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CTL Therapy

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Killer T Cells and the Risks of CTL Therapy

Date: August 13, 2003

by Chaya Venkat

How T Cells Kill – Serial Killers, Up Close and Personal

Related Article: Tregs

If you are a fan of active immunotherapy, as in gene therapy, T-cell therapy, stem cell transplants, etc., this article is a must-read for you.

Whether they are your own home-grown natural variety, or the high-tech armies grown for you outside your body by companies like Xcyte Therapies, activated T-cells primed for killing their targets are an important line of defense against cancer cells and cells infected by viruses. Activated T-cells are called Cytotoxic T Lymphocytes (CTLs) and they are quite different from naive T-cells prior to their call-up for active duty. CTLs kill target cells by forcing them to commit suicide. It might help us to learn a little bit about how these super efficient killers do their job, and equally important, why sometimes they fail to complete their mission.

T-Cells Attacking Tumor Cell

CTLs kill by two distinctly different mechanisms. One mechanism, called the Fas pathway, links the T-cell to the “death receptor” on the target cell. Under normal circumstances, cross-linking the death receptor makes the target cell an offer it cannot refuse. A chain of events is set in motion and the cascade ends with the destruction of the target. The actual killing of the cell is done by a system of proteases called caspases, within the target cell itself. Think of them as automated shears. Once activated by appropriate commands, they swing into action and literally cut up the DNA of the target cell. With enough damage done, the cell gives up the fight and dies quietly. Its innards are broken up into nice little packages for disposal, nothing leaking out and making a mess. This is apoptosis, orderly cell death programmed into each and every cell of your body. This type of death is a normal and natural function, and the mechanics, such as the caspase “shears” are built into each cell to ensure the end is a clean, non-messy operation.

The other kind of cell death, called necrosis, happens when the cell does not participate in its own death. Necrosis happens, for example, when there is tissue damage, when a whole bunch of cells in the neighborhood are bludgeoned to death, or burned to death. This is a messy affair, murder and mayhem as opposed to obedient suicide, with cell fragments all over the place leading to a huge clean up job. Lots and lots of cytokines are released, messengers calling up the cleaning crews. Inflammation, pain and potential sepsis — these are some of the results of necrotic cell death.

As I said, committing suicide upon receiving orders to do so is a normal function of all good and obedient cells. But, as we know by now, cancer cells are anything but "normal", and the Fas pathway used to give the execution order is often circumvented in many types of cancers. Over the long period of time that the cancer incubates in your body, cancer cells have a chance to undergo their own selection process, a type of Darwinian evolution at a cellular level. The malignant cells that are wide open to receive the execution orders through their death receptor have no choice in the matter, they die out quickly. Often, this process of selection weeds out the less efficient of the cancer cells; but it may leave behind a few cells, a smarter and more resistant group of cancer cells that have figured out a way of blocking the death receptor, how to ignore the command to die. 

One of the ways this is done by cancer cells is by developing a protein called c-FLIP. Pretty soon, all the cancer cells that did not have the c-FLIP protein have killed themselves on command, and the only subset of cancer cells left in the body are all cells that have learned to survive, by means of using this new technology. They are a tougher bunch than before, and harder to kill, since they have a survival advantage that the earlier and less robust cancer cells did not have. This is a type of "immune sculpting" of the cancer cells, in which the body's own immune system's attack on the cancer cells is the mechanism forcing the cancer cells to change into new and more resistant forms. This is one of the pivotal mechanisms that makes oncology such a complex and challenging field. The enemy is constantly changing, constantly learning new tricks, adapting to every new strategy we bring to the battlefield. Sort of like the Borg, for you Star Trek fans out there. 

c-FLIP is over-expressed in several types of melanoma , colon and cervical carcinomas, and other cancers, indicating they have found a way of blocking death signals through the Fas pathway. Many viruses have also evolved clever strategies with which they protect the infected cells that they call home. If the Fas pathway and use of the death receptor was the only way CTLs can kill cancer cells and virus infected cells, we would be in trouble, since it appears cancer cells and viruses are pretty good at figuring out this particular mode of attack, and protecting themselves against it. The second method used by CTLs to kill their targets is a lot more up-close and personal and a lot harder to resist. And this second mechanism is the main point of my article. 

