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Ira Pastan - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy.
Biomolecules, 2020Co-Authors: Michael Dieffenbach, Ira PastanAbstract: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.
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engineered anti gpc3 Immunotoxin hn3 abd t20 produces regression in mouse liver cancer xenografts through prolonged serum retention
Hepatology, 2020Co-Authors: Bryan D Fleming, Tim F Greten, Daniel J Urban, Matthew D Hall, Thomas Longerich, Ira PastanAbstract:BACKGROUND AND AIMS Treatment of hepatocellular carcinomas using our glypican-3 (GPC3)-targeting human nanobody (HN3) Immunotoxins causes potent tumor regression by blocking protein synthesis and down-regulating the Wnt signaling pathway. However, immunogenicity and a short serum half-life may limit the ability of Immunotoxins to transition to the clinic. APPROACH AND RESULTS To address these concerns, we engineered HN3-based Immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3-T20, which was modified to remove T-cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B-cell deimmunized Immunotoxin (HN3-mPE24) and our original HN3-Immunotoxin with a wild-type PE domain (HN3-PE38). All of our Immunotoxins displayed high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. HN3-T20 retained 73% enzymatic activity when compared with the wild-type Immunotoxin in an adenosine diphosphate-ribosylation assay. Interestingly, a real-time cell growth inhibition assay demonstrated that a single dose of HN3-T20 at 62.5 ng/mL (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20's serum retention, we tested the effect of adding a streptococcal albumin-binding domain (ABD) and a llama single-domain antibody fragment specific for mouse and human serum albumin. For the detection of Immunotoxin in mouse serum, we developed a highly sensitive enzyme-linked immunosorbent assay and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20 (326 minutes vs. 7.3 minutes); consequently, addition of an ABD resulted in HN3-ABD-T20-mediated tumor regression at 1 mg/kg. CONCLUSION These data indicate that ABD-containing deimmunized HN3-T20 Immunotoxins are high-potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.
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abstract cn07 02 Immunotoxins targeting gpc3 for liver cancer
Molecular Cancer Therapeutics, 2019Co-Authors: Bryan D Fleming, Tim F Greten, Ira PastanAbstract:Glypican-3 (GPC3) is a cell surface glypican that serves as a co-receptor for Wnt. Our laboratory has generated two high affinity antibodies targeting GPC3, HN3 and YP7. The HN3 human nanobody recognizes the N-lobe of GPC3, whereas the YP7 antibody recognizes the C-lobe of GPC3. Mutating residue F41 on the N-lobe of GPC3 inhibits binding of Wnt and the HN3 nanobody and inhibits activation of β-catenin. Both antibodies are fused to a fragment of Pseudomonas exotoxin A (PE38) to create recombinant Immunotoxins. Interestingly, the HN3-derived Immunotoxin (HN3-PE38) has superior antitumor activity as compared with the YP7-derived Immunotoxin (YP7-PE38). Intravenous administration of HN3-PE38 causes regression of hepatocellular carcinoma (HCC) tumor xenografts in mice. Our study establishes GPC3 as a promising target for Immunotoxin-based liver cancer therapy and demonstrates Immunotoxin-induced tumor regression via dual mechanisms: inactivation of Wnt signaling via the HN3 nanobody and inhibition of protein synthesis via the PE bacteria toxin. However, immunogenicity and a short serum half-life may limit the ability of Immunotoxins to transition to the clinic. To address these issues, we have recently engineered HN3-based Immunotoxins to contain various deimmunized PE toxins. These new Immunotoxins include HN3-T20, which is modified to remove domain II of the PE toxin and six T-cell epitopes. All of our Immunotoxins display high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. A real-time cell growth inhibition assay demonstrates that a single dose of HN3-T20 at 1.6 nM is capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20’s serum retention, we further engineer the HN3-T20 by adding a streptococcal albumin binding domain (ABD) and a llama single-domain