Date: August 28, 2003
by Chaya Venkat
Failures in Therapy: CD40 Ligand
Gene Therapy is the title that was chosen to describe a therapeutic approach pioneered at UCSD. Similar experiments have been identified as "vaccines" in clinical trials undertaken at other institutions. Variations on the theme are still being tried out in clinical trials recruiting patients in mid-2007. There is much interesting science involved in this approach but early results have been disappointing and the technology has yet to demonstrate therapeutic benefit.
One of the trickiest decisions that the immune system has to make on a continuous basis is telling apart friend and foe, "self" versus "non-self". Obviously, in a well functioning body, the immune system kills all pathogens, foreign invaders and the like, but leaves alone the body's own cells. This distinction between "self" and "non-self" is not always easy. Many pathogens have learnt over the millennia how to mimic our own cells, as a way of escaping detection by the immune system. Along the same lines, cancer cells are sometimes so much like perfectly good normal cells that it is impossible for the immune system to see them as a threat, and these malignant cells thereby slip under the radar of immune system surveillance and escape death. This is how cancers such as CLL gain a foothold in our bodies.
One of the ways in which the body protects against "Ooops! Friendly fire accident!!" type of errors is to have several layers of fail-safe mechanisms. It is not enough for the potential target to look like a bad guy, there has to be a secondary re-confirmation that this is indeed an enemy and must be killed. CLL cells display antigens on their surface in the necessary "major histocompatibility complex" (MHC) format . This should make them prime targets for attack by the body's immune system; but in reality, CLL cells are poor targets (poor "antigen-presenting cells") for cell kill because they do not have the second half of the equation, something called co-stimulatory molecules. One of the co-stimulatory molecules that gets a murderous response from T-cells is a marker called CD40. The gene therapy approach at UCSD attempts to up-regulate (increase) the expression of this marker on B-CLL cells, thereby making them better targets for cell kill by T-cells.
This is how it was done: CLL patients underwent leukapheresis and their blood mononuclear cells were isolated. In a nutshell, an adenovirus that has been doctored so that it cannot replicate is used as a vehicle to infect these mononuclear cells with CD154. Think of this protein CD154 as the "soul mate" for CD40. (The scientific name for 'soul mate' is "Ligand"). Another analogy would be CD40 and CD154 are two jigsaw pieces that fit perfectly well with each other, but do not fit with any other pieces. In some papers, CD154 does not even have its own name, it is simply called CD40L, where L stands for ligand. Don't let that confuse you, just remember CD154 is the same as CD40L.
The next step is to infuse the patient with his/her own mononuclear cells, infected with the CD154. (The adenovirus is just a way of infecting the cells, it is just the transportation method and is expected to have no other effect). In the presence of CD154, the B-CLL cells light up and increase the expression of its soul mate, CD40. As we mentioned above, now that the co-stimulatory molecule is also present, the B-CLL cells are confirmed and legitimate targets for T-cell attack. In the process of attacking the B-CLL cells that have been encouraged to express CD40 by means of this gene therapy infusion, the T-cells develop a little more enthusiasm for their work, and also kill some of the bystander CLL cells, even if they have not been tempted into expressing CD40. This is the "by-stander" effect the researchers were hoping for, because it would mean the cell kill would be a lot higher than just the actual CLL cells that were encouraged to express high levels of CD40.
The ultimate prize in these types of approaches is a clear and tempting one: eradication of all CLL cells in the body, cleanly, thoroughly and selectively; a lasting cure for this presently incurable cancer, with none of the toxic side effects of standard chemotherapy.
This is an extremely oversimplified version of the science, almost a cartoon version of it. You can find a lot more of the details in the plenary paper in Blood, describing the reaults of the Phase - 1 conducted earlier. You can access the the article free of charge: Plenary Paper in Blood (Full-text).
Any time I read about somehow subverting or getting around the body's failsafe mechanisms to prevent errors in the "self" versus "non-self" distinction, the first questions that pops into my mind is this: Great, the cancer cells will now be identified and killed, but what are the risks of things getting out of hand? Are we likely to get large numbers of perfectly good and necessary cell lines killed as well, creating auto-immune problems with this newly goosed up immune system? This is always an issue with any type of vaccine therapy. The CD40-CD154 pathway is an extremely potent and multifaceted system, controlling many different aspects of immune function. Triggering of this pathway is very stringently controlled by the body under normal conditions, in order to avoid inappropriate activation of the immune system. In fact, CD154 is expressed only very transiently by the body, just so as not to trigger the CD40 without real need. One of the questions in my mind is the effect of expression of CD154 marker, and hence the triggering of its mate CD40 over long periods of time. In the Phase -2 trial, the five infusions over 10 weeks means this CD40-CD154 pathway was activated strongly, five separate times. The Phase -1 trial confirmed that indeed this approach got the T-cells activated, and the activation lasted for more than a few weeks.
