Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p -Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2 + cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe–Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well–defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe–Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered.

No abstract

Summary We report a new strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.

Kinesin molecular motors harness the energy of ATP hydrolysis to transport cargo such as vesicles and organelles along intracellular microtubules. Purified components of this system can be used for nanoscale transport by integrating the motors and filaments into MEMS and NEMS devices. [1][2][3][4] Hence, it is important to understand the function of these proteins for biological, therapeutic, and nanotechnological applications. Existing techniques for studying motors include the microtubule gliding assay, [5] optical traps, [6] and ATPase assays. [7] Single-molecule visualization is crucial for investigating the motor mechanism and their ability to move and assemble nanoparticles. [8][9][10] In this report, we synthesize semiconductor nanocrystals, attach them to kinesins, demonstrate that single motors can be visualized by simple epifluorescence or evanescent wave microscopy, and show that motor function is unaffected by particle functionalization.Single kinesin motors functionalized with green fluorescent protein (GFP) or synthetic fluorophores can be imaged by total internal reflection fluorescence (TIRF) microscopy, [8] and their position resolved to within nearly one nanometer. [11] By tracking kinesins in which one of the two motor domains (heads) was labeled, this technique was used to show that at limiting ATP concentrations each head takes 16-nm steps along a microtubule, ruling out the "inchworm" model of kinesin motility. [11] However, because the spatial resolution is based on the number of photons collected, the temporal resolution using these fluorophores is limit-ed to roughly 300 ms. Brighter fluorophores are needed to measure faster events. While fluorescent beads have higher signal intensities, their size alters the diffusion properties of the tagged molecule and complicates intracellular experiments.Semiconductor nanocrystals (quantum dots) have great potential in biological imaging due to their small size ( % 5-10 nm radius with functionalization), high quantum yield, large excitation band, and negligible photobleaching. Quantum dots with different optical properties can be synthesized with ease by growing them to different sizes, [12] and single fluorophores can be visualized by simple epifluorescence microscopy rather than the evanescent wave microscopy that is generally required for GFP and other synthetic fluorophores. In addition, they can be introduced into cells by a variety of methods. [13] By synthesizing our own quantum dots, we have the advantage of being able to separately tune the emission wavelength and control the surface functionality.The goal of this study is to functionalize quantum dots with active kinesin biomolecular motors and transport these dots along immobilized microtubules. This new labeling approach will open up a number of avenues of investigation. First, it will enable more precise tracking of motors in vitro to understand motor stepping and detachment under controlled conditions. Second, these bright particles should enable individual kinesins to be followed in c...