Clinical-Scale Production and Characterization of Ntla-5001 - a Novel Approach to Manufacturing CRISPR/Cas9 Engineered T Cell Therapies

Abstract

Adoptive T cell therapy has shown exciting efficacy in the treatment of certain hematological malignancies, particularly B cell tumors. However, with other cancers there has been limited success to date, and there remain significant challenges to develop safe and effective advanced cell therapies. Therefore, Intellia Therapeutics is leveraging its proprietary genome editing and cell engineering capabilities to develop a next-generation T cell therapy for the treatment of acute myeloid leukemia (AML). NTLA-5001 is an autologous T cell drug product genetically modified using CRISPR/Cas9 to eliminate endogenous T cell receptor (TCR) expression and transduced with AAV to site-specifically integrate a transgene encoding a Wilms' Tumor 1 (WT1)-targeting TCR into the TRAC locus. The TCR recognizes an HLA-A*02:01 restricted epitope of WT1. To overcome manufacturing difficulties often seen in autologous cell therapies, we developed a robust, electroporation-free, functionally closed-system manufacturing process capable of producing large numbers of minimally differentiated T cells with high editing rates, robust transduction efficiency, low translocation rates, and high viability. The manufacturing process begins with the enrichment of CD8 + and CD4 + T cells from patient apheresis to facilitate an optimum CD8:CD4 ratio at culture initiation. After incubation, T cells are stimulated using an αCD3 αCD28 activation reagent followed by disruption of the TCRβ chain by CRISPR/Cas9 via a lipid nanoparticle (LNP) containing mRNA encoding SpCas9 and sgRNA targeting TRBC. TCRα is subsequently knocked out in the same manner using an LNP containing SpCas9 mRNA and a sgRNA targeting the TRAC locus followed by delivery of the WT1-TCR transgene using AAV-6 for site-specific integration of the WT1-TCR template into the TRAC locus via homology directed repair. T cells are then expanded for several days under constant perfusion using a chemically defined expansion media in a rocking bioreactor until harvest, formulation, and cryopreservation. To date, clinical-scale production of NTLA-5001 at Intellia using healthy donors (n = 6) averaged 9.2 days in length. In that time, the process produced an average of 24.3 × 10 9 total T cells with an average viability of 93%. Although T cells underwent rapid expansion, they retained a minimally differentiated phenotype, with >90% of T cells at harvest expressing CD62L. Using our novel LNP-mediated cell engineering approach, we were able to achieve an average of 98.0% knockout of the endogenous TCR while simultaneously expressing the WT1-TCR in an average of >50% of T cells. This sequential editing approach reduced TRBC/TRAC translocation rates to near background levels. In addition, NTLA-5001 drug product displayed cytotoxic functionality when exposed to cell lines presenting the target WT1 peptide. The NTLA-5001 clinical-scale manufacturing process is a controlled and robust platform for the generation of minimally differentiated T cells with high rates of editing and transgene expression. Disclosures Cossette: Intellia Therapeutics: Current Employment. Aiyer: Intellia Therapeutics: Current Employment. Kimball: Intellia Therapeutics: Current Employment. Luby: Intellia Therapeutics: Current Employment. Zarate: Intellia Therapeutics: Current Employment. Eng: Intellia Therapeutics: Current Employment. Doshi: Intellia Therapeutics: Current Employment. Cole: Intellia Therapeutics: Current Employment. Kolluri: Intellia Therapeutics: Current Employment. Jaligama: Intellia Therapeutics: Current Employment. Gardner: Intellia Therapeutics: Current Employment. Wood: Intellia Therapeutics: Current Employment. Clark: Intellia Therapeutics: Current Employment.

Challenges and solutions for therapeuticTCR‐based agents

“…310 TCRengineered T cells are also being investigated for treating hematologic malignancies, particularly for AML/MDS by targeting the differentially expressed TAA WT1. 65,[311][312][313] Interestingly, relapse after WT1 TCR therapy was associated with antigen escape not by WT1 mutation or HLA downregulation but by immunoproteasome regulation, 314 a challenge that can be overcome by informed epitope selection.…”

“…To demonstrate the feasibility of neoantigen calling and TCR identification at a scale to treat a large cohort of patients, a recent effort demonstrated the feasibility of identifying patient‐specific neoantigens, their cognate TCRs, and manufacture of neoantigen TCR‐engineered T cells, dosing 16 patients with various solid tumors 310 . TCR‐engineered T cells are also being investigated for treating hematologic malignancies, particularly for AML/MDS by targeting the differentially expressed TAA WT1 65,311–313 . Interestingly, relapse after WT1 TCR therapy was associated with antigen escape not by WT1 mutation or HLA downregulation but by immunoproteasome regulation, 314 a challenge that can be overcome by informed epitope selection.…”

CRISPR-Cas System: The Current and Emerging Translational Landscape

“…Recently, Intellia Therapeutics initiated a trial [ 64 ] for evaluating their autologous TCR therapy for treatment of acute myeloid leukemia. Their product, named NTLA-5001, comprised T cells that had their TRBC and TRAC loci disrupted sequentially using CRISPR-Cas9, and later, an exogenous TCR specific to Wilms Tumor 1-specific antigen was inserted into the TRAC loci using CRISPR-Cas9 [ 83 ]. As of October 2022, Intellia therapeutics has indicated that this trial has been stopped, and a similar allogenic product is under preclinical development.…”

“…Interestingly, Intellia Therapeutics unveiled their gene editing approach [ 83 ] for their NTLA-5001 product evaluated in a clinical trial [ 64 ]. Here, they employed a sequential editing approach to KO first the TRBC locus of TCR receptor using a lipid nanoparticle (LNP) containing Cas9 and a sgRNA.…”

A systematic review of gene editing clinical trials

“…During the trial related to Acute Myeloid Leukemia (NCT05066165), TRAC and TRBC genes are knocked out in T cells using CRISPR technology. It should be highlighted that TRAC is knocked out by introducing WT1-targeting TCR (a particular TCR against the antigen present on the surface of the tumor) into this gene locus (Cossette et al, 2021).…”