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Ira Pastan - One of the best experts on this subject based on the ideXlab platform.
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phase i study of mesothelin targeted immunotoxin lmb 100 in combination with tofacitinib in persons with pancreatobiliary cancer or other mesothelin expressing solid tumors
Journal of Clinical Oncology, 2021Co-Authors: Guillaume Joe Pegna, Raffit Hassan, David J Fitzgerald, Ira Pastan, Mehwish Iqra Ahmad, Cody J Peer, William D Figg, Seth M Steinberg, Christine AlewineAbstract:TPS452Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting with a modified Pseudomonas Exotoxin A payload. Formation of anti-drug antibodies (ADAs) is thou...
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immunogenicity of immunotoxins containing Pseudomonas Exotoxin a causes consequences and mitigation
Frontiers in Immunology, 2020Co-Authors: Ronit Mazor, Ira PastanAbstract:Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Pseudomonas Exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.
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Pseudomonas Exotoxin immunotoxins and anti tumor immunity from observations at the patient s bedside to evaluation in preclinical models
Toxins, 2019Co-Authors: Yasmin Leshem, Ira PastanAbstract:Immunotoxins are protein drugs composed of a targeting domain genetically fused to a protein toxin. One killing domain being explored is a truncated Pseudomonas Exotoxin A (PE). PE based immunotoxins are designed to kill cells directly by inhibiting their ability to synthesize proteins. However, observations from clinical trials suggest that this alone cannot explain their anti-tumor activity. Here we discuss patterns of clinical responses suggesting that PE immunotoxins can provoke anti-tumor immunity, and review murine models that further support this ability. In addition, we describe our preclinical effort to develop a combination therapy of local PE immunotoxins with a systemic anti-CTLA-4 immune check point blocking antibody. The combination eradicated murine tumors and prolonged the survival of mice. Clinical trials that test the ability of immunotoxins to augment immunotherapy have been recently opened.
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Pseudomonas Exotoxin Immunotoxins and Anti-Tumor Immunity: From Observations at the Patient’s Bedside to Evaluation in Preclinical Models
MDPI AG, 2019Co-Authors: Yasmin Leshem, Ira PastanAbstract:Immunotoxins are protein drugs composed of a targeting domain genetically fused to a protein toxin. One killing domain being explored is a truncated Pseudomonas Exotoxin A (PE). PE based immunotoxins are designed to kill cells directly by inhibiting their ability to synthesize proteins. However, observations from clinical trials suggest that this alone cannot explain their anti-tumor activity. Here we discuss patterns of clinical responses suggesting that PE immunotoxins can provoke anti-tumor immunity, and review murine models that further support this ability. In addition, we describe our preclinical effort to develop a combination therapy of local PE immunotoxins with a systemic anti-CTLA-4 immune check point blocking antibody. The combination eradicated murine tumors and prolonged the survival of mice. Clinical trials that test the ability of immunotoxins to augment immunotherapy have been recently opened
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improving the in vivo efficacy of an anti tac cd25 immunotoxin by Pseudomonas Exotoxin a domain ii engineering
Molecular Cancer Therapeutics, 2018Co-Authors: Gilad Kaplan, Yasmin Leshem, Ronit Mazor, Fred Lee, Youjin Jang, Ira PastanAbstract:Tac (CD25) is expressed on multiple hematologic malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac and a 38-kDa fragment of Pseudomonas Exotoxin A (PE38). Although LMB-2 has shown high cytotoxicity toward Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and nonspecific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T-cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site, which is required for cytotoxic activity. Although this strategy has been successfully implemented for mesothelin and CD22-targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of anti-Tac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II–deleted toxin variants. The domain II–deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored 5-fold of the cytotoxic activity and dramatically improved the MTD. Both of these improvements led to significantly increased antitumor efficacy in vivo. We conclude that the next-generation anti-Tac immunotoxin is an improved candidate for targeting Tac-expressing malignancies. Mol Cancer Ther; 17(7); 1486–93. ©2018 AACR.
Raj K Puri - One of the best experts on this subject based on the ideXlab platform.
