To see the other types of publications on this topic, follow the link: Immunotoxine.
Journal articles on the topic 'Immunotoxine'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Immunotoxine.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Zovickian, John, Virginia Gray Johnson, and Richard J. Youle. "Potent and specific killing of human malignant brain tumor cells by an anti-transferrin receptor antibody-ricin immunotoxin." Journal of Neurosurgery 66, no. 6 (June 1987): 850–61. http://dx.doi.org/10.3171/jns.1987.66.6.0850.
Full textAbstract:
✓ Immunotoxins are hybrid molecules which combine the exquisite selectivity of monoclonal antibodies with the potent toxicity of protein toxins. An immunotoxin was constructed by linking a murine monoclonal antibody against the human transferrin receptor (TR) to the plant toxin, ricin. The cytotoxic activity of the anti-TR-ricin immunotoxin was tested in vitro and demonstrated highly potent and cell type-specific killing of cells derived from human glioblastoma, medulloblastoma, and leukemia. The anti-TR-ricin immunotoxin killed more than 50% of “target” cells at a concentration of 5.6 × 10−13 M after an 18-hour incubation with the ionophore, monensin. This potency exceeds that of any other anti-TR immunotoxin reported in the literature. When the activity of the anti-TR-ricin immunotoxin against “target” tumor-derived cells was compared with the immunotoxin's activity against “non-target” cells, it could be predicted that a selective toxicity of anti-TR-ricin immunotoxin between tumor cells and normal brain was more than 150- to 1380-fold. Solid-phase indirect radioimmunoassay techniques were used to demonstrate significantly higher levels of TR in the glioblastoma- and medulloblastoma-derived cell lines, as well as in surgical tissue samples of medulloblastoma and glioblastoma, as compared to normal brain. Immunotoxins targeted to the TR may possess sufficient specificity to be of therapeutic importance, particularly to treat neoplastic disease of the central nervous system involving compartments (such as intrathecal, intraventricular, or cystic) where delivery of immunotoxins to tumor would not require transvascular transport.
2
Allahyari, Hossein, Sahar Heidari, Mehdi Ghamgosha, Parvaneh Saffarian, and Jafar Amani. "Immunotoxin: A new tool for cancer therapy." Tumor Biology 39, no. 2 (February 2017): 101042831769222. http://dx.doi.org/10.1177/1010428317692226.
Full textAbstract:
Cancer is one of the main reasons of death in the most countries and in Iran. Immunotherapy quickly became one of the best methods of cancer treatment, along with chemotherapy and radiation. “Immunotoxin Therapy” is a promising way of cancer therapy that is mentioned in this field. Immunotoxins are made from a toxin attaching to an antibody target proteins present on cancer cells. The first-generation immunotoxins were made of a full-length toxin attached to whole monoclonal antibodies. But, these immunotoxins could bind to normal cells. DAB389IL2 was the first immunotoxin approved by the Food and Drug Administration. Current trends and researches are ongoing on finding proteins that in combination with immunotoxins have minimal immunogenicity and the most potency for target cell killing.
3
Wu, Y. N., M. Gadina, J. H. Tao-Cheng, and R. J. Youle. "Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins." Journal of Cell Biology 125, no. 4 (May 15, 1994): 743–53. http://dx.doi.org/10.1083/jcb.125.4.743.
Full textAbstract:
All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.
4
Fleming, Bryan D., and Mitchell Ho. "Development of Glypican-3 Targeting Immunotoxins for the Treatment of Liver Cancer: An Update." Biomolecules 10, no. 6 (June 20, 2020): 934. http://dx.doi.org/10.3390/biom10060934.
Full textAbstract:
Hepatocellular carcinoma (HCC) accounts for most liver cancers and represents one of the deadliest cancers in the world. Despite the global demand for liver cancer treatments, there remain few options available. The U.S. Food and Drug Administration (FDA) recently approved Lumoxiti, a CD22-targeting immunotoxin, as a treatment for patients with hairy cell leukemia. This approval helps to demonstrate the potential role that immunotoxins can play in the cancer therapeutics pipeline. However, concerns have been raised about the use of immunotoxins, including their high immunogenicity and short half-life, in particular for treating solid tumors such as liver cancer. This review provides an overview of recent efforts to develop a glypican-3 (GPC3) targeting immunotoxin for treating HCC, including strategies to deimmunize immunotoxins by removing B- or T-cell epitopes on the bacterial toxin and to improve the serum half-life of immunotoxins by incorporating an albumin binding domain.
5
Dieffenbach, Michael, and Ira Pastan. "Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy." Biomolecules 10, no. 7 (June 30, 2020): 979. http://dx.doi.org/10.3390/biom10070979.
Full textAbstract:
Immunotoxins are a class of targeted cancer therapeutics in which a toxin such as Pseudomonas exotoxin A (PE) is linked to an antibody or cytokine to direct the toxin to a target on cancer cells. While a variety of PE-based immunotoxins have been developed and a few have demonstrated promising clinical and preclinical results, cancer cells frequently have or develop resistance to these immunotoxins. This review presents our current understanding of the mechanism of action of PE-based immunotoxins and discusses cellular mechanisms of resistance that interfere with various steps of the pathway. These steps include binding of the immunotoxin to the target antigen, internalization, intracellular processing and trafficking to reach the cytosol, inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (EF2), and induction of apoptosis. Combination therapies that increase immunotoxin action and overcome specific mechanisms of resistance are also reviewed.
6
Weldon, John E., Laiman Xiang, Oleg Chertov, Inger Margulies, Robert J. Kreitman, David J. FitzGerald, and Ira Pastan. "A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity." Blood 113, no. 16 (April 16, 2009): 3792–800. http://dx.doi.org/10.1182/blood-2008-08-173195.
Full textAbstract:
Abstract Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.
7
Zovickian, John, and Richard J. Youle. "Efficacy of intrathecal immunotoxin therapy in an animal model of leptomeningeal neoplasia." Journal of Neurosurgery 68, no. 5 (May 1988): 767–74. http://dx.doi.org/10.3171/jns.1988.68.5.0767.
Full textAbstract:
✓ Immunotoxins comprise a new class of cell-type specific cytotoxic reagents which consist of a monoclonal antibody linked to a protein toxin. This report examines the efficacy of intrathecal immunotoxin therapy for the treatment of tumors of the cerebrospinal fluid compartment. A syngeneic animal model of leptomeningeal neoplasia was developed in which percutaneous inoculation of L2C tumor cells into the cisterna magna of Strain 2 guinea pigs produced disseminated leptomeningeal and intraventricular leukemia and death. Percutaneous intracisternal injecton of 2 µg of an anti-idiotype monoclonal antibody (M6)-intact ricin immunotoxin 24 hours following intracisternal inoculation of 105 L2C cells (10,000 times the lethal dose) produced prolonged survival (p < 0.005) of tumor-bearing animals. The immunotoxin therapy caused no detectable toxicity. Intracisternal injection of either M6 monoclonal antibody alone or a nonspecific control immunotoxin had no therapeutic effect. The observed extension of survival times in immunotoxin-treated animals corresponds to a median 2- to 3-log (99% to 99.9%) and, in some animals, possibly a 5-log (99.999%) or greater kill of tumor cells. These results support a possible role for immunotoxins in the clinical treatment of central nervous system neoplastic disease involving compartments (intrathecal, intraventricular, or cystic tumor).
8
Guerrero-Ochoa, Patricia, Diederich Aguilar-Machado, Raquel Ibáñez-Pérez, Javier Macías-León, Ramón Hurtado-Guerrero, Javier Raso, and Alberto Anel. "Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology." International Journal of Molecular Sciences 21, no. 17 (August 26, 2020): 6165. http://dx.doi.org/10.3390/ijms21176165.
Full textAbstract:
Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.
9
Bregni, M., S. Siena, A. Formosa, DA Lappi, D. Martineau, F. Malavasi, B. Dorken, G. Bonadonna, and AM Gianni. "B-cell restricted saporin immunotoxins: activity against B-cell lines and chronic lymphocytic leukemia cells." Blood 73, no. 3 (February 15, 1989): 753–62. http://dx.doi.org/10.1182/blood.v73.3.753.753.
Full textAbstract:
Abstract B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39- SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.
10
Bregni, M., S. Siena, A. Formosa, DA Lappi, D. Martineau, F. Malavasi, B. Dorken, G. Bonadonna, and AM Gianni. "B-cell restricted saporin immunotoxins: activity against B-cell lines and chronic lymphocytic leukemia cells." Blood 73, no. 3 (February 15, 1989): 753–62. http://dx.doi.org/10.1182/blood.v73.3.753.bloodjournal733753.
Full textAbstract:
B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39- SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.
11
Winkler, U., C. Gottstein, G. Schon, U. Kapp, J. Wolf, ML Hansmann, H. Bohlen, P. Thorpe, V. Diehl, and A. Engert. "Successful treatment of disseminated human Hodgkin's disease in SCID mice with deglycosylated ricin A-chain immunotoxins." Blood 83, no. 2 (January 15, 1994): 466–75. http://dx.doi.org/10.1182/blood.v83.2.466.466.
