RÉSUMÉ
Folate receptor is over-expressed in a variety of carcinomas. To design a cytotoxic drug that would selectively target these carcinomas, we synthesized folate-maytansinoids. These drugs showed high affinity toward folate receptor, appeared to enter cells exclusively via the folate receptor-mediated caveolar pathway and displayed high cytotoxic potency (in the range of 10[-11] to 10[-10] M) and remarkable selectivity for folate receptor-expressing carcinoma cell lines. Folate-maytansinoids represent a new class of tumor-specific agents in which the targeting and the cytotoxic function can be altered independently.
Sujet(s)
Antinéoplasiques/métabolisme , Antinéoplasiques/pharmacologie , Maitansine/pharmacologie , Récepteurs de surface cellulaire , Antinéoplasiques/synthèse chimique , Protéines de transport/métabolisme , Tests de criblage d'agents antitumoraux , Technique d'immunofluorescence indirecte , Récepteurs des folates à ancre GPI , Humains , Cellules KB , Maitansine/analogues et dérivés , Maitansine/synthèse chimique , Maitansine/métabolisme , Cellules cancéreuses en cultureRÉSUMÉ
Improving the tumour selectivity of cytotoxic drugs through conjugation to tumour-reactive monoclonal antibodies may lead to novel, more potent agents for cancer therapy. The maytansinoid drugs are 100- to 1000-fold more cytotoxic in vitro than current clinical anticancer drugs. We recently demonstrated that conjugation of maytansinoid drugs to monoclonal antibodies renders them highly efficacious against cancers of breast and colon in both in vitro and in in vivo tumour models. Antibody-maytansinoids represent a new generation of immunoconjugates that may yet fulfil the promise of effective cancer therapy through antibody targeting of cytotoxic agents.
RÉSUMÉ
The maytansinoid drug DM1 is 100- to 1000-fold more cytotoxic than anticancer drugs that are currently in clinical use. The immunoconjugate C242-DM1 was prepared by conjugating DM1 to the monoclonal antibody C242, which recognizes a mucin-type glycoprotein expressed to various extents by human colorectal cancers. C242-DM1 was found to be highly cytotoxic toward cultured colon cancer cells in an antigen-specific manner and showed remarkable antitumor efficacy in vivo. C242-DM1 cured mice bearing subcutaneous COLO 205 human colon tumor xenografts (tumor size at time of treatment 65-130 mm3), at doses that showed very little toxicity and were well below the maximum tolerated dose. C242-DM1 could even effect complete regressions or cures in animals with large (260- to 500-mm3) COLO 205 tumor xenografts. Further, C242-DM1 induced complete regressions of subcutaneous LoVo and HT-29 colon tumor xenografts that express the target antigen in a heterogeneous manner. C242-DM1 represents a new generation of immunoconjugates that may yet fulfill the promise of effective cancer therapy through antibody targeting of cytotoxic agents.
Sujet(s)
Antibiotiques antinéoplasiques/administration et posologie , Anticorps antitumoraux/usage thérapeutique , Tumeurs colorectales/thérapie , Immunotoxines/toxicité , Maitansine/analogues et dérivés , Animaux , Anticorps monoclonaux/usage thérapeutique , Humains , Maitansine/administration et posologie , Souris , Souris SCID , Transplantation tumorale , Transplantation hétérologue , Cellules cancéreuses en cultureRÉSUMÉ
Bis-indolyl-(seco)-1,2,9a-tetrahydrocyclopropa[c]benz[e]indol-4-on e compounds are synthetic analogues of CC-1065 that are highly cytotoxic toward a broad spectrum of tumor cell lines. One of these compounds, called DC1, was conjugated to antibodies via novel cleavable disulfide linkers. Conjugates of DC1 with murine mAbs anti-B4 and N901 directed against tumor-associated antigens CD19 and CD56, respectively, proved to be extremely potent and antigen selective in killing target cells in culture. DC1 conjugates with humanized versions of anti-B4 and N901 antibodies were also constructed and demonstrated to be as cytotoxic and selective as the respective murine antibody conjugates. The anti-B4-DC1 conjugate showed antitumor efficacy in an aggressive metastatic human B-cell lymphoma survival model in SCID mice and completely cured animals hearing large tumors. Anti-B4-DC1 was considerably more effective in this tumor model than doxorubicin, cyclophosphamide, etoposide, or vincristine at their maximum tolerated doses.
