RESUMEN
PURPOSE: We have performed a phase I study of the cytotoxic immunoconjugate CMB-401 in women with epithelial ovarian cancer (EOC). CMB-401 is a directed chemotherapy that comprises a genetically engineered human antibody against polymorphic epithelial mucin, to which is attached covalently two to three molecules, on average, of the cytotoxic antibiotic calicheamicin. The primary objectives of this two-centre study were to identify end-organ toxicities and to establish the maximum tolerated dose (MTD). PATIENTS AND METHODS: Thirty-four patients aged 37-75 years with progressive EOC not amenable to platinum/standard therapy, and with satisfactory WHO performance status (0-2) were recruited. Patients had received a mean of 3.2 previous chemotherapeutic regimens with a median interval since last chemotherapy of 182 days (range 34-1217). Patients received up to four cycles of a dual infusion of 35 mg/m2 hCTMO1 'predose' followed by doses of CMB-401 which were increased for each cohort--a regimen which minimises drug uptake in normal tissues whilst enhancing delivery to the ovarian tumour. CMB-401 dosing commenced at 2 mg/m2 and progressed via seven cohorts to 16 mg/m2. RESULTS: CMB-401 was generally well tolerated. However, transient fever and emesis occurred, necessitating routine prophylaxis, and increasingly significant malaise was reported as the dose increased. WHO grade 3-4 toxicities, irrespective of causality, included: anaemia 21%, granulocytopenia 9%, thrombocytopenia 9%, liver transaminases 3%, sepsis 3%, haemorrhage 6%, nausea/vomiting 76%; pulmonary 6%, and conscious state/somnolence 6%. The MTD was reached at 16 mg/m2. During the study four patients had a greater than 50% reduction in CA125, and three patients had radiological evidence of reduction in tumour bulk. CONCLUSIONS: CMB-401 appears to have an acceptable toxicity profile with demonstrable activity against EOC.
Asunto(s)
Aminoglicósidos , Antibacterianos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Carcinoma/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Adulto , Anciano , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Antígeno Ca-125 , Carcinoma/mortalidad , Enediinos , Femenino , Humanos , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias Ováricas/mortalidad , Análisis de SupervivenciaRESUMEN
The monoclonal antibody A33 recognises a tumour-associated antigen on human colorectal carcinoma, and has undergone preliminary evaluation in the clinic where selective localisation to hepatic metastases has been demonstrated [Welt et al. (1994) J. Clin. Oncol. 12, 1561-1571]. A33 and an A33 tri-fab fragment (TFM) were labelled with 90Y via a stable macrocyclic ligand for biodistribution and therapy studies in nude mice bearing SW1222 colon carcinoma xenografts. Biodistribution studies demonstrated tumour localisation for both A33 IgG and TFM with low bone, liver and kidney levels. Clearance of TFM from the blood was much faster than IgG and this led to lower tumour accumulation for TFM but superior tumour-blood ratios. The maximum per cent injected dose per g localised to tumour was 35.9% +/- 5.3% for A33 IgG and 12.9% +/- 4.6% for A33 TFM with tumour-blood ratios at 48 h after administration of 5.6 +/- 1.8 and 29.2 +/- 9.8 respectively. Autoradiography studies with 125I-labelled A33 IgG and TFM demonstrated a homogeneous distribution within tumour tissue which was not observed with other anti-colorectal tumour antibodies. TFM penetrated into the tumour tissue more rapidly than IgG. In therapy studies, a single dose of 90Y-A33 IgG (250 microCi per mouse) or 90Y-A33 TFM (300 microCi per mouse) led to complete regression of 2-week-old tumour xenografts with long-term tumour-free survivors. A transient drop in white blood cell count was observed with both IgG and TFM but was significantly more pronounced with IgG. The cell count fell to 8.4% of control for IgG, whereas with TFM cell counts fell to 51% of control before recovery. These results indicate that the more rapid blood clearance of 90Y-TFM confers reduced toxicity compared with 90Y-IgG although similar therapeutic effects are achieved. When the dose of 90Y-IgG was adjusted to give the same dose to tumour achieved with 300 microCi 90Y-TFM, a lesser therapeutic effect was observed. This may be owing to more rapid tumour penetration achieved with TFM. Both A33 IgG and TFM demonstrated potent anti-tumour effects against human tumour xenografts in this mouse model system. The stability of these 90Y-labelled conjugates and their effective tumour penetration are promising for the development of humanised reagents for clinical studies.
