Engineered T cells directed at tumors with defined allelic loss

Hamburger, Agnes E.; DiAndreth, Breanna; Cui, Jiajia; Daris, Mark; Munguia, Melanie L.; Deshmukh, Kiran; Mock, Jee‐Young; Asuelime, Grace E.; Lim, Emily D.; Kreke, Michelle; Tokatlian, Talar; Kamb, Alexander

doi:10.1016/j.molimm.2020.09.012

Cited by 43 publications

(52 citation statements)

References 31 publications

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“…An independent study targeting CD19 with exogenously expressed A2 as the inhibitory antigen (non-genetically linked targeting; Fig. 5A) was published during final preparation of this manuscript and reached similar conclusions (122). These studies build on the clinical success of CAR T cells by targeting a unique class of tumor-specific antigens and lay the conceptual and practical foundation for harnessing the immune system to react against one of the most frequent types of genetic alterations in human cancers.…”

Section: Discussionmentioning

confidence: 59%

Targeting loss of heterozygosity for cancer-specific immunotherapy

Hwang

Mog

Douglass

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by “inverting” the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

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Section: Discussionmentioning

confidence: 59%

Targeting loss of heterozygosity for cancer-specific immunotherapy

Hwang

Mog

Douglass

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…All variants of A*02:01-NY-ESO-1 directed TCR were derived from 1G4 a95:LY/wtb (20). TCRa/b fusions containing the NY-ESO-1-responsive activating and inhibitory constructs were created as previously described (19). Briefly, ICDs from CD28, CD4, CD8a, 4-1BB, various truncations of Lck, Fyn, ZAP70, and LAT were fused to TCRa/b with either S, GGS, (G 4 S) 2 , (G 4 S) 3 , or (G 4 S) 4 as linkers.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Using the 10-ml format Neon Electroporation System, the Jurkat cells were transfected at 1400 V, 10 ms, three pulses. Transfected cells were immediately transferred to prewarmed RPMI 1640 supplemented with 20% HIA FBS and 0.1% pen/strep and incubated for 48 h at 37 C and 5% CO 2 (19).…”

Section: Generation Of Cd3-knockout Jurkat Nfat Luciferase Reporter Linesmentioning

confidence: 99%

“…Briefly, CD3d/e knockout cells were transfected using the 100-ml format Neon Electroporation System as described above with TCRa and TCRb subunits targeting A*02:01-NY-ESO-1 (clone 1G4 a95:LY) (20) with either CD3d alone, CD3e alone, or with both CD3d and CD3e. Rescue of functional activity was measured by coculturing with peptide-loaded T2s as was done previously (19) and described below. The pool of CD3d/e knockout cells generated was then further engineered to remove CD3g and CD3z as above and functionally validated (Fig.…”

Section: Generation Of Cd3-knockout Jurkat Nfat Luciferase Reporter Linesmentioning

confidence: 99%

“…16,17). These motifs have been co-opted in the design of artificial monomeric inhibitory receptors (iCAR and Tmod) that produce ligand-dependent inhibition of CAR and TCR signaling (18,19).…”

Section: Introductionmentioning

confidence: 99%

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Design of TCR Structural Variants That Retain or Invert the Normal Activation Signal

Mock

et al. 2021

ImmunoHorizons

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We designed variant human TCRs composed of the full-length TCRa/b or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR a-and b-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the a/b-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.

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High‐throughput screen to identify and optimize NOT gate receptors for cell therapy

Martire,

Wang,

McElvain

et al. 2024

Cytometry Pt A

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Logic‐gated engineered cells are an emerging therapeutic modality that can take advantage of molecular profiles to focus medical interventions on specific tissues in the body. However, the increased complexity of these engineered systems may pose a challenge for prediction and optimization of their behavior. Here we describe the design and testing of a flow cytometry‐based screening system to rapidly select functional inhibitory receptors from a pooled library of candidate constructs. In proof‐of‐concept experiments, this approach identifies inhibitory receptors that can operate as NOT gates when paired with activating receptors. The method may be used to generate large datasets to train machine learning models to better predict and optimize the function of logic‐gated cell therapeutics.

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Engineered T cells directed at tumors with defined allelic loss

Cited by 43 publications

References 31 publications

Targeting loss of heterozygosity for cancer-specific immunotherapy

Targeting loss of heterozygosity for cancer-specific immunotherapy

Design of TCR Structural Variants That Retain or Invert the Normal Activation Signal

High‐throughput screen to identify and optimize NOT gate receptors for cell therapy

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