antibody fragment (ALB1) specific for serum albumin. For the detection of Immunotoxin in mouse serum, we develop a highly sensitive ELISA and find that HN3-ABD-T20 has a 45-fold higher serum half-life than HN3-T20 (326 min vs 7.3 minutes); consequently, HN3-ABD-T20 shows the best serum retention and remains detectible even after a 24-hour period in mice. Furthermore, addition of an albumin binding domain results in HN3-ABD-T20 mediated tumor regression in mice at a low dose (1 mg/kg). These data show that the albumin binding deimmunized Immunotoxins are high potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer. Citation Format: Mitchell Ho, Bryan D Fleming, Tim F Greten, Ira Pastan. Immunotoxins targeting GPC3 for liver cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-02. doi:10.1158/1535-7163.TARG-19-CN07-02
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abstract cn07 03 recombinant Immunotoxins for hematologic malignancies
Molecular Cancer Therapeutics, 2019Co-Authors: Robert J Kreitman, Daniel Gorelik, Maryalice Stetlerstevenson, Constance M Yuan, Haowei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira PastanAbstract:Recombinant Immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant Immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant Immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant Immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant Immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant Immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant Immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant Immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03
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abstract 59 engineering glypican 3 targeting Immunotoxins for the treatment of liver cancer
Cancer Research, 2017Co-Authors: Bryan D Fleming, Brittany Nixon, Ira PastanAbstract:The expression of glypican-3 (GPC3) in hepatocellular carcinoma offers a target with high tissue specificity and cell signaling implications. A human single domain antibody discovered by phage display technology, HN3, was fused to domain II and domain III of Pseudomonas exotoxin A. This protein (HN3-PE38) showed a high level of cytotoxic activity with a IC50 of 0.4 nM on Hep3B cells, but a relatively low maximum tolerated dose of 0.8 mg/kg in mice. In order to produce an Immunotoxin with reduced toxicity, a new version was constructed that removed domain II and seven B cell epitopes from the Pseudomonas toxin. This deimmunized Immunotoxin (HN3-mPE24) was shown to have a similar IC50 of 0.2 nM on Hep3B cells. To determine if further deimmunization was possible, three new versions have been generated with T cell epitopes or a series of both B and T cell epitopes removed. These include the HN3-T20 Immunotoxin which had six T cell epitopes removed, HN3-T19 with 4 B cell, 4 T cell and 2 shared epitopes, and HN3-M11 with 5 B cell, 4 T cell and 2 shared epitopes. A comparative analysis of these Immunotoxins was made using in vitro cell proliferation assays using Hep3B. Both the HN3-T20 (IC50 = 0.6 nM) and HN3-T19 (IC50 = 0.8 nM) Immunotoxins had similar activity to HN3-mPE24 (IC50 = 0.7 nM) in a side by side comparison. The HN3-M11 variant had poor cytotoxicity and was excluded from in vivo examination. A Hep3B subcutaneous xenograft model was generated in athymic nude mice and was followed by nine rounds of intravenous Immunotoxin treatments. The mPE24, T20 and T19 Immunotoxins all showed an increase in average survival rate 70 days (mPE24), 76 days (T20) and 72 days (T19) when compared to the 55 days for PE38 and the 41 days for PBS alone. Additionally, the T20 and the T19 showed a maximum tolerated dose that was similar to that of the mPE24 with dosages as high as 10 mg/kg being well tolerated. This data would suggest that the HN3-T19 Immunotoxins has potential clinical applications because it represents the most deimmunized Immunotoxin to date. Citation Format: Bryan D. Fleming, Brittany Nixon, Ira Pastan, Mitchell Ho. Engineering glypican-3 targeting Immunotoxins for the treatment of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 59. doi:10.1158/1538-7445.AM2017-59
Zhirui Wang - One of the best experts on this subject based on the ideXlab platform.
