Fantastic Stuff

 

It certainly is for Judy Perkins, a lady from Florida, who is the subject of a research paper published last week in the journal Nature Medicine by Nikolaos Zacharakis, Steven Rosenberg and their colleagues at the National Cancer Institute in Bethesda, Maryland. Having reached a point where she was enduring pain and facing death from metastatic breast cancer, the paper notes that she has undergone “complete durable regression … now ongoing for over 22 months.”  Wow! Hard to even begin to imagine how she must feel — or, for that matter, the team that engineered this outcome.

How was it done?

Well, it’s a very good example of what I do tend to go on about in these pages — namely that science is almost never about ‘ground-breaking breakthroughs’ or ‘Eureka’ moments. It creeps along in tiny steps, sideways, backwards and sometimes even forwards.

You may recall that in Self Help – Part 2, talking about ‘personalized medicine’, we described how in one version of cancer immunotherapy a sample of a patient’s white blood cells (T lymphocytes) is grown in the lab. This is a way of either getting more immune cells that can target the patient’s tumour or of being able to modify the cells by genetic engineering. One approach is to engineer cells to make receptors on their surface that target them to the tumour cell surface. Put these cells back into the patient and, with luck, you get better tumour cell killing.

An extra step (Gosh! Wonderful GOSH) enabled novel genes to be engineered into the white cells.

The Shape of Things to Come? took a further small step when DNA sequencing was used to identify mutations that gave rise to new proteins in tumour cells (called tumour-associated antigens or ‘neoantigens’ — molecular flags on the cell surface that can provoke an immune response – i.e., the host makes antibody proteins that react with (stick to) the antigens). Charlie Swanton and his colleagues from University College London and Cancer Research UK used this method for two samples of lung cancer, growing them in the lab to expand the population and testing how good these tumour-infiltrating cells were at recognizing the abnormal proteins (neo-antigens) on cancer cells.

Now Zacharakis & Friends followed this lead: they sequenced DNA from the tumour tissue to pinpoint the main mutations and screened the immune cells they’d grown in the lab to find which sub-populations were best at attacking the tumour cells. Expand those cells, infuse into the patient and keep your fingers crossed.

Adoptive cell transfer. This is the scheme from Self Help – Part 2 with the extra step (A) of sequencing the breast tumour. Four mutant proteins were found and tumour-infiltrating lymphocytes reactive against these mutant versions were identified, expanded in culture and infused into the patient.

 

What’s next?

The last step with the fingers was important because there’s almost always an element of luck in these things. For example, a patient may not make enough T lymphocytes to obtain an effective inoculum. But, regardless of the limitations, it’s what scientists call ‘proof-of-principle’. If it works once it’ll work again. It’s just a matter of slogging away at the fine details.

For Judy Perkins, of course, it’s about getting on with a life she’d prepared to leave — and perhaps, once in while, glancing in awe at a Nature Medicine paper that does not mention her by name but secures her own little niche in the history of cancer therapy.

References

McGranahan et al. (2016). Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 10.1126/science.aaf490 (2016).

Zacharakis, N. et al. (2018). Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nature Medicine 04 June 2018.

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The Shape of Things to Come?

One of the problems of trying to keep up with cancer – and indeed helping others to do so – is that you (i.e. ‘I’) get really irritated with the gentlemen and ladies of the press for going over the top in their efforts to cover science. I have therefore been forced to have a few rants about this in the past – actually, when I came to take stock, even I was a bit shocked at how many. Heading the field were Not Another Great Cancer Breakthough, Put A Cap On It and Gentlemen… For Goodness Sake. And not all of these were provoked by The Daily Telegraph!

If any of the responsible reporters read this blog they probably write me off as auditioning for the Grumpy Old Men tv series. But at least one authoritative voice says I’m really very sane and balanced (OK, it’s mine). Evidence? The other day I spotted the dreaded G word (groundbreaking) closely juxtaposed to poor old Achilles’ heel – and yes, it was in the Telegraph – but, when I got round to reading the paper, I had to admit that the work referred to was pretty stunning. Although, let’s be clear, such verbiage should still be banned.

A Tumour Tour de Force

The paper concerned was published in the leading journal Science by Nicholas McGranahan, Charles Swanton and colleagues from University College London and Cancer Research UK. It described a remarkable concentration of current molecular fire-power to dissect the fine detail of what’s going on in solid tumours. They focused on lung cancers and the key steps used to paint the picture were as follows:

1. DNA sequencing to identify mutations that produced new proteins in tumour cells (called tumour-associated antigens or ‘neoantigens’ – meaning molecular flags on the cell surface that can provoke an immune response – i.e. the host makes antibody proteins that react with (stick to) the antigens). Typically they found just over 300 of these ‘neoantigens’ per tumour – a reflection of the genetic mayhem that occurs in cancer.