Once a naïve T-cell has been activated and targeted to kill, in other words when it has become a CTL, it starts the process of accumulating granules of two types of toxic materials within itself. Naïve T-cells do not carry these granules, the ammunition is issued only to CTLs, officers that have been called up for active duty, and given orders to kill specific targets. The CTL itself is protected from the action of these two toxic materials, since they are stored in an inactive state while they are within the T-cell. Once the target has been located, the CTL gets real close to the target, the killer and the target are almost in physical contact at the moment of assassination. Complex machinery within the T-cell makes sure the toxic granules are brought around and released just so, just between the T-cell and its victim. One of the toxic materials is called Perforin. It is released first: its job is to bore into the hide of the target cell (the cell membrane) and polymerize to form a tube or tunnel leading into the vulnerable innards of the target cell. Once the walls are breached, the CTL releases the second toxin, called Granzyme-B, right at the entrance to the tunnel built by its partner perforin. The open tunnel lets granzyme into the target cell, where it starts the process of initiating a series of reactions called "caspase cascade". Remember, we talked about caspase "shears" before. Pretty soon, the DNA of the target cell is torn apart, fragmented, and the cell has no choice but die. The CTL packs up, and moves on to the next target, ready to repeat the process of stab with perforin and poison with granzyme B, all over again. Neat trick!! Perforin makes a neat tunnel or perforation, I guess that is why it is called Perforin. Then granzyme-B goes inside for the cell kill. If enough holes can be punched into the target by perforin, that by itself may be enough to kill, sort of like stabbing the target to death. But working together with its partner granzyme-B, the job is a lot easier, sort of like a nice stiletto making a hole in the cell membrane, with granzyme-B poisoning the tip to drive the message home: time to die! Talk about chemical warfare! Granzyme B is one of the most toxic materials to living cells. All of us practice chemical warfare at a cellular level, every moment of our lives, our CTLs do that for us at the bidding of our bodies. 

Obviously, CTLs have to be careful not to nick themselves or their comrades, the other CTLs, with their own poison-tipped knives. Perforin and granzyme are not inactive, as they were when they were tucked away in granules within the CTL. Once released, the two poisons are in their active form. Unless the CTL has special protection, every time it releases its granules of perforin and granzyme, it is just as likely that the perforin will build a tunnel through its own hide, and the granzyme will kill the CTL!! Our fearless fighters of cancer cells and pathogens need protection from their own weapons. So, over the millennia that our immune systems have evolved, our bodies have developed an inhibitor that prevents granzyme-B from doing its job on cytotoxic cells. Think of this as an antidote for the poison, or a gas mask, if you will. This inhibitor, called PI9 (proteinase inhibitor 9), is expressed by all CTLs, NK-cells and neutrophils, standard issue for all troops messing with perforin and granzyme. 

If that was the end of the story, it might have a happy ending. Once activated and targeted to kill the cancer cells, the CTLs would hunt down and kill each and every cancer cell in your body. Even if the cancer cells became clever at blocking their death receptors and therefore could not be politely invited to kill themselves via the Fas pathway, they can still be killed by the more up-front and lethal attack of perforin and granzyme. If the body is running a little low on its cadre of assassins, we can always try to grow huge armies of trained and motivated killers outside the body and infuse the CTLs to finish the job. This is the great attractiveness of adoptive CTL therapy.

What Can Go Wrong with T-cell Therapy?

We know now that armed and targeted T-cells, "CTLs", are efficient killers of target cells. Two different mechanisms were discussed, the first using the Fas pathway to deliver the death signal to the target, forcing it to commit suicide. We also discussed that many cancer cells can get around the Fas mediated death signal. The second mechanism of cell kill used combination of two toxic substances, perforin and granzyme-B. Perforin builds a tunnel into the target, and granzyme-B goes in through this tunnel to finish the job. It was pointed out that killer cells such as CTLs, NK-cells and neutrophils have built in protection against granzyme-B, so that the killers do not kill themselves with their own weapons.

