{
  "abstract": "Background Viral nucleic acids are detected by TLR7/8 (RNA) and TLR9 (DNA). Recently, vidutolimod, the first CpG-A TLR9 agonist, showed systemic monotherapy activity (9/40 responses) in PD-1 blockade-resistant melanoma. 1 In the randomized ECOG-ACRIN EA6194 trial in neoadjuvant melanoma, the addition of vidutolimod induced a 20% increase in pathologic complete response compared to pembrolizumab alone, supporting earlier data in a non-randomized trial.2 3 Vidutolimod is delivered in a viral-like particle that induces neutralizing antibodies,4 5 and vidutolimod resistance in advanced melanoma is associated with TLR7/8+, TLR9- immune suppressive tumor-associated myeloid cells (TAM).6 We hypothesized that co-stimulating TLR7/8/9 with an RNA/DNA mimic of viral replicative complexes in a non-immunogenic LNP may activate TAM and overcome vidutolimod resistance, inducing more effective anti-tumor responses.Methods We designed and synthesized novel native backbone RNA/DNA hybrids to co-stimulate TLR7/8/9. Delivery using non-inflammatory LNP avoids the induction of neutralizing antibody responses seen with vidutolimod and oncolytic viruses. LNP were characterized in vitro in normal human PBMC and human tumor-associated immune cells, in vivo in mice and monkeys, and in several mouse tumor models. Controls included small molecule agonists of TLR7/8 (bind at 1 site), RNA agonists of TLR7/8 (bind at both sites), and CpG-A, CpG-B, and CpG-C TLR9 agonists.Results The small molecule TLR7/8 agonist R-848 induced high inflammatory and immunomodulatory cytokines with only modest secretion of IFN-a (e.g., figure 1). In contrast, RNA TLR7/8 agonists or RNA/DNA TLR7/8/9 agonists (e.g., Z-007, figures 1 and 2) induce remarkably high IFN-a secretion without inflammatory cytokines in human cells and in mice and monkeys. The combination of RNA/DNA TLR7/8/9 agonists showed synergy for IFN-a induction compared to TLR7/8 or TLR9 agonists alone, far exceeding (>50X higher) pharmacodynamic responses reported to vidutolimod, yet with minimal inflammatory cytokine secretion. RNA-seq showed systemic induction of transcriptional signatures associated with human clinical response in monkey liver, spleen, lymph nodes, and blood following IV injection of Z-007 over a 100-fold dose-range (weekly dosing X 4) with no clinical or laboratory toxicity. IV or IP dosing of Z-007 induced tumor regression in mouse models without apparent toxicity.Conclusions These data demonstrate that native backbone RNA/DNA TLR7/8/9 agonists (exemplified by Z-007) induce high levels of IFN-a secretion and promote antitumor immunity without a systemic inflammatory response. Considering the ability of Z-007 to activate TAM, and the role of high IFN-a in promoting anti-tumor CD8+ T cell induction, Z-007 appears highly promising for human cancer immunotherapy.Acknowledgements We thank Raphael Nir, Adam Nir, and Justin Rosenthal (SBH Sciences) for performing in vitro immune assays in PBMC and TLR reporter cell lines; Brad Walker and Dan Conlon (Pace Life Sciences) for preparing Z-007 LNPs; Joachim Confais and Marine Febre (Cynbiose) for managing the in vivo monkey studies including serum, blood, and tissue cytokine/chemokine, FACS, and RNA Seq analyses; Sabrina Torre and Karl-Rudolph Erlemann (TherAquila Inc) for managing the in vivo mouse pharmacodynamic and tumor model studies, and Frédéric Couture (TransBIOTech) for performing these.References Milhem MM, Zakharia Y, Davar D, et al, Intratumoral vidutolimod as monotherapy or in combination with pembrolizumab in patients with PD-1 blockade-resistant melanoma: final analysis from a phase Ib study. Cancer, in press.Davar D, Morrison RM, Dzutsev AK, et al. Neoadjuvant vidutolimod and nivolumab in high-risk resectable melanoma: a prospective phase II trial. Cancer Cell. 2024;42:1–21.Tarhini AA, Sandra JL, Davar D, et al, A phase 2 randomized trial of neoadjuvant pembrolizumab alone or in combination with TLR9 agonist vidutolimod in macroscopic, resectable stage III melanoma: ECOG-ACRIN EA6194. ASCO. 2025.Ribas A, Medina T, Kirkwood JM, et al, Overcoming PD-1 blockade resistance with CpG-a toll-like receptor 9 agonist vidutolimod in patients with metastatic melanoma. Cancer Discovery. 