Early Study Shows Feasibility of Single Vector Multiplexed shRNA for CAR T Cells

A first-in-human trial was presented during the 2020 American Society of Clinical Oncology (ASCO) Virtual Scientific Program, which is evaluating the first-generation single short-hairpin RNA (shRNA)-vector with BCMA-targeted chimeric antigen receptor (CAR) as a potential non-gene edited approach to utilizing allogeneic CAR T-cell therapy in patients with cancer. The trial was able to demonstrate that multiple shRNAs are active within 1 single vector, which takes away from the need for individual clinical reagents targeting multiple genes.

In this study, the multiplexed shRNA platform was able to provide a next-generation, allogeneic approach to the development of CAR T cells, which enables flexibility, versatility, and single-step engineering by utilizing an all-in-1 vector approach. This multiplexed shRNA vector system will continue to be developed as investigators evaluate whether this approach would enhance the therapeutic benefit of CAR T-cell therapy.

This research is expected to continue moving forward by the end of this year. The next wave of development will look at clinical candidates that could use multiple shRNA knockdowns. The preclinical development has demonstrated the proof of concept and feasibility for concurrent knockdown of up to 4 genes.

In an interview with Targeted Oncology, David E. Gilham, PhD, of the University of Manchester, discussed the findings from the single vector multiplexed shRNA approach to concurrently knockdown the expression of multiple genes in CAR T cells when used as treatment of patients with cancer.

TARGETED ONCOLOGY: Could you start by providing an overview of the study?

Gilham: This study itself is focused on a technology that we have been working with now for a few years, which is shRNA, and primarily using this to modulate and knockdown gene expression in our CAR T-cell therapy. In this iteration, the poster at ASCO that we're addressing, is based on our base technology where we're using a single shRNA combined with the CAR-targeting BDMA. We're using this as a means to generate an allogeneic T-cell product. The shRNA targets a gene called CD3ζ, which is an important part of the T cell receptor complex and in the allogeneic space, can cause graft versus host disease. By expressing this single shRNA, we can target the T cell receptor, reduce expression of that cluster, resulting in reduction of GVHD, and by co-expressing the BCMA CAR from a single vector, this is all based with the 1 piece of DNA, we can generate an allogeneic product that's also able to target through the CAR. The key aspect of the ASCO poster is it takes these technologies to the next stage through what we call multiplexing. The advantage is that we can include multiple shRNA into the same vector.

In addition to targeting CD3ζ, the T cell receptor complex, we can, for instance, also include another shRNA that targets MHC class 1 for instance. The next generation of our shRNA platform is to target multiple genes to give multiple functionalities and more power potentially to the allogeneic approach, but within all of this maintaining the expression of the various genes within a single vector are all in 1 vector approach, which keeps costs down effectively and facilitates manufacturing of the CAR T cells.

TARGETED ONCOLOGY: What were the methods behind this study?

Gilham: Our baseline is that we know, when working with our colleagues who generated the concept that we're working with, that the main reason why this technology works is that we express the shRNA, which is a short stretch of RNA, within the context of a microRNA framework. This microRNA framework allows the shRNA to be expressed within our CAR, and this is the key, the fact that we can have effectively 1 promoter that drives expression of all the genes within that particular vector, and so the shRNA, the CAR, and other genes are all expressed within that 1 vector.

What we've done specifically within this work is continue to work on the framework itself, so the initial construct was around 250 base pairs or so to express a single hairpin. We expressed 2 hairpins from a slightly larger construct, and then as in the ASCO poster, we've now moved up to 4 shRNA resulting in 4 independent genes. However, we worked very hard to keep the size of the transgene load of the shRNA framework down, and so it's around about 500 base pairs now, which is a fairly small imposition on the CAR vector, but we can target up to 4 shRNA, so 4 independent genes, also while coexpressing a CAR and the marker gene. This is this is an early stage, but going forward, our aim is to use multiple shRNAs in this multiplex format so we can target a potential array of genes to try and enhance the function of the CAR T cells.

TARGETED ONCOLOGY: What was found in this study, and what are the next steps?

Gilham: In terms of the practicality of the multiplex approach, 1 of the key things we've confirmed is that from a manufacturing perspective, because we're expressing everything from a single vector and we include a cell surface marker gene at the same point, we can carry out just a single selection, which shows that the cells that we isolate through that single procedure, express all of the shRNA and the CAR at the same time. The concept of using a single vector in expressing various components is to try and maintain simplicity in terms of the manufacturing process, and in particular, a single cell selection. We're trying to avoid ramping up costs of manufacturing, which are associated with having to carry out multiple manipulations. One of the key things of the paper that we presented shows that expressing multiple shRNA impact on multiple different types of gene expression profiles, but all this being carried out in a way that we think is relatively straightforward in terms of cell manufacturing, particularly from the level of manipulation that has to be done.

TARGETED ONCOLOGY: What is your take home message, and is there anything else you would like to highlight?

Gilham: Concerning this paper, in particular, it sets the scene for us to go forward. We're just about to submit our IND to test our first shRNA approach, which is the single therapy with BCMA, is a proof of principle and clinically testing the shRNA approach. Now we have high confidence, and so of course, we're thinking about how we can take this to the next level, which is where there's multiplexing. Being able to target elements such as MHC class 1 or kinases, perhaps, are very important for the functionality of the T cell, to my mind means that we can think in greater depth about manipulating the phenotype of the cells and optimizing their function, particularly when looking to challenge solid tumors, which is the holy grail of CAR T-cell therapy. I think this is an important technology that will help us and facilitators to drive down that lane.

_Reference

_{Gilham DE, Bornschein S, Springuel L, et al. Single vector multiplexed shRNA provides a non-gene edited strategy to concurrently knockdown the expression of multiple genes in CAR T cells. J Clin Oncol 38: 2020 (suppl; abstr 3103) doi:10.1200/JCO.2020.38.15_suppl.3103}