Full textAbstract:
Abstract To evaluate the effects of deglycosylated ricin A-chain (dgA) immunotoxins against disseminated Hodgkin's lymphoma, we used RFT5.dgA (CD25) and IRac.dgA (70 kD) to treat L540Cy Hodgkin cells in severely immunodeficient SCID mice. In this model, more than 90% of the animals developed multiple lymphomas in various organs such as the lymph nodes, liver, bone marrow, and extranodal sites that killed untreated animals after a mean survival time (MST) of 36.3 days. A single intraperitoneal injection of 8 micrograms of either immunotoxin rendered 95% (RFT5.dgA) and 93% (IRac.dgA), respectively, of mice tumor-free when applied 1 day after tumor challenge. The MST of the RFT5.dgA-treated group was extended by more than 80 days (P < .00001). SCID mice treated 12 days after tumor challenge had lower remission rates (46%), suggesting that the antitumor effect of the immunotoxins depends on the number of tumor cells present. We conclude that ricin A-chain immunotoxins have potent antitumor effects against disseminated Hodgkin's tumors in SCID mice and that this model is ideally suited for the evaluation of different immunotoxin treatment modalities.
12
Winkler, U., C. Gottstein, G. Schon, U. Kapp, J. Wolf, ML Hansmann, H. Bohlen, P. Thorpe, V. Diehl, and A. Engert. "Successful treatment of disseminated human Hodgkin's disease in SCID mice with deglycosylated ricin A-chain immunotoxins." Blood 83, no. 2 (January 15, 1994): 466–75. http://dx.doi.org/10.1182/blood.v83.2.466.bloodjournal832466.
Full textAbstract:
To evaluate the effects of deglycosylated ricin A-chain (dgA) immunotoxins against disseminated Hodgkin's lymphoma, we used RFT5.dgA (CD25) and IRac.dgA (70 kD) to treat L540Cy Hodgkin cells in severely immunodeficient SCID mice. In this model, more than 90% of the animals developed multiple lymphomas in various organs such as the lymph nodes, liver, bone marrow, and extranodal sites that killed untreated animals after a mean survival time (MST) of 36.3 days. A single intraperitoneal injection of 8 micrograms of either immunotoxin rendered 95% (RFT5.dgA) and 93% (IRac.dgA), respectively, of mice tumor-free when applied 1 day after tumor challenge. The MST of the RFT5.dgA-treated group was extended by more than 80 days (P < .00001). SCID mice treated 12 days after tumor challenge had lower remission rates (46%), suggesting that the antitumor effect of the immunotoxins depends on the number of tumor cells present. We conclude that ricin A-chain immunotoxins have potent antitumor effects against disseminated Hodgkin's tumors in SCID mice and that this model is ideally suited for the evaluation of different immunotoxin treatment modalities.
13
Glennie, M. J., H. M. McBride, F. Stirpe, P. E. Thorpe, A. T. Worth, and G. T. Stevenson. "Emergence of immunoglobulin variants following treatment of a B cell leukemia with an immunotoxin composed of antiidiotypic antibody and saporin." Journal of Experimental Medicine 166, no. 1 (July 1, 1987): 43–62. http://dx.doi.org/10.1084/jem.166.1.43.
Full textAbstract:
The potency and specificity of immunotoxins consisting of monoclonal antiidiotype conjugated to the ribosome-inactivating protein, saporin, have been evaluated in the treatment of guinea pig L2C B lymphocytic leukemia. The immunotoxins were therapeutically much more effective than their parent antibodies. Their specificity reflected that of their antiidiotype component. Although the leukemia emerged eventually in most animals treated with these conjugates, most of the cells showed altered Ig expression, which rendered them resistant to the therapy. Commonly, the emerging cells had lost mu heavy chain production, leaving them negative for intracellular, surface, and secreted IgM, but still positive for lambda light chain production. In addition, a minor group of L2C variants was identified in a protocol designed to detect mutants at very low frequency: here the cells were exposed in vitro to immunotoxin and, while still viable as judged by dye-exclusion, inoculated in large numbers into animals. In tumor that emerged under these circumstances, the majority of cells were again immunoglobulin-negative; however a minority exhibited IgM with an altered idiotype (Idiotope-loss variants), rendering them unreactive with immunotoxin. Immunotherapy with unmodified anti-Id antibody alone does not reveal these variants, and we suggest it is the increased selective force exerted by the highly potent immunotoxins that allow these minor nonreactive populations to emerge.
14
Sokolova, E. A., O. A. Stremovskiy, T. A. Zdobnova, I. V. Balalaeva, and S. M. Deyev. "Recombinant Immunotoxin 4D5scFv-PE40 for Targeted Therapy of HER2-Positive Tumors." Acta Naturae 7, no. 4 (December 15, 2015): 93–96. http://dx.doi.org/10.32607/20758251-2015-7-4-93-96.
Full textAbstract:
Recombinant immunotoxins are extremely promising agents for the targeted therapy of tumors with a certain molecular profile. In this work, we studied the properties of a new recombinant HER2-specific immunotoxin composed of the scFv antibody and a fragment of Pseudomonas exotoxin A (4D5scFv-PE40). High affinity of the immunotoxin for the HER2 tumor marker, its selective cytotoxicity against HER2-overexpressing cells, and its storage stability were demonstrated. The 50% inhibitory concentration (IC50) of the 4D5scFv-PE40 immunotoxin for HER2-overexpressing cancer cells was 2.5-3 orders of magnitude lower compared to that for CHO cells not expressing this tumor marker and was 2.5-3 orders of magnitude lower than IC50 of free PE40 for HER2-overexpressing cancer cells. These findings provide a basis for expecting in the long run high therapeutic index values of the 4D5scFv-PE40 immunotoxin for its use in vivo.
15
Park, Sangsu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Minh Tan Nguyen, Gunsup Lee, Chong Jai Kim, Yeon Jin Jang, and Han Choe. "Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion." International Journal of Molecular Sciences 22, no. 12 (June 17, 2021): 6483. http://dx.doi.org/10.3390/ijms22126483.
Full textAbstract:
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
16
Cizeau, Jeannick, Marianne G. P. Torres, Sharla G. Cowling, Stacy Stibbard, Arjune Premsukh, Joycelyn Entwistle, and Glen C. MacDonald. "Fusogenics." Journal of Biomolecular Screening 16, no. 1 (December 3, 2010): 90–100. http://dx.doi.org/10.1177/1087057110387425.
Full textAbstract:
Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer.
17
Pasetto, Matteo, Antonella Antignani, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Ira Pastan, Scott E. Martin, and David J. FitzGerald. "Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity." Proceedings of the National Academy of Sciences 112, no. 10 (February 23, 2015): E1135—E1142. http://dx.doi.org/10.1073/pnas.1501958112.
Full textAbstract:
Immunotoxins (antibody–toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (∼22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, aPseudomonasexotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients. High and low concentrations of SS1P were chosen to allow for the identification of both mitigators and sensitizers. As expected, silencing known essential genes in the immunotoxin pathway, such as mesothelin, furin, KDEL receptor 2, or members of the diphthamide pathway, protected cells. Of greater interest was the observation that many RNAi targets increased immunotoxin sensitivity, indicating that these gene products normally contribute to inefficiencies in the killing pathway. Of the top sensitizers, many genes encode proteins that locate to either the endoplasmic reticulum (ER) or Golgi and are annotated as part of the secretory system. Genes related to the ER-associated degradation system were not among high-ranking mitigator or sensitizer candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for chemical intervention that could increase immunotoxin killing of cancer cells and enhance our understanding of toxin trafficking.
18
Ruiz-de-la-Herrán, Javier, Jaime Tomé-Amat, Rodrigo Lázaro-Gorines, José G. Gavilanes, and Javier Lacadena. "Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins." Toxins 11, no. 10 (October 12, 2019): 593. http://dx.doi.org/10.3390/toxins11100593.
Full textAbstract:
Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo.
19
Siena, S., S. Villa, M. Bregni, G. Bonnadonna, and AM Gianni. "Amantadine potentiates T lymphocyte killing by an anti-pan-T cell (CD5) ricin A-chain immunotoxin." Blood 69, no. 1 (January 1, 1987): 345–48. http://dx.doi.org/10.1182/blood.v69.1.345.345.
Full textAbstract:
Abstract The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.
20
Siena, S., S. Villa, M. Bregni, G. Bonnadonna, and AM Gianni. "Amantadine potentiates T lymphocyte killing by an anti-pan-T cell (CD5) ricin A-chain immunotoxin." Blood 69, no. 1 (January 1, 1987): 345–48. http://dx.doi.org/10.1182/blood.v69.1.345.bloodjournal691345.
Full textAbstract:
The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.
21
Pincus, SETH H., Jan McClure, and Hua Fang. "Use of Anti-HIV Immunotoxins as Probes of the Biology of HIV-Infected Cells." Canadian Journal of Infectious Diseases 5, suppl a (1994): 23A—27A. http://dx.doi.org/10.1155/1994/576154.