The granzyme and perforin combination is one of the hardest to subvert. With the exception of the privileged killer cells with the antidote PI9, it was thought that few target cells are able to keep the granzyme B out. Once it is in the target, granzyme-B activates the caspase "shears", as do most other cell-kill mechanisms. On getting the right signals, the caspase "shears" cut up and fragment the DNA of the target cell and it dies as a result. Many different approaches to cell kill, such as chemotherapy, Fas pathway, granzyme/perforin attack, all of these depend to a great deal on the caspase "shears" to deliver the final death blow. The caspase cascade is at the crux of most avenues for orderly cell death, "apoptosis". 

Recently, a new defense mechanism used by some cancer cells was reported. It has been shown that PI9, the antidote for Granzyme-B, was not restricted to CTLs and NK cells and neutrophils. PI9 expression was observed specifically in B-cell NHLs of intermediate to high grade. Now, normal B-cells do not use perforin or granzyme weapons, there is no reason for these guys to be issued PI9 antidote! In one study, 43% of diffuse large B-cell lymphoma and 25% of Burkitt lymphoma tumor cells were found to express PI9. Looks like resistance to granzyme-B is no longer a state secret, an antidote available only to our select troops licensed to use perforin and granzyme weapons, to immunize them against their own weapons. Looks like some of the bad guys also have the same technology. Although there is still debate on exactly how this can happen, PI9 expression in tumors may reflect a selection process, just as in the other cases of drug resistance we discussed. Use a particular weapon often enough, pretty soon the only bad guys left alive are those that have figured out a way around it. And this smarter subset is the one that survives and breeds the future generation of resistant cancer cells. It has been speculated that in some cases the tumors that have been under chronic CTL-mediated apoptosis may develop resistance to this particular mode of attack, by learning to express high levels of PI9. This may be another mechanism used by tumor cells to escape your body's natural home-grown immune surveillance. 

The interesting observation has been made that in biopsy specimens, tumors are often seen to be infiltrated by many, many home grown CTLs and NK cells. It is as if these elite killers were crowding in, trying to kill the cancer cells, but obviously not succeeding in that task. In fact, tumors with heavy infiltration of CTLs were the very cancers that often exhibited PI9 protection. By the very process of bringing on more and more troops, trying to kill the cancer cells at any cost, the immunologic pressure of the CTLs may have helped create a more resistant form of the cancer. I used this concept in a previous article: necessity is the mother of invention. When natural processes or therapy push hard to kill the tumor but do not succeed in getting the job done completely, this cardinal sin of failure may carry the extra penalty of teaching the enemy some precious tricks and help them develop resistance to that particular mode of attack. (Notice I said may carry, not will carry, or must carry). A more aggressive and virulent variant does not have to develop each and every time there is immunologic pressure on cancer cells. This, like most other systems dealing with large numbers of cells, is a matter of statistics and odds. 

When cancer cells become resistant to the granzyme-B attack of CTLs by means of expressing the antidote PI9, something else may happen that can be a lot more sinister. Remember, we said that once it is inside, Granzyme-B kills by initiating a series of chemical reactions called caspase cascade. Well, guess what, many of our much beloved conventional chemotherapy drugs kill cancer cells by the same set of mechanisms, caspase cascades. OOPS! Is it possible that in the process of becoming resistant to chronic CTL attack and in the process learning how to block the signal that activates the caspase "shears", the cancer cells are now also more resistant to good old standard chemo, because the caspase shears no longer respond to signals? 

I think you can guess my question by now: is it possible that in some CLL patients, adoptive ex-vivo grown T-cell therapy may lead to selection of residual CLL cells with higher expression of PI9? Especially if the T-cell therapy just succeeds in teaching new tricks to the CLL cells and does not finish the job of killing them off? Does the moral of the tumor infiltrating CTLs apply here as well? Does the cardinal sin of failing to totally eradicate the cancer mean we might be selecting for a more resistant form of the cancer? A new variant of the CLL that is not only more resistant to CTL attack, but one that may also use the lessons learned in this context to block chemotherapy down the road?

"Normal" CLL cell lines as well as other low grade B-cell malignancies are rarely positive for PI9. Table 1 of the Blood article below (you can use the link provided to access the full article, it is free of charge) lists Diffuse Large B-cell lymphoma as one of the cancers with high levels of PI9. DLBL is an aggressive lymphoma, hard to treat and with a poorer prognosis than standard CLL. A more common name for this variant is Richter's transformation. A certain percentage of CLL patients (less than 5%) develop this variant over the course of their disease, and a diagnosis of Richter's transformation spells big trouble.

