{
  "abstract": "Background Thailanstatins are naturally occurring anti-proliferative compounds that target spliceosomes and pre-mRNA splicing. PH1 is a proprietary Thailanstatin analog that has anti-tumor activity in preclinical xenograft and syngeneic models as Her2 and Trop2 ADCs. 1 2 Treatment of NCI-N87 gastric cancer cells with PH1 stimulated intron retention and neopeptides with high affinity to MHC Class I, as predicted by MHC flurry analysis.1 Trastuzumab PH1 ADCs induced complete regression (CR) of MC38-hHer2 colon carcinoma tumors and CR mice rejected a rechallenge with a fresh bolus of tumor cells suggesting immune memory.1 The current work investigates immunomodulatory mechanisms of PH1 contributing to tumor regression and memory.Methods Pre-established MC38-hHer2 tumors were treated with Vehicle, anti-mPD-1, Tras PH1, Kadcyla, Tras PH1+mPD-1, Kadcyla+mPD-1 intravenously for 2 weeks. Immunoprofiling and immunohistochemistry was performed during CR. Survival was monitored over 5 months after which CR mice were rechallenged with tumor cells. Following demonstration of specific immune memory, mouse PBMCs were analyzed for clonal expansion and diversity by immune repertoire sequencing and lungs were analyzed for tissue-resident memory cells.Results The anti-splicing mechanism of action (MOA) of PH1 specifically increased tumor infiltration of neutrophils, polymorphonuclear MDSCs, and induced macrophage repolarization that was not associated with Kadcyla arms ( figure 1C). All CR mice from different treatment arms rejected tumor rechallenge with no further ADC treatment, however there were some similarities and differences in immunological responses between the treatment arms. The memory response was associated with IgM expansions in all treatment arms. However, the number/diversity of unique IgM clones expanded in Tras PH1 arm was significantly higher than Naïve and mPD-1 arms (figure 2C). Conversely, immune rejection in mPD-1 arms was linked to TCR beta expansions (figure 2E). Interestingly, TCR delta clonal expansions were observed only in Tras PH1+mPD-1 combo, but not in single agent arms, suggesting a gain-of-function (figure 2F). Similarly, binning circulating antibodies identified a significantly greater number of unique clones directed against frameshift peptide (FSP) neoepitope in the Tras PH1+mPD-1 combo.Conclusions When combined, the MOAs of the two agents complemented each other where Tras PH1 increased neoantigens, innate myeloid MOAs, and stimulated IgM antibodies whereas mPD-1 activated αβ T cells and promoted antibody class switching. The synergy of Tras PH1+mPD-1 may be due to each agent potentially improving the other’s MOA or may be due to expansion of MHC non-restricted γδ T cells which neither single agent was able to achieve on its own.Acknowledgements We acknowledge the contributions of Mary Do, Will Monteith, Greg Tuffy and Jeffrey Kang for generating PH1 ADCs for the studies in References 1, 2 and the current study. We thank our past and current CEOs- Sanjeev Satyal, Stephen LaMond, Hoyoung Huh, Samir Patel, and Abizer Gaslightwala for their continued support of ADC payloads with novel MOAs.References Mitra SK, Jammalamadaka V, Kang J, Losic T, Tuffy G, Liang TW, Tipton K, Lopez A, Savage S, Monteith W, Haskins WE, Jurica MS, Satyal S, Do M. Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy. Cancer Res. 2021;81(13_Supplement):1832.Mitra SK, Monteith W, Do M, Tuffy G, Savage S, Kang J, Abuhay M, Losic T, Ghone S, Haskins WE, Jammalamadaka V, Satyal S. Cancer Res. 2023;83(7_Supplement):6297.Abstract 951 Figure 1Immunological effects of PH1 ADCs singly/or in combination with Checkpoint Inhibitors. A. NCI-N87 cells were treated with PH1 payload or DMSO vehicle control. Alternatively spliced genes were identified using the ‘knownAlt’ track of UCSC Table Browser and differential spliced gene expression (DGE) is shown above. Overexpressed spliced genes are enriched in novel transcripts contributing to neoantigen whereas reduced genes are largely substrates of nonsense mediated decay (NMD). B Trastuzumab PH1 ADC was compared singly and in combination with anti-mouse PD-1 (mPD-1) against MC-38 human Her2 (MC-38hHer2) colon cancers in C57BL/6 syngeneic mouse model. DAR-matched Trastuzumab-DM1 (Kadcyla) was used as control. Significantly higher complete regression (CR) rates were observed with Tras PH1+mPD-1 combo. C. Immuno-profiling during early tumor regression identified macrophage repolarization, and granulocytic increases such as neutrophils and PMN MDSCs with low Arginase I expression. D. Kaplan Meier plot showing improved OS of Tras PH1 combination. E. Higher numbers of tissue-resident central and effector memory cells were observed in Tras PH1 combo as opposed to single agentAbstract 951 Figure 2Immunological changes associated with rejection of MC38 rechallenge of completely regressed mice: Rationale for PH1 ADC synergy with anti PD-1. A. Completely regressed (CR) mice were challenged with a fresh bolus of 5x105 MC38 parental cells (no hHer2) subcutaneously on the left flank and 2x105816-F10 cells on the right flank, resulting in specific rejection of MC38 cells indicative of immune memory and epitope spreading beyond Her2 target antigen. PBMCs were obtained and CDR3s were sequenced as described (iRepertoire protocol). B. Analysis of Heavy Chains revealed dominant non-class-switched clones across treatment arms largely contributed by IgM species. C. Notwithstanding clonal selection and amplification against antigen, that typically reduces diversity, there was greater number of unique IgM clones expanded in Tras PH1 arm (p<0.05). D. Increased diversity of IgG1 and IgG2 chains were also observed in Tras PH1 arm (p<0.05). E. Conversely, anti-mPD-1 affected clonal expansions of TCR beta chains. F. Neither single agent Tras PH1 or anti mPD-1 treatment induced clonal expansions of TCR delta chains, but the combination significantly did, suggesting expansions of TCR δγ clones in the combo arm",
  "authors": [
    {
      "affiliations": [
        "Akari Therapeutics, Plc, San Francisco, CA, USA"
      ],
      "name": "Satyajit K Mitra"
    }
  ],
  "title": "951 A novel splicing-targeted ADC payload drives immune activation, synergy with checkpoint inhibitors, and enhanced therapeutic potential beyond cytotoxicity",
  "uid": "21917988-c1dc-59eb-af11-ee42716a47ba"
}