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Biology subjects

Webber, B.

Publications and source records attributed to Webber, B..

3 recordsLinked to original sources

Modeling CISH-KO TIL therapy: T-cell persistence and endogenous competition determine response in gastrointestinal cancer

BackgroundTumor-infiltrating lymphocyte (TIL) therapies have shown promise in murine models; however, early-phase clinical trials reveal substantial heterogeneity in treatment response across patients. Elucidating the biological drivers of this variability is critical for improving patient selection and therapeutic design. MethodsWe developed a mechanistic mathematical model of Cish-inactivated TIL therapy capturing interactions among tumor cells, endogenous TILs, and infused CISH-KO (Cish gene knockout) T-cells. The model was calibrated to longitudinal tumor size data from two murine studies and also fitted to clinical data from a first-in-human phase 1 trial enrolling 22 and treating 12 patients with metastatic gastrointestinal cancers (27 independently tracked tumor lesions). We used the calibrated model to identify patient-level determinants of response and to simulate combination and fractionation treatment strategies in silico. FindingsFour mechanistically distinct parameters emerged as dominant determinants of therapeutic outcome: CISH-KO T-cell persistence (r = 0.827, p < 10-4) was the single strongest predictor of therapeutic failure; other major determinants included endogenous T-cell proliferation rate (r = 0.648, p < 10-4) , CISH-KO T-cell killing rate, and intrinsic tumor growth rate. In silico simulations predicted that the addition of anti-PD-1 checkpoint blockade to standard-dose CISH-KO TIL therapy achieved comparable or superior tumor control relative to dose doubling, while maintaining lower peak T-cell level. Additionally, fractionating the same total dose across multiple delayed infusions further improved predicted tumor control by prolonging CISH-KO T-cell persistence, again without raising peak effector burden. InterpretationThe efficacy of current CISH-KO TIL protocols is influenced not only by the cytotoxic potency of infused T-cells, but also by their persistence within the tumor microenvironment and competitive pressure from the reconstituting host immune compartment. These findings support strategies that enhance the functional efficiency and prolong the availability of infused T-cells--such as concurrent PD-1 blockade or fractionated dosing of the same total cell product--rather than dose escalation alone and identify CISH-KO T-cell persistence and endogenous immune competition as actionable targets for improving TIL-based immunotherapies.

Cancer Biology↗

Installation of Dominant-Negative Mutations in FAS and TGFβR2 via Base Editing in Primary T Cells

Adoptive cell transfer (ACT) of engineered T cells is effective against B-cell malignancies but has faltered against solid tumors due to the immunosuppressive tumor microenvironment (TME). FASL and TGF{beta} are key mediators of T cell dysfunction in the TME and overexpressing dominant negative (dn) forms of their receptors in T cells increases anti-tumor efficacy in solid tumor models. However, an approach which directly targets the endogenous genes would be more amenable to multiplex editing and reduce competition with WT alleles. Here, we employ base editing (BE) in primary human T cells to install naturally occurring dominant negative FAS and TGF{beta}R2 mutations. In vitro survival and proliferation assays demonstrate that BE T cells are resistant to pro-apoptotic and anti-proliferative effects of FAS and TGF{beta} signaling. CAR-T cells with BE-installed dn TGF{beta}R2 or dn FAS exhibit improvements in cytotoxicity, while dn TGF{beta}R2 CAR T demonstrate increased persistence and reduced expression of phenotypic markers of exhaustion compared to controls. Moreover, BE-engineered dn CAR T outperform lentiviral-engineered cDNA over expression counterparts in several functional assays. Considering the efficiency of BE and its amenability for multiplex editing, our novel approach lends itself to engineering strategies necessary to overcome T cell dysfunction in solid tumors.

immunology↗

EditR: A novel base editing quantification software using Sanger sequencing

CRISPR/Cas9-Cytidine deaminase fusion enzymes - termed Base Editors - allow targeted editing of genomic deoxcytidine to deoxthymidine (C[->]T) without the need for double stranded break induction. Base editors represent a paradigm-shift in gene editing technology, due to their unprecedented efficiency to mediate targeted, single-base conversion; however, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time consuming. To overcome these limitations, we developed a simple, cost effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed EditR. We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence (baseeditr.com). EditR is more accurate than enzymatic assays, and provides added insight to the position, type and efficiency of base editing. Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.

genetics↗