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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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A quick description of Lymphocyte Development and Activation

Lymphocytes Development and Activation

Lymphocytes (B cells and T cells – we’ll not talk about NK cells here) go through a generalizable sequence of maturation events. Each starts in the Bone Marrow (BM) as a Hematopoietic Stem Cell (HSC), where it starts its development. T cells leave the BM relatively sooner and go to the Thymus, while B cells remain in the BM for most of their development and then finalize development in the spleen.

 Positive Selection for BCR/TCR Development

Regardless of the type of cell (T or B), development occurs in two stages – First, each cell will attempt to make a unique lymphoid receptor (B cells have a BCR; T cells have a TCR). In order to do this, genetic material must be shuffled. While this shuffling does randomize the binding pocket of the lymphoid receptor, it may also destabilize these same receptors’ structure. To account for this, lymphocytes undergo a ‘positive selection’ period that ensures that a viable receptor is formed. This is actually done twice: once to ensure that a ‘Pre-Lymphoid Receptor’ is formed and again to for a ‘Mature Receptor.’  However, both are considered positive selection. Failure to pass this selection point leads to death of the cell. (In the figure below, Pre-Lymphocyte receptors are colored red, mature receptors are grey)

Negative Selection Against Self-Reactive BCR/ TCRs

Once cells have survived positive selection, they are considered Immature Lymphocytes. Although the terminology is poor here, these immature cells have mature lymphocyte receptors. At this point, these receptors have to be tested against all possible ‘Self ‘- antigens. In this case, binding means that these cells have the potential to react against the self – this is a no, no. The Immune System turned against the self is extraordinarily dangerous. Therefore, self-reactive cells are eliminated during this negative selection process. (In the figure below, mature lymphocytes have grey nucleus, all prior stages have red nuclei).

LymphosFollowing these developmental stages, the cells that have survived both positive and negative selection are 1) stable, mature lymphocyte receptors and 2) not reactive to ‘self’. These cells then enter the immune repertoire for that organism and are available to react against any foreign threats. Again, it is important to emphasize that each lymphocyte has a unique receptor and therefore will only get activated by a unique foreign antigen.

I may write more later to discuss some of the details that distinguish B and T cell development, but for now, this generalizable description will suffice.

Clonal Selection

Once a part of the immune repertoire, lymphocytes are on the lookout for foreign antigen that is capable of being bound by that cell’s receptor. Depending on the cell type, this interaction may be in one of several contexts (either in the context of MHC I, MHC II or as a naïve, soluble antigen), but regardless of the context, these ‘naïve’ lymphocytes will become activated by binding of their lymphocyte receptor. And once activated, lymphocytes will proliferate and differentiate. Differentiation typically goes in one of two general directions:

1)   generating activated effector cells (these secrete antibody if B cells, kill target cells, if CD8 T cells, or become helpers if CD4 T cells)

–or-

2)   generating memory cells, cells that act the same as the mature naïve cell that was activated, but are more numerous and can, themselves be activated upon stimulation.

An example of this activation is shown below in this HHMI video about how CD8 T cells can be stimulated to activate and then kill any target cells. This video also does a good job of illustrating how antigens get digested within a cell and expressed in the context of MHC I.

http://www.hhmi.org/biointeractive/media/antigen_ctl-lg.mov

 
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Posted by on April 25, 2013 in Uncategorized

 

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