Full textAbstract:
OBJECTIVE: Anti-human immunodeficiency virus (HIV) immunotoxins are potential treatments for HIV infection. but they may also be used as probes to study the relationship between HIV and the cell it infects. Data from the present study indicate the complexity of this relationship.DESIGN: A panel of monoclonal antibodies directed against different epitopes on the HIV envelope protein(s) gp l20 and gp 41 was conjugated to ricin A chain. The activity of these immunotoxins on HIV-infected cell lines was studied.RESULTS: The data demonstrate that HIV-infected cell lines may be killed by some, but not all, of these immunotoxins. The killing is not directly proportional to the binding of the antibody to the infected cell and is influenced by the viral strain. The immunotoxins were used to select persistently infected cell lines for immunotoxin-resistant variants: these demonstrate several different viral or cellular defects. The incubation of infected cells with a soluble form of the viral receptor increases the sensitivity of the cells to anti-gp41, but not anti-gpl20, immunotoxins by altering both the levels of expression and internalization of the viral envelope. Drugs that inhibit lysosomal degradation (ammonium chloride, monensin. chloroquine) enhance the efficacy of these immunotoxins.CONCLUSIONS: Because immunotoxins must be internalized to function, they may be used to study the intracellular trafficking of the target antigens. In the present study, this was done using the HIV-envelope protein as expressed in infected cells as the target antigen
22
Chavez-Cortez, Elda-Georgina, Gustavo Vargas Felix, Edgar Rangel López, Julio Sotelo, Carlos Martínez-Canseco, Verónica Pérez-de la Cruz, and Benjamin Pineda. "Production and Evaluation of an Avian IgY Immunotoxin against CD133+ for Treatment of Carcinogenic Stem Cells in Malignant Glioma: IgY Immunotoxin for the Treatment of Glioblastoma." Journal of Oncology 2019 (June 2, 2019): 1–15. http://dx.doi.org/10.1155/2019/2563092.
Full textAbstract:
Background. Glioblastoma is the most common malignant tumor of Central Nervous System. Despite the research in therapeutics, the prognosis is dismal. Malignant glioma stem cells (MGSCs) are a major cause of treatment failure and increasing tumor recurrence. In general, cancer stem cells (CSCs) express prominin-1 (CD133), considered as a potential therapeutic target. In this study, we produced an avian immunotoxin directed against the subpopulation of CD133+ CSCs within a malignant glioma. We used the avian IgY because it has various advantages as increased affinity to mammal antigens and inexpensive obtention of large amounts of specific antibodies (approximately 1 mg/per egg). The design, production, purification and use of IgY anti CD133 immunotoxin constitute an original goal of this research.Methods. The immunodominant peptide of CD133 was designed to immunize hens; also, the extracellular domain of CD133 was cloned to probe the IgY antibodies. In parallel, a recombinant abrin A chain was produced inE. coliin order to join it to the Fc domain of the anti-CD133 IgY to conform the immunotoxin. This anti-CD133 IgY anti-tumor immunotoxin was testedin vitroandin vivo. Results. The cytotoxicity of the immunotoxinin vitroshowed that IgY-abrin immunotoxin reduced 55% cell viability. After subcutaneous MGSCs implantation, the animals treated intraperitoneally or intratumorally with the IgY-abrin immunotoxin showed more than 50% decrease of tumor volume.Conclusion. Results showed that the IgY-abrin immunotoxin had cytotoxic activity against CD133+ MGSCs and provides a novel approach for the immunotherapy of glioblastoma.
23
Hertler, A. A., and A. E. Frankel. "Immunotoxins: a clinical review of their use in the treatment of malignancies." Journal of Clinical Oncology 7, no. 12 (December 1989): 1932–42. http://dx.doi.org/10.1200/jco.1989.7.12.1932.
Full textAbstract:
Immunotoxins are a new class of antitumor agents consisting of tumor-selective ligands (generally monoclonal antibodies [MoAbs]) linked to highly toxic protein molecules that have been modified to remove their normal tissue-binding domains. These immuno-conjugates combine the potency of the parent toxin with the specificity of the attached ligand. Toxins used in the construction of immunotoxins belong to a group of peptides that catalytically inhibit the elongation step of protein synthesis, and include ricin, abrin, pokeweed antiviral protein, gelonin, Pseudomonas exotoxin A, diptheria toxin, and alpha-sarcin. To synthesize immunotoxins, the normal cell-binding function must be removed by chemical cleavage or modification, or in the case of toxins that have been cloned, genetic engineering used to delete amino acids critical to cell binding. Covalent linkage of toxin to ligand generally involves a disulfide or thioether bond, though recently, recombinant toxin molecules with ligands that are genetically engineered into the protein have been made. The most successful clinical application of immunotoxins has been in the depletion of T cells from allogeneic bone marrow grafts to prevent graft-versus-host disease (GVHD). Clinical trials have been conducted using immunotoxins for the systemic treatment of chronic lymphocytic leukemia (CLL), GVHD, and selected solid tumors. With the possible exception of GVHD, responses have been limited. Obstacles have included rapid systemic clearance, poor delivery to extravascular tumor deposits, and humoral immune responses to the immunotoxin. Research to overcome these problems is in progress and should lead to a better definition of the role of immunotoxins in the therapy of malignancies.
24
Decker, Thomas, Madlene Oelsner, Robert J. Kreitman, Giuliana Salvatore, Qing-cheng Wang, Ira Pastan, Christian Peschel, and Thomas Licht. "Induction of caspase-dependent programmed cell death in B-cell chronic lymphocytic leukemia by anti-CD22 immunotoxins." Blood 103, no. 7 (April 1, 2004): 2718–26. http://dx.doi.org/10.1182/blood-2003-04-1317.
Full textAbstract:
Abstract B cells of chronic lymphocytic leukemia (CLL) are long-lived in vivo, possibly because of defects in apoptosis. We investigated BL22, an immunotoxin composed of the Fv portion of an anti-CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment. B cells from 22 patients with CLL were immunomagnetically enriched (96% purity) and were cultured with BL22 or an immunotoxin that does not recognize hematopoietic cells. The antileukemic activity of BL22 was correlated with CD22 expression, as determined by flow cytometry. BL22 induced caspase-9 and caspase-3 activation, poly(adenosine diphosphate [ADP]-ribose)polymerase (PARP) cleavage, DNA fragmentation, and membrane flipping. Cell death was associated with the loss of mitochondrial membrane potential and the down-regulation of Mcl-1 and X-chromosomal inhibitor of apoptosis protein (XIAP). Furthermore, BL22 induced a proapoptotic 18-kDa Bax protein and conformational changes of Bax. Z-VAD.fmk abrogated apoptosis, confirming that cell death was executed by caspases. Conversely, interleukin-4, a survival factor, inhibited spontaneous death in culture but failed to prevent immunotoxin-induced apoptosis. BL22 cytotoxicity was markedly enhanced when combined with anticancer drugs including vincristine. We also investigated HA22, a newly engineered immunotoxin, in which BL22 residues are mutated to improve target binding. HA22 was more active than BL22. In conclusion, these immunotoxins induce caspase-mediated apoptosis involving mitochondrial damage. Combination with chemotherapy is expected to improve the efficacy of immunotoxin treatment. (Blood. 2004;103:2718-2726)
25
Mazor, Ronit, Emily M. King, Masanori Onda, Nicolas Cuburu, Selamawit Addissie, Devorah Crown, Xiu-Fen Liu, Takashi Kei Kishimoto, and Ira Pastan. "Tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity." Proceedings of the National Academy of Sciences 115, no. 4 (January 8, 2018): E733—E742. http://dx.doi.org/10.1073/pnas.1717063115.
Full textAbstract:
Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.
26
Decker, Thomas, Michaela Wagner, Madlene Oelsner, Robert J. Kreitman, Ira Pastan, Christian Peschel, and Thomas Licht. "BL22, a Recombinant Anti-CD22 Immunotoxin, Induces Cell Cycle Arrest and Apoptosis in B-Cell Lymphoma." Blood 104, no. 11 (November 16, 2004): 4613. http://dx.doi.org/10.1182/blood.v104.11.4613.4613.
Full textAbstract:
Abstract A recombinant immunotoxin, BL22 has shown clinical efficacy against hairy cell leukemia and other B-cell non-Hodgkin’s lymphomas. BL22 contains an antibody-derived domain that recognizes CD22, and PE38, a truncated Pseudomonas exotoxin A domain, for inhibition of protein synthesis. Previous studies have shown that Pseudomonas exotoxin A-based immunotoxins exert their cytotoxicity by apoptosis and additional, yet uncharacterized mechanisms. We investigated the cytotoxicity of BL22 in two mantle cell lymphoma lines, NCEB-1 and Granta-519, and in the diffuse, large-cell lymphoma line SU-DHL-4. Presence of the CD22 target was confirmed by flow cytometry. Treatment of NCEB-1 cells to 1 μg/ml BL22 for 24-32h reduced the numbers of viable cells in comparison to cells exposed to a non-binding immunotoxin. Induction of apoptosis, determined by activation of caspase-3 and exposure of phosphatidylserine, was not detected before 48h of exposure. Incorporation of bromo-deoxyuridine into DNA was, however, almost completely inhibited after 24h. Correspondingly, the numbers of G1 phase cells were increased upon treatment with BL22 as determined by flow cytometry. G1-arrest resulted from decreased enzyme activities of cyclin-dependent kinases (cdk)-2 and 4 as assessed in vitro with respective substrates, histone H1 and the retinoblastoma susceptibility (RB) protein. Immunoblotting revealed markedly reduced amounts of cyclins A, E, D1 and D3 whereas cdk2 and cdk4 protein expression levels remained unchanged. Expression of the RB protein was decreased, and the protein was hypophosphorylated in immunotoxin-treated cells. Pulse-labeling with [35S]-labeled amino acids, followed by immunoprecitation of cyclins E and D1 confirmed that BL22 inhibited synthesis of these cyclins. Thus, immunotoxin-induced cell cycle arrest results from insufficient synthesis of regulatory cyclins required for progression through G1 into S-phase. Similar results were obtained in Granta-519 cells. SU-DHL-4 diffuse, large-cell lymphoma cells were highly sensitive to BL22, and G1 arrest was observed as early as after 8–24h in these cells. Expression of cyclins A, D3 and E was decreased while cyclin D1 was not expressed in SU-DHL-4 cells. Interestingly, cell cycle arrest did not prevent from subsequent induction of apoptosis in lymphoma cells continuously exposed to the immunotoxin. In conclusion, we have characterized cell cycle arrest as a previously unknown mechanism that contributes to the cytotoxicity of Pseudomonas exotoxin-based immunotoxins.
