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CTL Therapy

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Adoptive T-cell Immunotherapy

Date: August 8, 2003

by Chaya Venkat

Of Armies of Shock Troops

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Introduction to CTL Therapy

To simplify (perhaps ridiculously oversimplify) an extremely complex and rapidly moving field, CTL (Cytotoxic T-Lymphocyte) based immunotherapy works this way:

Cytotoxic T-cells (CTLs) are part of the immune system that are supposed to recognize foreign antigens displayed by infected cells, attack and kill them. The "foreign" antigens can be from virus infections, as in the case of EBV (Epstein-Barr Virus) or CMV (cytomegalovirus), or even cancer that has hijacked the particular cell.

The problem is that viruses and cancers have gotten good at evading surveillance in a number of ways. First of all, the antigens they display are often not immunogenic, i.e., they don't look like bad guys that need immediate killing. Second, they put out cytokines and the like that make the T-cells lazy, inactive, and not interested in doing their job. In some cases, the bad cells wage a kind of warfare, killing off the T-cells and keeping their population numbers down.

Here is how ex-vivo (out side the body) approaches work. T-cells are collected from the patient, and far away from the confusing cytokines put out by the cancer and/or viruses, they are correctly exposed to the tumor or viral antigens, along with the necessary co-stimulatory signals. Once their education is complete, literally billions of copies of these CTLs are grown and injected back into the patient.

Below is an abstract from Blood that is interesting.


Autologous Epstein-Barr Virus (EBV)-Specific Cytotoxic T Cells for the treatment of Persistent Active EBV Infection

Barbara Savoldo, M Helen Huls, Zhensheng Liu, Takayuki Okamura, Hans-Dieter Volk, Petra Reinke, Robert Sabat, Nina Babel, James F Jones, Jennifer Webster-Cyriaque, Adrian P Gee, Malcolm K Brenner, Helen E Heslop, and Cliona M Rooney

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA. Department of Medical Immunology, Charite Hospital, University of Berlin, Berlin, Germany. Department of Nephrology, Charite Hospital, University of Berlin, Berlin, Germany. Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO, USA. The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 

Chronic Active Epstein-Barr Virus-infection (CAEBV) syndrome is a heterogeneous EBV-related disorder characterized by chronic fatigue, fever, lymphadenopathy and/or hepato-splenomegaly, associated with abnormal patterns of antibody to EBV. CAEBV can range from disabling mild/moderate forms to rapidly lethal disorders. Even patients with mild/moderate disease frequently suffer adverse effects from long-term anti-inflammatory agents and have a quality of life that progressively deteriorates. It is still unknown why these individuals are unable to produce an effective immune-response to control EBV and no effective treatment is currently available. Since ex-vivo expanded EBV-specific cytotoxic T-lymphocytes (EBV-CTL) can safely restore EBV-specific cellular immune-responses in immunodeficient patients, we assessed the possibility that adoptive immunotherapy might also effectively treat CAEBV-infection. Following stimulation with irradiated EBV-transformed lymphoblastoid cell lines (LCL), EBV-CTL were successfully generated from 8/8 patients with the mild/moderate form of CAEBV-infection. These CTL were predominantly CD3+CD8+ cells and produced specific killing of the autologous LCL. Five of these patients, with 1 to 12-year histories of disease were treated with 1 to 4 injections of EBV-CTL. Following infusion, there was resolution of fatigue and malaise, disappearance of fever and regression of lymphadenopathy and splenomegaly. The pattern and titers of anti-EBV antibodies also normalized. No toxicity was observed. Four patients did not show any relapse of disease within 6-36 months follow-up; one patient had recurrence of fatigue and myalgia 1 year post-CTL infusion. We suggest that adoptive immunotherapy with autologous EBV- CTL may represent a safe and feasible alternative treatment for patients affected with mild/moderate CAEBV-infection and that this approach should be evaluated in the more  severe forms of the disease.

