Alejo Morales

Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies

A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellmans reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (microgram/mL). After subtraction of the background due to Ellmans reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 micrograms, the lowest detection limit for cysteine quantification was 0.03 microgram. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG(1)), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 microg/100 microCi of (99m)Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of (99m)Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significative accumulation was found in tumor (6.14 +/- 2.50 %ID/g, 5.06 +/- 2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99mTc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99mTc at high yield. The resulting 99mTc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

The pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t 1/2alpha) of 0.250 h and a mean elimination (t 1/2beta) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99mTc-labeled humanized MAb R3 conjugate in patients should be supported.

TECHNETIUM-99M DIRECT RADIOLABELING OF MONOCLONAL ANTIBODY IOR EGF/R3

Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99mTc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99mTc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellmans reagent and was found to be 6.65+/-0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.

A computer program for quantification of SH groups generated after reduction of monoclonal antibodies

Reduction of disulfide bonds to sulfhydryl (SH) groups for direct radiolabeling of antibodies for immunoscintigraphic studies of colorectal and other cancers continues to be of considerable research interest. We have developed a general strategy and a versatile computer program for the quantification of the number of SH per molecule of antibody (Ab) generated after the treatment of monoclonal antibodies (MAbs) with reducing agents such as 2-mercaptoethanol (2-ME), stannous chloride (SnCl2), dithiothreitol (DTT), dithioerythritol (DTE), ascorbic acid (AA), and the like. The program we describe here performs an unweighted least-squares regression analysis of the cysteine standard curve and interpolates the cysteine concentration of the samples. The number of SH groups per molecule of antibody in the 2-mercaptoethanol and in the other reducing agents was calculated from the cysteine standard curve using Ellmans reagent to develop the yellow color. The linear least-squares method fit the standard data with a high degree of accuracy and with the correlation coefficient r of 0.999. A program has been written for the IBM PC compatible computer utilizing a friendly menu to interact with the users. The package allows the user to change parameters of the assay, to calculate regression coefficients slope, intercept and its standard errors, to perform statistical analysis, together with detailed analysis of variance, and to produce an output of the results in a printed format.