Below are a number of PubMed abstracts, the first one is a very recent review paper that I bought. I will warn you though, it is not light reading. One line quote from this review paper that justifies my concerns regarding autoimmune disease: "However, the potential risk of systemic inflammation and autoimmune consequences remains a concern for systemic CD154-based experimental therapy". The second abstract below also voices the same caution, "Through B lymphocyte activation with CD154-transfected.. cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of auto antibodies". This paper also mentions up-regulation of CD23 as a result of this approach. CD23 on B-cells is strongly associated with cell proliferation, and could potentially cause more rapid accumulation of CLL cells.
The title of the third abstract "Triggering of CD40 antigen inhibits Fludarabine-induced apoptosis in B chronic lymphocytic leukemia cells" is pretty self-explanatory, and raises another question. The more I read, the more convinced I am that the CD40-CD154 pathway controls many different and vital functions of cell survival and proliferation. One question to ask ourselves is how many changes are brought about in the phenotype of the CLL clone, as a result of triggering of CD40 over a period of time as is done in this gene therapy trial? It would be interesting to see if there were any significant changes in the phenotype of the trial participants as a result of the gene therapy. In any case, reduced response to Fludarabine would be a huge issue, if indeed this is the case. But, like much else in cutting edge science in this area, there is still a lot of controversy and ambiguity in the research findings reported in different papers. I would not consider this a cast in concrete finding, just something to keep in mind.
My next comments are based on two quotations from the plenary paper itself, which therefore represent the researchers' own views:
"After treatment, a few patients developed mild, transient elevations in serum transaminase levels. Some patients had prolongation of prothrombin time, hypoalbuminemia, and/or a 40% or less reduction in platelet counts after treatment (data not shown)."
40% reduction in platelets? That certainly got my attention. Was this rather significant platelet reduction as a result of ITP (idiopathic thrombocytopenic purpura, destruction of platelets by antibodies generated against them in the patient's own body, a form of autoimmune disease)?? If after receiving one single shot of the gene therapy infusion, some Phase -1 patients ( how many??) had as much as 40% reduction in platelets (why?? and for how long??), how does one estimate the risk in the Phase -2 trial, where the patients got five infusions of the gene therapy over a period of roughly 10 weeks, at higher dosage levels?
"Patients with CLL are at high risk for spontaneously developing autoimmune hemolytic anemia or immune thrombocytopenic purpura. One model proposes that aberrant expression of CD154 may be responsible for such autoimmunity in patients with CLL. An alternative model proposes that such autoimmunity may reflect immune dysregulation caused by an acquired deficiency of CD154 activity. In this regard, it is noteworthy that patients with congenital lack of CD154 also are at increased risk for developing autoimmune hemolytic anemia and immune thrombocytopenic purpura despite having a profound immune deficiency. In any case, none of the treated patients developed any signs of such autoimmunity despite receiving relatively large numbers of cells expressing CD154."
In the second quote above from the plenary paper, the authors make it quite clear they are aware that one school of thought considers "aberrant expression of CD154" as potential cause of autoimmune disease, both AIHA and ITP, but the UCSD researchers believe this was not seen in their Phase - 1 trail patients. It would be interesting to see if they reach the same conclusion after the Phase -2 trial as well, based on actual results of tests for autoimmune antibodies against red blood cells and platelets, before, during and after the gene therapy. Also of interest would be any direct comparison of patients' CLL phenotypes, before and after the gene therapy, to see if the therapy caused significant changes in markers such as CD23. No doubt such meaningful and precious data was collected during this pivotal trial.
My final comments are these: the CD40-CD154 pathway is an extremely vital and complex system. We are only now beginning to understand some of the intricacies of this beautifully orchestrated and tightly regulated mechanism. Only time will tell if these early efforts at tweaking it to induce potential cures for diseases such as CLL will meet with success, or if we need to go back and fine tune our methods so that we do not run into serious problems of autoimmune disease and/or potential refractory response to important chemotherapy drugs such as Fludarabine. There is a lot of energy, enthusiasm and controversy in many aspects of this elegant and far-reaching research, but in the opinion of this layperson observer, the jury is still out on any definitive conclusions.
Cancer Gene Ther 2003 Jan;10(1):1-13
Prospects for CD40-directed experimental therapy of human cancer.
Tong AW, Stone MJ.
Cancer Immunology Research Laboratory, Baylor Sammons Cancer Center, Baylor University Medical Center, Dallas, TX.
CD40, a member of the tumor necrosis factor receptor (TNF-R) family, is a surface receptor best known for its capacity to initiate multifaceted activation signals in normal B cells and dendritic cells (DCs). CD40-related treatment approaches have been considered for the experimental therapy of human leukemias, lymphomas, and multiple myeloma, based on findings that CD40 binding by its natural ligand (CD40L), CD154, led to growth modulation of malignant B cells. Recent studies also exploited the selective expression of the CD40 receptor on human epithelial and mesenchymal tumors but not on most normal, nonproliferating epithelial tissues. Ligation of CD40 on human breast, ovarian, cervical, bladder, non small cell lung, and squamous epithelial carcinoma cells was found to produce a direct growth-inhibitory effect through cell cycle blockage and/or apoptotic induction with no overt side effects on their normal counterparts. CD154 treatment also heightened tumor rejection immune responses through DC activation, and by increasing tumor immunogenicity through up-regulation of costimulatory molecule expression and cytokine production of epithelial cancer cells. These immunopotentiating features can produce a "bystander effect" through which the CD40-negative tumor subset is eliminated by activated tumor-reactive cytotoxic T cells. However, the potential risk of systemic inflammation and autoimmune consequences remains a concern for systemic CD154-based experimental therapy. The promise of CD154 as a tumor therapeutic agent to directly modulate tumor cell growth, and indirectly activate antitumor immune response, may depend on selective and/or restricted CD154 expression within the tumor microenvironment. This may be achieved by inoculating cancer vaccines of autologous cancer cells that have been transduced ex vivo with CD154, as documented by recently clinical trials. This review summarizes recent findings on CD154 recombinant protein- and gene therapy-based tumor treatment approaches, and examines our understanding of the multifaceted molecular mechanisms of CD154-CD40 interactions.
