Immunotoxins

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Ira Pastan - One of the best experts on this subject based on the ideXlab platform.

  • Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy.
    Biomolecules, 2020
    Co-Authors: Michael Dieffenbach, Ira Pastan
    Abstract:

    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.

  • abstract cn07 03 recombinant Immunotoxins for hematologic malignancies
    Molecular Cancer Therapeutics, 2019
    Co-Authors: Robert J Kreitman, Daniel Gorelik, Maryalice Stetlerstevenson, Constance M Yuan, Haowei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan
    Abstract:

    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

  • abstract cn07 02 Immunotoxins targeting gpc3 for liver cancer
    Molecular Cancer Therapeutics, 2019
    Co-Authors: Bryan D Fleming, Tim F Greten, Ira Pastan
    Abstract:

    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

  • tolerogenic nanoparticles restore the antitumor activity of recombinant Immunotoxins by mitigating immunogenicity
    Proceedings of the National Academy of Sciences of the United States of America, 2018
    Co-Authors: Ronit Mazor, Emily M King, Masanori Onda, Nicolas Cuburu, Selamawit Addissie, Devorah Crown, Xiufen Liu, Takashi Kei Kishimoto, Ira Pastan
    Abstract:

    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 naive 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.

  • abstract 59 engineering glypican 3 targeting Immunotoxins for the treatment of liver cancer
    Cancer Research, 2017
    Co-Authors: Bryan D Fleming, Brittany Nixon, Ira Pastan
    Abstract:

    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

Robert J Kreitman - One of the best experts on this subject based on the ideXlab platform.

  • abstract cn07 03 recombinant Immunotoxins for hematologic malignancies
    Molecular Cancer Therapeutics, 2019
    Co-Authors: Robert J Kreitman, Daniel Gorelik, Maryalice Stetlerstevenson, Constance M Yuan, Haowei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan
    Abstract:

    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

  • Essential role for Bim in mediating the apoptotic and antitumor activities of Immunotoxins
    Oncogene, 2017
    Co-Authors: Antonella Antignani, D. Segal, D Huang, Robert J Kreitman, N. Simon, D J Fitzgerald
    Abstract:

    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.

  • effect of antigen shedding on targeted delivery of Immunotoxins in solid tumors from a mathematical model
    PLOS ONE, 2014
    Co-Authors: Youngshang Pak, Ira Pastan, Robert J Kreitman, Byungkook Lee
    Abstract:

    Most cancer-specific antigens used as targets of antibody-drug conjugates and Immunotoxins are shed from the cell surface (Zhang & Pastan (2008) Clin. Cancer Res. 14: 7981-7986), although at widely varying rates and by different mechanisms (Dello Sbarba & Rovida (2002) Biol. Chem. 383: 69–83). Why many cancer-specific antigens are shed and how the shedding affects delivery efficiency of antibody-based protein drugs are poorly understood questions at present. Before a detailed numerical study, it was assumed that antigen shedding would reduce the efficacy of antibody-drug conjugates and Immunotoxins. However, our previous study using a comprehensive mathematical model showed that antigen shedding can significantly improve the efficacy of the mesothelin-binding immunotoxin, SS1P (anti-mesothelin-Fv-PE38), and suggested that receptor shedding can be a general mechanism for enhancing the effect of inter-cellular signaling molecules. Here, we improved this model and applied it to both SS1P and another recombinant immunotoxin, LMB-2, which targets CD25. We show that the effect of antigen shedding is influenced by a number of factors including the number of antigen molecules on the cell surface and the endocytosis rate. The high shedding rate of mesothelin is beneficial for SS1P, for which the antigen is large in number and endocytosed rapidly. On the other hand, the slow shedding of CD25 is beneficial for LMB-2, for which the antigen is small in number and endocytosed slowly.

  • antibody fusion proteins anti cd22 recombinant immunotoxin moxetumomab pasudotox
    Clinical Cancer Research, 2011
    Co-Authors: Robert J Kreitman, Ira Pastan
    Abstract:

    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 .

