Conjugation of monoclonal antibodies with TETA using activated esters: biological comparison of 64Cu-TETA-1A3 with 64Cu-BAT-2IT-1A3.

A simple method for conjugation of monoclonal antibodies (mAbs) with the chelating agent 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), has been developed using commercially available reagents. This method involved activation of a single carboxyl group of TETA with N-hydroxysulfosuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The resulting activated ester of TETA was reacted with the anti-colorectal carcinoma mAb 1A3 at molar ratios ranging from 10:1 to 100:1 to give immunoconjugates modified with an average of 0.4 to 2.0 functional chelators per antibody molecule. The TETA-1A3 conjugate was labeled with 64Cu at specific activities as high as 15.4 microCi/microgram, and the radiolabeled mAb exhibited high in vitro serum stability and minimal loss of immunoreactivity. The biodistribution of 64Cu-labeled TETA-1A3 in hamsters bearing GW39 human colon carcinoma xenografts was compared to that of 64Cu-BAT-2IT-1A3 (BAT = 6-(p-bromoacetamidobenzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid; 2IT = 2-iminothiolane). Both conjugates showed high tumor uptake (6.60-9.05% injected dose/gram) from 24 to 48 h post-injection and generally similar blood clearance and non-target organ uptakes. Human absorbed dose estimates derived from the hamster biodistribution data showed the critical organs for both conjugates to be the large intestine and the red marrow. Our results suggest that the in vitro and in vivo performance characteristics of 64Cu-TETA-1A3 compare favorably with those of 64Cu-BAT-2IT-1A3 and that further evaluation of the diagnostic and therapeutic efficacy of 64Cu-TETA-1A3 is warranted.

Maximum tolerated dose and large tumor radioimmunotherapy studies of 64Cu-labeled monoclonal antibody 1A3 in a colon cancer model.

The purpose of this study was 2-fold: to determine the maximum tolerated dose (MTD) of 64Cu-bromoacetamidobenzyl- 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT)-2-iminothiolane (2IT)-monoclonal antibody (MAb) 1A3 in hamsters, and second, to determine the therapeutic efficacy of 64Cu-BAT-2IT-MAb 1A3 at various dose levels in hamsters with large (600 mg), 7-day-old GW39 human colorectal carcinoma tumors. In the MTD studies, non-tumor-bearing hamsters were injected with varying amounts of Cu-64-BAT-2IT MAb 1A3 (>10 mCi) normalized to mCi injected/kg of hamster body weight. Results indicated that the MTD was 150 mCi of Cu-64/kg of body weight. Hamsters receiving higher doses (170-190 mCi/kg) lost greater than 20% of their body weight, and all died between 8 and 13 days (n = 3). All hamsters receiving doses < or = 150 mCi/kg (120-150 mCi; n = 13) survived to the experimental end point (6 weeks) with an overall gain in weight. WBC and platelet counts were depressed in all animals 7 days after treatment but returned to normal values in the survivors by 2 weeks. For larger tumor therapy studies, 40% (8 of 20) of hamsters receiving a single dose of 7.0 mCi and 62.5% (5 of 8) of hamsters receiving 15 mCi of Cu-64-BAT-2IT-MAb 1A3 remained tumor free 4 months after treatment. In dose fractionation studies, hamsters received two 3.5 mCi doses separated by 24 or 48 h with 44% (4 of 9) and 25% (2 of 8) survival, respectively. In every large tumor experimental group, 100% of animals experienced tumor growth inhibition compared to saline control animals. Together, the MTD and the large tumor therapy studies confirm that 64Cu-labeled agents are excellent candidates for radioimmunotherapy trials.

HLA extended haplotypes in steroid-sensitive nephrotic syndrome of childhood.

Idiopathic nephrotic syndrome has been postulated to have an immunopathogenic basis. To determine whether steroid-sensitive nephrotic syndrome is associated with greater than expected frequencies of specific extended haplotypes of the major histocompatibility complex, we studied genetic markers (Class I, II, III HLA alleles and glyoxalase I) in 173 subjects in 42 families of patients with nephrotic syndrome of childhood. The single allele, DQW2, was found in 72% of steroid sensitive patients compared with only 35% of the controls (P = 0.003). In half of 32 steroid sensitive, but not 10 steroid resistant, patients, one or both of two specific extended haplotypes (alleles that segregate together) were identified. The first, [HLA-A1, B8, DR3, DRW52, SCO1], occurred in 11 of 64 haplotypes, or 17%, compared to 5% of controls (P = 0.017). The other, [HLA-B44, DR7, DRW53, FC31], occurred in 10 of 64 haplotypes, 16% compared to 3.8% of controls (P = 0.014). Five patients had both haplotypes. Patients with these specific extended haplotypes had a greater frequency of relapses than did those with other haplotypes. These data provide additional support for the hypothesis that steroid-sensitive nephrotic syndrome has an immunogenetic basis.

Molecular heterogeneity of creatine kinase isoenzymes.

Cytoplasmic creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a dimeric enzyme exhibiting three isoenzymes (MM, MB and BB). The two subunits have been reported to have identical molecular weights (Mr) of 41 000. We have demonstrated that the M subunits from human, canine, rabbit, mouse and bovine tissue have similar apparent Mr values of 43 000 as determined by SDS-polyacrylamide gel electrophoresis. In contrast, the Mr of the B subunits was different from that of the M subunit and varied with each species (human Mr 44 500; canine Mr 46 000; rabbit Mr 44 000 and mouse Mr 49 000). Cyanogen bromide cleavage showed all M subunits to have identical fragments, while B subunits exhibited cleavage products with patterns unique for each species. Despite the differences in Mr and cyanogen bromide fragment patterns, all B subunits were capable of producing enzymatically active hybrid (MB) molecules in combination with M subunits from any species tested. Mitochondrial creatine kinase subunits exhibited identical molecular weights and were similar to the M subunits and failed to combine with either the cytosolic M or B subunits. Thus, B subunits appear less conserved during evolution compared to M subunits, but have retained the amino acid sequences essential for subunit interaction and enzymatic activity.