Eur J Immunol 2001 Dec;31(12):3484-92
Enforced and prolonged CD40 ligand expression triggers autoantibody production in vivo.
Santos-Argumedo L, Alvarez-Maya I, Romero-Ramirez H, Flores-Romo L.
Department of Molecular Biomedicine, Centro de Investigacion y Estudios Avanzados, Mexico, Mexico.
CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39-kDa glycoprotein (CD154) expressed on the surface of activated T lymphocytes and is essential for thymus-dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40-CD40 ligand interaction in vitro and in vivo by using CD154-transfected L929 cells. In vitro assays showed that CD154-L929 cells can induce on B cells: IL-4-dependent proliferation, up-regulation of CD23, CD54 and class II molecules and can also rescue WEHI-231 B cell lymphoma from anti-IgM-induced apoptosis. Interestingly, in vivo assays revealed that when CD154-L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti-erythrocyte autoantibodies. Through B lymphocyte activation with CD154-transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40-CD154 in the induction of primary responses in vivo.
Article in Blood (Full-text)
Blood 1998 Aug 1;92(3):990-5
Triggering of CD40 antigen inhibits fludarabine-induced apoptosis in B chronic lymphocytic leukemia cells.
Romano MF, Lamberti A, Tassone P, Alfinito F, Costantini S, Chiurazzi F, Defrance T, Bonelli P, Tuccillo F, Turco MC, Venuta S.
Dipartimento di Biochimica e Biotecnologie Mediche, Universita Federico II, Napoli, Italia.
We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean +/- SE: 28.5 +/- 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% +/- 7.8% on average (P = .0077). Because the CD40 antigen activates NF-kappaB/Rel transcription factors in B cells, and NF-kappaB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-kappaB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-kappaB/Rel activity; p50, RelA, and c-Rel components of the NF-kappaB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-kappaB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-kappaB/Rel levels. To determine the involvement of NF-kappaB/Rel activity in the G28-5-mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-kappaB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-kappaB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-kappaB/Rel inhibitors, could improve the therapeutic effect of fludarabine. Copyright 1998 by The American Society of Hematology.
The "by-stander effect" observed in the gene therapy trial was most interesting. Good science, but a lot remains to be sorted out. For example, with this massive by-stander effect, where the impact of the CD154 transduction was multiplied ten thousand fold, and presumably there was a similar magnitude impact on the immune response, why was there not an equally large effect seen on the decrease in CLL cells? It was my understanding that in indolent diseases like CLL, the balance between proliferation and apoptosis is only slightly out of kilter. The immune system is not quite up to the job, and the equilibrium is shifted in favor of accumulation of the clonal cells. Well, with this kind of a swift kick in the pants, the immune system should be revving up nicely, and there should be a substantial and sustained decrease in lymphocytes. Some of the patients did not have a very high tumor burden to start with, see Table 1 in the paper. Patients 1, 3, 4, 6, and 9, for example, all had low lymphocyte count prior to treatment, and none of them had required any prior chemotherapy. All were in Rai-stage II, presumably in a W&W mode. One would expect that their immune systems were still pretty intact, just not functioning at par.
I will be the first to admit, this is all very complex stuff, with many inter-linked cascades. Gene therapy is interesting to me because it represents a fundamental shift in the way cancer is viewed: cancer does not have to be killed with massive doses of toxic materials that almost end up killing the patient, but can be destroyed using the body's own subtle yet extremely powerful immune system to fight the battle.
Dr. Kipps did an interview with Andrew Schorr on HealthTalk, where I believe he discussed his take on nurse-like cells that create a protective microenvironment for the CLL cells and help them avoid attack by the immune system. The question I had was how this ties into his gene therapy trial, what impact if any these nurse-like cells have on making the CLL cells better antigen presenting cells by the gene therapy approach.
There was an uproar several months ago about two children with severe combined immune deficiency disease (SCID) who were treated with gene therapy to try and correct the genetic abnormality that led to their condition. While the therapy seemed to be effective, in the case of two of the "bubble boys" there were dangerous side-effects. The therapy used a "gutted" retrovirus vector to carry the genetic information. In theory, the de-activated retrovirus was no more than a vehicle to carry the necessary genetic code to the cells of the patients. In actuality, in the case of these two small patients, the viral vector incorporated itself into an inappropriate location of the cell's genes, causing them to mutate. Both children developed cancer (leukemia).
