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    LMB-2

    Date: April 13, 2024

    by Chaya Venkat

    New Phase II Clinical Trial

    Related Articles: Monoclonal Antibody Review

    CAT8015 – A Trojan Horse

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    I would like to bring to your attention a brand new Phase - II clinical trial that has just been announced (Published: 2024-03-24). I have done a little bit of digging and I like what I see thus far. It uses a monoclonal antibody that targets interleukin-2 receptors (IL-2R) on B-cells, tethered to an immunotoxin which kills the cells once they have been tagged by the monoclonal part.

    Rituxan and Campath are two monoclonals that are familiar to most of us, which target the CD20 and CD52 markers, respectively, on CLL cells. A limitation in the use of unmodified monoclonal antibodies such as Rituxan and Campath is that they are relatively ineffective at the actual cell kill. They need assistance from the body's own immune system to finish the job. That is exactly where this new drug called LMB-2 struts its stuff. For starters, it carries its own cell kill mechanism with it. Think of the legendary Trojan Horse - this new monoclonal drug comes close to that concept.

    I like this clinical trial because it is innovative without being wild-eyed: it packs sufficient punch to actually have a chance of getting good responses and it is not yet one more combination or permutation of Rituxan with purine analogs and alkylating agents. I like it that it already in phase -II trials after very positive results in other blood cancers such as Hairy Cell Leukemia (a close cousin of CLL) and I particularly like it that they are already finding ways of improving its usefulness in CLL. The technology patents are owned by the National Institute of Health, but available for licensing to drug companies. Do take the time to read about LMB-2. This is one clinical trial we should be following closely. The clinical trial location, as well as eligibility criteria and contact information are given at the end of this article, in case you would like to participate.

    Background

    Interleukin -2 (IL-2) is a cytokine that exerts its effect by attaching itself strongly to its receptors (IL-2R) on activated T cells, natural killer (NK) cells and B cells. One segment of the IL-2R is of particular interest to us because resting normal cells do not express it whereas it is heavily expressed by the abnormal cells in certain forms of lymphoid cancers such as adult T cell leukemia/lymphoma (ATL), B-cell hairy cell leukemia, and Hodgkin's disease. This cancer-specific part of the IL-2 receptor is called by several names: IL-2R, Tac, CD25. We will use the name CD25 for this important marker on cancer cells. If you see the other names in literature, do not let it flummox you. Antibodies targeted against this marker on cancer cells are referred to as anti-IL-2R, anti-tac, or anti-CD25 respectively. They mean the same thing.

    Several monoclonal antibodies that attach to CD25 have been made (daclizumab, Zenapax). If you have read some of our extensive list of articles on Rituxan, you know by now that monoclonal antibodies are "Y" shaped molecules with very shape-selective "pincers" at the tips of the arms of the "Y", such that they will grab on to and hold only one type of molecule or marker that fits the particular shape of their pincers. Monoclonal antibodies tend to be pretty large molecules, and sometimes this can contribute to problems in getting the drug where it is needed most, in bulky lymph nodes or packed bone marrow. The imagery that comes to mind is a fat lady trying to squeeze through a narrow doorway into a crowded room with lots of bulky furniture around. Not a pretty sight.

    The drug we are discussing here, LMB-2, uses just one of the arms of the "Y". It is therefore shaped like a slim and slender stick, instead of a bulky "Y", with one pincer at one end of its svelte shape. The pincer is shaped just right to attach to the CD25 markers on cancer cells. The other end of this abbreviated monoclonal is attached to a fragment of a toxin from a bacteria called Pseudomonas. A very common bacteria, it lives in the soil all around us. The toxin fragment is carefully chosen not to bind to any of our cells by itself, it just goes along for the ride with the LMB-2 monoclonal. In other words, it is a passive back seat passenger that is unable to attach to, or damage any cell by itself. The toxin fragment can go into action only when the LMB-2 part attaches to a cell that expresses CD25. As usual, this is a bit of an over simplification but close enough for government work.