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diphtheria toxin based anti human cd19 Immunotoxin for targeting human cd19 tumors
Molecular Oncology, 2017Co-Authors: Qian Zheng, Qi Huang, Zhaohui Wang, Huiping Zhang, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19+ tumors. We have developed a diphtheria toxin-based anti-human CD19 Immunotoxin for targeting human CD19+ tumors. We have constructed three isoforms of the CD19 Immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 Immunotoxins was assessed using human CD19+ JeKo-1 tumor-bearing NOD/SCID IL-2 receptor γ-/- (NSG) mouse model. In these animals, CD19 Immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 Immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19+ tumors.
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diphtheria toxin based anti human ccr4 Immunotoxin for targeting human ccr4 cells in vivo
Molecular Oncology, 2015Co-Authors: Zhaohui Wang, Min Wei, Huiping Zhang, Hongyuan Chen, Sharon Germana, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4+ tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 Immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 Immunotoxins bound to the human CCR4+ tumor cell line as well as CCR4+ human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4+ tumor cells compared to the monovalent anti-human CCR4 Immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4+ tumor-bearing mouse model. The Immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with human CCR4+ acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 Immunotoxin is a promising drug candidate for targeting human CCR4+ tumor cells and Tregs in vivo.
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Diphtheria-toxin based anti-human CCR4 Immunotoxin for targeting human CCR4(+) cells in vivo.
Molecular oncology, 2015Co-Authors: Zhaohui Wang, Min Wei, Huiping Zhang, Hongyuan Chen, Sharon Germana, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4+ tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 Immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 Immunotoxins bound to the human CCR4+ tumor cell line as well as CCR4+ human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4+ tumor cells compared to the monovalent anti-human CCR4 Immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4+ tumor-bearing mouse model. The Immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with human CCR4+ acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 Immunotoxin is a promising drug candidate for targeting human CCR4+ tumor cells and Tregs in vivo.
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Effect of pre-existing anti-diphtheria toxin antibodies on T cell depletion levels following diphtheria toxin-based recombinant anti-monkey CD3 Immunotoxin treatment.
Transplant immunology, 2012Co-Authors: Abraham J Matar, David H. Sachs, Zhirui Wang, R Crepeau, Vimukthi Pathiraja, Raimon Duran-struuck, Ashley Gusha, Masayuki Tasaki, Christene A. HuangAbstract:Diphtheria toxin (DT)-based anti-CD3 Immunotoxins have clinical relevance in numerous applications including autoimmune disease therapies and organ transplantation tolerance protocols. Pre-existing anti-DT antibodies acquired either by vaccination against diphtheria toxin or infections with C. diphtheriae may interfere or inhibit the function of these anti-CD3 Immunotoxins. Previously, a full-length anti-rhesus monkey CD3 Immunotoxin, FN18-CRM9, was shown to be less effective at depleting circulating T cells in animals with pre-existing anti-DT antibody titers than in animals without antibodies, and subsequent doses were ineffective. In this study, the T cell depletion function of a truncated DT based recombinant anti-monkey CD3 Immunotoxin, A-dmDT390-scfbDb (C207), as part of a reduced intensity conditioning regimen prior to hematopoietic cell transplantation, was compared between two groups of monkeys: those with and without pre-existing anti-diphtheria titers. T cell depletion was comparable in both groups of monkeys, and therefore appeared to be unaffected by the presence of moderate levels of pre-existing anti-diphtheria antibodies.
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development of a diphtheria toxin based antiporcine cd3 recombinant Immunotoxin
Bioconjugate Chemistry, 2011Co-Authors: Zhirui Wang, David M Neville, David H. Sachs, Raimon Duranstruuck, R Crepeau, Abraham J Matar, Isabel Hanekamp, Srimathi Srinivasan, Christene A. HuangAbstract:Anti-CD3 Immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant Immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant Immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant Immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 Immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of Immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.
Robert J Kreitman - One of the best experts on this subject based on the ideXlab platform.