Of course, the process is not simple. One has to first find the right antigens on the CLL cells, and then train the T-cells to attack all cells exhibiting those specific antigens. The abstract below talks about the work underway at Dana Farber, under John Gribben, to identify the right antigens on CLL cells. I cannot swear to it, but I do believe we will be seeing solid clinical trials for CLL using CTL technology within the next year. My guess would be that Anderson and Dana Farber already have some such plans under consideration. Again, I would expect that initially these approaches would be used as adjuncts to other therapy modalities, first to drastically reduce the tumor burden by more conventional methods, and then use CTL therapy to do the mop-up job of cleaning out the last remaining CLL cells. I am willing to bet dollars for donuts that we will start to see higher percentage of PCR negative responders with these double-barreled approaches. Whether this translates into reasonably long term “cures” is any body's guess.


Identification of Novel Tumor-Associated Antigens in CLL

John G Gribben MD DSc Dana Farber Cancer Institute, Harvard Medical School, Boston, MA

The impressive responses that can be seen in CLL patient to donor lymphocyte infusion and to the use of allogeneic stem cell transplantation after non-myeloablative conditioning regimens have demonstrated that CLL can be a target of T cell mediated response. However, this comes at the cost of considerable morbidity and mortality from graft versus host disease. The generation of a T cell mediated response targeted at a tumor associated antigen would likely reduce this morbidity. In addition, there is considerable evidence that it is possible to generate autologous T cell responses against CLL cells, particularly once the CLL cells have been activated via CD40 or after gene transfection. As a model antigen, we examined CD8+ T cell responses against immunoglobulin derived peptides. We have previously shown that cytotoxic T cell responses can be generated native and heteroclitic Ig derived peptides. These responses are mostly weak and we are therefore seeking to identify additional candidate tumor antigens using SEREX. Eight cDNA expression libraries have been constructed from untreated CLL patients with different disease stages. After screening of over 30 million clones with autologous and allogeneic serum from CLL patients. Sera from other lymphoma patients and from healthy donors were used as controls. Using this approach we identified 25 different clones containing 1485 to 3990 bp and representing 15 different genes designated as KW-1 to KW-15. Ten of these antigens are novel and five have been detected previously using SEREX in other malignancies although none had previously been described in CLL. Five of the antigens were detected only by the sera of CLL patients. An additional 6 were detected by the sera of patients with CLL and with B cell lymphomas. The last 3 antigens were also detected by the sera of some of the normal donors. Of note, those patients whose sera detected multiple different antigens tended to have early stage disease, normal serum immunoglobulins, low expression of CD38 on CLL cells and were not previously treated. Using RT-PCR, we identified 6 clones with restricted expression pattern or over-expression in tumor samples, including KW-2 which has a cancer-testis-like expression profile. KW- 2 is expressed in all CLL cells tested to date, lymphoblastic leukemias, Burkitt's lymphomas, but it is not expressed in normal B cells. In addition, we detected novel sequences encoding previously described genes but representing novel splice variants with restricted expression pattern. The functional properties of these alternatively spliced and restrictively expressed genes are currently under investigation. In conclusion, we identified a number of novel tumor antigens that are potential targets for the development of polyvalent immunization strategies in patients with CLL. The identified genes and splice variants might be involved in the pathogenesis of CLL and other cancers as well as representing novel targets for development of specific immunological or molecular therapy strategies. As major humoral responses against these antigens were detected mostly in patients with early stage disease these patients might be ideal candidates for immunization approaches.

Adoptive T Cell Therapy

Adoptive T cell therapy involves the ex-vivo (out of the body) selection and expansion of antigen-specific T cell clones. It provides a means of increasing antigen-specific immunity without the in-vivo constraints that can accompany vaccine-based strategies.

There are many possible ways to select, train and expand these T-cells. The technology involved can be complex and obscure. A biotech firm that finds a cost-effective way to do it and can demonstrate efficacy in live patients would indeed have hit the jackpot. One can expect to see periodic announcements of some proprietary technology or the other getting a try out in the real world.

You can read about the saga of Xcyte Therapies and its technology in T-cell Therapy at UCSD.

Another effort is highlighted below:

In 2002, t he University of Washington and an affiliated company "Targeted Genetics" were granted a broad patent for a cost-effective method for making billions of copies of patient's own cytotoxic T Cells (CTLs) geared to fight specific cancers and infectious agents. Their press release is below. I took the liberty of moving the "Background" section to the front, just so we all know what they are talking about.

