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Cancer Immunotherapy Continued: Non-transgenic T Cell Therapy

A number of adoptive T cell therapies are being examined for cancer treatment including isolation and culturing tumor infiltrating lymphocytes (TILs), isolating and expanding a specific T cell or clone, and generating novel T cells with chimeric receptors designed to target tumor cells and provide robust activation signals to the T cell. 1,2

Recently, I wrote a short essay about CAR T Cell therapy and how this therapy uses genetically modified T Cells to generate a large number of your own cells, capable of targeting tumors bearing a known antigen (e.g. CD19 as a Lymphoma marker).

T Cells are one of the immune system’s specific attackers, capable of recognizing cells bearing specific antigen only. They are engaged and activated via interactions with APCs presenting antigen bound to MHC molecules as well as other ‘secondary’ signals.  For a more complete description, see

In the case when the T Cell recognizes the antigen, it proliferates and activates if provided sufficient secondary signals in addition to TCR stimulation. In the absence of recognition, the T Cells will not stably bind the APC and therefor not receive sufficient signaling to activate.

T Cells ‘see’ antigen through presentation in the context of MHC molecules on the surface of Antigen Presenting Cells (APCs)

T Cells ‘see’ antigen through presentation in the context of MHC molecules on the surface of Antigen Presenting Cells (APCs)

Some benefits to that therapy include incorporation of a well-designed chimeric antigen receptor capable of providing normal T Cell Receptor (TCR) signals as well as signals from co-receptors required to generate mature effector cells. Because this construct targets the CD19 molecule directly, it does not require processing and presentation of antigen via MHC I by the tumor cells (important because one strategy tumor cells use to evade immune detection is to down-regulate MHC I). Using the patient’s own cells also means that immunosuppressive drugs aren’t required to prevent the body from rejecting the therapy.

One drawback though, is that the construct is made synthetically and can only include antibody binding regions specific to known cell surface antigens. So, if you know the cells you want to get rid of, and you can make an antibody to bind those cells preferentially, CAR T Cells are a good therapy for you.

Using Non-Transgenic T cells, similar effects can be obtained with an inverse set of pros and cons. Because this therapy does not utilize chimeric receptors, cells specific for a known  antigen aren’t singularly generated. Rather, a diverse array of cells is generated against tumor targets without requiring the isolation and characterization of one particular antigen. As opposed to the CAR T Cells these cells can only interact with target cells that present antigen via their MHC I molecules, which can be a drawback in situations where the tumor cells have downregulated antigen presentation molecules.

The Non-transgenic cells used may be generated in several ways. One method includes the harvest of tumor tissue from the patient, followed by killing these cells and re-injecting them (possibly in the presence of an adjuvant) to illicit a targeted immune response. 7-10 days later, peripheral T Cells enhanced for target specificity by the vaccine can be harvested and amplified outside of the body. In this way, cells can be amplified to numbers far outpacing what might be found in the patient, while also providing additional activation signals to promote effector cell development.

A second way of utilizing non-Transgenic T Cells in therapy is to isolate only those T Cells found to be actively invading the tumor. This biases toward cells already selected for by the immune system that may simply not be able to keep pace with the tumor’s growth. Ex vivo amplification can provide these cells the boost in numbers required to tip the balance in favor of the patient.

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Coupling any of these therapies with other treatments, such as the human monocloncal antibody anti-CTLA-4 (ipilimumab) 4, can further support T Cell efficacy – in this case by blocking checkpoints used to dampen the immune response following a period of activation. In healthy patients, these checkpoints allow the immune system to revert to a state of homeostasis once pathogens have been cleared. In cancer patients, the tumor may not yet be eradicated before checkpoint molecules begin to dampen the response. By interrupting these, the window during which T Cells are most effective is widened — at least in some patients.

This article has been cross-posted on Medium

A Few References:
1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315690/
2. https://depts.washington.edu/tumorvac/research/t-cell-therapy
3. My Medium Post
4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951510/

 
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Posted by on November 15, 2015 in Uncategorized

 

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The Skinny on Cancer Immunotherapy: focus on CAR T Cells

Screen Shot 2015-10-22 at 9.47.44 AMOne of the more interesting modern therapies being used to fight cancer aims to coax, or engineer a patient’s own T Cells to fight disease.
In very basic terms, the principle is not dissimilar to vaccine strategies used against infectious disease. That is, they direct and boost the patient’s immune system against target cells. One reason vaccinations have been so successful in fighting disease is that they leave much of the hard work to nature – the same nature that has been keeping you and your ancestors healthy enough to successfully reproduce for millions of years. Give the immune system a push in the right direction with a well designed, safe vaccine and the body does the rest leading to (at least theoretically) life-long protection. At this point, the most limiting factor to how long protection lasts is because we live so much longer than humans have ever lived before.

