To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast. Smc2p and Smc4p form a stable heterodimer, the “Smc2/4 complex,” which upon analysis by sedimentation equilibrium appears to reversibly self-associate to form heterotetramers. Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins. Hydrolysis activity is unaffected by the presence of DNA. Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide. High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase. Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge. Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible. Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA. To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer. Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA.

Background. The murine A6H monoclonal antibody targets a cell surface antigen associated with renal cell carcinoma with high specificity and excellent biodistribution properties. Tumor to blood ratios of >40:1 have been achieved in clinical studies. Objectives. In order to generate an antibody engineering system that would allow the construction of improved derivatives for diagnostics and therapeutics, a single-chain Fv antibody (scFv) derived from A6H was constructed. The initial single-chain Fv, constructed with a cysteine residue and hexa-histidine sequence at the C-terminus, displayed a limited solubility of 100 μg/ml at pH 7.4. The low solubility and refolding yield of the original single-chain Fv required that a more soluble variant be designed and constructed. Study design. We hypothesized that lowering the pI of the scFv antibody away from the physiological range would yield a more soluble antibody. A derivative was thus subsequently engineered with five glutamic acid residues followed by the cysteine and hexa-histidine residues. The cysteine was included to provide a conjugation site for future radiolabeling studies. Results. The redesigned A6H single-chain Fv has a predicted pI of 6.1, relative to 7.5 for the native scFv. The redesigned A6H scFv displayed a greatly enhanced solubility of >15 mg/ml at pH 7.4. Both the original scFv and the redesigned single-chain Fv exhibited a strong tendency to form dimers and soluble high molecular weight aggregates. The monomer and disulfide bonded dimer were separated from the aggregates and complete cell binding isotherms were obtained, demonstrating that the purified A6H scFv retains much of the activity of the parent monoclonal. Conclusion. The addition of glutamic acid to the C-terminus of poorly soluble scFv antibodies could provide a straightforward avenue for improving their solubility properties. The increased solubility of the A6H scFv allowed the purification of the monomeric and dimeric species from the soluble aggregated species.

In this investigation, a comparison of wild type recombinant streptavidin (r-SAv) with two genetically engineered mutant r-SAv proteins was undertaken. The investigation also included a comparison of the r-SAv with two streptavidin (SAv) proteins from commercial sources. In vitro characterization of the SAv proteins was conducted by HPLC, SDS-PAGE, IEF, and electrospray mass spectral analyses. All SAv proteins studied appeared to be a single species by size exclusion chromatography (HPLC) and SDS-PAGE analyses, but multiple species were noted in the IEF and MS analyses. In vivo comparisons of the SAv proteins were accomplished with dual isotope-labeled SAv in athymic mice. In an initial experiment, tissue localization of r-[131I]SAv directly radiolabeled using chloramine-T was compared with r-SAv radiolabeled with the N-hydroxysuccinimidyl p-iodobenzoate conjugate ([125I]-PIB), a radioiodination reagent that has been shown to result in iodine-labeled proteins which are stable to in vivo deiodination. The data obtained indicated that there is little difference in the distribution (except kidney localization) when r-SAv labeled by the two methods. Data obtained from comparison of r-[131I]SAv with a disulfide-stabilized r-SAv mutant (r-SAv-H127C), a C-terminal cysteine-containing r-SAv mutant (r-[125I]SAv-S139C), and two 125I-labeled SAv proteins obtained from commercial sources indicated that their distributions were quite similar, except the kidney concentrations were generally lower than that of r-[131I]SAv. On the basis of the similar distributions of the SAv proteins studied, it appears that the r-SAv mutants may be interchanged for the (wild type) r-SAv in pretargeting studies. Further, the similarity of distributions with two commercially available SAv proteins suggests that the results obtained in our studies and those of other groups may be directly compared (with consideration of animal model, sacrifice time, etc.).

We have used the polysome-selection method to isolate peptide ligands that bind with high affinity to Prostate-Specific Antigen (PSA), an important prostate-cancer marker. Two random libraries, each encoding approximately 10(12) random peptides, mere transcribed into RNA and translated in vitro. Polysomes were planned by affinity selection of the nascent peptides against immobilized PSA. Over 30% of the selected species had significant affinity for PSA; the dissociation constant of the complex formed by the best isolate with PSA was < 10(-9) M. Formation of streptavidin conjugates of selected peptides improved their affinities and, in one case, virtually eliminated non-specific binding. The polysome-selection method can be used to produce high-affinity peptide, ligands of potential use in diagnostic and therapeutic procedures. (C) 1997 Academic Press.

