Genetic Engineering Publications - GEG Tech top picks

In most cases, the number of naturally occurring cancer-targeting T cells will be far too low to trigger an immune response capable of eradicating a patient's tumor(s). PACT Pharma is a privately held biopharmaceutical company developing personalized, neo-antigen-specific T-cell receptor (TCR)-T therapies to treat a range of solid tumors.  Patient T cells are isolated and CRISPR-modified by electroporation with Cas9 protein, guide RNAs to knock out endogenous TCR genes (TCRα ( TRAC) and TCRβ ( TRBC)) and an HR template plasmid encoding the transgenic neoTCR. The results of a phase 1 clinical trial that were published in Nature. The researchers report that CRISPR-modified T cells were preferentially directed to the tumor and could be recovered from post-infusion biopsies in all patients for whom biopsies were available. They also note that CRISPR-edited T cells frequently accounted for the top 2-20% of immune cells in the tumor, and a reduction in tumor size was observed in some lesions in a single lung cancer patient.

For 17 years, Thomas Blankenstein has been working on his Berlin-based start-up T-knife and is finally going to be able to test his T-cell receptor (TCR) cancer immunotherapy. To make this immunotherapy, they replaced the TCR genes of a mouse with those of a human and inserted genes to encompass both the human TCR repertoire and human major histocompatibility complex (MHC) molecules. They eliminated genes from related mice, generating mouse lines carrying human genes, and then subjected them to a succession of crosses to bring all the genes together in a single mouse, the HuTCR mouse. Designing transgenic mice with such efficient humanized TCRs has been very difficult and laborious. However, TCR-T cells are capable of more extensive signaling and killing than CAR-T cells. The engineered TCRs integrate seamlessly into the signal transduction pathways of T cells: there are ten subunits in a TCR versus one subunit in a CAR, they have ten immunoreceptor tyrosine activation motifs versus three in a CAR, and they are associated with more co-stimulatory receptors: CD3, CD4, CD2. In addition, unlike CARs that only bind to antigens on the cell surface, TCRs can target intracellular as well as extracellular tumor antigens.

In the new study, Dr. Sykulev and colleagues in Takami Sato's lab engineered CAR-T cells to recognize an antigen on melanoma cells called high molecular weight melanoma-associated antigen (HMW-MAA). Melanoma cells express varying amounts of HMW-MAA on their cell surfaces. In their research, the researchers evaluated the extent to which CAR-T cells killed melanoma cells. They found that CAR-T cells effectively killed melanoma cells expressing high levels of HMW-MAA, but not those with lower levels of the antigen. The researchers then tested how well another type of immunotherapy, known as TCR-T cells that uses T cells engineered to express a specific T-cell receptor, killed the target cells. When the researchers treated melanoma cells with TCR-T cells, they found that the treatment readily killed tumor cells, even in melanoma cell lines that expressed far less HMW-MAA antigens than needed for CAR-T. The comparison thus revealed that TCR-T is superior to CAR-T therapy for melanoma.

In this work, the authors  assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) for CRISPR system to knock out endogenous TCR expression. Using nuclease-expressing plasmid DNA, they achieved up to 19.9% and 12.2% knockout of TCR expression in primary T cells with CRISPR/Cas9 and TALENs, respectively. In contrast, delivery of TALEN mRNA by electroporation resulted in high viability and TCR knockout efficiencies of up to 78.8% for the TCR α chain and 81.2% for the β chain on day 6 after electroporation.