Biology subjects

Kotanchek, T.

Publications and source records attributed to Kotanchek, T..

3 recordsLinked to original sources

bioRxiv2025

Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells

Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations within a final product, with many cells lacking desired of receptor expression or those that have exhausted or other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via nonviral CRISPR genome-editing to eliminate the T cell receptor (TCR) alpha chain (TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. Within these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which subsequently triggers CAR-mediated killing by a separate subset of cells within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-GD2 CAR T cell products up to [≥]99% CAR+/TCR-, favoring a stem cell memory-like phenotype (CD45RAhigh/CD62Lhigh). Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/684419v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@199d85forg.highwire.dtl.DTLVardef@14c638aorg.highwire.dtl.DTLVardef@1b3322org.highwire.dtl.DTLVardef@1ba253d_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Predicting T Cell Quality During Manufacturing Through an Artificial Intelligence-based Integrative Multi-Omics Analytical Platform

Large-scale, reproducible manufacturing of therapeutic cells with consistently high quality is vital for translation to clinically effective and widely accessible cell therapies. However, the biological and logistical complexity of manufacturing a living product, including challenges associated with their inherent variability and uncertainties of process parameters, currently make it difficult to achieve predictable cell-product quality. Using a degradable microscaffold-based T cell process as an example, we developed an Artificial Intelligence (AI)-driven experimental-computational platform to identify a set of critical process parameters (CPP) and critical quality attributes (CQA) from heterogeneous, high dimensional, time-dependent multi-omics data, measurable during early stages of manufacturing and predictive of end-of-manufacturing product quality. Sequential, Design-of-Experiment (DOE)-based studies, coupled with an agnostic machine-learning framework, were used to extract feature combinations from media assessment that were highly predictive of total live CD4+ and CD8+ naive and central memory (CD63L+CCR7+) T cells and their ratio in the end-product. This computational workflow could be broadly applied to any cell therapy and provide a roadmap for discovering CQAs and CPPs in cell manufacturing.

bioengineering↗

bioRxiv2019

Functionalized microcarriers improve T cell manufacturing by facilitating migratory memory T cell production and increasing CD4/CD8 ratio

Adoptive cell therapies (ACT) using chimeric antigen receptor (CAR) T cells have shown promise in treating cancer, but manufacturing large numbers of high quality cells remains challenging. Critically, current T cell expansion technologies only partially recapitulate the in vivo microenvironment found in the human lymph nodes. In these organs, T cells expand at high cell density with autocrine/paracrine signaling, as well as signals from the extracellular matrix (ECM). Here we describe a T cell expansion system using degradable gelatin microcarriers functionalized with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), which address several of these shortcomings. We show that using this system, we can achieve approximately 2-fold greater expansion compared to functionalized magnetic beads, the current industry standard. Furthermore, carriers generated higher numbers of CCR7+CD62L+ migratory, central memory T cells and CD4+ T cells across multiple donors. Both these phenotypes have emerged as important for establishing durable and effective responses in patients receiving T cell immunotherapies. We further demonstrate that carriers can achieve greater memory cell yield compared to beads across a range of IL2 concentrations from 20 U/mL to 100 U/mL. These differences were greater at lower IL2 concentrations, indicating that the carriers are more efficient. We optimized this system using a design of experiments (DOE) approach and found that the carrier concentration affects the memory cell yield in a quadratic manner, where high or low concentrations are detrimental to memory formation. Finally, we show that carriers do not hinder CAR transduction and can maintain the CD4 and memory phenotype advantages in CAR-transduced T cells.

bioengineering↗