Sujet(s)
Antibiotiques antinéoplasiques/pharmacologie , Immunotoxines/pharmacologie , Indoles , Leucomycines/pharmacologie , Animaux , Antigènes CD/analyse , Antigènes CD/immunologie , Antigènes CD19 , Antigènes de différenciation des lymphocytes B/analyse , Antigènes de différenciation des lymphocytes B/immunologie , Antigènes de différenciation des lymphocytes T/immunologie , Antigènes CD56 , Duocarmycines , Femelle , Humains , Lymphome B/thérapie , Souris , Souris SCID , Cellules cancéreuses en cultureRÉSUMÉ
The potential of immunoconjugates of cytotoxic drugs for the treatment of cancer has not yet been realized owing to the difficulty of delivering therapeutic concentrations of these drugs to the target cells. In an effort to overcome this problem we have synthesized maytansinoids that have 100- to 1000-fold higher cytotoxic potency than clinically used anticancer drugs. These maytansinoids are linked to antibodies via disulfide bonds, which ensures the release of fully active drug inside the cells. The conjugates show high antigen-specific cytotoxicity for cultured human cancer cells (50% inhibiting concentration, 10 to 40 pM), low systemic toxicity in mice, and good pharmacokinetic behavior.
Sujet(s)
Immunotoxines/usage thérapeutique , Maitansine/usage thérapeutique , Animaux , Anticorps monoclonaux/métabolisme , Anticorps monoclonaux/usage thérapeutique , Fixation compétitive , Tests de criblage d'agents antitumoraux , Immunotoxines/composition chimique , Immunotoxines/métabolisme , Maitansine/analogues et dérivés , Maitansine/composition chimique , Maitansine/métabolisme , Souris , Cellules cancéreuses en cultureRÉSUMÉ
We prepared an immunoconjugate consisting of a monoclonal antibody recognizing the Thy-1 antigen and the ribosome-inactivating protein gelonin linked by a disulfide bond. This immunotoxin preparation was judged to contain less than 5% free antibody or gelonin. It was highly toxic in vitro in an antigen-specific fashion to the Thy-1 expressing RADA leukemia of A/J mice. The IC50 of this preparation on RADA in vitro was 10(-12) M, while the IC50 on the Thy-1 negative S1509a fibrosarcoma of A/J mice was 10(-7) M. The toxicity of this immunoconjugate was also measured in a direct proliferation and it was found that a 4-h exposure and a 24-h exposure of RADA cells to a 1 nM concentration of immunotoxin killed 90% and 99.9% of cells, respectively. Furthermore, efficacy in vitro was not due to the intrinsic susceptibility of RADA cells to tis type of immunotoxin, as one prepared with gelonin and an antibody recognizing the TLa determinant on this leukemia had no efficacy in vitro. Clearance of the anti-Thy-1-gelonin immunoconjugate from the circulation of A/J mice after i.v. injection was rapid, especially during the first 8 h after injection, possibly because of binding to Thy-1 expressing tissue. Delivery of immunoconjugate to ascitic tumor in vivo was substantially better if the immunoconjugate was given by i.p. injection, rather than by the i.v. route. When given either i.v. or i.p. at the time of i.p. tumor inoculation in vivo, the anti-Thy-1-gelonin immunotoxin showed potency in an antigen-specific fashion; while this immunoconjugate prolonged survival and frequently cured RADA-inoculated mice, neither anti-Thy-1 antibody, gelonin, a combination of the two, nor immunotoxin of irrelevant specificity had any significant effect on survival. Anti-Thy-1-gelonin also had no effect on survival of A/J mice inoculated i.p. with S1509a. Furthermore, it was determined that a single i.p. dose of anti-Thy-1-gelonin killed 90% to 99% cells in vivo, and that the immunoconjugate was about as effective in this model as either adriamycin or cytoxan.