  • Recombinant Immunotoxins Containing Truncated Bacterial Toxins for the Treatment of Hematologic Malignancies
    BioDrugs, 2009
    Co-Authors: Robert J Kreitman
    Abstract:

    Immunotoxins are molecules that contain a protein toxin and a ligand that is either an antibody or a growth factor. The ligand binds to a target cell antigen, and the target cell internalizes the immunotoxin, allowing the toxin to migrate to the cytoplasm where it can kill the cell. In the case of recombinant Immunotoxins, the ligand and toxin are encoded in DNA that is then expressed in bacteria, and the purified immunotoxin contains the ligand and toxin fused together. Among the most active recombinant Immunotoxins clinically tested are those that are targeted to hematologic malignancies. One agent, containing human interleukin-2 and truncated diphtheria toxin (denileukin diftitox), has been approved for use in cutaneous T-cell lymphoma, and has shown activity in other hematologic malignancies, including leukemias and lymphomas. Diphtheria toxin has also been targeted by other ligands, including granulocyte-macrophage colony-stimulating factor and interleukin-3, to target myelogenous leukemia cells. Single-chain antibodies containing variable heavy and light antibody domains have been fused to truncated Pseudomonas exotoxin to target lymphomas and lymphocytic leukemias. Recombinant Immunotoxins anti-Tac(Fv)-PE38 (LMB-2), targeting CD25, and RFB4(dsFv)-PE38 (BL22, CAT-3888), targeting CD22, have each been tested in patients. Major responses have been observed after failure of standard chemotherapy. The most successful application of recombinant Immunotoxins today is in hairy cell leukemia, where BL22 has induced complete remissions in most patients who were previously treated with optimal chemotherapy.

David J Fitzgerald - One of the best experts on this subject based on the ideXlab platform.

  • abstract a67 the mtor inhibitor everolimus enhances the cytotoxicity of pseudomonas based Immunotoxins mechanistic insights
    Molecular Cancer Research, 2016
    Co-Authors: Antonella Antignani, Ira Pastan, Lesley Mathewsgriner, Mark Ferrer, Nathan Simon, Evan Angelus, Manjie Huang, Craig J Thomas, David J Fitzgerald
    Abstract:

    Abstracts: AACR Special Conference: Metabolism and Cancer; June 7-10, 2015; Bellevue, WA Pseudomonas exotoxin (PE)-based Immunotoxins (antibody-toxin fusion proteins) have produced frequent complete remissions in patients with hairy cell leukemia but far fewer responses in other cancers. PE and PE-Immunotoxins kill cells via a pathway that includes endocytosis, proteolytic processing, transport to the ER and translocation of an enzymatically active toxin fragment from the ER to the cytosol. In the cytosol the toxin ADP-ribosylates EF2 which results in the cessation of protein synthesis. Using two cell lines (KB or Nalm6) that exhibit moderate resistance to immunotoxin killing, we screened 500 compounds from the MIPE-3 library seeking compounds that enhanced the cytotoxicity of Immunotoxins targeting either mesothelin or CD22. Results indicated that as few as 20 compounds could enhance cytotoxicity and only a few appeared potentially useful in a clinical setting. One compound of particular interest was the mammalian target of rapamycin (mTOR) inhibitor, everolimus. The mTOR pathway plays a pivotal role in cell signaling and proliferation and ultimately tumorigenesis. Moreover, protein synthesis is the best characterized process controlled by the mTORC1 pathway, making everolimus an excellent candidate to enhance the cytotoxic activity of PE-Immunotoxins. We tested combinations of PE-Immunotoxins and everolimus on various tumor cell lines (Nalm-6, Daudi, MD-MBA-468 and KB) using different concentration of both compounds. In all cell lines, the cytotoxicity of the combination was greater than either compound alone. As could be anticipated, the increased cytotoxicity was associated with enhanced reductions in cellular protein synthesis: confirming synergy between the immunotoxin and everolimus. To define the mechanism for the observed synergy, we evaluated changes in the levels of the principal constituents of the mTORC1 pathway. We noted that in 24 hours the immunotoxin drastically reduced the level of the p70S6 kinase in a dose dependent manner. No significant changes were seen in the levels of the TSC1, TSC2, FKBP12, mTOR, 4EBP1 or S6 proteins. Reduced levels of p70S6 kinase resulted in the absence of phospho-S6 protein from cells treated with everolimus and immunotoxin compared to the reduced phosphorylation observed with everolimus alone. We suggest that the lack of the p70S6K caused by the immunotoxin-mediated inhibition of protein synthesis is an essential element of the synergy with everolimus-mediated inhibition of mTORC1. Further, results of synergy in vitro were replicated in xenograft models where combinations exhibited enhanced anti-tumor action compared to either agent alone. Citation Format: Antonella Antignani, Lesley Mathews-Griner, Mark Ferrer, Nathan Simon, Evan Angelus, Manjie Huang, Ira Pastan, Craig Thomas, David J. FitzGerald. The mTOR inhibitor, everolimus, enhances the cytotoxicity of pseudomonas-based Immunotoxins: Mechanistic insights. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A67.

