RESUMO
The two naturally occurring forms of ricin A chain, Mr 33,000 and Mr 30,000 (RTA33 and RTA30) have been purified, and their chemical compositions, toxicities, and tissue distributions have been determined. As reported previously, the in vitro and in vivo toxicities of RTA30 and RTA33 are similar. However, RTA30, which contains less carbohydrate with a lower mannose content than RTA33, accumulated less in the liver than did RTA33. Monoconjugate immunotoxins (i.e., containing one RTA per monoclonal antibody molecule) were constructed between RTA30 or RTA33 and the antitumor monoclonal antibody 791T/36, which recognizes a Mr 72,000 antigen on osteosarcoma and colon carcinoma cells. The two immunotoxins had similar cytoxicities in vitro but differed substantially in their pharmacokinetics and tissue distributions in vivo in nude mice bearing C170 human colorectal carcinoma xenografts. The immunotoxin derived from RTA30 (IT30) accumulated less in the liver than the immunotoxin derived from RTA33 (IT33) and cleared more slowly from the blood; the alpha and beta half-lives for IT30 and IT33 were 0.50 and 20.5 versus 0.17 and 14.6 h, respectively. As a probable consequence, IT30 accumulated to approximately 3-fold higher levels in the C170 xenografts than IT33. The reduced clearance of IT30 by the reticuloendothelial system thus resulted in prolonged survival in the blood and enhanced tumor localization relative to IT33.
Assuntos
Imunotoxinas/farmacocinética , Osteossarcoma/tratamento farmacológico , Ricina/farmacocinética , Sarcoma Experimental/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Imunotoxinas/síntese química , Imunotoxinas/farmacologia , Imunotoxinas/uso terapêutico , Leucemia de Células T , Camundongos , Camundongos Nus , Peso Molecular , Transplante de Neoplasias , Osteossarcoma/metabolismo , Ricina/farmacologia , Ricina/uso terapêutico , Sarcoma Experimental/metabolismo , Distribuição Tecidual , Transplante HeterólogoRESUMO
Preparation of the chelating agent (S)-4-[2,3-bis[bis(carboxymethyl)am ino]propyl]phenyl isothiocyanate is reported. Procedures for conjugation of this and (S)-N-4-[2,3-bis[bis-(carboxymethyl)amino] propyl]phenyl bromoacetamide to monoclonal antibodies and other proteins are described. The conjugates may be purified quickly by centrifugation through Sephadex G-50. The number of protein-bound chelating groups may be measured by titration with standard 57Co2+, using thin-layer chromatography to monitor binding. The labeled products retain their immunoreactivity, as illustrated by experiments in vivo with chelate-conjugated antibody to mouse I-AK antigen.
Assuntos
Anticorpos Monoclonais , Quelantes , Reagentes de Ligações Cruzadas , Metais , Animais , Afinidade de Anticorpos , Linfócitos B/imunologia , Quelantes/síntese química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Radioisótopos de Cobalto , Reagentes de Ligações Cruzadas/síntese química , Ácido Edético/análogos & derivados , Ácido Edético/síntese química , Índio , Linfonodos/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Radioisótopos , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
Because monoclonal antibodies can recognize and bind to specific groups of atoms such as tumour antigens, they have promise for use in vivo as carriers of radionuclides, drugs or other appended molecules for diagnosis and treatment of disease. Attachment of metal ions to antibodies by means of bifunctional chelating agents can add the diverse nuclear, physical and chemical properties of the metallic elements to these specific binding proteins (ref. 4 and refs therein). With the ultimate aim of engineering probe-binding properties into the antibodies themselves, we have now prepared monoclonal antibodies against the EDTA chelate of indium. These antibodies show a remarkable preference for indium chelates; changing to another metal such as scandium or gallium can decrease the antibody-binding constant by more than three orders of magnitude. These antibodies also introduce a new degree of control over the biological distributions of chelated radionuclides, markedly altering their uptake in tumours and normal organs.
Assuntos
Anticorpos Monoclonais/imunologia , Ácido Edético/imunologia , Índio/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Cátions , Haptenos , Relação Estrutura-AtividadeRESUMO
The murine IgM monoclonal antibody (mAb) E5 was produced by a hybridoma derived from spleen cells of a mouse immunized with the J5 rough mutant of Escherichia coli O111:B4. In a multicenter randomized placebo-controlled clinical trial, E5 has been shown to reduce significantly the mortality and morbidity of patients with Gram-negative sepsis. The characteristics of E5 binding to endotoxin were studied in vitro. We report here the results of binding to an extensive panel of rough lipopolysaccharide (LPS) and lipid A preparations. Using standard immunologic techniques, including enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), as well as an antibody capture assay using immobilized antibody and a chromogenic Limulus amebocyte lysate (LAL) detection system, E5 was shown to bind to all rough LPS (chemotypes Ra through Re from Salmonella minnesota and E. coli J5) and lipid A preparations tested. E5 displayed a Kd for Ra LPS of approximately 6.5 nM. These results confirm and extend those reported previously and provide evidence that E5 binds specifically to lipid A and to the lipid A moiety of rough LPS.