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combination immunotherapy with il 4 Pseudomonas Exotoxin and ifn α and ifn γ mediate antitumor effects in vitro and in a mouse model of human ovarian cancer
Immunotherapy, 2019Co-Authors: Daniel S Green, Raj K Puri, Bharat H. Joshi, Syed R Husain, Chase L Johnson, Yuki Sato, Jing Han, Stephen M Hewitt, Kathryn C ZoonAbstract:Aim We have shown that IL-4 fused to Pseudomonas Exotoxin (IL-4-PE) is cytotoxic to ovarian cancer cell lines. The antineoplastic properties of IFN-α, IFN-γ and IL-4-PE have been studied and showed some promise in the clinical trials. Here, we investigated whether the combination of IL-4-PE, IFN-α and IFN-γ will result in increased ovarian cancer cell death in vitro and in vivo. Materials & methods Ovarian cancer cells were tested in vitro to analyze the cytotoxic effects of IL-4-PE, IFN-α and IFN-γ, and the combination of all three. Tumor-bearing xenograft mice were treated with the combination of IL-4-PE, IFN-α and IFN-γ to monitor their overall survival. The JAK/STAT phosphorylation signaling pathways were studied to delineate the mechanism of synergistic antitumor activity. Results The combination of IL-4-PE with IFN-α and IFN-γ resulted in increased ovarian cancer cell death in vitro and in vivo. Mechanistically, the synergistic antitumor effect was dependent on interferon signaling, but not IL-4-PE signaling as determined by signaling specific chemical inhibitors. The combination therapy induced the activation of critical mediators of apoptosis. Conclusion The combination of IL-4-PE with interferons increased overall survival of mice with human ovarian cancer xenograft. These data suggest that this novel combination could provide a unique approach to treating ovarian cancer.
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phase i trial of intravenous il 4 Pseudomonas Exotoxin protein nbi 3001 in patients with advanced solid tumors that express the il 4 receptor
Journal of Immunotherapy, 2005Co-Authors: Linda L Garland, Raj K Puri, Barbara J Gitlitz, Scot W Ebbinghaus, Henry Pan, Hans De Haan, Daniel D Von Hoff, Robert A FiglinAbstract:NBI-3001 is a novel immunotoxin of attenuated Pseudomonas Exotoxin fused to circularly permutated IL-4, which has shown some antitumor effects in glioblastoma multiforme with intratumoral administration. The authors evaluated the safety and tolerability of NBI-3001 administered intravenously in a dose-escalation design to patients with renal cell and non-small cell lung carcinoma whose tumors showed at least 10% IL-4 receptor expression. Cohorts of three to six patients were treated at dose levels of 0.008, 0.016, and 0.027 mg/m2 daily x 5 days every 28 days. Neutralizing antibody (NAB) titers, plasma levels of NBI-3001, and patient tolerability were monitored sequentially. 14 patients received a total of 36 cycles of NBI-3001 (range 1-6). No dose-limiting toxicities were noted at dose levels 0.008 and 0.016 mg/m2. At 0.027 mg/m2, two patients developed self-limiting, grade 3 or 4 transaminase elevation during cycle 1. NAB titers of more than 1:100 were detected in five of the seven patients treated with at least two cycles; the median titer after cycle 1 and the median maximum titer in subsequent cycles were 1:50 and approximately 1:1,710, respectively. No objective tumor responses were noted. Eight of 12 evaluable patients with renal cell carcinoma had stable disease; four patients had disease progression. High NAB titers resulted in four patients being withdrawn from the study. The dose-limiting toxicity for intravenous NBI-3001 was transaminase elevation at 0.027 mg/m2. NBI-3001 at 0.016 mg/m2 was well tolerated. Low circulating levels of NBI-3001, coupled with rising NAB titers, may have contributed to the lack of response in tumors that express IL-4R.
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neuroradiographic changes following convection enhanced delivery of the recombinant cytotoxin interleukin 13 pe38qqr for recurrent malignant glioma
Journal of Neurosurgery, 2005Co-Authors: Ian F Parney, Raj K Puri, Mitchel S Berger, Sandeep Kunwar, Michael T Mcdermott, Michael D Prados, Soonmee Cha, David Croteau, Susan M ChangAbstract:Object. Convection-enhanced delivery (CED) is a novel method for delivering therapeutic agents to infiltrative brain tumor cells. For agents administered by CED, changes on magnetic resonance (MR) imaging directly resulting from catheter placement, infusion, and the therapeutic compound may confound any interpretation of tumor progression. As part of an ongoing multiinstitutional Phase I study, 14 patients with recurrent malignant glioma underwent CED of interleukin (IL) 13—PE38QQR, a recombinant cytotoxin consisting of human IL-13 conjugated with a truncated Pseudomonas Exotoxin. Serial neuroradiographic changes were assessed in this cohort of patients. Methods. Patients were treated in two groups: Group 1 patients received IL13—PE38QQR before and after tumor resection; Group 2 patients received infusion only after tumor resection. Preoperative and postinfusion MR images were obtained prospectively at specified regular intervals. Changes were noted along catheter tracks on postresection MR images obtaine...
