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.

The development of protein conjugate therapeutics requires control over the site of modification to allow for reproducible generation of a product with the desired potency, pharmacokinetic, and safety profile. Placement of a single nonnatural amino acid at the desired modification site of a recombinant protein, followed by a bioorthogonal reaction, can provide complete control. To this end, we describe the development of copper-catalyzed azide-alkyne cycloaddition (CuAAC, a click chemistry reaction) for site-specific PEGylation of interferon β-1b (IFNb) containing azidohomoalanine (Aha) at the N-terminus. Reaction conditions were optimized using various propargyl-activated PEGs, tris(benzyltriazolylmethyl)amine (TBTA), copper sulfate, and dithiothreitol (DTT) in the presence of SDS. The requirement for air in order to advance the redox potential of the reaction was investigated. The addition of unreactive PEG diol reduced the required molar ratio to 2:1 PEG-alkyne to IFNb. The resultant method produced high conversion of Aha-containing IFNb to the single desired product. PEG-IFNbs with 10, 20, 30, and 40 kDa linear or 40 kDa branched PEGs were produced with these methods and compared. Increasing PEG size yielded decreasing in vitro antiviral activities along with concomitant increases in elimination half-life, AUC, and bioavailability when administered in rats or monkeys. A Daudi tumor xenograft model provided comparative evaluation of these combined effects, wherein a 40 kDa branched PEG-IFNb was much more effective than conjugates with smaller PEGs or unPEGylated IFNb at preventing tumor growth in spite of dosing with fewer units and lesser frequency. The results demonstrate the capability of site-specific nonnatural amino acid incorporation to generate novel biomolecule conjugates with increased in vivo efficacy.

[reaction: see text] Furanomycin is a Streptomyces metabolite that substitutes for isoleucine in protein translation. We report a concise and modular synthesis starting from the Garner aldehyde and proceeding in seven steps to furanomycin. The key steps include a stereoselective acetylide addition and the Ag+-mediated cyclization of an alpha-allenic alcohol to construct the trans-2,5-dihydrofuran. The efficiency (12% overall yield) and flexibility of the route will provide ample quantities of furanomycin and analogues for protein engineering.

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.

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.