A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.

Animals↗

PubMedDate not supplied

A facile, water-soluble method for modification of proteins with DOTA. Use of elevated temperature and optimized pH to achieve high specific activity and high chelate stability in radiolabeled immunoconjugates.

We have developed a method for attachment of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid (DOTA) to proteins by activation of a single carboxyl group with N-hydroxysulfosuccinimide (sulfo-NHS). The sulfo-NHS active ester of DOTA was prepared in a single step using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and DOTA conjugates of cytochrome c and the anti-carcinoembryonic antigen chimeric monoclonal antibody cT84.66 were prepared by adding the DOTA active ester reaction mixture to the proteins at pH 8.5-9.0. Mass spectrometry of the cytochrome c conjugates showed that as the molar ratio of DOTA active ester to protein in the reaction mixture was increased from 10:1 to 100:1, the average number of chelators attached to the protein molecule increased from 2.64 to 8.79. When DOTA active ester reacted with the antibody at a molar ratio of 100:1, the conjugate averaged 3.8 chelates per antibody. Immunoreactivity of the antibody conjugate radiolabeled with 111In(III) and 90Y(III) remained quantitative. Variation of the DOTA:sulfo-NHS:EDC activation stoichiometry from 2:2:1 to 10:10:1 revealed that the kinetic stability of the radioconjugates increased as the molar ratio of carbodiimide, relative to DOTA and sulfo-NHS, was decreased. Radiolabeling of the protein conjugates with 111In(III) and 90Y(III) proved to be sensitive to pH, buffer, and temperature effects. The optimum pH for the labeling reaction was different for each protein and may be related to the isoelectric point of the protein. Radiometal incorporation at high specific activity was accomplished in acetate and Tris buffers, but the presence of citrate inhibited the labeling reaction. Increasing the temperature of the radiolabeling reaction from 25 to 43 degrees C greatly increased both the efficiency of radiometal incorporation and the kinetic stability of the radioconjugates. Stability studies of the conjugates in human serum and in the presence of a 5000- to 250,000-fold excess of diethylenetriaminepentaacetic acid (DTPA) demonstrated that the radiolabeled proteins are kinetically inert under physiological conditions. In serum, the 111In(III)-labeled antibody showed a rate of radiometal loss of approximately 0.08% per day. In the presence of excess DTPA, both conjugates lost 111In(III) at a rate of about 0.3% per day. No loss of 90Y(III) from the conjugates was observed in serum, but in excess DTPA, both 90Y(III) labeled proteins showed a rate of radiometal loss of approximately 0.2% per day. Therefore, kinetic analysis of metal loss from a radiolabeled immunoconjugate in the presence of a vast excess of DTPA may provide a better indication of the in vivo stability of that immunoconjugate than serum stability studies.

Antibodies, Monoclonal↗

PubMedDate not supplied

Maleimidocysteineamido-DOTA derivatives: new reagents for radiometal chelate conjugation to antibody sulfhydryl groups undergo pH-dependent cleavage reactions.

We have synthesized two bifunctional derivatives of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid (DOTA) equipped with maleimide groups for conjugation to reduced disulfide bonds of monoclonal antibodies. Using water-soluble carbodiimide chemistry, DOTA was coupled to L-cysteine to incorporate both a "pendant-type" carboxyl group for metal coordination and an orthogonal thiol group for protein attachment. The homobifunctional reagent 1,6-bis(maleimido)hexane was then used to introduce the maleimide functionality via a sulfide linkage to the macrocycle, and alternatively, the sulfide group was converted to a sulfone side chain. Both maleimide derivatives were conjugated to the anticarcinoembryonic antigen chimeric monoclonal antibody cT84.66 after light reduction of the mAb with dithiothreitol. In this manner, antibody conjugates were prepared which afforded near-quantitative labeling with the radiometals 111In(III) and 90Y(III) as well as quantitative immunoreactivity. Radioimmunoconjugates prepared with the sulfide and sulfone compounds exhibited relatively rapid linker-dependent radiometal loss when incubated in human serum and aqueous solutions at physiological temperature and pH. The unconjugated maleimidocysteineamido-DOTA derivatives and their Y(III) complexes were incubated in aqueous solution at 37 degrees C, and the resulting decomposition products were analyzed by HPLC and mass spectrometry. These studies revealed that the two bifunctional chelating agents underwent linker-specific cleavage reactions which were considerably faster at pH 7.4 than at pH 5.4. The chemically labile linker systems are expected to release chelated radiometal from mAb conjugates in a pH-dependent manner. This property may impart favorable tumor uptake and normal tissue clearance on radioimmunoconjugates prepared with these reagents, on the basis of the observation that many solid tumors are significantly more acidic than normal tissues.

Antibodies, Monoclonal↗

PubMedDate not supplied

Biodistribution of 111In- and 90Y-labeled DOTA and maleimidocysteineamido-DOTA conjugated to chimeric anticarcinoembryonic antigen antibody in xenograft-bearing nude mice: comparison of stable and chemically labile linker systems.