Sujet(s)
Anticorps monoclonaux/administration et posologie , Immunotoxines/administration et posologie , Alloanticorps/administration et posologie , Leucémie expérimentale/thérapie , Protéines végétales/administration et posologie , Animaux , Survie cellulaire , Souris , Souris de lignée A , Protéines inactivant les ribosomes de type 1RÉSUMÉ
The p-aminophenyl gamma-ester of 7-methylguanosine 5'-triphosphate (m7GTP) was synthesized and coupled to Sepharose 4B. A 0.5 M salt extract of rabbit reticulocyte ribosomes was passed over a column containing the affinity medium. After extensive washing, a solution of m7GTP was passed through the column, and a single polypeptide species of 24 kilodaltons (kDa) was eluted. This had an electrophoretic mobility identical with that of the mRNA cap-binding protein. This assignment was confirmed by the fact that the eluted material was enriched nearly 200-fold in the ability to specifically bind 32P-labeled capped oligonucleotides. A control affinity medium consisting of GTP similarly coupled to Sepharose failed to retain the 24-kDa species. The postribosomal supernatant fraction yielded slightly more of the 24-kDa species than the ribosomal wash fraction when passed over this affinity medium.
Sujet(s)
Protéines de transport/isolement et purification , Guanosine triphosphate/analogues et dérivés , Coiffes des ARN , Animaux , Chromatographie d'affinité/méthodes , Guanosine triphosphate/synthèse chimique , Indicateurs et réactifs , Protéines de liaison à la coiffe de l'ARN , Lapins , Réticulocytes/analyse , Ribosomes/analyseRÉSUMÉ
The paramagnetic effects of Mn2+ . glyoxalase I on the 13C relaxation rates of the reaction product, S-(D-lactoyl)glutathione, separately enriched in the lactoyl carbonyl (C-1) and hydroxymethylene (C-2) carbons, have been measured at 62.8 MHz. The 1/fT1p values of C-1 (1100 +/- 120 s-1) and C-2 (712 +/- 290 s-1) and the previously determined tau c (0.74 ns) yield Mn2+ to carbon distances of 5.7 +/- 0.3 and 6.1 +/- 0.5 A, respectively. These distances, together with previously determined Mn2+-proton distances (Sellin, S., Rosevear, P.R., Mannervik, B., and Mildvan, A.S. (1982) J. Biol. Chem. 257, 10023-10029) constrain the thioester carbonyl group of the product to point toward the metal, with the oxygen positioned to accept a hydrogen bond from a water ligand, in a kinetically competent, second sphere complex. Model-building studies indicate that any averaging of multiple second sphere complexes would require as a major contributor at least one conformation with the lactoyl carbonyl oxygen within hydrogen-bonding distance of an intervening water ligand. Such a structure would facilitate polarization of the carbonyl group in the reverse glyoxalase reaction.
Sujet(s)
Lactoyl glutathione lyase/sang , Lyases/sang , Érythrocytes/enzymologie , Glutathion/analogues et dérivés , Humains , Ligands , Spectroscopie par résonance magnétique , Manganèse , Liaison aux protéinesRÉSUMÉ
Fluoromethylglyoxal in the presence of glutathione has been shown to undergo a novel glyoxalase I-catalyzed product partitioning to S-fluorolactoylglutathione and S-pyruvylglutathione with fluoride elimination. While the partition ratio (fluoride eliminated/total fluoride) was insensitive to pH 5.5-7.5 and concentrations of substrate and glyoxalase I, it was species-dependent. When [1-2H]fluoromethylglyoxal was reacted with glyoxalase I, an increase in the partition ratio and retention of deuterium in the product fluorolactate was observed. This result can only be explained by a selective primary isotope effect on the protonation of an enediol intermediate relative to fluoride elimination.