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abstract cn07 03 recombinant Immunotoxins for hematologic malignancies
Molecular Cancer Therapeutics, 2019Co-Authors: Robert J Kreitman, Daniel Gorelik, Maryalice Stetlerstevenson, Constance M Yuan, Haowei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira PastanAbstract:Recombinant Immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant Immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant Immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant Immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant Immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant Immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant Immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant Immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03
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Essential role for Bim in mediating the apoptotic and antitumor activities of Immunotoxins
Oncogene, 2017Co-Authors: Antonella Antignani, D. Segal, D Huang, Robert J Kreitman, N. Simon, D J FitzgeraldAbstract:Protein synthesis is crucial for regulating cell homeostasis and, when unrestricted, it can lead to tumorigenesis. Immunotoxins derived from Pseudomonas exotoxin are antibody–toxin fusion proteins that inhibit protein synthesis of mammalian cells via ADP-ribosylation of the eukaryotic elongation factor-2. Here we investigate the role of the Bcl-2 family proteins in the response of cancer cells to Immunotoxin challenge. Besides the well-known reduction of the prosurvival Bcl-2 family member, Mcl-1, following inhibition of protein synthesis, we show for the first time that Immunotoxins also reduce the levels of selected proapoptotic BH-3-only proteins. Among these, only Bim protein levels correlated with the ability of Immunotoxins to induce an apoptotic response. To support our findings, we verified that a Bim knockout completely abolished Immunotoxin-mediated apoptosis. Further, mice bearing either wild-type or Bid knockout tumors responded to Immunotoxin treatment with a decrease in growth kinetics, whereas mice engrafted with Bim knockout tumors showed no reduction in tumor size or prolongation of survival following Immunotoxin treatment. From these results, we conclude that Bim expression is a major susceptibility factor for tumor cell death and, as such, constitutes a potential biomarker that could be evaluated before Immunotoxin treatment. In support of this hypothesis, clinically, we analyzed patient cells before Immunotoxin treatment and report that samples of hairy cell leukemia with high levels of Bim protein responded with a greater decrease in leukemic cell count compared with those samples expressing a low level of Bim.
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major cancer regressions in mesothelioma after treatment with an anti mesothelin Immunotoxin and immune suppression
Science Translational Medicine, 2013Co-Authors: Raffit Hassan, Robert J Kreitman, Andrew C Miller, Elad Sharon, Anish Thomas, James C Reynolds, Alexander Ling, Markku Miettinen, Seth M Steinberg, Daniel H FowlerAbstract:Immunotoxins are potent anticancer agents with an unusual mechanism of action: inhibition of protein synthesis resulting in apoptotic cell death. Immunotoxins have produced many durable complete responses in refractory hairy cell leukemia, where patients rarely form antibodies to the bacterial toxin component of the Immunotoxin. Patients with mesothelioma, however, have normal immune systems and form antibodies after one cycle, and tumor responses to the Immunotoxin have not been observed in this disease. We describe the results of a trial in which major antitumor responses were seen in patients with advanced mesothelioma who received the anti-mesothelin Immunotoxin SS1P, together with pentostatin and cyclophosphamide, to deplete T and B cells. Of 10 patients with chemotherapy-refractory mesothelioma, 3 have had major tumor regressions with 2 ongoing at 15 months, and 2 others responded to chemotherapy after discontinuing Immunotoxin therapy. Antibody formation was markedly delayed, allowing more SS1P cycles to be given, but this alone does not appear to account for the marked antitumor activity observed.
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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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a protease resistant Immunotoxin against cd22 with greatly increased activity against cll and diminished animal toxicity
Blood, 2009Co-Authors: John E. Weldon, Robert J Kreitman, David J Fitzgerald, Laiman Xiang, Oleg Chertov, Inger Margulies, Ira PastanAbstract: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.
Christene A. Huang - One of the best experts on this subject based on the ideXlab platform.