The distinction has been made between the retrovirus used in the SCID children therapy and de-activated adenoviral vectors used in many of the other gene therapies, that the latter was a lot "safer". For example, the recent Phase - II gene therapy trials for CLL patients at UCSD used a gutted adenoviral vector to transfect the patient's B-cells with CD154. Independent of the efficacy of the CLL gene therapy itself, (please see previous articles on risks associated with chronic CD40 ligation), the expectation was that de-activated adenovirus (basically a 'cold' virus) is a safe vector. Recent research at Stanford suggests otherwise. Attached below is a report of their findings.
There is no indication yet, on the level of potential risk associated with deactivated adenoviral vectors. Upon more investigation, it may indeed prove to be small. But the Stanford researchers have found that the adenoviral vector used in the CLL gene therapy is also capable of causing similar sort of cell damage that led to cancer in the SCID children. In the case of the SCID children, the development of leukemia was an unexpected and startling wake-up call to the researchers. In the case of CLL patients, development of another cancer or more virulent from of CLL may just be chalked up as secondary cancers and transformations often seen in CLL patients. Would these trial participants be monitored and studied over the long term? Would such adverse events, should they happen, even be remarked upon, or examined in detail to see if the adenoviral vector had anything to do with it? I would sincerely hope so.
The biggest value of early phase clinical trials is the information and knowledge we gain from them. Negative results teach us just as much, if not more, as positive results.
Here is an article reporting on the safety of a gene therapy experiment at Stanford.
Harmful potential of viral vectors fuels doubts over gene therapy.
By: Erika Check
Doctors at Stanford treat a haemophiliac in a gene-therapy trial — but how safe is the procedure?
The troubled field of gene therapy was dealt a fresh blow this week, after a study suggested that modified viruses used in some trials might cause health problems.
The study, led by geneticist Mark Kay at Stanford University, California, examined a modified virus used in gene-therapy trials to treat haemophilia and cystic fibrosis. It revealed that the virus has the potential to cause the same problems that led to cancer in an unrelated gene-therapy trial last year.
In gene therapy, doctors use a gutted virus as a 'vector' to transfer corrective genes into a patient's cells. But if the vector stitches itself into a cell's genes, it can cause the cell to mutate and become cancerous. This was demonstrated last year, when two children who had gene therapy for severe combined immunodeficiency disease (SCID) developed leukaemia (see Nature 419, 545–546; 2002).
Scientists are still trying to establish exactly why the SCID patients developed cancer, and will discuss the trial at this week's meeting of the American Society of Gene Therapy in Washington DC. But most agree that gene therapy was the cause.
Kay's study focused on a vector made from an adeno-associated virus — an organism that does not cause disease in people, but which can be engineered to infect human cells. In a paper published online on 1 June (H. Nakai et al. Nature Genet. doi:10.1038/ng1179; 2003), Kay and his colleagues show that the vector used in the haemophilia and cystic fibrosis trials integrates itself more often into genes than it does into regions of DNA that do not contain genes. The finding suggests that the vector could potentially cause the sort of cellular defects that led to cancer in the SCID patients.
But researchers caution that the vector used in the SCID trials, which was based on a retrovirus, is very different from the adeno-associated vector. For instance, retroviruses must insert themselves into human DNA to work, but adeno-associated viral vectors integrate themselves into the genome much less often.
"Adeno-associated vectors clearly have a better safety profile than retroviral vectors," says David Russell, a geneticist at the University of Washington in Seattle. "But we really can't say yet that adeno-associated vectors won't cause cancer."
Kay's team, which is running a gene-therapy trial for haemophilia, tracked the adeno-associated viral vectors in mice. They extracted liver cells whose DNA contained the vector and then sequenced the DNA around the vector. They then analysed the sequences to see whether they matched a known gene. The team found that 72% of the time, the vector had interrupted a gene. Had it inserted itself randomly, the vector would have interrupted a gene no more than 40% of the time.
Last August, Frederic Bushman and his colleagues at the Salk Institute for Biological Sciences in La Jolla, California, suggested that retroviruses also insert themselves into genes more often than into other regions of DNA (A. R. Schroder et al. Cell 110, 521–529; 2002).
Such results are leading researchers to seek better ways to target vectors to specific regions of DNA, and to develop vectors that don't integrate into DNA at all. But in the meantime, Kay says that he has taken numerous precautions to protect the 14 haemophiliacs he has treated.
"I don't think we need to modify anything at this point," Kay says. "But this is a risk we'll have to address before the vector is in widespread use."
The following correspondence was received from Dr. Thomas J. Kipps at the University of California at San Diego on September 24, 2003, addressing certain topics discussed above. It is reproduced here with his permission. We appreciate his feedback. Discussion such as this with leaders in the research community offers valuable insight for our members. It is a priority for us to develop such dialogue with the entire research community. We would like to thank Dr. Kipps for taking the time to write.