William-Coley_206x236Immunotherapy against cancer has been an area of interest since the 1890s, when William Coley observed that cancer patients who had infections at the site of surgical resection fared better than those without infections. Rather than dismissing this observation as uninformative, he speculated that the immune system plays an active role in preventing or regressing tumors.

In fact, the immune system is constantly performing ‘immune surveillance’ to prevent newly-generated cancer cells from developing into tumors. Direct evidence for this involves ‘knocking out’ elements of the immune system and watching for cancer. As predicted by the theory, immunodeficient animals develop spontaneous tumors at a higher rate, and earlier than do immune-competent animals.

The pudding: (from : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1857231/)

Evidence for Immuno Surveillance

Evidence for Immuno Surveillance

But vaccinations used against infectious diseases are given before the patient is infected (known as prophylactic vaccination).

How can we immunize people against all the cancers that may crop up in all their various forms?

The answer is – we don’t. In the case of cancer, we perform vaccinations ‘therapeutically’, or after disease has started. Otherwise there really would simply be too many possible targets.
So, we wait, and help the body to fight the challenges that actually do arise.
A number of methods have been developed and tested to accomplish this, here, I want to specifically address a personalized therapy that takes cells from the patient, ‘aggravates’ and expands them, and then re-infuses them into the same patient.
Currently, there are several ways this is being done with various outcomes.

One method involves immunizing the patient against killed cancer cells isolated from the themselves (via surgery), then harvesting the reacting cells and expanding them to numbers much higher than those reached in vivo, and then re-administering to the patient as a jump-start to immunity. The advantages are that these immune cells are ‘self’ and therefore do not have to be ‘matched’ to the recipient a la transplantation surgery. It is also possible to remove any regulatory cells (T regs), that often impair immune responses, prior to re-administration.
A more engineered response has been investigated by investigators such as Carl June, of the Abramson Cancer Center at the University of Pennsylvania. These cells, known as CAR T Cells express ‘Chimeric Antigen Receptors’ directly target tumor cells using transgenic antibodies that incorporate the intracellular signaling domains of up to three immune-activating receptors. See the illustration below for details of this receptor’s design (taken from ‘Breakthroughs in Cancer Immunotherapy webinar by Dr. June )
Screen Shot 2015-10-21 at 7.20.04 PM
In the case of CAR T Cells, most have been made to fight B Cell Chronic Lymphocytic Leukemia (B Cell CLL). These cells are a good test case for the technique for a number of reasons, including the fact that they uniformly* express a marker called CD19 on their surface and also because they are a ‘liquid tumor’ – meaning that the cancer cells are individual cells moving through the body (at least many are). Treatment of solid tumors can bring added complications such as the need to infiltrate the tumor in order to find target cells.
As I said, CD19 is a common protein expressed on these cells. Therefore, at least the CAR receptor part is standardized between patients – this is the piece that is added to cells transgenically so that they will bear a receptor known to engage the target cells with high affinity. Because it must be added to the patient’s own cells, this is accomplished using a viral vector that infects the T Cells in culture and provides the DNA required to make the receptor. (In case you’re worried about the virus, these are engineered to only infect the first cell they encounter, they cannot reproduce themselves and continue an infection)
So, let’s walk through it:

Screen Shot 2015-10-21 at 7.20.04 PM
1. Blood cells are isolated from a patient
2. T Cells are purified (i.e. isolated)
3. T Cells are infected with virus in culture.
4. T Cells grow up with the chimeric antigen receptor expressed on their surface
5. These cells are then re-injected into the patient via I.V. drip over about 30 minutes time.
6. Let the cells do the work