Is there a chance that a garden variety CLL with low PI9 expression can transform into a high PI9 expression disease as a result of selection, through long term attack by home grown CTLs? Is this what happened in the small percentage of CLL patients that were unfortunate enough to go through Richter's transformation? They were the unlucky few in whose case the immunologic pressure selected for a variant of the original CLL cells, a variant that is PI9 positive? Is it because the malignant B-cells have developed PI9 antidote to granzyme-B attack by CTLs that Richter's transformation so hard to treat, why it carries a significantly poorer prognosis than CLL? And in the process of doing that, do they also develop a level of resistance to standard chemotherapy drugs?

As you may guess, during my conversation with Dr. Frolich of Xcyte Therapies I asked this question. Does therapy with Xcyted CTLs carry the potential risk of selecting for a more resistant form of CLL, one where the cancer cells now have higher levels of the PI9 inhibitor? Dr. Frolich did not think this was very likely, but he did not rule it out 100%, either. I suggested to him that part of the clinical study should include monitoring the PI9 levels expressed by CLL cells in patients, before and after Xcyte T-cell therapy. My impression was that he thought it might be a good idea, but they have so many other equally good markers to measure and monitor, PI9 may or may not be the most relevant or important one, from their perspective.

Let me be very clear about one thing. These are my concerns, not proven facts. Moreover, they are the concerns of a lay person, yours truly. Who knows, I may have all this garbled up to no end. Even if I got the facts straight, these may be no more than nit-picking caveats, of no real significance in the real world. The two papers I cite below are worth reading, both are free. If not the whole papers, then at least read the abstracts. Then you can make up your own mind.

At the very least, I will no longer consider immunotherapy of the type discussed here, using adoptive CTLs grown ex-vivo to be entirely risk-free. I suppose it never was, and no researchers claimed it to be risk free. It is just that there is such a "buzz" in patient communities about these sexy new targeted therapies. There is an implicit assumption that biotherapies and immunotherapies are not capable of harming you. Rubbish. Selection of more dangerous variants of the original cancer can happen with immunotherapy techniques, just as it can with chemotherapy. 70% of all CML patients who responded to Gleevec were out of remission 2 years later. All of these patients now had a variant of their original cancer phenotype, one that could no longer be controlled by Gleevec. There is no doubt about this part, "immune sculpting" is a fact of life, even if the sculptor is named Gleevec. How about a sculptor named Xcyte ???

Abstract:

Blood. 2002 Jan 1;99(1):232-7

Expression of the granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system? 

Bladergroen BA, Meijer CJ, ten Berge RL, Hack CE, Muris JJ, Dukers DF, Chott A, Kazama Y, Oudejans JJ, van Berkum O, Kummer JA. 

Department of Pathology, Free University Medical Center, Amsterdam, The Netherlands. 

Blood Journal Article 

In tumor cells, the serine protease granzyme B is the primary mediator of apoptosis induced by cytotoxic T lymphocytes (CTLs)/natural killer (NK) cells. The human intracellular serpin proteinase inhibitor 9 (PI9) is the only known human protein able to inhibit the proteolytic activity of granzyme B. When present in the cytoplasm of T lymphocytes, PI9 is thought to protect CTLs against apoptosis induced by their own misdirected granzyme B. Based on the speculation that tumors may also express PI9 to escape CTL/NK cell surveillance, immunohistochemical studies on the expression of PI9 in various lymphomas were performed. Ninety-two cases of T-cell non-Hodgkin lymphoma (NHL), 75 cases of B-cell NHL, and 57 cases of Hodgkin lymphomas were stained with a PI9-specific monoclonal antibody. In T-cell NHL, highest PI9 expression was found in the extranodal T-cell NHL. In nearly 90% of enteropathy-type T-cell NHLs and 80% of NK/T-cell, nasal-type lymphomas, the majority of the tumor cells expressed PI9. In nodal T-anaplastic large cell lymphomas and peripheral T-cell lymphomas (not otherwise specified), PI9 expression occurred less frequently. In B-cell NHL, PI9 expression was associated with high-grade malignancy; 43% of diffuse large B-cell lymphomas showed PI9(+) tumor cells. Finally, PI9 expression was also found in 10% of Hodgkin lymphomas. This is the first report describing the expression of the granzyme B inhibitor PI9 in human neoplastic cells in vivo. Expression of this inhibitor is yet another mechanism used by tumor cells to escape their elimination by cytotoxic lymphocytes. 