Please refer to several other articles on adoptive CTL therapy in this website. I am getting to be quite optimistic about the potential use of this technology, initially in combination with other treatment modalities such as BMT and RFC, but eventually, when we get all the detail right, this method could become the non-toxic, do-no-harm, high quality remission approach we are all rooting for. There are rumors of several phase-1 clinical trials on the drawing boards already, and we need to be on the look out for these.

Press Release from Targeted Genetics:

Adoptive Immunotherapy Background

The human body is bombarded daily with infectious agents and potentially cancer-causing DNA damage. Normally, these assaults do not lead to chronic diseases such as cancer or hepatitis but usually cause an acute, self-limited disease that is resolved within a matter of days. The cellular immune system is responsible for keeping these assaults in check. The cellular immune system is comprised of two subsets of T cells: CD4+ T helper cells and CD8+ cytotoxic T cells (CTLs). Targeted CTLs recognize and eliminate virally infected or malignantly transformed cells. Activation and expansion of targeted CTLs requires a complex set of cellular interactions including cytokines and other factors produced by antigen-specific CD4+ helper T cells. Chronic viral infection or cancer occurs when these assaults overwhelm the ability of CTLs to eliminate diseased cells. Adoptive immunotherapy is a process in which researchers remove a small amount of blood from the patient and isolate the very small number of CTLs specific for the disease present in the patient. Once isolated, these cells are expanded to numbers (billions) necessary to combat the overwhelming disease burden in the patient. These targeted CTLs are then infused into the patient to cure or contain the disease. Animal models of current expansion technologies result in only a seven to 10 fold increase in cell number in a seven to 10 day period and require reagents not available for human use. Targeted Genetics' REM technology overcomes these hurdles for treating human disease, allowing billions of cells to be produced in less than two weeks.

The majority of therapies on the market or under development for chronic viral and malignant disease act to stimulate a targeted CTL response (therapeutic vaccines) or manage disease burden (protease inhibitors) in an effort to stimulate in vivo expansion of the patients' own targeted CTLs. Targeted Genetics is the only company developing direct approaches to harness the power of the immune system via the REM technology. Application of the REM technology to adoptive immunotherapy may enable the potential of this approach to be realized.

Targeted Genetics receives exclusive license to technology

SEATTLE -- Fred Hutchinson Cancer Research Center (FHCRC) and Targeted Genetics Corporation (Nasdaq: TGEN) announce that they have received notification from the U.S. Patent and Trademark Office regarding the issuance of patent #5,827,642 entitled " Rapid Expansion Method ("REM") for in vitro Propagation of T Lymphocytes."

The patent covers technology used to expand antigen-specific T cells for use in adoptive immunotherapy techniques, which has the potential to treat diseases such as cancer as well as viral infections. This technology was developed by Stanley R. Riddell, M.D., and Philip D. Greenberg, M.D., Ph.D., researchers in the Hutchinson Center's Clinical Research Division and professors at the University of Washington. Greenberg is also a member of Targeted Genetics' Scientific Advisory Board. The Hutchinson Center has exclusively licensed this technology to Targeted Genetics.

"Adoptive immunotherapy represents a potentially powerful tool in the treatment of infectious disease and cancer," says Riddell. "Previous studies have demonstrated that T lymphocytes play a crucial role in eliminating virally infected or cancerous cells from the body. However, clinical application of adoptive immunotherapy has been difficult because the technology to expand antigen-specific cells while retaining function has not been readily available. The REM technology enables the production of sufficient numbers of cells capable of eliminating infected or malignant cells to make adoptive immunotherapy a viable therapeutic approach."

Researchers at Targeted Genetics and the Hutchinson Center have leveraged the REM technology to develop methods for genetically modifying cytotoxic T cells (CTL) to enhance their function by providing key proteins that may be lacking in certain disease settings, such as AIDS. Additionally, REM expansion modified CTLs could be used to deliver cytokines to diseased target cells without the toxicities observed when cytokines are administered systemically.

Targeted Genetics has supported two Phase I studies of HIV-specific CTLs in HIV infected patients conducted by Riddell and Greenberg at the Hutchinson Center. Additional Phase I/II studies of adoptive immunotherapy using CTLs expanded with the REM technology are ongoing at the Hutchinson Center for the precaution of cytomegalovirus (CMV) disease in bone marrow transplant patients and for the treatment of patients with late stage melanoma.

