A standardized method for T cell receptor (TCR) replacement through CRISPR-Cas9 mediated editing and retroviral transduction of primary murine naïve CD8 T cells

Goal-oriented transgenic TCR-T cell design for efficient cancer immunotherapy 2266120

Adoptive cell transfer (ACT) therapy is a promising approach for cancer treatment facing several limitations. Tumor-infiltrating lymphocyte (TILs) therapy is limited by patient condition and tumor accessibility. Peripheral lymphocyte redirection strategies by low transduction efficiency and potency. We previously showed that transgenic TCR-T cells suffer inconsistent expression of transgenic receptor and surface dilution by endogenous TCR, both limiting effective avidity for antigen. In this work we compare 3 general strategies for transgenic TCR-T cell engineering: Viral transduction, viral transduction coupled with endogenous TCR knock-out and Crispr-cas9 knock-in under endogenous TCR promoter. Thus, we identify their use cases for goal oriented therapeutic T-cells design. We use pMIGII retroviral vector for viral transduction. For endogenous TCR knock-out we deliver CRISPR-CAS9-gRNA RNP targeting TRAC and TRBC loci, using NEON system. For CRISPRS-CAS9 knock-in we use the above method followed by AAV6 transduction to deliver HDR template targeting TRAC. For Cytolytic Assay we use Chromium 51 release. Regular viral transduction resulted in transgenic TCR-T cells with low cytolytic activity, that were rescued by CRISPR-CAS9 knock-out of endogenous TCR. Both these approaches, however, resulted in loss of cytolytic activity and receptor expression with subsequent restimulations, due to semi-random gene insertion by viral vector. CRISPR-CAS9 HDR knock-in under endogenous TCR promoter produced consistent near-physiological transgenic TCR expression level, and cytolytic activity, maintained over multiple restimulations. Viral Transduction couples with endogenous TCR knock-out is sufficient to produce highly potent transgenic TCR -T cells for short-term use, with repeated infusions or in the context of in vivo transduction. Precision knock-in under endogenous TCR locus produces TCR-T cells more suitable for off-the-shelf reagent with long lasting cytolytic activity. MRIE at SKCC, DOD CDMP HT9425-25-1-0902 Technological Innovations in Immunology (TECH)

Engineering antigen-driven co-stimulation and T helper cell activity into TCR-T cells with CD8-41BB fusion receptors enhances anti-tumor activity

Applications and Future Perspectives of CRISPR/Cas9 Gene Editing Technologies in CAR-T Cell Therapy

A recent major breakthrough in cancer immunotherapy is the Chimeric antigen receptor-T cell (CAR-T cell) therapy, which has shown significant clinical efficacy in haematological malignancy treatment. Nonetheless, its application in a more general way is limited by a number of challenges, such as T-cell exhaustion, off-target associated toxicities, and the difficulty of personalised manufacturing. Recently, new opportunities have come into solving these problems with the introduction of clustered regularly interspaced short tandem repeats (CRISPR)-Cas9 genome editing, which has made it possible to perform precise and combinatorial genetic editing in CAR-T cells. The important applications of CRISPR in CAR-T cell engineering, which include the disruption of inhibitory immune checkpoints to enhance antitumour activity, the generation of universal allogeneic CAR-T cells by deletion of T-cell receptor and human leukocyte antigen (HLA) genes, and the modulation of cytokine signalling pathways to reduce toxicity are discussed in this review. Moreover, novel approaches, including targeted CAR integration and multiplex gene editing, are discussed as having the potential to enhance the therapeutic efficacy and scalability. In spite of these improvements, there are issues of off-target effect, delivery efficacy, genomic instability, and unaddressed issues of long-term safety. CAR-T cell therapies are likely to be improved further in future through advancements in genome editing technology, delivery methods, and synthetic biology. In general, CRISPR/Cas9-based engineering is a promising way of developing the next generation of precision cancer immunotherapy.

Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells

An industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs) and provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.

A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.

Engineering T cells in vivo to express a synthetic cytokine receptor enables preferential expansion and enrichment of anti-CD22 CAR T cells.

Ex vivo-manufactured chimeric antigen receptor (CAR) T cells targeting CD19 have transformed treatment strategies for patients with B cell malignancies and shown promising results in autoimmune disease. Yet despite their success, significant barriers remain that impede patient access to these therapies, including their complex manufacturing and the need for lymphodepleting chemotherapy. Moreover, many patients who receive ex vivo-manufactured CD19 CAR T cells do not achieve durable responses, suggesting that the expansion and persistence of CAR T cells may need improvement, and/or that strategies targeting alternative antigens may be needed for patients who are refractory to or relapse following treatment with CD19-directed therapies. Umoja is developing a surface-engineered lentiviral vector (LVV) platform capable of generating CAR T cells in vivo without lymphodepletion. UB-VV400 delivers a payload comprised of a fully human anti-CD22 CAR and the rapamycin-activated cytokine receptor (RACR), which enable preferential expansion and enrichment of CAR T cells in response to rapamycin. Here, we describe preclinical studies demonstrating that UB-VV400 generates anti-CD22 CAR T cells that kill CD22-expressing B cells and tumor cells in vitro and in vivo, and that engagement of RACR results in the selective expansion and enrichment of CAR T cells in vivo leading to complete tumor clearance and B cell depletion.