27
Mansfield, Elizabeth, Peter Amlot, Ira Pastan, and David J. FitzGerald. "Recombinant RFB4 Immunotoxins Exhibit Potent Cytotoxic Activity for CD22-Bearing Cells and Tumors." Blood 90, no. 5 (September 1, 1997): 2020–26. http://dx.doi.org/10.1182/blood.v90.5.2020.
Full textAbstract:
Abstract Many B-cell malignancies express the CD22 antigen on their cell surface. To kill cells expressing this antigen, the RFB4 monoclonal antibody (MoAb) has been linked chemically with either deglycosylated ricin A chain or truncated versions of Pseudomonas exotoxin. These immunotoxins exhibited selective cytotoxic activity for CD22+ cells and antitumor activity in nude mouse models bearing human B-cell lymphomas. To construct a recombinant immunotoxin targeted to CD22, we first cloned the variable portions of the heavy and light chains of RFB4. The cloned Fv fragments were joined by a newly created disulfide bond to form a disulfide stabilized (ds) construct. The RFB4 construct was combined by gene fusion with PE38, a truncated version of PE. The recombinant immunotoxin was then expressed in Escherichia coli, purified by column chromatography and tested for cytotoxicity activity. RFB4(dsFv)PE38 retained its binding activity for CD22, was very stable at 37°C and exhibited selective cytotoxic activity for CD22+-cultured cell lines. Because of its favorable binding characteristics and potency for CD22-positive cell lines, RFB4(dsFv)PE38 was tested for antitumor activity in a nude mouse model of human lymphoma. CA46 cells were injected subcutaneously and then treated with the RFB4(dsFv)PE38 immunotoxin. Antitumor activity was dose responsive and was not evident when an irrelevant immunotoxin was administered on the same schedule.
28
Byers, VS, PJ Henslee, NA Kernan, BR Blazar, R. Gingrich, GL Phillips, CF LeMaistre, G. Gilliland, JH Antin, and P. Martin. "Use of an anti-pan T-lymphocyte ricin a chain immunotoxin in steroid- resistant acute graft-versus-host disease." Blood 75, no. 7 (April 1, 1990): 1426–32. http://dx.doi.org/10.1182/blood.v75.7.1426.1426.
Full textAbstract:
Abstract Acute steroid-resistant graft-versus-host disease (AGVHD) after allogeneic bone marrow transplantation is frequently fatal. A new treatment for this T-lymphocyte-mediated condition uses an immunotoxin, H65-RTA, comprised of a monoclonal antibody that recognizes the CD5 lymphocyte differentiation antigen coupled to ricin A chain, a cytotoxic enzyme that inhibits protein synthesis. The safety and efficacy of this lymphocyte-targeted immunotoxin was evaluated in patients with severe AGVHD in a phase I-II dose escalation study with group expansion at the two middle doses. Thirty-four patients received up to 14 daily intravenous infusions of the immunotoxin. The principal side effects were constitutional symptoms such as fatigue and myalgias, and hypoalbuminemia with weight gain was seen at all doses. Thirty-two patients were evaluated for improvement or resolution of disease. Durable complete or partial responses were not dose-related and were seen in 16 patients. Skin GVHD had the highest incidence of response (73%), although improvement or resolution in gastrointestinal tract (45%) and liver (28%) GVHD was also noted. Survival in responding patients was significantly prolonged at all times as compared with those with no response (P = .03). Treatment was associated with a rapid decrease in peripheral blood T lymphocytes, which persisted for greater than 1 month after therapy. Anti-immunotoxin antibodies were seen in 6 of the 23 patients tested; these were of low titer and did not block immunotoxin binding to T cells. Results of this study indicate that anti-T-lymphocyte immunotoxins may form a new class of immunosuppressive agents useful in T-lymphocyte-mediated diseases.
29
Byers, VS, PJ Henslee, NA Kernan, BR Blazar, R. Gingrich, GL Phillips, CF LeMaistre, G. Gilliland, JH Antin, and P. Martin. "Use of an anti-pan T-lymphocyte ricin a chain immunotoxin in steroid- resistant acute graft-versus-host disease." Blood 75, no. 7 (April 1, 1990): 1426–32. http://dx.doi.org/10.1182/blood.v75.7.1426.bloodjournal7571426.
Full textAbstract:
Acute steroid-resistant graft-versus-host disease (AGVHD) after allogeneic bone marrow transplantation is frequently fatal. A new treatment for this T-lymphocyte-mediated condition uses an immunotoxin, H65-RTA, comprised of a monoclonal antibody that recognizes the CD5 lymphocyte differentiation antigen coupled to ricin A chain, a cytotoxic enzyme that inhibits protein synthesis. The safety and efficacy of this lymphocyte-targeted immunotoxin was evaluated in patients with severe AGVHD in a phase I-II dose escalation study with group expansion at the two middle doses. Thirty-four patients received up to 14 daily intravenous infusions of the immunotoxin. The principal side effects were constitutional symptoms such as fatigue and myalgias, and hypoalbuminemia with weight gain was seen at all doses. Thirty-two patients were evaluated for improvement or resolution of disease. Durable complete or partial responses were not dose-related and were seen in 16 patients. Skin GVHD had the highest incidence of response (73%), although improvement or resolution in gastrointestinal tract (45%) and liver (28%) GVHD was also noted. Survival in responding patients was significantly prolonged at all times as compared with those with no response (P = .03). Treatment was associated with a rapid decrease in peripheral blood T lymphocytes, which persisted for greater than 1 month after therapy. Anti-immunotoxin antibodies were seen in 6 of the 23 patients tested; these were of low titer and did not block immunotoxin binding to T cells. Results of this study indicate that anti-T-lymphocyte immunotoxins may form a new class of immunosuppressive agents useful in T-lymphocyte-mediated diseases.
30
Sapra, Puja, Chien-Hsing Chang, Sailaja Vanama, Sharon Singh, Hans J. Hansen, Ivan D. Horak, and David M. Goldenberg. "Preclinical Safety and Efficacy of Two Novel Immunotoxins Consisting of Ranpirnase (Rap) Fused to an Internalizing Anti-CD74 Humanized IgG4 Antibody in Human Non-Hodgkin’s Lymphoma Xenografts." Blood 106, no. 11 (November 16, 2005): 346. http://dx.doi.org/10.1182/blood.v106.11.346.346.