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analysis of antitumor activity of an interleukin 13 il 13 receptor targeted cytotoxin composed of il 13 antagonist and Pseudomonas Exotoxin
Clinical Cancer Research, 2004Co-Authors: Mitomu Kioi, Koji Kawakami, Raj K PuriAbstract:We have shown previously that a chimeric fusion protein composed of human interleukin-13 (IL-13) and Pseudomonas Exotoxin (PE), termed IL-13 cytotoxin (IL13-PE38), is specifically cytotoxic to various cancer cell lines and primary cell cultures derived from a variety of solid cancers. In addition, we have shown that IL-13 mutant IL-13E13K, in which glutamic acid (E) residue at position 13 of IL-13 molecule was substituted by a lysine (K), is a powerful antagonist of IL-13 and binds to IL-13 receptor with a higher affinity compared with wild-type IL-13. In this study, we have generated an IL-13 cytotoxin IL13E13K-PE38, in which IL-13 antagonist is fused to PE to determine whether this molecule has improved cytotoxicity to tumor cells compared with wild type (wt)IL13-PE38. Highly purified IL13E13K-PE38 was tested in various tumor cell lines including seven glioblastoma multiforme cell lines to compare its binding to the cells, in vitro cytotoxicity, in vivo antitumor activity, and safety in mouse model with wtIL13-PE38. IL13E13K-PE38 bound to U251MG and IL-13Ralpha2 chain-transfected tumor cell lines with 3 to 10 times higher affinity compared with wtIL13-PE38. However, IL13E13K-PE38 did not show higher cytotoxicity compared with wtIL13-PE38 in glioblastoma multiforme or any other cell lines tested. The antitumor activity of IL13E13K-PE38, when administered intraperitoneally to nude mice bearing U251 tumors, was also similar to wtIL13-PE38. Some improvement in antitumor activity was observed when lower doses of IL13E13K-PE38 were injected intratumorally in subcutaneous tumors. These results indicate that in general, IL13E13K-PE38 mediates similar cytotoxicity and antitumor activity to wtIL13-PE38 despite its improved binding affinity to IL-13 receptors.
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intratumoral administration of recombinant circularly permuted interleukin 4 Pseudomonas Exotoxin in patients with high grade glioma
Clinical Cancer Research, 2000Co-Authors: Robert W Rand, Robert J Kreitman, Ira Pastan, Nicholas J Patronas, Frederick Varricchio, Raj K PuriAbstract:Human glioblastoma but not normal brain cells express numerous receptors for the cytokine interleukin (IL)-4. To target these receptors, we have investigated the safety and activity of directly infusing IL-4(38-37)-PE38KDEL, a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas Exotoxin (PE), into recurrent malignant high-grade gliomas. IL-4(38-37)-PE38KDEL (IL-4-toxin) was infused over a 4-8-day period into gliomas of nine patients by one to three stereotactically placed catheters. No apparent systemic toxicity occurred in any patient. The infusion of IL-4-toxin in six of nine patients showed glioma necrosis as evidenced by diminished gadolinium enhancement on magnetic resonance imaging. Seven of nine patients underwent craniotomy because of increased intracranial pressure at 16-101 days after the beginning of infusion. In six of these seven patients, partial-to-extensive tumor necrosis with edema was confirmed pathologically. No histological evidence of neurotoxicity to normal brain was identified in any patient. Two patients were not operated on; by magnetic resonance imaging, one showed mottled gadolinium enhancement, and the other showed extensive necrosis of tumor leading to complete remission; this patient remains disease-free > 18 months after the procedure. We conclude that direct glioma injection of IL-4(38-37)-PE38KDEL is safe without systemic toxicity. Local toxicity seemed attributable mainly to tumor necrosis or occasionally to the volume of infusion. Histological evidence of toxicity to normal brain was not observed and in many patients, could be pathologically excluded. Additional patients are being treated to determine the maximal tolerated concentration and volume of IL-4(38-37)-PE38KDEL.
David J Fitzgerald - One of the best experts on this subject based on the ideXlab platform.