Targeted Genetics has several issued and pending patents in this area. Prior to development of the REM technology, technical and regulatory hurdles made adoptive immunotherapy too costly for commercialization. Targeted Genetics has further developed this technology to facilitate commercial development by focusing on scalability of costs and reagents. Targeted Genetics has filed additional patents regarding improvements to the REM technology.

"In addition to its significant potential in adoptive immunotherapy, Targeted Genetics' REM technology also is a strategic core technology for research, development, and commercialization of other technologies aimed at treating chronic viral and malignant disease," said H. Stewart Parker, President and Chief Executive Officer of Targeted Genetics. "The REM process is applicable to broad-based technologies such as genomics and to the identification of new proteins or small molecules with therapeutic potential. This patent, in concert with Targeted Genetics' other patents and intellectual property, forms a powerful core technology platform either as a stand alone therapeutic or in concert with existing technologies."

Targeted Genetics Corporation develops gene and cell therapy products for the treatment of certain acquired and inherited diseases. The company has three lead development programs targeting cystic fibrosis, cancer, and infectious diseases as well as an extensive gene delivery and cellular therapy technology platform.

The Fred Hutchinson Cancer Research Center is an independent, non- profit research institution dedicated to the development and advancement of biomedical technology to eliminate cancer and other potentially fatal diseases. Recognized internationally for its pioneering work in bone marrow transplantation, the Center has four scientific divisions collaborating to form a unique environment for conducting basic and applied science. One of 35 National Cancer Institute-designated comprehensive cancer centers in the country, it is the only one in the Northwest.

T-cell Therapy with a Bispecific Antibody

Here is an ASH 2001 abstract that talks about both of my favorite subjects, CTL therapy and bispecific antibodies. Part of the problem with the CTL therapy that I have been discussing in previous articles is that each t-cell can kill at the most one b-cell. The actual kill ratio achieved is a lot less than that. So, we have to grow literally billions of CTLs to go in and do the mop-up job. That is also why CTL therapy is typically considered to be effective after more conventional therapies like chemo or monoclonals or combinations like RFC, when the vast majority of tumor cells have already been destroyed. The logic is that the externally trained CTLs can hope to do the job only when they are not outnumbered. The higher the ratio of CTLs compared to b-cells left over as "Minimal Residual Disease", the better the chance of full PCR negative status (dare I say "cure") after the mop-up CTL therapy. Clinicians typically hope for this ratio to be far better than 100 killer CTL cells for every 1 target cancer b-cell.

Now the new bispecific antibody spin, added on top of CTL approach: the experiment described below compared b-cell kill rates at a lowly ratio of 10: 1 CTL versus b-cell ratio. As expected, the kill rate of b-cells at this low ratio was only 20%. Now they added CD19XCD3 bispecific antibody to the mix. This has the effect of cross linking the CTL cells (via the CD3 hook) to the b-cell (via the CD19 hook). Forcing this kind of close proximity between the killer and the target increased the kill rate by more than 4 fold, to 81%.

To my mind, the odds are constantly improving on this approach, day by day: with better ways of growing the patient's own t-cells to be effective CTLs outside the body, better ways of using dendritic cells to be more effective antigen presenting cells and therefore better ways of indoctrinating the t-cells, better ways of culturing and growing the billions of copies needed, and now better ways of getting higher kill rates, more bang for the buck. All of this with minimal toxicity, no worries about graft-versus-host disease and the like since the patient's own cells are used.

Pretty exciting results, to my mind. So all you folks out there, who are approaching end of w&w and looking at first therapy decisions, whether you are looking at frontline Rituxan type approaches, or go the Full Monty for combos like RFC, keep an eye out for Autologous CTL therapy of the types mentioned in my past few articles as a mop-up procedure, after you have completed the necessary rounds of chemo or what ever. The seed t-cells etc will have to be harvested from your blood probably prior to going into chemotherapy, so you need to plan for it ahead of time. The problem is going to be finding locations where you have access to CTL and/or CTL/bispecific antibody type therapy options. I have a hunch these approaches are at least in
the planning stage at most of the CLL consortium centers, but we need to find out more.

