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Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Mutations have been constructed that delete either one or two base pairs near position -19 in the lac pS promoter. Deletion of either of two adjacent base pairs increases the rate of open complex formation by nearly an order of magnitude. Two promoters that have different single-base deletions are indistinguishable by either their rates of open complex formation or stability of the open complexes once formed. However, simultaneous deletion of both base pairs produces a promoter that forms complexes at a rate similar to that of the unmodified DNA sequence. The maximal rate of open complex formation is achieved at a spacer length of 17 base pairs, the most frequently occurring spacer length among promoters. These results suggest that the spacing between the two strongly conserved regions of sequence homology is an important determinant of the rate of open complex formation. A model is suggested that proposes that three important promoter elements, the -10 region, the -35 region, and the spacer region, act simultaneously to facilitate open complex formation by RNA polymerase.The promoter is a DNA sequence that directs RNA polymerase to bind and initiate transcription specificially; Comparison of prokaryotic promoter sequences has shown two regions of sequence homology located -10 and 35 base pairs prior to the start point of transcription (1, 2). The precise roles of each of these regions in promoter function are unknown. The importance of these homologies is supported by the concentration of promoter mutations in these sequences. In general, sequence changes that decrease homology to the consensus tend to be down promoter mutations.The region between the -10 and -35 sequences has not been shown to be of importance. These sequences are poorly conserved and few mutations exist in this region. Nevertheless, the number of intervening nucleotides is highly conserved (1). This suggests that proper spacing between the -10 and -35 sequences may be functionally important and prompts us to identify this as a third region of the promoter, the "spacer" region.To test the importance of this conserved spacer length, we have constructed mutations that change the spacer length in the lac pS promoter. This promoter has a spacer region one nucleotide longer than the consensus length. Deletion in vitro yields promoter DNA that is of consensus length and shorter. The properties of these strains lend support to the hypothesis that the spacer region is a third important element in promoter function. MATERIALS AND METHODSRNA polymerase holoenzyme was prepared according to Gonzales et at (3). Nuclease S1 was purchased from Miles and purified on sulfopropyl-Sephadex by a modification of Vogt (4).Bovine serum albumin (fraction V; Pentex) was purified by passage over a heparin agarose column. Other materials were as described (5).For construction of deletion mutants (see Fig. 1), EcoRI restriction fragments containing the ps promoter (5.5 ,ug; ref. 6) were ligated overnight at 15'C. The resultant polymer was cleaved with Hpa II. Ha...

The rates of productive and abortive initiation of transcription in vitro at the lac UV5 promoter have been determined and compared to values determined for phage lambda and T7 promoters. The rate constants for productive initiation of lac transcript are consistently lower over a range of low to moderate concentration of initiating nucleoside triphosphate (ATP). Abortive initiation of lac dinucleoside tetraphosphate is also slower at low to moderate concentrations of ATP. These data demonstrate the existence of significant differences in initiation rate among promoters. We suggest that these differences may be a consequence of the initial mRNA sequences and extents of RNA polymerase cycling during initiation of promoter-specific transcription.

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