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diphtheria toxin based anti human cd19 Immunotoxin for targeting human cd19 tumors
Molecular Oncology, 2017Co-Authors: Qian Zheng, Qi Huang, Zhaohui Wang, Huiping Zhang, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19+ tumors. We have developed a diphtheria toxin-based anti-human CD19 Immunotoxin for targeting human CD19+ tumors. We have constructed three isoforms of the CD19 Immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 Immunotoxins was assessed using human CD19+ JeKo-1 tumor-bearing NOD/SCID IL-2 receptor γ-/- (NSG) mouse model. In these animals, CD19 Immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 Immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19+ tumors.
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diphtheria toxin based anti human ccr4 Immunotoxin for targeting human ccr4 cells in vivo
Molecular Oncology, 2015Co-Authors: Zhaohui Wang, Min Wei, Huiping Zhang, Hongyuan Chen, Sharon Germana, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4+ tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 Immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 Immunotoxins bound to the human CCR4+ tumor cell line as well as CCR4+ human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4+ tumor cells compared to the monovalent anti-human CCR4 Immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4+ tumor-bearing mouse model. The Immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with human CCR4+ acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 Immunotoxin is a promising drug candidate for targeting human CCR4+ tumor cells and Tregs in vivo.
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Diphtheria-toxin based anti-human CCR4 Immunotoxin for targeting human CCR4(+) cells in vivo.
Molecular oncology, 2015Co-Authors: Zhaohui Wang, Min Wei, Huiping Zhang, Hongyuan Chen, Sharon Germana, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4+ tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 Immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 Immunotoxins bound to the human CCR4+ tumor cell line as well as CCR4+ human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4+ tumor cells compared to the monovalent anti-human CCR4 Immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4+ tumor-bearing mouse model. The Immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with human CCR4+ acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 Immunotoxin is a promising drug candidate for targeting human CCR4+ tumor cells and Tregs in vivo.
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Effect of pre-existing anti-diphtheria toxin antibodies on T cell depletion levels following diphtheria toxin-based recombinant anti-monkey CD3 Immunotoxin treatment.
Transplant immunology, 2012Co-Authors: Abraham J Matar, David H. Sachs, Zhirui Wang, R Crepeau, Vimukthi Pathiraja, Raimon Duran-struuck, Ashley Gusha, Masayuki Tasaki, Christene A. HuangAbstract:Diphtheria toxin (DT)-based anti-CD3 Immunotoxins have clinical relevance in numerous applications including autoimmune disease therapies and organ transplantation tolerance protocols. Pre-existing anti-DT antibodies acquired either by vaccination against diphtheria toxin or infections with C. diphtheriae may interfere or inhibit the function of these anti-CD3 Immunotoxins. Previously, a full-length anti-rhesus monkey CD3 Immunotoxin, FN18-CRM9, was shown to be less effective at depleting circulating T cells in animals with pre-existing anti-DT antibody titers than in animals without antibodies, and subsequent doses were ineffective. In this study, the T cell depletion function of a truncated DT based recombinant anti-monkey CD3 Immunotoxin, A-dmDT390-scfbDb (C207), as part of a reduced intensity conditioning regimen prior to hematopoietic cell transplantation, was compared between two groups of monkeys: those with and without pre-existing anti-diphtheria titers. T cell depletion was comparable in both groups of monkeys, and therefore appeared to be unaffected by the presence of moderate levels of pre-existing anti-diphtheria antibodies.
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development of a diphtheria toxin based antiporcine cd3 recombinant Immunotoxin
Bioconjugate Chemistry, 2011Co-Authors: Zhirui Wang, David M Neville, David H. Sachs, Raimon Duranstruuck, R Crepeau, Abraham J Matar, Isabel Hanekamp, Srimathi Srinivasan, Christene A. HuangAbstract:Anti-CD3 Immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant Immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant Immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant Immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 Immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of Immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.
David H. Sachs - One of the best experts on this subject based on the ideXlab platform.