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phase i study of mesothelin targeted immunotoxin lmb 100 in combination with tofacitinib in persons with pancreatobiliary cancer or other mesothelin expressing solid tumors
Journal of Clinical Oncology, 2021Co-Authors: Guillaume Joe Pegna, Raffit Hassan, David J Fitzgerald, Ira Pastan, Mehwish Iqra Ahmad, Cody J Peer, William D Figg, Seth M Steinberg, Christine AlewineAbstract:TPS452Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting with a modified Pseudomonas Exotoxin A payload. Formation of anti-drug antibodies (ADAs) is thou...
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Research Article ABT-737 Overcomes Resistance to Im Apoptosis and Enhances the Delivery Exotoxin–Based Proteins to the
2016Co-Authors: Cell C, Antonella Antignani, Roberta Traini, Diana V Pastrana, Gal Ben-josef, Elizabeth M, David J FitzgeraldAbstract:Pseudomonas Exotoxin (PE)–based immunotoxins (anti complete remissions in patients with hairy cell leukemia b address possible mechanisms of resistance, we investigat comp errin s, the notox other fold, endo ted. A in the cate f Immun geted to antibody directed or antige tibody F Exotoxin of hema against c ture of p with the nificant h toxin. this or-slocate rg-Glu-o traffic n active sing the system cally to hesis. It se toxin to kill a n early ith the been developed. These have led to the appreciation that ause prosurvival-2 family of pro-ce of harsh treat-of Bcl-xl was the ith 122 standar
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abstract 5490 the cytotoxic activity of the anti mesothelin immunotoxin ss1p is enhanced by the pkc inhibitor enzastaurin
Cancer Research, 2013Co-Authors: Abid R. Mattoo, Ira Pastan, David J FitzgeraldAbstract:The activation of protein kinase C (PKC) contributes to tumor survival and proliferation provoking the development of inhibitory agents that may function as cancer therapeutics. Pseudomonas Exotoxin-based immunotoxins are antibody-based recombinant proteins that employ an ADP-ribosylation domain for the enzymatic inhibition of protein synthesis of target cancer cells. Combining PKC inhibitors with immunotoxins was undertaken to explore possible synergy. Enzastaurin but not two other PKC inhibitors (Sortastaurin and G06976) combined with immunotoxins directed to surface mesothelin or the transferrin receptor to produce synergistic or additive cell death via apoptosis. Enhancement of killing was noted with enzastaurin concentrations of 5 uM or greater. Mechanistic insights of the combined immunotoxin-enzastaurin killing centered on the rapid complete loss of the prosurvival Bcl2 protein, Mcl-1, and the activation of caspase 3/7. Moreover, efficient cell killing was associated with loss of Akt and the BH3-only protein Bad. Enzastaurin-mediated enhanced cell killing was specific to Pseudomonas Exotoxin-based immunotoxins as there was no enhancement with other agents that inhibit protein synthesis such as cycloheximide and diphtheria toxin. Immunotoxin-enzastaurin combinations could be useful for the treatment of human cancers that express mesothelin and are not efficiently killed by SS1P alone. Citation Format: Abid R. Mattoo, Ira Pastan, David Fitzgerald. The cytotoxic activity of the anti-mesothelin immunotoxin SS1P is enhanced by the PKC inhibitor enzastaurin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5490. doi:10.1158/1538-7445.AM2013-5490
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Pseudomonas Exotoxin a mediated apoptosis is bak dependent and preceded by the degradation of mcl 1
Molecular and Cellular Biology, 2010Co-Authors: Richard J Youle, David J Fitzgerald, Ira PastanAbstract:Pseudomonas Exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax(-/-)) MEFs but failed in Bak knockout (Bak(-/-)) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax(-/-) MEFs, but not in Bak(-/-) or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x(L), was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x(L) in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x(L) inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.