While prostate‐specific antigen (PSA) is already an invaluable marker for prostate cancer, there is continuing demand for new anti‐PSA antibodies with specific characteristics, e.g., high sensitivity and specificity and equimolar binding to free PSA (f‐PSA) and the PSA‐α‐1‐antichymotrypsin complex (PSA‐ACT), as well as the ability to distinguish between these 2 immunoreactive forms of PSA. We have therefore generated and characterized 10 anti‐PSA monoclonal antibodies (MAbs). Apparent dissociation constants (Kd) of MAbs were determined by direct ELISA yielding Kd‐0.2‐164.0 nM. Western blots suggested that 3 of the MAbs (60‐1A2, 60‐8A2 and 17‐1A2) bind to linear epitopes. Sandwich assays identified 5 major antigenic regions as binding targets of the MAbs. Three combinations of MAbs recognize f‐PSA and PSA‐ACT in equimolar fashion with high sensitivity. Two of the MAb combinations are specific for f‐PSA. Physical analysis of the new antibodies has allowed us to assign the MAbs to binding classes (based on their sandwiching capabilities) and to determine accurate apparent dissociation constants. Int. J. Cancer 71: 1019‐1028, 1997. © 1997 Wiley‐Liss Inc.

An evaluation of the use of a biotinylated monoclonal antibody Fab' fragment in tumor pretargeting was conducted. As a model system, tumor colocalization of avidin or recombinant streptavidin (r-streptavidin) and the biotinylated Fab' fragment (Fab'-S-biotin) of A6H, an antirenal cell carcinoma antibody, was evaluated in athymic mice bearing human renal cell carcinoma xenografts. A new water soluble sulfhydryl reactive biotinylation reagent, N-(13-N-maleimdo-4, 7,10-trioxatridecanyl)-biotinamide, was synthesized and used for biotinylation of Fab'. A biodistribution of ChT-labeled A6H Fab'-S-biotin was conducted. Data from that distribution indicated that the Fab'-S-biotin localized well (i.e. 28% ID/g at 24 h) to human tumor xenografts in athymic mice. Subsequently, a biodistribution study involving pretargeting radioiodinated A6H Fab'-S-biotin to tumor xenografts, followed by administration of r-streptavidin at 4 or 20 h, was conducted. Specific colocalization of r-streptavidin to tumors containing the A6H Fab'-S-biotin was evident from the data obtained. In a similar biodistribution study, specific colocalization of avidin to tumors pretargeted with A6H Fab'-S-biotin was also observed. The avidin used in the study was radioiodinated with the N-hydroxysuccinimidyl ester of p-[125I]iodobenzoate ([125I]PIB-NHS). Very low concentrations (e.g. 0.35% ID/g) of avidin colocalized at the tumor. To further show that specific colocalization within the tumor xenografts had occurred with biotinylated A6H Fab', radioiodinated avidin and r-streptavidin were co-injected into athymic mice bearing tumor xenografts to obtain their distributions without having biotinylated Fab' present. At 20 h postinjection, only small differences in the blood and tumor concentrations of either protein were observed, indicating that the specific tumor colocalization seen in the previous two biodistributions must have been due to the presence of Fab'-S-biotin. Calculations were conducted to estimate how much r-streptavidin (as a molar ratio) was colocalized. From the data obtained it was estimated that 36-61% of the tumor-localized Fab'-S-biotin molecules were bound with r-streptavidin and 4-23% bound with avidin, under the conditions studied.