Full textAbstract:
Abstract Rap, an amphibian ribonuclease, is a single-chain protein of 104 amino acids that kills cells by degrading t-RNA upon internalization. CD74 is a rapidly internalizing type-II transmembrane chaperone molecule associated with HLA-DR, and has high expression on hematological malignancies including B-cell non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM). We have constructed and evaluated two novel immunotoxins, 2L-Rap-hLL1-γ 4P and 2L-Rap(N69Q)-hLL1-γ 4P, each composed of two Rap molecules fused to hLL1, an internalizing anti-CD74 humanized monoclonal antibody. The Rap gene was inserted at the N-terminus of the light chain in the expression vector of hLL1 and expressed in NS0 mouse myeloma cells. To reduce unwanted cytotoxicity, the CH1, CH2, CH3 and the hinge regions of the γ 1 chain of hLL1 were replaced with those of γ 4. Additionally, the serine residue in the hinge region was converted to proline to prevent the formation of IgG4 half-molecules. Noting that Rap contains a potential N-glycosylation site at the 69th residue of asparagine(N69), a variant of Rap, referred to as Rap(N69Q), was constructed by changing N to Q (glutamine) and this variant was used to make 2L-Rap(N69Q)-hLL1-γ 4P. Purified recombinant immunotoxins were shown to be a single peak by SE-HPLC and their MW determined by MALDI-TOF to be 177,150, which is in agreement with the MW of one IgG (150,000) plus two Rap molecules (24,000). In vitro, both immunotoxins retained RNase activity, specific binding to CD74, and were significantly more potent against CD74-positive NHL and MM cell lines (Daudi, Raji and MC/CAR) than naked hLL1 or non-specific control immunotoxin, 2L-Rap(N69Q)-hRS7(immunotoxin against EGP-1). In Raji and Daudi Burkitt’s lymphoma xenograft models, treatment with a single 5- to 50-μg dose of 2L-Rap-hLL1-γ 4P, given as early or delayed treatment, resulted in cures of most animals. Additionally, treatment with a single 15-μg dose of 2L-Rap(N69Q)-hLL1-γ 4P 1-day post injection of cells resulted in 100% cures. Treatment with 2L-Rap-hLL1-γ 4P or 2L-Rap(N69Q)-hLL1-γ 4P was significantly better than all controls, including saline, naked hLL1 and non-specific immunotoxin. The maximum tolerated dose of 2L-Rap-hLL1-γ 4P or 2L-Rap(N69Q)-hLL1-γ 4P in SCID or BALB/c mice was 50 μg/mouse and the dose-limiting toxicity was hepatic. In our preliminary studies, we have observed that treating animals with NSAID’s, such as indomethacin, can ameliorate the hepatoxicity of 2L-Rap-hLL1-γ 4P. All animals that were injected with 100 μg/mouse 2L-Rap-hLL1-γ 4P alone died with a median survival time of 7 days; however, animals treated with 1.25mg/kg indomethacin prior and post-treatment of 2L-Rap-hLL1-γ 4P survived the duration of study (day 40). Experiments to determine the possible causes of liver toxicity produced by 2L-Rap-hLL1-γ 4P and to determine the MTD of Rap-immunotoxins in mice after treatment with indomethacin are ongoing. In conclusion, we have constructed two CD74-targeted novel recombinant immunotoxins containing Rap molecules that have demonstrated curative therapeutic effects in animal models of human B-cell lymphoma, and thus could be potential therapeutics for CD74-postive lymphomas and myelomas.
31
Goulet, Anne-Christine, Victor S. Goldmacher, John M. Lambert, Chantal Baron, Denis-Claude Roy, and Edouard Kouassi. "Conjugation of Blocked Ricin to an Anti-CD19 Monoclonal Antibody Increases Antibody-Induced Cell Calcium Mobilization and CD19 Internalization." Blood 90, no. 6 (September 15, 1997): 2364–75. http://dx.doi.org/10.1182/blood.v90.6.2364.
Full textAbstract:
Abstract CD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4–blocked ricin (anti-B4–bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4–bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4–bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20–bR and anti-CD38–bR, were devoid of the calcium increasing effect of anti-B4–bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4–bR effect. Anti-B4–bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4–bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4–bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4–bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.
32
Goulet, Anne-Christine, Victor S. Goldmacher, John M. Lambert, Chantal Baron, Denis-Claude Roy, and Edouard Kouassi. "Conjugation of Blocked Ricin to an Anti-CD19 Monoclonal Antibody Increases Antibody-Induced Cell Calcium Mobilization and CD19 Internalization." Blood 90, no. 6 (September 15, 1997): 2364–75. http://dx.doi.org/10.1182/blood.v90.6.2364.2364_2364_2375.
Full textAbstract:
CD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4–blocked ricin (anti-B4–bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4–bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4–bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20–bR and anti-CD38–bR, were devoid of the calcium increasing effect of anti-B4–bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4–bR effect. Anti-B4–bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4–bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4–bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4–bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.
33
Wensley, Harrison J., Wendy S. Smith, Suzanne E. Holmes, Sopsamorn U. Flavell, and David J. Flavell. "The Effect of Small Molecule Pharmacological Agents on the Triterpenoid Saponin Induced Endolysosomal Escape of Saporin and a Saporin-Based Immunotoxin in Target Human Lymphoma Cells." Biomedicines 9, no. 3 (March 15, 2021): 300. http://dx.doi.org/10.3390/biomedicines9030300.
Full textAbstract:
Triterpenoid saponins augment the cytotoxicity of saporin based immunotoxins. It is postulated that this results from a saponin-mediated increase in the endolysosomal escape of the toxin to the cytosol, but this remains to be confirmed. To address this issue, we used a number of pharmacological inhibitors of endocytic processes as probes to investigate the role played by saponin in the endolysosomal escape of fluorescently labeled saporin and a saporin based immunotoxin targeted against CD38 on human lymphoma and leukemia cell lines. Endolysosomal escape of the toxin was measured by flow cytometric pulse shape analysis. These results were compared to the effects of the various inhibitors on the saponin-mediated augmentation of toxin and immunotoxin cytotoxicity. Inhibitors of clathrin-mediated endocytosis, micropinocytosis, and endosomal acidification abrogated the saponin-induced increase in the endolysosomal escape of the toxin into the cytosol, suggesting that these processes may be involved in the internalization of saponin to the same endolysosomal vesicle as the toxin. Alternatively, these processes may play a direct role in the mechanism by which saponin promotes toxin escape from the endolysosomal compartment to the cytosol. Correlation with the effects of these inhibitors on the augmentation of cytotoxicity provides additional evidence that endolysosomal escape is involved in driving augmentation.
34
Desjardins, Annick, Dina Randazzo, Vidya Chandramohan, Katherine Peters, Margaret Johnson, Daniel Landi, Mustafa Khasraw, et al. "CTIM-23. A PHASE 1 TRIAL OF D2C7-IT IN COMBINATION WITH ATEZOLIZUMAB IN RECURRENT WHO GRADE IV MALIGNANT GLIOMA (MG)." Neuro-Oncology 22, Supplement_2 (November 2020): ii38. http://dx.doi.org/10.1093/neuonc/noaa215.157.
Full textAbstract:
Abstract BACKGROUND D2C7 immunotoxin (D2C7-IT) is a dual-specific recombinant immunotoxin comprising an EGFR-wt and mutant-specific (EGFRvIII) monoclonal antibody fragment and a genetically engineered form of the pseudomonas exotoxin. When injected directly into the tumor mass by convection enhanced delivery (CED), in addition to direct tumor cell killing, immunotoxins induce secondary immune responses by the activation of CD4+ and CD8+ T-cells. We completed a phase 1 dose escalation study of D2C7-IT injected by CED into recurrent WHO grade III-IV MG and identified the phase 2 dose (6,920 ng/mL). Three patients remain in partial response more than 58, 38, and 32 months after a single D2C7-IT intratumoral infusion. As optimal induction of anti-tumor immune responses by immunotoxins is impeded by potent MG-mediated immunosuppression, we are assessing the toxicity of the combination of D2C7-IT with atezolizumab in patients with recurrent WHO grade IV MG. METHODS Eligibility includes adult patients with recurrence of a solitary supratentorial WHO grade IV MG; ≥4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS >70%. Patient receives an intratumoral infusion of D2C7-IT and initiates two weeks later atezolizumab at 1200mg, followed by atezolizumab every 3 weeks for up to 2 years. Two cohorts of 3 patients are initially accrued to assess the toxicity of the combination. Assuming accrual continues after the initial two cohorts of 3 patients, an additional 12 patients will be accrued to the study. RESULT The first enrolled patient experienced a grade 3 DLT (grade III ALT elevation) after the first infusion of atezolizumab, but showed a more extensive immunotherapeutic effect by imaging than observed with patients on the D2C7-IT monotherapy trial. Enrollment is ongoing. CONCLUSION D2C7-IT monotherapy has shown prolonged survival and disease control in some patients. We are now evaluating the combination of D2C7-IT with checkpoint inhibition.
35
Bao, Xuhui, Stephen T. Keir, Smita K. Nair, Ira Pastan, Darell D. Bigner, and Vidyalakshmi Chandramohan. "A combinatorial immunotherapy for malignant brain tumors: D2C7 immunotoxin and immune checkpoint inhibitors." Journal of Clinical Oncology 35, no. 7_suppl (March 1, 2017): 102. http://dx.doi.org/10.1200/jco.2017.35.7_suppl.102.
Full textAbstract:
102 Background: Immunotoxins can induce direct and rapid cytotoxicity by targeting specific tumor antigens. D2C7 is a unique recombinant immunotoxin targeting EGFRwt/EGFRvIII, two frequently overexpressed proteins on gliomas, and is currently being tested in a phase I/II clinical trial (NCT02303678) for recurrent malignant gliomas. Immunotoxins have also been shown to induce a secondary antitumor immune response via stimulation of cytotoxic T lymphocytes (CTLs). Immune checkpoint inhibitors, which have successfully treated several advanced tumors by promoting the antitumor function of CTLs, may further enhance this immunotoxin-induced antitumor response. Thus, we hypothesize that combining D2C7 with immune checkpoint inhibitors will promote long-term tumor regression due to primary cytotoxicity and enhanced anti-tumor immunity. Methods: We developed a CT2A-mD2C7 mouse glioma cell line with robust in vitro cytotoxicity of D2C7 (IC50= 0.47 ng/mL). In vivo anti-tumor efficacy was evaluated by intratumoral injection of D2C7 combined with intraperitoneal injection of anti-CTLA4 or anti-PD1 antibodies in single-side and bilateral subcutaneous (SC) CT2A-mD2C7 glioma models in C57BL/6 immunocompetent mice. Results: In the single-side model, D2C7 monotherapy and combinatorial therapy showed a significant tumor growth delay (P < 0.01). Complete regression ( ≥ 40%) was only observed in combinatorial therapy groups. All cured mice rejected the rechallenging of CT2A parental cells in the contralateral flank and the subsequent rechallenging of CT2A-mD2C7 cells in the brain. In the bilateral model, the larger right tumors were treated with D2C7/anti-CTLA4/anti-PD1 monotherapy or D2C7+anti-CTLA4/PD1 combinatorial therapy, while the left tumors were untreated by D2C7. In the groups where the right tumors were treated with monotherapy or combinatorial therapy, the left untreated tumors also grew much slower. Furthermore, the combinatorial therapy led to the most significantly delayed growth of the left untreated tumors (P < 0.05). Conclusions: Immune checkpoint inhibitors can enhance D2C7-induced anti-tumor immunity to achieve a synergistic long-term tumor regression.