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abt 737 overcomes resistance to immunotoxin mediated apoptosis and enhances the delivery of Pseudomonas Exotoxin based proteins to the cell cytosol
Molecular Cancer Therapeutics, 2010Co-Authors: Roberta Traini, Antonella Antignani, Gal Benjosef, Diana V Pastrana, Elizabeth Moskatel, Ashima K Sharma, David J FitzgeraldAbstract:Pseudomonas Exotoxin (PE)-based immunotoxins (antibody-toxin fusion proteins) have achieved frequent complete remissions in patients with hairy cell leukemia but far fewer objective responses in other cancers. To address possible mechanisms of resistance, we investigated immunotoxin activity in a model system using the colon cancer cell line, DLD1. Despite causing complete inhibition of protein synthesis, there was no evidence that an immunotoxin targeted to the transferrin receptor caused apoptosis in these cells. To address a possible protective role of prosurvival Bcl-2 proteins, the BH3-only mimetic, ABT-737, was tested alone or in combination with immunotoxins. Neither the immunotoxin nor ABT-737 alone activated caspase 3, whereas the combination exhibited substantial activation. In other epithelial cell lines, ABT-737 enhanced the cytotoxicity of PE-related immunotoxins by as much as 20-fold, but did not enhance diphtheria toxin or cycloheximide. Because PE translocates to the cytosol via the endoplasmic reticulum (ER) and the other toxins do not, ABT-737-mediated effects on the ER were investigated. ABT-737 treatment stimulated increased levels of ER stress response factor, ATF4. Because of its activity in the ER, ABT-737 might be particularly well suited for enhancing the activity of immunotoxins that translocate from the ER to the cell cytosol.
Robert J Kreitman - One of the best experts on this subject based on the ideXlab platform.
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Confirmation and prevention of targeted toxicity by a recombinant fusion toxin
2016Co-Authors: Robert J KreitmanAbstract:To target malignant cells by exploiting their display of limited numbers of tumor associated antigens, ligands for these antigens have been connected to protein toxins capable of killing a cell with a single molecule. Toxins that show this high potency include ricin, diphtheria toxin, and Pseudomonas Exotoxin (1 –3). A variety of antibodies and growth factors have been chemically conjugated or genetically fused to mutant forms of these toxins, and found to produce major responses in clinical trials (4–14). One such agent, denileukin diftitox, contains human interleukin-2 fused with amino acids 1 to 388 of diphtheria toxin and is approved for the treatment of a subset of patients with cutaneous T-cell lymphoma (14). Another agent under clinical development, BL22, contains an Fv fragment of an anti-CD22 monoclonal antibody fuse
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antibody fusion proteins anti cd22 recombinant immunotoxin moxetumomab pasudotox
Clinical Cancer Research, 2011Co-Authors: Robert J Kreitman, Ira PastanAbstract:Recombinant immunotoxins are fusion proteins that contain the cytotoxic portion of a protein toxin fused to the Fv portion of an antibody. The Fv binds to an antigen on a target cell and brings the toxin into the cell interior, where it arrests protein synthesis and initiates the apoptotic cascade. Moxetumomab pasudotox, previously called HA22 or CAT-8015, is a recombinant immunotoxin composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38-kDa fragment of Pseudomonas Exotoxin A, called PE38. Moxetumomab pasudotox is an improved, more active form of a predecessor recombinant immunotoxin, BL22 (also called CAT-3888), which produced complete remission in relapsed/refractory hairy cell leukemia (HCL), but it had a Clin Cancer Res; 17(20); 6398–405. ©2011 AACR .
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recombinant immunotoxin against b cell malignancies with no immunogenicity in mice by removal of b cell epitopes
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Masanori Onda, Robert J Kreitman, Richard Beers, Byungkook Lee, John E Weldon, Laiman Xiang, Ira PastanAbstract:Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas Exotoxin A, has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas Exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines, has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia, and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.
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characterization of the b cell epitopes associated with a truncated form of Pseudomonas Exotoxin pe38 used to make immunotoxins for the treatment of cancer patients
Journal of Immunology, 2006Co-Authors: Masanori Onda, David J Fitzgerald, Satoshi Nagata, Richard Beers, Robert J Fisher, James J Vincent, Byungkook Lee, Michihiro Nakamura, Jaulang Hwang, Robert J KreitmanAbstract:Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas Exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.
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Anti-tumor activity of K1-LysPE38QQR, an immunotoxin targeting mesothelin, a cell-surface antigen overexpressed in ovarian cancer and malignant mesothelioma.