PMID: 11756176
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Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11515-20. Epub 2001 Sep 18

Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. 

Medema JP, de Jong J, Peltenburg LT, Verdegaal EM, Gorter A, Bres SA, Franken KL, Hahne M, Albar JP, Melief CJ, Offringa R. 

Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands. 

http://www.pnas.org/cgi/content/full/98/20/11515

The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer. 
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Potential Risks of CTL Therapy - Cartoon Version

They say a picture is worth a thousand words. Jargon and acronyms are not as pretty as pictures, but they too serve the same purpose. When experts talk to each other, a great deal of complex detail is encapsulated in one quick word of jargon. Since all the members of the club know exactly what the term means, no one has to go into elaborate detail, every one gets the same "picture", quickly and efficiently.

Well, we are not medical experts. All of the jargon and acronyms sound like gibberish to us, and we don't get the pretty picture, just a headache that needs an extra dose of NSAIDs (aspirin, in lay terms). We can try to explain each term, every time, in excruciating detail. I do try some of that, which is what makes my articles so long and boring. Another approach is to use our own brand of pictures instead of words. We may not know medical jargon, but we know what sump pumps are, what bullet proof vests are, how they work and why one needs them. Analogies sound a little silly, but they achieve the same purpose as jargon. Analogies use our everyday and commonly shared understanding of things and how they work, to paint pictures that explain more complex issues. 
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Law and Disorder Under the Microscope

The setting is a large city police department. The citizens are concerned about increasing corruption and graft in their law enforcement body. By now you have seen enough of my analogies, you can guess the police department is a surrogate for your immune system, the good cops are the good B-cells, and CLL is the corruption of good-cops-gone-bad that we wish to eradicate. And the watch-dog Internal Affairs guys are the T-cells, keeping an eye on things, maintaining immune surveillance. 

In this scenario the Internal Affairs guys (local T-cells) have let things get out of control (ineffective immune surveillance by home-grown CTLs) and now there is a real problem, corruption has been confirmed in the police department (CLL has been diagnosed). But the Police Chief convinces the Mayor to give the Internal Affairs guys another chance. "May be they can clean it out, may be they can only do part of the job, but what is the harm in giving them another chance?" he pleads. (Immunotherapy may or may not be able to do the full job, "but it can't hurt you", right ?!?) "After all, who knows these crooked cops in the department better than our own IA guys?" argues the Chief.

The Mayor agrees. Some IA guys are pulled out of the department (T-cells are removed from the patient via leukapheresis) and sent off to a special training camp (Company Lab), to get them far removed from the contamination in the department (away from the influence of subversive cytokines produced by cancer cells). They are given motivational lectures, their hot buttons pushed (CD3 and CD28 markers activated), and re-focused on their mission (targeted to tumor antigen). Their numbers are vastly increased by new recruitment (ex-vivo expansion of cytotoxic T-cells). Finally, they are ready to go back to the department (infused back into the body of the patient), motivated, trained and ready to destroy corrupt cops (CLL cells) where ever they may hide in the department. 

All this takes a long time. "Far too long", the Chief mutters, "and meanwhile our problems get worse and worse". Day by day, the ranks of the good cops become smaller and the legions of the cops-on-the-take increase. Pretty soon, it is hard to find a good cop anywhere in the department! (The tumor burden grows as the patient waits. The CLL cells keep proliferating, the WBC ticks up higher each month. The vast majority of B-cells are now CLL cells). Will the rescuing cavalry get here in time? 