2 tumoursVariation in neoantigen profile between two multi-region sequenced non-small cell lung tumours. There were approximately 400 (left) and 300 (right) neoantigens/tumour

  • Blue: proportion of clonal neoantigens found in every tumour region.
  • Yellow: subclonal neoantigens shared in multiple but not all tumour regions.
  • Red: subclonal (‘private’) neoantigens found in only one tumour region.
  • The left hand tumour (mostly blue, thus highly clonal) responded well to immunotherapy (from McGranahan et al. 2016).

2. Screening the set of genes that regulate the immune system – that is, make proteins that detect which cells belong to our body and which are ‘foreign.’ This is the human leukocyte antigen (HLA) system that is used to match donors for transplants – called HLA typing.

3. Isolating specialised immune cells (T lymphocytes) from samples of two patients with lung cancer, growing them in the lab to expand the population and testing how good these tumour-infiltrating cells were at recognizing the abnormal proteins (neo-antigens) on cancer cells.

4. Detecting proteins released by different types of infiltrating T cells that regulate the immune response. These include so-called immune checkpoint molecules that limit the extent of the immune response. This showed that T cell subsets that were very good at recognizing neo-antigens – and thus killing cancer cells (they’re CD8+ T cells or ‘killer’ T cells) also made high levels of proteins that restrain the immune response (e.g., PD-1).

5. Showing that immunotherapy (using the antibody pembrolizumab that reacts with PD-1) could significantly extend survival of patients with advanced non-small cell lung cancer. We’ve already met this approach in Self-help Part 1.

The critical finding was that the complexity of the tumour (called the clonal architecture) determines the outcome. Durable benefit from this immunotherapy requires a high level of mutation but a restricted range of neo-antigens. Put another way, tumours that are highly clonal respond best because they have common molecular flags present on every tumour cell.

6. Using the same methods on some skin cancers (melanomas) with similar results.

What did this astonishing assembly of results tell us?

It’s the most detailed picture yet of what’s going on in individual cancers. As one of the authors, Charles Swanton, remarked “This is exciting. This opens up a way to look at individual patients’ tumours and profile all the antigen variations to figure out the best ways for treatments to work. This takes personalised medicine to its absolute limit where each patient would have a unique, bespoke treatment.”

He might have added that it’s going to take a bit of time and a lot of money. But as a demonstration of 21st century medical science it’s an absolute cracker!

References

McGranahan et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 10.1126/science.aaf490 (2016).

 

Gosh! Wonderful GOSH

Anyone who reads these pages will long ago, I trust, have been persuaded that the molecular biology of cells is fascinating, beautiful and utterly absorbing – and all that is still true even when something goes wrong and cancers make their unwelcome appearance. Which makes cancer a brilliant topic to talk and write about – you know your audience will be captivated (well, unless you’re utterly hopeless). There’s only one snag, namely that – perhaps because of the unwelcome nature of cancers – it’s tough to make jokes. One of the best reviews I had for Betrayed by Nature was terrifically nice about it but at the end, presumably feeling that he had to balance things up, the reviewer commented that it: “..is perhaps a little too light-hearted at times…” Thank you so much anonymous critic! Crikey! If I’d been trying to do slap-stick I’d have bunged in a few of those lewd chemicals – a touch of erectone, a bit of PORN, etc. (btw, the former is used in traditional Chinese medicine to treat arthritis and the latter is poly-ornithinine, so calm down).

I guess my serious referee may have spotted that I included a poem – well, two actually, one written by the great JBS Haldane in 1964 when he discovered he had bowel cancer which begins:

I wish I had the voice of Homer

To sing of rectal carcinoma,

Which kills a lot more chaps, in fact,
Than were bumped off when Troy was sacked.

Those couplets may reflect much of JBS with whom I can’t compete but, nevertheless, in Betrayed by Nature I took a deep breath and had a go at an update that began:

Long gone are the days of Homer
But not so those of carcinoma,
Of sarcoma and leukemia

And other cancers familia.
But nowadays we meet pre-school
That great and wondrous Molecule.
We know now from the knee of Mater
That DNA’s the great creator.

and went on:

But DNA makes cancer too

Time enough—it’ll happen to you.
“No worries sport” as some would say,
These days it’s “omics” all the way.

So heed the words of JBS

Who years ago, though in distress,
Gave this advice on what to do

When something odd happens to you:
“Take blood and bumps to your physician
Whose only aim is your remission.”

I’d rather forgotten my poem until in just the last week there hit the press a story illustrating that although cancer mayn’t be particularly fertile ground for funnies it does gloriously uplifting like nothing else. It was an account of how science and medicine had come together at Great Ormond Street Hospital to save a life and it was even more thrilling because the life was that of a little girl just two years old. The saga brought my poem to mind and it seemed, though I say it myself, rather spot on.

The little girl, Layla, was three months old when she was diagnosed with acute lymphoblastic leukemia (ALL) caused by a piece of her DNA misbehaving by upping sticks and moving to a new home on another chromosome – one way in which genetic damage can lead to cancer. By her first birthday chemotherapy and a bone marrow transplant had failed and the only remaining option appeared to be palliative care. At this point the GOSH team obtained special dispensation to try a novel immunotherapy using what are being called “designer immune cells“. Over a few months Layla recovered and is now free of cancer. However, there are no reports of Waseem Qasim and his colleagues at GOSH and at University College London dancing and singing the Trafalgar Square fountains – they’re such a reserved lot these scientists and doctors.

