{
  "abstract": "Background Immune checkpoint blockade (ICB), despite being approved for use in many solid tumor indications, is ineffective in immunologically ‘cold’ tumors, which contain few tumor infiltrating lymphocytes (TILs) owing to an immunosuppressive tumor microenvironment (TME). Recent data implicate tertiary lymphoid structures (TLSs) as critical players in modulating antitumor immunity in the TME, and their presence correlates with improved response to anti-PD-(L)1 ICB. 1–3 TLSs are ectopic lymphoid aggregates resembling secondary lymphoid organs in structure and function that arise in nonlymphoid tissues in response to a coordinated cascade of inflammatory signals defined by a hallmark 12-chemokine gene signature.4 TLSs vary in composition, organization, and maturation states, but generally contain TILs (both T and B cells), dendritic cells (DCs), and specialized blood vessels known as high endothelial venules (HEVs). VAX014 is a novel oncolytic agent based on recombinant bacterial minicells currently undergoing a Phase 1 study in combination with PD-1-directed ICB (NCT_05901285). Several published nonclinical studies demonstrate VAX014 synergizes with anti-PD-(L)1 in vivo in multiple anti-PD-(L)1 resistant tumor models.5 6 As such, we now explore whether local intratumoral (i.t.) treatment with VAX014 results in the formation of TLSs in the immune competent MB49 and MC38 murine tumor models.Methods In both models, we performed a longitudinal immunotranscriptome analysis on bulk RNA purified from tumors receiving a single i.t. dose of saline or VAX014 at 4 hours, 24 hours, and 3 days post treatment (n=3/treatment group/timepoint) to evaluate the expression of the 12-chemokine signature. In a second set of experiments, we evaluated saline- and VAX014-treated tumors 5 days post-treatment (n=3-9/treatment group) by immunohistochemistry (IHC) against CD11c, CD19, CD8, CD4, and PNAd to quantify TLSs, and where present, determine TLS immune composition and intratumoral location. 7 8 Results Immunotranscriptome data revealed robust upregulation of the 12-chemokine TLS gene signature in response to VAX014 in both models, suggesting the possible formation of TLSs. Accordingly, IHC analysis revealed a significant increase in the number of TLSs in VAX014-treated tumors compared to saline-treated tumors in both models (mean of 0 vs. 2.5 for MB49 and 0 vs. 2.2 for MC38) which were comprised of varied aggregates of CD11c+ DCs, CD4+ and CD8+ TILs, and PNAd+ HEVs within the tumor parenchyma. CD19+ B-cells were present but not always associated with immune aggregates.Conclusions These findings support that i.t. VAX014 induces TLS formation, suggesting TLS formation may contribute to the substantiated ability of VAX014 to potentiate response to anti-PD-(L)1 ICB.References Li Z, Liu S, Liu D, et al. Multiple mechanisms and applications of tertiary lymphoid structures and immune checkpoint blockade. J Exp Clin Cancer Res. 2025;44:84. https://doi.org/10.1186/s13046-025-03318-6Jiang Q, Tian C, Wu H, Min L, Chen H, Chen L, Liu F, Sun Y. Tertiary lymphoid structure patterns predicted anti-PD1 therapeutic responses in gastric cancer. Chin J Cancer Res. 2022 Aug 30;34(4):365–382. doi: 10.21147/j.issn.1000-9604.2022.04.05. PMID: 36199531; PMCID: PMC9468020.Vanhersecke L, Brunet M, Guégan JP, et al. Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression. Nat Cancer. 2021;2:794–802. https://doi.org/10.1038/s43018-021-00232-6Coppola D, Nebozhyn M, Khalil F, Dai H, Yeatman T, Loboda A, Mulé JJ. Unique ectopic lymph node-like structures present in human primary colorectal carcinoma are identified by immune gene array profiling. Am J Pathol. 2011 Jul;179(1):37–45. doi: 10.1016/j.ajpath.2011.03.007. Epub 2011 May 3. PMID: 21703392; PMCID: PMC3123872.Reil KA, Tsuji S, Molina E, et al. Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors. Journal for ImmunoTherapy of Cancer. 2023;11:e006749. doi: 10.1136/jitc-2023-006749Tsuji S, Reil K, Nelson K, Proclivo VH, McGuire KL, Giacalone MJ. Intravesical VAX014 synergizes with PD-L1 blockade to enhance local and systemic control of bladder cancer. Cancer Immunol Res. 2022 Aug 3;10(8):978–995. doi: 10.1158/2326-6066.CIR-21-0879. PMID: 35679299; PMCID: PMC9357178.Dobosz P, Stępień M, Golke A, Dzieciątkowski T. Challenges of the immunotherapy: perspectives and limitations of the immune checkpoint inhibitor treatment. Int J Mol Sci. 2022 Mar 5;23(5):2847. doi: 10.3390/ijms23052847. PMID: 35269988; PMCID: PMC8910928.Ouyang P, Wang L, Wu J, Tian Y, Chen C, Li D, Yao Z, Chen R, Xiang G, Gong J, Bao Z. Overcoming cold tumors: a combination strategy of immune checkpoint inhibitors. Front Immunol. 2024 Mar 13;15:1344272. doi: 10.3389/fimmu.2024.1344272. PMID: 38545114; PMCID: PMC10965539.Ethics Approval These studies were conducted under animal protocol number 22-05-004Mc, approved by the Institutional Animal Care and Use Committee at San Diego State University.",
  "authors": [
    {
      "affiliations": [
        "Vaxiion Therapeutics, San Diego, CA, USA"
      ],
      "name": "Amanda M Parikh"
    },
    {
      "affiliations": [
        "Vaxiion Therapeutics, San Diego, CA, USA"
      ],
      "name": "Katherine A Reil"
    },
    {
      "affiliations": [
        "Vaxiion Therapeutics, San Diego, CA, USA"
      ],
      "name": "Shingo Tsuji"
    },
    {
      "affiliations": [
        "Vaxiion Therapeutics, San Diego, CA, USA"
      ],
      "name": "Kinsey L Nelson"
    },
    {
      "affiliations": [
        "San Diego State University, San Diego, CA, USA"
      ],
      "name": "Carrie D House"
    },
    {
      "affiliations": [
        "Vaxiion Therapeutics, San Diego, CA, USA"
      ],
      "name": "Matthew J Giacalone"
    }
  ],
  "title": "885 Intratumoral administration of VAX014 induces tertiary lymphoid structure formation",
  "uid": "8d0767b6-579b-596c-b24e-90e9ddccae90"
}