From: Thomas Kipps, MD
Sent: Wednesday, September 24, 2003 11:07 PM
To: Chaya Venkat
Subject: Website article 8/28/03
I had the pleasure of visiting your website as it was brought to my attention by a patient with CLL. I agree with you that it is important to recognize that clinical trials, particularly phase I/II trials involving highly novel therapies, have unknown risks. However, I hope you agree that it is important to keep to a minimum speculation on risks that is based upon inaccurate information or misleading associations. In your article posted on 8/28/03, you address the risks of adenoviral vectors, citing work done at Stanford. However, the work done at Stanford is on adeno-associated virus vectors, which are quite different from adenovirus vectors. The former has the capacity to integrate into the host cell's genome and thereby potentially cause insertional mutagenesis. Such integration into the genome has not been observed with adenovrius vectors, which maintain themselves in an episomal state and become diluted out with subsequent cell replication. In the gene therapy trial at UCSD we used adenovirus vectors, not adeno-associated virus vectors.
The problem observed in the SCID gene therapy trial resulted from the fact that the French investigators used retrovirus vectors. Retrovirus vectors insert their genetic material into the host cell's genome. Thus the genetic material is passed on from cell to daughter cell in perpetuity, allowing for long term transgene expression. However, as noted with adeno-associated viruses, this runs the risk for insertional mutatagenesis, which apparently was the cause for the leukemias identified in the SCID gene therapy trial. This risk has not been encountered with adenovirus vectors.
Evaluating new approaches to treat CLL (e.g. new drugs, mAbs, gene therapy, T cell therapy) is important, as what currently is available does not appear satisfactory. I hope we can define better and curative treatments for this disease. To do this, we need to develop new treatment strategies that unfortunately will have unknown risks despite our best efforts in pre-clinical pharmacologic/toxicology testing. With each new approach there are pros and cons along with regulatory hurdles and reasons for not moving forward. On top of this is the cost for conducting the trial. However, it is only through clinical testing that we can identify new therapies that potentially can cure this disease.
By evaluating the responses observed in clinical trials, we hopefully can improve on any one approach that proves to have clinical/biologic activity and mitigate some of the toxicity associated with its use.
Sometimes one approach might ultimately only prove effective when used in combination with another. However, it is generally necessary to test each approach alone before using it in combination with something else so as to learn about its own potential for toxicity and/or clinical activity.
I agree that the patients undergoing clinical trials are the true heros in this whole story. I admire the fortitude of the patients who enroll in such trials and am very concerned about their welfare. The CRC is committed to moving this field forward and as fast as possible. However, I still am not satisfied with the pace of things and wish we could do more to benefit our patients. I appreciate your help and support in this important effort.
Thomas J. Kipps, M.D., Ph.D.
Director, CLL Research Consortium (CRC)
Professor and Deputy Director for Research
UCSD Cancer Center
University of California, San Diego
Here is a abstract on another version of Gene Therapy for CLL. There are a lot of similarities between this one and the trial at UCSD. Both use viral vectors to transport the genetic material into the cell of choice. In the case of the Kipps' trial, the target cells were B-cells, and a de-fanged adenovirus was used as the vector. The genetic information transduced into the cells is CD154. In the case of this new approach, the viral vector is a retrovirus, and the cells infected ("transduced") are the T- cells, and they are transduced with an artificial T-cell receptor called 19z1. The idea is that T-cells armed with this new receptor will attack B-cells carrying the CD19 marker. As you are probably aware, most B-cells display the CD19 marker, but not stem cells. So presumably little harm is done to the stem cells as a result of this approach. So far, the new approach has been limited to CLL cell lines. There are many steps and much time between that and human trials. The abstract below is from ASCO 2000.
I continue to be impressed by how much we are learning about the genetic fundamentals of this disease. Someday, not too far in the future, we might actually be able to cure it through these sorts of approaches. I just wish it is not so hard to wait, when we have people we love whose lives are on the line.
Genetic modification of peripheral blood T cells from patients with chronic lymphocytic leukemia results in cytotoxic activity against autologous tumor cells.
Year: 2002 Abstract No: 1100 Category: Leukemia, Adult
Author(s): Renier J Brentjens, Jean Baptiste Latouche, Mark Weiss, Isabelle Riviere, Michel Sadelain, Memorial Sloan-Kettering Cancer Center, New York, NY.
Indolent B cell malignancies remain largely incurable. Currently, various immuno-therapies, including anti-idiotype immunization and antibody therapy, are being developed to treat these cancers. CD19 is expressed by a majority of both acute and chronic B cell malignancies, but not by hematopoietic stem cells and is therefore an attractive target for immune-based therapy. We have recently developed an artificial T cell receptor, termed 19z1, that recognizes CD19. Human peripheral blood T cells transduced with a retroviral vector encoding 19z1 lyse CD19+ tumor cells in vitro and are readily expanded by co-cultivation on fibroblasts that express CD19 and the co-stimulatory molecule B7.1. We have established that SCID-Beige mice, bearing systemic Raji Burkitt lymphoma tumors, are cured when treated intravenously with 19z1 transduced T cells. In order to further demonstrate that adoptive T cell therapy with genetically modified T cells is feasible, we isolated primary peripheral blood lymphocytes from 4 patients with advanced chronic lymphocytic leukemia (CLL). Isolated T cells rapidly expanded after stimulation with PHA, and were subsequently readily transduced with the 19z1 retroviral vector. 19z1+ T cells obtained from 4 of 4 patients specifically lysed both Raji as well as autologous CLL tumor cells in vitro. These data demonstrate that the 19z1 receptor is functional in T cells from patients with CLL, and further indicate that adoptive therapy with 19z1-transduced autologous T cells can be developed into an effective treatment for patients with B cell tumors.