  • Combination treatments with the PKC inhibitor, enzastaurin, enhance the cytotoxicity of the anti-mesothelin immunotoxin, SS1P.
    PLOS ONE, 2013
    Co-Authors: Abid R. Mattoo, Ira Pastan, David J Fitzgerald
    Abstract:

    Activated protein kinase C (PKC) contributes to tumor survival and proliferation, provoking the development of inhibitory agents as potential cancer therapeutics. Immunotoxins are antibody-based recombinant proteins that employ antibody fragments for cancer targeting and bacterial toxins as the cytotoxic agent. Pseudomonas exotoxin-based Immunotoxins act via the ADP-ribosylation of EF2 leading to the enzymatic inhibition of protein synthesis. Combining PKC inhibitors with the immunotoxin SS1P, targeted to surface mesothelin, was undertaken to explore possible therapeutic strategies. Enzastaurin but not two other PKC inhibitors combined with SS1P to produce synergistic cell death via apoptosis. Mechanistic insights of the synergistic killing centered on the complete loss of the prosurvival Bcl2 protein, Mcl-1, the loss of AKT and the activation of caspase 3/7. Synergy was most evident when cells exhibited resistance to the immunotoxin alone. Further, because PKC inhibition by itself was not sufficient to enhance SS1P action, enzastaurin must target other kinases that are involved in the immunotoxin pathway.

  • abt 737 overcomes resistance to immunotoxin mediated apoptosis and enhances the delivery of pseudomonas exotoxin based proteins to the cell cytosol
    Molecular Cancer Therapeutics, 2010
    Co-Authors: Roberta Traini, Antonella Antignani, Gal Benjosef, Diana V Pastrana, Elizabeth Moskatel, Ashima K Sharma, David J Fitzgerald
    Abstract:

    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.

  • differential cellular internalization of anti cd19 and cd22 Immunotoxins results in different cytotoxic activity
    Cancer Research, 2008
    Co-Authors: Richard Beers, David J Fitzgerald, Ira Pastan
    Abstract:

    B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the Immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two Immunotoxins, FMC63(Fv)-PE38–targeting CD19 and RFB4(Fv)-PE38 (BL22)–targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, ∼7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized Immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for Immunotoxins and probably for other immunoconjugates that act inside cells. [Cancer Res 2008;68(15):6300–5]

  • Improved design of Pseudomonas exotoxin A-based Immunotoxins
    Molecular Cancer Therapeutics, 2007
    Co-Authors: John E. Weldon, David J Fitzgerald, Laiman Xiang, Ira Pastan
    Abstract:

    A73 Immunotoxins are engineered proteins that combine antibodies with protein toxins. They are used therapeutically to target and kill cancer cells expressing specific cell surface antigens. Immunotoxins based on Pseudomonas exotoxin A (PE) consist of the FV portion of an antibody fused to domains Ib, II, and III of PE. Clinical trials are currently in progress to assess PE Immunotoxins for the treatment of CD22-expressing lymphomas and leukemias, and mesothelin-expressing solid tumors. Broad therapeutic application of PE Immunotoxins, however, has been limited by recurring problems of immunogenicity and nonspecific toxicities. In an effort to reduce the immunogenicity of PE Immunotoxins, we have pursued a strategy of identifying and removing lysosomal protease cleavage sites in the toxin. This approach has two potential benefits. Firstly, we diminish MHC class II-associated immune response by preventing efficient lysosomal processing of the immunotoxin. Secondly, by removing protease-accessible regions that are not essential for cytotoxicity, we begin to approach the minimum effective size of the toxin, eliminating potential epitopes. We have identified lysosomal protease cleavage sites in Immunotoxins by N-terminal sequence analysis of peptide fragments generated by digestion of an immunotoxin with either purified lysosomal proteases or the lysosomal fraction of Raji B cells. Cleavage sites were found only within domains II and Ib of the toxin. None were found in domain III or the FV. A series of anti-CD22 immunotoxin mutants were produced that contain large deletions of domain II in order to both remove protease sites and minimize the size the toxin. Analyses of the in vitro cytotoxicity of these mutants revealed that much of domain II and all of domain Ib are not essential for immunotoxin activity. Currently, the most promising compromise between reducing the size of the immunotoxin and preserving its cytotoxicity is a mutant construct, termed PE25 HA22, which consists of an anti-CD22 FV linked to domain III of PE by a short stretch of domain II. In vitro cytotoxicity testing shows that PE25 HA22 has similar activity to standard HA22. In vivo testing of PE25 HA22 anti-tumor activity in a mouse xenograft model shows that the mutant protein is slightly less active than standard HA22, but has greatly reduced nonspecific toxicity. Mice can tolerate doses of PE25 HA22 that are 10-fold higher than HA22, which significantly enhances the anti-tumor response. We conclude that PE25 HA22 represents a promising step forward in the development of PE Immunotoxins. The focus of future research is the evaluation of PE25 HA22 immunogenicity by measuring the antibody response in mice.

Ulrich Brinkmann - One of the best experts on this subject based on the ideXlab platform.

  • Recombinant Immunotoxins for cancer therapy
    Expert opinion on biological therapy, 2001
    Co-Authors: Ulrich Brinkmann, Andrea Keppler-hafkemeyer, Peter Hafkemeyer
    Abstract:

    Recombinant Immunotoxins consist of Fv regions of tumour-selective antibodies fused to toxins found in bacteria, plants or fungi. These toxins must be modified to remove normal-tissue binding sites but to retain all other functions of cytotoxicity. The recombinant antibody fragments target the modified toxin to cancer cells which are killed, either by direct inhibition of protein synthesis, or by concomitant induction of apoptosis. Cells that are not recognised by the antibody fragment because they do not carry the tumour antigen, are spared. Many factors influence the in vivo antitumour activity of recombinant Immunotoxins. Among them are considerations of which types of cancer may be the best targets for immunotoxin therapy as well as tumour specificity of the antigen that is targeted by the recombinant antibody. Other relevant issues are the affinity of Immunotoxins and their ability to enter and penetrate into tissues and tumours, which in turn is dependent on the size of the protein. A great deal of ...