Screen Shot 2015-10-22 at 10.33.53 AM
This therapy has an impressive track record so far with studies with success rates from ~60%- 90% of patients responding and remaining disease free for years (Maude et al).
Following the initial infusion of cells, CAR T Cells proliferate in vivo to very high numbers and can even form immunological memory cells to come to the rescue in the event of a relapse.
So, what next?
A number of startup companies have emerged to tackle the logistics of bringing this type of therapy – an extreme example of personalized medical care – into being. Unlike traditional drug therapies where a single compound is mass produced and distributed world-wide, each patient must have their own cells processed and returned to them for infusion. This therapy is much more of a service, and as such, will require physical locations across the country that can manage the handling of cells.
The up side, however, is potentially transforming fatal diseases into manageable ones with a high quality of life after therapy.
Just ask Emma:
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*Well, most do, anyway.

 
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Posted by on October 22, 2015 in Uncategorized

 

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Cell Division, Contact Inhibition and Cancer

imagesIn my general biology class we are now reading chapter 5 of Mader et al, on Cell Division. This chapter is a bridge between those chapters on descriptive cell biology and those describing the activities of the cell and how we explain the inheritance of traits from one generation to another.

We focused our attention on Mitosis and Meiosis of diploid Eukaryotic cells and followed how these two types of nuclear division manage the sorting of genetic material into daughter cells ensuring that each cell gets an appropriate set of instructions for life.

The body is comprised of somatic cells that include everything from skin to muscle and nerve cells. These are called somatic because soma comes from the greek word for body.

The other type of cell is the gametic cell, referring to germ-line, or sex, cells.

Each of these type of cells goes through its own type of division in order to end up with the correct amount of genetic material ( or ‘ploidy’) in the resulting cell or organism. Ploidy refers to the number of complete sets of genetic material a cell has. We, humans, are diploid organisms having two sets of genetic material in each cell.

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Mitosis in Diploid Cells

Somatic cells undergo mitosis in a way that maintains the diploid state of cells creating two exact replicas of the parent cell.

This mechanism makes sense, because one skin cell might replace a neighboring skin cell following its demise in order to maintain a confluent layer. We would expect every skin cell to be genetically identical to every other and mitosis delivers just that.

However, if we imagine sex cells that were made from mitosis, these would also be diploid (2n). Then a diploid sperm would fuse with a diploid egg and make a tetraploid offspring. Then that organism would have octaploid offspring and so on. This, of course, does not happen.

ImageSex cells are instead produced by a different kind of division called meiosis. Meiosis is merely a specialized form of mitosis in which the genetic material (ploidy) is ‘halved’. The resulting cells are then haploid (1n or n). As part of the specialization, meiosis occurs in two steps so instead of producing two cells, it produces four (at least theoretically). Also, instead of being identical, each of the resulting sex cells is unique.

But this discussion is supposed to be about cancer, so let’s ignore meiosis for now. I’ve discussed cancer before here, but I just found a couple of good animations that I wanted to include.

The first is an excellent animation on cell cycle and contact inhibition. See how cancer is defined here as the lack of respect for cell-cell signaling, that would otherwise result in a healthy monolayer of cells.

The second, discusses how cancer cells would need to alter their environment in order to get the nutrients they need to survive. It can be tempting to think that cancer cells don’t need these things, but they certainly do.

http://bcove.me/uc5vydod

Once cells mutate in a way that initiates cancer, the constant struggle with the immune system amounts to a selective pressure allowing only the strongest cells to survive. 😦 A brief article describing this battle can be found on the HHMI website. A more thorough treatment of the subject can be found in a freely available review by my friend Dr. Ezekiel Fuentes-Panana.

Here’s one last animation showing a tumor mass producing metastatic cells that leave the mass and migrate to new locations within the body. I’m not that fond of this video, but it does communicate the message I wanted to get across.

Alltogether, cancer cells are those that no longer obey the rules of polite cellular society and continue to reproduce through unchecked mitosis when such division is not in the best interest of the organism as a whole. One way these cells do this is to cease responding to contact inhibition signals. This results in the production of a tumor mass that will need to obtain energy and will often do so by sending out pro-angiogenic signals resulting in new blood vessel formation. As the tumor continues to grow, it may invade neighboring tissue and ultimately even metastasize into the blood or lymph leading to a number of secondary tumors throughout the body.