The goal of gene therapy is to insert a healthy copy of a gene into a cell where it can take over for a faulty version, or a missing gene.
Current gene therapy approaches use an inactivated virus to carry the therapeutic gene into the host cell. However, the gene inserts itself into the chromosome at random positions. This means there is a chance that if the gene lodges in the wrong position it may trigger unwanted and dangerous side effects. This is what is thought to have happened in the French "bubble boy" case, where insertion of the therapeutic gene into the wrong position turned "on" a cancer gene, and the patient developed leukemia.
The new technique is able to introduce genetic material into a host in a more precise way. It also does away with the need to use a virus to transport genetic material into the body. The technique is based on mechanism used by a virus called a bacteriophage that infects bacteria. It does this by producing a protein that guides its DNA to a specific site on the bacteria chromosome.
If proven in human clinical trials, this process can be extremely valuable for treatment of cancers, viral infections and many genetic disorders. For example, you are aware of the p53 gene, that is a tumor suppressor gene. A substantial percentage of CLL patients have defective p53 gene, or it is entirely absent, as a result of genetic mutation. CLL patients with p53 mutation or deletion have a poorer prognosis, and respond less effectively to therapy. This is also true of many other cancers besides CLL, including a large number of solid cancers.
A company called Introgen Therapeutics (historically affiliated with M. D. Anderson) has been exploring p53 gene insertion, but using the usual adenovirus as the carrier. The first URL link below discusses their technology in various cancers. The second URL link takes you to the BBC site that discusses the new approach to inserting the desired genes, but without using viruses as carriers.
A member raised a question regarding the differences between the UCSD gene therapy trials and the protocol used in the French "bubble-boy" experiment with specific regard to the question of the vaccine DNA lodging in an untargeted region.
Gene therapy protocols are very complex, and indeed the devil is in the details, so I cannot say that the protocol used in the French "bubble boy" trial is similar in all respects to the gene therapy trials under way at UCSD for CLL patients. Yes, the UCSD trial also uses adenoviral vector to get the genetic information into the cells. I suppose in this respect the two trials have something in common. However, I do not know enough about the rest of the details to know if a similar risk profile exists in the UCSD trial as well. The quote below is from the plenary paper on the phase-I trial, by Dr. Weirda, confirming the use of an adenoviral vector. Please refer to earlier articles in CLL Topics when this paper was discussed in great detail.
"The CLL B cells can be transduced in vitro with high-titer, replication-defective adenovirus vector to express high levels of transgene.21 A replication-defective adenovirus vector was constructed using serotype 5 adenovirus in which the E1 region of the virus genome was replaced with the gene encoding murine CD154"
I would like to point out a couple of things:
I suppose that is the role of patient advocacy groups such as this one, to collect and provide the information in a manner that is digestible to the layperson, and give human context to the clinical results. The job would be a lot easier, if there was more direct contact and cooperation between the patient groups and clinical researchers. If I were in charge of the world (thank heavens I am not!!), I would make sure all clinical trial and technology review boards had patient representatives on them. Taking nothing away from our hard working scientists and researchers, or their life long devotion to their chosen fields, still when rubber meets the road, it is the patients who risk their lives in clinical trials. Surely they should have some say in the management of the process.
I have written before about potential T-cell therapy (see CTL Therapy). The idea is to grow large populations of T-cells and/or NK cells ("Natural Killer" cells) outside the body, then inject these fighting armies back into the patient to attack the cancer cells. Since the T-cells and/or NK cells are grown from the patient's own system, there is little danger of graft versus host type of problems. But one of the concerns has been that T-cells and NK cells grown from CLL patients are somehow not very efficient in killing the patient's cancer cells. For this approach to work, they need to be made more effective in some fashion.
Here is a very interesting take on this idea. We know now that Rituxan works by attaching itself to the CD20 marker on B-cells, and thereby painting a big bulls-eye on these cells. While some percentage of the cell killing may be happening by action of the Rituxan alone, the bulk of the cell kill is brought about by the T- cells and NK cells of the patient attacking the B-cells "lit up" by the Rituxan molecule attached to them. This is the so called "Complement Dependent Cytotoxcity" (CDC) and "Antibody Dependent Cellular Cytotoxicity" (ADCC). Part of the reason not to delay Rituxan therapy till too late is that when the tumor burden is high, the numbers and efficiency of the T-cells and NK cells is compromised, and while the Rituxan dutifully "lights up" the CLL cells, there are few efficient T-cells and NK cells to carry out the actual assassinations.
This ASH abstract below describes a neat way of solving both problems at once. The Italian team has succeeded in culturing and expanding populations of NK cells outside the body. The idea is to combine infusion of these armies of NK cells with simultaneous treatment with Rituxan, so that the combination can bring about the required level of cell kill. The Rituxan helps in making the CLL cells easier to kill, and the externally grown army of NK cells means there are enough of them to do the job. The idea seems to work thus far in in-vitro experiments, with a dramatic increase of seven times higher cell kill when Rituxan is combined with the expanded NK-cell army in fighting the CLL cells. The Italian team says they are about to embark on clinical trials soon, based on these results.