So, here is some home work for you all. If you have contacts, if you presently see one of the CLL experts at Consortium centers, ask about CTL therapy. Ask about CD3XCD19 bispecific antibody therapy. Read up about it as much as you can before you ask, so that you can make the most sense of the answer. Last but not least, don't forget to write, and share your information with the rest of us. We are all in this together.

Above all, stay as healthy as you can, as long as you can. There is a dawn coming, I can sense it, and I want you all to be there for the new day.


[1546] Apoptosis in CLL-Cells Induced by Autologous NK T Cells Is Enhanced by a Recombinant Bispecific Tandem Diabody.

Martin Kornacker, Kipriyanov M. Sergey, Little Melvyn, Moldenhauer Gerhard, Edeltraud Weilguni, Markus Herbst, Manfred Hensel, Anthony D. Ho. 

Dep. of Internal Medicine V, University of Heidelberg, Heidelberg, Germany; Affimed Therapeutics, Ladenburg, Germany; Division of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany.

Natural killer T cells (NK T cells) are defined by the coexpression of T- and NK-markers. Two types of NK T cells exist: One type is selected by CD1d, expresses a skewed T-cell receptor repertoire and is found primarily in thymus and liver. A second type is independent of CD1d, has an unbiased T-cell receptor repertoire and is found in spleen and bone marrow. A cell population similar to CD1d independent NK T cells can be expanded from peripheral blood mononuclear cells by the timed addition of g-IFN, IL-2 and OKT-3. In vitro culture leads to a 900 fold expansion of CD3+56+ NK T cells in three weeks. MHC independent cytolysis of  various syngeneic tumor cell lines could be demonstrated in a mouse model (Baker et al., Blood 97(10), 2001). Bispecific antibodies or their recombinant analogues crosslink effector cells with their targets and may enhance their cytotoxicity. We used MACS to separate CD3+ cells for expansion of NK T cell effectors and CD19+ targets from peripheral blood of CLL patients. Because primary CLL cells cannot be labelled sufficiently with 51Cr they are not suitable as targets in a standard 51Cr-release cytotoxicty assay. Therefore, we assessed cytotoxicity by measuring apoptosis in CLL cells. Targets were labelled with the red membrane dye PKH26, incubated with effector cells and analyzed for annexinV- FITC positivity by flow cytometry. After 4 hour incubation of NK T- with CLL cells at an effector to target ratio of 10 : 1, specific apoptosis in CLL cells was 20%. Programmed cell death in target cells could be increased by using a recombinant CD3 x CD19 bispecific tandem diabody generated by functional dimerization of a single chain molecule that contained four antibody variable domains (VH3-VL19-VH- 19-VL3) in an orientation preventing intramolecular pairing. Adding CD19xCD3 bispecific tandem diabody at a concentration of 1 mg/ml to the assay caused specific apoptosis in 81% of CLL cells. These results could be reproduced in primary CLL cells from 2 other patients. In conclusion, NK T cells induce apoptosis in primary autologous CLL cells without antigenic priming. Retargeting of NK T cells with a recombinant bispecific tandem diabody increased target cell death significantly. The fact that these ex vivo activated and expanded effector cells do not cause GVHD in a cross major transplantation model (Baker et al.) holds promise for their use in cellular therapy after allogeneic stem cell transplantation in CLL and lymphoma.

Keywords: CLL\ Antibody\ Killer cells

Can Adoptive T-cell Immunotherapy be Curative?