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diphtheria toxin based anti human cd19 Immunotoxin for targeting human cd19 tumors
Molecular Oncology, 2017Co-Authors: Qian Zheng, Qi Huang, Zhaohui Wang, Huiping Zhang, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19+ tumors. We have developed a diphtheria toxin-based anti-human CD19 Immunotoxin for targeting human CD19+ tumors. We have constructed three isoforms of the CD19 Immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 Immunotoxins was assessed using human CD19+ JeKo-1 tumor-bearing NOD/SCID IL-2 receptor γ-/- (NSG) mouse model. In these animals, CD19 Immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 Immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19+ tumors.
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diphtheria toxin based anti human ccr4 Immunotoxin for targeting human ccr4 cells in vivo
Molecular Oncology, 2015Co-Authors: Zhaohui Wang, Min Wei, Huiping Zhang, Hongyuan Chen, Sharon Germana, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4+ tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 Immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 Immunotoxins bound to the human CCR4+ tumor cell line as well as CCR4+ human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4+ tumor cells compared to the monovalent anti-human CCR4 Immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4+ tumor-bearing mouse model. The Immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with human CCR4+ acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 Immunotoxin is a promising drug candidate for targeting human CCR4+ tumor cells and Tregs in vivo.
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Diphtheria-toxin based anti-human CCR4 Immunotoxin for targeting human CCR4(+) cells in vivo.
Molecular oncology, 2015Co-Authors: Zhaohui Wang, Min Wei, Huiping Zhang, Hongyuan Chen, Sharon Germana, Christene A. Huang, Joren C. Madsen, David H. Sachs, Zhirui WangAbstract:CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4+ tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 Immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 Immunotoxins bound to the human CCR4+ tumor cell line as well as CCR4+ human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4+ tumor cells compared to the monovalent anti-human CCR4 Immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4+ tumor-bearing mouse model. The Immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with human CCR4+ acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 Immunotoxin is a promising drug candidate for targeting human CCR4+ tumor cells and Tregs in vivo.
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Effect of pre-existing anti-diphtheria toxin antibodies on T cell depletion levels following diphtheria toxin-based recombinant anti-monkey CD3 Immunotoxin treatment.
Transplant immunology, 2012Co-Authors: Abraham J Matar, David H. Sachs, Zhirui Wang, R Crepeau, Vimukthi Pathiraja, Raimon Duran-struuck, Ashley Gusha, Masayuki Tasaki, Christene A. HuangAbstract:Diphtheria toxin (DT)-based anti-CD3 Immunotoxins have clinical relevance in numerous applications including autoimmune disease therapies and organ transplantation tolerance protocols. Pre-existing anti-DT antibodies acquired either by vaccination against diphtheria toxin or infections with C. diphtheriae may interfere or inhibit the function of these anti-CD3 Immunotoxins. Previously, a full-length anti-rhesus monkey CD3 Immunotoxin, FN18-CRM9, was shown to be less effective at depleting circulating T cells in animals with pre-existing anti-DT antibody titers than in animals without antibodies, and subsequent doses were ineffective. In this study, the T cell depletion function of a truncated DT based recombinant anti-monkey CD3 Immunotoxin, A-dmDT390-scfbDb (C207), as part of a reduced intensity conditioning regimen prior to hematopoietic cell transplantation, was compared between two groups of monkeys: those with and without pre-existing anti-diphtheria titers. T cell depletion was comparable in both groups of monkeys, and therefore appeared to be unaffected by the presence of moderate levels of pre-existing anti-diphtheria antibodies.
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development of a diphtheria toxin based antiporcine cd3 recombinant Immunotoxin
Bioconjugate Chemistry, 2011Co-Authors: Zhirui Wang, David M Neville, David H. Sachs, Raimon Duranstruuck, R Crepeau, Abraham J Matar, Isabel Hanekamp, Srimathi Srinivasan, Christene A. HuangAbstract:Anti-CD3 Immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant Immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant Immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant Immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 Immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of Immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.
