Enhancement of Immunotoxin Efficacy by Acid-cleavable Cross-linking Agents Utilizing Diphtheria Toxin and Toxin Mutants

Neville, David M.; Srinivasachar, Kasturi; Stone, Rebecca; Scharff, Joshua

doi:10.1016/s0021-9258(18)63747-0

Cited by 36 publications

(6 citation statements)

References 29 publications

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“…Youle et al (Green¢eld et al 1987;Johnson et al 1988Johnson et al , 1989 developed the strategy of mutating DT in order to create immunotoxins with equal potency to those made with wild-type DT but reducing the toxicity associated with wild-type DT. Neville et al (1989) showed that it was the intracellular routing of DT-based immunotoxins by intracellular DT receptors or alternative receptors that determine their e¤cacy. For instance, the CRM9 immunotoxin, lacking the DT-binding site, nevertheless routed appropriately, resulting in cell killing when directed at the CD3 or the transferrin receptor (Neville et al 1989).…”

Section: Construction Of Anti-cd3 Immunotoxinmentioning

confidence: 99%

“…Neville et al (1989) showed that it was the intracellular routing of DT-based immunotoxins by intracellular DT receptors or alternative receptors that determine their e¤cacy. For instance, the CRM9 immunotoxin, lacking the DT-binding site, nevertheless routed appropriately, resulting in cell killing when directed at the CD3 or the transferrin receptor (Neville et al 1989). Subsequently, Neville et al evaluated an anti-human CD3^CRM9 immunotoxin to kill human T-cell subcutaneous tumours in N:NIH/bg/nu/xid mice which were de¢cient in their killer cell immune resp onsiveness.…”

Section: Construction Of Anti-cd3 Immunotoxinmentioning

confidence: 99%

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Treatment with immunotoxin

Knechtle

2001

Phil. Trans. R. Soc. Lond. B

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T-cell depletion prior to or beginning at the time of transplantation has been shown to be a valuable adjunct to the induction of immunological unresponsiveness. Both total lymphoid irradiation and anti-lymphocyte globulin have been used for this purpose in experimental models of transplantation as well as in human organ transplant recipients. However, these methods of T-cell depletion are limited in their ability to deplete T cells selectively due to non-specific targeting and limited efficacy. A new anti-CD3 immunotoxin has been developed with a far more potent ability to deplete T cells selectively as measured by flow cytometry analysis of peripheral blood T lymphocytes as well as lymph node lymphocytes. This immunotoxin is well tolerated by rhesus monkeys when administered in vivo. When administered as a single immunosuppressive agent pretransplant, it substantially promotes allograft survival, inducing tolerance in at least one-third of recipients as measured by subsequent acceptance of donor skin grafts and rejection of third-party skin grafts. When administered on the day of transplant in combination with steroid pretreatment and a brief course of deoxyspergualin or mycophenolate mofetil (4 to 14 days), long-term unresponsiveness is also produced and in a more reliable manner than using immunotoxin alone. A new immunotoxin directed at the human CD3epsilon has been developed with excellent potency in T-cell killing and lacking the Fc portion of the CD3 antibody. This construct may be useful for T-cell depletion in humans and has a potential application in tolerance induction in human organ transplantation. Lessons learned from anti-CD3 immunotoxin in the non-human primate model to date include (i) profound (2-3 log) depletion of T-cells can be accomplished safely without inducing lymphoma or infection, (ii) such depletion is a useful adjunct for tolerance induction to allogeneic organ transplants, and (iii) tolerance to both allogeneic renal transplants and xenogeneic islet transplants has been accomplished using such strategies to date in non-human primates and in pigs. Immunotoxin may be useful for the induction of chimerism using strategies that include donor bone marrow infusion. Successful strategies for tolerance induction have also been developed using immunotoxin without the adjunct of donor bone marrow or stem cell infusion. Clinical application of immunotoxin will use a newly engineered construct with the potential for causing cytokine release, less susceptibility to neutralization by anti-diphtheria antibody and not dependent on chemical conjugation of an antibody and toxin. The usefulness of immunotoxin is directly related to its tremendous potency for depleting T cells. Based on results in nonhuman primates, it is anticipated that it will become a useful agent in tolerance induction in humans.

