Supplementary Figure 6 from Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors

2024-11-01

<p>Supplementary Figure 6 | a-b, Cytotoxicity of a, miTCR1 and b, miTCR2 variants against Nalm6. c-d, Long-term flow cytometry-based cytotoxicity of lead c, miTCR1 and d, miTCR2 variants. e, Quantification of cytokines in supernatants 24h after co-culture of engineered T cells with Nalm6. f, Expression of TCRC chains in αCD22 miTCR1, CD22 miTCR2 and CD19 miTCR1 engineered Jurkat cells. g, Induction of reporter NFAT-GFP upon exposure to CD19+CD22+ Nalm6 in Jurkat cells. Screening studies in a-b performed using two independent donors; c-d, representative data from three independent donors; e, data from one donor. ****P < 0.0001 by two-way ANOVA (c-d).</p>

Supplementary Figure 4 from Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors

2024-11-01

<p>Supplementary Figure 4 | Names and amino acid changes of miTCR variant panel. PD insertions are highlighted in blue.</p>

Supplementary Figure 3 from Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors

2024-11-01

<p>Supplementary Figure 3 | a, Predicted structures of miTCR1 and miTCR2 α and β chains overlaid on resolved structures of TCR α and β chains. b, Overlay of five highest-ranking predictions for miTCR1 and miTCR2 variable-constant chain interface. c, Resolved native TCR α chain V-C interface structure and predicted V-C interface structures of miTCR2 WT and modified α chains. d, Resolved native TCR β chain V-C interface structure and predicted V-C interface structures of miTCR1 WT, miTCR2 WT and modified β chains.</p>

Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors

Cancer Immunology Research · 2024-06-12 · 2 citations

Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of nonmalignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing, and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and preclinical T-cell products at critical stages of manufacturing.

Rational protein engineering to enhance MHC-independent T cell receptors

bioRxiv (Cold Spring Harbor Laboratory) · 2024-02-23

Abstract Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity against cancer, however remain limited in their scope and long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the endogenous T cell response, we designed hybrid MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant domains. While functional, we observed stark differences in miTCR-driven T cell function that were dependent on receptor orientation. Using predictive structural modeling, we observed significant biochemical conflicts at the hybrid variable-constant domain interface. To overcome this, we performed iterative sequence modifications and structural modeling to design a panel of miTCR variants predicted to have improved interface stability. Functional screening nominated a variant with superior efficacy to all other miTCRs as well as a standard CAR against high burdens of leukemia. Statement of Significance Improving the durability of engineered T cell immunotherapies is critical to enhancing efficacy. We used structure-informed design to evolve MHC-independent T cell receptors that drive improved tumor control. This work underscores the central role of synthetic receptor structure on T cell function and provides a framework for improved receptor engineering.

Supplementary Figure 2 from Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors

2024-09-03

<p>A, Expansion of T cells in which stimulation cultures did or did not contain IL7/IL15. B, CD4 and CD8 composition of and C, memory composition of T cell products at the conclusion of manufacturing when stimulation cultures did or did not include IL7/IL15.</p>

Supplementary Figure 1 from Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors

2024-09-03

<p>A, Representative ow cytometry plots of transduction efficiency of various synthetic receptors in control (unedited) and TRAC+TRBCKO T cells when editing is performed pre-stimulation. B, Stability of CRISPR editing over-time when performing post-stimulation editing.</p>

Regulatory Considerations for Genome-Edited T-cell Therapies

Cancer Immunology Research · 2024-07-16 · 8 citations

Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.

Supplementary Figure 7 from Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors

2024-11-01

<p>Supplementary Figure 7 | a, Overlay of predicted mut031 α chain variable region and resolved FMC63 variable chain structure. Modeling performed using AlphaFold2. b, Memory lineages of human T cells engineered to express miTCR1 WT or mut035 at the conclusion of manufacturing cultures. c-d, Proportion of human T cells that were early lineage (naïve or central memory) c, throughout 6 day stimulation manufacturing cultures or d, one day after clearance of Nalm6. e, Expansion of human T cells expressing miTCR1 WT, mut035 or 19/BBζ CAR from two donors. b-d, data from one donor.</p>

Data from Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors

2024-11-01

<div>Abstract<p>Chimeric antigen receptor (CAR)–based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T-cell activation causes T-cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T-cell response, we designed MHC-independent T-cell receptors (miTCR) by linking antibody variable domains to T-cell receptor constant chains. Using predictive modeling, we observed that this standard “cut and paste” approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable–constant domain interface. Through iterative modeling and sequence modifications, we developed structure-enhanced miTCRs which significantly improved receptor-driven T-cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T-cell persistence and enabled improved leukemic control <i>in vivo</i> compared with classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function.</p><p><b>Significance:</b> Improving the durability of engineered T-cell immunotherapies is critical to enhancing efficacy. We used a structure-informed design to evolve improved miTCR function across several models. This work underscores the central role of synthetic receptor structure in T-cell function and provides a framework for improved receptor engineering.</p></div>