A bone marrow transplant is, at the present moment, the only procedure that holds the promise of a potential "cure". I say potential because there is no guarantee that even a successful BMT will not relapse, for no obvious reason. In recent months we have had a couple of leading citizens of our CLL community that relapsed after several years of apparently fully successful BMT procedures. Not the kind of odds to gladden a patient's heart. Until recently, BMTs were undertaken only in relatively young patients, because the process required very stressful and heavy duty chemotherapy to wipe out completely the patient's own immune system, to make room for the new and healthy donor bone marrow. Now several cancer centers undertake what are called "mini-transplants", which are not as tough on the patient as the full blown variety. This has meant that the age limit has been stretched, and patients in their seventies may now be potential candidates. The juggling act in all these procedures is to maximize the impact of the graft versus leukemia effect, and minimize the graft versus host disease. Put in simpler language, the aim is for the transplant to kill the cancer before it kills you. We are learning more about how to match donor with recipient, but even today there is a substantial risk of morbidity associated with this procedure. There is also the not so little problem of finding a donor who is a good match, not every one is fortunate enough to have a family member who just happens to be a perfect match. Matched Unrelated Donors ("MUDs") are also harder to find for patients of minority ethnicity, since there are that many fewer registered donors that belong to those groups. I am beginning to wonder if the problems associated with BMTs will go away relatively soon, simply because the procedure itself will become obsolete, replaced by less dangerous and more successful immunotherapies. Think about it, the major problem with BMTs is that no matter how well we think we are matching the donor with the recipient, the match is never quite perfect, there is always the risk of rejection of the graft, or graft-versus-host-disease. These particular problems are erased in one stroke, if we use the patient's own immune cells. This is the logic behind the ex-vivo grown CTL (cytotoxic T-cells) therapy approaches I have described in recent articles. We are coming up the learning curve rapidly in how and when to collect the necessary cells from the patient, how to grow billions of copies of them outside the body, away from the influence of all the suppressive signals put out by the cancer, and how to train them to be efficient killers of the cancer cells once they are put back into circulation within the patient. Many of the problems associated with a donor based immune replacement are short-circuited in this approach, since the immune cells put back into the patient are his/her own. This also means that the draconian preparatory regime that had to be endured prior to a standard BMT may no longer necessary, a simpler and safer de-bulking of the cancer (to give the new CTLs a fighting chance, not get overwhelmed by too many enemy cells to kill) may be sufficient to do the job. 

Let me be the first to acknowledge, we are not quite there yet. There is a lot to be learned yet, and many crucial details to be worked out before these CTL type approaches begin to live up to their promise. With any immunotherapy approach, there is always the danger of solving one problem just to create another, our understanding of the extremely complex interactions within the immune system is far from perfect. For example, we want the hyped-up CTLs to kill the cancer cells, and only the cancer cells. Otherwise we may run into situations of auto-immune problems, where the newly energized immune system starts killing off perfectly healthy cells. The other issue to solve is how to get the immune system from forgetting the lessons learned, how to maintain a supply of the CTLs as "memory" T-cells, so that if the cancer were ever to rear its ugly head, the memory cells quickly grow a large army of daughter cells capable of going out and doing battle. I fully expect that in addition to the T-cell therapy clinical trial announced by Dr. Kipps, similar trials will soon be announced at the other CLL Consortium centers. Each will have a slightly different take on it, slightly different ways of growing the T-cell  population, different ways of educating them, different adjuvants that may be co-administered to improve the efficiency of the tumor recognition and killing of the tumor. It is an exciting time, with new options opening up. Below is a brief PubMed abstract that talks about use of Interleukin-2 in generating "memory" T-cells. 


Full IL-2 during in vitro priming promotes subsequent engraftment and successful adoptive tumor immunotherapy by persistent memory phenotypic CD8(+) T cells. 

Bathe OF, Dalyot-Herman N, Malek TR. 

Department of Surgery, University of Miami School of Medicine, Miami, FL 33101

Adoptive T cell tumor immunotherapy potentially consists of two protective components by the transferred effector cells, the immediate immune response and the subsequent development of memory T cells. The extent by which adoptively transferred CD8(+) CTL are destined to become memory T cells is ambiguous as most studies focus on the acute effects on tumor shortly following adoptive transfer. In this study we show that a substantial fraction of the input CTL develop into memory cells that reject a s.c. tumor challenge. The use of exogenous IL-2 or a combination of IL-2 and IL-4, but not solely IL-4, during the ex vivo culture for the CTL inoculation was necessary for efficient development of CD8(+) memory T cells. Thus, an important component of adoptive immunotherapy using CTL is the production of CD8(+) Ag-specific memory cells which is primarily favored by IL-2 receptor signaling during ex vivo generation of the effector CTL.

The Chemokine Trail

Earlier this week I had the opportunity to discuss some of the issues relevant to one half of this equation, the adoptive T-cell therapy part, with Dr. Mark Frolich, VP and Medical Director of Xcyte Therapies, during an hour long phone conversation. This article and a few more in the next week or so are based on impressions I gathered during that conversation, as well as my own research into published literature. While I have made every effort to be accurate, please remember these comments are my perspective of the technology, not direct quotes attributable to Dr. Frolich or his company. Since Dr. Frolich was kind enough to give me advance warning before he called me, I was ready with a laundry list of questions for him, probably many more than he wanted!  