    This is where the Trojan Horse part comes in. Normally, CD25 markers are anchored on the surface of cancer cells - sitting on the front porch and watching the world go by, as it were. But if they are tagged by the appropriate molecules, LMB-2 in our case, they get into a panic and scuttle off the porch and into the house, slamming the front-door shut. In this process of withdrawing into the innards of the cell, they take with them the LMB-2 now attached to them. Once inside, the toxic payload carried by the LMB-2 can do its work.

    In real life, each cancer cell has hundreds, even thousands, of the CD25 markers. As each one of these is tagged by an LMB-2 molecule, it withdraws into the cell and brings inside the toxin attached to it. Pretty soon the toxin load gets to be too much for the cell. It turns out that the Pseudomonas toxin fragment prevents the cell from synthesizing proteins it needs to live, literally starving the cell in a matter of hours. The cell is killed from the inside, quickly and efficiently. Since this mechanism of cell kill is quite novel, even cancer cells that have learned to subvert and get around other cell kill mechanisms are vulnerable. In other words, patients who have become resistant to other drugs, or have poor cytogenetics that prevent normal apoptosis (suicide) pathways working in such cancer cells, are hard to treat. But even they are likely to respond to LMB-2.

    LMB-2 is very cytotoxic to various cells expressing CD25. Most HCL and T-cell leukemia cells express high numbers of CD25 molecules and are therefore very responsive to LMB-2. However, B-CLL cells tend to be generally less sensitive to LMB-2, partly because the numbers of CD25 sites per cell are considerably lower in B-CLL compared with HCL. The good news is that there are ways of getting around this, as we discuss below.

    Early Clinical Trial Results

    Below is an abstract that describes the pivotal Phase -1 clinical trial done at the NCI, Bethesda, looking at the efficacy of LMB-2 in a number of blood cancers. If you are interested in reading the full text of this article, write to us at email and we will help you locate it.

    The main purpose of Phase -1 clinical trials is to establish the right dosage, get a fix on potential toxicity and determine if the drug has sufficient activity to merit further study. Researchers also want to know how long the drug hangs around in the patient ("pharmacokinetics"), whether it reaches all the nooks and crannies of bulky lymph nodes (bioavailability and tissue distribution) and if patients are likely to develop allergic reactions and antibodies to the drug itself. In this study, 35 patients with a mix of blood cancers were enrolled. All of the participants had become refractory and had even failed salvage therapies. Typically these are the hardest patients to treat and any kind of a response to therapy is good news. The results with LMB-2 were surprisingly good, given the nature of the patient group selected and the Phase - I stage of the study.

    Out of the 8 CLL patients enrolled, only one got a good remission. But boy, was this case history a doozy! The patient's WBC count was high, at 230,000, and he had had relapsed after treatment with chlorambucil, chlorambucil/prednisone, cyclophosphamide/ vincristine/prednisone, and fludarabine. Phew! This guy had been through the chemo wars, had every chemotherapy drug known to man (almost), and had full-blown stage 4 disease with complicating factors such as autoimmune anemia (AIHA), splenectomy, frequent infections, massive lymph nodes, some liver problems as well as multiple pulmonary emboli that required use of blood thinners. The first cycle of LMB-2 (50 mg/kg repeated three times per week) was complicated by fever brought on by pneumonia. But the WBC decreased by 50% during even this first cycle and stabilized there. The patient's massive cervical lymph nodes also resolved after the first cycle. The next two cycles of LMB-2 were well tolerated, without further opportunistic infections. After three rounds, the patient got a response of 70% to 80% as well as getting rid of all his symptoms. At that point he elected followed-up with no further therapy. His WBC increased very slowly during the 5 months follow-up. Not bad!!

    Abstract

    J Clin Oncol. 2024 Apr;18(8):1622-36.

    Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies.

    Kreitman RJ, Wilson WH, White JD, Stetler-Stevenson M, Jaffe ES, Giardina S, Waldmann TA, Pastan I.

