Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis

Goldmacher, Victor S.; Scott, Charles F.; Lambert, John M.; McIntyre, Gordon D.; Blättler, Walter A.; Collinson, Albert R.; Stewart, Jean K.; Chong, Lisa D.; Cook, Sherrilyn; Slayter, Henry S.; Beaumont, Elisabeth; Watkins, Simon C.

doi:10.1002/jcp.1041410129

Cited by 51 publications

(21 citation statements)

References 71 publications

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“…It is unlikely that recycled CD22 is involved in the internalization, because the amount of internalized RFB4 is roughly equal to surface CD22 plus the decrease of intracellular CD22. Transferrin receptor (TfR) has been shown to be able to internalize 2-4 times the number of cell surface bound anti-TfR ITs into cells and these ITs showed potent cytotoxicity (37). TfR is known for its recycling ability and intracellular pool, thus may share a similar mechanism as intracellular CD22 utilized to internalize large amount of ITs.…”

Section: Discussionmentioning

confidence: 99%

Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

et al. 2008

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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, f7 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]

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Section: Discussionmentioning

confidence: 99%

Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

et al. 2008

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“…Most of these methods were by linking gelonin to a protein or peptide ligands (e.g., monoclonal antibodies [mAb], growth factors, cytokines, etc. ), so that it could enter cells via the receptor‐mediated endocytosis pathway . The success of such methods, however, relied heavily on the extent of internalization of the ligand–receptor complex, which could vary significantly dependent upon the type of ligands and receptors involved .…”

Section: Introductionmentioning

confidence: 99%

“…), so that it could enter cells via the receptor-mediated endocytosis pathway. 1,[10][11][12] The success of such methods, however, relied heavily on the extent of internalization of the ligand-receptor complex, which could vary significantly dependent upon the type of ligands and receptors involved. 12 Moreover, once endocytosed, the drugs would be entrapped in the endosomes and, unless finding a way to escape, they would not have access to the cell cytoplasm-a pre-requisite for gelonin to exert cytotoxic effects.…”

Section: Introductionmentioning

confidence: 99%

Recombinant TAT-gelonin fusion toxin: Synthesis and characterization of heparin/protamine-regulated cell transduction

Shin

Zhao

Zhang

et al. 2014

J. Biomed. Mater. Res.

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Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TATGel displayed up to 177-fold lower IC50 (avg. 54.3 nM) than rGel (avg. IC50: 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TATGel in achieving a highly effective yet safe drug therapy for the treatment of tumors.

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“…It has been shown, however, that in most cases IT containing ricin A chain are less cytotoxic than native ricin. It is believed that the B chain is necessary for optimal internalization or transmembrane migration [13,27,40,43,45]. In this re-gard, we have used blocked ricin as the cytotoxic effector molecule, which consists of an intact ricin molecule in which the galactose-binding sites of the B chain have been blocked with covalently attached affinity ligands [21].…”

Section: Introductionmentioning

confidence: 99%

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

Rasmussen

Counts

Lambert

et al. 1992

Cancer Immunol Immunother

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The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked ("blocked ricin"), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC50) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC50 values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%-45% viable cells remained after 2 days yet only 3%-9% remained if phorbol ester and Ca2+ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 x 10(-3); when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10(-1). As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.

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Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis

Cited by 51 publications

References 71 publications

Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

Recombinant TAT-gelonin fusion toxin: Synthesis and characterization of heparin/protamine-regulated cell transduction

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

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