Date: 28th January 2020
The latest, most innovative wave of immunotherapies to reach the clinic are undergoing a revolution of personalisation and at the heart of these lies CAR-T cell therapy. However, providing individualised medicines is time-consuming and expensive, and to date they are able to target only a few (non-solid) cancers. Now scientists have exploited CRISPRs to discover T-cells equipped with a new type of T-cell receptor (TCR) which recognises and kills most human cancer types, leading to the hope of a single go-to cancer treatment.
Cancer is the second leading cause of death globally, and was responsible for an estimated 9.6 million deaths in 2018. With around 1 in 6 deaths occurring globally and with more than 200 types of human cancers currently identified, finding effective treatments and ultimately a cure for cancer is highly sought after with synthetic biologists and bioengineers joining the race towards developing safe, effective and alternative solutions.
How do T–cells work?
T-cells play a central role in the immune system, and protect the body from infections and diseases, including cancer. They possess on their surface T-cell receptors (TCRs), which are responsible for recognising fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules.
To be recognised by T cells, antigens must be loaded on human leucocyte antigen (HLA) molecules. HLA is the human version of the major histocompatibility complex (MHC) of vertebrates, a group of cell-surface proteins coded by more than 250 genes on chromosome 6.
The T-cells scan the surface of cells to determine which are ‘foreign’ or unhealthy and which are ‘normal’. Once activated, T-cells divide rapidly and target the cell for destruction or they secrete cytokines that regulate or assist the immune response, to eliminate the target cells.
We inherit many different HLA genes from our parents, and as they are highly polymorphic they widely differ between individuals. Our individual HLA type is, for example, what will determine tissue rejection following organ or stem cell transplantation. We now know recipients and donors must be HLA-matched otherwise the immune system deems the donor cells or organs as foreign and eliminates them.
Currently chimeric antigen receptor cell therapy (CAR-T) has been employed as an effective tool against cancer. Patient or donor T-cells are genetically engineered to produce an artificial T-cell receptor which targets specific proteins indicative of the cancer type. When introduced back into the body, the CAR-T cells more readily recognise specific cancer cells and more effectively target and destroy them.
It is here that the HLA’s step back into the story, they are also pivotal in cancer immunotherapy, as cancer-specific antigens are presented on HLA molecules. Therefore, one of the major limitations of CAR-T is that therapy needs to be HLA-matched for different individuals.
Currently only a minor number of patients with a minor number of cancers can be treated using CAR-T and to date only a few FDA approved CAR-T therapies exist. This makes a one-size-fits-all single T-cell-based treatment up until now impossible.
Now researchers from Cardiff University, UK, led by Andrew Sewell, have discovered a new type of TCR which recognises and targets most human cancer types, whilst ignoring healthy cells. The new TCR is hoped to provide a different route to target and destroy a wide range of cancers in all individuals.
Whilst the most studied ‘conventional’ T cells are those mentioned above, there are others that have been described as ‘unconventional’. One such group is MR1-restricted mucosal associated invariant T-cells (MAIT cells) which recognise lipids, small-molecule metabolites and specially modified peptides, and are considered HLA-independent.
The team, therefore, postulated that this mechanism of HLA-independent T-cell targeting would enable the immune destruction of a more broad-ranging set of cancer types.
T-cell populations were therefore proliferated in response to cancer cells (adenocarcinomic human alveolar basal epithelial cells) in conditions that favoured cells using an HLA-independent mechanism and from this the authors identified one T-cell clone, MC.7.G5. Further experiments showed that this clone killed multiple cancer cell lines such as lung, melanoma, leukaemia, colon, breast, prostate, bone and ovarian; cancer types that did not share a common HLA. Importantly, MC.7.G5 did not kill healthy cells and remained inert to them.
To unravel the mechanism of action of these ‘new, unconventional’ T-cells the authors turned to CRISPRs.