Here is a crazy idea that struck me as I was reading the abstract. We have seen in the Gene Therapy plenary paper published last year that in the Phase-1 study T-cell populations are increased as much as ten times their pre-treatment levels, as a consequence of the Gene therapy. The peak in the T-cell concentration happened a couple of weeks after the infusion. If we were to assume that the Phase-2 trial with five infusions of the GT has had a similar and hopefully even more substantial increase in the T-cell (and NK cell) populations in the GT trial participants, these guys may now prime candidates for Rituxan therapy.
Using the analogy of striking while the iron is hot, it seems to me that the Rituxan therapy should be started when the T-cell and NK cell populations are at their peak, to get maximum bang for the buck. What do you guys think, does this sound reasonable? Perhaps here is one way for our GT participants to get a solid, long lasting remission out of their participation in the GT trial, by taking advantage of the boosted numbers of T-cells and NK-cells. The more I think about it, the better I like it. But then, I am not a doctor, who knows what else is involved that I am missing completely.
In Vitro Expanded NK Cells Combined with Rituximab Can Efficiently Lyse Autologous B-CLL Cells and Overcome the Natural Resistance to NK Cell Mediated Killing.
Raewyn Broady, Josée Golay, Rosanna Gramigna, Valeria Facchinetti, Gianmaria Borleri, Anna Salvi, Tiziano Barbui, Martino Introna, Alessandro Rambaldi.
Hematology Division, Ospedali Riuniti, Bergamo, Italy; Laboratory of Molecular Immunohematology, Istituto Ricerche Farmacologiche Mario Negri, Milano, Italy
We have previously reported that in vitro, the chimeric anti CD20 monoclonal antibody Rituximab and complement can efficiently lyse freshly isolated B cells from some patients with PLL and B-NHL but is less active against B-CLL (Golay J. et al: Blood 2001). In addition to this low complement dependent cytotoxicity (CDC) we also documented that antibody dependent cellular cytotoxicity (ADCC) is similarly modest. Results of clinical trials of Rituximab in B-CLL are in keeping with these laboratory data. Leukemic cells from patients with B-CLL appear to be totally resistant to autologous NK effectors and in most cases are also resistant to normal allogeneic NK cells. Given that the action of Rituximab is thought to involve ADCC through NK cells, combining Rituximab with ex vivo expanded, activated NK cells may increase the efficacy of killing through enhancing ADCC. In this study we assessed (1) the potential of expanded and purified NK cells to lyse B-CLL targets in vitro, (2) the possibility to expand and purify NK cells from B-CLL patients and (3) whether in vitro expanded NK cells are cytotoxic against autologous leukemic cells alone or in the presence of Rituximab. NK cells were expanded and enriched by co-culturing blood mononuclear cells from normal donors with an irradiated feeder layer (EBV+ lymphoblastoid B-cell line RPMI 8866) which allowed an average 43 fold increase in NK cells with a final purity of >90%. Using standard 51Cr release assays, immunomagnetically purified CD56+/CD3- NK cells exhibited modest activity against a B-CLL cell line (MEC 2) but cytotoxicity was increased by the addition of Rituximab. Similar results were obtained when using the NK cell line, NKL. Using freshly isolated B-CLL cells as targets and purified allogeneic NK cells as effectors, the lytic activity was low (<20% at E:T ratio of 4:1). Addition of Rituximab significantly enhanced killing of the CLL targets (>60% at E:T ratio of 4:1). In contrast, none of the samples examined were lysed by PBMCs or the NKL cell line either in the presence or absence of Rituximab. Using the same co-culture method we could further demonstrate that NK cells could also be expanded from the peripheral blood of B-CLL patients in the presence of active disease. This approach allowed a 5-10 fold NK cell expansion with an average purity prior to immunomagnetic selection of 80%, with no B-CLL cells remaining at the end of the 10 day culture period. These NK cells did not show a significant cytotoxic potential against autologous B-CLL cells (<10% at E:T ratio of 4:1) but their lytic activity was dramatically increased by the addition of Rituximab (>70% at E:T ratio of 4:1). These data suggest that the administration of monoclonal antibody with NK cells may enhance the efficacy of autologous NK cells in B-CLL. On the basis of this in vitro data, a therapeutic trial is planned to determine if the combination of Rituximab and ex vivo expanded autologous NK cells will be efficacious for patients with B-CLL.
It must be admitteed that this business of growing NK cells or T-cells outside the body is still an art form. Many different approaches are being tried, both to grow large populations of the immune system cells, as well as training them to kill cancer cells efficiently. I think we will see well constructed and large scale clinical trials based on the best of these approaches in the next couple of years.
The abstract below was written about a month back, and deals with the same sort of concept. I am reproducing it, and some of the discussion that went with it, because I think it is of topical interest in view of our discussions on Gene Therapy. The abstract deals with combining Interleukin - 2 (IL-2) with Rituxan. The patient group is NHL, and the results are encouraging, given the patients were refractory to start with.