36
Du, Xing, Satoshi Nagata, Tomoko Ise, Maryalice Stetler-Stevenson, and Ira Pastan. "FCRL1 on chronic lymphocytic leukemia, hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins." Blood 111, no. 1 (January 1, 2008): 338–43. http://dx.doi.org/10.1182/blood-2007-07-102350.
Full textAbstract:
FCRL1 (Fc receptor–like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC50s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL.
37
Jegalian, Armin G., Alan S. Wayne, Robert J. Kreitman, Francis J. Mussai, Ira Pastan, Constance M. Yuan, and Maryalice Stetler-Stevenson. "CD22 Expression in Pediatric B-Lineage Acute Lymphoblastic Leukemia." Blood 114, no. 22 (November 20, 2009): 4119. http://dx.doi.org/10.1182/blood.v114.22.4119.4119.
Full textAbstract:
Abstract Abstract 4119 While the majority of pediatric patients with newly diagnosed B-lineage acute lymphoblastic leukemia (ALL) are cured with standard chemotherapy regimens, treatment is associated with multiple toxicities, and ALL remains the most frequent cause of cancer mortality in childhood. CD22, a B-lineage surface glycoprotein involved in B cell signaling and adhesion, is expressed in most cases of B-lineage ALL. We are conducting clinical trials of anti-CD22 immunotoxins [RFB4(dsFv)-PE38] for pediatric ALL. To assess eligibility for such targeted therapy, CD22 expression by ALL cells was studied in peripheral blood and/or bone marrow aspirate samples from 50 patients with relapsed ALL. The level of CD22 expression by ALL cells was quantitated by measuring mean anti-CD22 antibody binding per ALL cell (ABC) under saturating conditions using flow cytometry and the BD Biosciences QuantiBRITE system for fluorescence quantitation. Patients ranged in age from 3 to 22 years (median 10 years) and included 27 males and 23 females. CD22 expression was detected in all samples, and the vast majority of cases demonstrated expression of CD22 in 100% of leukemic blasts. CD22 antigen density in ALL cells varied widely among patients at baseline (range 451 - 14,519; mean 4276; median 3824; standard deviation 2976; see graph). CD22-directed immunotoxin therapy was initiated in 29 of the 50 patients, 19 of whom had samples quantitated for CD22 expression levels both before and after immunotoxin therapy. Most patients exhibited limited variation in the mean number of anti-CD22 molecules bound per ALL cell when comparing multiple specimens. In conclusion, CD22 expression varies widely in pediatric B-lineage ALL and persists despite repeated exposure to CD22-directed therapy. (MedImmune, LLC, sponsored the clinical studies of anti-CD22 immunotoxin CAT-8015.) Disclosures: No relevant conflicts of interest to declare.
38
Gramatzki, Dorothee, Emese Szabo, Martin Gramatzki, Matthias Peipp, and Michael Weller. "Targeting of CD317 by the immunotoxin HM1.24-ETA’ to allow immunotherapy in glioblastoma patients." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e13560-e13560. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13560.
Full textAbstract:
e13560 Background: Glioblastoma is the most common primary malignant brain tumor with a poor prognosis. CD317 (HM1.24) is a transmembrane protein and may exist in differently spliced variants. It is highly expressed on plasma cells in multiple myeloma, as well as in certain solid tumor types. While several antibody drug conjugates are already in clinical practice, small immunotoxins with a different intracellular mode of action are only established in hairy cell leukemia. The immunotoxin HM1.24-ETA’ protein is a CD317 single chain Fv (scFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’). Methods: In vivo CD317 mRNA expression in human glioma of different grades and survival probabilities of glioblastoma patients based on CD317 mRNA expression were analyzed using the database of the Cancer Genome Atlas network (TCGA). CD317 protein expression was analyzed by immunohistochemistry in a human tissue microarray (TMA). In vitro CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in several human glioblastoma cell lines. A cytotoxicity assay after treatment with HM1.24-ETA’ immunotoxin was performed in human glioblastoma cell lines. Results: Data on mRNA expression from the TCGA database demonstrated, that CD317 was upregulated in human glioblastomas compared to lower grade gliomas. In the group of glioblastoma patients increased CD317 mRNA expression was associated with decreased probability of survival ( p< 0.001). CD317 protein levels correlated directly with the tumor grade of astrocytic gliomas in the TMA. CD317 was expressed heterogeneously on mRNA and protein levels in the tested cell-lines in vitro. HM1.24-ETA’ induced cytotoxicity in CD317-positive glioblastoma cells in a concentration-dependent manner. Animal experiments currently performed suggest activity in glioblastoma xenografted mice. Conclusions: These data highlight CD317 as an interesting target antigen and HM1.24-ETA’ immunotoxin as a strategy for immunotherapy of glioblastoma patients.
39
Frankel, A. E., J. Woo, S. L. Zuckero, A. A. Mankin, M. Grable, K. Mitchell, Y. Lee, and D. M. Neville. "CD3 immunotoxin therapy of cutaneous T cell lymphoma." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e19511-e19511. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e19511.
Full textAbstract:
e19511 Background: T cell lymphomas represent 12% of lymphoma cases in the U.S. Cytotoxic chemotherapies, radiation therapies, monoclonal antibodies, transcription modulators and topical therapies yield remissions, but over half of patients relapse and die with progressive disease. Patients ineligible for allogeneic transplant need additional therapeutic options. One class of T cell directed agents are immunotoxins composed of protein synthesis inactivating peptide toxins covalently linked to antibodies or hormone ligands. We prepared a new immunotoxin, A-dmDT390-bisFv(UCHT1) composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain antibody fragments reactive with an acidic loop on the extracellular domain of CD3epsilon. We prepared a clinical batch of drug and obtained FDA approval for a phase I trial (IND#100712). Methods: Cohort of three-six CTCL patients were treated with immunotoxin via 15min IV infusion twice daily for four days at 2.5–5ug/kg. CTCAEv3.0 toxicity grading, blood samples counts, chemistries, CMV/EBV PCR, PK, immune response and flow cytometry, and disease assessments by CT scans, skin mapping and bone marrow biopsies were done. Results: Six patients received all eight 2.5ug/kg doses. Toxicities were mild- moderate with fever, chills, nausea, transaminasemia, hypoalbuminemia, lymphopenia and reactivation of EBV and CMV. Side effects responded to antipyretics, anti-emetics, albumin infusions, rituximab and valgancyclovir. Lymphopenia was marked at the end of treatment (99.9% reduction) and was followed by partial recovery at two weeks. Circulating T regulatory cells doubled. Cmax occured 5 min post-infusion and was 18ng/mL. Half-life was 49 min. All patient had anti-immunotoxin antibodies at a median of 1.3ug/mL which increased after 30 days to 9–1700ug/mL. Two of five evaluable patients had PRs lasting one and 6+ months. Conclusions: This novel immunotoxin has dramatic clinical activity even at the lowest dose in CTCL patients and merits applications at higher doses in CTCL and other CD3+ T cell leukemia/lymphoma patients. The lymphodepletion with recovery of T regulatory cells suggests the drug may be beneficial for immunosuppression of T cell autoimmune disorders. No significant financial relationships to disclose.
40
Yu, Xin, Stephen T. Keir, Scott Szafranski, Steven Clayton, Ira Pastan, Darell D. Bigner, and Vidyalakshmi Chandramohan. "Immunotoxin and bcl-2 inhibitor combination therapy targeting chondroitin sulfate proteoglycan 4." Journal of Clinical Oncology 35, no. 7_suppl (March 1, 2017): 74. http://dx.doi.org/10.1200/jco.2017.35.7_suppl.74.
