{
  "abstract": "Background Logic-gated cell therapy systems are considerably more complicated than conventional therapeutics and, therefore, more challenging to optimize. One type of logic gate, the NOT gate, responds to two inputs: the presence of antigen A and absence of antigen B. Perhaps the best studied NOT gate is called Tmod TM which consists of an activator, such as a chimeric antigen receptor (CAR), and an inhibitory receptor or blocker.1 To enable a high-throughput screening of Tmod modules, we developed a functional screen technology that utilizes a fluorescence-activated cell sorter (FACS) combined with fluorescent reporters to select cells based on desired function(s). Moreover, we adapted the functional screen platform to identify additional modules such as boosters, which increase the complexity of the system even further.Methods A FACS-based screening system was designed to rapidly select functional blockers and inducible boosters from a pooled library of candidates. This screen incorporates an NFAT-responsive GFP reporter Jurkat cell line for iterative selection of GFP(+) and (-) cell populations by FACS after exposure to different stimuli ( figure 1). The selected populations were analyzed by DNA sequencing to identify candidate blocker or booster sequences enriched during the process.Results To develop the functional screen platform and establish its workflow, a library of 46 blocker variants with varying intracellular domains was designed to demonstrate enrichment of active blockers. 2 Moreover, selected features were used to successfully predict the blocker behavior with 70-80% accuracy by a machine-learning algorithm. The functional screen platform was extended to screen for a signal 1 booster that activated T cell signaling in the presence of a small molecule (rimiducid). A library of ~30,000 booster variants, containing combinations of intracellular protein-protein interaction and signaling domains and motifs, was screened in the Jurkat reporter cell line and selected for GFP(+) cells in the presence of rimiducid. After three rounds of induction with rimiducid, >100 variants were strongly enriched (figure 2). A subset of these were shown to activate Jurkat cells in a rimiducid-dependent manner. Following the functional screen in Jurkat cells, a more complex booster library of ~100,000 variants was designed and screened directly in primary T cells to identify variants with proliferative and/or survival advantage.Conclusions The screening system described here identifies active blockers and boosters from large libraries of variants. Its throughput is sufficient to generate datasets to train machine learning models to help predict and optimize behavior of logic-gated cell therapeutics.References DiAndreth B, Hamburger AE, Xu H, Kamb A. The Tmod cellular logic gate as a solution for tumor-selective immunotherapy. Clin. Immunol. 2022; 241:109030.Martire S, Wang X, McElvain M, Suryawanshi V, Gill T, DiAndreth B, Lee W, Riley TP, Xu H, Netirojjanakul C, Kamb A. High-throughput screen to identify and optimize NOT gate receptors for cell therapy. Cytometry. 2024;105(10):741–751.Abstract 259 Figure 1An example flow schema of the functional screen for booster identificationAbstract 259 Figure 2Normalized count distribution of ~30,000 booster constructs in the GFP(+) Jurkat cells after three rounds of induction with rimiducid compared to the input. The top hits selected for further evaluation are shown in black",
  "authors": [
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Sara Martire"
    },
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Vasantika Suryawanshi"
    },
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Michele Mcelvain"
    },
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Mark Daris"
    },
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Tanveer Gill"
    },
    {
      "affiliations": [
        "310.ai, CA, USA"
      ],
      "name": "Timothy P Riley"
    },
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Alexander Kamb"
    },
    {
      "affiliations": [
        "A2 Biotherapeutics, Inc., Agoura Hills, CA, USA"
      ],
      "name": "Chawita Netirojjanakul"
    }
  ],
  "title": "259 Genetic screens to identify novel functional modules for a NOT gate",
  "uid": "01b8e10e-1781-5099-99f5-851802577687"
}