{
  "abstract": "Background Acute myeloid leukemia (AML) accounts for 25% of acute leukemias in children and 33% in adults. CD33 is overexpressed in AML blasts and natural killer (NK) cells engineered with anti-CD33 chimeric antigen receptor (CD33.CAR NK) using a Cas9/AAV6 system exhibit significantly enhanced cytotoxicity against AML. 1 Separately, chronic TGFβ stimulation during NK expansion (TGFβ imprinting or TGFβi) induces NK cells to secrete high levels of IFNγ and TNF, and confer resistance to TGFβ-induced suppression in tumor microenvironment.2 The goal of this study is to determine whether TGFβi would further enhance anti-AML efficacy of CD33.CAR NK cells and whether NKTR-255 (polymer conjugated IL15)3 further augments these effects.Methods Unmodified NK, NK TGFβi, CD33.CAR NK, CD33.CAR NK TGFβi were evaluated with and without NKTR-255 (40ng/ml, generously provided by Nektar) in long-term cytotoxicity assays against GFP-luciferase labeled Molm13 AML cell line. NK cells were co-cultured at 2:1 ratio with target cells, and fresh tumor cells were added every 24 hours. Cytotoxicity and IFNγ secretion were assessed 24 hours after each cycle as previously described. 4 Results CD33.CAR NK cells exhibited superior cytotoxicity as well as IFNγ secretion compared to unmodified NK all throughout (p<0.01) but the potency declined with repeated tumor challenges (p < 0.01). TGFβ imprinting significantly preserved both cytotoxicity and IFNγ secretion in CD33.CAR NK cells at later cycles. Conversely, TGFβ imprinting alone initially reduced the anti-AML effects (66.3 ± 2.3% of NK TGFβi vs 83.7 ± 0.8% of NK, p<0.01) ( figure 1A, B).NKTR-255 significantly enhanced cytotoxicity and IFNγ levels in all NK groups. The combination of TGFβ imprinting and NKTR-255 induced the most sustained cytotoxicity for CD33.CAR NK TGFβi. Notably, this combination also significantly rescued the declining cytotoxicity of NK TGFβi alone, rendering a cytotoxicity of 83.8 ± 0.9% at cycle 4, vs 58.2 ± 7.2% for NK + NKTR-255, and 40.7 ± 4.7% for NK TGFβi (p<0.01), suggesting synergism of TGFβ imprinting and NKTR-255 (figure 1A, B).Consistently, flow cytometry demonstrated higher NK persistence and fewer AML cells in TGFβi and NKTR-255 modified conditions at cycle 5 (figure 1C). The improved NK survival correlated with an increase in pSTAT5 and CD25 in TGFβi NK at cycle 1 (figure 1D).Conclusions TGFβ imprinting and NKTR-255 synergistically enhanced and sustained anti-AML activity of NK and CD33.CAR NK cells, supporting a novel strategy to improve NK-based immunotherapy for AML.Acknowledgements Supported by DOD W81XWH-21-1-0841.References Naeimi Kararoudi MS Likhite E Elmas, Yamamoto K, Schwartz M, Sorathia K, de Souza Fernandes Pereira M, Sezgin Y, Devine RD, Lyberger JM, Behbehani GK, Chakravarti N, Moriarity BS, Meyer K, Lee DA. Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV. Cell Rep Methods 2022;2(6):100236.Foltz JA, Moseman JE, Thakkar A, Chakravarti N, Lee DA. TGFbeta imprinting during activation promotes natural killer cell cytokine hypersecretion. Cancers (Basel) 2018;10(11).Luo W, Gardenswartz A, Hoang H, Chu Y, Tian M, Liao Y, Ayello J, Rosenblum JM, Mo X, Marcondes AM, Overwijk WW, Cripe TP, Lee DA, Cairo MS. Combinatorial immunotherapy of anti-MCAM CAR-modified expanded natural killer cells and NKTR-255 against neuroblastoma. Mol Ther Oncol 2024;32(4):200894.Chu Y, Nayyar G, Tian M, Lee DA, Ozkaynak MF, Ayala-Cuesta J, Klose K, Foley K, Mendelowitz AS, Luo W, Liao Y, Ayello J, Behbehani GK, Riddell S, Cripe T, Cairo MS. Efficiently targeting neuroblastoma with the combination of anti-ROR1 CAR NK cells and N-803 in vitro and in vivo in NB xenografts. Mol Ther Oncol 2024;32(2):200820.Abstract 642 Figure 1A) AML cell death (% of killing) and B) IFNγ in longterm cytotoxicity assay. C) GFP and CD56 Flow cytometry analysis at the end of 5 cycles. D) pSTAT5 and CD25 flow analysis on NK TGFβi and NK only, 24 hours post incubation with AML cells",
  "authors": [
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Yanling Liao"
    },
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Mishel Ramirez"
    },
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Meijuan Tian"
    },
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Shaimaa AbdAllah"
    },
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Morgan Anderson-Crannage"
    },
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Edo Schaefer"
    },
    {
      "affiliations": [
        "Nektar Therapeutics, San Francisco, CA, USA"
      ],
      "name": "Mario A Marcondes"
    },
    {
      "affiliations": [
        "Nationwide Children’s Hospital, Columbus, OH, USA"
      ],
      "name": "Yasemin Sezgin"
    },
    {
      "affiliations": [
        "Nationwide Children’s Hospital, Columbus, OH, USA"
      ],
      "name": "Dean Anthony Lee"
    },
    {
      "affiliations": [
        "New York Medical College, Valhalla, NY, USA"
      ],
      "name": "Mitchell S Cairo"
    }
  ],
  "title": "642 Synergistic anti-leukemic effect of NKTR-255 and TGFβ-imprinted CD33.CAR natural killer cells",
  "uid": "6b9e244e-d094-5c42-a999-1116579ab834"
}