapeutics, resulting in the development of a first-in-class immunotherapeutic for TLS induction in solid tumors. Citation Format: Anne R. Diers, Qiuchen Guo, John D. Christie, David Krisky, Fabrina M. Gaspal, Erin Richardson, Charlotte G. Smith, David R. Withers, Adam P. Croft, Paul P. Tak, Francesca Barone. A first-in-class multimodal immunotherapy for induction of tertiary lymphoid structures as a novel therapeutic strategy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB263.