Asunto(s)
Neoplasias Colorrectales/radioterapia , Radioinmunoterapia , Radioisótopos de Itrio , Animales , Anticuerpos Monoclonales/farmacocinética , Autorradiografía , División Celular , Neoplasias Colorrectales/patología , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G , Recuento de Leucocitos , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Factores de Tiempo , Distribución Tisular , Trasplante Heterólogo , Pérdida de Peso , Radioisótopos de Itrio/farmacocinéticaRESUMEN
A humanised IgG1/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg l-1 in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocyclic chelator 12N4 (DOTA) was shown to localise very effectively to human colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab' variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has also been constructed and expressed with yields of 500 mg l-1. Trimaleimide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity than hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have been used to produce hA33 TFM labelled stably and site specifically with yttrium-90 or indium-111 respectively. These molecules have been used to demonstrate that hA33 TFM is cleared more rapidly than hA33 IgG from the circulation of animals but does not lead to accumulation of these metallic radionuclides in the kidney. 90Y-labelled hA33 TFM therefore appears to be the optimal form of the antibody for radioimmunotherapy of colorectal carcinoma.
Asunto(s)
Inmunoconjugados/metabolismo , Inmunoconjugados/farmacología , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/radioterapia , Cricetinae , ADN Complementario/genética , Ensayos de Selección de Medicamentos Antitumorales , Expresión Génica , Genes de Inmunoglobulinas , Humanos , Hibridomas/metabolismo , Hibridomas/fisiología , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Radioisótopos de Indio/farmacocinética , Radioisótopos de Indio/farmacología , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Trasplante de Neoplasias , Distribución Tisular , Trasplante Heterólogo , Células Tumorales Cultivadas , Radioisótopos de Itrio/farmacocinética , Radioisótopos de Itrio/farmacologíaRESUMEN
The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Fragmentos de Inmunoglobulinas/uso terapéutico , Neoplasias Experimentales/radioterapia , Radioinmunoterapia , Animales , Células CHO , Bovinos , Cricetinae , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Trasplante Heterólogo , Radioisótopos de Itrio/uso terapéuticoRESUMEN
A novel 111In ligand (a C-functionalised derivative of 1,4,7-triazacyclononanetriacetic acid), termed 9N3, was covalently attached to chimeric B72.3, labelled with 111In and compared with 111In-labelled chimeric B72.3 diethylenetriaminepentaacetic acid (DTPA) cyclic anhydride conjugate (cDTPA) and a C-linked derivative of DTPA (CT-DTPA) in athymic mice bearing human colon carcinoma xenografts. Significant differences in biodistribution were observed between 9N3 and cDTPA conjugates especially in the tumour uptake and blood, liver, femur and colon levels at 24, 48 and 144 h. Significantly higher tumour uptake was observed for 111In-cB72.3-9N3 compared with 111In-cB72.3-cDTPA at all time points. Radiolocalisation (RI) indices increased with time for the 9N3 conjugate but remained constant for the cDTPA conjugate. The biodistribution of 111In-labelled cB72.3-CT-DTPA was similar to that of 111In-labelled cB72.3-9N3 except for elevated kidney levels. A 12N4 macrocycle (a C-functionalised derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid) was also tested for its ability to chelate 111In and its biodistribution examined. Labelled conjugates with this macrocycle were more difficult to prepare in a stable form but gave a very similar biodistribution to the 9N3 macrocycle conjugate. Macrocycle-antibody conjugates of this type offer considerable promise for tumour imaging in patients.
Asunto(s)
Anticuerpos Monoclonales , Quelantes/síntesis química , Quelantes/farmacocinética , Neoplasias Colorrectales/diagnóstico por imagen , Compuestos Heterocíclicos con 1 Anillo , Radioisótopos de Indio , Ligandos , Oligopéptidos , Ácido Pentético/análogos & derivados , Aminas/química , Animales , Cromatografía Líquida de Alta Presión , Femenino , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacocinética , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Cintigrafía , Proteínas Recombinantes de Fusión , Distribución Tisular , Células Tumorales Cultivadas/trasplanteRESUMEN
The chemically averaged molecular weights of a variety of native and conjugated monoclonal antibodies, approximately 150,000, were measured by matrix-assisted UV-laser desorption/ionization mass spectrometry. The average mass of the carbohydrate present in a monoclonal antibody was estimated from the difference between the measured mass of the monoclonal antibody and the mass of the protein present in the monoclonal antibody computed from the amino acid translation of the DNA sequence. The loading of chelators and anticancer drugs conjugated to a monoclonal antibody was quantitated from the difference in the measured masses for the conjugated and untreated monoclonal antibody relative to the expected mass change upon conjugation of 1 mol of chelator or drug. The loading results obtained by mass spectrometry were consistent in most cases with measurements obtained by radioactivity trace assay or UV spectrometry. Similar matrix-assisted UV-laser desorption/ionization mass spectrometric studies were also made after reducing untreated and conjugated monoclonal antibodies with dithiothreitol to determine the distribution of carbohydrate and chelator between the light and heavy chains of the molecules. Matrix-assisted UV-laser desorption/ionization mass spectra were used to compute loading values for covalently bound drugs and proteins, while the loading values obtained by use of gel-filtration HPLC and UV spectrometry cannot distinguish between covalently and noncovalently bound drugs and proteins.