Full textAbstract:
74 Background: Immunotoxins (ITs) are a class of bifunctional chimeric proteins composed of an antibody fragment linked to a toxin. When ITs internalize into target cells, they induce protein synthesis inhibition and apoptosis. While ITs are highly specific and potent, the efficacy of IT-based therapies in some tumor cells is limited by hyperactive anti-apoptotic pathways and inefficient translocation of ITs from the endoplasmic reticulum to the cytosol. Therefore, to improve the efficacy of IT-based therapies, we evaluated a dual-pathway therapy that combines an IT with the ABT-737, ABT-263, or ABT-199 small molecule Bcl-2 inhibitor. Methods: The immunotoxin 9.2.27-PE38KDEL (9.2.27-IT) was generated by fusing a truncated mutant form of Pseudomonas exotoxin A to a single-chain variable fragment antibody. It targets human chondroitin sulfate proteoglycan 4 (CSPG4), an antigen highly expressed in a variety of cancer cells. We screened and identified 3 human glioblastoma xenografts, 3 human melanoma cell lines, and 5 human breast cancer cell lines resistant to the 9.2.27-IT despite their high levels of cell surface expression of CSPG4 (IC50 of IT alone was >100 ng/ml in all cell lines except for one melanoma cell line). In vitro cytotoxicity of the 9.2.27-IT —alone or in combination with the individual Bcl-2 inhibitors ABT-737, ABT-263, or ABT199—was assessed. Concentrations of ABT analogues were chosen so that ABT alone did not induce cytotoxicity. Results: The treatment groups that responded to the combination therapy yielded IC50 values ranging from 0.04 – 9 ng/ml for glioblastoma xenografts, 0.21-15 ng/ml for melanoma cell lines, and 4.5-50 ng/ml for breast cancer cell lines. The most potent combination group showed >1000 fold improvement of IC50 compared to using the immunotoxin alone. ABT-737 produced the strongest synergistic effects among the ABT analogues. Preliminary results from in vivo studies further demonstrated that this approach engendered a synergistic response and delayed tumor growth in immunotoxin-resistant mouse tumor models. Conclusions: This new combinatorial approach will potentially help to overcome immunotoxin resistance in cancer patients and provide better therapeutic outcomes.
41
Teo, Michelle Yee Mun, Jeremy Jeack Ceen Ng, Jung Yin Fong, Jung Shan Hwang, Adelene Ai-Lian Song, Renee Lay Hong Lim, and Lionel Lian Aun In. "Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells." PeerJ 9 (April 15, 2021): e11063. http://dx.doi.org/10.7717/peerj.11063.
Full textAbstract:
Background KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.
42
Zhang, Hongyu, Mi Deng, Fen Pei, Shouye Wang, and Mitchell Ho. "Next-Generation Antibody Therapeutics: Discovery, Development and Beyond: highlights of the third annual conference of the Chinese Antibody Society." Antibody Therapeutics 2, no. 4 (October 1, 2019): 99–107. http://dx.doi.org/10.1093/abt/tbz012.
Full textAbstract:
ABSTRACT The Chinese Antibody Society (CAS) convened the third annual conference in Cambridge, Massachusetts, USA on April 7, 2019. More than 600 global members attended the meeting. The theme of this conference was Next-Generation Antibody Therapeutics: Discovery, Development and Beyond. The meeting covered a vast variety of topics including cancer immunotherapy, single-domain antibodies as well as bispecific antibodies, immunotoxins, transgenic mouse platforms for next-generation monoclonal antibody discovery and antibody chemistry, manufacturing and controls (CMCs). Two hot topics were comprehensively discussed by the prestigious panelists and hosts at the panel discussions during the conferences, i.e., bispecific antibodies and antibody CMC. Statement of Significance: The Chinese Antibody Society convened the third annual conference in Cambridge, Massachusetts, USA on 7 April 2019. The meeting covered a variety of topics, including cancer immunotherapy, single-domain antibody, bispecific antibody, immunotoxin, transgenic mouse platforms for next-generation monoclonal antibody discovery and antibody CMC.
43
Siena, S., DA Lappi, M. Bregni, A. Formosa, S. Villa, M. Soria, G. Bonadonna, and AM Gianni. "Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates." Blood 72, no. 2 (August 1, 1988): 756–65. http://dx.doi.org/10.1182/blood.v72.2.756.756.
Full textAbstract:
Abstract The authors conjugated, by a disulphide bond, the antihuman T- lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen- induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double- antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.
44
Siena, S., DA Lappi, M. Bregni, A. Formosa, S. Villa, M. Soria, G. Bonadonna, and AM Gianni. "Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates." Blood 72, no. 2 (August 1, 1988): 756–65. http://dx.doi.org/10.1182/blood.v72.2.756.bloodjournal722756.
Full textAbstract:
The authors conjugated, by a disulphide bond, the antihuman T- lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen- induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double- antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.
45
Pichinuk, Edward, Itai Benhar, Oded Jacobi, Ravit Ziv, Nechama Smorodinsky, Daniel B. Rubinstein, and Daniel H. Wreschner. "Targeting of tumor cells and tumor stem cells by antibodies specific for the cell-bound alpha/beta junction of MUC1." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e13018-e13018. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13018.
Full textAbstract:
e13018 Background: MUC1 is a glycoprotein over-expressed on the cell surface of a variety of malignancies and has long been a subject of interest in targeted therapy.Cleavage of MUC1 yields two unequal chains, a large extracellular a subunit non-covalently bound to a smaller b subunit in an on-and-off mechanism. Because it is released into the peripheral circulation, the MUC1 a subunit readily sequesters antibodies directed against it. In order to avoid such peripheral sequestration, Abs directed against MUC1 must be engineered as to target cell bound moieties of MUC1. Methods: Seven anti-MUC1 monoclonal antibodies were generated, five Ig-gamma1 and two IgA, all directed against the MUC1 a/b junction, a structure which remains cell bound at all times. Results: In addition to their binding differentiated breast cancer cells at pico Molar concentrations, the anti-MUC1 mAbs strongly bind breast cancer stem cells. With a view for clinical application, the DMB5F3 anti-MUC1 mAb was partially humanized with resultant production of chimeric IgG1. In order to demonstrate that chimeric anti-MUC1 antibodies can be used in tumor cell killing, mAb DMB5F3 was linked to the pseudomonas bacterial exotoxin ZZ-PE38. The resultant anti-MUC1 immunotoxin not only efficiently internalized the toxin, it resulted in strong cytotoxicity of MUC1+ breast cancer. In fact, DMB5F3:PE38 immunotoxin bound MUC1+ cancer cells with an affinity (Ab concentrations as low as 20pM) exceeding that seen with cetuximab (anti-EGFR1)-PE38 and tratuzumab (anti-erbB2)-PE38 immunotoxins. Conclusions: Taken together, these findings indicate [1] the therapeutic use of targeting cell-bound MUC1 [2] high affinity activity against both tumor and tumor stem cells suggests that the anti-MUC1 mAbs target not only 'mature' differentiated malignancy but self-reproducing cells giving rise to new malignant growth as well [3]tumor can be targeted directly by anti-MUC1 antibodies or by linking anti-MUC1 to toxin, resulting in efficient immunotoxin cytotoxicity [4] as it was shown to be a targetable entity, the SEA domain represents a promising immunogen for MUC1 tumor vaccines.
46
Ahuja, Yachna, Maryalice Stetler-Stevenson, Robert J. Kreitman, Ira Pastan, and Alan S. Wayne. "Pre-Clinical Evaluation of the Anti-CD22 Immunotoxin CAT-8015 in Combination with Chemotherapy Agents for Childhood B-Precursor Acute Lymphoblastic Leukemia (Pre-B ALL)." Blood 110, no. 11 (November 16, 2007): 865. http://dx.doi.org/10.1182/blood.v110.11.865.865.
Full textAbstract:
Abstract Despite advances in curative treatment, childhood ALL remains a leading cause of cancer-related mortality in pediatrics and survivors are at risk of multiple late effects. Targeted therapies that overcome chemotherapy resistance and decrease nonspecific toxicities are needed. The B-lineage differentiation antigen CD22 represents a relevant target for pre-B ALL. Monotherapy with the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) has been well tolerated in a pediatric Phase I trial. To date however, only transient clinical activity has been observed with this agent in children with ALL. We conducted cytotoxicity studies to evaluate combination regimens of a higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) with standard ALL chemotherapeutic agents. Methods: CD22+ human malignant cell samples studied included cell lines of Burkitt lymphoma (CA46) and pre-B ALL (REH and KOPN-8), as well as primary lymphoblasts from 2 children with multiply relapsed pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. Asparaginase, cytarabine, doxorubicin, methotrexate, and vincristine were added to cells individually and in various combinations with CAT-8015. Total incubation time was 2 days for cell lines and 5 days for primary patient blasts. Cell viability was assessed using WST-8. The Chou-Talalay median-effect method was used for data analysis (Chou TC, Talalay P. Adv Enzyme Regul1984;22:27–55). Results: All samples were sensitive to CAT-8015 and to most chemotherapy agents tested. Simultaneous addition of a fixed concentration of CAT-8015 with varying concentrations of each chemotherapy drug led to a 2- to 4-fold reduction in IC50 in comparison to individual agents. Assays with simultaneous addition of equipotency ratios of CAT-8015 and chemotherapy agents had synergistic or additive interactions. These interactions were sequence dependent, and the addition of CAT-8015 followed by chemotherapy led to greater positive interactions than the reverse order. Further assays with simultaneous addition of CAT-8015 and doublet chemotherapy drugs showed enhanced synergistic and additive interactions compared to assays with only doublet agents. Conclusions: CAT-8015 combined with standard chemotherapy agents results in synergistic or additive cytotoxicity against CD22-positive Burkitt lymphoma and pre-B ALL cell lines and primary blasts from children with pre-B ALL. High affinity anti-CD22 recombinant immunotoxins have the potential to overcome chemotherapy resistance and to decrease nonspecific treatment-associated toxicities. In addition, CAT-8015 might synergistically enhance chemotherapy-induced cytotoxicity, thus improving efficacy and allowing the use of less toxic regimens in the future. Clinical trials of recombinant immunotoxins targeting CD22 in combination with standard ALL chemotherapy regimens are planned.
