Immunotoxins: A New Approach to Cancer Therapy

Vitetta, Ellen S.; Krolick, Keith A.; Miyama-Inaba, Muneo; Cushley, William; Uhr, Jonathan W.

doi:10.1126/science.6218613

Cited by 242 publications

(56 citation statements)

References 39 publications

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“…The inclusion of whole ricin in these constructs is complicated by the presence of the B chain galactose-binding sites, which override the specificity conferred by the antibody molecules [33]. The presence of the B chain seems to be necessary, however, to ensure efficient delivery of the A chain into the cytoplasm [33,34]. Site-specific mutation of cDNA clones, affecting amino acids involved in B chain galactose binding, may ultimately lead to a modified ricin and an increased specificity of ricin-based immunotoxins.…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of cloned cDNA coding for preproricin

Lamb

Roberts

Lord

1985

European Journal of Biochemistry

262

137

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The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain (267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-aminoacid linking region (molecular mass 1385 Da).Ricin, the toxic lectin of Ricinus communis seeds, is a heterodimer whose subunits (designated the A and B chains) are joined by a disulphide bond [l]. The amino acid sequence of both subunits has been determined [2-41. The molecular mass of the A polypeptide is 30522 Da and it contains one N-linked oligosaccharide (on asparagine residue lo), while the B chain, molecular mass 29082 Da, contains two oligosaccharide chains (asparagine residues 93 and 133). The B chain also contains two galactose-binding sites [5]. Interaction of these binding sites with exposed galactose residues on cell surface components leads to the translocation of the toxic A chain into the cytoplasm, where it irreversibly inactivates 60s ribosomal subunits [l].We have recently shown that the A and B chains of ricin are synthesized, both in vivo and in vitro, in the form of a single precursor polypeptide tentatively assumed to contain one copy of each subunit [6]. This assumption has been confirmed in the present study by cloning and sequencing cDNAs encoding a ricin precursor. MATERIALS AND METHODS Muter iuls Isolation of m R N ATotal RNA was isolated from maturing castor bean (Ricinus communis) seeds taken from greenhouse-grown plants [7]. Poly(A)-rich RNA was purified by chromatography on oligo(dT)-cellulose [8] and was fractionated on the basis of size by sucrose density gradient centrifugation [9]. mRNA was translated in the nuclease-treated rabbit reticulocyte lysate system [lo].cDNA synthesis 3.0 pg of poly(A)-rich RNA, enriched in ricin precursor mRNA, was used as a template for oligo(dT)-primed synthesis of cDNA using avian myeloblastosis virus reverse transcriptase followed by second-strand synthesis with DNA polymerase according to published procedures [I I]. After treatment of the double-stranded cDNA with Aspergillus oxyzae S 1 nuclease, homopolymeric poly(dC) tails were added to the 3' ends using calf thymus terminal deoxynucleotidyltransferase [ 121. cDNA cloning and library screeningPoly(dC)-tailed cDNA was fractionated by ...

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Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of cloned cDNA coding for preproricin

Lamb

Roberts

Lord

1985

European Journal of Biochemistry

262

137

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“…The production and testing of a number of these compounds which recognize surface structures of specific normal or tumor cell populations is currently underway (reviewed in [1][2][3][4][5]. In vitro studies with cultured cells have shown that some ofthese immunotoxin conjugates can kill the specific cell population recognized by the constituent monoclonal antibody, but are relatively nontoxic to cells which do not bind that antibody (1)(2)(3)(4)(5). While these findings are encouraging, considerably less experimental data are available regarding the systemic toxicity and the efficacy of these compounds when used in vivo.…”

Section: Introductionmentioning

confidence: 99%

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. IV. Cytotoxic effect of an anti-T11-gelonin immunotoxin.

Reimann¹,

Goldmacher²,

Lambert³

et al. 1988

J. Clin. Invest.

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The cytotoxic effect of a lymphocyte-specific immunotoxin formed by disulfide conjugation of an anti-Ti 1 monoclonal antibody with the ribosome-inactivating protein gelonin was assessed in vitro on peripheral blood T cells and in vivo on splenic and lymph node T cells of macaque monkeys. This immunotoxin was cytotoxic to proliferating peripheral blood T cells in vitro as measured by both direct and indirect assays. Two sequential intravenous infusions into macaque monkeys achieved plasma concentrations of immunotoxin far in excess of those shown to be cytotoxic for cultured T cells and coated all T cells in lymph nodes and spleen with intact immunotoxin for four days. However, the cytotoxic effect of the immunotoxin on T cells in vivo was considerably less than that predicted by the in vitro studies. Further experiments suggested that the state of activation of the targeted T cell population in vivo, or the appearance of anti-immunotoxin antibodies, which occurred in all infused monkeys, might attenuate immunotoxin-mediated cell killing in vivo. These studies illustrate the significant differences between the action of immunotoxin conjugates in vitro, and those seen when these conjugates are utilized as therapeutic agents in vivo.

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“…After entry into the cytosol, the A chain catalytically inactivates the 60S ribosomal subunit, thereby inhibiting protein synthesis. A single molecule of A chain in the cytosol may be sufficientto kill a cell (2,3,5).…”

mentioning

confidence: 99%

“…Each chain has a molecular weight of 32 kD; the A chains attack the 60S ribosomal subunit inside the cell, and the B chains bind galactose. Diphtheria toxin, abrin, and ricin are examples of naturally occurring toxins (2)(3)(4). Some plant toxins consist only of A chains (gelonin and pokeweed antiviral protein).…”

mentioning

confidence: 99%