Finally, the large army of re-trained, re-focused and re-motivated Internal Affairs guys return (no ordinary and naive T-cells, these are CTLs, cytotoxic T-cells, equipped with perforin and granzyme granules, and able to hit the Fas pathway, a huge army of CTLs). Citizens cheer as the army of IA guys go in (CTLs are infused back into patient), local papers carry photographs of the Chief and Mayor, all smiles in front of the Department. After all, these IA guys are from the same department (the T-cells were obtained from the patient) and they know the ropes. Plus they are now in larger numbers, and they know more tricks (once naïve T-cells become CTLs, they are able to develop and use perforin and granzyme-B toxic granules). 

Alas. Our intrepid warriors face an enormous problem, too big even for them. Sure, they catch some of the bad cops, especially the ones lurking in the very offices of the Internal Affairs division (CLL cells are destroyed most readily in the lymph nodes, since this is where the CTLs home in most readily). But there are just too many of the bad cops (the tumor burden is too high). It is clearly a case of out-numbered armies. The brazen bad-cops roam the corridors in broad day light (high CLL cell count in the peripheral blood). Some of the villains hide out in remote buildings and file rooms, making them hard to find (bone marrow and other specific locations like spleen and liver may be hard to reach by the CTLs, without specific homing chemokines to direct them there). 

But that is not the worst of it. As days go by, and the problem is not handled expeditiously and thoroughly (CLL cells are not eradicated, quickly and effectively), pretty soon the old friendships between the AI guys and the bad cops re-surface. Old boy Network is a fact of life between cops and IA guys (There is a lot of cross-talk between T-cells and B-cells). All cops talk to each other, they gossip, they share information and donuts, brag about new techniques they have learned. (B-cells and T-cells cross-talk means each influences the other group strongly, each can modulate and control to some degree the behavior of the other group). After all, they share the same offices, walk the same hallways, hang out in the same bars (T-cells and B-cells share lymph nodes, peripheral blood, spleen, liver etc). They talk the same language (similar sets of cytokines) and share the same perspectives (many matching receptors and ligand pairs on B-cells and T-cells). Heck, they all came from the same village! (T-cells and B-cells all originate from the same lymphoid hematopoietic precursor in the bone marrow).

What happens when the hunter gets too cozy with his target? Some of the motivation to make the kill disappears (lots of cross-talk between CTLs and B-cells, makes the T-cells anergic, unable to act). Pretty soon a few of the crooked cops figure out all the new techniques the IA guys have learned (after prolonged CTL attack, sometimes there is a selection of new variant of the original cancer cells, and this new variants is the one to survive and breed, if it has developed resistance to the major cell kill mechanisms of CTLs: Fas pathway, and perforin / granzyme B pathway). It is clear that any small victory that the new, improved IA guys achieved early on is quite temporary. They are now back into their previous role of ineffective internal security. 

Our Chief is worried, the Mayor holds him personally responsible for this fiasco. Calling in for outside help, the big bully boys from the anti-corruption Feds (Fludarabine) seems to be the next thing to do, one of few remaining choices. But at the back of the Chief's mind there is a nagging worry. Will the Feds (Fludarabine) be able to do the job now? After all they use some of the same techniques in their line of work as the newly trained IA guys had tried, (most chemotherapy drugs and CTLs use the same set of caspase cascade pathways to kill cancer cells), what if the bad cops who have learned how to get around the efforts of the IA army can now get around the best the Feds can do as well? (In developing resistance to CTL attack, the cancer cells may, repeat, may also become resistant to common chemotherapy drugs such as Fludarabine). That would be bad news indeed! Not only did all that training and troop expansion of the IA guys do little good (very partial response to ex-vivo grown T-cell therapy in this scenario), it is expensive as all heck (new immunotherapies are rarely inexpensive), it might have taught the bad cops a few new tricks (Granzyme B resistance). Heck, these bad cops are almost a new breed, they have changed their tactics so much (increased drug resistance, potential development of a new and more refractory variant, resistant to Fas pathway and granzyme-B). 

Chief wonders how long he will be able to keep his job (future prognosis) if his worst nightmares come true. 
_______

I will be the first to admit, this is an unusually dark scenario. And none of it has to come true. I clearly slanted the scenario to give the Chief as much of a problem as I could and cut him no slack at all. 

