Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

Preparation of bis(beta-trimethylsilylethanesulfonyl)imide and its use in the synthesis of protected amine derivatives.

Bis(beta-trimethylsilylethanesulfonyl)imide (SES(2)NH) can be easily prepared in 85% yield by alkylation of the trianion of bismethanesulfonimide with 2 equiv of commerically available (iodomethyl)trimethylsilane. This synthon undergoes effective Mitsunobu alkylation reactions with both primary and secondary alcohols to afford the corresponding bis-SES imides. These imides can be selectively cleaved to the mono-SES-protected amines, and in addition undergo a one-pot cleavage/N-alkylation to afford SES derivatives of secondary amines.

A mild, convenient, and inexpensive procedure for conversion of vinyl halides to alpha-haloketones.

Treatment of a vinyl chloride with commercially available aqueous sodium hypochlorite solution in a 2:5 mixture of acetic acid/acetone at 0 degrees C for about 1 h cleanly leads to the corresponding alpha-chloroketone. Similarly, if a vinyl bromide is exposed to sodium hypobromite (freshly prepared from bromine and sodium hydroxide) at 0 degrees C in 2:5 acetic acid/acetone as solvent, an alpha-bromoketone is produced. This methodology has been applied to a number of vinyl chlorides and vinyl bromides, and the transformations generally proceed in high yields. The mild reaction conditions are compatible with a variety of functional groups including amides, esters, and imines.