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Section: Construction Of Anti-cd3 Immunotoxinmentioning

confidence: 99%

Section: Construction Of Anti-cd3 Immunotoxinmentioning

confidence: 99%

Treatment with immunotoxin

Knechtle

2001

Phil. Trans. R. Soc. Lond. B

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“…In addition, acetals can be formed using a variety of types of hydroxyl groups including primary, secondary, tertiary, and syn-1,2- and -1,3-diols, and the rate of hydrolysis can be tuned by varying the structure of the acetal. Despite this, there are only a few reports of acetals applied as protein cross-linking agents ( , ) and as pH-sensitive protecting groups masking anticancer drug activity ( − ). Therefore, the goal of this work is to broadly explore the usefulness of acetals as pH-sensitive linkages for drug delivery.…”

Section: Introductionmentioning

confidence: 99%

Acetals as pH-Sensitive Linkages for Drug Delivery

2004

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pH-Sensitive linkages designed to undergo hydrolysis at mildly acidic pH can trigger the release of therapeutics selectively at targets such as tumor and inflammatory tissues and in the endosomes and lysosomes of cells. Acetals have the potential to be used as linkages for a range of alcohol functionalities, and, by altering their chemical structure, it is possible to tune their hydrolysis rate. The syntheses of four conjugates of model drug molecules with PEO using acetals of varying chemical structure are described herein. Primary and secondary alcohols, as well as syn-1,2-diols, were incorporated in the conjugates. The hydrolysis kinetics were investigated by HPLC, and the conjugates had half-lives ranging from less than 1 min to several days at pH 5.0, with slower hydrolysis at pH 7.4 in all cases. These acetal linkages are therefore promising for use in a variety of drug delivery applications ranging from polymer-drug conjugates to pH-sensitive micelles and nanoparticulate systems.

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“…Immunotoxin Expression and Purification. The FN18-CRM9 immunotoxin was conjugated as previously described by thiolating both the mAb FN18 moiety and the toxin CRM9 moiety and then cross-linking with bis-(maleimido)hexane (5).…”

Section: Cloning Of Anti-monkey Cd3 Variable Regions and Construction...mentioning

confidence: 99%

“…Diphtheria toxin (DT, 1 535 amino acids, 59 kDa) is composed of three functionally distinct regions: an enzymatically active domain, a cell receptor binding domain, and a domain for translocation of the toxin across the cell membrane (1). Because of its efficiency at crossing eukaryotic cell membranes and initiating cell death, it has been used to construct immunotoxins (ITs) to target and kill specifically selected cells (2)(3)(4)(5). ITs were initially generated by chemically conjugating the toxin to a targeting protein [such as a monoclonal antibody (mAb)].…”

Section: Introductionmentioning

confidence: 99%

Genetic Construction and Characterization of an Anti-Monkey CD3 Single-Chain Immunotoxin with a Truncated Diphtheria Toxin

Thompson

et al. 1997

Bioconjugate Chem.

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We have previously developed a chemically conjugated anti-rhesus monkey CD3 immunotoxin FN18-CRM9 that can deplete in vivo T cells and induce long term tolerance of mismatched renal allograft in rhesus monkeys. This immunotoxin is a monkey analogue of anti-human CD3 immunotoxin UCHT1-CRM9. In this study, we cloned the light and heavy chain variable regions of anti-monkey CD3 monoclonal antibody FN18 and constructed a single-chain Fv (sFv) by linking variable light and variable heavy regions with a (Gly4Ser)3 linker. The single-chain immunotoxin DT390-FN18sFv was constructed by ligating the sFv to the carboxyl terminus of DT390, a truncated form of diphtheria toxin. The DT390-FN18sFv fusion protein was expressed in Escherichia coli and purified with Ni-RTA affinity and anion exchange columns. Similar to the chemically conjugated immunotoxin FN18-CRM9, DT390-FN18sFv can also specifically inhibit protein synthesis in primary monkey T cells in a dose-dependent manner. DT390-FN18sFv at 10(-7) mol/L or FN18-CRM9 at 10(-8) mol/L is sufficient to reduce protein synthesis of monkey primary T cells to less than 5% of the control. The 50% inhibition dosage (IC50) of FN18-CRM9 is 1 x 10(-10) mol/L, while the IC50 of DT390-FN18sFv is 1 x 10(-8) mol/ L, reflecting the lowered affinity of monovalent Fab' FN18 to its parental divalent antibody. The availability of functional FN18sFv will provide the basis for the construction of divalent anti-CD3 immunotoxins for preclinical studies on the induction of tolerance in organ transplantation and experimental autoimmune diseases.

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Enhancement of Immunotoxin Efficacy by Acid-cleavable Cross-linking Agents Utilizing Diphtheria Toxin and Toxin Mutants

Cited by 36 publications

References 29 publications

Treatment with immunotoxin

Treatment with immunotoxin

Acetals as pH-Sensitive Linkages for Drug Delivery

Genetic Construction and Characterization of an Anti-Monkey CD3 Single-Chain Immunotoxin with a Truncated Diphtheria Toxin

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