2021;11:1–10.Sabree SA, Lemke-Miltner CD, Blackwell SE, et al, Monocytes exposed to immune complexes reduce pDC type 1 interferon response to vidutolimod. Vaccines, 2021;9:982–994.Liu H, Zhao L, Zheng P, et al, Novel transcriptional signatures associated with antitumor activity in vidutolimod-treated patients with anti-PD-(L)1-refractory melanoma and non-small cell lung cancer. AACR. 2022.Ethics Approval Patient ascites samples were collected at the University of Iowa Holden Comprehensive Cancer Center under IRB#201202743 with informed consent. The monkey study was reviewed by the Animal Welfare Body of Cynbiose and the Ethics Committee of VetAgro-Sup (1 avenue Bourgelat, 69 280 Marcy l’Étoile, France) and approved under number 2444 (MESRI number: 2024060609066896/#50229). All monkey experiments were conducted in accordance with the European Directive 2010/63/UE. The monkey animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) since 2015, an accreditation renewed in 2021. Mouse pharmacodynamic studies were performed in accordance to local animal welfare committee of the University of Montreal (Comité de Déontologie en Expérimentation Animale, CDEA) in agreement with regulations of the Canadian Council on Animal Care (CCAC). Mouse tumor models were performed at TransBIOTech, which is accredited by the Canadian Council on Animal Care (CCAC), and the studies were approved by the Cégep de Lévis Animal Care Committee and complied with CACC standards and regulations governing the use of animals for research.Abstract 919 Figure 1Human tumor-associated immune cells secrete high IFN-α without inflammatory/immunomodulatory response to Z-007 but not small molecule TLR7/8 agonist R848. Methods: Frozen ascites cells collected from patients with peritoneal tumors (N=3; representative sample shown) were cultured for 24 hr with various TLR agonists (0.5 μg/nL), and supernatants assayed by luminexAbstract 919 Figure 2Weekly dose-escalation of IV Z-007 induces unprecedented IFN-a and CXCL10 in cynomolgus monkeys with low inflammation. Methods: male and fmale cynomolgus monkeys (n=2/group) were injected IV weekly X 4 with the indicated of an RNL TLR7/8 agonist (green lines) or the RNA/DNA Z-007 TLR7/8/9 agonists (synthetic 32 base oligonucleotide froumulated in LNP; blue lines). For pharmacodynamics comparison, the corresponding serum IFN-α and CXCL 10 levels in responding humans undergoing vidutolimod therapy (‘vidu’) are shown in purple. Monkeys showed no fever or other clinical or laboratory signs of toxicity, all doses were well tolerated",
  "authors": [
    {
      "affiliations": [
        "Zola Therapeutics, Needham, MA, USA"
      ],
      "name": "Arthur M Krieg"
    },
    {
      "affiliations": [
        "Zola Therapeutics, Needham, MA, USA"
      ],
      "name": "Evan Walters"
    },
    {
      "affiliations": [
        "Zola Therapeutics, Needham, MA, USA"
      ],
      "name": "James Barsoum"
    },
    {
      "affiliations": [
        "Zola Therapeutics, Needham, MA, USA"
      ],
      "name": "Thomas Broome"
    },
    {
      "affiliations": [
        "Zola Therapeutics, Needham, MA, USA"
      ],
      "name": "Richard Fahrner"
    },
    {
      "affiliations": [
        "Pace Life Sciences, Woburn, MA, USA"
      ],
      "name": "Jonathan Neidigh"
    },
    {
      "affiliations": [
        "Pace Life Sciences, Woburn, MA, USA"
      ],
      "name": "Michael Mellas"
    },
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "Sue E Blackwell"
    },
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "George J Weiner"
    },
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "Carlos Chan"
    }
  ],
  "title": "919 Preclinical characterization of a first-in-class RNA/DNA hybrid TLR7/8/9 agonist, Z-007, ex vivo in human tumor-associated cells, in vivo in mice and primates, and tumor regression in mice",
  "uid": "64f49426-5505-5a5e-b11a-545379467d9d"
}