Here are some of the points one could take away from this cartoon analogy: 

  1. Immunotherapy of this type (ex-vivo adoptive T-cell therapy) may have the best chance of success when there is an overwhelming advantage in the numbers of the CTLs, not in favor of the CLL cells. Patients with low tumor burden, or in the early part of a good remission are likely to have the best chance; also, once the dose-dependent toxicity has been worked out, it makes sense to use the highest possible numbers of CTLs. Just as a comparison, almost all of the clinical trials of idiotype vaccine immunotherapy in NHL have recruited only patients who have achieved a full CR with prior chemotherapy and had minimum residual disease going into the vaccine therapy. Any army commander will tell you, if you are going into war, make sure you have more than enough ammunition, enough to "Shock and Awe", to use a recent phrase from our military history.  
  2. Crosstalk between B-cells and T-cells
    There is an inherent problem with T-cell based immunotherapy for B-cell cancers. The very high level of cross-talk between these two compartments of the immune system makes for a tricky situation, in my opinion. I do not have facts to back me up on this, but my sense of it is that in T-cell based therapies, the immunological pressure is more likely to develop variants of B-cell cancers. Since B-cells are also immune system cells, it is not as big a stretch for them to learn the tricks of their fellow immune system cells, the T-cells. This may be less of a problem in the case of other cancers, say for example, in the use of T-cell therapy for renal carcinoma, or skin cancer. Incidentally, adoptive T-cell therapy approaches have shown their best results yet in skin cancer.
  3. In the absence of orchestrated chemokines to lay out the trails of bread crumbs, it is not easy to see how the CTLs will get to the different tumor sites in a hurry. This is another front on which CLL is a tough nut to crack. CLL is a widely dispersed disease, not confined to a single location as is the case with many solid tumors in early stage. We can think of CLL as "metastasized" cancer from day one! Even compared to NHL, the other B-cell blood cancer, the higher level of bone marrow involvement makes CLL more of a challenge. 
  4. Certain approaches may increase our chances of an outright victory, 100% killing of the cancer cells and none left over to grow back into a more virulent strain. Some of these are discussed above. Another item to remember: it does not help to depend on just one antigen as the marker for identifying the targets to be killed by the CTLs. Using a more diverse set of markers may give the cancer a harder time to adapt, a harder time shedding the markers, or changing them so they look different, or just internalizing them into the interior of the cell, where they can no longer be spotted. On the other side of the coin, a wider set of antigens for the CTLs to target may increase the risk of something going awry, triggering autoimmune disease. Even in the success stories of CTL therapy in skin cancer, "vitiglio" is a fact of life, where autoimmune attack on skin pigment cells leaves discolored patches of skin.
  5. Fundamentally, the price one pays for failure in the cancer game is development of new variants, "sculpted" into new forms by the very therapy that tried to control them, tried but failed. The first abstract below from researchers at National Cancer Institute says it all, in plain English !!, and it reiterates each of these points I made above. Bottom line, this report says, and I quote: "Tumor escape variants are likely to emerge after treatment with increasingly effective immunotherapies". 

The ultimate Catch-22? Who said life (or CLL) was simple or fair?
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Abstracts:

Nat Immunol. 2002 Nov;3(11):999-1005.

Natural selection of tumor variants in the generation of "tumor escape" phenotypes. 

Khong HT, Restifo NP. 

National Cancer Institute, National Institutes of Health, Bethesda, MD

The idea that tumors must "escape" from immune recognition contains the implicit assumption that tumors can be destroyed by immune responses either spontaneously or as the result of immunotherapeutic intervention. Simply put, there is no need for tumor escape without immunological pressure. Here, we review evidence supporting the immune escape hypothesis and critically explore the mechanisms that may allow such escape to occur. We discuss the idea that the central engine for generating immunoresistant tumor cell variants is the genomic instability and dysregulation that is characteristic of the transformed genome. "Natural selection" of heterogeneous tumor cells results in the survival and proliferation of variants that happen to possess genetic and epigenetic traits that facilitate their growth and immune evasion. Tumor escape variants are likely to emerge after treatment with increasingly effective immunotherapies. 

PMID: 12407407
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BMC Cancer. 2003 Jul 28 [Epub ahead of print]. 

Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment. 

Bathe OF, Dalyot-Herman N, Malek TR. 