    Laboratory of Clinical Pathology, Metabolism Branch, Medicine Branch, and Biopharmaceutical Development Program, Science Applications International Corporation Frederick, National Cancer Institute, National Institute of Health, Bethesda, MD 20242, USA.

    PURPOSE: To evaluate the toxicity, pharmacokinetics, immunogenicity, and antitumor activity of anti-Tac(Fv)-PE38 (LMB-2), an anti-CD25 recombinant immunotoxin that contains an antibody Fv fragment fused to truncated Pseudomonas exotoxin.
    PATIENTS AND METHODS: Patients with CD25(+) hematologic malignancies for whom standard and salvage therapies failed were treated with LMB-2 at dose levels that ranged from 2 to 63 microg/kg administered intravenously over 30 minutes on alternate days for three doses (QOD x 3).
    RESULTS: LMB-2 was administered to 35 patients for a total of 59 cycles. Dose-limiting toxicity at the 63 microg/kg level was reversible and included transaminase elevations in one patient and diarrhea and cardiomyopathy in another. LMB-2 was well tolerated in nine patients at the maximum-tolerated dose (40 microg/kg QOD x 3); toxicity was transient and most commonly included transaminase elevations (eight patients) and fever (seven patients). Only six of 35 patients developed significant neutralizing antibodies after the first cycle. The median half-life was 4 hours. One hairy cell leukemia (HCL) patient achieved a complete remission, which is ongoing at 20 months. Seven partial responses were observed in cutaneous T-cell lymphoma (one patient), HCL (three patients), chronic lymphocytic leukemia (one patient), Hodgkin's disease (one patient), and adult T-cell leukemia (one patient). Responding patients had 2 to 5 log reductions of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous masses and splenomegaly. All four patients with HCL responded to treatment.
    CONCLUSION: LMB-2 has clinical activity in CD25(+) hematologic malignancies and is relatively nonimmunogenic. It is the first recombinant immunotoxin to induce major responses in cancer. LMB-2 and similar agents that target other cancer antigens merit further clinical development.

    PMID: 10764422
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    Improving Response of CLL to LMB-2

    In preclinical studies with cell lines and mice as well as in clinical trials with actual cancer patients, LMB-2 has demonstrated high cytotoxic activity against tumor cells that express CD25. HCL is particularly sensitive to LMB-2, mainly because these cells express very high levels of the CD25 marker. However, LMB-2 is not as toxic to CLL samples.

    The good news is that expression of CD25 on B-CLL cells is not cast in concrete. It can be changed during the course of immune response to pathogens. We have discussed in previous articles the effect of bacterial DNA on human immune systems (Enhancing the Effectiveness of Rituxan with Bacterial DNA). Over the millennia the immune systems of mammals have developed a healthy respect for and fear of bacterial invasion. Since bacterial DNA is different from our own, it is immediately recognized as foreign and very dangerous. Unmethylated CpG dinucleotide motifs (just jargon words, you do not need to know what exactly they mean) are present in bacterial DNA but not in mammalian DNA. Our bodies have evolved to recognize this difference and mount an immediate and potent immune response at the first sight of bacterial DNA. Part of this immune response is an inflammatory process, which activates monocytes, dendritic cells and B cells. Since they are good at getting the immune system primed and ready to kill, CpG-ODNs have been used to help boost monoclonal antibody therapy. In fact, CpG-ODNs in combination with Rituxan are currently being evaluated in phase I/II clinical studies in CLL and NHL.

    The abstract below describes the effect of a CpG-ODN drug called DSP30 on CD25 expression of B-CLL cells (and you can read the full text for free - just click on the link provided in the abstract). Pre-treatment of CLL cells with this bacterial DNA snippet increased CD25 expression several fold. There was clear and conclusive evidence that this up-regulation of the CD25 marker made the CLL cells that much more responsive to LMB-2 treatment. It is a little bit of a one-two punch, the DSP-30 sets up the CLL cells: the bacterial DNA fragment scares the living daylights out of the immune system, forcing the CLL cells to kick into an activated state ready to do battle with the presumed hordes of invading bacteria. One of the consequences of immune activation is strong up-regulation of the crucial CD25 marker. The scene is now set for the second punch: LMB-2 attaches itself to the thousands of CD25 markers on CLL cells. As we discussed above, the CD25 marker is promptly drawn into the inside of the cell, along with the LMB-2 and its payload of Pseudomonas toxin. Enough of the toxin accumulating within the CLL cell soon causes breakdown in all protein synthesis and the cell has no choice but to die.