  • stabilization of the fv fragments in recombinant Immunotoxins by disulfide bonds engineered into conserved framework regions
    Biochemistry, 1994
    Co-Authors: Yoram Reiter, Robert J Kreitman, Ulrich Brinkmann, Sunhee Jung, Byungkook Lee, Ira Pastan
    Abstract:

    Abstract Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved framework positions of VH and VL. We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin [Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542]. This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart. To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing Immunotoxins. One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor. Comparison of the specificity and activity of these Immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart. These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding. Thus, it should be feasible to use the positions we have identified in the conserved framework region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)

  • engineering interchain disulfide bonds into conserved framework regions of fv fragments improved biochemical characteristics of recombinant Immunotoxins containing disulfide stabilized fv
    Protein Engineering, 1994
    Co-Authors: Yoram Reiter, Ulrich Brinkmann, Keith O Webber, Sunhee Jung, Byungkook Lee, Ira Pastan
    Abstract:

    Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant Immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv Immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv Immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv Immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv Immunotoxins as therapeutic and diagnostic agents.

  • 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, 1992
    Co-Authors: Ulrich Brinkmann, Johannes Buchner, Ira Pastan
    Abstract:

    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.

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  • effect of antigen shedding on targeted delivery of Immunotoxins in solid tumors from a mathematical model
    PLOS ONE, 2014
    Co-Authors: Youngshang Pak, Ira Pastan, Robert J Kreitman, Byungkook Lee
    Abstract:

    Most cancer-specific antigens used as targets of antibody-drug conjugates and Immunotoxins are shed from the cell surface (Zhang & Pastan (2008) Clin. Cancer Res. 14: 7981-7986), although at widely varying rates and by different mechanisms (Dello Sbarba & Rovida (2002) Biol. Chem. 383: 69–83). Why many cancer-specific antigens are shed and how the shedding affects delivery efficiency of antibody-based protein drugs are poorly understood questions at present. Before a detailed numerical study, it was assumed that antigen shedding would reduce the efficacy of antibody-drug conjugates and Immunotoxins. However, our previous study using a comprehensive mathematical model showed that antigen shedding can significantly improve the efficacy of the mesothelin-binding immunotoxin, SS1P (anti-mesothelin-Fv-PE38), and suggested that receptor shedding can be a general mechanism for enhancing the effect of inter-cellular signaling molecules. Here, we improved this model and applied it to both SS1P and another recombinant immunotoxin, LMB-2, which targets CD25. We show that the effect of antigen shedding is influenced by a number of factors including the number of antigen molecules on the cell surface and the endocytosis rate. The high shedding rate of mesothelin is beneficial for SS1P, for which the antigen is large in number and endocytosed rapidly. On the other hand, the slow shedding of CD25 is beneficial for LMB-2, for which the antigen is small in number and endocytosed slowly.

  • lowering the isoelectric point of the fv portion of recombinant Immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity
    Cancer Research, 2001
    Co-Authors: Masanori Onda, Robert J Kreitman, Byungkook Lee, Satoshi Nagata, Yasuo Tsutsumi, James J Vincent, Qingcheng Wang, Ira Pastan
    Abstract:

    Recombinant Immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with Immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other Immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant Immunotoxins and other Fv fusion proteins without losing antitumor activity.

  • stabilization of the fv fragments in recombinant Immunotoxins by disulfide bonds engineered into conserved framework regions
    Biochemistry, 1994
    Co-Authors: Yoram Reiter, Robert J Kreitman, Ulrich Brinkmann, Sunhee Jung, Byungkook Lee, Ira Pastan
    Abstract:

    Abstract Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved framework positions of VH and VL. We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin [Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542]. This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart. To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing Immunotoxins. One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor. Comparison of the specificity and activity of these Immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart. These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding. Thus, it should be feasible to use the positions we have identified in the conserved framework region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)

  • engineering interchain disulfide bonds into conserved framework regions of fv fragments improved biochemical characteristics of recombinant Immunotoxins containing disulfide stabilized fv
    Protein Engineering, 1994
    Co-Authors: Yoram Reiter, Ulrich Brinkmann, Keith O Webber, Sunhee Jung, Byungkook Lee, Ira Pastan
    Abstract:

    Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant Immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv Immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv Immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv Immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv Immunotoxins as therapeutic and diagnostic agents.

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