Effect of Chemical Deglycosylation on the in vivo Fate of Ricin A-Chain

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.

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The Effect of Chemical Deglycosylation of Ricin A-Chain on the Therapeutic Potential of Ricin A-Chain Immunotoxins

Lectins and Glycoconjugates in Oncology ◽

10.1007/978-3-642-73662-9_9 ◽

1988 ◽

pp. 97-102

Author(s):

D. C. Blakeyl ◽

G. J. Watson ◽

P. P. Knowles ◽

P. E. Thorpe

Keyword(s):

Therapeutic Potential ◽

Ricin A Chain ◽

A Chain ◽

Chemical Deglycosylation ◽

Ricin A

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Immunotoxins up to the present day

Bioscience Reports ◽

10.1007/bf01115993 ◽

1989 ◽

Vol 9 (2) ◽

pp. 139-156 ◽

Cited By ~ 10

Author(s):

Francis A. Drobniewski

Keyword(s):

Clinical Studies ◽

Polyclonal Antibodies ◽

Cell Binding ◽

Ricin A Chain ◽

Future Role ◽

A Chain ◽

Ricin A

Immunotoxins consist of monoclonal or polyclonal antibodies conjugated to bacterial or plant toxins. The toxins used are typically of the A-B type in which a toxic A chain is coupled to a B chain responsible for cell binding and facilitation of A chain entry into the cytosol. Two broad strategies have been followed: coupling intact toxins, or A chains alone, to antibodies. This review examines current progress in in vitro and in vivo research, including recent clinical studies, concentrating principally on ricin or ricin A chain conjugates. The future role of conjugates using membrane-acting toxins, immunolysins, is also discussed.

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Cytotoxicity of a cell-reactive immunotoxin containing ricin A chain is potentiated by an anti-immunotoxin containing ricin B chain.

Journal of Experimental Medicine ◽

10.1084/jem.160.1.341 ◽

1984 ◽

Vol 160 (1) ◽

pp. 341-346 ◽

Cited By ~ 46

Author(s):

E S Vitetta ◽

R J Fulton ◽

J W Uhr

Keyword(s):

Cell Surface ◽

Cell Line ◽

Ricin A Chain ◽

Daudi Cell ◽

A Cell ◽

A Chain ◽

Ricin A

In vitro killing of the human Daudi cell line by either univalent [F(ab')] or divalent (IgG) forms of rabbit anti-human Ig (RAHIg) coupled to ricin A chain can be specifically potentiated by a "piggyback" treatment with ricin B chain coupled to goat anti-rabbit Ig (GARIg). When cells are treated with univalent immunotoxin (IT) [F(ab') RAHIg-A] and then cultured, IT can be detected on the cell surface for at least 5 h, since GARIg-B can still enhance killing at this time. These results provide a strategy for in vivo use of A chain- and B chain-containing IT.

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In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain

Cancer Immunology Immunotherapy ◽

10.1007/bf01741320 ◽

1991 ◽

Vol 34 (1) ◽

pp. 24-30 ◽

Cited By ~ 2

Author(s):

Odile Rostaing-Capaillon ◽

Pierre Casellas

Keyword(s):

Ricin A Chain ◽

A Chain ◽

Ricin A

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Effects of therapy with T101 ricin A-chain immunotoxin in two leukemia patients

Blood ◽

10.1182/blood.v67.6.1680.1680 ◽

1986 ◽

Vol 67 (6) ◽

pp. 1680-1687 ◽

Cited By ~ 65

Author(s):

G Laurent ◽

J Pris ◽

JP Farcet ◽

P Carayon ◽

H Blythman ◽

...

Keyword(s):

Target Cells ◽

Pharmacokinetic Studies ◽

Facs Analysis ◽

Ricin A Chain ◽

Blast Cells ◽

Rapid Clearance ◽

A Chain ◽

Free Antigen ◽

Ricin A

Abstract Two leukemia patients, refractory to chemotherapy, were treated with T101-ricin A-chain immunotoxin (T101 IT). Patient 1 (T-ALL) received a single 13.5 mg dose of T101 IT IV (12-hour infusion). Patient 2 (B-CLL) was treated with a daily 25 mg dose of T101 IT IV (two-hour infusion) over three consecutive days. Patient 2 also received 300 mg of chloroquine IM on days two and three as enhancer. In vivo binding of T101 IT was demonstrated by FACS analysis using either an antimouse Ig- FITC or anti-A-chain-FITC antibodies. Following IT therapy, the expression of T65 antigen on target cells dropped to 50% and 20% of pretreatment levels, respectively. In patient 1, circulating blast cells remained unsaturated during therapy while in patient 2, cells were fully saturated for four to six hours following each infusion. Pharmacokinetic studies showed a rapid clearance of T101 IT after IV administration. Antimouse and anti-A-chain antibodies could not be detected. There were no treatment-related adverse effects. In patient 1 a rapid but transient decrease of target cells was observed, possibly related to the administration of the antibody part of T101 IT. In contrast, patient 2 showed a 40% reduction of the lymphocyte count, which remained stable over a period of 2 weeks. Such a clinical benefit following IT therapy in patient 2 could be ascribed to the absence of circulating free antigen and the complete saturation of target cells.

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In vivo therapy of a murine B cell tumor (BCL1) using antibody-ricin A chain immunotoxins.

Journal of Experimental Medicine ◽

10.1084/jem.155.6.1797 ◽

1982 ◽

Vol 155 (6) ◽

pp. 1797-1809 ◽

Cited By ~ 77

Author(s):

K A Krolick ◽

J W Uhr ◽

S Slavin ◽

E S Vitetta

Keyword(s):

Tumor Cells ◽

Blood Cells ◽

Residual Tumor ◽

Cell Tumor ◽

Combined Approach ◽

Ricin A Chain ◽

Total Lymphoid Irradiation ◽

A Chain ◽

Ricin A

Prolonged remissions were induced in mice bearing advanced BCL1 tumors by the combined approach of nonspecific cytoreductive therapy and administration of a tumor-reactive immunotoxin. Thus, the vast majority of the tumor cells (approximately 95%) were first killed by nonspecific cytoreductive therapy using total lymphoid irradiation (TLI) and splenectomy. The residual tumor cells were then eliminated by intravenous administration of an anti-delta immunotoxin. In three of four experiments, all animals treated in the above fashion appeared tumor free 12-16 wk later. In one experiment, blood cells from the mice in remission were transferred to normal BALB/c recipients, and the latter animals have not developed detectable tumor for the 6 mo of observation. Because 1-10 adoptively transferred BCL1 cells will cause tumor in normal BALB/c mice by 12 wk, the inability to transfer tumor to recipients might indicate that the donor animals were tumor free. In the remainder of the animals treated with the tumor-reactive immunotoxin there was a substantial remission in all animals, but the disease eventually reappeared. In contrast, all mice treated with the control immunotoxin or antibody alone relapsed significantly earlier (3-4 wk after splenectomy).

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