T-cells, like many of the other immune system cell lines, follow a complex migratory path. They travel through the blood, tissues, bone marrow, lymph nodes etc, obeying a 'bread-crumb-trail' of chemokines. These "chemokines" are chemical signals that change over time, directing the T-cells to move from one location to another, as the demands of the body dictate. B-cells follow similar patterns of chemokine signals. By now we are all aware that CLL takes different forms in different patients, some of us have very high peripheral blood lymphocyte counts with relatively low involvement in bone marrow and lymph nodes. Others have deceptively low lymphocyte numbers in CBC, but have bulky disease in lymph nodes, spleen, bone marrow etc. Even in just one patient, swollen lymph nodes are known to wax and wane, peripheral blood lymphocyte counts go up and then down over time, for no apparent reason. Chemokine signaling is at the root of this preference for one location over another, at a given time. 

One of our members wrote post regarding her response to Xcyte T-cell therapy in the ongoing Phase - I clinical trial at UCSD. She seems to have had good response in her lymph nodes, the location that T-cells home-in most readily. But interim indications are that her bone marrow and peripheral blood are still infiltrated with CLL cells. My sense of Dr. Frolich's response is that this is still an area where our knowledge is incomplete, the science of getting the CTL armies to go exactly where we want them is yet in its infancy. The delicate manipulation of various chemokine homing mechanisms are not yet within our control. This is one of the many important hurdles to effective adoptive CTL therapy. At this point, our approach seems to be to throw in so many of the activated T-cell troops into the fray as possible, hope that we can defeat the enemy by sheer force of numbers, even if we do not have fine-tuned control of troop mobilization in the war theatre.  The abstract below suggests additional details on the subject of chemokine control of T-cell migration.


Immunology. 2003 Mar;108(3):296-304

Memory T-cell competition for bone marrow seeding.

Di Rosa F, Santoni A.

Department of Experimental Medicine and Pathology, University of Rome La Sapienza, Rome, Italy.

The presence in the bone marrow of memory CD8 T cells is well recognized. However, it is still largely unclear how T-cell migration from the lymphoid periphery to the bone marrow is regulated. In the present report, we show that antigen-specific CD4 T cells, as well as antigen-specific CD8 T cells, localize to the bone marrow of immunized mice, and are sustained there over long periods of time. To investigate the rules governing T-cell migration to the bone marrow, we generated chimeric mice in which the lymphoid periphery contained two genetically or phenotypically distinct groups of T cells, one of which was identical to the host. We then examined whether a distinct type of T cell had an advantage over the others in the colonization of bone marrow. Our results show that whereas ICAM1 and CD18 molecules are both involved in homing to lymph nodes, neither is crucial for T-cell bone marrow colonization. We also observed that memory-phenotype CD44high T cells, but not virgin-type CD44-/low T cells, preferentially home to the bone marrow upon adoptive transfer to normal young mice, but not to thymectomized old recipients where an existing memory T-cell pool precludes their free access. Thus, T-cell colonization of the bone marrow uses distinct molecules from those implicated in lymph node homing, and is regulated both by the properties of the T cell and by the competitive efficacy of other T cells inhabiting the same, saturable niche. This implies that the homing potential of an individual lymphocyte is not merely an intrinsic property of the cell, but rather a property of the lymphoid system taken as a whole. 

PMID: 12603595

How Many Troops Are Needed to Win the War?

Using the analogy of armies, one is inevitably confronted with the question of relative strengths of the armies. I questioned Dr. Frolich as to why patients with relatively high tumor load were chosen for their Phase -1 clinical trial at UCSD. Especially in this dose escalation study, some of the earlier cohorts of patients had only a billion T-cells infused back. Sounds like a lot, but not when you compare against the numbers of CLL cells present in a patient with high tumor load. Some of the later groups of patients may get as many as 50 billion or more of the T-cells infused back. There is little doubt in my mind that this technology has a lot better chance of success if it is used in early stage patients, or those in first remission. When faced with insurmountable odds, out numbered human armies and T-cell armies both seem to suffer the same fate. 