First a genome-wide CRISPR–Cas9 approach, using the GeCKO (genome-scale CRISPR-Cas9 knockout) v.2 library, was employed. The lentivirus libraries targeted every protein-coding gene in the human genome with six different single guide RNAs (sgRNAs), and was used to identify genes essential for recognition of target cells by MC.7.G5. Human cells (HEK293T) were infected with the viruses, creating a library of ‘mutated’ cells and, when screened with the new T cells, only the cells no longer expressing the proteins required for recognition by clonal cells from MC.7.G5 remained alive. Then by sequencing the CRISPR sgRNAs in the MC.7.G5-resistant HEK293T cells, the team revealed that MR1, an antigen-presenting molecule that presents microbial metabolites, was the target candidate molecule. Furthermore, deletion of MR1 using CRISPR-mediated knockout in several cancer cells lines demonstrated decreased MC.7.G5 destruction of cells. This, along with other data, demonstrated MR1 as the molecule that mediates cancer targeting by the T-cells.
An important characteristic of immunotherapies is safety; healthy cells and tissues should elude detection and destruction. After several lines of investigation the team deemed that healthy cells were incapable of activating MC.7.G5, even when stressed or damaged. Thus, the potential for MC.7.G5 as an investigative immunotherapy remained high.
The real challenge was to test the ‘new’ T-cells in vivo. In order to do this the team used leukaemic cells engrafted into immunodeficient mice, followed by adoptive transfer of MC.7.G5. Mice treated with MC.7.G5 had significantly fewer leukaemic cells present. This was most strikingly was at day 18, with controls exhibiting >78% cancerous cells in the bone marrow whilst those treated with MC.7.G5 had <7.2%. Furthermore, the life expectancy was far greater in those mice treated with MC.7.G5.
To explore the therapeutic potential of MR1 the team harvested T-cells from patients with a specific melanoma. The T-cells were then engineered to express MC.7.G5 TCR and then tested against both autologous and nonautologous melanomas. The engineered TCR-T cells were shown to be very efficient at killing both types of cancer and this cytotoxicity was ablated in MR1 knockout melanomas, demonstrating the specificity of MR1 in this response.
The authors conclude that the MC.7.G5 TCR can redirect the T-cells of patients to kill cancer cells without the requirement of a specific HLA, making this an attractive system for targeting a wide range of cancers, in a wide range of patients.
The work presented here in Nature Immunology, is a truly exciting step in the quest to find a ‘universal’ cure for cancer. As MR1 does not vary in the human population this make for a very attractive new target for immunotherapies. However, it is still early days, and whilst there have recently been advances made in our knowledge of how MR1 may function, there are still many holes to be plugged in our understanding before we can begin to think therapy. However, with the work presented here there isn’t any doubt that this represents hope for the wave of next generation therapies.
Once again we also see here the power of the CRISPR system. Its use here – the genome-wide CRISPR–Cas9 screening- provided a potent discovery platform for this unconventional T-cell ligand and associated receptor.
In terms of the short term future work by Sewell and his team, they hope to elucidate the precise molecular mechanism by which the new TCR distinguishes between healthy cells and cancer. The hypothesis is that the mechanism involves changes in cellular metabolism, which may translate to changes in metabolites expressed at the cell surface by MR1.
With such high expectations for a powerful pan-cancer weapon at stake, the group are aiming to bring the approach to trial in patients by the end of the year. We are hoping that they overcome any hurdles and challenges that awaits…
For more information please read the press release from Cardiff University
Crowther, M. D., G. Dolton, M. Legut, M. E. Caillaud, A. Lloyd, M. Attaf, S. A. E. Galloway, C. Rius, C. P. Farrell, B. Szomolay, A. Ager, A. L. Parker, A. Fuller, M. Donia, J. McCluskey, J. Rossjohn, I. M. Svane, J. D. Phillips and A. K. Sewell (2020). “Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.” Nature Immunology.