A new method of preparing radiolabeled cobalamin derivatives has been developed. The method involves the use of cobalamin-tri-n-butylstannyl hippurate conjugates as intermediates to obtain radioiodinated cobalamin-iodohippurate conjugates. The arylstannyl functionality was used as an exchangeable group to obtain high specific activity radioiodinations and to circumvent some deleterious side reactions common to cobalamins under electrophilic iodination conditions. The first step in the synthesis of tri-n--butylstannyl hippurate conjugates was to obtain free carboxylate groups on the cobalamin moiety. This was accomplished by mild acid hydrolysis of the b-, d-, or e-propionamide side chains on the corrin ring, followed by careful separation of the isomeric products. The second step was to couple a linking molecule (diaminododecane) to the carboxylate. The final step was to conjugate p-tri-n-butylstannyl hippurate to the cobalamin-diaminododecane adduct. All three isomeric cobalamin-p-tri-n-butylstannyl hippurate conjugates were prepared, as were the corresponding cobalamin-p-iodohippurate conjugates (HPLC standards). Radioiodination reactions were conducted with N-chlorosuccinimide and Na[*I]I in Me OH using conditions previously developed for arylstannylations. However, unlike the previous reactions, a key factor in obtaining the desired radioiodinated cobalamins was that the reaction be conducted under neutral conditions. Isolated yields of 40-65% were obtained for all three cobalamin isomers. Specific activities of 10-33% theoretical were obtained for the radioiodinated cobalamins. Evaluation of competitive binding of (nonradioactive) cobalamin-iodohippurate conjugates with recombinant human transcobalamin II showed that the e-isomer bound nearly as well as [57Co]cyanocobalamin (50%), whereas the b-isomer had decreased binding (6%) and the d-isomer was significantly decreased in its binding (0.7%). Two biodistributions of the radioiodinated e-isomer were conducted in athymic mice. One biodistribution investigated tissue localization in mice bearing a renal cell carcinoma xenograft, and the other biodistribution investigated tissue localization when the radioiodinated cyanocobalamin was mixed with 1% BSA prior to injection. A comparison of the results of the two biodistributions and a discussion of how they relate to previous [57/60Co]cyanocobalamin biodistributions are provided.

An investigation was conducted to evaluate the feasibility of site-selective addition of diagnostic and therapeutic agents to monoclonal antibody F(ab')2 fragments through cross-linking of antibody Fab' fragments. In the investigation, trifunctional equilibrium transfer alkylation cross-link (ETAC) reagents, 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid, 1a, N-[4-[2,2bis[(p-tolylsulfonyl)methyl]acetyl]-benzoyl]-4- (tri-n-butylstannyl)phenethylamine, 3a, and N-[4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]- benzoyl]-4-[125,131I]iodophenethylamine, 3b, were synthesized. The ETAC derivatives were reacted with Fab' fragments of an antirenal cell carcinoma antibody (A6H) produced from reduction of F(ab')2 using 1,4-dithiothreitol. Cross-linking of Fab' was obtained to yield a radioiodinated modified F(ab')2, [mF(ab')2], fragment. The cross-linking reaction produced mixed addition products, requiring the desired mF(ab')2 to be separated from radioiodinated Fab' by size exclusion HPLC. Tumor cell binding immunoreactivities varied (60-90%) for five isolated mF(ab')2 preparations but were consistent with other radiolabeled antibody preparations tested on the same day. In vitro stability testing indicated that the mF(ab')2 was reasonably stable toward loss of the ETAC cross-linking reagent, except under strongly basic conditions. Under reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, protein bands believed to be cross-linked heavy chain dimers were observed. Biodistribution of purified radioiodinated A6H mF(ab')2 was conducted in athymic mice bearing a renal cell carcinoma xenograft (TK-82). A nonmodified control A6H F(ab')2, radioiodinated as a p-[125,131I]-iodobenzoyl conjugate, was coinjected for comparison. The radioiodionated mF(ab')2 had a similar distribution to the radioiodinated control at 3.5, 19, and 43 h postinjection. In another study, the distribution of radioiodinated A6H Fab' was evaluated at 4 and 24 h to establish clearance and pharmacokinetics for comparison with the data obtained from the mF(ab')2. The biodistribution data indicated that A6H mF(ab')2 was quite different from that of A6H Fab'. The results from this preliminary study suggest that it may be possible to attach (large polymeric) diagnostic or therapeutic agents to monoclonal antibody F(ab')2 fragments through the use of ETAC reagents.

A preliminary investigation of an 211At labeled anti-renal cell carcinoma antibody fragment, A6H F(ab′)2, was conducted. In the investigation, A6H F(ab′)2 was labeled by conjugation with N-succinimidyl p-[211At]astatobenzoate, and the in vivo biodistribution was evaluated in athymic mice bearing TK-82 renal cell carcinoma xenografts. As a control, p-[125I]iodobenzoyl labeled A6H F(ab′)2 was coinjected with the astatinated F(ab′)2. The data obtained demonstrated that the two radiolabels (211At and 125I) had quite similar distributions, providing evidence that the 211At remained attached to the A6H F(ab′)2in vivo. Further, the astatinated antibody attained a 2:1 tumor-to-blood ratio, and greater than 35:1 tumor-to-muscle ratio, at 4 h post-injection, suggesting that this antibody conjugate could be used to evaluate treatment of metastatic renal cell carcinoma in a mouse model.