    Sensitization of B-CLL cells by means of bacterial DNA to the ravages of an immunotoxin such as LMB-2 could be a potentially harmful treatment approach if the toxicity toward normal lymphocytes were also increased. After all, our goal is to kill CLL cells but spare good B-cells as well as other innocent cell lines. One of the conclusions of this paper is that the CD25 up-regulation by DSP30 was much stronger in B-CLL cells than in normal B cells. In other words, DSP30 gives us a specific way in which to increase the sensitivity of CLL cells to death by LMB-2, while sparing the good B-cells. This sounds very good to me. Of course, an added benefit is that CpG-ODN's such as DSP30 can also result in additional antitumor effects. The antitumor activity is most likely related to activation of NK cells, and production of cytokines with anticancer activity such as interleukin-12, tumor necrosis factor-alpha. Every little bit counts - it is in a good cause.

    Abstract

    Blood. 2024 Feb 15;99(4):1320-6.

    Blood Journal Article

    Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides.

    Decker T, Hipp S, Kreitman RJ, Pastan I, Peschel C, Licht T.

    3rd Department of Medicine, Technical University of Munich, Munich, Germany

    A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC(50)) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.

    PMID: 11830482
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    Potential Toxicity with LMB-2

    Anytime something sounds this good, I get a little anxious about potential side effects. It is great to kill CLL cells in droves, but not if at the same time perfectly good cells of the immune system are also killed in large numbers. Campath, the only monoclonal antibody therapy that is currently approved by the FDA as frontline CLL therapy, kills all B-cells, all T-cells, as well as several other cell lines that exhibit the CD52 marker, the target of Campath. As we discussed in previous articles ("Campath Looking Better and Better"), this causes profound immune suppression and it is very important to protect the patient from opportunistic infections during this window of vulnerability. Standard chemotherapy drugs such as fludarabine have no problem killing cancer cells (until the cancer cells get smart and learn how to avoid getting killed) by mangling the DNA of the cells. The worry with them is the potential for mutagenicity and secondary cancers down the road, as well as the inevitable hematological toxicity and immune suppression.

    Looking over the Phase - I study we discussed above, I did not see any major red flags. No heavy immune suppression, no high grade hematological toxicity. The only flag is with regard to potential liver toxicity. This issue is discussed in detail in the abstract below. The full article is also available free of charge if you are interested, just click on the link provided in the abstract. There is mounting evidence that liver toxicity in LMB-2 therapy is due to the production of large amounts of TNF-alpha in the liver, and this toxicity can be controlled and prevented by appropriate use of TNF-alpha blocking drugs such as indomethacin. Something to remember and be sure to watch out for if you ever embark on LMB-2 therapy. Frankly, I did not see much else that caused a high degree of concern regarding toxicity, even in the heavily pre-treated set of patients chosen for the Phase - I trial.

    Abstract

    J Immunol. 2024 Dec 15;165(12):7150-6.

    Article from Journal of Immunology

    Inhibition of TNF-alpha produced by Kupffer cells protects against the nonspecific liver toxicity of immunotoxin anti-Tac(Fv)-PE38, LMB-2.

    Onda M, Willingham M, Wang QC, Kreitman RJ, Tsutsumi Y, Nagata S, Pastan I.

    Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD

    LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas: exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-alpha by these cells. Following LMB-2 administration to mice, the levels of TNF-alpha in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-alpha in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-alpha produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.

