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.

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oximebonded, site-specific NDCs were even more apparent when lowantigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.A ntibody drug conjugates (ADCs) are emerging as a new class of anticancer therapeutics that combine the efficacy of small-molecule therapeutics with the targeting ability of an antibody (Ab) (1, 2). By combining these two components into a single molecular entity, highly cytotoxic small-molecule drugs (SMDs) can be delivered to cancerous target tissues, thereby enhancing efficacy while reducing the potential systemic toxic side effects of the SMD. Conventional ADCs are typically produced by conjugating the SMD to the Ab through the side chains of either surface-exposed lysines or free cysteines generated through reduction of interchain disulfide bonds (3, 4). Because antibodies contain many lysine and cysteine residues, conventional conjugation typically produces heterogeneous mixtures that present challenges with respect to analytical characterization and manufacturing. Furthermore, the individual constituents of these mixtures exhibit different pharmacology with respect to their pharmacokinetic, efficacy, and safety profiles, hindering a rational approach to optimizing this modality (5).Recently, it was reported that the pharmacological profile of ADCs may be improved by applying site-specific conjugation technologies that make use of surface-exposed cysteine residues engineered into antibodies (THIOMABS) that are then conjugated to the SMD, resulting in site-specifically conjugated ADCs (TDCs) with defined Ab-drug ratios. Rel...

Integrins are a diverse family of heterodimeric (alphabeta) adhesion receptors recently shown to be concentrated within synapses and involved in the consolidation of long-term potentiation. Whether neuronal types or anatomical systems in the adult rat brain are coded by integrin type was studied in the present experiments by mapping the relative densities of mRNAs for nine alpha and four beta subunits. Expression patterns were markedly different and in some regions complementary. General results and areas of notable labeling were as follows: alpha1-limited neuronal expression, neocortical layer V, hippocampal CA3; alpha3 and alpha5-diffuse neuronal and glial labeling, Purkinje cells, hippocampal stratum pyramidale, locus coeruleus (alpha3); alpha4- discrete limbic regions, olfactory cortical layer II, hippocampal CA2; alpha6-most prominently neuronal, neocortical subplate, endopiriform, subiculum; alpha7-discrete, all neocortical layers, hippocampal granule cells and CA3, cerebellar granule and Purkinje cells, all efferent cranial nerve nuclei; alpha8-discrete neuronal, deep cortex, hippocampal CA1, basolateral amygdala, striatum; alphaV-all cortical layers, striatum, Purkinje cells; beta4-dentate gyrus granule cells; beta5-broadly distributed, neocortex, medial amygdala, cerebellar granule and Purkinje cells, efferent cranial nerve nuclei; alpha2, beta2, and beta3-mRNAs not detected. These results establish that brain subfields express different balances of integrin subunits and thus different integrin receptors. Such variations will determine which matrix proteins are recognized by neurons and the types of intraneuronal signaling generated by matrix binding. They also could generate important differences in synaptic plasticity across brain systems.

Fibroblast growth factor 21 (FGF21) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF21 analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF21 for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF21 ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF21 analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF21 at a specific and preferred site confers superior metabolic pharmacology.

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

The identification of leptin as a mediator of body weight regulation provided much initial excitement for the treatment of obesity. Unfortunately, leptin monotherapy is insufficient in reversing obesity in rodents or humans. Recent findings suggest that amylin is able to restore leptin sensitivity and when used in combination with leptin enhances body weight loss in obese rodents and humans. However, as the uniqueness of this combination therapy remains unclear, we assessed whether co-administration of leptin with other weight loss-inducing hormones equally restores leptin responsiveness in diet-induced obese (DIO) mice. Accordingly, we report here the design and characterization of a series of site-specifically enhanced leptin analogs of high potency and sustained action that, when administered in combination with exendin-4 or fibroblast growth factor 21 (FGF21), restores leptin responsiveness in DIO mice after an initial body weight loss of 30%. Using either combination, body weight loss was enhanced compared with either exendin-4 or FGF21 monotherapy, and leptin alone was sufficient to maintain the reduced body weight. In contrast, leptin monotherapy proved ineffective when identical weight loss was induced by caloric restriction alone over a comparable time. Accordingly, we find that a hypothalamic counter-regulatory response to weight loss, assessed using changes in hypothalamic agouti related peptide (AgRP) levels, is triggered by caloric restriction, but blunted by treatment with exendin-4. We conclude that leptin re-sensitization requires pharmacotherapy but does not appear to be restricted to a unique signaling pathway. Our findings provide preclinical evidence that high activity, long-acting leptin analogs are additively efficacious when used in combination with other weight-lowering agents.

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5 leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the ␣ subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5 untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and ␣-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5 untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5 leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

We recently reported restoration of leptin responsiveness in diet-induced obese (DIO) mice using a pharmacologically optimized, polyethylene-glycolated (PEG)-leptin analog in combination with exendin-4 or FGF21. However, the return of leptin action required discontinuation of high-fat diet (HFD) exposure. Here we assess whether a single peptide possessing balanced coagonism at the glucagon-like peptide 1 (GLP-1) and glucagon receptors can restore leptin responsiveness in DIO mice maintained on a HFD. DIO mice were treated with PEG-GLP-1/glucagon (30 nmol/kg every fourth day) to induce an ∼15% body weight loss, upon which they were randomized to continue PEG-GLP-1/glucagon therapy or reassigned to receive supplemental daily PEG-leptin (185 nmol/kg/day). The addition of PEG-leptin to PEG-GLP-1/glucagon resulted in an ∼18% greater weight loss as compared with PEG-GLP-1/glucagon alone and was accompanied by further decreases in food intake and improved glucose and lipid metabolism. The beneficial effect of PEG-leptin supplementation occurred after an initial body weight loss similar to what we previously reported following reduced dietary fat along with PEG-leptin and exendin-4 or FGF21 cotreatment. In summary, we report that GLP-1/glucagon coagonism restores leptin responsiveness in mice maintained on a HFD, thus emphasizing the translational value of this polypharmacotherapy for the treatment of obesity and diabetes.