Biodistributions of two radiometal chelate conjugates of the human/murine chimeric anticarcinoembryonic antigen monoclonal antibody cT84.66 were obtained in nude mice bearing LS174T human colorectal carcinoma xenografts. Derivatives of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) were covalently attached to the antibody by a stable amide linkage and by a maleimidocysteineamido side chain (MC-DOTA) that has been shown to be chemically labile at physiological temperature and pH. Biodistributions of both 111In and 90Y labels were obtained in these studies. At common biodistribution time points, it was found that the 111In label had greater uptake in the liver than 90Y for both conjugates. No significant differences were found with respect to bone uptake of 90Y using either chelate. Blood curves were generally lower at comparable time points for MC-DOTA, indicative of faster clearance as compared to DOTA. Tumor uptake was high for both conjugates (57-68% ID/g at 48 h), with a longer tumor residence time in the case of the DOTA conjugate, probably a result of its longer blood circulation times. We conclude that bone uptake of 90Y would be minimal if either DOTA or MC-DOTA were used as the bifunctional chelator. This would imply preference for these macrocyclic ligands if radiation doses to the bone marrow would be considered to be dominated by skeletal uptakes. Alternatively, if bone marrow radiation dose is dominated by circulating antibody, the chemically labile linker system employed by the MC-DOTA conjugate offers the advantage of enhanced blood clearance.

Animals↗

PubMedDate not supplied

Acquisition of cytomegalovirus infection among female employees at a pediatric hospital.

Of 842 female employees at a pediatric hospital who were tested for cytomegalovirus antibody, 487 (57.8%) were seronegative. We also studied 228 females who had been employed for 3 or more months, and 139 (61.0%) were seronegative. One year after the initial serum 173 seronegative women provided a second sample. Eleven (6.4%) had seroconverted to cytomegalovirus, and none gave a history of significant illness in the interval. Of the 173 women 138 had patient contact jobs and 35 had noncontact jobs. In the contact group 10 of 138 (7.2%) seroconverted including 5 of 46 (10.9%) intensive care nurses, 2 of 11 (18.2%) women on the blood drawing or intravenous insertion team and 3 of 81 (3.7%) nurses on medical and surgical wards. In the noncontact group 1 of 35 (2.9%) seroconverted. Among female employees at a pediatric hospital the risk of cytomegalovirus is substantial and appears to be related to the type of patient contact.

Cytomegalovirus Infections↗

PubMedDate not supplied

A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.

We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.66 was expressed in high yield in a bioreactor and purified to homogeneity in a single step on an anti-idiotypic antibody affinity column. The molecular size of the antibody was 150 kDa as judged by gel filtration, SDS gel electrophoresis under non-reducing conditions, and MALDI-TOF/MS. The 150 kDa antibody had nearly identical kinetic (Kon = 1.53 x 10(6) M-1 s-1, .koff = 1.14 x 10(-5) s-1) and affinity constants (Kaff = 1.34 x 10(11) M-1) compared to the parent murine (Kaff = 1.25 x 10(11) M-1) and chimeric (Kaff = 1.16 x 10(11) M-1) antibodies when tested on biosensor chips. When delta SSchT84.66 was conjugated to the isothiocynato derivative of DTPA, radiolabeled with 111In, and injected into either normal or nude mice bearing tumor xenografts, it gave nearly identical biodistributions to chT84.66. delta SSchT84.66 and chT84.66 antibodies gave a maximum tumor uptake of 48 and 74% ID/g, and tumor to blood ratios of 5.3 and 6.2 at 48 h, respectively. We conclude that delta SSchT84.66 irreversibly associates into H2L2 dimers after concentration, that the dimers are stable under both the in vitro and in vivo conditions used in this study, and the properties of the antibody are virtually indistinguishable from the parent chT84.66 antibody.

Animals↗

PubMedDate not supplied

The growth and development status of homeless children entering shelters in Boston.

In order to characterize the children who enter emergency shelters in Boston, we reviewed the data collected at intake interviews by the pediatric nurse practitioner visiting 10 family shelters and one hotel in Boston as part of the Boston Health Care for the Homeless Project. Families were interviewed soon after their entry into the shelter. Children were weighed and measured, and the Denver Developmental Screening Test (DDST) was administered. From November 1986 to November 1987, 133 families with 213 children were interviewed. Ninety-four percent of the children were in the care of their mothers, and 92 percent were younger than 5 years of age. Sixty-five percent of the families were black, 20 percent were white, and 11 percent were Hispanic. Eighty-nine percent of the families were receiving Aid to Families with Dependent Children benefits, 90 percent were receiving Medicaid benefits, 72 percent were receiving food stamps, and 52 percent were receiving benefits under the Special Supplement Food Program for Women, Infants, and Children. Eighty-five percent of the children were reported to have a regular source of primary pediatric care, and 23 percent were reported to have medical problems. Weight-for-age, weight-for-height, and height-for-age measurements were similar to those reported for national samples of low income children. Ten children (4.7 percent) were found to have abnormal or questionable DDST examinations.

Boston↗