Cardiff Crock of Gold?

 

One of the oddities of science is that we are aware that we know little and understand less and yet manage to be surprised at frequent intervals when some bright spark discovers something new. So, surprised most of us indeed were by a paper from Andrew Sewell and colleagues at Cardiff University who have tracked down a hitherto unknown sub-population of white blood cells that may turn out to be extremely useful.

Before we get to the really exciting bit we need a follow-up word on CRISPR-Cas9, because that was what the Cardiff group used, and a clear picture of how the immune system works in cancer.

CRISPR-Cas in short

This method adapts a bacterial defence system for detecting and destroying invading viruses. It uses RNA guides to locate specific bits of DNA inside a cell, enabling molecular scissors to cut that section of DNA. This can disable a specific gene or allow a new gene to be inserted — described in Sharpening CRISPR and Re-writing the Manual of Life.

However, as well as being able to knock out genes or insert new ones, CRISPR has another feature. By using designer guide RNAs, CRISPR can scan the entire range of the genome. This DNA scanning feature can be scaled up to screen many genomic sites in parallel in one experiment. Synthesis of short fragments of nucleic acids (oligonucleotides) is carried out automatically using computer-controlled instruments (oligonucleotide synthesizers). The scale is astonishing: high-throughput DNA synthesis platforms can produce libraries of oligos (millions of them), each encoding a different guide RNA sequence and hence a different DNA target. Oligo libraries can be cloned into a lentiviral (a retrovirus) vector system for delivery to cells. This generates parallel, high-throughput, loss-of-function of specific genes from which their function can be inferred.

The immune system and cancer

The immune system can recognise cancer cells as abnormal and kill them. This happens because cancer cells (and cells infected by pathogens) break down proteins made within the cell and display those fragments on their surface. Thus cancer cells can ‘present’ their own antigens thereby stimulating an immune response that leads to their elimination by the immune system. Antigens on the cell surface bind to killer T cells (aka cytotoxic T cells) via the T-cell receptor (a complex of proteins on the T cell surface). This provokes the release of perforin that makes a pore, or hole, in the membrane of the infected cell. Cytotoxins then pass into the cell through this pore, destroying it. Almost all cell types can present antigens in some way and the loss of ‘antigen presentation’ is a major escape mechanism in cancer. It allows tumour cells to become ‘invisible’ and avoid immune attack by anti-tumour white blood cells.Scheme showing a cytotoxic T cell, (a type of lymphocyte aka a killer T cell, cytolytic T cell or CD8+ T cell), that kills cancer cells, interacting via its TCR with an antigenic peptide attached to an MHC molecule on the surface of a target cell. Granzymes are enzymes that cause apoptosis in targets cells.

What is the major histocompatibility complex?

Antigen-presenting cell (APCs) display antigen on their surface attached to major histocompatibility complexes (MHCs). MHCs are essential for the adaptive immune system to work, i.e. the sub-system of the immune response that eliminates pathogens. Human MHCs are also called the HLA (human leukocyte antigen) complex to distinguish it from other vertebrates. They’re encoded by a group of genes that are highly polymorphic — meaning that there are many different variant forms of the genes (alleles). The upshot of this is that no two individuals have exactly the same set of MHC molecules, with the exception of identical twins. This is the cause of transplant rejection wherein an immune response is switched on against HLA antigens expressed on APCs transferred along with the transplanted organ.

And now for the exciting news

The CRISPR screen used by Andrew Sewell and colleagues turned up a new type of T cell — one that differs from conventional T cells by presenting fragments of tumour proteins attached not to HLA proteins but to a different a receptor called MR1. The difference is critical because MR1 doesn’t vary between humans, unlike the highly variable HLAs. This appears to be why, in laboratory experiments, T cells with the MR1-seeking receptor can mediate killing of most types of human cancer cells without damaging healthy cells.

What they did was to take a sample of peripheral blood and select lymphocytes that proliferated in the presence of a cancer cell line (derived from a human lung cancer). They found that this cell clone kills a wide range of cancer cells in culture — so they used the CRISPR screening method to track down what the clone was targetting on cancer cells. Answer: MR1.

The novel T cell clone kills a broad range of tumour cells but does not kill cancer cell lines lacking MR1 or a range of healthy cells from various tissues. From Crowther et al., 2020.

The Cardiff group were further able to show that T-cells of melanoma patients modified to express this new TCR could destroy not only the patient’s own cancer cells, but also other patients’ cancer cells in the laboratory, regardless of HLA type (see Self Help – Part 2 and Gosh! Wonderful GOSH for how adoptive cell transfer works).

Transfer of the clone carrying the novel T cell receptor redirects patient T cells to recognize their own melanoma cells. Normal cells are unaffected. Black dots: + MR1; Grey dots: – MR1. From Crowther et al., 2020.

The data show (left) two T cell populations from two patients with metastatic melanoma. T cells transduced with the T cell receptor that binds MR1 recognized their own melanoma cells and killed them. Normal cells were unaffected regardless of MR1 expression.

These findings describe a TCR that exhibits pan-cancer cell recognition via the invariant MR1 molecule. Engineering T cells from patients that lacked detectable anti-cancer cell activity rendered them capable of killing the patients’ melanoma cells. However, these cells did not attack healthy cells so this method of genetic engineering, coupled with adoptive cell transfer, offers exciting opportunities for pan-cancer, T cell–mediated immunotherapy.

This discovery is most timely because, although CAR-T therapy is personalised to each patient, it targets only a few types of cancers and thus far has not worked for solid tumours.

CRISPR and related technologies are leading us into a new world in which Chinese scientists have already made the first CRISPR-edited human embryos and the first CRISPR-edited monkeys and, very recently in the first human trial of cells modified with CRISPR gene-editing technology, shown that the treatment is safe and lasting. This work, by You Lu at the West China Hospital in Chengdu, took immune cells from people with aggressive lung cancer and disabled the PD-1 gene. The PD-1 protein normally attenuates the immune system to prevent it attacking its own tissues but, as this reduces its anti-cancer capacity, knocking out PD-1 should overcome that restriction.

These advances are remarkable but we are still at the very beginning of gene therapy for cancer and the promise is almost limitless.

Reference

Crowther, M.D., Sewell, J.D. et al., (2020). Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nature Immunology  21,  178–185.

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