I guess companies do not always have total control over the details of the trial, some of the details are mandated by regulatory agencies, and some are constrained by available volunteer pool. But it does underline a fundamental flaw in the present mechanism of recruiting candidates for clinical trials. With the advent of new biotherapies such as adoptive T-cell therapy, various monoclonal antibody therapies etc, the regulatory agencies need to re-examine their guidelines. This is necessary in my opinion, both in terms of who can be recruited for these trials, and what end-point constitutes "success".  In the prior era of relatively toxic chemotherapy drug candidates, the principle of "Do no harm" meant only patients in late stage disease could be inducted into clinical trials, since these heavily pre-treated folks had fewer choices left, and even an early phase clinical trial offered some hope that was not there otherwise. "Success" was measured by increased overall survival. It is surely a good end-point, no patient would disagree with that. After all, when rubber meets the road, the one thing we care to know about a new therapy is, will it prolong life for the patient? But this worthwhile end-point can become a real bottle neck in developing new therapies for relatively indolent diseases like CLL. How does one prove absolute and incontrovertible survival advantage for a given new therapy in CLL, without prohibitively expensive trials running for almost a decade? A significant portion of the patient population live that long with existing therapies, especially with the addition to monoclonal antibodies. Perhaps it is time to come up with surrogate end points such as changes in phenotype, bone marrow clearance, lack of myelosuppression, time to relapse etc. As a patient advocate, I would consider a new therapy at least a partial success if it is able to delay and control of the disease, without toxicity and risk of "burned bridges" in terms of becoming more refractory to conventional therapy. 

Prostate cancer is one of the most popular cancers with drug companies to test the efficacy of a new drug. It helps that the potential market value of a new therapy for prostate cancer is huge, many more patients have this form of cancer than, say, CLL. But another reason for selecting prostate cancer as the model to test new drugs is that the PSA level measured by a simple blood test is the accepted surrogate for disease load. Clinical trials need to do little more than follow this simple variable in their patient cohorts, to judge efficacy. I wish it were as easy to follow efficacy of new drugs in CLL, but CBC information can be downright misleading, if it is the only parameter followed. Bone marrow and phenotype information is a lot harder to get, a lot more expensive, and yet this is the only way to get a good fix on state of the disease in CLL. 

In any case, Dr. Frolich commented that the intent of Phase - I trials is not curative, but to show proof of principle. That phrase, early phase trials are not meant to be curative for the individual patient, is worth repeating. And it does appear that the technology has succeeded in that goal, the infused "Xcyted" T-cells seem to be able to survive and kill CLL cells in-vivo. The second abstract below outlines some of the problems with "out-numbered armies" of the good-guys. 


Blood. 2003 Jan 15;101(2):766-70. Epub 2002 Aug 22.

Tissue distribution of target antigen has a decisive influence on the outcome of adoptive cancer immunotherapy. 

Meunier MC, Roy-Proulx G, Labrecque N, Perreault C.

Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, PQ, Canada. 

Adoptive transfer of allogeneic T cells has unmatched efficacy to eradicate leukemic cells. We therefore sought to evaluate in kinetic terms interactions between T cells and allogeneic leukemic cells. T cells primed against the model B6(dom1) minor histocompatibility antigen were adoptively transferred in irradiated B10 (B6(dom1)-positive) and congenic B10.H7(b) (B6(dom1)-negative) recipients, some of which were also injected with EL4 leukemia/lymphoma cells (B6(dom1)-positive). A key finding was that the tissue distribution of the target epitope dramatically influenced the outcome of adoptive cancer immunotherapy. Widespread expression of B6(dom1) in B10 recipients induced apoptosis and dysfunction of antigen-specific T cells. Furthermore, in leukemic B10 and B10.H7(b) hosts, a massive accumulation of effector/memory B6(dom1)-specific T cells was detected in the bone marrow, the main site of EL4 cell growth. The accumulation of effector/memory cells in recipient bone marrow was EL4 dependent, and its kinetics was different from that observed in recipient spleen. We conclude that strategies must be devised to prevent apoptosis of adoptively transferred T cells confronted with a high antigen load and that local monitoring of the immune response at the site of tumor growth may be mandatory for a meaningful assessment of the efficacy of adoptive immunotherapy.  

PMID: 12393700



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