47
Leonard, JE, R. Taetle, D. To, and K. Rhyner. "Preclinical studies on the use of selective antibody-ricin conjugates in autologous bone marrow transplantation." Blood 65, no. 5 (May 1, 1985): 1149–57. http://dx.doi.org/10.1182/blood.v65.5.1149.1149.
Full textAbstract:
Abstract Whole-ricin immunoconjugates were synthesized with the pan-T cell antibodies T101 and 3A1 and assayed in the presence of 0.1 mol/L lactose. Their toxicity for cell lines, peripheral blood T lymphocytes, and normal bone marrow progenitors was compared with that of whole ricin. In the presence of 0.1 mol/L lactose, normal cells and cell lines exhibited the following sensitivities to ricin: 8392 (human malignant B cell line) less than E rosette-positive lymphocytes less than bone marrow progenitors less than 8402 (human T ALL) less than CEM (human T ALL). Ricin sensitivities correlated with ricin binding as determined by immunofluorescence. In the presence of lactose, peripheral blood T cells were resistant to 0.1 nmol/L ricin, but a similar concentration of T101-ricin inhibited normal and malignant T colony formation by greater than 98%. 3A1-ricin was slightly less effective. At a conjugate concentration of 0.1 nmol/L, bone marrow progenitor colony formation was inhibited by 30% or less; T101-positive cells were at least tenfold more sensitive than normal progenitors. When mixtures of 10% CEM cells and marrow cells were incubated with T101-ricin, 95% of CEM colonies were killed, and 96% of marrow granulocyte/ macrophage progenitors survived. Some free ricin was released from immunotoxin-treated cells, producing minimal inhibition of protein synthesis or cell growth. We conclude that (a) normal blood cells and malignant cell lines exhibit varying degrees of ricin sensitivity in the presence of lactose; (b) T101-ricin is at least tenfold more toxic to T lymphocytes than to bone marrow progenitor cells and is effective in mixtures of normal and malignant cells; and (c) treatment of infiltrated marrow with anti-T cell immunotoxins should safely remove target T cells without excessively damaging normal progenitors or producing excessive free ricin. Anti-T cell, whole-ricin immunotoxins merit trials for autologous transplantation.
48
Leonard, JE, R. Taetle, D. To, and K. Rhyner. "Preclinical studies on the use of selective antibody-ricin conjugates in autologous bone marrow transplantation." Blood 65, no. 5 (May 1, 1985): 1149–57. http://dx.doi.org/10.1182/blood.v65.5.1149.bloodjournal6551149.
Full textAbstract:
Whole-ricin immunoconjugates were synthesized with the pan-T cell antibodies T101 and 3A1 and assayed in the presence of 0.1 mol/L lactose. Their toxicity for cell lines, peripheral blood T lymphocytes, and normal bone marrow progenitors was compared with that of whole ricin. In the presence of 0.1 mol/L lactose, normal cells and cell lines exhibited the following sensitivities to ricin: 8392 (human malignant B cell line) less than E rosette-positive lymphocytes less than bone marrow progenitors less than 8402 (human T ALL) less than CEM (human T ALL). Ricin sensitivities correlated with ricin binding as determined by immunofluorescence. In the presence of lactose, peripheral blood T cells were resistant to 0.1 nmol/L ricin, but a similar concentration of T101-ricin inhibited normal and malignant T colony formation by greater than 98%. 3A1-ricin was slightly less effective. At a conjugate concentration of 0.1 nmol/L, bone marrow progenitor colony formation was inhibited by 30% or less; T101-positive cells were at least tenfold more sensitive than normal progenitors. When mixtures of 10% CEM cells and marrow cells were incubated with T101-ricin, 95% of CEM colonies were killed, and 96% of marrow granulocyte/ macrophage progenitors survived. Some free ricin was released from immunotoxin-treated cells, producing minimal inhibition of protein synthesis or cell growth. We conclude that (a) normal blood cells and malignant cell lines exhibit varying degrees of ricin sensitivity in the presence of lactose; (b) T101-ricin is at least tenfold more toxic to T lymphocytes than to bone marrow progenitor cells and is effective in mixtures of normal and malignant cells; and (c) treatment of infiltrated marrow with anti-T cell immunotoxins should safely remove target T cells without excessively damaging normal progenitors or producing excessive free ricin. Anti-T cell, whole-ricin immunotoxins merit trials for autologous transplantation.
49
Uckun, FM, LM Chelstrom, JD Irvin, D. Finnegan, R. Gunther, J. Young, V. Kuebelbeck, DE Myers, and LL Houston. "In vivo efficacy of B43 (anti-CD19)-pokeweed antiviral protein immunotoxin against BCL-1 murine B-cell leukemia." Blood 79, no. 10 (May 15, 1992): 2649–61. http://dx.doi.org/10.1182/blood.v79.10.2649.2649.
Full textAbstract:
Abstract We show that a highly aggressive subclone of murine BCL-1 B-lineage leukemia expresses a single 2.4-kb transcript hybridizing to the human CD19 cDNA probe and reacts strongly with the anti-human CD19 monoclonal antibodies (MoAb) B43, B4, Leu-12, and J3–119. In contrast to their strong reactivity with anti-human CD19 MoAb, BCL-1 cells show no reactivity with MoAb directed against human CD22, CD72, HLA-DR, IgD, or IgM. Western blot analysis of BCL-1 whole cell lysates with the anti- human CD19 MoAb J3–119 showed a single 69-Kd protein band, which was not detected by the negative control MoAb G19.4 (anti-CD3). In contrast to BCL-1 cells, normal BALB/c splenocytes or mouse splenocyte/myeloma hybridoma cell lines did not (1) express any transcripts that hybridized to the human CD19 cDNA probe, (2) react with B43/anti-CD19 MoAb, or (3) express the 69-Kd protein that reacts with the anti-human CD19 MoAb J3–119. Murine BCL-1 B-cell leukemia thus provides a unique model of disseminated B-lineage leukemia to evaluate the antileukemic efficacy of anti-CD19 immunotoxins. This model was subsequently used to evaluate the in vivo homing ability, pharmacokinetics, and antileukemic efficacy of B43 MoAb conjugated to the plant hemitoxin pokeweed antiviral protein (PAP). B43-PAP immunotoxin (1) showed strong and antigen-specific reactivity with BCL-1 cells, (2) promptly penetrated the spleens of leukemic mice, (3) rapidly reduced the BCL-1 leukemia burden of leukemic mice and, most importantly, (4) improved survival. Finally, B43-PAP immunotoxin was more effective against BCL-1 leukemia than 700 cGy (LD100/30) total body irradiation (TBI) followed by syngeneic bone marrow transplantation (BMT).
50
Uckun, FM, LM Chelstrom, JD Irvin, D. Finnegan, R. Gunther, J. Young, V. Kuebelbeck, DE Myers, and LL Houston. "In vivo efficacy of B43 (anti-CD19)-pokeweed antiviral protein immunotoxin against BCL-1 murine B-cell leukemia." Blood 79, no. 10 (May 15, 1992): 2649–61. http://dx.doi.org/10.1182/blood.v79.10.2649.bloodjournal79102649.
Full textAbstract:
We show that a highly aggressive subclone of murine BCL-1 B-lineage leukemia expresses a single 2.4-kb transcript hybridizing to the human CD19 cDNA probe and reacts strongly with the anti-human CD19 monoclonal antibodies (MoAb) B43, B4, Leu-12, and J3–119. In contrast to their strong reactivity with anti-human CD19 MoAb, BCL-1 cells show no reactivity with MoAb directed against human CD22, CD72, HLA-DR, IgD, or IgM. Western blot analysis of BCL-1 whole cell lysates with the anti- human CD19 MoAb J3–119 showed a single 69-Kd protein band, which was not detected by the negative control MoAb G19.4 (anti-CD3). In contrast to BCL-1 cells, normal BALB/c splenocytes or mouse splenocyte/myeloma hybridoma cell lines did not (1) express any transcripts that hybridized to the human CD19 cDNA probe, (2) react with B43/anti-CD19 MoAb, or (3) express the 69-Kd protein that reacts with the anti-human CD19 MoAb J3–119. Murine BCL-1 B-cell leukemia thus provides a unique model of disseminated B-lineage leukemia to evaluate the antileukemic efficacy of anti-CD19 immunotoxins. This model was subsequently used to evaluate the in vivo homing ability, pharmacokinetics, and antileukemic efficacy of B43 MoAb conjugated to the plant hemitoxin pokeweed antiviral protein (PAP). B43-PAP immunotoxin (1) showed strong and antigen-specific reactivity with BCL-1 cells, (2) promptly penetrated the spleens of leukemic mice, (3) rapidly reduced the BCL-1 leukemia burden of leukemic mice and, most importantly, (4) improved survival. Finally, B43-PAP immunotoxin was more effective against BCL-1 leukemia than 700 cGy (LD100/30) total body irradiation (TBI) followed by syngeneic bone marrow transplantation (BMT).