    PMID: 11120846
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    Just Announced Phase - II Clinical Trial with LMB-2

    This one is hot off the presses, the clinical trial was first published March 24 of this year. You can get all the details by going to the link at the bottom of this section. This Phase - II study of LMB-2 for CLL patients is to determine the response rates to this drug. The secondary aims are to determine length of remissions, obtain more information on toxicity and a better fix on the mechanism of action, such as correlation with intensity of CD25 expression.

    Patients will receive LMB-2 as an intravenous infusion (40 micro grams/Kg dose over 30 minutes) on alternate days, three times a week. The treatment will be repeated once a month for a maximum of 6 months. Patients who achieve a complete response will quit after no more than 2 courses. A total of 16-27 patients will be recruited. Eligibility criteria require CD25-positive CLL, intermediate or high risk disease requiring treatment, and patients who have relapsed within 2 years of fludarabine therapy. Reasonable liver function is a requirement, in view of the potential for liver damage. Prior monoclonal therapy with Rituxan, etc., is not a problem. As in the Phase - I clinical trial, it seems to me that here too the researchers are looking to get hard-to-treat cases, give themselves a real challenge. It also helps make the results that much more credible. I am always suspicious of clinical trials that appear to be "cherry-picking", which makes the statistics hard to interpret.

    The good news with LMB-2 is that it is not a chemotherapy drug and it does not need to break the DNA strands as does fludarabine. It is a classic monoclonal antibody, very much along the lines of Rituxan and Campath. The difference between it and the other two monoclonals is that it carries a payload of toxin to make it very much more effective in the cell kill department. Rituxan, for example, does not work very well with patients who have been through heavy chemotherapy regimes because their immune systems are no longer able to assist in the cell kill, and Rituxan is not very good at doing the job by itself. Not so with LMB-2. Remember the patient we discussed above, the one who had every thing and the kitchen sink thrown at him? LMB-2 was able to kill more than 80% of the tumor cells in his case, even though his immune system was in shambles by that time. I have not discussed it in this article, but LMB-2 is also being looked at for solid cancers, since it has a very good bioavailability profile, it is able to penetrate and be selectively absorbed by solid tumor masses. This is an improvement over Rituxan or Campath, neither of which is able to deal very well with bulky lymph nodes.

    The big fly in the ointment is the expression of CD25 in your particular brand of CLL. I can understand that the researchers have to learn to walk before they can run, and they have to test LMB-2 by itself first in the various phases of clinical trials before they can add booster drugs such as DSP30 to improve its efficacy. Hopefully they will get around to these more meaningful combinatory clinical trial sooner rather than later. I understand the NCI is willing to license this technology. If you have several millions of dollars of spare change laying around, how about licensing this drug so that we the patients can speed up its development? Right. In my dreams.

    If you satisfy the eligibility criteria, and you have already had a few rounds of chemo and are once again looking for a therapy option that does not include bone marrow transplants, you owe it to yourself to review the details of this clinical trial. You also have the choice of several alphabet soup of chemotherapy combinations out there with ever increasing hematological toxicity and immune suppression to match. I think this trial compares well because fundamentally it is an immunotherapy approach, and if it works for you then you would have bought yourself some time without having paid for it with more irreversible toxicity. If you are interested, ask your oncologist about it. I believe it is also possible to self-nominate yourself for these clinical trials.

    But do remember, this is a clinical trial. Not quite as iffy as a Phase - I trial, but not as solid as a full blown multi-center and large scale Phase - III trial either. I like it that the work done thus far has been fully published in peer reviewed and prestigious journals, details for every one to see and judge. But no doubt there are still unknown effects and surprises out there with this technology. If it was a slam dunk or a guaranteed thing, it would hardly be necessary to conduct a clinical trial. They could just hand it out to all CLL patients. You get my drift - the term "buyer beware" applies here as well as in most things in life.

    Contact Information

    Warren Grant Magnuson Clinical Center
    NCI Clinical Studies Support
    Bethesda, Maryland, 20242-1182, United States
    Recruiting now.
    Patient Recruitment 888-NCI-1937
    http://clinicaltrials.gov/show/NCT00080821
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