Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogenous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60−90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo, and are well tolerated at efficacious doses.Monoclonal antibodies (mAbs) have been used extensively as carriers of fluorophores, radionuclides, cytotoxic agents, and enzymes, yielding conjugates that find utility in therapeutic (1-3) and imaging applications (4,5), ELISA-based assays (6), as well as for the investigation of protein structure and dynamics (7). The methods employed for making mAbbased conjugates can be classified in two general categories: those that involve the random modification of mAb amino acid residues, and those that are highly regioselective. Examples of random modification procedures include the acylation of lysine ε-amino groups (8), alkylation of tyrosines (9), and amidation of carboxylates (10). The biological and functional properties of these conjugates are often acceptable, however random modification of mAbs may impair antigen binding and leads to conjugate heterogeneity.In the past several years, a number of selective methods have been described to introduce molecules of interest onto mAbs. The ability to control the location and stoichiometry of conjugation can significantly improve the properties of mAb conjugates in some applications. The greatest selectivities are obtained using recombinant technologies for the production of fusion proteins (11)(12)(13)(14). Selective modification has also been reported for such chemically based methods as reductive amination of oxidized mAb carbohydrates (15), photoaffinity labeling of unconventional mAb binding sites (16), and reduction-alkylation of antibody interchain disulfides (17,18 We have previously described the preparation of mAb-drug conjugates for use as antitumor agents (17,19). The potent a...

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We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the ␤-and di-␣-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNA Leu , similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)