Department of Surgery, University of Calgary, Calgary, AB, Canada. 

BACKGROUND. Adoptive immunotherapy with cytotoxic T lymphocytes (CTL) represents an alternative approach to treating solid tumors. Ideally, this would confer long-term protection against tumor. We previously demonstrated that in vitro-generated tumor-specific CTL from the ovalbumin (OVA)-specific OT-I T cell receptor transgenic mouse persisted long after adoptive transfer as memory T cells. When recipient mice were challenged with the OVA-expressing E.G7 thymoma, tumor growth was delayed and sometimes prevented. The reasons for therapeutic failures were not clear.
METHODS. OT-I CTL were adoptively transferred to C57BL/6 mice 21 - 28 days prior to tumor challenge. At this time, the donor cells had the phenotypical and functional characteristics of memory CD8+ T cells. Recipients which developed tumor despite adoptive immunotherapy were analyzed to evaluate the reason(s) for therapeutic failure.
RESULTS. Dose-response studies demonstrated that the degree of tumor protection was directly proportional to the number of OT-I CTL adoptively transferred. At a low dose of OT-I CTL, therapeutic failure was attributed to insufficient numbers of OT-I T cells that persisted in vivo, rather than mechanisms that actively suppressed or anergized the OT-I T cells. In recipients of high numbers of OT-I CTL, the E.G7 tumor that developed was shown to be resistant to fresh OT-I CTL when examined ex vivo. Furthermore, these same tumor cells no longer secreted a detectable level of OVA. In this case, resistance to immunotherapy was secondary to selection of clones of E.G7 that expressed a lower level of tumor antigen.
CONCLUSIONS. Memory engraftment with tumor-specific CTL provides long-term protection against tumor. However, there are several limitations to this immunotherapeutic strategy, especially when targeting a single antigen. This study illustrates the importance of administering large numbers of effectors to engraft sufficiently efficacious immunologic memory. It also demonstrates the importance of targeting several antigens when developing vaccine strategies for cancer. 

PMID: 12882650
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Clin Invest. 2003 May;111(10):1487-96.

Article from the Journal of Clinical Investigations

Antigenic drift as a mechanism for tumor evasion of destruction by cytolytic T lymphocytes. 

Bai XF, Liu J, Li O, Zheng P, Liu Y. 

Division of Cancer Immunology, Department of Pathology, Ohio State University Medical Center, Columbus, Ohio  

It is established that mutations in viral antigenic epitopes, or antigenic drifts, allow viruses to escape recognition by both Ab's and T lymphocytes. It is unclear, however, whether tumor cells can escape immune recognition via antigenic drift. Here we show that adoptive therapy with both monoclonal and polyclonal transgenic CTLs, specific for a natural tumor antigen, P1A, selects for multiple mutations in the P1A antigenic epitope. These mutations severely diminish T cell recognition of the tumor antigen by a variety of mechanisms, including modulation of MHC:peptide interaction and TCR binding to MHC:peptide complex. These results provide the first evidence for tumor evasion of T cell recognition by antigenic drift, and thus have important implications for the strategy of tumor immunotherapy. 

PMID: 12750398
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Tissue Antigens. 2002 Oct;60(4):273-81. 

Tumor escape from killing: role of killer inhibitory receptors and acquisition of tumor resistance to cell death. 

Chouaib S, Thiery J, Gati A, Guerra N, El Behi M, Dorothee G, Mami-Chouaib F, Bellet D, Caignard A. 

Inserm U487, IFR 54, Institut Gustave Roussy, France.

Immunotherapy of cancer has always been a very attractive fourth-modality therapeutic approach. Over the past few years, advances in the identification of tumor antigens have offered new perspectives and provided new opportunities for more accurate immunotherapy for cancer. However, when applied to patients with established tumors, it rarely leads to an objective response. This is partly due to the fact that tumors evade host immunity at both the induction and effector phases. Thus, understanding tumor escape mechanisms may be the key to successful immunotherapy for cancer. In the present review, we will focus on how the expression of killer Ig receptors (KIR) on tumor infiltrating lymphocytes can compromise their function and how tumors evade apoptotic death - two additional mechanisms of tumor escape. 

PMID: 12472656
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