Journal of Immunotherapy, 2000Co-Authors: Raffit Hassan, Jaye L. Viner, Inger Margulies, Robert J KreitmanAbstract:Summary: Mesothelin, a differentiation antigen, is a 40-kD glycosylphosphatidylinositol-linked cell-surface glycoprotein, that is present on the surface of normal mesothelium and is overexpressed in many patients with epithelial ovarian cancer and malignant mesotheliomas. Monoclonal antibody K1 is a murine immunoglobulin G1 that recognizes mesothelin. LysPE38QQR is a truncated form of Pseudomonas Exotoxin that lacks the cell-binding domain, but retains the translocation and adenosine diphosphate–ribosylation domains. It has a single lysine residue near the amino terminus that is available for conjugation to antibodies. To prevent chemical conjugation of the antibody to lysine residues at the C-terminus of Pseudomonas Exotoxin, the two lysine residues at positions 590 and 606 were mutated to glutamine, and the lysine residue at position 613 was mutated to arginine. Monoclonal antibody K1 was chemically conjugated with LysPE38QQR , by modifying the antibody with sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate and coupling it with SPDP N-succinimidyl 3-(2-pyridyldithio)propionate–modified LysPE38QQR. The resulting immunotoxin K1-LysPE38QQR was highly toxic to A431-K5 cells (a human epidermoid carcinoma cell line transfected with a mesothelin expression plasmid) with a half-maximal inhibitory concentration of 3–6 ng/mL. The immunotoxin had negligible activity against A431 cells, which do not express mesothelin (median inhibitory concentration > 100 ng/mL). This immunotoxin also caused complete regression of tumors in nude mice that received xenografts of mesothelin-positive human carcinomas. These results show that immunotoxins directed against mesothelin are a therapeutic option that merits further investigation for the treatment of ovarian cancer and malignant mesotheliomas.
Ulrich Brinkmann - One of the best experts on this subject based on the ideXlab platform.
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prospects of bacterial and plant protein based immunotoxins for treatment of cancer
Cancer Genomics & Proteomics, 2014Co-Authors: Ulrich H Weidle, Georg Tiefenthaler, Christian Schiller, Elisabeth H Weiss, Guy Georges, Ulrich BrinkmannAbstract:Bacterial- and plant-derived immunotoxins have documented potential for treatment of cancer. We discuss Anthrax toxin, ribosome inactivating-toxins, such as saporin and ricin, and ADP-ribosylating toxins such as Diphtheria toxin and Pseudomonas Exotoxin, with focus on the latter, which has been most thoroughly investigated. Regarding their potential as anticancer agents, critical issues such as immunogenicity and toxicity are outlined. We describe different generations of immunotoxins, the pathways for the delivery of the cytotoxic 'warheads', molecular parameters modulating efficacy, and combination therapy with other anticancer agents. Finally, we discuss deimmunization strategies based on the removal of B- and T-cell epitopes from the cytotoxic component, and highlight promising clinical proof-of-concept studies.
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role of cas a human homologue to the yeast chromosome segregation gene cse1 in toxin and tumor necrosis factor mediated apoptosis
Biochemistry, 1996Co-Authors: Ulrich Brinkmann, Elisabeth Brinkmann, Maria Pia Gallo, Uwe Scherf, Ira PastanAbstract:We have previously isolated by expression/selection cloning plasmids containing human cDNAs that rendered MCF-7 breast cancer cells resistant to immunotoxins, Pseudomonas Exotoxin (PE), and diphthe...
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cloning and characterization of a cellular apoptosis susceptibility gene the human homologue to the yeast chromosome segregation gene cse1
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Ulrich Brinkmann, Elisabeth Brinkmann, Maria Pia Gallo, Ira PastanAbstract:Abstract We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas Exotoxin, Pseudomonas Exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.
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independent domain folding of Pseudomonas Exotoxin and single chain immunotoxins influence of interdomain connections
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Ulrich Brinkmann, Johannes Buchner, Ira PastanAbstract:Abstract We have studied the refolding of completely unfolded and reduced Pseudomonas Exotoxin (PE) and of recombinant single-chain immunotoxins made with monoclonal antibody B3 that are composed of a heavy-chain variable region connected by a flexible linker to the corresponding light-chain variable region (Fv), which is in turn fused to a truncated form of PE. We have found by direct activity assays that different functional domains of these multifunctional proteins fold independently with different kinetics. The ADP-ribosylation domain of PE and of the recombinant immunotoxin fold rapidly, whereas the assembly of the binding and/or translocation domains is regained more slowly. The complete refolding of native PE occurs more rapidly than the refolding of the recombinant immunotoxins. To determine the influence of the connector region between the B3(Fv) moiety and the toxin on the folding process of the recombinant immunotoxin B3(Fv)-PE38KDEL, we have made two different mutations in the peptide that connects the single-chain Fv domain to domain II of PE. These molecules show different folding kinetics, differences in their propensity to aggregate, and different yields of correctly folded molecules. A mutation that decreases aggregation increases the rate of formation and the yield of active immunotoxin molecules.