 
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Posted by on October 14, 2013 in Uncategorized

 

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Cancer and the immune system (briefly)

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Macrophage engulfing bacteria

 What a breath of fresh air! A good old friend of mine, who I met while in graduate school and is now living in Mexico city has been working on a couple of papers that he is submitting to some English language journals. I’ve only read one of them so far – it’s an interesting review of work that suggests that tumors actively co-opt processes of the immune system to their own advantage. His spoken English is quite good, but it’s another thing altogether to write well for a scientific publication. Lucky for me, I guess, because it gives me a way to be involved.

It is well established that the immune system functions to prevent tumor formation known as immunosurveillance. This is pretty consistent with the basic role of defending the self against any non-self target it encounters. If you’re unfamiliar with immunology and want one thing to learn, that’s it: The immune system is there to recognize a black and white world of self vs non-self. The details are complicated, but it’s fairly well worked out that through a series of positive and negative selection events you can train your immune cells to be tolerant of you (self), but reactive against anything new (non-self).

With respect to cancer, it’s important to recognize that these cells start out as self and are ignored by the immune system, but they change in a way that they are not acting the way they should. The problem for the immune system is that these changes typically just mean that the cells are acting abnormally, but they don’t necessarily look foreign. Despite this, we know that animals that lack a functional immune system will succumb to tumors at higher frequency earlier in life than those with competent immunity.

My friend’s article extends this relationship beyond immunosurvellience and suggests that the tumor cells undergo a selection process by the immune system that will eliminate weaker cells, leaving only cells that either escape the notice of the immune system entirely or are extraordinarily resistant to attacks. Further, he describes that the remaining cells will often co-opt signals of the immune system to advance their own function and survival. 

I look forward to finishing up this paper and hope to be able to point you toward a journal that it is published in sometime in the near future. Until then, it’s so refreshing to think about immunology again. I miss it.

 

 
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Posted by on November 3, 2012 in Uncategorized

 

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Regulating Cell Cycle

ImageCell cycle progression is regulated by the interplay of two major types of proteins. One protein promotes cell cycle progression (i.e. its effect is to push cells through the checkpoints); the other type of protein inhibits cell cycle progression. Together, these proteins establish a balance that permits cell cycle progression under typical conditions  – where there is no DNA damage. However, if there is DNA damage, proteins that sense this damage will turn on proteins that inhibit cell cycle and also those that are responsible for repairing the damage.

If all goes well and the damage is repaired, then the delaying proteins turn off and cell cycle is allowed to proceed. If the damage is NOT repaired, then the cell is instructed to commit suicide by apoptosis (cell death without inflammation).

The proteins that push cells through cycle are called proto-Oncogenes. The ones that inhibit cell cycle are called tumor suppressors.

That’s how things work when cell cycle is being regulated properly. But sometimes, the DNA damage may affect the genes for these two types of proteins specifically. When this happens, cell cycle regulation may be compromised. When that happens, cancer can develop.

Why? Because cancer is a disease of cell cycle regulation. The basic problem in cancer is that cells have ceased to have their cell cycle regulated properly and are dividing out of control. Remember, multicellular organisms like us thrive because we have delegated different behaviors to different cells / tissues / organs and all the cells are co-operating in a way that promotes the health of the individual organism ahead of the health of each individual cell.

So, what kind of mutation causes this to happen? Well, it depends upon which protein has been mutated. If pro to-oncogenes have a ‘gain of function’ mutation, this will make them work extra hard to push cells through cell cycle checkpoints – even when there are signals to prevent this. (Think of a gas pedal that gets stuck to the floor. It doesn’t matter if you hit the brakes, you’re still moving forward) If this same protein undergoes a ‘loss of function’ mutation, then the cell can never enter cell cycle and will probably die off.

Contra-wise, if tumor suppressor proteins have a ‘gain of function’ mutation, then cell cycle cannot proceed and the cell will probably die. (In a car analogy, tumor suppressor proteins are the brake pedal. If the break is always on, then you can never go forward. If the tumor suppressor protein has a ‘loss of function’ mutation, then cell cycle can never stop (our brakes are broken).

So, each type of protein has to have a specific type of mutation to lead to unregulated cell cycle.

Keep in mind, that the purpose of cell cycle regulation is to protect the integrity of the genome (all the DNA of the cell). If this regulation breaks, then more and more mutations can occur over time.

 
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Posted by on October 15, 2012 in Uncategorized

 

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