The downside of IL-2 therapy in the early days was its toxicity, which is dose dependent. In the early years of AIDS therapy, patients died due to IL-2 toxicity, because the doses were too high. We now know a lot more about dosages and how to control the side effects. These side effects include fevers, chills, rigors, sweats, muscle and joint pains, nausea, vomiting and fluid retention. All of the usual fun stuff that we have come to enjoy and appreciate in therapy. Actually, some one made the comment that the side-effects of the Gene Therapy at UCSD were startlingly similar to the side effects of IL-2 therapy!
The thought crossed my mind, perhaps it may be that the Gene Therapy increases levels of IL-2 within the body, which then led to increased production of T-cells, which led to CLL cell kill. In fact, the plenary paper describing the Phase-1 Gene Therapy trial reported substantial increases in various interleukins, and boosted T-cell populations, after the Gene Therapy infusion.
It would be ironic if one could have achieved much of the benefits of the Gene Therapy by just taking IL-2 injections: a heck of a lot cheaper, less risk of the unknown, no waiting and hoping to get into the trial, and better understood therapy in terms of dosage and toxicity. But I will be the first to admit, perhaps the Gene Therapy achieved much more complex goals, and I am not competent to either understand or comment on these intricacies. This is not false modesty on my part, the more I read of immunobiology, the more amazed I am by the tremendous complexity.
I understood that Dr. Kipps was speculating about the potential of combination of Gene Therapy and Rituxan, at some presentation or the other. If that is the case, tacking on Rituxan therapy at the end of the Gene Therapy protocol may be just the right thing to do for some patients. I know a lot of people were disappointed because they could not get into the Gene Therapy trial. Wouldn't it be great if they could get much of the same effect with a simpler and more accessible IL-2 plus Rituxan therapy?
Rituxan as frontline single agent therapy is very attractive to a lot of us, because it is not your standard issue chemotherapy drug. The side effects from the infusion are relatively mild, and generally limited to the first infusion for most patients. Unlike older chemotherapy drugs such as Fludarabine and Cyclophosphamide, Rituxan is thought to be far less damaging to the bone marrow. The problem is that Rituxan does not do a thorough job by itself. Complete remissions are few and far between, and there are no PCR negative responses in CLL patients using Rituxan as single and frontline therapy, not to my knowledge. The punch is much, much stronger when Rituxan is combined with other chemotherapy drugs such as the famous RFC (Rituxan, Fludarabine and Cyclophosphamide) combo. But this gets us right back to worrying about the toxicity of the "F" and "C".
Remember, majority of the cancer cell killing in Rituxan therapy is done by the body's own immune system, the monoclonal tags the cancer cells and thereby paints a big "kill me" sign on them, making them more vulnerable and more readily recognized and attacked by the immune system. Well, it stands to reason that in CLL patients with compromised immune systems, anything that boosts the immune system is going to help the efficiency of Rituxan. There seem to be many approaches to doing just that, boosting the population numbers and effectiveness of the T-cells and NK-cells, prior to use of Rituxan: one approach is to grow these immune system cells outside the body and then inject them back. Another approach is using things like IL-2. Perhaps a third approach is the Gene Therapy. Of the three, it seems to me that the combination of Rituxan with other immunomodulatory drugs such as IL-2 (or GM-CSF, as in a previous article) is the approach that is within our reach right now.
Br J Haematol 2002 Jun;117(4):828-34
Combination immunotherapy with rituximab and interleukin 2 in patients with relapsed or refractory follicular non-Hodgkin's lymphoma.
Friedberg JW, Neuberg D, Gribben JG, Fisher DC, Canning C, Koval M, Poor CM, Green LM, Daley J, Soiffer R, Ritz J, Freedman AS.
Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.
Rituximab has significant activity as a single agent in the treatment of follicular non-Hodgkin's lymphoma (NHL). Interleukin 2 (IL-2) is a lymphokine that increases effector cell number. In an effort to augment antibody-dependent cell-mediated cytotoxicity (ADCC) associated with rituximab therapy, low-dose IL-2 was added to a standard rituximab regimen and patients were evaluated for safety and efficacy. Twenty patients with relapsed or refractory follicular NHL were treated with IL-2 (1.2 MIU/m(2)/d for 56 d subcutaneously) as outpatients. Rituximab (375 mg/m(2)) was given on d 15, 22, 29 and 36. The regimen was well tolerated and only three patients required dose adjustments in IL-2. Infusional toxicity associated with rituximab was not exacerbated by IL-2. Peripheral blood immunophenotyping demonstrated significant increases in circulating CD8+ and CD56+ lymphocytes in all evaluable patients (P = 0.0002). Increases in total eosinophil number were observed in all patients. Eleven patients responded to therapy, for an overall response rate of 55%. Four additional patients had stable disease. For these 15 patients, the median time to progression exceeded 13 months. We conclude concomitant cytokine therapy to enhance ADCC with monoclonal antibody therapy was well tolerated and did not exacerbate antibody- related infusional toxicity. Further studies of this rational combination are warranted and ongoing.
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Topic: Therapy Choices