How did they do it?

In principle they used the gene therapy approach that, helpfully, we described recently (Self Help Part 2). T cells isolated from a blood sample have novel genes inserted into their DNA and are grown in the lab before infusing into the patient. The idea is to improve the efficiency with which the T cells target a particular protein (CD19) present on the surface of the leukemia cells by giving them artificial T cell receptors (also known as chimeric T cell receptors or chimeric antigen receptors (CARs) – because they’re made by fusing several bits together to make something that sticks to the target ‘antigen’ – CD19). The engineered receptors thereby boost the immune response against the leukemia. The new genetic material is inserted into a virus that carries it into the cells. So established is this method that you can buy such modified cells from the French biotech company Cellectis.

105 picAdoptive cell transfer immunotherapy. T cells are isolated from a blood sample and novel genes inserted into their DNA. The GOSH treatment also uses gene editing by TALENs to delete two genes. The engineered T cells are expanded, selected and then infused into the patient.

Is that all?

Not quite. To give themselves a better chance the team added a couple of extra tricks. First they included in the virus a second gene, RQR8, that encodes two proteins – this helps with identifying and selecting the modified cells. The second ploy is, perhaps, the most exciting of all: they used gene editing – a rapidly developing field that permits DNA in cells to be modified directly: it really amounts to molecular cutting and pasting. Also called ‘genome editing’ or ‘genome editing with engineered nucleases’ (GEEN), this form of genetic engineering removes or inserts sections of DNA, thereby modifying the genome.

The ‘cutting’ is done by proteins (enzymes called nucleases) that snip both strands of DNA – creating double-strand breaks. So nucleases are ‘molecular scissors.’ Once a double-strand break has been made the built-in systems of cells swing into action to repair the damage (i.e. stick the DNA back together as best it can without worrying about any snipped bits – these natural processes are homologous recombination and non-homologous end-joining, though we don’t need to bother about them here).

To be of any use the nucleases need to be targeted – made to home in on a specific site (DNA sequence) – and for this the GOSH group used ‘transcription activator-like effectors’ (TALEs). The origins of these proteins could hardly be further away from cancer – they come from a family of bacteria that attacks hundreds of different types of plants from cotton to fruit and nut trees, giving rise to things like citrus canker and black rot. About six years ago Jens Boch of the Martin-Luther-University in Halle and Adam Bogdanove at Iowa State University with their colleagues showed that these bugs did their dirty deeds by binding to regulatory regions of DNA thereby changing the expression of genes, hence affecting cell behavior. It turned out that their specificity came from a remarkably simple code formed by the amino acids of TALE proteins. From that it’s a relatively simple step to make artificial TALE proteins to target precise stretches of DNA and to couple them to a nuclease to do the cutting. The whole thing makes a TALEN (transcription activator-like effector nuclease). TALE proteins work in pairs (i.e. they bind as dimers on a target DNA site) so an artificial TALEN is like using both your hands to grip a piece of wood either side of the point where, using your third hand, you make the cut. The DNA that encodes the whole thing is inserted into plasmids that are transfected into the target cells; the expressed gene products then enter the nucleus to work on the host cell’s genome. There are currently three other approaches to nuclease engineering (zinc finger nucleases, the CRISPR/Cas system and meganucleases) but we can leave them for another time.

The TALENs made by the GOSH group knocked out the T cell receptor (to eliminate the risk of an immune reaction against the engineered T cells (called graft-versus-host disease) and CD52 (encodes a protein on the surface of mature lymphocytes that is the target of the monoclonal antibody alemtuzumab – so this drug can be used to prevent rejection by the host without affecting the engineered T cells).

What next?

This wonderful result is not a permanent cure for Layla but it appears to be working to stave off the disease whilst she awaits a matched T cell donor. It’s worth noting that a rather similar approach has been used with some success in treating HIV patients but it should be born in mind that, brilliant though these advances are, they are not without risks – for example, it’s possible that the vector (virus) that delivers DNA might have long-term effects – only time will tell.

Almost the most important thing in this story is what the GOSH group didn’t do. They used the TALENs gene editing method to knock out genes but it’s also a way of inserting new DNA. All you need to do is add double-stranded DNA fragments in the correct form at the same time and the cell’s repair system will incorporate them into the genome. That offers the possibility of being able to repair DNA damage that has caused loss of gene function – a major factor in almost all cancers. Although there is still no way of tackling the associated problem of how to target gene editing to tumour cells, it may be that Layla’s triumph is a really significant step for cancer therapy.

Reference

Smith, J. et al. (2015). UCART19, an allogeneic “off-the-shelf” adoptive T-cell immunotherapy against CD19+ B-cell leukemias. Journal of Clinical Oncology 33, 2015 (suppl; abstr 3069).